mohamad hamshou- thesis- 2012
TRANSCRIPT
ir. Mohamad HAMSHOU
Thesis submitted in fulfillment of the requirements for the degree of
Doctor (PhD) in Applied Biological Sciences
Toxicity and mode of action of fungal lectins
in pest insects important in agriculture
Promoters: Prof. dr. ir. Guy Smagghe
Ghent University
Department of Crop Protection
Laboratory of Agrozoology
Prof. dr. Els J.M. Van Damme
Ghent University
Department of Molecular Biotechnology
Laboratory of Biochemistry and Glycobiology
Dean: Prof. dr. ir. Guido Van Huylenbroeck
Rector: Prof. dr. Paul Van Cauwenberge
Mohamad Hamshou (2012). Toxicity and mode of action of fungal lectins in pest insects
important in agriculture. PhD thesis, Ghent University, Ghent, Belgium.
ISBN-number 978-90-5989-525-6
The author and the promoters give the authorization to consult and to copy parts of this work
for personal use only. Any other use is limited by the Laws of Copyright. Permission to
reproduce any material contained in this work should be obtained from the author.
The promoters: The author:
Prof. dr. ir. Guy Smagghe Prof. dr. Els JM Van Damme ir. Mohamad Hamshou
Members of the examination committee
Prof. dr. ir. Guy Smagghe (promoter)
Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Belgium
Prof. dr. Els JM Van Damme (promoter)
Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Ghent University,
Belgium
Prof. dr. ir. Patrick Van Damme (chairman)
Department of Plant Production, Faculty of Bioscience Engineering, Ghent University, Belgium
Prof. dr. ir. Monica Höfte
Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Belgium
Prof. dr. ir. Marie-Christine Van Labeke
Department of Plant Production, Faculty of Bioscience Engineering, Ghent University, Belgium
Prof. dr. Jozef Vanden Broeck
Department of Biology, Animal Physiology and Neurobiology, Zoological Institute, Katholieke
Universiteit Leuven
Prof. dr. ir. Peter Bossier
Department of Animal Production, Laboratory of Aquaculture & Artemia Reference Center,
Faculty of Bioscience Engineering, Ghent University, Belgium
ACKNOWLEDGMENT
I acknowledge the presence of God who created me and gave me this rare privilege to
achieve my dream of attaining the highest qualification. This thesis is an output of several
years of research that has been done since I came to Ghent. Since that time, I have worked
with many people whose support and collaboration in various and diverse ways contributed
to this great success of my thesis. It is a pleasure to convey my gratitude to them all in my
humble acknowledgment.
I am highly indebted to my supervisors Prof. Dr. ir. Guy Smagghe and Prof. Dr. Els Van
Damme who taught and supervised me during these years of unraveling the mysteries behind
lectin-insect interactions in the Laboratory of Agrozoology and the Laboratory of
Biochemistry and Glycobiology. Guy and Els, it is a great honor to work with you. Without
any doubt, your efforts were putting me on the right path. I will never forget your guidance
and the help you gave me even during weekends, holidays and all other opportunities “Heel
Hartelijk Bedankt”.
My kind regards to Prof. Dr ir Hussein AL-Mohammad (Aleppo University) and Prof. Dr ir.
Roland Verhé who first introduced me to UGent and helped me to find the opportunity to do
my PhD in UGent.
I sincerely thank the chairman of the jury committee, Prof. Dr. ir. Patrick Van Damme and
the other jury members, Prof. Dr. ir. Monica Höfte, Prof. Dr. ir. Marie-Christine Van
Labeke, Prof. Dr. Jozef Vanden Broeck, Prof. Dr. ir. Peter Bossier.
I wish to express my profound appreciation to my colleagues at the laboratory of
Agrozoology for the friendly atmosphere and cooperation, Prof. dr. ir. L. Tirry, Prof. dr. ir. P.
De Clercq, S. Shahidi-Noghabi, A. Sadeghi, A. Jalali, E. De Geyter, S. Bahrami-Kamangar,
J. Maharramov, S. Jacques, O. Christiaens, R. De Wilde, T. Walski, Na Yu, N. De Zutter, A.
Billiet, N. Shoker, N. Berkvens, M. Bonte, T. Soin, Yves Verhaegen, Katrien Michiels, B.
Ingels, P. Demaegt, T. Machtelinckx, H. Huvenne, J. Bonte, A. Bryon, W. Dermauw, T. Van
Leeuwen, S. Maes, J. Moens, G. Herregods, J. Liu, K. Maebe, D. Staljanssens, P. Van
Nieuwenhuyse, I. Meeus, H. Mosallanejad and S. Caccia.
I express my deep gratitude to every member of the Departement of Molecular
Biotechnology, N. Lannoo, E. Fouquaert, G. Vandenborre, B. Al Atalah, A. Delporte, Ch.
Shang, K. Stefanowicz, D. Schouppe, B. Nagels, J. Van Hove and W. De Vos.
I must thank D. Van De Velde, R. Van Caenegem, L. Dierick, B. Vandekerkhove, R.
Termote-Verhalle, K. Plas, I. Tilmant, G. Meesen, S. De Schynkel and F. De Block, and the
technical and assistant staffs of the laboratories.
Hereby I also thank Prof. Dr. Kris Gevaert and Bart Ghesquière (VIB, Department of
Medical Protein Research) for their help with the proteomics analysis.
I sincerely thank Ruben De Wilde for his kind help of Dutch translation of the thesis
summary.
I would like to express my gratitude to all my Syrian friends in Belgium and their families
who have helped me during my study, especially Tarad, Abd Al Karim, M. Khlosy, M. AL-
Abed, Tamer, Kosy, M. Shehab, M. Moslet, M. Al- Shoker, M. Akash, M. AL-Hazaa, Hanan,
Fateh, Raki, Ammar, Ehab, Ola and many other friends of the Syrian community in Ghent.
I also wish to send my sincere gratitude to the General Commission for Scientific
Agricultural Research and the Ministry of Higher Education (especially Mrs. Eyman & Heba)
in Syria who supported me to pursue my stay and education in Belgium. I would like also to
send my gratitude to the Syrian embassy in Belgium (especially Mr. Yamen & Fayez).
I am very grateful to my mother and father. Their prayers, passionate encouragements and
generosities have followed me everywhere to give me a lot of power. My deepest gratitude
goes to my sisters and brothers. I wish to send my best regards to my wife’s family especially
my mother and father-in-low. I wish all of you a prosperous life full of happiness and health.
My lovely wife “Dalal” and my adorable children “Maria, Ahmad and Wesam”, you were
the main supporters of me along my entire PhD thesis. I am deeply grateful for your patience
and sacrifices. I hope I can compensate you with all my love for all the moments which I
spent far away from you.
Mohamad
May 2012
Table of content
List of abbreviations
Scope
Chapter 1: literature
1.1 AGRICULTURE 2
1.2 INSECTS 2
1.2.1 Hemiptera 4
1.2.2 Lepidoptera 6
1.2.3 Insect gut 8
1.2.4 Insect cell lines 10
1.3 CROP PROTECTION 11
1.3.1 Current control strategies 12
1.3.1.1 Lectins as bio insecticidal agent 12
1.4 INSECT GLYCOSYLATION PATTERNS 15
1.5 APOPTOSIS 17
1.5.1 The insect caspases 20
1.6 FUNGAL LECTINS: their toxicity and antiproliferative activity 24
1.6.1 Basidiomycota 30
1.6.2 Ascomycota 42
1.6.3 Discussion 45
1.6.3.1 Classification 45
1.6.3.2 Localization 47
1.6.3.3 Specificity 48
1.6.3.4 Molecular mass and subunit composition 48
1.6.3.5 Biological activity 48
1.6.3.5.1 Anti-virus activity 48
1.6.3.5.2 Anti-fungal activity 49
1.6.3.5.3 Anti-amoeba activity 49
1.6.3.5.4 Anti-nematode activity 49
1.6.3.5.5 Anti-insect activity 50
1.6.3.5.6 Anti-mice/rat activity 50
1.6.3.5.7 Cytotoxicity and antiproliferative activity 50
1.6.3.6 Mechanisms of fungal lectin activity 51
1.6.4 Conclusions 51
Chapter 2: Analysis of lectin concentrations in different Rhizoctonia solani strains
2.1 ABSTRACT 54
2.2 INTRODUCTION
…………………………………………………………….……………. 55
2.3 MATERIALS AND METHODS 57
2.3.1 Isolates and growth conditions 57
2.3.2 Protein extraction 57
2.3.3 Determination of total protein content 58
2.3.4 Analysis of lectin activity in different Rhizoctonia strains 58
2.3.5 Gel electrophoresis 58
2.4 RESULTS 58
2.4.1 Agglutination assays 58
2.4.2 Protein analysis 59
2.5 DISCUSSION 60
Chapter 3: Entomotoxic effects of fungal lectin from Rhizoctonia solani
towards Spodoptera littoralis
3.1 ABSTRACT 64
3.2 INTRODUCTION
…………………………………………………………….……………. 65
3.3 MATERIALS AND METHODS 66
3.3.1 Isolation of RSA 66
3.3.2 Insects 67
3.3.3 Effects of RSA feeding on insect survival, growth and development 67
3.3.4 Effect of RSA combined with Bt toxin 68
3.3.5 Statistical analysis 68
3.4 RESULTS 69
3.4.1 Effects of RSA feeding on insect survival, growth and development 69
3.4.2 Effects of RSA combined with Bt toxin 73
3.5 DISCUSSION 73
Chapter 4: Insecticidal properties of Sclerotinia sclerotiorum agglutinin and its
interaction with insect tissues and cells
4.1 ABSTRACT 78
4.2 INTRODUCTION
…………………………………………………………….……………. 79
4.3 MATERIALS AND METHODS 81
4.3.1 Pea aphids 81
4.3.2 Insect midgut CF-203 cell line and culture conditions 81
4.3.3 Purification of SSA 81
4.3.4 FITC-labeling of SSA 81
4.3.5 Treatment of A. pisum with SSA via artificial liquid diet 82
4.3.6 Histofluorescence for localization of SSA in aphid body tissues 83
4.3.7 Cytotoxic effect of SSA in insect midgut CF-203 cells 83
4.3.8 DNA fragmentation analysis 84
4.3.9 Caspase-3 activity assay 84
4.3.10 Uptake of SSA in midgut CF-203 cells 84
4.3.11 Effect of saponin on toxicity and uptake of SSA in midgut CF-203 cells 85
4.3.12 Effect of carbohydrates and glycoprotein on toxicity of SSA in midgut
CF-203 cells 85
4.4 RESULTS 86
4.4.1 Insecticidal effects of SSA on pea aphids 86
4.4.2 Localization of SSA upon feeding in aphid body tissues 86
4.4.3 Cytotoxicity of SSA in insect midgut CF-203 cells 87
4.4.4 DNA fragmentation and caspase-3 activity in midgut CF-203 cells upon
exposure to SSA 87
4.4.5 Internalization of SSA in midgut CF-203 cells 89
4.4.6 Inhibitory effect of carbohydrates and glycoprotein on SSA toxicity in
midgut CF-203 cells 90
4.5 DISCUSSION 91
Chapter 5: High entomotoxic activity of the GalNAc/Gal-specific Rhizoctonia solani
lectin in pest insects relies on caspase 3-independent midgut cell apoptosis
5.1 ABSTRACT 100
5.2 INTRODUCTION
…………………………………………………………….……………. 101
5.3 MATERIALS AND METHODS 102
5.3.1 Insects 102
5.3.2 Purification of RSA and labeling with FITC 102
5.3.3 Treatment of S. littoralis with RSA via artificial diet 102
5.3.4 Treatment of A. pisum with RSA via artificial diet 103
5.3.5 Histofluorescence procedures 103
5.3.6 Bioassay with insect midgut cell cultures 103
5.3.7 Effect of sugars on cell toxicity of RSA in midgut CF-203 cells 103
5.3.8 Uptake of RSA in CF-203 cells 103
5.3.9 Primary cell cultures 104
5.3.10 Effect of saponin on the cytotoxicity and uptake of RSA in CF-203 cells 104
5.3.11 DNA fragmentation and nuclear staining with Hoechst in the midgut cells 104
5.3.12 Caspase activity assay in midgut cells 105
5.3.13 Isolation of binding partners of RSA from the membrane of midgut cells 105
5.4 RESULTS 107
5.4.1 Insecticidal effects of RSA on cotton leafworm caterpillars and pea aphids 107
5.4.2 Localization of RSA in the insect body of caterpillars and aphids 107
5.4.3 Cellular toxicity of RSA in midgut cells 110
5.4.4 Effect of carbohydrates on RSA toxicity in midgut CF-203 cells 111
5.4.5 Uptake of RSA in the midgut cells 111
5.4.6 DNA fragmentation analysis and nuclear condensation in midgut cells by
RSA 113
5.4.7 Caspase activity in midgut cells upon exposure to RSA 114
5.4.8 Proteomic analysis of soluble and membrane proteins of midgut cells bound
to RSA column 115
5.5 DISCUSSION 118
Chapter 6: GalNAc/Gal-binding Rhizoctonia solani agglutinin has antiproliferative
activity in Drosophila melanogaster S2 cells via MAPK and JAK/STAT signaling
pathways
6.1 ABSTRACT 124
6.2 INTRODUCTION
…………………………………………………………….……………. 125
6.3 MATERIALS AND METHODS 126
6.3.1 Isolation of lectins and labeling with FITC 126
6.3.2 Cell proliferation assay 127
6.3.3 Effect of carbohydrates on RSA antiproliferative activity on S2 cells 128
6.3.4 RSA activity in S2 cells following pre-incubation with kinase inhibitors 128
6.3.5 Internalization assay 128
6.3.6 DNA fragmentation analysis in S2 cells 128
6.3.7 Nuclear staining with Hoechst dyes 129
6.3.8 Proteomic analysis of the RSA binding proteins in the membrane of S2 cells 129
6.4 RESULTS 131
6.4.1 RSA causes inhibition of cell proliferation in S2 cells 131
6.4.2 Importance of carbohydrate binding for antiproliferative activity of RSA 131
6.4.3 Binding and internalization of RSA compared to plant lectins 133
6.4.4 RSA treatment does not induce apoptosis 134
6.4.5 Effect of kinase inhibitors on RSA activity 134
6.4.6 Proteomic analysis of membrane proteins of S2 cells retained on RSA
affinity column 135
6.5 DISCUSSION 136
Chapter 7: GENERAL DISCUSSION, CONCLUSIONS AND PERSPECTIVES FOR
FUTURE RESEARCH
7.1 GENERAL DISCUSSION 146
7.1.1 Fungi as a source for bioactive compound 146
7.1.2 Fungal lectins as bio-insecticidal proteins 146
7.1.3 The midgut as primary target for RSA and SSA 149
7.1.4 Study of RSA and SSA binding at cellular level 150
7.1.5 Investigation of the mode of action of RSA and SSA at cellular level 151
7.1 GENERAL CONCLUSIONS 156
7.2 PERSPECTIVES FOR FUTURE RESEARCH 158
Summary/Samenvatting 161
Summary 162
Samenvatting 165
References 169
Curriculum Vitae 205
Appendix 211
List of abbreviations
AAL Agaricus arvensis lectin
ABL Agaricus bisporus lectin
ACL Agrocybe cylindracea lectin
AG Anastomosis group
ALG-2 Apoptosis-linked gene-2
ANOVA Analysis of variance
APA Allium porrum agglutinin
ASAL Allium sativum leaf agglutinin
BEL Boletus edulis lectin
Bm5 Ovarian insect cells
BPA Bauhinia purpurea agglutinin
Bt Bacillus thuringiensis
BVL Boletus venenatus lectin
CARD Caspase recruitment domain
CF-203 Midgut insect cells
CGL2 Coprinopsis cinerea galectin
CHAPS 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate
CL Confidence limits
CNL Clitocybe nebularis lectin
ConA Canavalia ensiformis agglutinin
CPB Fat body insect cells
cry Crystal toxin of Bacillus thuringiensis
Cut Outer cuticle
DAP 1,3-diaminopropane
DED Death effector domain
DISC Death-inducing signal complex
DMSO Dimethylsulfoxide
DNA Deoxyribonucleic acid
DTT Dithiothreitol
EDTA Ethylenediaminetetraacetic acid
FADD Fas-associated death domain
FAF-1 Fas-associated protein factor-1
Fas Death receptor on the cell surface
FBS Fetal bovine serum
FDR false discovery rate
FIP Fungal Immunomodulatory Protein
FITC Fluoresceine isothiocyanate
FVL Flammulina velutipes lectin
Gal Galactose
GalNAc N-acetylgalactosamine
GCL Ganoderma capense lectin
GFL Grifola frondosa lectin
GLL Ganoderma lucidum lectin
GNA Galanthus nivalis agglutinin
GPCR G-protein-coupled receptor
H2O2 Hydrogen peroxide
HEA Hericium erinaceum lectin
HEPES 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, N-(2-
Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid)
HIV-1 Human Immunodeficiency Virus 1
HPLC High performance liquid chromatography
IC50 The half maximal inhibitory concentration
IPM Integrated Pest Management
IRA Iris hybrid agglutinin
IUL Inocybe umbrinella lectin
JAK Janus kinase
KDa Kilodalton
KL-15 Boletopsis leucomelas lectin
LC50 The median lethal dose
LD50 The median lethal dose
LT50 Median lethal time
Lum Insect gut lumen
MEK MAP kinase
MG Midgut
MIC Minimum Inhibitory Concentration
mM Millimolar
MOA Marasmius oreades lectin
MTT (3-(4,5)dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
PAL Pholiota adiposa lectin
PBS Phosphate buffered saline
PCD Programmed Cell Death
PCL Pleurotus citrinopileatus lectin
PeCL Penicillium chrysogenum lectin
PHA Phaseolus vulgaris agglutinin
PJL Paecilomyces japonica lectin
PM Peritrophic membrane
PMSF Phenylmethylsulphonyl fluoride
PNA Peanut agglutinin
POL Pleurotus ostreatus lectin
RBL Rhizoctonia bataticola lectin
RDL Russula delica lectin
RFU Relative fluorescence units
RLL Russula lepida lectin
RLU Relative luminescence units
RSA Rhizoctonia solani agglutinin
RTK Receptor tyrosine kinases
S2 Embryonic insect cells
SCL Schizophyllum commune lectin
SD Standard error
SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis
SEM Standard Error of the Means
SNA-I’ Sambucus nigra agglutinin I’
SNA-II Sambucus nigra agglutinin II
SPSS Statistical Product and Service Solutions (formerly Statistical
Package for the Social Sciences)
SRL Sclerotium rolfsii lectin
SSA Sclerotinia sclerotiorum agglutinin
TAP Sordaria macrospora lectin
TML Tricholoma mongolicum lectin
TMV Tobacco mosaic virus
TNF Tumor necrosis factor
VVL Volvariella volvacea lectin
WGA Wheat germ agglutinin
XCL Xerocomus chrysenteron lectin
XHL Xylaria hypoxylon lectin
μM Micromolar
Scope
Several plant lectins have been reported to possess insecticidal activity towards different pest
insects. However, until now very little is known about the insecticidal activity of fungal
lectins. Therefore the main aim of this PhD research was to investigate the insecticidal
activity of some fungal lectins and to study their mode of action.
Chapter 1 gives a literature review about insects and lectins. The first part presents a survey
on the control of pest insects, the insect midgut, glycosylation in insects, regulation of cell
death in insects and the pest insects used in this project. In the second part of this chapter, an
overview is presented on fungal lectins with emphasis on the toxicity and antiproliferative
activity of these lectins towards different organisms.
The first aim of this work was to find a Rhizoctonia solani strain(s) that expresses a high
concentration of Rhizoctonia solani agglutinin (RSA) which would allow to purify sufficient
amounts of RSA for (bio)assays with insects and insect cells. In chapter 2 ten R. solani
strains belonging to different anastomosis groups were screened for the presence and the
amount of RSA in their mycelium as well as in the sclerotia. The major aim was to identify a
Rhizoctonia strain expressing high levels of lectin.
The second aim was to determine the insecticidal activity of RSA. In chapter 3 the effects of
RSA on the growth, development and survival of an economically important caterpillar in
agriculture and horticulture, the cotton leafworm, Spodoptera littoralis were investigated by
rearing this insect on an artificial diet containing different concentrations of RSA.
The third aim of this project was to study the insecticidal activity and the mode of action of
the fungal lectin isolated from Sclerotinia sclerotiorum (SSA). In chapter 4 the activity of
SSA on the survival of the piercing-sucking pest insect pea aphid Acyrthosiphon pisum was
studied using a liquid artificial diet. Moreover, binding of SSA to different tissues in the pea
aphid body was investigated upon oral exposure to FITC-labeled SSA. Further assays were
done at the cellular level using the insect midgut cell line (CF-203) to answer several
questions related to the toxicity of SSA. Answering these questions will help to understand
the mechanism of action of SSA.
The fourth aim was to investigate the mode of action of RSA. Since some proteins are active
against Lepidopteran insects but not to Hemipterans RSA was also tested for toxicity against
the pea aphid, A. pisum in chapter 5. Moreover, the target sites for RSA in the pea aphid as
well as in the cotton leafworm were analyzed using FITC-labeled RSA. To better understand
the mode of action of RSA, in vitro experiments were done using midgut CF-203 cells. First
the activity and interaction of RSA with CF-203 cells were investigated. Several experiments
were performed to examine the dependency of RSA activity on apoptosis induction including
DNA fragmentation, nuclear condensation and caspase activation. Second, RSA affinity
chromatography of soluble and membrane extracts of CF-203 cells was performed to identify
putative glycosylated proteins as potential binding partners for RSA.
To determine whether or not the activity of RSA is cell or organism dependent, in chapter 6
the activity and the interaction of RSA was investigated in a different insect cell line, S2 cells
derived from embryos of Drosophila melanogaster, by doing some similar assays as
mentioned in chapter 5. In addition the effect of several kinase inhibitors on RSA activity
against S2 cells was investigated. Moreover, the potential binding partners for RSA on S2
cells were identified using RSA affinity chromatography.
The obtained results allowed us to draw a working hypothesis to explain the mode of action of
RSA in both cell lines (CF-203 and S2 cells).
1
Chapter 1
Chapter 1
Literature
2
Chapter 1
1.1. AGRICULTURE
Estimations by the United Nations have predicted that the global human population will be
over 7 billion people in 2012 and the population is expected to exceed 9 billion in 2050 and
10 billion in 2100. These increases in the human population are also increasing the
requirement for food. Agriculture is considered the main source of food and also the main
economy of the less developed countries. Agriculture is facing many problems which lead to
losses in the crop production, such as insects, weeds and diseases.
1.2. INSECTS
Insects are invertebrate animals belonging to the arthropods. They are one of the most diverse
organisms on the Earth. Insects include more than a million described species and represent
more than 90% of the different metazoan life forms on our planet. The ability of insects to
live in almost each environment makes them the most successful organisms occupying this
planet and in this way they can affect many aspects of our lives.
Insects are considered worldwide as one of the biggest problems in agriculture by attacking
and damaging different crops. Losses in agricultural production due to insect pests have been
estimated at 16% of the total production worldwide (Oerke et al., 1994). An attempt to
minimize crop losses due to insects was concerned by many researchers and entomologists.
Insect‟s bodies can be divided into three distinct parts: head, thorax and abdomen (Fig. 1.1).
The head carries the compound eyes and two antennae. While the thorax carries three pairs of
segmented legs and two or four wings. More than 60% of all known herbivorous insect
species are leaf-eating beetles (Coleoptera) or caterpillars (Lepidoptera) that cause damage
with their biting-chewing mouthparts (Fig. 1.2A) (Schoonhoven et al., 1998). In contrast,
Hemipteran insects have different piercing-sucking mouthparts that include a needle-like
stylet bundle consisting of two mandibular and two maxillary stylets (Cranston et al., 2003)
(Fig. 1.2B). Taxonomically, insects (Insecta class) belong to the subphylum Hexapoda, the
phylum Arthropoda within the Animal kingdom (Fig. 1.3). The class Insecta is subdivided
into orders, for example the order Lepidoptera and Hemiptera. Orders are divided into
families, families into genera, and genera are divided into species.
3
Chapter 1
Figure 1.1. Schematic representation of insect‟s morphology
Figure 1.2. (A) Schematic representation of Lepidopteran mouthparts (http://www.amentsoc.org/
insects/fact-files/mouthparts.html). (B) Schematic representation of Hemipteran mouthparts
(http://insected.arizona.edu/enforcers/resource/hemipteran.html)
4
Chapter 1
Figure 1.3. Taxonomy scheme of the insects used in this thesis. The taxonomy of the insects was
obtained from the following website (http://www.ncbi.nlm.nih.gov/Taxonomy/).
1.2.1. Hemiptera
Hemiptera (called also Rhynchota or true bugs) is one of the largest orders of insects. This
order consists of about 50.000-80.000 species. Many species of this order are considered
economically important pests, which are causing direct damage to plants by feeding or
causing an indirect effect by transmitting many plant virus diseases (Hogenhout et al., 2008).
Based on the differences in wing structure, the order of Hemiptera has been divided into two
distinct suborders. The first suborder is the Homoptera with insects where the front wing pair
may be uniformly membranous or stiffened throughout: good examples are aphids,
whiteflies, mealybugs, scale insects, froghoppers or spittlebugs, leafhoppers and treehoppers.
The second suborder is the Heteroptera (with the front wings clearly divided into two regions,
a hardened, leathery basal area and a membranous tip) such as shield bugs or stink-bugs,
capsid bugs, bedbugs, assassin bugs and water bugs.
Aphids are small hemimetabolous piercing-sucking insects, usually less than 5 mm, and
members of the Aphididae family, one family of the suborder Homopteran. Aphids are one of
the most destructive insect pests on the world agriculture (Pang et al., 2009). Aphids have a
pearlike shape and a pair of tubelike cornicles that can be found on the back of the abdomen.
This insect secretes honeydew which is known as a sugary liquid secreted through the anus.
5
Chapter 1
Wings are not always present; winged aphids are called "alates", while the wingless aphids
are known as "apterous” (Fig. 1.4).
Figure 1.4. Different forms of aphids: (A) wingless; (B) newborn nymph; (C) and (D) winged; (E)
nymph. (http://www.iranicaonline.org/uploads/files/Pests_Agricultural/pests_agric_fig_2.jpg)
1.2.1.1 Acyrthosiphon pisum
The pea aphid, A. pisum is a Hemipteran insect belonging to the Aphididae family. This aphid
is known to have a wide range of hosts from different legume species such as peas, alfalfa,
clover, and fresh beans, both snap and lima (Stoltz and McNea, 1982; Losey and Eubanks,
2000). Note that, the name “pea aphids” refers to the fact that pea crops are the major hosts in
the fields while other crops are considered as minor hosts (Hill, 1997). Pea aphids suck juice
from the phloem of their host plants by inserting their stylet (Fig. 1.2B) into the phloem
tissue. Then, the internal pressure inside the phloem helps to pump the sap into the aphid's
gut (Dixon, 1985). The wide host range and parthenogenic reproduction have made these
aphids one of the important migratory pests (Losey and Eubanks, 2000). The pea aphids have
a short and complex life cycle which includes two types of reproduction: the asexual and the
sexual reproduction (Fig. 1.5). Usually, eggs are laid in winter time and they enter a diapause
period. In the spring, these eggs hatch to asexual females which begin producing offspring
after 1-2 weeks following the hatching. After that, the aphids reproduce via parthenogenesis
by producing genetically identical nymphs that pass through four nymphal instars during
about 12 days before molting into an adult (Sharma et al., 1976; Blackman, 1987). In the fall,
the aphids develop to sexual females and males, and the mating results in overwintering eggs
6
Chapter 1
(Brisson and Stern, 2006). Pea aphids can easily be maintained in incubators to be used in
different laboratory bioassays.
Figure 1.5. The life cycle of the pea aphid (Brisson and Stern, 2006)
1.2.2. Lepidoptera
Lepidoptera is one of the largest orders of the class Insecta and belong to the most
widespread insects in the world. This order which is also called lepidopterans includes moths
and butterflies. The order of Lepidoptera consists of 47 superfamilies which consist of 128
families that have more than 180.000 species (http://www.ucl.ac.uk/taxome/). Insects of this
order are holometabolous and they are going through four stages in their life cycle: egg,
larva, pupa and adult (Powell, 2003) (Fig. 1.6). Among the Lepidoptera, adults commonly
feed on pollen or nectar while the larvae, called „caterpillars‟, are in many cases highly
phytophagous which makes this order one of the most destructive worldwide (Common,
1990).
7
Chapter 1
Figure 1.6. Different stages of Lepidoptera order. (A) egg; (B) larva; (C) pupa; (D) adult
http://ipm.ncsu.edu/ag271/peanuts/black_cutworm.html
1.2.2.1. Spodoptera littoralis
The cotton leafworm, Spodoptera littoralis (Fig. 1.7) belongs to the family Noctuidae and is
one of the most important lepidopterans in agriculture and horticulture, and has a wide host
range including at least 87 economically important plant species belonging to 40 families
distributed worldwide such as cotton, alfalfa, vegetables, maize, rice, soybeans, ornamentals,
weeds, etc. (Hill, 1987; Alford, 2003). This insect is one of the major insects in cotton fields
and can feed almost on all parts of cotton plants including the leaves, fruits, flower buds and
occasionally also on bolls. However, one of the problems to control this insect is its high
ability to develop relatively quickly resistance to most conventional insecticides. Each female
lays several hundred of eggs in clusters on the plant surface and covers them with orange-
brown hairs from the abdomen. The size of the egg is about 5 ± 2 mm diameter. Females of S.
littoralis have high fecundity and they can lay 2.000-3.000 eggs during 6-8 days. These eggs
hatch to larvae after 2-5 days after oviposition and immediately spread over the host plant.
The young caterpillars are gregarious but from 4th instar they become solitary and usually
they feed only at night and shelter in the soil during the day. Normally, the larvae develop
through six larval instars before inter the pupal stage. Pupation takes place in the soil inside a
loose cocoon and the pupae emergence and become adults (butterfly) after 7-10 days.
Butterflies are active at night and mate several times before laying eggs.
8
Chapter 1
The larvae of S. littoralis feed voraciously on almost all plant organs. Usually, they prefer
feeding on the young leaves, but when these leaves have been consumed the larvae can attack
also other parts such as stems, buds or pods. An infestation frequently leads to that all leaves
are devoured and plant development is affected by destroying growth points and flowers.
Figure 1.7. Larval stage of cotton leafworm, Spodoptera littoralis. Photo: M. Hamshou.
1.2.3. Insect gut
The insect gut is divided into three parts, the fore-, mid- and hindgut (Fig. 1.8A). The foregut
starts at the mouth and includes the cibarium, the pharynx, the esophagus, and the crop. The
latter is a storage organ in many insects and also serves as a site for digestion in others. In
most insects, foregut ends with the proventriculus, a valve to control the entry of food into the
midgut which is the main site for digestion and absorption of nutrients. The midgut consists
of the ventriculus, a simple tube from which blind sacs (gastric or midgut ceca) are branched.
The midgut epithelium of insects has several functions such as enzyme production, digestion,
and secretion (Chapman, 1998). These functions are probably because of the characteristic
structure of epithelial cells which form the midgut epithelium. Usually, the cytoplasm of the
epithelial cells has distinct regionalization in organelle arrangements, and as a consequence,
basal, perinuclear, and apical regions appear (Rost-Roszkowska et al., 2007; Rost-
Roszkowska and Undrul, 2008). Usually, the peritrophic membrane (PM), a film-like
anatomical structure is lining the midgut and separates the luminal contents into two places:
the endoperitrophic space and the ectoperitrophic space (Lehane, 1997). It is thought that the
PM plays a role to protect the gut surface from damage caused by abrasive food material and
to limit the access of microorganisms. In addition, it allows the transfer of liquid and digested
9
Chapter 1
substances to the midgut epithelial cells, but prevents the passage of larger food particles. The
columnar cells with a brush border (Fig. 1.8B) are the most common midgut epithelial cells
that are adjacent to the gut lumen. Although this membrane was found in most insects, it does
not occur in some insect orders such as Hemiptera, which are instead covered with
perimicrovillar membranes (PMVM) (Andries and Torpier 1982; Silva et al., 2004). The
domains of the microvilli are set in position by columns obliquely disposed between them
and the microvillar membrane (Fig. 1.8C) (Lane and Harrison, 1979). PMVMs maintain the
compartmentalization of digestion as an alternative to the peritrophic membrane (Ferreira et
al., 1988, Silva et al., 1995).
Figure 1.8. Schematic representation of insect gut compartments. (A) Different part of the midgut,
(B) Columnar cells, (C) Microvillus and (D) Glycocalyx: the carbohydrate moiety of intrinsic proteins
and glycolipids occurring in the luminal face of microvillar membranes (Terra and Ferreira, 2005).
10
Chapter 1
The glycoproteins and glycolipids on the luminal side of microvillar membranes are
decorated with a variety of carbohydrates (Fig. 1.8D) that play a role in mediating different
cellular and developmental events (Haltiwanger and Lowe, 2004). At the end of the midgut,
there is the sphincter or pylorus, a valve which locates between the midgut and the hindgut.
The hindgut consists of the ileum, colon and rectum and terminates with the anus (Fig. 1.8A).
The hindgut is involved in uptake of digested material, although to a lesser extent than the
midgut.
1.2.4. Insect cell lines
About half century ago, the first insect cell line was established from ovaries of the
diapausing silkmoth, Antheraea eucalypti (Grace, 1962). In the 50 years since that
achievement, many insect cell lines have been added to the list, to reach more than 500
established cell lines as depicted in Figure 1.9. This figure shows also that most of the insect
lines have been derived from Lepidoptera and Diptera (Lynn, 2001; Lynn, et al., 2005,
Smagghe, 2007).
Figure 1.9. The number of established invertebrate cell lines developed from 1962 to 2000
categorized by insect orders. (Source: Smagghe, 2007)
These cell lines were considered a useful research tool for screening of the biological efficacy
of novel pesticide candidates and their mode of action at the cellular level. In addition, cell
lines can provide large amounts of homogenous material in which the selected target sites are
directly present for the candidate insecticides. Insect cell lines have been derived from
different parts of the insect‟s body such as ovaries, embryos, hemocytes, imaginal discs, fat
body as well as from the midgut. These cells can easily be maintained in a laboratory by use
of specific culture medium. Recently, insect cell lines were used widely to investigate the
11
Chapter 1
toxicity of lectins and elucidate their mechanism of action. For example, the lepidopteran
midgut cell line (CF-203) was used to investigate the activity of different lectins (Smagghe et
al. 2005). Using CF-203 cells, Vandenborre et al. (2006) studied the interaction of a lectin
with receptor proteins in an attempt to determine the possible signal transduction pathways.
More recently, Shahidi-Noghabi et al. (2010a, 2011) did several assays using the same
midgut cells to determine the activity and the mode of action of Sambucus nigra agglutinin.
1.3. CROP PROTECTION
Up to date chemical insecticides are the most common compounds used to control insects.
These insecticides have been considered as one of the major factors involved in increasing
agricultural productivity in the 20th century. The world global pesticide market was about
US$ 40 billion in 2008 and it is expected to increase about 20 % in 2014 to reach 51 billion
(Fig. 1.10).
Figure 1.10. Global pesticide market by Segment (2008-2014)
http://www.bccresearch.com/report/biopesticides-market-chm029c.html
However, extensive use of chemical control has led to many problems including; (a) toxic
effects on humans, (b) developing resistance against these compounds by many pests, (c)
killing beneficial organisms such as pollinators, predators and parasitoids, (d) pesticide
residues in food, (e) harmful effects on nutrient cycling, (f) bad effects on soil, water and air
quality, and (f) reduction of biodiversity and impact on non-target species including some
mammals, birds, fishes, etc. through food chains. These problems pushed researchers to find
safer alternative methods to control pests.
12
Chapter 1
1.3.1. Current control strategies
In fact, the best way to control insects is the integrated pest management (IPM) which is
defined as using multiple tactics to control insect pests and to keep their abundance and
damage under the economic significance levels. IPM could include a combination of
practices such as the wisely use of pesticides, crop rotation, biological control and the use of
resistant plant varieties to suppress insect pest damage. The last category is one of the best
options which can be used and also includes the use of genetically engineered insect-resistant
crops.
The resistance of plants to insects is related to several defensive mechanisms which could be
separated to physical and chemical mechanisms. Proteins are one of most important
macromolecules which could be involved in the defensive mechanisms. Up to date, there are
many different proteins possessing an insecticidal activity which could be expressed in
transgenic plants including lectins, ribosome-inactivating proteins, protease inhibitors, α-
amylase inhibitors, arcelins, canatoxin-like proteins, ureases and chitinases (Carlini et al.,
2002; Vasconcelos et al, 2004; Karimi et al., 2010). The Bacillus thuringiensis (Bt) endotoxin
was the first protein that was expressed in tobacco plants (Vaeck et al., 1987). These plants,
engineered with truncated genes encoding Cry1A (a) and Cry1A (b) toxins, showed
resistance towards the larvae of the chewing tobacco hornworm Manduca sexta (Barton et al.,
1987, Vaeck et al., 1987). Since then, the transgenic crops that produce B. thuringiensis (Bt)
toxins are grown widely for pest control (Tabashnik et al., 2011). Two main problems were
faced using the Bt toxin based technology: it did not show protection towards sucking insects
and many insects developed a resistance to Bt toxin (Tabashnik et al., 1990; McGaughey and
Whalon, 1992; Ferre and Rie, 2002; Janmaat and Myers, 2003; Price and Gatehouse, 2008).
Because of these problems the interest grows to look for alternative strategies based on the
use of plant defence proteins such as lectins.
1.3.1.1. Lectins as bio-insecticidal agent
During the recent decades, many studies have focused on the investigation of the insecticidal
activity of different lectins, especially plant lectins, and the elucidation of their mechanism of
action while there were only very few studies on lectins from fungi (which is discussed in
fungal lectins part below). The insecticidal activity of plant lectins has been reported towards
different pest insects belonging to the orders Lepidoptera, Coleoptera, Diptera and
Homoptera (Vandenborre et al. 2009; Michiels et al. 2010). This activity of lectins and the
13
Chapter 1
potential of several plant lectins as insecticidal proteins was demonstrated both by in vitro
assays, using lectins incorporated into artificial diets (Sadeghi et al., 2009c; Shahidi-Noghabi
et al., 2010b), and in vivo assays, with transgenic plants expressing a foreign lectin gene
(Sadeghi et al., 2008; Shahidi-Noghabi et al., 2009).
1.3.1.1.1. Toxic effects of lectins towards Hemiptera
Plant lectins have been reported to possess insecticidal activity towards different insects
belonging to the Hemiptera order as demonstrated by using artificial diets incorporated with
lectins or transgenic plants expressing lectins. For instance, the lectin from Galanthus nivalis
(GNA) exerted toxic effects against different Hemipteran insects such as the pea aphid A.
pisum (Rahbé et al., 1995), the glasshouse potato aphid Aulacorthum solani (Down et al.,
1996), and the red cotton bug Dysdercus cingulatus (Roy et al., 2002) when it was
incorporated into the artificial diet. In addition, transgenic plants expressing GNA also
affected the growth and survival of some insects belonging to Hemiptera. For example,
genetically modified rice plants showed insecticidal activity against the green rice leafhopper
Nephotettix virescens (Ramesh et al., 2004), the brown planthopper Nilaparvata lugens (Saha
et al., 2006b) and the small brown planthopper Laodelphax striatellus (Sun et al., 2002).
Moreover, the peach aphid Myzus persicae was shown to be sensitive to the Allium sativum
lectin when the lectin was added to the artificial diet (Sauvion et al., 1996) or expressed in
tobacco plants (Dutta et al., 2005b).
1.3.1.1.2. Toxic effects of plant lectins towards Lepidoptera
Many plant lectins have been reported to affect insect growth, development, and fecundity of
a wide range of Lepidopteran insects when these insects were fed on an artificial diet
supplemented with lectins and/or on transgenic plants overexpressing the lectin genes.
For example, the cotton bollworm Helicoverpa armigera was found to be affected by
different plant lectins isolated from Galanthus nivalis, Triticum aestivum, Canavalia
ensiformis, Arachis hypogea, Artocarpus integrifolia, Cicer arietinum and Lens culinaris
when the larvae of H. armigera were fed on an artificial diet containing different
concentrations of these lectins (Shukla et al., 2005). Furthermore, larvae of the European corn
borer (Ostrinia nubilalis) were found to be sensitive to lectins from Triticum aestivum,
Ricinus communis and Bauhinia purpurea (Czapla & Lang, 1990).
In addition, transgenic plants expressing lectins (mainly GNA) exerted an insecticidal activity
towards different insects from the order Lepidoptera. For instance, GNA expressed in tomato,
14
Chapter 1
tobacco, rice and sugarcane showed toxic effects towards Lacanobia oleracea (Wakefield et
al., 2006), Helicoverpa assulta (Zhang et al., 2007), Chilo suppressalis (Loc et al., 2002) and
Eoreuma loftini (Setamou et al., 2002). Furthermore, transgenic rice expressing Allium
sativum leaf agglutinin exhibited entomotoxic activity against different sap-sucking pests
(Yarasi et al., 2008). In addition transgenic tobacco plants expressing A. sativum lectin or
leek lectin demonstrated entomotoxic activity against S. littoralis (Sadeghi et al., 2007;
Sadeghi et al., 2009a).
1.3.1.1.3. Interaction of lectins with receptors in insect
The biological activity of lectins depends on their ability to bind carbohydrates which are all
present on the surface of cells, such as the epithelial cells of animal digestive tracts (Villalobo
and Gabius, 1998). The importance of lectin binding to a sugar moiety of a glycosylated
protein in the insect gut has been suggested to be the prerequisite factor for the insecticidal
activity of any lectin (Peumans and Van Damme, 1995a; Peumans and Van Damme, 1995b).
For example, toxicity of Phaseolus vulgaris agglutinin (PHA) on the midgut epithelial cells
of the bruchid Callosobruchus maculatus was proposed to depend on the binding of PHA to
these cells (Gatehouse et al., 1984). Moreover, the correlation between binding and
insecticidal activity of PHA against different insects was reported (Habibi et al., 1998; Habibi
et al., 2000; Fitches et al., 2001; Bandyopadhyay et al., 2001). In contrast, lack of binding of
PHA to the midgut cells of bean weevil (Acanthoscelides obtectus) could explain the non-
toxic effect of PHA towards this insect (Gatehouse et al., 1989). In fact, the correlation
between the binding of the lectins and their insecticidal activity is not general for all lectins.
For instance, detailed studies on the mechanisms of two lectins from Sambucus nigra (SNA-I
and SNA-II) on the insect midgut CF-203 cells, revealed that both lectins did not bind the
cells but they got internalized in the cells which resulted in strong toxicity (Shahidi-Noghabi
et al., 2011). The importance of the carbohydrate-binding domain for the insecticidal activity
has been demonstrated by two different methods (i) mutation of Griffonia simplicifolia lectin
to eliminate the carbohydrate-binding activity reduced the toxicity of this lectin towards the
cowpea bruchid, C. maculatus (Zhu-Salzman et al., 1998); (ii) incubation of different lectins
with their specific sugar reduced the binding and toxicity of these lectins on different cell
lines (Kuramoto et al., 2005).
15
Chapter 1
1.4. INSECT GLYCOSYLATION PATTERNS
Membrane proteins were reported to serve as transport systems, light-transducing agents,
antigens and receptors. Plasma membranes contain carbohydrates as glycoproteins and
glycolipids. In general, glycosylation occurs on the extracellular surface of the plasma
membrane. Glycosylation is defined as a covalent attachment of an oligosaccharide chain to a
protein and is considered to be a very common protein modification. The composition of the
carbohydrate chain is very diverse and can modify the characteristics of a protein. The two
major forms of this protein modification are N-glycans and O-glycans which refers to the
type of glycosidic linkage of this carbohydrate structure to the amino acids Asn and Ser/Thr,
respectively. Glycosylation of proteins can mediate different processes such as subcellular
localization, protein quality control, cell-cell recognition and cell-matrix binding events in
addition to other rules which are not fully understood. In fact, most studies on glycobiology
have focused on mammals although insect glycobiology is a promising research field because
they are the most diverse organisms and have a wide genetic diversity. Up to date, almost all
information concerning glycobiology in insects was obtained from studies with the fruit fly,
Drosophila melanogaster (Diptera).
Studies on D. melanogaster have shown that glycans could affect developmental processes as
demonstrated by using lectins to study the variation of glycosylation as a function of organ,
cell type, and developmental stage in this insect (Fredieu and Mahowald, 1994; D'Amico and
Jacobs, 1995). Moreover, glycosylation was reported to contribute to the function(s) of some
proteins with important roles in development (O'Tousa, 1992; Kaushal et al., 1994)
Drosophila proteins were shown to be decorated with high-mannose oligosaccharides and
core fucosylated pauci-mannose glycans as demonstrated by different N-linked glycans
studies (Seppo and Tiemeyer, 2000; Fabini et al., 2001; Sarkar et al., 2006). Furthermore, the
N-glycan profile of the fly was found to change according to the developmental stages which
suggests specific roles of certain glycan structures during different stages of development
(Seppo and Tiemeyer, 2000; Aoki et al., 2007; Ten Hagen et al., 2009). Recently a protein
modified by a mucin type O-linked glycosylation was identified from Drosophila
(Schwientek et al., 2007; Tian and Hagen, 2009). The recent progress in using lectins in
glycoproteomics and insect glycobiology will provide new insights in the interactions
between lectins and insects, which in turn will help to better understand the mode of action
behind the lectin activity.
16
Chapter 1
1.4.1. Gal/GalNAc Linkage residues
Compared with the wide heterogeneity observed in most animals, insects seem to synthesize
a surprisingly low number of very simple O-glycans. So far, studies conducted on several
Lepidopteran cell lines suggested that the O-glycosylation in insects was restricted to
GalNAc-α-Ser/Thr and Galβ1–3GalNAc-α-Ser/Thr (Thomsen et al., 1990; Kramerov et al.,
1996; Lopez et al., 1999; Maes et al., 2005; Garenaux et al., 2011). In addition, the most
abundant O-glycan structure in Drosophila is the mucin type O-glycosylation. As shown in
figure 1.11, this type of glycosylation involves in the addition of GalNAc to Ser/Thr to form
the Tn antigen (GalNAcα1-S/T), often extended with galactose (Gal) (Tian and Hagen,
2009).
Figure 1.11. Biosynthesis of the most common mucin-type O-glycans in D. melanogaster (Tian and
Hagen, 2009).
17
Chapter 1
Moreover, investigation of the involvement of glycosyltransferases in complex-type N-
glycosylation in different Lepidopteran insect cell lines suggested the ability of these cell
lines to synthesize complex type carbohydrate chains containing GalNAc β14GlcNAc units
(Van Die et al., 1996; Tran et al., 2012). The presence of fucosylated, sialylated, hybrid,
biantennary complex, and triantennary complex glycans in Drosophila embryos was
demonstrated (Varki et al., 2008). Interestingly, some lectins which can recognize and bind to
Gal/GalNAc have been reported to possess high insecticidal activity, such as lectin from
Sambucus nigra (Shahidi-Noghabi et al., 2010b) and Glechoma hederacea lectin (Wang et
al., 2003).
1.5. APOPTOSIS
For all the living organisms, including the life cells in the earth and universe itself, there is a
time to live and afterwards a time to die. There are two ways in which cells die as a response
to a variety of stimuli, such as toxins, genotoxic compounds, tumor necrosis factor and
various environmental stresses: (i) Killing the cells by injury or disease, which is
uncontrolled cell death or (ii) Programmed Cell Death (PCD) or apoptosis, which is a
regulated cell suicide. Eventually, the term apoptosis was used in order to describe the
morphological processes that lead to controlled cellular self-destruction. This term was first
used in a publication by Kerr et al. (1972).
Apoptosis is a normal component of the development and health in the multicellular
organisms by which cells undergo death to control cell proliferation or in response to DNA
damage. A good example for the involvement of apoptosis in animal development is a
massive cell death in the interdigital mesenchymal tissue to form free and independent digits
(Zuzarte-Luis and Hurle, 2002). Another example, during nervous system development,
about 1.5 times the adult number of neurons will die by apoptosis in later stages when the
adult nervous system is formed (Hutchins, 1998). The apoptosis has several characteristics
such as shrinkage of cells, chromatin condensation, blebbing, formation of membrane-bound
apoptotic bodies that contain organelles, cytosol and nuclear fragments (Fig. 1.12). And
finally the cells suicide and died (Gewies, 2003; Ma et al., 2005). Three different mechanisms
of apoptosis have been described. A first mechanism occurs as a response to internal by
signals in a cell such as Bcl-2, Apaf-1 (apoptotic protease activating factor-1), Bax,
cytochrome c, caspase 9, ATP, etc. A second mechanism is caused by external signals such
18
Chapter 1
as Fas, FasL, TNF, TNF receptor, etc. and a third mechanism is triggered by toxic factors
(Ma et al., 2005).
Apoptosis is of widespread biological significance and could be involved in several
biological processes such as development, differentiation, proliferation, regulation and
function of the immune system and in the removal of defect and therefore harmful cells
(Gewies, 2003). Thus, dysfunction or dysregulation of apoptosis can result in a variety of
pathological conditions. For instance, defects in the apoptotic process can cause cancer,
autoimmune diseases and spreading of viral infections, while excessive apoptosis can
enhance neurodegenerative disorders, AIDS and ischaemic diseases (Fadeel, 1999).
Moreover, apoptosis is also considered as a defense mechanism against virus infection
directly interfering with virus multiplication (Clem and Miller, 1993) and also against
bacterial pathogens by eliminating the infected cells via programmed cell death (Böhme and
Rudel, 2009).
Actually, the central executioners of the apoptotic signaling pathway are caspases which are
activated in most cases of apoptotic cell death (Bratton, 2000; Olsson and Zhivotovsky,
2011).
Figure 1.12. Cellular changes during apoptotic cell death. The changes include cellular shrinking,
chromatin condensation and margination at the nuclear periphery with the eventual formation of
membrane-bound apoptotic bodies that contain organelles, cytosol and nuclear fragments and are
phagocytosed without triggering inflammatory processes. The photo is modified from Gewies (2003).
It is worth mentioning that there are caspase-independent apoptosis pathways which could
depend on calpains, cathepsins, endonucleases, and other proteases. These proteins can
initiate and execute programmed cell death that can be regulated by several cellular
19
Chapter 1
organelles such as mitochondria, lysosomes, and the endoplasmic reticulum (ER), which can
work together or independently (reviewed by Bröker et al., 2005).
About 50 years ago, the involvement of apoptosis in insect development has been reported by
Lockshin and Williams (1964). The first ecdysone peak during metamorphosis of the wild
silkmoths and the tobacco hawkmoth induces apoptotic degeneration of the larval
intersegmental muscles, proleg motoneurons, and labial glands (Lockshin and Williams,
1964; Lockshin and Zakeri, 1994). Moreover, apoptosis can be induced as result of the
decrease in the ecdysone titer shortly before hatching degeneration of abdominal neurons and
intersegmental muscles (Truman, 1984). Important changes in food habits between larval and
adult stages show large modifications in the digestive tract. For instance, the larval midgut of
the greater wax moth, Galleria mellonella, undergoes apoptosis during metamorphosis (Uwo
et al., 2002). Moreover, apoptosis of the larval midgut of Heliothis virescens was correlated
with higher caspase expression shortly before and after pupation (Parthasarathy and Palli,
2007). A recent study demonstrated that apoptosis is a fundamental host defense mechanism
against Parachlamydiaceae in insect cells (Sixt et al., 2012).
It is worth mentioning that Apoptosis which is reported in the salivary gland of Apis mellifera
larvae was found to lie between the classical apoptosis and autophagy because it exhibited
some characteristics of both phenomena (Silva-Zacarin et al., 2007).
Two types of PCD have been reported during Drosophila development: (i) apoptosis which is
characterized by membrane blebbing, nuclear condensation and DNA fragmentation, and (ii)
autophagy which is distinguished by the destruction of the whole tissues and the presence of
autophagic vacuoles (Abrams et al., 1993). Interestingly, PCR cloning studies as well as the
analysis of the complete Drosophila euchromatic genomic sequence showed that there are
insect homologs for many of the mammalian PCD genes (Rubin et al., 2000; Vernooy et al.,
2000). Drosophila was considered a good and easy way to investigate the function of these
PCD genes in vivo (Hay and Guo, 2006).
The release of cytochrome c from the mitochondria by various apoptotic stimuli initiates the
major caspase activation pathway(s) in mammalian cells (He et al., 2000; Arnoult et al.,
2002; Jiang and Wang, 2004). In insects (Fig 1.13), cytochrome c was found to be involved
in apoptosis of many lepidopteran cell lines such as Sf9 cells (Sahdev et al. 2003), Sl-1 cells
(Malagoli et al., 2005) and LdFB cells (Shan et al., 2009). In contrast, the majority of studies
20
Chapter 1
on Drosophila showed that there is no evidence for the involvement of cytochrome c in
apoptosis of this insect (Liu et al., 2012).
Figure 1.13 Model for the role of cytochrome c during insect cell apoptosis (from: Liu et al., 2012).
1.5.1. The insect caspases
Caspases (cysteine aspartate-specific proteinases) are one of the main executors of the
apoptotic process in mammals and insects. They belong to a family of cysteine proteases and
exist within the cell as inactive pro-forms or zymogens. These zymogens can be cleaved to
form active enzymes following the induction of apoptosis. There are two types of apoptotic
caspases, based on their place of entry into the cell death pathway: initiator (apical) caspases
and effector (executioner) caspases. The prodomain of the initiator caspases contains the
death effector domain (DED) in procaspase-8 and -10, or the caspase recruitment domain
(CARD) in procaspase-2 and procaspase-9 (Thornberry and Lazebnik, 1998; Earnshaw et al.,
1999; Fuentes-Prior and Salvesen, 2004). Both DED and CARD are involved in procaspase
activation and downstream caspase-cascade regulation through protein-protein interactions
(Fuentes-Prior and Salvesen, 2004; Ho and Hawkins, 2005). The activation of caspases is
usually occurring through two pathways: the death signal-induced or death receptor-mediated
pathway and the stress-induced or mitochondrion-mediated pathway (i.e. a caspase-9-
dependent pathway) (Fan et al., 2005).
21
Chapter 1
In mammals, the death receptors, such as Fas or TNF, can specifically recognize cell death
signals, such as FasL (Fas ligand) or TNF (tumor necrosis factor). This binding activates the
death receptors. Then, Fas can bind to the Fas-associated death domain (FADD) (or TNFR-
associated death domain, TRADD) and cause FADD aggregation and the emergence of
DEDs which interact with the DEDs in the prodomain of procaspase-8/-10. The result of this
interaction is formation of the death-inducing signal complex (DISC) that activate
the initiator caspases-8, -9, -10 (Fig. 1.14) (Boatright and Salvesen, 2003; Alenzi et al.,
2010). Subsequently, the initiators activate the effector caspases, caspase-3, -6, -7 (Boatright
and Salvesen, 2003). Then, the effector caspases cleave key cellular substrates such as protein
kinases, signal transduction proteins and DNA repair proteins (Fischer et al., 2003).
Apoptosis can also occur via intrinsic pathways which are triggered in response to a wide
range of intracellular signals, such as oncogene activation and DNA damage. Those
intracellular signals are altering the permeability of the mitochondrial outer membrane which
in turn leads to the release of several proteins to the cytosol, such as Smac/Diablo and
cytochrome c. Cytochrome c forms an apoptosome, a catalytic multiprotein platform that
activates caspase-9. Subsequently, activation of caspase-8 and/or caspase-9 leads to activate
the effector caspase-3, -6 and -7 (Fig. 1.14) (Czerski and Nuñez, 2004).
Figure 1.14. Schematic representation of caspase-dependent apoptosis pathways in mammals and the
main regulating factors in apoptotic pathways (Fan et al., 2005).
22
Chapter 1
Various molecules were reported to regulate the activation and inactivation of caspases such
as IAP, Bcl-2 family proteins, calpain, Ca2+
, Gran B and cytokine response modifier A (Crm
A) (Fig. 1.14) (Launay et al., 2005).
Caspases have been characterized and studied well in mammals but they are less documented
in insects. In fact, the insect caspases were described mainly in D. melanogaster (Kumar and
Doumanis, 2000; Cooper and Granville, 2009) and recently in Lepidopteran insects
(Courtiade et al., 2011). In Drosophila, some caspases were reported to have a homologue
with mammalian caspases, while others have none. For instance, Dredd (a Drosophila
caspase) has similarity with mammalian caspase-8 and Dronc (a Drosophila caspase) is a
homologue of the mammalian caspase-9 and the human caspase-2 (Kumar and Doumanis,
2000). Strica (a Drosophila caspase) has no similarity to any other characterized motifs such
as CARD and death inducing domain, DID.
Interestingly, some homologues of Drosophila proteins involved in apoptosis have been
recognized in other insects. Aedes aegypti Dredd (AeDredd) was found to have the highest
sequence similarity with Drosophila Dredd and with human caspase-8 (Cooper et al., 2007a).
Aedes Dronc (AeDronc), is a homologue of the Drosophila Dronc (Cooper et al., 2007b).
Homologues of Drosophila Strica/Dream have been identified in the genome of both A.
aegypti and A. gambiae (Bryant et al., 2008). In addition, the homologeus of Drosophila
Dredd have been identified in Tribolium castaneum (Zou et al., 2007).
In Lepidoptera, several caspases have been identified. Sf-caspase-1 was the first insect
caspase identified from the lepidopteran Spodoptera frugiperda. This caspases was found to
be similar to Drosophila Drice and mammalian caspase-3 (Ahmad et al., 1997). Later, a
caspase called Sl-caspase-1 was found in S. littoralis cells which showed similarity with Sf-
caspase-1 (Liu et al., 2005). Moreover, Tn-caspase-1 was characterized in Trichoplusia ni
and found to be the main effector caspase in T. ni cells (Hebert et al., 2009). Recently, Hearm
caspase-1, an effector caspase identified from the cotton bollworm, Helicoverpa armigera,
has been found to be homologous to Sf-caspase-1 and Drosophila Drice (Yang et al., 2008)
and the homologue of Drosophila Dredd was identified in Bombyx mori (Xia et al., 2008). In
a recent study, 63 caspase genes were identified from 27 different lepidopteran species.
Phylogenetic analyses demonstrated that Lepidoptera possess at least 5 caspases (Courtiade et
al., 2011). Lep-Caspase-1, -2 and -3 were found to be putative effector caspases, while Lep-
Caspase-5 and -6 are reported to be putative initiator caspases in homology to Drosophila
23
Chapter 1
caspases. However, these caspases need further study to clarify the exact function and their
potential interactions (Courtiade et al., 2011). Figure 1.15 shows a comparative analysis of
the different homologues of caspases in the apoptotic pathway in mammals, Drosophila and
Lepidoptera.
Figure 1.15. Apoptotic pathway in mammals, Drosophila and Lepidoptera. Homologs of caspases
and caspase regulators across species are indicated by the same color. Initiator and effector caspases
are colored in blue and red respectively. The death receptor is colored in grey, the adaptor protein in
orange, the protein forming the apoptosome in yellow, the apoptotic inducers in purple, and the
caspase inhibitors in brown. (from: Courtiade et al., 2011).
24
Chapter 1
1.6. FUNGAL LECTINS: their toxicity and antiproliferative activity
Lectins are carbohydrate-binding proteins of non-immune origin possessing at least one non-
catalytic domain, which binds reversibly and non-covalently to mono- or oligosaccharides,
glycoproteins and glycolipids (Goldstein et al., 1980; Peumans and Van Damme, 1995a).
More than a century ago the first lectin was described by Stillmark who discovered lectin
activity in the seeds of castor tree, Ricinus communis (Stillmark, 1888). Since then, many
new lectins from various sources have continuously been added to the list of carbohydrate-
binding proteins. Due to their ability to bind carbohydrates, most of these proteins can also
agglutinate erythrocytes, a reaction which can be inhibited by using a specific sugar (Sumner
and Howell, 1936). Lectins are ubiquitously distributed in nature and can be found in plants,
fungi, bacteria, viruses, invertebrates and vertebrates (Vandenborre et al., 2009; Khan and
Khan, 2011; Vasta and Ahmed, 2008; Hartmann and Lindhorst, 2011). They are valuable
proteins not only because they are found in all organisms, but especially because their
reversible interaction with specific carbohydrates allows them to bind to glycoconjugates that
play an important role in cell physiology. All these properties have made lectins as one of the
most studied groups of proteins which are used as tools in biological and biomedical
research, especially in studies related to cell-cell interactions, cancer invasion and metastasis,
inflammation, and immunology.
In the past decades plant lectins have been studied in much more detail than any of the lectins
from other sources. Many plant lectins have been found in storage tissues where they
represent 0.1–10% of the total protein in the tissue. Therefore it has been proposed that these
lectins could serve as plant storage proteins (Van Damme et al., 1998). Furthermore, owing to
their ability to recognize specific carbohydrates it was suggested that these lectins may act as
defense proteins (Peumans and Van Damme 1995a). This hypothesis was shown to be correct
for several plant lectins (Michiels et al., 2010; Vandenborre et al., 2011b).
Lectins from fungi are far less documented than the plant lectins. Phallin was the first fungal
lectin that was discovered in Amanita phalloides in 1891 (Kobert, 1893) and later in 1910,
the second fungal lectin was reported from the mushroom Amanita muscaria (Ford, 1910).
To date more than 350 fungal lectins have been reported. The majority of these lectins was
detected in mushrooms (which can be defined as a macrofungi with a distinctive fruiting
body) and the rest was isolated from microfungi (which can be distinguished
from macrofungi only by the absence of a large fruiting body). Lectins have been isolated
25
Chapter 1
from the orders Agaricales, Boletales, Russulales, Cantharellales, Atheliales, Polyporales and
Thelephorales.All these orders belong to the class Agaricomycetes and the phylum
Basidiomycota. In addition, a few fungal lectins were purified from the orders Eurotiales,
Helotiales, Pezizales, Sordariales and Xylariales which belong to different fungal classes
within the phylum Ascomycota. Both phyla Basidiomycota and Ascomycota belong to the
subkingdom Dikarya within the kingdom of Fungi (Fig. 1.16).
Figure 1.16. Overview of the taxonomy of fungi from which lectins were isolated and will be
discussed in this review. The taxonomy of the fungi was obtained from the following website
(http://www.ncbi.nlm.nih.gov/Taxonomy/).
Fungal lectins have been reviewed in several recent papers (Guillot and Konska, 1997; Wang
et al., 1998; Konska, 2006; Singh et al., 2010; Khan and Khan, 2011a, Singh et al., 2011).
This chapter will give an overview only of those fungal lectins that were shown to possess
toxic properties and/or antiproliferative activity (Table 1.1).
26
Chapter 1
Ta
ble
1.1
. O
ver
vie
w o
f th
e fu
ngal
lec
tins
that
hav
e bee
n s
tudie
d i
n m
ost
det
ail
for
thei
r ac
tivit
y t
ow
ard
s so
me
org
anis
ms
or
cell
s.
(ND
= n
ot
det
erm
ined
).
Ref
.
Lec
tins
wit
h a
nti
vir
al a
ctiv
ity (
mai
nly
HIV
-1)
Sun e
t al
., 2
003
Zhen
g e
t al
., 2
007
Li
et a
l., 2010
Zhao
et
al., 2
009b
Zhan
g e
t al
., 2
009
Li
et a
l., 2008
Zhao
et
al., 2
010
Han
et
al., 2
005
Lec
tins
wit
h a
nti
fungal
act
ivit
y
Gir
jal
et a
l., 2011
Lec
tins
wit
h t
oxic
ity a
gai
nst
Am
oeb
a
Ble
ule
r-M
artí
nez
et
al., 2
011
Ble
ule
r-M
artí
nez
et
al., 2
011
Wohls
chla
ger
et
al., 2
01
1
Ble
ule
r-M
artí
nez
et
al., 2
011
3D
str
uct
ure
GA
LE
CT
IN
XC
L
- - - - -
RIC
IN
FIP
β-p
ropel
ler
RIC
IN
RIC
IN
RIC
IN
Sp
ecif
icit
y
gly
copro
tein
s
mel
ibio
se, xylo
se
inuli
n
sever
al s
ug
ars
inuli
n
sever
al s
ug
ars
inuli
n
lact
ose
gly
copro
tein
s
fuco
se
Gal
NA
c
Gal
/Gal
NA
c
Gal
/Gal
NA
c
MW
(k
Da)
Su
bun
it m
ass
* n
o o
f su
bun
its
15.8
* 2
16.3
* 2
51 *
1
17 *
1
16 *
2
16.2
* 2
30 *
2
32 *
2
15 *
1
33.4
* 2
15.5
* 2
33 *
1 +
23 *
1
17 *
2
Lec
tin
sou
rce
Agro
cybe
aeg
erit
a
Bole
tus
eduli
s
Her
iciu
m e
rinace
um
Inocy
be
um
bri
nel
la
Pholi
ota
adip
osa
Ple
uro
tus
citr
inop
ilea
tus
Russ
ula
del
ica
Sch
izop
hyl
lum
com
mun
e
Ganoder
ma l
uci
dum
Ale
uri
a a
ura
nti
a
Cli
tocy
be
neb
ula
ris
Mara
smiu
s ore
ades
Scl
eroti
nia
scl
eroti
oru
m
27
Chapter 1
Tab
le 1
.1.
conti
nued
Ref
.
Ble
ule
r-M
artí
nez
et
al., 2
011
Lec
tins
wit
h t
oxic
ity a
gai
nst
inse
cts
Ble
ule
r-M
artí
nez
et
al., 2
011
Pohle
ven
et
al., 2
011
Künzl
er e
t al
., 2
010
Fra
nci
s et
al.
, 2011
Ble
ule
r-M
artí
nez
et
al., 2
011
Tri
guer
os
et a
l., 2003
Lec
tins
wit
h t
oxic
ity a
gai
nst
Nem
ato
de
Zhao
et
al., 2
009a
Ble
ule
r-M
artí
nez
et
al., 2
011
Zhao
et
al., 2
009a
Pohle
ven
et
al., 2
012
Buts
chi
et a
l., 2010
Zhao
et
al., 2
009a
Wohls
chla
ger
et
al., 2
01
1
Bhat
et
al., 2
010
3D
str
uct
ure
XC
L
β-p
ropel
ler
RIC
IN
GA
LE
CT
IN
-
XC
L
XC
L
GA
LE
CT
IN
β-p
ropel
ler
XC
L
RIC
IN
GA
LE
CT
IN
FIP
RIC
IN
XC
L
Sp
ecif
icit
y
Gal
/Gal
NA
c
fuco
se
Gal
NA
c an
d o
ther
sugar
s
β-g
alac
tosi
de
man
nose
Gal
/Gal
NA
c
Gal
/Gal
NA
c
lact
ose
, si
alic
aci
d
Fuco
se
mel
ibio
se, xylo
se
Lac
diN
Ac
β-g
alac
tosi
de
Ara
bin
ose
Gal
/Gal
NA
c
Gal
/Gal
NA
c
MW
(k
Da)
Su
bun
it m
ass
* n
o o
f su
bun
its
16.1
* 1
33.4
* 2
15.5
* 2
16.7
* 1
40 *
1 +
31 *
1
16.1
* 1
15 *
1
16 *
2
33.4
* 2
17 *
1
15.5
* 2
16.7
* 1
15.5
* 1
33 *
1 +
23 *
1
17 *
2
Lec
tin
sou
rce
Sord
ari
a m
acr
osp
ora
Ale
uri
a a
ura
nti
a
Cli
tocy
be
neb
ula
ris
Copri
nopsi
s ci
ner
ea
Pen
icil
lium
chry
sogen
um
Sord
ari
a m
acr
osp
ora
Xer
oco
mus
chry
sente
ron
Agro
cybe
cyli
ndra
cea
Ale
uri
a a
ura
nti
a
Bole
tus
eduli
s
Cli
tocy
be
neb
ula
ris
Copri
nopsi
s ci
ner
ea
Ganoder
ma l
uci
dum
Mara
smiu
s ore
ades
Scl
eroti
um
rolf
sii
28
Chapter 1
Tab
le 1
.1.
conti
nued
Ref
.
Ble
ule
r-M
artí
nez
et
al., 2
011
Zhao
et
al., 2
009a
Ble
ule
r-M
artí
nez
et
al., 2
011
Zhao
et
al., 2
009a
Lec
tins
wit
h t
oxic
ity a
gai
nst
mic
e/ra
ts
Sun e
t al
., 2
003
Hori
be
et a
l., 2010
Lec
tins
wit
h a
ctiv
ity a
gai
nst
dif
fere
nt
cell
lin
es
Zhao
et
al., 2
011
Yu e
t al
., 1
993
Zhao
et
al., 2
003
Anto
nyuk e
t al
., 2
010
Fen
g e
t al
., 2
006
Koyam
a et
al.
, 2002
Bovi
et a
l., 2011
Pohle
ven
et
al., 2
009
Ng e
t al
., 2
006
3D
str
uct
ure
XC
L
-
XC
L
-
GA
LE
CT
IN
- -
XC
L
GA
LE
CT
IN
- - -
XC
L
RIC
IN
FIP
Sp
ecif
icit
y
Gal
/Gal
NA
c
gal
acto
se
Gal
/Gal
NA
c
xylo
se, in
uli
n
gly
copro
tein
s
asia
lofe
tuin
Inuli
n
Gal
β-1
,3-G
alN
Ac
gly
copro
tein
s
com
ple
x s
ugar
s
Inuli
n
Glc
NA
c
mel
ibio
se, xylo
se
lact
ose
, as
ialo
fetu
in
lact
ofe
rrin
MW
(k
Da)
Su
bun
it m
ass
* n
o o
f su
bun
its
16.1
* 1
17.5
* 2
15 *
1
14.4
* 2
15.8
* 2
11 *
3
15.2
* 2
16 *
4
15.8
* 2
18 *
2
14.7
* 2
15 *
1
16.3
* 2
15.9
* 2
12 *
1
Lec
tin
sou
rce
Sord
ari
a m
acr
osp
ora
Tri
cholo
ma m
ongoli
cum
Xer
oco
mus
chry
sente
ron
Xyl
ari
a h
ypoxy
lon
Agro
cybe
aeg
erit
a
Bole
tus
venen
atu
s
Agari
cus
arv
ensi
s
Agari
cus
bis
poru
s
Agro
cybe
aeg
erit
a
Am
anit
a v
irosa
Arm
illa
ria l
ute
o-v
iren
s
Bole
topsi
s le
uco
mel
as
Bole
tus
eduli
s
Cli
tocy
be
neb
ula
ris
Fla
mm
uli
na v
eluti
pes
29
Chapter 1
Tab
le 1
.1.
conti
nued
Ref
.
Ngai
and N
g, 2004
Nag
ata
et a
l., 2005
Li
et a
l., 2010
Ble
ule
r-M
artí
nez
et
al., 2
011
Zhao
et
al., 2
009b
Par
k e
t al
., 2
004
Zhan
g e
t al
., 2
009
Li
et a
l., 2008
Wan
g e
t al
., 2
000
a
Nag
re e
t al
., 2
010
Zhao
et
al., 2
010
Zhan
g e
t al
., 2
010b
Chum
khunth
od e
t al
., 2
006
Wan
g e
t al
., 1
996
Lin
and C
hou, 1984
Mar
ty-D
etra
ves
et
al., 2
004
Liu
et
al., 2
006
3D
str
uct
ure
FIP
Jaca
lin
-
XC
L
- - - -
β-p
ropel
ler
- - -
RIC
IN
- -
XC
L
-
Sp
ecif
icit
y
Gal
/Gal
NA
c
muci
n
Inuli
n
Gal
/Gal
NA
c
sever
al s
ug
ars
sial
ic a
cid
Inuli
n
sever
al s
ug
ars
mel
ibio
se
com
ple
x s
ugar
s
Inuli
n
Inuli
n
Gal
NA
c
sever
al s
ug
ars
thyro
glo
buli
n
Gal
/Gal
NA
c
xylo
se, in
uli
n
MW
(k
Da)
Su
bun
it m
ass
* n
o o
f su
bun
its
18 *
1
24 *
1
51 *
1
16.1
* 1
17 *
1
16 *
ND
16 *
2
16.2
* 2
40 *
1 +
41 *
1
11 *
4
30 *
2
16 *
2
31.5
* 2
17.5
* 2
13 *
2
15 *
1
14.4
* 2
Lec
tin
sou
rce
Ganoder
ma c
ap
ense
Gri
fola
fro
ndosa
Her
iciu
m e
rinace
um
Sord
ari
a m
acr
osp
ora
Inocy
be
um
bri
nel
la
Paec
ilom
yces
japo
nic
a
Pholi
ota
adip
osa
Ple
uro
tus
citr
inop
ilea
tus
Ple
uro
tus
ost
reatu
s
Rhiz
oct
onia
bata
tico
la
Russ
ula
del
ica
Russ
ula
lep
ida
Sch
izop
hyl
lum
com
mun
e
Tri
cholo
ma m
ongoli
cum
Volv
ari
ella
volv
ace
a
Xer
oco
mus
chry
sente
ron
Xyl
ari
a h
ypoxy
lon
30
Chapter 1
1.6.1. Basidiomycota
1.6.1.1. Lectins from the fungal order Agaricales
1.6.1.1.1. Agaricus arvensis lectin
A. arvensis lectin (AAL) is an inulin specific lectin purified from the dried fruiting bodies of
the wild edible mushroom A. arvensis. AAL has a molecular weight of 30.4 kDa and is
composed of two subunits of 15.2 kDa each (Zhao et al., 2011). The lectin exhibits potent
antiproliferative activity towards HepG2 and MCF-7 tumor cells with an IC50 of 1.64 and 0.82
μM, respectively.
1.6.1.1.2. Agaricus bisporus lectin
Four A. bisporus lectins (ABL) were found in the common commercial golden white
mushroom A. bisporus. They have molecular weights ranging between 64 and 85 kDa, and
are made up of identical subunits of 16 kDa (Presant and Kornfeld, 1972; Ahmad et al., 1984;
Sueyoshi et al., 1985). The biological activity of all these ABLs cannot be inhibited by any
simple sugar but is inhibited by Gal β-1,3-GalNAc (Yu et al., 1993).
Incubation of ABL with different cell lines (HT29 human colorectal carcinoma cells, Caco-2
human colorectal cancer cells, human breast cancer MCF-7 cells, and rat mammary
fibroblasts Rama-27 cells) revealed an inhibitory effect of the lectin in all these cells in a
dose-dependent manner. For instance, 50% inhibition of HT29, MCF-7 and Rama-27 cells
was achieved by 3, 5 and 25 μg/ml ABL, respectively, while this value was more than 50
μg/ml for Caco-2 cells (Yu et al., 1993). ABL also exerted a dose-dependent proliferation
inhibitory effect on human ocular fibroblasts. This inhibition was recorded to be 40% when
ABL was dosed at 100 μg/ml (Batterbury et al., 2002). It was shown that FITC-ABL was
bound to the cell surface and was then internalized in the cells and accumulated around
the nuclear envelope. Furthermore, ABL induced a strong antiproliferative activity against
human retinal pigment epithelial cells with an inhibition of 80% at 60 µg/ml ABL. It was
proposed that ABL could block the antigenic sites which resulted in the inhibition of cell
proliferation (Kent et al., 2003). ABL also caused lymphocyte (T cells) death in a dose- and
time-dependent manner with a reduction in the cell viability of about 50% after a 2h
incubation with 100 nM lectin. Most of the cells died after 24 h (Ho et al., 2004).
31
Chapter 1
1.6.1.1.3. Agrocybe aegerita lectin
A. aegerita lectin was isolated from the fruiting bodies of the mushroom A. aegerita. The
lectin is a homodimeric protein and consists of two subunits of 15 kDa. The activity of A.
aegerita lectin was inhibited by lactose and some glycoproteins such as bovine submaxillary
mucin, glycophorin A, and hog gastric mucin (Sun et al., 2003). The lectin agglutinates
erythrocytes of all human types (A, B and O) and 12 different animal species.
The A. aegerita lectin was reported to have high inhibitory activity towards human and mouse
tumour cells. For instance Zhao et al. (2003) reported a strong inhibitory effect of the A.
aegerita lectin against seven different tumour cell lines (SW480, HeLa, SGC-7901, MGC80-
3, BGC-823, HL-60 and S-180 cells). The effects of A. aegerita lectin in all these cell lines
were dose-dependent with inhibition effects between 42.8% and 82.6% as determined by
MTT assay when the lectin was dosed at 100 µg/ml (Zhao et al., 2003). Moreover, in vivo
studies showed that when A. aegerita lectin was injected into tumour-bearing mice the lectin
reduced the tumour growth by 36.36%, which also significantly reduced the death ratio of the
treated group by 80% compared with the control group (Zhao et al., 2003). Interestingly, the
A. aegerita lectin exerted toxicity towards mice with an LD50 value of 15.85 mg/kg (Sun et al.,
2003).
It was shown that the activity of the A. aegerita lectin in HeLa cells was due to apoptosis
induction which depends mainly on the internalization of the lectin into the cells and its
nuclear localization (Liang et al., 2009). In addition, DNase activity was also proposed as a
mechanism behind the A. aegerita lectin activity (Zhao et al., 2003). Similar to the native A.
aegerita lectin, the recombinant A. aegerita lectin also induced apoptosis in HeLa cells (Yang
et al., 2005a).
In addition to the activity of the A. aegerita lectin on tumour cells, the lectin showed antiviral
activity towards tobacco mosaic virus (TMV) (Sun et al., 2003). The 50% inhibition dose of
the lectin for TMV infection was determined to be 35 ± 5 µg/ml. To explain the mode of
action of the A. aegerita lectin on TMV it was suggested that the lectin attaches to TMV
which leads to blocking of the infection sites (Sun et al., 2003).
1.6.1.1.4. Agrocybe cylindracea lectin
A lectin named ACL was purified from the fruiting bodies of the edible mushroom A.
cylindracea (Yagi et al., 1997). ACL was found to be a heterodimeric lectin with a molecular
32
Chapter 1
weight of 31.5 kDa and has specificity towards lactose, sialic acid and inulin. ACL was
reported to have potent mitogenic activity towards mouse splenocytes (Wang et al., 2002a).
ACL exhibited potent anti-nematode toxicity against two plant parasitic nematodes
Ditylenchus dipsaci and Heterodera glycines (Zhao et al., 2009a). The effect of ACL was
concentration-dependent as well as time-dependent with an LC50 of 1.4 mg/ml when D.
dipsaci was incubated for 48 h with the lectin. A 4.5-fold lower toxicity of ACL was recorded
on H. glycines (LC50 = 6.3 mg/ml) (Zhao et al., 2009a). The toxic effect of ACL was reduced
about 40 % in both nematodes after adding a specific sugar (lactose).
1.6.1.1.5. Amanita virosa lectin
A 36 kDa lectin was isolated from the fruiting bodies of the mushroom A. virosa. The lectin
was characterized as a homodimeric protein composed of two subunits with a molecular mass
of 18 kDa. The activity of this lectin was not inhibited by any simple sugar (Antonyuk et al.,
2010). This lectin exerted a cytotoxic effect towards CEM T4 and Jurkat human cells with
respective LD50 values of 0.72 and 0.44 μg/ml, respectively, while less toxicity was found in
the mammalian leukemia L1210 cells, the LD50 being 3.42 μg/ml (Antonyuk et al., 2010).
1.6.1.1.6. Armillaria luteo-virens lectin
A lectin called ALL has been found in dried fruiting bodies of the A. luteo-virens mushroom.
It is a dimeric protein with a molecular weight of 29.4 kDa. ALL shows specificity towards
inulin (Feng et al., 2006). The lectin showed antiproliferative activity against MBL2, L1210
and HeLa tumor cells with IC50 values of 2.5, 5, and 10 μM, respectively.
1. 6.1.1.7. Clitocybe nebularis lectin
Different lectins have been found in the C. nebularis fruiting bodies. The molecular mass of
these lectins ranged between 15.5 and 31 kDa. These proteins show specificity mainly for
asialofetuin and lactose (Pohleven et al., 2011). CNLs belong to the ricin B-like lectin
superfamily (Pohleven et al., 2009).
CNL was reported to have antiproliferative activity towards leukemic Mo-T cells as
determined by the MTS assay (Pohleven et al., 2009). The effect of CNL was dose-dependent
and the reduction in cellular proliferation was about 60 % at 100 µg/ml CNL. Interestingly the
activity of CNL was abolished after preincubation of the lectin with its specific sugar (lactose)
which most probably means that binding of CNL to a specific sugar is the first step in starting
33
Chapter 1
the biological effect of the lectin (Pohleven et al., 2009). A similar inhibition was reported in
Jurkat cells after incubation with recombinant CNL (Pohleven et al., 2012).
Feeding of the nematode Caenorhabditis elegans on Escherichia coli expressing CNL
inhibited the development of the larvae by approximately 50% and none of these larvae
developed to adult whereas about 80% of these larvae became an adult in the control
treatment (Pohleven et al., 2012).
CNL exhibits insecticidal activity towards different insects. Feeding of the fruit fly
(Drosophila melanogaster) on a diet containing CNL resulted in a significant mortality with
an LC50 about 48 μg/ml. In addition, CNL showed an important anti-nutritional effect towards
the Colorado potato beetle (Leptinotarsa decemlineata) and this effect was concentration-
dependent. For example feeding the larvae on 0.02% CNL for 10 days reduced the larval
weight about 50% compared to the control larvae (Pohleven et al., 2011). Moreover a 10-fold
higher toxicity towards D. melanogaster was observed with another lectin isolated from
Clitocybe nebularis (called CnSucL) but this lectin did not show any toxic effect in L.
decemlineata (Pohleven et al., 2011).
Feeding of Aedes aegypti on a diet containing E. coli BL21 (DE3) cells expressing CNL
reduced the survival of second instar larvae for about 80% (Bleuler-Martínez et al., 2011).
The same authors also showed that CNL has toxicity towards the amoeba Acanthamoeba
castellanii.
1.6.1.1.8. Coprinopsis cinerea galectin (CGL2)
Several lectins are present in the fruiting bodies of the mushroom C. cinerea. They are called
CGL1, CGL2 and CGL3 and are genetically related to family of β-galactoside-binding lectins
(Cooper et al., 1997; Boulianne et al., 2000; Walti et al., 2008).
Both CGL1 and CGL2 show nematotoxic activity towards C. elegans (Butschi et al., 2010).
Practically, L1 larvae of C. elegans were fed on a diet containing E. coli cells expressing
either the CGL1 or the CGL2 proteins. After 72h the number of L4 larvae was recorded. Only
10 ± 10% larvae in both treatments reached the L4 stage while all the larvae in the control
treatment became L4. Further analysis on CGL2 showed that the effect was dose-dependent
with an LD50 value of 350 mg/ml (Butschi et al., 2010).
The toxicity of CGL2 was dependent on its ability to bind carbohydrate moieties mainly on
the intestinal epithelium of C. elegans while no activity was detected with the mutant CGL2
34
Chapter 1
protein (W72G) which does no longer possess β-galactoside binding activity (Butschi et al.,
2010).
In addition to the anti-nematode activity, CGL2 also has anti-insect activity. Feeding of A.
aegypti on recombinant CGL2 expressed in E. coli reduced the larval survival for about 80%
(Künzler et al., 2010).
1.6.1.1.9. Flammulina velutipes lectin
FVL is a hemagglutinin composed of one subunit of 12 kDa found in the fruiting bodies of
the mushroom F. velutipes. The hemagglutinating activity of FVL was inhibited by
lactoferrin, a milk glycoprotein (Ng et al., 2006). FVL exerted a dramatic antiproliferative
activity against L1210 cells with an IC50 of 13 μM. Moreover 40 μM FVL inhibited the
cellular proliferation completely.
1.6.1.1.10. Grifola frondosa lectin
The lectin named GFL was isolated from the fruiting bodies of the mushroom G. frondosa.
GFL has high affinity for GalNAc and a molecular mass between 30-52 kDa (Kawagishi et
al., 1990). More recently, Nagata et al. (2005) extracted another lectin from G. frondosa with
a molecular weight of 24 kDa. In contrast to the GFL isolated by Kawagishi et al. (1990), the
activity of GFL was not affected by any monosaccharide but was only inhibited by porcine
stomach mucin (Nagata et al., 2005).
GFL exerted a strong cytotoxicity towards HeLa cells. The minimum GFL concentration
necessary to kill all the cells was 25 μg/ml (Kawagishi et al., 1990). Interestingly this toxicity
of GFL for HeLa cells was inhibited by preincubation of the lectin with its specific sugar
(GalNAc).
1.6.1.1.11. Inocybe umbrinella lectin
A lectin with a molecular weight of 17 kDa was extracted from the fruiting bodies of the toxic
mushroom I. umbrinella and named IUL (Zhao et al., 2009b). Several sugars could inhibit the
hemagglutinating activity of IUL such as raffinose, melibiose, lactose and galactose.
HIV-1 reverse transcriptase was inhibited by IUL with an IC50 of about 5 mM. Moreover,
IUL exhibited an antiproliferative effect towards hepatoma HepG2 and breast cancer MCF-7
cells. The IC50 values determined were 3.5 and 7.4 mM, respectively (Zhao et al., 2009b).
35
Chapter 1
1.6.1.1.12. Marasmius oreades lectin
A lectin called MOA was found in the fairy ring mushroom M. oreades. MOA has specificity
towards Galα1,3Gal/GalNAc. MOA is a heterodimeric protein of 50 kDa, with two subunits
of 33 and 23 kDa, respectively (Winter et al., 2002; Wohlschlager et al., 2011). MOA was
shown to possess a strong toxicity towards the nematode C. elegans and the amoeba A.
castellanii when both organisms were incubated in the presence of MOA-expressing E. coli.
Although all the C. elegans become L4 in the control treatment none of these worms reaches
the L4 stage when fed on MOA. In addition, MOA inhibited the growth of A. castellanii
(Wohlschlager et al., 2011).
1.6.1.1.13. Pholiota adiposa lectin
The P. adiposa lectin (PAL) is a homodimeric protein composed of two identical subunits of
16 kDa each. The plant polysaccharide inulin was the only carbohydrate compound which
inhibited the hemagglutinating activity of PAL (Zhang et al., 2009). The lectin induced strong
inhibitory activity against the cellular proliferation of HepG2 and MCF-7 tumor cells with an
IC50 value of 2.1 and 3.2 μM, respectively. Furthermore, PAL also potently inhibited the HIV-
1 reverse transcriptase with an IC50 value of 1.9 μM (Zhang et al., 2009).
1.6.1.1.14. Pleurotus citrinopileatus lectin
A 32.4 kDa lectin was extracted from fresh fruiting bodies of the edible mushroom P.
citrinopileatus. The hemagglutinating activity of PCL was inhibited be several sugars such as
maltose and insulin.
PCL showed potent antitumor effect in mice bearing sarcoma 180 with approximately 80%
inhibition of the tumor growth after 20 days treatment of the mice with PCL (5 mg/kg body).
Moreover the lectin exerted inhibitory activity against HIV-1 reverse transcriptase with an
IC50 of 0.93 μM (Li et al., 2008).
1.6.1.1.15. Pleurotus ostreatus lectin
POL is a melibiose-specific lectin isolated from the fruiting bodies of the oyster mushroom P.
ostreatus. The lectin is composed of two subunits with a molecular mass of 40 and 41 kDa,
respectively. Injection of POL into mice for 20 days at the dose of 1.5 mg/kg body weight
inhibited tumor growth of sarcoma S-180 and hepatoma H-22 cells by 88 and 75%,
respectively (Wang et al., 2000a).
36
Chapter 1
1.6.1.1.16. Schizophyllum commune lectin
A homodimeric lectin (SCL) was purified from the edible split gill mushroom S. commune.
The protein has a molecular mass of 64 kDa and is composed of two subunits of 32 kDa.
Lactose potently inhibited the activity of SCL (Han et al., 2005). SCL exerted a dramatic
inhibition against HIV-1 reverse transcriptase with an IC50 of 1.2 μM (Han et al., 2005).
Chumkhunthod et al. (2006) isolated another lectin from S. commune with a subunit of 31.5
kDa and different sugar specificity. This lectin showed high affinity towards GalNAc. The
GalNAc-specific SCL showed a potent cytotoxic effect towards human epidermoid carcinoma
cells with an IC50 value of 20 μg/ml (Chumkhunthod et al., 2006).
1.6.1.1.17. Tricholoma mongolicum lectin
Two lectins, named TML-1 and TML-2, have been purified from the mycelium of the edible
mushroom T. mongolicum. Both proteins are built up of two subunits with a similar molecular
weight of 17.5 kDa. The activity of TML1 and TML2 was abolished by several sugars such as
lactose, GalNAc and galactose (Wang et al., 1995). Both lectins exhibited antiproliferative
effects towards mouse monocyte-macrophage PU5-1.8 cells and mouse mastocytoma P815
cells. In addition, TML-1 and TML-2 inhibited the growth of sarcoma 180 cells by 69 % and
92%, respectively (Wang et al., 1996).
Feeding of the plant nematodes D. dipsaci and H. glycines on a diet containing TML-1 and
TML-2 revealed that both lectins possess nematotoxic activity (Zhao et al., 2009a). The effect
of TML-1 and TML-2 was time- and dose-dependent in both nematodes with LC50 values of
6.3 and >10 mg/ml, respectively, for D. dipsaci, while these values were 6.4 and 1.7 mg/ml,
respectively, for H. glycines.
Incubation of human hepatoma (H3B), human choriocarcinoma (JAr), mouse melanoma
(B16) and rat osteosarcoma (ROS) cell lines with TML-1 and TML-2 decreased the cell
viability in all cell lines as shown in Table 1.2 (Wang et al., 2000b).
37
Chapter 1
Table 1.2. Decrease in viability of different tumor cell lines after exposure to 1μM of TML-1 or TML-
2. The table was adapted from Wang et al. (2000b).
Tumor cell line % decrease in tumor cell viability
TML-1 (1μM) TML-2 (1μM)
H3B 58 ± 6 44 ± 3
B16 39 ± 3 56 ± 6
Jar 37 ± 2 26 ± 2
ROS 35 ± 1.4 41 ± 11
1.6.1.1.18. Volvariella volvacea lectin
The lectin VVL was isolated from the fruiting bodies as well as from cultured mycelia of the
edible mushroom, V. volvace. VVL is a homodimeric protein with a molecular mass of 32
kDa. The hemagglutinating activity of VVL was not inhibited by simple carbohydrates but it
was inhibited by thyroglobulin (She et al.,1998).
VVL was reported to exert a toxic effect towards mice with an LD50 of 17.5 mg/kg mice. In
addition, VVL showed antitumor activity against Sarcoma 180 cells. When mice were
inoculated with these tumor cells their lifespan was 12.5 ± 5 days but when these mice were
injected with 85 or 175 μg VVL per mouse the lifespan increased for 63 and 110 %,
respectively, which demonstrated the strong activity of VVL towards Sarcoma 180 cells (Lin
and Chou, 1984).
VVL exerted a strong reduction of the cell viability of T cells. The cell viability was reduced
by approximately 50% when the cells were incubated for 2h with 10 nM VVL and all the
cells died after 24 h incubation with 125 nM VVL (Ho et al., 2004).
1.6.1.2. Lectins from the fungal order Atheliales
1.6.1.2.1. Sclerotium rolfsii lectin
SRL is a lectin extracted from the sclerotial bodies of the soil-borne phytopathogenic fungus
S. rolfsii. The lectin was described as a homodimeric protein made up of two subunits of 17
kDa. SRL has high affinity towards Gal/GalNAc (Wu et al., 2001). SRL showed anti-
nematode activity against the common root knot nematode, Meloidogyne incognita.
Incubation of M. incognita juveniles with 47 μg/ml SRL for 48h resulted in 36 % mortality
38
Chapter 1
which increased to 48% with a 5-fold higher dose of SRL (Bhat et al., 2010). It was proposed
that binding of SRL to glycoproteins present on the digestive tract of the nematode might
explain the toxicity of SRL.
1.6.1.3. Lectins from the fungal order Boletales
1.6.1.3.1. Boletus edulis lectin
BEL was purified from fresh fruiting bodies of B. edulis. The lectin has specificity for
melibiose and xylose. It is a homodimeric lectin that is built of two subunits of 16.3 kDa
(Zheng et al., 2007).
BEL exerted anti-nematode activity towards D. dipsaci and H. glycines (both plant parasitic
nematodes). For example feeding of both nematodes on a diet containing 10 mg/ml BEL for
48h resulted in 34 and 59% mortality, respectively (Zhao et al., 2009a).
BEL also showed an inhibitory effect towards human immunodeficiency virus-1 reverse
transcriptase with an IC50 of 14.3 μM (Zheng et al., 2007).
Furthermore BEL was reported to inhibit the proliferation of human carcinoma cell lines
dramatically. The proliferation of the colon cancer cells HT29 was inhibited for 92% at a
concentration of 10 µg/ml BEL. Less inhibition was observed in liver cancer cells (HepG2)
and breast cancer cells (MCF-7) with 79% and 77% inhibition, respectively, at the same
concentration (Bovi et al., 2011).
1.6.1.3.2. Boletus venenatus lectin
The BVLs are a family of isolectins that were purified from the mushroom B. venenatus and
were named BVL-1 to -8, respectively. All BVLs have a similar molecular weight (33 kDa)
and are composed of three identical subunits of 11 kDa. Mono- and oligosaccharides failed to
inhibit BVL activity, but the lectin activity was strongly inhibited by glycoproteins such as
asialofetuin (Horibe et al., 2010). BVLs exert high toxicity towards mice and rats. Injection of
BVLs into mice at a ratio of 0.5 mg/mouse resulted in killing of all the mice within a day after
the injection. Moreover, although oral feeding of rats on a diet containing 40 mg BVLs/kg
body did not kill these animals, they suffered from diarrhea about 4h after lectin application.
Interestingly, using an anti-diarrheal before BVL treatment prevented the rats to suffer from
diarrhea (Horibe et al., 2010).
39
Chapter 1
1.6.1.3.3. Xerocomus chrysenteron lectin
XCL is a lectin identified from the edible mushroom X. chrysenteron. The lectin is specific
for GalNAc and Gal and has a molecular weight of 15 kDa (Trigueros et al., 2003). XCL
exerted toxic effects on fruit fly, D. melanogaster and pea aphid, Acyrthosiphon pisum with
an LC50 of 0.4 and 0.7 mg/ml, respectively (Trigueros et al., 2003). XCL was shown to be
internalized in insect (SF9) or mammalian (NIH-3T3 and Hela) cell lines via a clathrin-
dependent pathway (Francis et al., 2003). Moreover, feeding of Myzus persicae nymphs on an
artificial diet containing different concentrations of XCL for 24h resulted in a significant
mortality of the insects with an LC50 of 0.46 mg/ml. In addition the lectin also exerted toxic
effects on other biological parameters such as development time, weight and fecundity
(Karimi et al., 2007).
A recent report showed that XCL has a highly significant effect on the growth of the
nematode C. elegans and the mosquito A. aegypti (Bleuler-Martínez et al., 2011). At the time
when all the larvae of C. elegans and A. aegypti reached L4 and L2, respectively, in the
control treatment, 0 and 6% of the respective larvae fed on E. coli cells expressing XCL
reached the same stage.
XCL also caused a dose-dependent inhibition of cellular proliferation of two mammalian cell
lines, namely Hela and NIH-3T3 cells (Marty-Detraves et al., 2004), and it was proposed that
XCL interferes with the cell adhesion process by binding to receptors on the cell surface.
1.6.1.4. Lectins from the fungal order Cantharellales
1.6.1.4.1. Rhizoctonia bataticola lectin
RBL is a lectin isolated from the mycelium of the phytopathogenic fungus R. bataticola. The
lectin shows high affinity towards complex sugars (Nagre et al., 2010). The molecular mass
of RBL is about 44 kDa and the protein consists of four subunits of 11 kDa.
RBL exerted a significant cytotoxic effect on the human ovarian cancer cell line PA-1 in a
concentration-dependent manner with an LC50 of 0.15 µM (Nagre et al., 2010).
1.6.1.4.2. Rhizoctonia solani agglutinin
R. solani agglutinin, known as RSA, is a lectin that was purified from the soil pathogen R.
solani (Vranken et al., 1987). RSA was found to be a homodimeric protein consisting of two
identical subunits of 15.5 kDa. The lectin has high affinity for GalNAc/Gal and more complex
40
Chapter 1
glycoproteins (Candy et al., 2001). RSA is structurally and evolutionary related to the family
of proteins possessing a ricin-type lectin motif (Candy et al., 2001). R. solani produces black
sclerotia in harsh conditions. Since RSA is an abundant protein in these sclerotia the lectin
was proposed to play role as a storage protein (Kellens and Peumans, 1990).
1.6.1.5. Lectins from the fungal order Polyporales
1.6.1.5.1. Ganoderma capense lectin
GCL is a lectin isolated from the medicinal mushroom G. capense. The lectin has a molecular
mass of 18 kDa and its activity can be inhibited by Gal/GalNAc (Ngai and Ng, 2004). GCL
induced proliferation inhibitory activity against three cancer cell lines L1210, M1 and HepG2
with IC50 values of 8 μM, 12.5 μM and 16.5 μM, respectively (Ngai and Ng, 2004).
1.6.1.5.2. Ganoderma lucidum lectin
GLL is a lectin isolated from the fruiting bodies of the mushroom G. lucidum. The lectin was
found to be a hexameric protein with subunits of 18.5 kDa. Simple sugars failed to inhibit the
hemagglutinating activity of GLL which was inhibited by glycoproteins such as fetuin and
fibrinogen (Thakur et al., 2007). A different lectin was purified from G. lucidum and was
found to be a monomer with a molecular mass of 15 kDa (Girjal et al., 2011)
A strong toxicity (LC50=1.7 mg/ml) of GLL was induced when the plant nematode H.
glycines was fed on GLL for 48h. A lower toxicity was observed with the nematode D.
dipsaci. When this worm was fed on diet containing 10 mg/ml GLL for 48h the mortality rate
was about 34% (Zhao et al., 2009a).
Interestingly, GLL possesses a significant antifungal effect towards several phytopathogens
and dermatophytic fungi. The activity was determined as the Minimum Inhibitory
Concentration (MIC) of GLL against different fungi (Table 1.3) (Girjal et al., 2011). To our
knowledge no fungal lectins with antifungal activity except for GLL have been reported. So
far only very few plant lectins with antifungal activity have been reported, such as the potato
tuber lectin, the stinging nettle lectin, the wheat germ lectin and the flageolet bean lectin
(Broekaert et al., 1989;Gozia et al., 1995; Ciopraga et al., 1999; Xia and Ng, 2005).
41
Chapter 1
Table 1.3. Minimum Inhibitory Concentration (MIC) of GLL against pathogenic fungi causing plant
diseases (Phytopathogens) and skin diseases (Dermatophytes). The table was adapted from (Girjal et
al., 2011).
Phytopathogenic fungi Dermatophytic fungi
Fungal strains MIC
( μg/ml)
Fungal strains MIC
( μg/ml)
Fusarium oxysporum 20 Trichophyton rubrum 65
Penicillium chrysogenum 35 Trichophyton tonsurans 20
Aspergillus niger 50 Trichophyton interdigitale 20
Colletotrichum musae 60 Epidermophyton floccosum 15
Botrytis cinerea 65 Microsporum canis 70
1.6.1.6. Lectins from the fungal order Russulales
1.6.1.6.1. Hericium erinaceum lectin
The H. erinaceum agglutinin (HEA) was extracted from the fruiting bodies of the monkey
head mushroom H. erinaceum. The lectin has a molecular mass of 51 kDa and has high
affinity towards inulin (Li et al., 2010). HEA exhibited potent inhibition of the cellular
proliferation of hepatoma (HepG2) and breast cancer (MCF-7) cells with IC50 values of
56 and 77 μM, respectively. The lectin also exerted high inhibition activity against HIV-1
reverse transcriptase with an IC50 of 32 μM.
1.6.1.6.2. Russula delica lectin
RDL is a dimeric lectin found in the fresh fruiting bodies of the mushroom R. delica. The
lectin consists of two identical subunits of 30 kDa. RDL showed high specificity towards
inulin and o-nitrophenyl-β-D-galactopyranoside (Zhao et al., 2010). RDL manifested high
HIV-1 reverse transcriptase inhibitory activity with an IC50 of 0.26 μM. Furthermore, the
proliferation of MCF-7 breast cancer cells and HepG2 hepatoma cells was strongly inhibited
by RDL with IC50 values of 0.52 and 0.88 µM, respectively.
1.6.1.6.3. Russula lepida lectin
The R. lepida lectin (RLL) was isolated from dried fruiting bodies of the mushroom R. lepida.
The lectin is composed of two subunits with a mass of 16 kDa each. Inulin and O-
42
Chapter 1
nitrophenyl-β-D-galacto-pyranoside inhibited the hemagglutinating activity of RLL. In
addition, RLL demonstrated antiproliferative activity towards two tumor cell lines, MCF-7
and Hep G2 with IC50 values of 0.9 and 1.6 mM, respectively (Zhang et al., 2010b).
1.6.1.7. Lectins from the fungal order Thelephorales
1.6.1.7.1. Boletopsis leucomelas lectin
KL-15 is a lectin isolated from the edible mushroom Kurokawa (B. leucomelas). The lectin
consists of a single polypeptide of 15 kDa (Koyama et al., 2002). The cellular proliferation of
human monoblastic leukemia U937 was inhibited by KL-15 in a dose-dependent manner with
an IC50 of approximately 15 mg/ml. The effect of KL-15 in U937 cells was apoptosis-
dependent as was clearly determined via observation of typical apoptosis features such as
formation of apoptotic bodies, nuclear condensation, and DNA fragmentation (Koyama et al.,
2002).
1.6.2. Ascomycota
1.6.2.1. Lectins from the fungal order Eurotiales
1.6.2.1.1. Paecilomyces japonica lectin
PJL is a sialic acid-specific lectin that was extracted from the mushroom P. japonica. The
molecular mass of PJL is 16 kDa (Park et al., 2004). PJA decreased the cell viability of
human stomach cancer SNU-1 cells, human pancreas cancer AsPc-1 cells, and human breast
cancer MDA-MB-231 cells by 65, 46 and 30%, respectively, when PJA was dosed at 1 μM.
In contrast only a small effect was observed on human colon cancer SNU-C1cells, human
lung cancer A549 cells, human bladder cancer T24 cells, and human liver cancer Hep3B cells
with toxicity about 7 ± 2% in all cell lines (Park et al., 2004).
1.6.2.1.2. Penicillium chrysogenum lectin
The P. chrysogenum lectin, abbreviated as PeCL, is a lectin produced in the mycelium of the
fungus P. chrysogenum. The activity of PeCL was counteracted by mannose and the lectin has
a molecular mass of 71 kDa divided on two subunits of 31 and 40 kDa, respectively (Francis
et al., 2011).
PeCL exerted significant differences in mortality of the green peach aphid, M. persicae when
this aphid was fed on an artificial diet containing PeCL with an LC50 value of 0.4 mg/ml
43
Chapter 1
(Francis et al., 2011). Interestingly, PeCL showed a 2-fold higher toxicity than the plant lectin
concanavalinA.
1.6.2.2. Lectins from the fungal order Helotiales
1.6.2.2.1. Sclerotinia sclerotiorum agglutinin
The lectin abbreviated as SSA is a lectin extracted from the sclerotia of the soil-borne plant
pathogen S. sclerotiorum, a fungus with a wide range of hosts. SSA was characterized as a
homodimeric protein made up of two subunits of approximately 17 kDa. The lectin has
specificity towards Gal/GalNAc (Candy et al., 2003).
The crystal structure of SSA was determined at 1.6 Å resolution and confirmed the β-trefoil
fold for SSA similar to other ricin B-like lectins. SSA contains a single carbohydrate-binding
site per monomer and reveals a novel dimeric assembly markedly dissimilar from those
described for other ricin-type lectins (Sulzenbacher et al., 2010).
Recently, SSA was also shown to have a dramatic toxicity towards the insect A. aegypti and
the amoeba A. castellanii when they were fed on E. coli cells expressing SSA. 45% of the A.
aegypti larvae fed on SSA failed to reach the L2 stage while all of these larvae developed to
L2 in the control treatment. Moreover, the growth of A. castellanii was inhibited by 76% after
6 days treatment with the recombinant SSA (Bleuler-Martínez et al., 2011).
1.6.2.3. Lectins from the fungal order Pezizales
1.6.2.3.1. Aleuria aurantia lectin
A fucose binding lectin was extracted from the fruiting bodies of the orange peel mushroom
A. aurantia. The lectin was reported to have a molecular weight of 72 kDa composed of two
identical subunits (Kochibe and Furukawa, 1980). The A. aurantia lectin expressed in E. coli
demonstrated a high toxicity for different organisms. For example larval feeding of the
nematode C. elegans and the mosquito A. aegypti on E. coli expressing the A. aurantia lectin
showed a drastic effect on the survival and development of these larvae. When all untreated
C. elegans larvae reached the fourth instar only 1.6 % of the treated larvae had become L4. In
addition only 4.4 % of the treated A. aegypti larvae developed to the second instar while all
the untreated larvae reached this instar. Moreover, incubation of the amoeba A. castellanii
with this lectin reduced its growth by 40% (Bleuler-Martínez et al., 2011). It was reported that
glycans of nematodes, insects and amoebae are rich in terminal fucose residues (Ma et al.,
44
Chapter 1
2006; Paschinger et al., 2008) which could explain the toxicity of the A. aurantia lectin
towards all these organisms.
1.6.2.4. Lectins from the fungal order Sordariales
1.6.2.4.1. Sordaria macrospora lectin (TAP1)
TAP1 is a protein isolated from the mushroom S. macrospora. TAP1 was considered to be a
lectin based on its sequence similarity to the X. chrysenteron lectin (Nowrousian & Cebula
2005). TAP1 exerted a dramatic toxicity towards the nematode C. elegans, the insect A.
aegypti and the amoeba A. castellanii when they were incubated with E. coli cells expressing
TAP1 (Bleuler-Martínez et al., 2011). The highest effect was observed against C. elegans
larvae which failed to reach the L4 stage after 72h feeding on TAP1 while all the larvae in the
control treatment developed to L4. As for insecticidal activity, at the time point when all the
larvae of A. aegypti developed to second instar in the control treatment 40% of these larvae
which were fed on TAP1 expressed in E. coli did not reach this instar. In addition, TAP1
reduced the growth of A. castellanii by 33% after 6 days treatment (Bleuler-Martínez et al.,
2011).
1.6.2.5. Lectins from the fungal order Xylariales
1.6.2.5.1. Xylaria hypoxylon lectin
A xylose and inulin specific lectin was detected in the inedible mushroom X. hypoxylon. The
lectin, designated as XHL, has two subunits of 14.4 kDa (Liu et al., 2006).
XHL showed a high toxic effect towards two plant nematodes namely D. dipsaci and H.
glycines. The respective mortality rates were 81 ± 6 and 59 ± 5% when both nematodes were
fed on a diet containing 10 mg/ml XHL, and the respective LC50 values were 0.3 and 2.2
mg/ml, after 48h incubation with the lectin (Zhao et al., 2009a).
XHL showed highly potent antimitogenic activity in mouse splenocytes with an IC50 value
below 0.5 μM. In addition, XHL exerted highly potent antiproliferative activity towards
leukemia M1 and hepatome HepG2 cells with IC50 values less than1 μM (Liu et al., 2006).
45
Chapter 1
1.6.3. Discussion
In the past 20 years many lectins have been reported in fungi. Nevertheless only a small
fraction of the thousands of species of mushrooms have been examined for lectin activity.
Although many fungal lectins have been purified and characterized only some of them have
been studied in detail for their biological activity. Table 1.1 gives an overview of the lectins
that have been studied in most detail for their activity towards some organisms or cells.
Starting from these data a comparative analysis was made for these lectins. Since structural
data are also available for a number of fungal lectins with interesting biological properties,
this study allows investigating whether some structural characteristics could be important for
biological activity.
1.6.3.1. Classification
All fungal lectins with reported toxicity or antiproliferative activity have been isolated mainly
from two phyla. More than two-thirds of these lectins were isolated from the Basidiomycota.
The other lectins were mainly purified from the Ascomycota. Within the group of lectins from
Basidiomycota, 62% of the lectins was purified from the order Agaricales, 10% from
Boletales, 10% Russulales, 7% from Cantharellales and the rest from the orders Atheliales,
Polyporales and Thelephorales. The lectins purified from species within the phylum
Ascomycota were obtained from species within 5 different orders including Eurotiales,
Helotiales, Pezizales, Sordariales and Xylariales.
In recent years a few attempts have been made to classify the fungal lectins. Similarly to the
group of plant lectins fungal lectins have been divided into six different families of lectins
with structural relationships (based on X-ray crystallographic data) and/or evolutionary
relationships (based on homology between the amino acid sequences) (Table 1.4) (Goldstein
and Winter, 2007; Khan and Khan, 2011b).
According to Goldstein and Winter (2007) the group of fungal lectins can be divided into 6
families. Each of these lectin families will be discussed briefly.
1- Ricin family: This group contains lectins which show the presence of a ricin-B like domain
such as the C. nebularis lectin and the M. oreades lectin (Khan and Khan, 2011). In fact, the
ricin-like domains in fungal lectins display structural similarity to domains from plant toxins
such as ricin, abrin and mistletoe lectin.
46
Chapter 1
2- Fungal Immunomodulatory Protein (FIP) family: Unique folds were observed in
Flammulina velutipeslectin which were never reported before in lectins (Paaventhan et al.,
2003).
3- XCL family: This family was first described in the Xerocomus chrysenteron lectin and it
does not show any significant sequence similarity to any known protein in the databases but it
has structural similarity to actinoporins (Birck et al., 2004).
4- β-propeller family: The crystal structure of the lectins in this family consists of a six-bladed
β-propeller fold and a small antiparallel two-stranded β-sheet which plays a role in
dimerization (Wimmerova et al., 2003). The first crystal structure for a fungal lectin was that
fromthe Aleuria aurantia lectin and was found to have the six-bladed β-propeller fold.
5- Galectin family: This family contains all lectins which possess a galactose binding lectin
domain such as lectins from A. aegerita, A. cylindracea and C. cinerea (Khan and Khan,
2011). Actually, the galectin family was first described for the animal lectins that bind β-
galactosides (Barondes et al., 1994).
6- Jacalin family: This family includes the lectins which show sequence similarity to the
sequence of jacalin, a lectin isolated from jackfruit, Artocarpus heterophyllus (Kabir, 1998).
For instance, the Grifola frondosa lectin is a jacalin-related lectin.
A detailed analysis of the structural and evolutionary relationships among fungal lectins and
the taxonomy of the species where they were purified from revealed that there is no
agreement between the classification system of fungal lectins in different lectin families and
the taxonomical classification of the fungi. For instance, the lectins from A. bisporus, A.
aegerita, C. nebularis, F. velutipes, G. frondosa and P. ostreatus represent six lectins isolated
from species belonging to the same fungal order (Agaricales) but each lectin belongs to a
different lectin family (Table 1.4). Moreover, within the same lectin family we can find
lectins from fungal species belonging to different orders but also from different phyla. For
example within the family of Ricin-related lectins we find the lectin from R. solani, a species
belonging to the Basidiomycota as well as the lectin from S. sclerotiorum a species from the
Ascomycota (Table 1.4). Since the grouping of lectins in different lectin families is based on
structural and sequence similarity all lectins within one lectin family have a similar 3D fold.
47
Chapter 1
Table 1.4. Classification of the fungal lectins according to Goldstein and Winter, 2007. Some
examples of fungal lectins with toxic properties and/or antiproliferative activity are given.
Lectin Family Example(s)
Ricin
Clitocybe nebularis lectin
Marasmius oreades lectin
Rhizoctonia solani agglutinin
Sclerotinia sclerotiorum agglutinin
Schizophyllum commune lectin
FIP Flammulina velutipes lectin
XCL
Agaricus bisporus lectin
Sordaria macrosporalectin
Xerocomus chrysenteron lectin
β-propeller Aleuria aurantia lectin
Pleurotus ostreatus lectin
Galectin Agrocybe aegerita lectin
Coprinopsis cinerea lectin
Jacalin Grifola frondosa lectin
1.6.3.2. Localization
Most of the fungal lectins that showed toxic or antiproliferative activity were isolated from
the fruiting bodies of different mushrooms. Some of these lectins were found in the mycelium
of the mushroom (e.g. T. mongolicum and the phytopathogenic P. chrysogenum and R.
bataticola) (Wang et al., 1995; Francis et al., 2011; Nagre et al., 2010) while other lectins
were isolated from the sclerotial bodies (e.g. of three plant pathogens, S. rolfsii, S.
sclerotiorum and R. solani) (Wu et al., 2001; Candy et al., 2003; Vranken et al., 1987). In the
case of R. solani it was shown that the lectin is present both in the mycelium and in the
sclerotia, but lectin concentrations in sclerotia are considerably higher (at least 8 times) than
in the mycelium (Kellens et al., 1992). Furthermore lectin concentrations in R. solani were
shown to be developmentally regulated (Kellens and Peumans, 1990).
48
Chapter 1
1.6.3.3. Specificity
Fungal lectins with toxic or antiproliferative activity exhibit a wide range of carbohydrate
binding specificities, varying from simple monosaccharides to complex sugars or
glycoproteins. About one third of these lectins have high affinity for Gal/GalNAc. In addition
an important fraction of fungal lectins revealed specificity towards complex carbohydrates or
inulin (Table 1.1). Inulins are polymers composed mainly of fructose units, and typically
these have a terminal glucose residue. In contrast to what has been observed in plants, the
mannose-specific lectins are rare within the class of fungal lectins. However, recently Francis
et al., (2011) reported a fungal lectin with high affinity for mannose from P. chrysogenum. It
should also be acknowledged that the specificity reported for some fungal lectins is
questionable (Winter and Goldstein, 2007). For instance, it is difficult to interpret the binding
activity of some fungal lectins that can be inhibited by very different sugars, such as the
lectins isolated from I. umbrinella and P. citrinopileatus (Zhao et al., 2009b; Li et al., 2008).
1.6.3.4. Molecular mass and subunit composition
The molecular mass of fungal lectins showing toxic effects ranges from 12 kDa in the F.
velutipes lectin (Ng et al., 2006) to 114 kDa in the G. lucidum lectin (Thakur et al., 2007), but
the majority of the fungal lectins have a molecular mass of approximately 30 kDa. Some of
these lectins are composed of only one subunit but the majority of fungal lectins consist of
more than one subunit. The number of lectin subunits can reach up to 6 as shown for the
lectin isolated from G. lucidum (Thakur et al., 2007). It is striking that the molecular mass of
the lectin subunits is approximately 16 kDa for a large numbers of fungal lectins (Table 1.1).
1.6.3.5. Biological activity
Fungal lectins were shown to exhibit different activities against various organisms including
viruses, fungi, nematodes, insects, amoebae and different types of cell lines.
1.6.3.5.1. Anti-virus activity:
Several fungal lectins inhibit the reverse transcriptase activity of the Human
Immunodeficiency Virus 1 (HIV-1), with IC50 values ranging between 0.26 μM and 5mM.
Unfortunately it is hard to find a relation between the fungal lectins with inhibitory activity
against HIV-1 reverse transcriptase and their sugar specificity, but it seems that 50% of these
lectins interact with inulin.
49
Chapter 1
Moreover, the fungal lectin from the mushroom A. aegerita exerted an antiviral activity
towards the tobacco mosaic virus (TMV) with an IC50 value of 1.1 ± 0.2 µM (Sun et al.,
2003).
1.6.3.5.2. Anti-fungal activity:
Within the group of fungal lectins only one lectin (GLL, G. lucidum lectin) was found to
display activity towards some phytopathogens and dermatophytic fungi (Girjal et al., 2011).
The Minimum Inhibitory Concentration (MIC) of GLL against the different fungi tested
ranged between 15-70 µg/ml (Table 1.3). Moreover, many fungal lectins were found to be
devoid of antifungal activity, such as for instance lectins from A. aegerita, B. edulis, S.
commune, P. ostreatus, P. citrinopileatus, A. arvensis, H. erinaceum, G. capense, P. adiposa,
I. umbrinella (Sun et al., 2003; Zheng et al., 2007; Chumkhunthod et al., 2006; Wang et al.,
2000a; Li et al., 2008; Zhao et al., 2011; Li et al., 2010; Ngai and Ng, 2004; Zhang et al.,
2009; Zhao et al., 2009b).
1.6.3.5.3. Anti-amoeba activity:
The lectin called CGL2 isolated from the mushroom C. cinerea showed high toxicity towards
A. castellanii with a growth inhibition of 81%. Moderate activity (33-57%) was observed for
some other fungal lectins including the lectins from C. nebularis, A. aurantia, S. macrospora
and S. sclerotiorum. In contrast, CGL3, another lectin isolated from the C. cinerea mushroom
and XCL, a lectin isolated from X. chrysenteron did not show any significant effect towards
A. castellanii (Bleuler-Martínez et al., 2011).
1.6.3.5.4. Anti-nematode activity:
Some fungal lectins showed toxic effects against several nematodes, including the model
worm C. elegans but also parasitic nematodes such as D. dipsaci and H. glycines.
From all fungal lectins tested against the plant parasitic nematodesD. dipsaci and H. glycines,
the X. hypoxylon lectin was the most potentagainst both nematodes (Zhao et al., 2009a). In
addition, these nematodes were also affected by other fungal lectins such A. cylindracea
lectin, T. mongolicum lectin (TML-1 and TML-1), G. lucidumand Boletus edulis (Zhao et al.,
2009a).
Rearing of the nematode C. elegans on E. coli expressing different fungal lectins showed
different responses. Some fungal lectins inhibited the larval growth of C. elegans by 85-100%
including lectins from C. cinerea (CGL2), X. chrysenteron, A. aurantia and S. macrospora
50
Chapter 1
but C. nebularis lectin and S. sclerotiorum agglutinin did not show any activity (Bleuler-
Martínez et al., 2011).
S. rolfsii lectin killed about 50% of the common root knot nematode, M. incognita upon
incubation of the nematode with 6.6 μM SRL for 48 h (Bhat et al., 2010).
1.6.3.5.5. Anti-insect activity:
Many fungal lectins have been reported to possess an insecticidal activity against
economically important insect pests.
For instance, C. nebularis lectin exerted toxic activity towards different insects includingthe
mosquito A. aegypti, the fruit fly D. melanogaster and the Colorado potato beetle L.
decemlineata (Bleuler-Martínez et al., 2011; Pohleven et al., 2011). The X. chrysenteron
lectin was found to be toxic towards D. melanogaster, the pea aphid, A. pisum and green
peach aphid, M. persicae (Trigueros et al., 2003; Karimi et al., 2007).
1.6.3.5.6. Anti-mice/rat activity:
The lectins from A. aegerita and V. volvace induced an almost similar toxicity against mice
(LD50 = 16 and 18 mg/kg, respectively) (Sun et al., 2003; Lin and Chou, 1984). In addition,
injection of 0.5 mg B. venenatus lectin per mouse resulted in killing of all the mice within one
day. Moreover, rats suffered from diarrhea when they were fed on a diet containing the B.
venenatus lectin (Horibe et al., 2010).
1.6.3.5.7. Cytotoxicity and antiproliferative activity:
Fungal lectins showed activity towards different cell lines including human, animal and insect
cell lines. For instance, the growth of the Hela tumor cell line was dramatically inhibited by
the lectins from A. aegerita, X. chrysenteron and A. luteo-virens (Zhao et al., 2003; Marty-
Detraves et al., 2004; Feng et al., 2006). The liver cancer cells (HepG2) were affected by
several fungal lectins. Within these lectins the B. edulis lectin induced the highest inhibition
rate (Bovi et al., 2011). Moreover, several lectins inhibited cellular growth of the breast
cancer cells (MCF-7) such as the lectins from B. edulis; A. bisporus, A. arvensis, R. delica
and R. lepida (Bovi et al., 2011; Yu et al., 1993; Zhao et al., 2011; Zhao et al., 2010; Zhang et
al., 2010b).The survival of the mammalian leukemia L1210 cells was reduced dramatically by
different fungal lectins including the lectins from A. virosa, A. luteo-virens, G. capense and F.
velutipes (Feng et al., 2006; Ngai and Ng, 2004; Ng et al., 2006; Antonyuk et al., 2010).
51
Chapter 1
In vivo assays confirmed that injection of some fungal lectins into mice for 20 days inhibited
the tumor growth of sarcoma S-180 cells significantly. The most potent fungal lectin with
anti-tumor activity was the P. ostreatus lectin (Wang et al., 2000a) followed by lectins from
T. mongolicum (TML1 andTML2) and P. citrinopileatus (Wang et al., 1996; Li et al., 2008).
1.6.3.6. Mechanism of fungal lectin activity
In fact, very little is known about the mechanism behind the toxicity or antiproliferative
activity of fungal lectins. Interestingly, the activity was associated with apoptosis induction of
several lectins. For instance, apoptosis was induced in HeLa and U937 cells after treatment
with A. aegerita lectin (Liang et al., 2009) and B. leucomelas lectin (Koyama et al., 2002)
respectively. More research is necessary to unravel the mode of action of fungal lectins.
Therefore it will be interesting to know how and where the fungal lectin is binding with cells.
1.6.4. Conclusions
Although only a limited number of fungal lectins have been studied in great detail for their
biological activities it is clear that several of these lectins possess interesting properties.
Similar to plant and vertebrate lectins it can be envisaged that fungal lectins act as general
recognition molecules in the interaction between cells or in the interaction of cells with other
molecules. Most probably the physiological role of fungal lectins is related to the
identification of glycosylated structures at the level of cells, tissues or whole organisms.
Several reports have proven that fungal lectins can elicit diverse biological responses in
various cells and organisms.
Despite the fact that very little is known with respect to the working mechanism of fungal
lectins, several lectins have proven useful tools for biomedical applications such as the
Cordyceps militaris lectin which has been used as a nutraceutical and in traditional Chinese
medicine for cancer patients in Eastern Asia (Jung et al., 2007).
Since there is still a vast number of fungi that has never been checked for lectin activity, it is
reasonable to assume that many more interesting fungal lectins will still be discovered in the
future. In addition there are also several fungal lectins which until now have only been
cursorily characterized and need to be investigated in much more detail. Anyway from the
available information it is clear that fungi are a rich source of carbohydrate binding proteins
and lectins present in the fungal kingdom most probably play an important role in various
biological processes.
52
Chapter 1
53
Chapter 2
Chapter 2
Parts of this chapter are published in:
Hamshou M, Smagghe G, Van Damme EJM (2007) Analysis of lectin concentrations in
different Rhizoctonia solani strains. Communications in Agricultural and Applied Biological
Sciences, Ghent University 72, 639-644.
Analysis of lectin concentrations in different
Rhizoctonia solani strains
54
Chapter 2
2.1 ABSTRACT
Lectins are carbohydrate-binding proteins that contain at least one carbohydrate binding
domain which can bind to a specific mono- or oligosaccharide. These proteins are widely
distributed in plants. However, over the last decade evidence is accumulating that lectins
occur also in numerous fungi belonging to both the Ascomycota and Basiodiomycota.
Rhizoctonia solani is known to be an important pathogen to a wide range of host plants. In
this study, isolates of R. solani from different anastomosis groups have been screened for the
presence of lectin using agglutination assays to detect and quantitate lectin activity. The
evaluation included determination of the lectin content in mycelium as well as in sclerotia.
The amount of lectin in the sclerotia was higher than in the mycelium of the same strains. The
R. solani strains with the highest amounts of lectin have been selected for cultivation,
extraction and purification of the lectin.
55
Chapter 2
2.2 INTRODUCTION
Lectins constitute a group of (glyco) proteins of non-immune origin, which bind reversibly to
specific carbohydrates or more complex glycans. Plant lectins were defined by Peumans and
Van Damme (1995b) as ‘all proteins possessing at least one non-catalytic domain, which
binds reversibly to a specific mono or oligosaccharide’. Because of their interaction with
carbohydrate structures many lectins are able to agglutinate cells or precipitate
polysaccharides and glycoconjugates. As a consequence of their biochemical properties, they
have become a useful tool in several fields of biological research such as immunology, cell
biology and cancer research (Van Damme et al., 1998).
Living organisms of almost every taxonomic classification ranging from bacteria to higher
animals contain carbohydrate binding proteins known as agglutinins or lectins (Van Damme
et al., 1998). In recent years evidence has also accumulated that many fungi contain
agglutinating substances. Fungal lectins have been isolated and characterized from fruiting
bodies of several higher fungi such as commercial mushroom Agaricus bisporus (Presant and
Kornfeld, 1972) and from mycelium of lower fungi such as the parasitic Phycomycetes
conidiobolus (Ishikawa et al., 1979) and the soil born plant pathogen Rhizoctonia solani
(Kellens and Peumans, 1990b). R. solani is the asexual stage (anamorph) of the fungus,
whereas the sexual stage (teleomorph) is named Thanatephorus cucumeris. In nature R. solani
primarily survives asexually and exists as vegetative mycelium and/or sclerotia (Meyer,
2002). It is a destructive plant pathogen and can cause damage worldwide to more than 142
plant species, including many agricultural and horticultural crops (Guillemaut et al., 2003). In
addition, it is a selective species which is considered to survive in soil in the form of sclerotia
associated with organic matter (Kumar et al., 2002). The relative importance of sclerotia in
the life cycle of the fungus varies with different anastomosis groups. Sclerotia play an
important source of inoculum for some anastomosis groups. However, the mycelium infests
plant debris, and forms the main component of soil-borne inoculum (Kumar et al., 2002). The
sexual fruiting structures and basidiospores were firstly observed and described in detail by
Prillieux and Delacroiz in 1891 (Meyer, 2002). The variability in disease symptoms, host
range, and geographical location of R. solani isolates suggest that there are several strains of
the species. At present, 13 different anastomosis groups of the fungus (AG-1 through AG-13)
have been recognized (Mghalu et al., 2004) based on affinities for hyphal fusion
56
Chapter 2
(anastomosis), a genetic feature that results in exchange of nuclei and the combining of
different genotypes (Blazier and Conway, 2004).
The presence of lectins on the hyphal surface and tissues of R. solani has been reported
(Mghalu et al., 2004). It was shown that the amount of lectin in R. solani is dependent of the
stage of the life cycle. Whereas in young mycelium the lectin concentration is very low, the
amount of lectin increases gradually until formation of sclerotia and reaches its maximum in
adult sclerotia. At the start of the mycelioginic germination, the lectin content in sclerotia
rapidly decreases. The high concentration of R. solani lectin in the sclerotia, and its
developmental regulation, indicate that the lectin probably serves as a storage protein in the
resting structures of this fungus (Kellens and Peumans, 1990a).
Until now there have been a few reports that document the occurrence, purification and
characterization of the R. solani lectin or agglutinin (abbreviated as RSA). The lectin is a
dimeric protein composed of two identical subunits of 13 kDa, and exhibits specificity
towards N-acetylgalactosamine (GalNAc) and several other simple sugars (Vranken et al.,
1987; Kellens and Peumans, 1991). The lectin is present in R. solani strains of different
anatomosis groups. RSA agglutinates both human and rabbit erythrocytes (Kellens and
Peumans, 1990b). However, the lectin exhibits a much higher activity with rabbit red blood
cells. With respect to human erythrocytes RSA prefers blood type A over type B and type O
erythrocytes. The concentrations of lectin ranged from 0.05 to 30 mg/g lyophilized mycelium
(Kellens and Peumans, 1990b). At present the physiological role of Rhizoctonia lectin
remains unclear. Elad et al. (1983) suggested that the lectin may play a role in specific
recognition of the fungus by mycoparasites. Later Kellens and Peumans (1990a) suggested
that the lectin could play a role as a storage protein and potentially as defence protein against
herbivorous insects.
The objectives of this study were to screen for a R. solani strain that expresses a high amount
of lectin. In future experiments the lectin of this strain can be purified in sufficient amounts to
test the insecticidal activity of the lectin towards biting-chewing insects (e.g. cotton leaf
worm, Spodoptera littoralis) as well as piercing-sucking insects (e.g. pea aphid,
Acyrthosiphon pisum) (see chapters 3 and 5).
57
Chapter 2
2.3 MATERIALS AND METHODS
2.3.1 Isolates and growth conditions
Ten Rhizoctonia solani strains of different anastomosis groups (AG) (Pannecoucque et al.,
2008) were obtained from the Laboratory of Phytopathology and Virology at Ghent
University (Table 2.1) and grown on two different media, being potato dextrose agar (for use
in petri dishes) and potato dextrose broth (for liquid cultures in erlenmeyer flasks). The liquid
cultures were started by inoculating 200 ml of liquid medium with a few pieces of
approximately 1 cm² agar covered with mycelium from a 5-day old Rhizoctonia culture grown
on potato dextrose agar. All fungal cultures were incubated in a growth chamber at a
temperature of 25-27°C.
Table 2.1. Different strains of Rhizoctonia solani tested
No. Anastomosis group Strain Host Origin
1 3 ST-11-6 Potato Japan
2 1-1C BV-17 Endive Belgium
3 4 HGI AH-1 Peanut Japan
4 10 91778 Lettuce Belgium
5 1-1A CS-KA Rice Japan
6 6 GV NKN-2-1 Soil Japan
7 1-1B B-17 Lettuce Belgium
8 1-1C BV-17 Endive Belgium
9 2-2LP SLP 3-3 Saint Augustine grass Japan
10 8 (ZG-1-2) 92630 Barley West-Australia
2.3.2 Protein extraction
Mycelium from cultures of about 70-days old was collected and strongly squeezed by hand
before determining the wet weight. Subsequently samples were lyophilized and dry weight
was determined. Protein extracts were made in phosphate buffered saline (PBS; 170 mM
NaCl, 3 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, pH 7.4) using mortar and pestle.
Approximately 0.1 g of dry mycelium was extracted in 3 ml of PBS. The liquid was
58
Chapter 2
transferred to eppendorf tubes and centrifuged for 2 min at 12,000 g. Similarly, sclerotia were
harvested from the petri dishes and homogenized using a mortar and pestle (0.1 g in 3 ml
PBS). After centrifugation, the supernatants were transferred to new eppendorf tubes and
stored in the freezer at -20 °C until use.
2.3.3 Determination of total protein content
Total protein content was determined using the method of Bradford (Bradford, 1976).
Therefore 10 μl of each sample was mixed with 250 µl of the Coomassie Reagent in the wells
of a microtiter plate. Each sample was analysed in triplicate. The absorbance of the samples
was measured in a microtiter plate reader (BioTek Instruments, Inc., Winooski, USA).
2.3.4 Analysis of lectin activity in different Rhizoctonia strains
Lectin activity in extracts from mycelium and sclerotia was analyzed using agglutination
assays with trypsin-treated rabbit erythrocytes (BioMérieux, Marcy L'Etoile, France)
Therefore 10 µl of the protein extract was mixed with 10 µl of ammonium sulphate and 30 µl
of red blood cells in small glass tubes. The agglutination reaction was assessed after
incubation for 30 min at room temperature. For those samples that showed good agglutination
activity a dilution series was made in order to quantify the amount of lectin.
2.3.5 Gel electrophoresis
Crude extracts and pure lectin were analyzed by SDS-PAGE in 15 % (w/v) acrylamide gels as
described by Laemmli (1970). Approximately 22 µg of total protein from each sample and
12.5 µg of RSA were loaded on the gel.
2.4 RESULTS
2.4.1 Agglutination assays
Using agglutination assays with trypsin-treated rabbit erythrocytes lectin activity was checked
in extracts from mycelium as well as sclerotia (if available) of all Rhizoctonia strains under
59
Chapter 2
study. Only Rhizoctonia strains 5 (AG 1-1A), 7 (AG 1-1B) and 8 (AG 1-1C) gave a clear
positive result with protein extracts from mycelium. The lectin in the extracts was quantified
using a dilution series and comparison to a lectin preparation with known concentration.
Strain 5 (AG 1-1A) yielded the highest lectin concentration, representing approximately 2%
of the total protein. The minimal concentration of lectin that could be detected was 0.097
µg/ml. Only strains 2 (AG 1-1C), 6 (AG 6GV), 7 (AG 1-1B) and 8 (AG 1-1C) yielded
sclerotia. Although extracts from all these strains showed agglutination activity, strains 7 (AG
1-1B) and 8 (AG 1-1C) clearly had the highest lectin content (Table 2.2). As shown in Table
2.2 the lectin concentration in the sclerotia is considerably higher than in the mycelium of the
same strain. Sclerotia of strain 7 (AG 1-1B) contain about 8 times higher lectin content than
in the mycelium, sclerotia of strain 8 (AG 1-1C) contain about 128 times the lectin
concentration found in the mycelium.
Table 2.2 Quantification of total protein and lectin content in Rhizoctonia strains
Strain
No.
Anastomosis
group
Lectin content
(mg/g tissue)
Protein content
(mg/g tissue)
% lectin of
total protein
5 1-1A 3.750 181.62 2.06
Mycelium
7 1-1B 0.9375 68.31 1.37
8 1-1C 0.0585 66.24 0.088
Sclerotia 7 1-1B 7.500 282.45 2.65
8 1-1C 7.500 246.42 3.04
2.4.2 Protein analysis
Total protein content was determined in extracts from mycelium and sclerotia of all
Rhizoctonia strains which showed lectin activity. As shown in table 2.2, sclerotia of strains 7
(AG 1-1B) and 8 (AG 1-1C) show a high concentration of total protein. The concentration of
protein in the sclerotia is considerably higher than in the mycelium of the same strains. R.
solani strain 5 (AG 1-1A) reveals a high concentration of total protein in the mycelium.
Crude protein extracts from different Rhizoctonia strains were analysed by SDS-PAGE and
compared to a sample of the pure R. solani lectin (RSA). All strains that yielded lectin activity
also revealed a clear band in the protein extract at the same position as that of RSA (Fig. 2.1).
60
Chapter 2
Figure 2.1. SDS-PAGE of total protein extracts from mycelium (M) and sclerotia (S) of different
Rhizoctonia strains. RSA refers to the pure lectin of R. solani, whereas R shows a reference marker (ß-
galactosidase, 116.0 kDa; bovine serum albumin, 66.2 kDa; ovalbumin, 45.0 kDa; lactate
dehydrogenase, 35.0 kDa; restriction endonuclease Bsp981, 25.0 kDa; ß-lactoglobulin, 18.4 kDa;
lysozyme, 14.4 kDa).
It is clear from the gel that the lectin represents an important fraction of the total protein.
Furthermore it is evident that the lectin band in extracts from sclerotia represents a more
prominent band than in extracts from mycelia, which is in agreement with the results
presented in Table 2.2.
2.5 DISCUSSION
A screening of different strains of Rhizoctonia demonstrated that although many strains reveal
lectin activity, the lectin activity in the different strains/anastomosis groups is quite variable.
The lectin concentration ranged from 0.058 to 7.5 mg/g lyophilized mycelium or sclerotia,
which is in agreement with the data reported by Kellens and Peumans (1990b). The latter
authors have shown that the R. solani lectin concentrations ranged from 0.05 to 30 mg/g
lyophilized mycelium. The same authors also mentioned that the amount of lectin in sclerotia
is higher than in mycelium. Our experiment revealed that the amount of lectin in sclerotia is
8-128 times higher than in the mycelium of the same strain.
Mghalu et al. (2004) performed a large screening of 81 R. solani isolates for lectin activity in
mycelium. However, they did not check the presence of lectin activity in sclerotia. It was
shown that all R. solani strains of especially anastomosis groups 1 and 2 yielded high lectin
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activity. Within anastomosis group 1, AG 1-1A yielded the highest activity. Similarly, our
screening revealed that mycelium of strain 5 of anastomosis group 1-1A yielded the highest
lectin concentration. In addition, R. solani strains of anastomosis groups 1-1B (strain 7) and
1-1C (strain 8) also yielded high lectin activity both in the mycelium and in the sclerotia. At
present we cannot explain why R. solani strain 2 which also belongs to anastomosis group 1-
1C, did not show lectin activity.
From our comparative analysis we can conclude that Rhizoctonia strain 7 (AG 1-1B; Origin:
Belgium) has the highest amount of lectin in the sclerotia and also contains a high lectin
concentration in the mycelium. Since this strain also shows very good growth, strain 7 (AG 1-
1B) was selected for cultivation, extraction and purification of the lectin. In future
experiments the insecticidal activity of the lectin will be tested towards biting-chewing insects
(e.g. cotton leaf worm, Spodoptera littoralis) as well as piercing-sucking insects (e.g. pea
aphid, Acyrthosiphon pisum).
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63
Chapter 3
Chapter 3
Parts of this chapter are published in:
Hamshou M, Smagghe G, Van Damme EJM (2010) Entomotoxic effects of fungal lectin from
Rhizoctonia solani towards Spodoptera littoralis. Fungal Biology 114, 34-40.
Entomotoxic effects of fungal lectin from
Rhizoctonia solani towards Spodoptera littoralis
64
Chapter 3
3.1 ABSTRACT
The effects of the Rhizoctonia solani lectin (RSA) on the growth, development and survival
of an economically important caterpillar in agriculture and horticulture, the cotton leafworm,
Spodoptera littoralis were studied. The high lectin concentration present in the sclerotes of
the soil pathogen R. solani allowed the purification of large amounts of the pure lectin for
feeding experiments with cotton leafworm. Rearing of insects on a diet containing different
concentrations of RSA exerted a strong effect on the larval weight gain. This effect was
visible at the lowest concentration of 1 mg/g RSA at day 8 and day 11. Interestingly with 10
mg/g RSA, there was a dramatic reduction in larval weight of 89% at the end of L6 which was
followed by a high mortality rate of 82% in the treated larvae. Furthermore, the other
developmental stages of pupation and adult formation were also affected. In addition, the data
demonstrated that the combination of RSA with Bt toxin yielded synergistic effects. For
instance, 0.3 mg/g RSA + 0.05 mg/g Bt toxin caused reduced growth rate and higher
mortalities. These findings suggest that RSA is an interesting tool that can be used for
bioengineering insect resistance in important agronomical crops.
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3.2 INTRODUCTION
Problems associated with widespread insecticide usage, together with the development of
insect resistance to Bacillus thuringiensis (Bt) toxins in genetically engineered crops, have
resulted in a greater interest of scientists to exploit the potential of plant defensive proteins,
such as lectins, to help in combating crop damage. Lectins are a ubiquitous group of proteins
and several hundreds of these molecules have been isolated so far from plants, viruses,
bacteria, fungi, invertebrates and vertebrates including mammals (Carlini and Grossi-de-Sa,
2002). Plant lectins are defined as proteins possessing at least one non-catalytic domain,
which binds reversibly to specific mono- or oligosaccharides (Van Damme et al., 2008). One
of the roles attributed to plant lectins is their involvement in plant defense against pathogens
and phytophagous insects (Peumans and Van Damme, 1995b). This protective activity is in
accordance with the observation that most plant lectins are not targeted against plant
carbohydrates, but preferentially bind foreign glycans (Peumans et al., 2000). Next to plants,
it is of great interest that also mushrooms as well as other non-fruiting body forming fungi
contain lectins. Although many carbohydrate-binding proteins from fungi have been reported,
very little is known with respect to their physiological role (Wang et al., 1998).
The Rhizoctonia solani agglutinin, abbreviated as RSA, is a lectin that is synthesized by the
soil pathogen R. solani (Class: Basidiomycetes; Order: Cantharellales). This plant pathogenic
fungus has an asexual life cycle and survives as vegetative mycelium and sclerotia. These
sclerotia enable the fungus to survive in the soil under harsh conditions. In 1987, Vranken and
coworkers first reported the purification and characterisation of RSA. This lectin is a
homodimeric protein composed of 15.5 kDa subunits that show high affinity for N-
acetylgalactosamine (GalNAc) and more complex glycoproteins (Vranken et al., 1987). It was
shown that high concentrations of the lectin accumulate in the sclerotes (2-3% of the total
soluble protein), whereas the lectin concentration in the mycelium is usually rather low (0.1-
2% of the total soluble protein) (Chapter 2). At present, the complete RSA sequence is not
known. However, judging from the N-terminal sequence of 60 amino acids, it can be
predicted that RSA shows no important sequence homology to other known lectin sequences
(Candy et al., 2001), making this lectin a potentially highly novel compound for research into
innovative methods for the control of insect pests.
Because of the high lectin concentration in the sclerotes, it has been suggested that RSA could
play a role as a storage protein and could be involved in the defense of the fungus against
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predators (Kellens and Peumans, 1990a). This hypothesis was put forward based on some
striking similarities with plant lectins that have a dual role. Indeed, for many highly abundant
plant lectins, it was shown that they combine a role in storage with a role in plant defense
whenever the plant is under attack by predators (Peumans and Van Damme, 1995b). Indeed,
many plant lectins have been shown to have toxic effects towards insects (Van Damme, 2008,
Vandenborre et al., 2009). Using experiments in which purified lectins were added to an
artificial diet or transgenic plants were used to overexpress a lectin gene it was clearly shown
that carbohydrate-binding proteins interfere with the growth and reproduction of insects from
different orders. Although evidence shows that the carbohydrate-binding activity of plant
lectins is necessary for their insecticidal activity, the mode of action of lectins in insects
remains enigmatic. Over the last decade lectins particularly those binding mannose, have
received significant attention, predominantly Galanthus nivalis agglutinin (GNA) (Sauvion et
al., 1996; Down et al., 1996; Couty et al., 2001). Since then the potential use of mannose-
binding lectins in plant protection against several insects has been investigated in detail (Van
Damme, 2008; Vandenborre et al., 2009). In addition, some reports have shown that the
combination of two different insecticidal proteins in a single system provides an effective
insect control and also reduces the potential for development of resistant insects. For instance,
the combination of GNA with Bt toxin resulted in synergistic effects (Maqbool et al., 2001;
Zhang et al., 2007).
The present chapter reports the effects of RSA delivered via artificial diet on the survival and
growth of the cotton leafworm Spodoptera littoralis (Order: Lepidoptera; Family: Noctuidae).
This polyphagous noctuid species is an economically important caterpillar in agriculture and
horticulture, damaging at least 87 economically important plant species belonging to 40
families distributed worldwide (Smagghe and Degheele, 1994; Sadek, 2001).
3.3 MATERIALS AND METHODS
3.3.1 Isolation of RSA
R. solani strain AG 1-1B was grown on autoclaved wheat grains. To produce large quantities
of sclerotia, 25 g of wheat kernels and 60 ml of water were mixed in 250 ml Erlenmeyer
flasks. After autoclaving, small pieces of approximately 1 cm² agar covered with mycelium
from a 5-day old culture of R. solani grown on potato dextrose agar were added and the
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Chapter 3
fungal cultures were incubated in a growth chamber at a temperature of 25-27°C. After 4-5
weeks the sclerotia were harvested and used for lectin extraction. Sclerotia were lyophilized
and ground to a fine powder using a coffee mill. The dry powder was extracted in phosphate
buffered saline (PBS, 25 ml per g dry weight material) for approximately 12 h at room
temperature. Then the mixture was centrifuged at 3,000 g for 10 minutes and remaining debris
removed by passing the supernatant through filter paper (Whatmann 3MM). Affinity
chromatography was performed on a galactose column equilibrated with PBS. After loading
the extract, the affinity column was washed with PBS until the absorbance of the effluent at
280 nm was <0.1. Subsequently, the lectin bound to the column was eluted with 20 mM 1,3-
diaminopropane (DAP). The lectin fractions obtained after the first affinity chromatography
were brought to pH 7.0 and run on the galactose column for a second time. The RSA
preparation obtained after the second affinity chromatography was loaded on an anion
exchange chromatography column of Q Fast Flow, equilibrated with DAP. After washing
with DAP the lectin was eluted using 0.1 M Tris-HCl (pH 7.0) containing 0.5 M NaCl. If
necessary, these chromatography steps on the galactose and Q Fast Flow columns were
repeated in order to obtain high-purity lectin preparations. Finally, the lectin fractions were
dialyzed against water and lyophilized. The purity of the lectin was analysed by SDS-PAGE.
3.3.2 Insects
An established colony of the cotton leafworm S. littoralis was reared under standard
conditions of 23-25°C, 60-70% relative humidity and a 16:8 (light:dark) photoperiod in the
Laboratory of Agrozoology at Ghent University as described before (Lemeire et al., 2008).
Larvae were fed on artificial diet (Stonefly Heliothis diet, Ward’s Natural Science, Rochester,
NY), an artificial diet for Lepidopteran larval insects that can be prepared by adding cold
water. Under these conditions, the duration of each of the first five larval instars is about three
days each, whereas the sixth and last larval stage (L6) takes approximately six days. Larval
instars were determined on the basis of their respective head capsule width.
3.3.3 Effects of RSA feeding on insect survival, growth and development
Newborn (0-6h) first instar larvae of S. littoralis were selected from the continuous stock
colony and RSA was fed using Stonefly Heliothis diet. Based on previous range finding tests,
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Chapter 3
RSA was mixed at three concentrations of 1, 5 and 10 mg/g during the preparation of the diet.
Controls were fed with untreated diet. The experiment was set up in a 24-well plate and about
0.75 g of the diet was added to each well, and ten first instar larvae were transferred to each
well. Fresh diet was prepared every 4-5 days and stored at 4ºC in airtight containers. After 10
days the larvae were transferred to 9-mm Petri dishes. At different time points during the
experiment the weights of all larvae were monitored. The fresh weight of the resulting pupae
and adults was determined on the first day following pupation and upon adult eclosion,
respectively. Per treatment, three replications of 10 insects each were performed. The
experiment was done twice. Hence, a total of 60 insects were analyzed for each lectin
concentration.
3.3.4 Effect of RSA combined with Bt toxin
In a similar experimental setup we evaluated the combinatorial effect of RSA and the Bt
preparation Delfin (32,000 UI/mg, Sandoz, Brussels, Belgium). Three concentrations of RSA
(0.05, 0.1 and 0.3 mg/g) and Bt toxin (0.05, 0.1 and 0.3 mg/g) were tested together with five
combinations of RSA and Bt toxin (0.05 mg/g RSA+0.05 mg/g Bt, 0.3 mg/g RSA+0.05 mg/g
Bt, 0.1 mg/g RSA+0.1 mg/g Bt, 0.3 mg/g RSA+0.1 mg/g Bt, and 0.3 mg/g RSA+0.3 mg/g Bt).
At different time points during the experiment the larval weight as well as the percentages of
mortality and growth retardation were determined. For each treatment, three replicates of 10
insects each were performed, and the experiment was done twice. A total of 60 insects were
monitored for each treatment.
3.3.5 Statistical analysis
For each treatment and control, data on reduction in weight gain of the larvae, pupae and
adults are expressed as means ± SEM. To detect significant differences between treatments,
data were analyzed by one-way analysis of variance (ANOVA) and then means ± SEM were
separated using a post-hoc Tukey-Kramer test (p = 0.05) in SPSS v15.0 (SPSS Inc., Chicago,
IL). Medium toxicity concentration LC50 and medium effect period LT50 and corresponding
95% confidence intervals were calculated using GraphPad Prism v4 (GraphPad Software, San
Diego, CA); the goodness that the data fit to the probit curve model was evaluated based on
R2 values.
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Chapter 3
3.4 RESULTS
3.4.1 Effects of RSA feeding on insect survival, growth and development
The high lectin concentration present in the sclerotes of R. solani allowed the purification of
sufficient amounts of the pure lectin for testing of its insecticidal activity. The effect of the
RSA delivered via artificial diet, on survival, growth and development of caterpillars was
followed for first instar larvae of S. littoralis until they became pupa and then adult. Insect
survival was evaluated to analyze the toxicity potential of the lectin, and in addition, weight
gain of the larvae, pupae and adults, as well as percentage and time of pupation were
determined as biological endpoints to investigate lectin effects on insect growth and
development.
As shown in figure 3.1A-B, RSA exerted a strong effect on the larval weight gain and this
effect was clearly concentration-dependent. At day 8, the weight of S. littoralis larvae fed on a
diet containing 10 mg/g RSA was dramatically (p<0.01) reduced with 81±4%. The two lower
lectin concentrations of 1 and 5 mg/g RSA also significantly (p<0.05) reduced larval weight
by 34±6% and 60±5%, respectively, compared to the controls. As a result of the lectin effects
on larval development, the majority of the population of the insects treated with RSA at 1
mg/g (44%, 23/52), 5 mg/g (60%, 33/55) and 10 mg/g (64%, 36/56) was in the third instar,
while the majority of the control insects had developed into the fifth instar (32%, 19/60) and
some into the sixth instar (12%, 7/60). With 10 mg/g RSA, none of the insects had entered in
the fifth instar. Later on at day 11, these entomotoxic effects were maintained with a
respective significant (p<0.05) reduction of larval weights by 34±7%, 74±4% and 89±3% for
treatments with 1, 5 and 10 mg/g RSA (Fig. 3.1B). This reduction in larval weight was
accompanied with a dramatic retardation of development as the majority of the population
was stuck in the fourth instar (70%, 38/54) with 10 mg/g RSA and in the fifth instar (75%,
39/52) with 5 mg/g RSA. With 1 mg/g RSA, 30% (15/50) and 70% (35/50) were in the fifth
and sixth instar respectively, while in the control groups nearly all insects (87%, 52/60) were
already L6 larvae. The latter observations demonstrate that the entomotoxic effects caused by
RSA could not be counteracted by the intoxicated larvae. With the use of sigmoid regression,
the LT50, which is the time of feeding treated diet needed to reduce the larval weight by 50%,
could be determined, being 7.8 days for 5 mg/g RSA (95% confidence interval of 6.4-9.3
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Chapter 3
days; R2=0.78) and 7.4 days for 10 mg/g RSA (95% confidence interval of 6.4-8.3 days;
R2=0.97). Finally, most of these intoxicated insects died. With 10 mg/g RSA, 82±3% of the
treated larvae were killed and failed to reach the pupal stage, and with 1 and 5 mg/g RSA the
larval mortality reached about 28% (Fig. 3.2). To confirm the entomotoxic potential for RSA,
sigmoid regression estimated an LC50 value of 0.32% (95% confidence interval=0.11-0.95%;
R2=0.87), that is the concentration of RSA in the diet needed to kill 50% of S. littoralis larvae.
Figure 3.1. Concentration-dependent effect of the fungal lectin RSA against larval growth and
development of the cotton leafworm Spodoptera littoralis. RSA caused a strong reduction of larval
weight after 8 days (A) and 11 days (B) of feeding on treated diet as compared to controls. Data are
presented as mean fresh larval weight ± SEM; the numbers of insects per treatment are given with the
data. ANOVA resulted in 4 groups for ‘A’ (F=58.529, df=23, p<0.001), in 3 groups for ‘B’
(F=94.131, df=23, p<0.001). Values per graphic that are followed by a different letter (a-d) are
significantly different (post-hoc Tukey-Kramer test with p=0.05).
Control 1 5 10 RSA conc. (mg/g)
Control 1 5 10 RSA conc. (mg/g)
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Chapter 3
Figure 3.2. Concentration-dependent effect for induction of larval and pupal mortality in the cotton
leafworm Spodoptera littoralis by different concentrations of RSA when fed via treated diet from first
instar to pupation. Mortality percentages ± SEM are based on two repeated experiments, each
consisting of 30 insects per concentration; a total of 60 larvae were treated per lectin
concentration.ANOVA resulted in 3 groups (F=93.704, df=3, p<0.0001). Values per graphic that are
followed by a different letter (a-c) are significantly different (post-hoc Tukey-Kramer test with
p=0.05).
All individuals that survived and developed into pupae showed entomotoxic effects of the
accumulated RSA. First, there was a clear effect on insect development with a strong
retardation of 67±9% (p<0.0001) with 10 mg/g RSA as the developmental time from neonate
(L1) to pupation took 27.3±0.4 days as compared with 16.5±1.3 days in the controls. The
retardation effect (p=0.001) was 39.3±7.7% when RSA was dosed at 5 mg/g in the diet.
Second, the surviving pupae were negatively affected as they were smaller in size and weight
(Fig. 3.3A-B). With 10 mg/g RSA, the surviving pupae represented only 18% (11/60) of the
total number of treated larvae. They were smaller in size and weight with a reduction of
39±3% (p<0.05). With 5 mg/g RSA, the percentage of successful pupation was only 37%
(44/60) and their pupal weight was significantly (p<0.05) reduced by 22±5% (Fig. 3.3B). In
addition, lethal larval-pupal intermediates were observed in 4% of the insects treated with 1
and 5 mg/g RSA (Fig. 3.3C).
At the end of the experiment and with completion of the insect life cycle from the first larval
instar to the adult stage, 21% (9/43) and 27% (12/44) of the pupae that had developed from
larvae fed on 1 and 5 mg/g RSA, respectively, died before reaching the adult stage. In contrast
there was 92% successful development into the adult stage in the control (55/60).
Control 1 5 10 RSA conc. (mg/g)
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Figure 3.3. Concentration-dependent effect of the fungal lectin RSA against pupal and adult growth
and development of the cotton leafworm Spodoptera littoralis resulting from larvae fed on treated
diet. RSA caused a marked reduction of the weight of the resulting pupae (A) and adults (D) as
compared to controls. Panel (B) shows a pupa with reduced size in the treatment with 5 mg/g RSA
(left) compared to a normal pupa (right) in the control, and (C) a lethal larva-pupa intermediate. Data
are presented as mean fresh larval weight ± SEM compared to the control; the number of insects per
treatment is given with the data. ANOVA resulted in 3 groups for ‘A’ (F=19.300, df=22, p<0.001), in
3 groups for ‘D’ (F=27.643, df=22, p<0.001). Values per graphic that are followed by a different letter
(a-c) are significantly different (post-hoc Tukey-Kramer test with p=0.05).
Control 1 5 10 RSA conc. (mg/g)
Control 1 5 10 RSA conc. (mg/g)
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Hence due to the lower pupal weight in the RSA treatments, the resulting adults were smaller
in size and weight (Fig. 3.3D). The weight of the adults was significantly (p<0.05) reduced by
28±5% and 44±2% with RSA at 5 and 10 mg/g, respectively. In addition, we observed lethal
pupa-adult intermediates in 4% of the pupae in the treatments with 1 and 5 mg/g RSA.
3.4.2 Effects of RSA combined with Bt toxin
Combinations of RSA and Bt toxin showed higher entomotoxic effects against the larval
weight than with Bt toxin alone (Fig 3.4). When the diet was supplemented with low
concentrations of 0.05, 0.1 and 0.3 mg/g RSA and fed for 8 days, the negative effect on larval
weights was 7±6%, 21±10% and 25±11%, respectively. With Bt toxin at 0.05, 0.1 and 0.3
mg/g this was 12±6%, 35±3% and 79±5%. Typically when 0.05 mg/g Bt toxin was combined
with 0.3 mg/g RSA the entomotoxic effect yielded 49±1%, demonstrating an increase in
entomotoxic effect of 4-fold over Bt toxin alone. At higher concentrations of RSA and Bt
toxin the negative larval effect was also visible. Combining 0.1 mg/g RSA with 0.1 mg/g Bt
toxin inhibited to a higher extent (p<0.05) the larval weight reduction, being 67±2%. At
higher doses of Bt toxin (0.3 mg/g), there was no increased effect when combined with RSA
(0.05, 0.1 and 0.3 mg/g) (Fig 3.4).
Figure 3.4. Effect of RSA and Bt on larval weight. The larvae were fed 8 days on 0.05, 0.1 and 0.3
mg/g RSA or Bt toxin individually in addition to five combinations of RSA and Bt toxin (0.05 mg/g
RSA+0.05 mg/g Bt, 0.3 mg/g RSA+0.05 mg/g Bt, 0.1 mg/g RSA+0.1 mg/g Bt, 0.3 mg/g RSA+0.1
mg/g Bt, and 0.3 mg/g RSA+0.3 mg/g Bt). Values are given as means ± SEM based on two
independent repetitions. Values are followed by a different letter (a-e) are significantly different (post
hoc Tukey-Kramer test with p=0.05).
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For example, 0.3 mg/g Bt toxin combined with 0.3 mg/g RSA caused 74±4% weight
reduction which was equal (p>0.05) to 0.3 mg/g Bt toxin alone. Moreover, addition of 0.05
and 0.1 mg/g of Bt toxin to different concentrations of RSA (0.05, 0.1 and 0.3 mg/g) resulted
in an increased mortality of treated larvae of 1.5 to 2 fold over that with the Bt toxin alone.
For instance, the mortality at day 11 of treatment increased to 32% with 0.3 mg/g RSA + 0.1
mg/g Bt, while the mortality was only 10% and 16% when RSA and Bt were dosed alone in
the diet. There was no increase in mortality when the higher concentration of 0.3 mg/g Bt
toxin was combined with different concentrations of RSA (0.05, 0.1 and 0.3 mg/g).
3.5 DISCUSSION
In 1996, Mier and co-workers extensively screened of 175 different species of fungi for their
activity against insects such as Drosophila melanogaster and S. littoralis. Therefore,
powdered fungi were added to the rearing medium of both insects and the development of the
progeny was monitored. About 45% of the fungi (79 fungi out of 175) were shown to be toxic
to D. melanogaster, with an LC100 ranging between 0.1 and 60 mg/ml. Most of the
mushrooms that were highly toxic to D. melanogaster were also toxic to S. littoralis but in
most cases the LD100 was higher (Mier et al., 1996). These results indicated that many fungi
possibly contain interesting proteins and secondary metabolites with insecticidal activity. One
group of potentially interesting proteins is the group of lectins found in many fungi.
Unfortunately, although many fungal lectins have been identified and characterized in some
detail, very little is known with respect to their biological activities and their function in
fungi. Until now only very few reports have considered the insecticidal activity of purified
fungal lectins. Trigueros et al. (2003) reported the insecticidal activity of XCL, a lectin from
the edible mushroom Xerocomus chrysenteron (Class: Basidiomycetes, Order: Boletales)
towards the aphid Acyrthosiphon pisum (Hemiptera) and D. melanogaster (Diptera). More
recently a more extensive study of the effects of XCL on the aphid Myzus persicae was
performed, confirming the effect of the lectin on aphid development and mortality (Karimi et
al., 2007). To our knowledge, there are no reports dealing with the effects of XCL on
caterpillars.
The present study provides a detailed analysis of the effects of RSA, a fungal lectin from the
pathogen R. solani against the cotton leafworm S. littoralis. Feeding S. littoralis larvae on an
artificial diet containing different concentrations of RSA resulted in a significant reduction in
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larval weight. The reduction in larval weight was about 89% when the larvae were fed on a
diet containing 10 mg/g RSA, and 74% with 5 mg/g RSA for 11 days. Due to the lower
weight of the larvae that developed on the artificial diet containing RSA, the resulting pupae
and adults were smaller in size. Since RSA adversely affected the weight gain in the larval
stage, the development into pupae was also retarded. It is a well known phenomenon that
final instar larvae have to reach a minimum critical fresh weight before entering the pupal
stage (Davidowitz et al., 2003). As a consequence, RSA caused about 81% larval mortality
when tested at a concentration of 10 mg/g. So feeding the diet with RSA to S. littoralis larvae
resulted in a clear reduction in larval weight and a significant larval mortality. A good
example for comparison of insecticidal potential is the mannose-binding lectin GNA. Fitches
et al. (1997) reported a 32% and 23% reduction in the larval biomass of the tomato moth
(Lacanobia oleracea) fed on an artificial diet containing 20 mg/g GNA or excised leaves of
transgenic potato expressing GNA at 0.07% of total soluble protein, respectively. In addition,
these authors observed that GNA retarded larval development and affected pupation.
However, a significant effect of GNA on larval survival was only recorded in a glasshouse
experiment using transgenic potato plants (with GNA expression at 0.6% of total soluble
protein), which resulted in a decrease in larval survival of approximately 40%. Using a
similar bioassay as the one used in this study with RSA, Sadeghi et al. (2009a) recently
reported that feeding of larvae of S. littoralis with tobacco leaves expressing another
mannose-binding lectin from leek (Allium porrum) APA at 0.7%, reduced the larval weight
gain by 15-27%, and 28% of the intoxicated larvae were killed before pupation. Although
these results also show clear effects of the mannose-binding lectins on caterpillar
development, the percentages of weight reduction and mortality are much lower than
observed in this study for RSA. Therefore the results reported here demonstrate the high
potential of the fungal lectin RSA in the control of important pest Lepidoptera.
Currently, XCL and RSA are the only two known fungal lectins purified from species
belonging to the order of Basidiomycetes, and these clearly exhibit specificity for
Gal/GalNAc residues. However, both lectins are not related evolutionary. The XCL sequence
shows significant sequence homology to the group of Agaricus bisporus related lectins
(Trigueros et al., 2003), whereas the N-terminal 60 amino acid sequence of RSA shows some
residual sequence similarity to the ricin-B homologs (Candy et al., 2001).
Insecticidal activity of plant lectins has been studied in detail especially for mannose-binding
lectins (Van Damme, 2008; Vandenborre et al., 2009). At present the insecticidal properties
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of galactose-binding lectins are far less documented. Only a few galactose-binding lectins
were reported to have toxic effects against caterpillars. For instance, the Bauhinia purpurea
lectin and the Ricinus communis agglutinin revealed entomotoxic properties against Ostrinia
nubilalis (Czapla and Lang, 1990), whereas peanut (Arachis hypogeae) lectin showed activity
against Helicoverpa armigera (Shukla et al., 2005). More recently, the Annona coriacea
lectin was shown to be active against larvae of two pyralid moths, Ephestia (=Anagasta)
kuehniella and Corcyra cephalonica (Coelho et al., 2007). Bauhinia monandra leaf lectin was
toxic to Anagasta kuehniella (Macedo et al., 2007). Similar to our results with the fungal
lectin RSA, all these studies report entomotoxic effects by galactose-binding plant lectins on
insect survival, growth and development.
As already mentioned above, RSA is probably distantly related to the B-chain of ricin and
other type-2 ribosome-inactivating proteins (Candy et al., 2001). In a recent report, Shahidi-
Noghabi et al. (2009) have shown that treatment of caterpillars of the beet armyworm
(Spodoptera exigua) with the type-2 ribosome-inactivating protein SNA-I from elderberry
(Sambucus nigra) resulted in a significant reduction in fresh weight, retardation in
development as well as a significant increase in mortality of S. exigua larvae fed on
transgenic lines as compared to wild type plants. Furthermore these authors have shown that
the carbohydrate-binding properties of the elderberry lectins are a determining factor in their
insecticidal properties, since mutant lectins that have lost their ability to recognize
carbohydrate structures also loose their insecticidal activity (Shahidi-Noghabi et al., 2008).
Finally, we also tested the effect of RSA in combination with a Bt toxin preparation (Delfin).
The results show a greater reduction in larval weight when low concentrations of RSA and Bt
toxins were used. However, at a higher concentration (0.3 mg/g) of RSA and Bt toxin this
combinatorial effect was less clear and the effect was almost equal to that of the Bt treatment
alone. Delfin contains Cry1Aa, Cry1Ab, Cry1Ac and Cry2Aa proteins (Singh et al., 2008).
Previously it was shown that the Cry1Ac toxin specifically recognizes GalNAc and has a
binding pocket for this sugar (Burton et al., 1999). CryAc1 was shown to bind to a specific
receptor in the lepidopteran gut, namely aminopeptidase N (Hakim et al., 2010). This binding
can be inhibited by GalNAc (Gill et al., 1995). However, the toxicity of Cry1Ac is not solely
dependent on GalNAc-mediated binding (Masson et al., 1995; Banks et al., 2001), and
therefore it seems likely that Bt toxin will bind a broader range of putative target proteins in
the insect midgut than RSA. These results demonstrated that lectins like RSA can be
combined with Bt toxin without impairing insecticidal activity. In addition, it was of great
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interest that synergistic activities were observed when RSA was tested in combination with Bt
toxin in the lower concentration range.
The entomotoxic activity of RSA in diet is a good indication that this fungal lectin can be a
useful tool for bioengineering insect resistance in crops of agronomic importance. Clearly the
carbohydrate specificity of this lectin is very different from the mannose-binding lectins
which have been studied in detail for their insecticidal properties. Therefore, it can be
envisaged that RSA will target other proteins in the insect body, allowing to control important
and resistant pest insects. In addition, our data support that RSA can be combined with Bt
toxin. Genetic engineering of plants for the expression of two lectins with different
carbohydrate-binding specificity could enable to target a broader range of receptor proteins. It
was already shown that transgenic lines expressing GNA in combination with Bt toxin
(CrylAc and Cry2A) showed greater reduction in insect survival and greater reduction in plant
damage compared with single treatments (Maqbool et al., 2001; Zhang et al., 2007).
Similarly, engineering of plants with a fusion protein combining the Cry1Ac toxin with the
galactose-binding domain of the nontoxic ricin B-chain resulted in a stronger activity of the
fusion proteins against insects than for Cry1Ac alone (Mehlo et al., 2005).
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Chapter 4
Parts of this chapter are published in:
Hamshou M, Smagghe G, Shahidi-Noghabi S, De Geyter E, Lannoo N, Van Damme EJM
(2010) Insecticidal properties of Sclerotinia sclerotiorum agglutinin and its interaction with
insect tissues and cells. Insect Biochemistry and Molecular Biology 40, 883-890.
Insecticidal properties of Sclerotinia sclerotiorum
agglutinin and its interaction with
insect tissues and cells
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4.1 ABSTRACT
This project studied in detail the insecticidal activity of a fungal lectin from the sclerotes of
Sclerotinia sclerotiorum, referred to as Sclerotinia sclerotiorum agglutinin or SSA. Feeding
assays with the pea aphid (Acyrthosiphon pisum) on an artificial diet containing different
concentrations of SSA demonstrated a high mortality caused by this fungal lectin with a
median insect toxicity value (LC50) of 66 (49-88) µg/ml. In an attempt to unravel the mode of
action of SSA the binding and interaction of the lectin with insect tissues and cells were
investigated. Histofluorescence studies on sections from aphids fed on an artificial liquid diet
containing FITC-labeled SSA, indicated the insect midgut with its brush border zone as the
primary target for SSA. In addition, exposure of insect midgut CF-203 cells to 25 µg/ml SSA
resulted in a total loss of cell viability and the median cell toxicity value (EC50) was 4.0 (2.4-
6.7) µg/ml. Interestingly, cell death was accompanied with DNA fragmentation, but the effect
was caspase-3 independent. Analyses using fluorescence confocal microscopy demonstrated
that FITC-labeled SSA was not internalized in the insect midgut cells, but bound to the cell
surface. Prior incubation of the cells with saponin to achieve a higher cell membrane
permeation resulted in an increased internalization of SSA in the insect midgut cells, but no
increase in cell toxicity. Furthermore, since the toxicity of SSA for CF-203 cells was
significantly reduced when SSA was incubated with GalNAc and asialomucin prior to
treatment of the cells, the data of this project provide strong evidence that SSA binds with
specific carbohydrate moieties on the cell membrane proteins to start a signaling transduction
cascade leading to death of the midgut epithelial cells, which in turn results in insect
mortality. The potential use of SSA in insect control is discussed.
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4.2 INTRODUCTION
Although the application of insecticides in crop protection enables the control of insect
populations, their intensive use has also created many problems. Some insecticides are
recalcitrant and pollute the environment, and kill not only the pest insects, but also affect non-
target insects and vertebrates, including humans (Colosio and Moretto, 2008). Moreover, as a
consequence of the increased insecticide use, many insects have acquired resistance to
insecticides (Hemingway et al., 2002; Li et al., 2007a). These problems have forced
entomologists to look for other methods to control insect pests. Nowadays biological control
is used as an alternative to traditional insecticides for crop protection and is considered safe
for people as well as for the environment. Hence, some naturally occurring proteins that are
toxic to insect pests can be used in agriculture as biological insecticides via spraying or
transgenic plants (Van Rie, 2000; Van Damme, 2008).
Many proteins have been reported to possess toxic effects when ingested by insects (ffrench-
Constant and Bowen, 2000; Carlini and Grossi-de-Sá, 2002; Sanchis and Bourguet, 2008).
Several of these insecticidal proteins belong to the class of lectins (Gatehouse et al., 1995;
Czapla, 1997; Vasconcelos and Oliveira, 2004; Vandenborre et al., 2009) that groups all
carbohydrate-binding proteins of non-immune origin that contain at least one non-catalytic
domain which enables them to bind reversibly to specific mono- or oligosaccharides
(Peumans and Van Damme, 1995b). These proteins are widely distributed in nature and are
found in all living organisms ranging from viruses, bacteria, fungi to plants and animals
(Kilpatrick, 2002; Van Damme et al., 2008; Singh et al., 2010).
Toxicity assays with artificial diets containing lectins or transgenic plants overexpressing a
specific lectin demonstrated that lectins reduce the performance of several insect species
belonging to different orders (Michiels et al., 2010). For example the snowdrop lectin (GNA)
shows toxic effects on development and fecundity of the peach-potato aphid Myzus persicae
(Sauvion et al., 1996), the pea seed lectin reduces growth rate of pollen beetle larvae
(Melander et al., 2003), and elderberry lectin (SNA-I) exerts toxic effects on the larval growth
and development of the beet armyworm Spodoptera exigua (Shahidi-Noghabi et al., 2009).
At present the mode of action of lectins remains enigmatic. It is not known how the binding of
a lectin to tissue or cell surface carbohydrates affects growth and development of the insect.
Similar to plant lectins some fungal lectins have been reported to possess insecticidal
properties. However, at present only two reports describe detailed information about the
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entomotoxic properties of fungal lectins. The Xerocomus chrysenteron lectin (XCL) was first
reported to have insecticidal activity towards the pea aphid Acyrthosiphon pisum and the
fruitfly Drosophila melanogaster (Trigueros et al., 2003). Later the same lectin was shown to
exert toxic effects towards the aphid M. persicae (Jaber et al., 2008). Recently the Rhizoctonia
solani agglutinin was shown to exert noxious effects on the larval growth and development of
the cotton leafworm Spodoptera littoralis (Chapter 3). Until now no reports are available
regarding the mode of action of purified fungal lectins.
The Sclerotinia sclerotiorum agglutinin, abbreviated as SSA, is a lectin purified from
Sclerotinia sclerotiorum, a soil borne fungus with a wide range of hosts. The mycelium
produces numerous sclerotia, black seed-like reproductive structures that enable the fungus to
survive under harsh conditions and are the source of inoculum to infect different crops. In
2003, Candy et al. (2003) reported the purification and characterization of SSA as a
homodimeric protein built up of subunits of approximately 17 kDa with high affinity for
galactose/N-acetylgalactosamine (Gal/GalNAc). Molecular cloning of SSA demonstrated that
the lectin shares no sequence similarity with any known fungal lectin or protein (Van Damme
et al., 2007). However, molecular modeling suggested that the lectin is structurally related to
the ricin-B superfamily. Recently the crystal structure of SSA was determined at 1.6 Å
resolution and confirmed the β-trefoil fold for SSA, as previously identified for other ricin B-
like lectins. However, unlike other structurally related lectins, SSA contains a single
carbohydrate-binding site per monomer and reveals a novel dimeric assembly markedly
dissimilar from those described for other ricin-type lectins (Sulzenbacher et al., 2010).
In this chapter, the insecticidal activity of SSA on the survival of the pea aphid A. pisum was
studied using an artificial diet supplemented with pure SSA. A. pisum has a worldwide
distribution and is a representative of the important order of Hemiptera with piercing-sucking
pest insects. Pea aphids are found on many leguminous plants and transmit many plant viruses
(Brisson and Stern, 2006). Furthermore, to better understand the mode of action of SAA,
binding of FITC-labeled SSA to different tissues in the pea aphid body was investigated upon
oral exposure. Since SSA clearly interacts with the midgut epithelium, the cellular effects of
SSA in insect midgut CF-203 cells were also tested. To investigate the involvement of cell
membrane binding and internalization in the cell toxicity, the uptake of SSA in these insect
midgut cells was studied. Furthermore, SSA was incubated with GalNAc and the glycoprotein
asialomucin to investegated the effect of these complementary carbohydrate structures on the
toxicity of SSA against midgut CF-203 cells. These data provide strong indications that SSA
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interacts with specific carbohydrate moieties on the surface (brush border zone) of the midgut
epithelial cells to start a signaling transduction cascade leading to cell death and insect
mortality.
4.3 MATERIALS AND METHODS
4.3.1. Pea aphids
All stages of A. pisum are maintained in the laboratory on young broad bean (Vicia faba)
plants under standard conditions of 23-25ºC; 60-70% relative humidity and a 16:8 (light:dark)
photoperiod, as previously reported (Christiaens et al., 2010).
4.3.2. Insect midgut CF-203 cell line and culture conditions
The insect midgut cell line CF-203 that originated from the midgut of the eastern spruce
budworm Choristoneura fumiferana (Palli et al., 1997), was cultured in Insect-Xpress
medium (BioWhittaker-Cambrex Bioscience, Walkersville, MD) supplemented with 2.5%
heat-inactivated fetal bovine serum (FBS) (Sigma-Aldrich, Bornem, Belgium) as reported
before (Vandenborre et al., 2008).
4.3.3 Purification of SSA
Wheat medium was used to produce large quantities of sclerotia of S. sclerotiorum (isolated
from lettuce in Belgium; Van Beneden et al., 2005) as previously described in chapter 3.
4.3.4. FITC-labeling of SSA
Approximately 5 mg SSA were dissolved in 500 µl sodium borate buffer (50 mM, pH 8.5),
and mixed with 1.17 mg FITC dissolved in 117 µl dimethylformamide. After incubation for 1
h at room temperature in the dark, the free label was removed by gel filtration on a Sephadex
G25 column, using PBS as the running buffer. Lectin activity in the eluted fractions was
checked using an agglutination assay. Therefore, 10 µl of the eluted lectin was mixed with 10
µl of ammonium sulphate and 30 µl of trypsin-treated rabbit erythrocytes (BioMérieux,
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Marcy L'Etoile, France) in small glass tubes and the agglutination was observed after 15 min.
The lectin protein concentrations were determined with the method of Bradford (1976). The
FITC-labeled SSA fractions were stored in the freezer at -20°C.
4.3.5. Treatment of A. pisum with SSA via artificial liquid diet
In the insect bioassay, 15 neonates of A. pisum were fed on an artificial liquid diet (Febvaye et
al., 1988) that was supplemented with SSA at different concentrations (1-800 µg/ml),
essentially as previously described (Shahidi-Noghabi et al., 2008) (Fig 4.1). In the controls, the
diet was supplemented with an equivalent volume of PBS. In these experiments, survival of
treated nymphs of A. pisum was scored after 24, 48 and 72 h. Data were expressed as means ±
SE based on three repeats and the experiment was repeated two times. After Abbott’s
correction (Abbott, 1925) for mortality in the controls (<15%), the toxicity results were
analyzed and the median toxicity concentration (LC50) together with the 95% confidence limits
(95% CL) calculated in Prism v4 (GraphPad Prism, La Jolla, CA).
Figure 4.1. Scheme of the experimental setup of the aphids on artificial diet
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4.3.6. Histofluorescence for localization of SSA in aphid body tissues
Fourth-instar aphids were fed for 24 h on an artificial diet containing FITC-labeled SSA at
1000 µg/ml. Afterwards the insects were fixed at room temperature in Carnoy solution
(ethanol:chloroform:acetic acid, 6:3:1) for 24 h, dehydrated in an ethanol series and butanol,
and finally embedded in paraffin. Serial sections of 10 µm thickness were cut using a
microtome (Jung AG, Heidelberg, Germany) essentially as described before (Smagghe and
Degheele, 1994). After dewaxing and mounting, the location of FITC-labeled SSA in the
aphid body tissues was analyzed under an Olympus BX51 fluorescence microscope
(Olympus, Aartselaar, Belgium). Digital images were acquired using an Olympus Color View
II camera (Olympus, Belgium) and further processed with Olympus analySIS cell^F software
(Olympus Soft Imaging Solutions, Münster, Germany).
4.3.7. Cytotoxic effect of SSA in insect midgut CF-203 cells
To study the effect of SSA 20,000 midgut CF-203 cells were seeded in 100 µl Insect-Xpress
culture medium per well of a multiwell plate, and then incubated with different concentrations
of SSA (1, 10 and 25 µg/ml) for 4 days at 27°C. Control cells were treated with PBS using the
same volume that was used in the treated cells. For every treatment, four replicates were
prepared and each experiment was repeated two or three times.
After incubation, the cell numbers were counted using 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide (MTT) (Sigma-Aldrich) as a substrate according to Decombel
et al. (2005). The MTT assay is based on the enzymatic conversion of a yellow tetrazolium
salt to an insoluble formazan product by the mitochondria of viable cells. Absorbance of the
produced formazan was measured in a microtiter plate reader (PowerWave X340, Bio-Tek
Instruments Inc., Winooski, VT) at 560 nm.
The obtained data on cell toxicity were analyzed by one-way analysis of variance (ANOVA)
to detect significant differences between treatments, and then means ± SEM were separated
using a post hoc Tukey-Kramer test (p = 0.05) in SPSS v15.0 (SPSS Inc., Chicago, IL).
In addition, as for the insect toxicity bioassy, a concentration-response curve, a median
response concentration (EC50) and the corresponding 95% CL were estimated with Prism v4.
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4.3.8. DNA fragmentation analysis
As described above, midgut CF-203 cells were incubated with 25 µg/ml SSA (or PBS buffer
for the control) for 24 h at 27°C and then DNA was extracted as described in Shahidi-
Noghabi et al. (2010a). DNA samples were run on a 2% agarose gel at 100 V and visualized
by staining with ethidium bromide.
4.3.9. Caspase-3 activity assay
The caspase-3 activity was measured using Ac-DEVD-AFC as a synthetic tetrapeptide
fluorometric substrate (Sigma-Aldrich) as described before (Shahidi-Noghabi et al., 2010a).
Midgut CF-203 cells were incubated for 24 h at 27°C with 25 µg/ml SSA. PBS was used as a
negative control, while SNA-II, a plant lectin from elderberry Sambucus nigra, was used as a
positive control at concentration 10 µg/ml (Shahidi-Noghabi et al., 2010a). Afterwards, cells
were harvested by centrifugation at 1000 g for 5 min at 4°C, washed with PBS twice and
stored at -80°C until use. Cells were lysed in caspase assay buffer (50 mM HEPES, pH 7.4,
0.1 mM EDTA, 0.1% CHAPS, 5 mM DTT) for 5 min in an ice bath, and total protein
concentrations determined in according with Bradford (1976). To measure caspase-3 activity,
20 µg protein was dissolved in 50 mM HEPES (pH 7.4) containing 100 mM NaCl, 0.1%
CHAPS, 10 mM DTT, 1 mM EDTA and 10% glycerol, and 20 µM caspase-3 substrate Ac-
DEVD-AFC was added to start the reaction. The solution was incubated at 27°C and the
intensity of fluorescence was measured at an excitation wavelength of 400 nm and an
emission wavelength of 505 nm using a spectrofluorometer (TECAN, Infinite M200,
Switzerland). The reaction was followed every min for 60 min.
4.3.10. Uptake of SSA in midgut CF-203 cells
For microscopic quantification of cellular uptake of FITC-labeled SSA 50,000 midgut CF-203
cells were seeded onto poly-L-lysine coated glass slides. After 24 h, cells were washed with
serum-free medium and incubated for 1 h at 27°C with 50 µg/ml FITC-labeled SSA (prepared
in PBS). After three washes with PBS, cells were fixed with 4% formaldehyde for 15 min,
followed by another three washes with PBS. Slides were mounted with Vectashield (Vector
Labs), covered with a cover glass and sealed with nail polish. Cells were inspected with a
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Nikon eclipse TE2000-e epifluorescence microscope (Nikon, France) using a 40Х Plan Fluor
(NA 1.30) oil immersion lens and appropriate fluorescence filters. Quantitative visualization
of stained cells was performed on a Biorad Radiance 2000 confocal microscope mounted on a
TE300 epifluorescence body (Nikon Instruments, Paris, France). Experiments were carried
out with a Nikon S Fluor 40x (NA 1.3) objective and Lasersharp 2000 software. A 488 Argon
laser was used for simultaneous excitation of FITC and transmission imaging. FITC
fluorescence was detected on the photomultiplier tube through a 528/50 bandpass filter.
Images were sampled at 512 x 512 pixels with a physical pixel size of 335 nm x 335 nm.
4.3.11. Effect of saponin on toxicity and uptake of SSA in midgut CF-203 cells
To achieve a higher cell membrane permeation CF-203 cells were preincubated for 30 min
with 0.001% Quillaja bark saponin (Sigma Co, St Louis, MO) in the culture medium. This
saponin concentration was not toxic for the midgut cells based on own preliminary cell
viability assays with different concentrations. Afterwards, the cell toxicity caused by SSA and
the internalization of FITC-labeled SSA in the cells were analyzed as described above.
4.3.12. Effect of carbohydrates and glycoprotein on toxicity of SSA in midgut CF-203
cells
To demonstrate the carbohydrate-binding dependence of the interaction of SSA with cells, the
inhibitory effects of carbohydrates and glycoproteins on the SSA cell toxicity towards midgut
CF-203 cells was studied in a manner similar as described above. Before exposure to the cells,
10 µg/ml SSA was incubated for 1 h with GalNAc (50 and 100 mM, Sigma-Aldrich) or
porcine asialomucin (5 and 10 mg/ml, Sigma-Aldrich). In a parallel experiment the non-
binding sugar mannose (100 mM) was also evaluated. Data were compared to control cells
that were not exposed to SSA. After preincubation of the lectin together with the carbohydrate
or glycoprotein, the mixture was added to CF-203 cells and incubated for 24 h at 27°C. Then
the cell toxicity was determined with an MTT assay as described above.
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4.4 RESULTS
4.4.1. Insecticidal effects of SSA on pea aphids
Feeding of pea aphids with an artificial diet containing different concentrations (1-800 µg/ml)
of pure SSA for 3 days revealed increased mortality of pea aphids compared to a control diet.
As depicted in Fig. 4.2, the toxicity was concentration-dependent and followed a sigmoid
curve; the median LC50 toxicity value was 66 µg/ml (95% CL: 49-88 µg/ml; R2 = 0.93).
4.4.2. Localization of SSA upon feeding in aphid body tissues
As depicted in Fig. 4.3, cross sections on the aphids fed for 24 h on artificial liquid diet
containing FITC-SSA demonstrated an intense fluorescence at the microvilli (brush border
zone) at the apical surface side of the epithelial cells of the midgut. In addition, no
internalization of FITC-SSA was observed in the cytoplasm of these midgut cells.
Figure 4.2. Effect of different concentrations of SSA on the survival of pea aphids (A. pisum). Dose
response curve of mortality of pea aphids challenged for 3 days with an artificial diet containing
different concentrations of SSA. Data are corrected for mortality in the controls (0-15%) using
Abbott’s formula.
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Figure 4.3. Localization of SSA in the aphid body. (A) Transverse section of 4th-instar nymphs of pea
aphids A. pisum fed for 24 h on artificial diet containing FITC-labeled SSA at 1000 µg/ml, showing
binding of the lectin to the epithelium cells of the midgut (MG). Cut = outer cuticle. (B) Magnification
of the midgut showing that SSA bound to the microvilli (brush border zone) at the apical surface of the
midgut epithelium, but is not internalized in the cells. Lum = insect gut lumen.
4.4.3. Cytotoxicity of SSA in insect midgut CF-203 cells
Exposure of insect midgut CF-203 cells to SSA for 4 days resulted in clear signs of cell
toxicity, resulting in cell debris (Fig. 4.4A-B). Highly significant differences were observed in
cell viability between treatments and control cells (p < 0.0001).
As depicted in Fig. 4.4C, the effect was concentration-dependent; the loss of cell viability was
29 ± 1 % at 1 µg/ml SSA and 97 ± 1 % at 25 µg/ml. The median effective concentration
(EC50) was 4.0 (2.4-6.7) µg/ml after successful sigmoid regression curve fitting (R2 = 0.92)
with use of Prism v4 software (data not shown).
4.4.4. DNA fragmentation and caspase-3 activity in midgut CF-203 cells upon exposure
to SSA
Analysis of DNA extracted from midgut CF-203 cells after 24 h incubation with 25 µg/ml
SSA showed a clear ladder pattern, while no DNA fragmentation was observed in the control
cells (Fig. 4.5A). In addition, there was no induction of caspase-3 activity in these SSA-
treated cells as was the case with non-treated cells (negative control) (p = 0.698), while a clear
induction of caspase-3 protease activity was detected in the cells treated with SNA-II (as a
positive control) (Fig. 4.5B).
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Figure 4.4. Effect of different concentrations of SSA on insect midgut CF-203 cells. Cells were
incubated for 4 days at 27°C. (A) Control, (B) Treated cells with 25 µg/ml SSA, (C) SSA toxicity
towards CF-203 midgut cells. Cell toxicity was measured using an MTT assay after 4 days of
exposure to SSA at various concentrations. Data are presented as mean percentages of cell toxicity ±
SEM compared to the control, and based on four repeats and the experiments were repeated two or
three times. Values with a different letter are significantly different after a post hoc Tukey-Kramer test
(p = 0.05).
Figure 4.5. (A) DNA fragmentation in midgut CF-203 cells after treatment with 25 µg/ml SSA for 24
h. Approximately 5 µg of DNA of treated and non-treated (control) cells was analyzed on a 2%
agarose gel. (B) Shows caspase activity expressed as relative fluorescence units (RFU), and means ±
SD are based on n=3.
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4.4.5. Internalization of SSA in midgut CF-203 cells
Confocal microscopy analysis of midgut CF-203 cells exposed to FITC-labeled SSA for 1 h,
demonstrated that the fungal lectin SSA was not internalized, but was only bound to the cell
surface (Fig. 4.6A). Longer exposure times of cells to FITC-labeled SSA yielded similar
results. In addition, preincubation of the cells with 0.001% saponin from Quillaja bark for
obtaining a better cell permeation, resulted in a higher uptake of SSA in the cells but did not
increase the cell toxicity of SSA (Fig. 4.6B).
In addition, as depicted in Fig. 4.7, SSA at 1, 2 and 5 µg/ml in combination with 0.001%
saponin caused 29 ± 1%, 36 ± 3% and 50 ± 2% cell toxicity, respectively; the respective
toxicity was 28 ± 2%, 33 ± 2% and 47 ± 2% when SSA was dosed alone at the same lectin
concentrations. Treatment with 0.001% saponin alone did not show toxicity, as was the case
in the controls.
Figure 4.6. Fluorescence microscopy of CF-203 cells. (A) Cells were incubated with FITC-labeled
SSA (50 µg/ml) for 1 h. It is clear that SSA is bound to the cell surface but is not internalized. (B)
Cells were incubated with 0.001% saponin for 30 min prior to treatment with SSA as in A. Scale bar =
10 µm.
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Figure 4.7. Effect of saponin on toxicity of SSA in midgut CF-203 cells. Different concentrations of
SSA (1, 2 and 5 µg/ml) were tested alone and in combination with 0.001% saponin. Data are presented
as mean percentages of cell toxicity ± SEM compared to the control, and based on four replicates.
Each experiment was repeated two or three times. No significant differences were found for the
different SSA lectin concentration comparing exposure to SSA alone and SSA combined with 0.001%
saponin after ANOVA and post hoc Tukey-Kramer test (p = 0.05).
4.4.6. Inhibitory effect of carbohydrates and glycoprotein on SSA toxicity in midgut CF-
203 cells
Preincubation of SSA with carbohydrates or glycoproteins that bind to the lectin prior to
treatment of midgut CF-203 cells demonstrated that GalNAc and asialomucin reduced the cell
toxicity of SSA significantly (p < 0.0001). The reduction in toxicity of SSA towards CF-203
cells was 25 ± 3% and 35 ± 2% when SSA was incubated with 50 and 100 mM GalNAc,
respectively. A higher inhibition of SSA toxicity was observed with use of 5 and 10 mg/ml
asialomucin, resulting in a respective reduction of 52 ± 2% and 66 ± 2% in SSA toxicity. In
contrast, preincubation with mannose (100 mM) which does not bind to SSA, did not affect
the cell toxicity of SSA (Fig. 4.8).
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Figure 4.8. Inhibitory effect of carbohydrates and glycoprotein on the toxicity of SSA towards CF-203
cells. SSA (10 µg/ml) was preincubated for 1 h with different concentrations of GalNAc (50 and 100
mM) or asialomucin (5 and 10 mg/ml), and a non-binding sugar mannose (100 mM) was used as a
negative control. PBS was used in the control treatments. After preincubation of the lectins and the
carbohydrate or the glycoprotein for 1 h the mixtures were added to CF-203 cells and incubated for 24
h at 27°C. Cell toxicity was measured using an MTT assay. Data are presented as the mean
percentages of cell toxicity ± SEM compared to the control, and based on four repeats. Values with a
different letter are significantly different after a post hoc Tukey-Kramer test (p = 0.05).
5. DISCUSSION
Several in vitro and in planta studies have demonstrated the insecticidal activity of lectins on
growth and development of different insect species (reviewed in Vandenborre et al., 2009;
Michiels et al., 2010). However, over the years especially plant lectins with various
carbohydrate specificities have been tested in detail. At present only very few reports are
available with respect to the insecticidal activity of lectins from other sources, such as fungal
lectins. Therefore we will focus the discussion mainly on lectins from different origins with
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carbohydrate binding specificities similar to that of SSA.
Recently Shahidi-Noghabi et al. (2008, 2009) reported toxicity for the Gal/GalNAc-specific
SNA-I lectin from S. nigra, which is similar to SSA and belongs to the class of ricin-B related
lectins. Experiments with transgenic tobacco plants expressing SNA-I or incorporation of the
purified SNA-I into an artificial diet confirmed the insecticidal activity of this lectin in
Hemiptera like Myzus nicotianae and A. pisum. Concentrations of >100 µg/ml SNA-I in the
diet revealed a clear toxic effect on the pea aphid with an LC50 of 374 µg/ml after 18 days of
feeding. Interestingly, the present data show for the first time the high intrinsic insecticidal
activity of the fungal lectin SSA against A. pisum, a representative of sucking-piercing pest
Hemiptera, with an LC50 of 66 µg/ml. Although, the chimerolectin SNA-I of elderberry
previously showed clear aphicidal activity, SSA was about 5-6 times stronger and active at
lower concentrations. In addition, the effects were detected more rapidly, after 3 days with
SSA instead of after 18 days in case of SNA-I. The GalNAc-binding lectin from Amaranthus
caudatus seeds was also highly toxic to M. persicae with an LC50 of 68 µg/ml after 7 days of
feeding (Rahbé et al., 1995). Although this lectin belongs to the amaranthin lectin family, it
consists of β-trefoil domains similar to the ricin-B lectins (Transue et al., 1997). Previously,
Trigueros et al. (2003) reported strong insecticidal activity for the fungal lectin (XCL) from
the edible mushroom Xerocomus chrysenteron with an LC50 of 230 µg per ml of artificial diet
after 7 days of feeding to the pea aphid A. pisum. In our bioassays with pea aphids under
similar conditions, SSA was about 3.5 times more toxic than XCL and this effect was already
observed after 3 days. Finally, comparative analyses with the well known mannose-binding
lectin GNA and a novel commercial aphicide flonicamid, using the same feeding bioassay,
yielded an LC50 of 350 µg/ml for GNA (Sadeghi et al., 2009c) and 20.4 µg/ml for flonicamid
(Sadeghi et al., 2009d).
The strong effects observed for the fungal lectin SSA against aphids is of particular interest
in view of the fact that aphids are important pests of crops and ornamental plants, and are not
sensitive to Bacillus thuringiensis (Bt) delta-endotoxins. Furthermore since SSA is active
against Hemiptera at µg/ml level, we believe that fungal lectins like SSA have the potential to
play an important role in the development of integrated pest management strategies to control
pest insects. However more in-depth studies are necessary to investigate the toxicity of lectins
from different sources towards aphids and other pest insects, and to understand their mode of
action.
Next to an interesting insecticidal activity for SSA, this study also demonstrated that the
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fungal lectin is clearly targeting the insect midgut. Transverse sections of the body of A.
pisum aphids fed for 24 h on an artificial liquid diet containing FITC-SSA, clearly showed
that SSA is binding to the surface of the midgut epithelial cells, particularly at the microvilli
of the brush border zone. Conspicuously, there was no uptake of fluorescence in the
cytoplasm. This is in contrast to previous results reported by Fitches et al. (2001, 2004) who
have shown that binding of the mannose-binding lectins GNA and ConA to microvilli is
followed by transport of the proteins into the cells of the gut and Malpighian tubules.
Nonetheless, the current results resemble the observations by Sauvion et al. (2004) who
showed that ConA interacts with glycosylated receptors present at the cell surface of the
midgut epithelium cells of A. pisum. Later Cristofoletti et al. (2006) identified one of these
receptors for ConA in A. pisum as an aminopeptidase. Similarly, the mannose-binding lectin
from garlic leaves, ASAL, was shown to interact with glycosylated receptors in the epithelial
membrane of the pea aphid midgut (Majumder et al., 2004). Also from the Bt field there is
striking evidence that GalNAc plays a role in the activity of Cry1Ac endotoxin in pore
formation and binding at receptors in the brush border zone of the midgut epithelial cells
(Rodrigo-Simon et al., 2008). All these data provide evidence of a multifaceted involvement
of carbohydrates and the glycosylation pattern of binding receptors at the brush border zone
of the insect midgut epithelium in the mechanism of action of entomotoxic proteins like Cry
toxins and lectins. In any case, it is of great interest to target the insect gut as any impairment
will kill or affect the insect seriously (Hakim et al., 2010). Furthermore, taken into account
that aphids are not sensitive to Bt endotoxins, it is of great fundamental and commercial
interest to understand the mechanism of binding of SSA in the aphid gut. Therefore further
studies will envisage the identification of the carbohydrate-specific binding proteins and their
glycosylation pattern in the cell membrane at the microvilli of the epithelial midgut cells.
Here we hypothesize that interaction of SSA with Gal/GalNAc structures in the insect midgut
will play an important role in the insecticidal activity of the lectin. It is already shown in this
project that specific carbohydrates or glycoproteins that interact with the binding site of SSA
strongly reduced the cell toxicity of SSA for CF-203 cells. This effect was clearly dose-
dependent and was not observed with carbohydrates that do not interact with SSA.
As indicated above SSA was toxic to whole pea aphids. In addition, treatment of midgut CF-
203 cells with SSA also revealed a significant toxicity, resulting in clear cell debris. The
intense fluorescence observed at the cell membrane and the fact that there was no
internalization of FITC-SSA agree with earlier results on toxicity and binding at the brush
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border zone in A. pisum aphids. As a consequence, we hypothesize that binding of SSA to the
midgut epithelial cells is an important factor in the lectin toxicity. Although our knowledge
about the exact mechanism of action of lectins is still limited, lectin activity has been
associated with binding to the midgut epithelium which can cause damage to epithelial cells
and disruption of nutrient assimilation (Zhu-Salzman et al., 1998; Michiels et al., 2010).
Furthermore Miyake et al. (2007) reported that lectins such as WGA and ConA bind to the
surface of gut cells and inhibit membrane repair by inhibition of exocytosis, and this
inhibition is dependent on surface binding but not on other longer-term events. In addition,
these authors postulated that lectins such as WGA can potentially also inhibit the mucus
secretion (Hakim et al., 2010).
DNA fragmentation is considered a hallmark of apoptosis (programmed cell death) and
results from the activation of nucleases in cells undergoing apoptosis (Wyllie, 1980). Our
results showed DNA fragmentation in midgut CF-203 cells treated with SSA for 24 h.
Recently Shahidi-Noghabi et al. (2010a) also reported that the Gal/GalNAc-specific plant
lectins SNA-I and SNA-II from elderberry caused high toxicity in the CF-203 midgut cells
and this process included DNA fragmentation as well as induction of caspase-3 activity. In the
case of SSA, however, the lectin effect on DNA fragmentation was caspase-3 independent
since no obvious increase in caspase-3 activity was observed. Previously, caspase-3 was
reported not to be essential for DNA fragmentation during apoptosis in MCF-7 cells since
DNA fragmentation was also induced by other caspases (McGee et al., 2002) or through a
caspase-independent apoptotic pathway (Li et al., 2001). In this respect, it should be
mentioned that some Gal/GalNAc-specific lectins have been reported to exhibit potent
hemolytic activity and cytotoxicity, and membrane damage through the formation of ion-
permeable pores in the plasma membrane (Hatakeyama et al., 1995; Oda et al., 1997).
However, the formation of permeable pores in the plasma membrane does not seem to be the
mechanism of toxicity for SSA, since no morphological changes have been observed in the
treated midgut CF-203 cells. In addition, partial internalization of FITC-labeled SSA in the
midgut CF-203 cells was achieved using 0.001% saponin. Interestingly, this internalization of
SSA in the cells was not accompanied by an increase in cell toxicity. At present, we can only
speculate that binding of SSA to the cell surface can trigger a set of reactions/responses that
can ultimately provoke the toxicity of SSA for the insect cells and ultimately the aphid
organisms. Future studies will be necessary to identify and characterize the exact binding
target receptor(s) for SSA on the cell membrane and unravel the signal transduction cascade
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that is induced by SSA to cause cell death and insect mortality. In this context, modern and
advanced proteomic tools to isolate and identify glycoproteins (Vandenborre et al., 2010) and
the recent availability of the pea aphid genome (The International Aphid Genomics
Consortium, 2010) and the crystal structure of SSA (Sulzenbacher et al., 2010) will certainly
help to unravel the mechanisms behind the strong insecticidal action of this fungal lectin.
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Chapter 5
Parts of this chapter have been prepared for:
Hamshou M, Van Damme EJM, Caccia S, Vandenborre G, Ghesquière B, Gevaert K,
Smagghe G. High entomotoxic activity of the GalNAc/Gal-specific Rhizoctonia solani lectin
in pest insects relies on caspase 3-independent midgut cell apoptosis. Insect Biochemistry and
Molecular Biology. Submitted
High entomotoxic activity of the GalNAc/Gal-
specific Rhizoctonia solani lectin in pest insects
relies on caspase 3-independent
midgut cell apoptosis induction
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5.1 ABSTRACT
Whole insect assays where Rhizoctonia solani agglutinin (RSA) was fed to larval stages of the
cotton leafworm Spodoptera littoralis and the pea aphid Acyrthosiphon pisum demonstrated a
high concentration-dependent entomotoxicity, suggesting that this GalNAc/Gal-specific
fungal lectin might be a good control agent for different pest insects. Feeding of RSA at 10
mg/g in the solid diet of 2nd
-instar caterpillars caused 84% weight reduction after 8 days with
none of the caterpillars reaching the 4th
-instar stage. In sucking aphids 50% mortality was
achieved after 3 days of feeding with 9 µM of RSA in the liquid diet. Feeding of FITC-
labeled RSA to both pest species revealed lectin binding only to the apical/luminal side of the
midgut epithelium with the brush border zone, without lectin uptake in the midgut cells.
Lectin binding to the microvillar zone was also confirmed with primary cultures of larval
midgut columnar cells of S. littoralis.
In vitro assays with insect midgut CF-203 cell cultures indicated high cellular toxicity by
RSA with an EC50 of 0.3 µM. Preincubation of CF203 cells with GalNAc reduced the
cytotoxicity, confirming the importance of carbohydrate recognition for entomotoxicity of
RSA. CF203 cells treated with RSA showed symptoms of apoptosis with typical nuclear
condensation and DNA fragmentation, which was independent of caspase-3, but dependent of
caspase-7, -8 and -9 activities. Finally, RSA affinity chromatography of soluble and
membrane extracts of CF203 cells was performed to identify putative glycosylated proteins,
as potential binding partners for RSA.
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5.2 INTRODUCTION
Lectins or agglutinins are a very diverse group of carbohydrate-binding proteins that are
widely distributed throughout the living organisms, including plants, animals, fungi, bacteria
and viruses (Vasta and Ahmed, 2008; Michiels et al., 2010; Khan and Khan, 2011; Hartmann
and Lindhorst, 2011). Each lectin contains at least one non-catalytic domain which can
reversibly bind to a specific carbohydrate (Loris, 2002). Lectins play a role in various
biological processes such as cell-cell recognition, defense reactions or storage (Van Damme,
2008). In the last decade many plant lectins were shown to exert entomotoxic properties by
affecting the survival or development of pest insects belonging to different orders
(Vandenborre et al., 2011b). Furthermore several plant lectins with different carbohydrate
specificities were found to be toxic to insect cell lines originating from lepidopteran tissues
(Smagghe et al., 2007).
To our knowledge only very few fungal lectins have been reported to have insecticidal
activity towards pest insects. One of them is the Xerocomus chrysenteron lectin (XCL), a
lectin isolated from the edible mushroom Xerocomus chrysenteron, which showed high
toxicity towards the fruit fly Drosophila melanogaster and the aphids Acyrthosiphon pisum
and Myzus persicae (Trigueros et al., 2003; Jaber et al., 2008). Recently, insecticidal activity
was also reported for the fungal lectin isolated from the phytopathogenic fungi Sclerotinia
sclerotiorum. This Sclerotinia sclerotiorum agglutinin (SSA) showed high toxicity towards
the pea aphid A. pisum and the insect midgut cell line CF-203 (Chapter 4).
Rhizoctonia solani agglutinin (RSA) is a lectin that was isolated from the soil pathogen
Rhizoctonia solani (Vranken et al., 1987). This fungus produces black sclerotia which enables
the fungus to survive in the soil under harsh conditions for a long time. RSA is a homodimeric
protein consisting of two non-covalently associated subunits of 15.5 kDa with high affinity
for N-acetylgalactosamine (GalNAc), galactose (Gal) and more complex glycoproteins
(Candy et al., 2001). RSA is structurally and evolutionary related to the family of proteins
possessing a ricin-type lectin motif (Candy et al., 2001). Since RSA is abundantly present in
the sclerotes, it has been proposed that the lectin serves as a storage protein (Kellens and
Peumans, 1990).
In the present study, the toxicity of RSA was investigated towards the larval stages of the
cotton leafworm Spodoptera littoralis and A. pisum pea aphids. Both insect species are
important pest insects causing high damage in agriculture worldwide and are representatives
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for two important groups of pest insects with biting-chewing and piercing-sucking
mouthparts, respectively. To study the target tissue for the lectin after uptake in both
caterpillars and aphids, feeding experiments were performed with FITC-labeled lectin.
Furthermore, analyses were done with primary midgut cell cultures from S. littoralis larvae
and the midgut cell line CF-203. Finally, potential target proteins were identified from midgut
CF203 cells using RSA affinity chromatography followed by LC-MS/MS.
5.3 MATERIALS AND METHODS
5.3.1 Insects
A continuous colony of the cotton leafworm S. littoralis was kept on an agar-based artificial
diet (Iga and Smagghe, 2011), and of the pea aphid A. pisum on young broad bean (Vicia
faba) plants (Nachman et al., 2011). Both insects were maintained under standardized
conditions of 23-25ºC, 60-70% relative humidity and a 16:8 (light:dark) photoperiod.
5.3.2 Purification of RSA and labeling with FITC
RSA was purified from sclerotes of the R. solani strain AG 1-1B using a combination of
affinity chromatography on Gal-Sepharose 4B and ion exchange chromatography on a Q Fast
Flow column (HE Healthcare, Uppsala, Sweden) as described previously (Chapter 3). After
purification, RSA was labeled with FITC using the method described in Chapter 4.
5.3.3 Treatment of S. littoralis with RSA via artificial diet
Newborn (0-6 h old) 1st-instar larvae of S. littoralis were fed on Stonefly Heliothis artificial
diet containing different concentrations of purified RSA (1, 5 and 10 mg/g) for 8 days. At the
end of this experiment, the individual larval weight was determined and total larval mortality
in each treatment was recorded. Per treatment, three replications of 10 insects each were
performed, and the experiment was repeated twice. A total of 60 insects were analyzed for
each lectin concentration.
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5.3.4 Treatment of A. pisum with RSA via artificial diet
In the aphid bioassay, 15 neonates (0-12 h old) of A. pisum were fed on an artificial liquid diet
supplemented with purified RSA at different concentrations (1.5-35 µM), essentially as
described previously in Chapter 4.
5.3.5 Histofluorescence procedures
Second-instars of the cotton leafworm and 4th
-instars of the pea aphid were fed for 24 h on an
artificial diet containing FITC-labeled RSA at concentrations of 2 mg/g and 30 µM,
respectively. Afterwards the insects were fixed, dehydrated, embedded in paraffin and then
serial sections of 10 µm thickness were cut using a microtome as described previously in
Chapter 4.
5.3.6 Bioassay with insect midgut cell cultures
The cytotoxic effect of RSA was investigated towards the midgut CF-203 cells as was
described previously in Chapter 4.
5.3.7 Effect of sugars on cell toxicity of RSA in midgut CF-203 cells
The effects of sugars on cell toxicity of RSA towards midgut CF-203 cells were investigated.
Therefore, 0.15 µM RSA was incubated for 1 h with GalNAc (100 mM) and asialomucin (10
mg/ml), while the non-specific sugar mannose (100 mM) was used as a negative control.
Afterwards, the mixture was added to the CF-203 cells and incubated for 24 h at 27°C. An
MTT assay was used to calculate the cell toxicity as described in Chapter 4.
5.3.8 Uptake of RSA in CF-203 cells
For microscopic quantification of cellular uptake of FITC-labeled RSA in the midgut CF-203
cells, a similar experimental setup was used as described in Chapter 4.
Cells were inspected with a Nikon eclipse TE2000-e epifluorescence microscope (Nikon,
France) using a 40Х Plan Fluor (NA 1.30) oil immersion lens and appropriate fluorescence
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filters. Quantitative visualization of stained cells was performed on a BioRad Radiance 2000
confocal microscope mounted on a TE300 epifluorescence body (Nikon Instruments, Paris,
France) as described by Staljanssens et al. (2011).
5.3.9 Primary cell cultures
Primary midgut cell cultures were prepared from actively eating 4th
instars of S. littoralis.
Briefly, dissected midguts were obtained as described in Cermenati et al. (2007) and cells
dissociated for 1.5 h with 2 mg/ml of collagenase (Type I-AS, Sigma) in insect physiological
solution. Cells were recovered and re-suspended in the same solution and incubated for 1 h
with 0.9 µM of FITC-labeled RSA (as prepared above); control cells were incubated with
equal amounts of PBS. After incubation, for microscopic analysis, cells were fixed for 15 min
with 4% paraformaldehyde in PBS. After 3 rinses with PBS, samples were mounted in
Vectashield Mounting Medium (Vector Laboratories) and examined under a confocal laser
scanning microscope (Nikon A1R; Nikon Instruments Inc., Paris, France) as described in
Chapter 4.
5.3.10 Effect of saponin on the cytotoxicity and uptake of RSA in CF-203 cells
The effect of RSA in combination with Quillaja saponaria bark saponin (Sigma Co, St Louis,
MO) was tested in CF-203 cells. Therefore 0.3 µM of RSA and 0.001% of saponin were
tested in combination and compared to CF-203 cells in the controls (0.3 µM of RSA or
0.001% of saponin alone). Afterwards, the cell toxicity was measured using MTT assay. As
reported by De Geyter et al. (2012), CF-203 cells were incubated, under experimental
conditions as described in Chapter 4, during 30 min with 0.001% of Q. saponaria bark
saponin in the culture medium to obtain a higher cell membrane permeation. After that, cells
were incubated with FITC-labeled RSA as described in Chapter 4.
5.3.11 DNA fragmentation and nuclear staining with Hoechst in the midgut cells
DNA fragmentation was analyzed as described previously in Chapter 4. For nuclear staining
with Hoechst, CF-203 cells grown on poly-L-lysine coated glass were incubated with 0.7 µM
of RSA for 24 h at 27°C. The cells were washed with PBS and fixed with 2%
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paraformaldehyde for 20 min. After washing with PBS, the cell nuclei were stained with
Hoechst/PBS (1:1000, v/v) for 15 min. After washing with PBS, slides were mounted with
Vectashield (Vector Labs), covered with a cover glass. The cells were visualized under a
Nikon Ti florescence microscope (Nikon Benelux) using a 40Х oil immersion lens and the
appropriate filters to visualize Hoechst.
5.3.12 Caspase activity assay in midgut cells
The activities of four different caspases (-3, -7, -8 and -9) were investigated in CF-203 cells
after incubation with RSA. The caspase-3 like activity was measured using the fluorometric
substrate Ac-DEVD-AFC as described previously in Chapter 4.
The activation of caspases-3, -7, -8 and -9 was measured using the Caspase-Glo®3/7, 8 and 9
reagents (Promega) in the midgut CF-203 cells respectively. Practically, 100 µl of a cell
culture containing 2 x 105
cells per ml was loaded to a white-walled 96-well luminometer
plate. The cells were treated with 0.3 µM of RSA and 50 µM of hydrogen peroxide (H2O2)
(Sigma-Aldrich) for caspase-8 and -9 or 1% DMSO for caspase-3/7 as positive control.
Untreated cells were used as a negative control. The plates were incubated for 24 h at 27°C.
Three replicates for each treatment were performed. Then 100 µl of the Caspase-Glo® 3/7, 8
and 9 reagents was added to each well. The contents of the wells were mixed gently using a
plate shaker at 500 rpm for 0.5-2 min, and the plate was subsequently incubated at room
temperature for 1-2 h. Finally, the luminescence of each sample was measured using a
luminometer (TECAN, Infinite M200, Switzerland).
5.3.13 Isolation of binding partners of RSA from the membrane of midgut cells
Midgut CF-203 cells were collected by centrifugation at 500 × g for 10 min at 4°C. The
supernatant was removed carefully and PBS containing 2 mM of phenylmethylsulphonyl
fluoride (PMSF) was added to the pellet. The sample was vortexed and frozen at -80°C
overnight. Next day, the extract was thawed at 4°C and the solution was homogenized in an
Eppendorf tube with a pestle. After vortexing for 1-2 min, the extract was centrifuged at
16,000 × g for 2 h at 4°C and the supernatant containing the soluble proteins was collected.
The extract with the soluble protein fraction was stored at -80°C. The pellet was re-suspended
in 10 mM of HEPES buffer (pH 7.4), containing 1.5% Triton X-100 and incubated for 1h at
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4°C. In between the sample was vortexed every 15 min. After the incubation period, the
sample was centrifuged at 20,000 × g for 30 min at 4°C and the supernatant containing the
membrane proteins was collected and frozen at -80°C.
An RSA column was prepared and equilibrated with 0.2 M NaCl before loading with the
soluble protein fraction as described by Vandenborre et al. (2010b). After washing the RSA
column with 0.2 M NaCl, the proteins captured by RSA were eluted with 20 mM unbuffered
1,3-diaminopropane. Peak fractions were pooled, adjusted to 0.2 M NaCl and pH 7.6, and re-
loaded and separated on the RSA column. After elution of this second chromatographic
separation, the peak fractions were sent out for MS analysis. For the sample containing the
membrane proteins an identical RSA-affinity chromatography strategy was used. Both soluble
and membrane glycoproteins captured by RSA were analyzed by LC-MS/MS as described in
Schouppe et al. (2011) using an Ultimate 3000 HPLC system (Dionex) in-line connected to a
LTQ OrbiTRAP XL mass spectrometer (Thermo Electron).
The identified protein sequences were annotated by performing a BLAST search (EMBL-
EBI) against Genbank (http://blast.ncbi.nlm.nih.gov). For each amino acid sequence,
parameters were set on D. melanogaster as reference organism and only matches with an e-
value <0.5 were withheld. To subdivide the identified proteins into functionally related
subfamilies, the PANTHER database (http://www.pantherdb.org) was used. In addition, the
number of predicted N-glycosylation sites present on the polypeptide backbone was calculated
using the NetNGlyc 1.0 server (http://www.cbs.dtu.dk/services/NetNGlyc). Only Asn-X-
Ser/Thr sequences (where X is any amino acid except proline) with a prediction score >0.5
were withheld as potential N-glycosylation sites. Potential O-glycosylation sites in protein
sequences were predicted using NetOGlyc 3.1 Server
(http://www.cbs.dtu.dk/services/NetOGlyc-3.1/). Only sequences with a prediction score >0.5
were withheld as potential O-glycosylation sites. Finally, the location or orientation of the
predicted N- and/or O-glycosylation sites on the cell membrane was determined using the
TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/). An enrichment analysis
was performed on the glycoprotein dataset for annotation terms using the Database for
Annotation, Visualization and Integrated Discovery (DAVID)
(http://www.david.abcc.ncifcrf.gov). Statistically overrepresented annotation terms were
detected using the Benjamini statistics for multiple comparison corrections to calculate the
false discovery rate (FDR).
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5.4 RESULTS
5.4.1 Insecticidal effects of RSA on cotton leafworm caterpillars and pea aphids
RSA has high toxicity towards larvae of S. littoralis. There was a strong effect on the larval
weight gain and this effect was concentration dependent. At day 8, the weight of larvae fed on
a solid diet containing RSA at 10 and 5 mg/g was dramatically (p<0.0001) reduced with 84 ±
2% and 72 ± 5%, respectively, compared to the controls (Fig. 5.1A) without significant
differences between these two treatments (p=0.293). The lower lectin concentrations of 1
mg/g reduced larval weight by 12 ± 5% which was not significant compared to the controls
(p=0.312).
This reduction in larval weight was accompanied with effects on larval development. The
majority of the control larvae developed into the 4th
instar (78%), while only 38 ± 2, 15 ± 5
and 5 ± 5% of the larvae entered in the 4th
instar after treatment with RSA at 1, 5 and 10
mg/g, respectively (Fig. 5.1B). In addition, many of these intoxicated larvae died. At the
highest RSA concentration (10 mg/g), 48 ± 5% of the treated larvae were killed while in
treatments with RSA at 5 and 1 mg/g the mortality was only 15 ± 2 and 5 ± 2, respectively
(Fig. 5.1C).
Similarly, feeding of pea aphids on a liquid artificial diet containing increasing concentrations
(1.5-35 µM) of RSA resulted in a clear mortality compared to a control diet. Typically, high
nymphal mortality was observed at lectin concentrations of 7 µM and higher. As depicted in
Fig. 5.2A, the toxicity was concentration-dependent and followed a sigmoid curve; the
median LC50 toxicity value was 9 µM (95% CL: 7-12 µM; R2=0.81).
5.4.2 Localization of RSA in the insect body of caterpillars and aphids
A feeding experiment with caterpillars showed strong binding of RSA to the brush border
zone (apical microvillar side of the epithelium) in the midgut of 2nd
instars of S. littoralis fed
for 24 h on diet containing FITC-labeled RSA (Fig. 5.1D), but no FITC signal was seen in the
cytoplasm of the midgut cells.
Furthermore, pea aphid A. pisum 4th
instars fed for 24 h on a diet containing FITC-RSA also
demonstrated an intense fluorescence at the microvilli (brush border zone) of the epithelial
midgut cells, and typically there was no internalization of FITC-RSA in the cytoplasm of
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these cells (Fig. 5.2B,C).
Figure 5.1. Entomotoxic effects of the
fungal lectin RSA on larval growth and
development of the cotton leafworm
Spodoptera littoralis fed on a solid
artificial diet supplemented with
different concentrations of RSA for 8
days.
(A) Effect of RSA on larval weight as
compared to controls. Data are
presented as mean fresh larval weight ±
SEM.
(B) Effect of RSA on the larval
developmental stage. Data are presented
as percentage of the larvae that reach
the L4 stage after 8 days.
(C) Effect of RSA on larval survival.
Data are presented as the percentage of
mortality after 8 days. The number of
insects for each treatment is indicated.
Values per graphic followed by a
different letter (a-b) are significantly
different (post-hoc Tukey-Kramer test
with p=0.05).
(D) Transverse section of a 2nd
instar
larvae of cotton leafworm fed for 24 h
on an artificial diet containing 2 mg/g of
FITC-labeled RSA. The lectin bound to
the tips of the microvilli at the apical
surface of the midgut epithelium, but is
not internalized in the cells. Gut lumen
= Lum.
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Figure 5.2. Interaction of RSA in pea aphids (Acyrthosiphon pisum). (A) Dose response curve of
mortality of pea aphids challenged for 3 days with a liquid artificial diet containing different
concentrations of RSA. Data are corrected for mortality in the controls (0-20%) using Abbott’s
formula. (B) Transverse section of 4th instars of pea aphid A. pisum fed for 24 h on an artificial diet
containing FITC-labeled RSA at 30 µM, showing binding of the lectin to the epithelium cells of the
midgut (MG). Cut=outer cuticle. (C) Magnification of the midgut showing that RSA bound to the tips
of the microvilli (brush border zone) at the apical surface of the midgut epithelium, but is not
internalized in the cells. Gut lumen = Lum.
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5.4.3 Cellular toxicity of RSA in midgut cells
Exposure of CF-203 cells to purified RSA for 4 days revealed toxic effects, resulting in cell
debris (Fig. 5.3B). Treatment with the lowest lectin concentration of 0.03 µM yielded low cell
toxicity of 5 ± 1% without significant differences (p=0.8) (Fig. 5.3C). This toxicity increased
significantly (p<0.0001) with a 10-fold higher concentration of RSA (0.3 µM), resulting in 41
± 8%. Interestingly, at the highest lectin concentration tested (0.7 µM) there was a dramatic
increase in the cellular effects (p<0.0001) towards the midgut CF-203 cells (86 ± 2%) (Fig.
5.3C).
In addition, the median effect concentration EC50 (with 95% CL) that kills 50% of the midgut
CF-203cells exposed to RSA, was 0.3 µM (0.25-0.35 µM). The quality of the fitting to a
sigmoid curve was high with an R2=0.9.
Figure 5.3. Effect of RSA on CF-203 (A) control cells and (B) cells treated with 0.7 µM of RSA for 4
days at 27°C. (C) Cytotoxic effect of different concentrations of RSA (0.03, 0.3 and 0.7 µM) on
midgut CF-203 cells. The loss of viability was determined using of an MTT assay after 4 days of
lectin exposure.Data are presented as mean percentages of cytotoxicity ± SEM compared to the
control. Values are followed by a different letter (a-c) are significantly different (post hoc Tukey-
Kramer test with p=0.05).
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5.4.4 Effect of carbohydrates on RSA toxicity in midgut CF-203 cells
Incubation of RSA with specific and non-specific sugars for 1 h prior to treatment of the
midgut CF-203 cells showed that 100 mM GalNAc reduced the toxicity of RSA towards CF-
203 cells significantly (p<0.0001) with 55 ± 2% (Fig. 5.4). A lower inhibition of RSA toxicity
was observed when RSA was incubated with 10 mg/ml asialomucin (i.e. glycoprotein with
complex glycans) which exerted 23 ± 1% reduction in cytotoxicity (p=0.003). No inhibitory
effects on cell toxicity of RSA towards CF-203 cells were observed after incubation of the
cells with mannose which is a non-specific sugar for RSA (Fig. 5.4).
Figure 5.4. Inhibitory effect of sugars on the cell toxicity of RSA for midgut CF-203 cells. The lectin
(0.15 µM) was preincubated with a specific sugar (100 mM of GalNAc), a glycoprotein (10 mg/ml of
asialomucin), a non-specific sugar (100 mM of mannose) and PBS in the control treatments for 1 h.
Then the mixtures of RSA and sugar/glycoprotein were added to CF-203 cells and incubated for 24 h
at 27°C. Cell toxicity was measured using an MTT assay. Data are presented as mean percentages of
toxicity ± SE based compared to the control, and based on four repeats. Values are followed by a
different letter (a-c) are significantly different (post hoc Tukey-Kramer test with p=0.05).
5.4.5 Uptake of RSA in the midgut cells
Confocal microscopy analysis of midgut CF-203 cells exposed to FITC-labeled RSA
demonstrated that the fungal lectin RSA was not internalized by the cells but was only bound
to the cell surface (Fig. 5.5A).
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Figure 5. 5. Fluorescence microscopy of midgut CF-203 cells. (A) Binding of RSA on the cell surface
of CF-203 cells treated with 0.7 µM of RSA for 1 h. (B) Midgut CF-203 cells were incubated with
0.001 % saponin for 30 min prior to RSA treatment as in A. Saponin treatment allowed partial
internalization of RSA in CF-203 cells. (C) Bright field and (D) confocal laser scanning images of a
primary midgut columnar cell culture from S. littoralis. There is apparent binding of FITC-labeled
RSA to the microvillar region (indicated by the arrow). Scale bar=10 µm.
Moreover, incubation of midgut CF-203 cells with 0.001% of Q. saponaria bark saponin prior
to addition of RSA, revealed that saponin treatment resulted in partial internalization of RSA
in the cells while most of the lectin was still present on the cell surface (Fig. 5.5B). However,
the partial uptake of RSA in the cells was not accompanied by an increase in toxicity of RSA
towards CF-203 cells. The combination of 0.3 µM of RSA with 0.001% of saponin caused 49
± 2% cell toxicity, while the respective cytotoxic effect was only 51 ± 3% and 6 ± 3% when
RSA and saponin were dosed alone.
In a separate experiment with primary midgut cell cultures from S. littoralis larvae, incubation
of these midgut cells with FITC-labeled RSA demonstrated the binding of the fungal lectin to
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the microvilli of columnar cells, but no internalization was observed in the midgut cells (Fig.
5.5 C-D). In a few cases minor binding to the basolateral membrane was also detected.
5.4.6 DNA fragmentation analysis and nuclear condensation in midgut cells by RSA
Analysis of DNA extracted from CF-203 midgut cells after 24 h incubation with 0.7 µM of
RSA showed a clear DNA ladder pattern when analyzed on a 2% agarose gel. In contrast no
DNA fragmentation was observed in DNA extracted from the control treatment (Fig. 5.6A).
Figure 5.6. (A) DNA fragmentation in midgut CF-203 cells treated with 0.7 µM of RSA compared to
control cells. Ten micrograms of DNA was loaded on the 2% agarose gel. Nuclear condensation/
fragmentation in CF-203 (B) control cells and (C) cells treated with 0.7 µM RSA. A clear DNA ladder
pattern was showen in treated cells compared to untreated cells (control).
Fluorescence microscopy of CF-203 cells incubated for 24 h with 0.7 µM of RSA revealed
clear characteristic changes in nuclear morphology after RSA treatment with condensed and
fragmented nuclei and apoptotic bodies (Fig. 5.6C). In contrast, the control cells showed a
normal nucleus (Fig. 5.6B).
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5.4.7 Caspase activity in midgut cells upon exposure to RSA
The activity of four different caspases (-3, -7, -8 and -9) was investigated in CF-203 cells after
24 h of incubation with RSA. There was no induction of caspase-3 activity in these RSA-
treated midgut cells as was the case with non-treated cells (negative control). However, when
cells were incubated with SNA-II (used as a positive control), a clear induction of caspase-3
activity was detected (Fig. 5.7A).
Figure 5.7. Caspase activities in midgut CF-203 cells treated with RSA. (A) Caspase-3 like activity:
Cells were exposed to 0.7 µM of RSA, 0.2 µM of SNA-II (positive control) and PBS (negative
control) for 24 h. Data are expressed as mean relative fluorescence units (RFU) ± SD after 1 h reaction
with caspase-3 substrate. (B), (C) and (D) Caspase-7, -8 and -9 activities in midgut CF-203 cells: Cells
were treated with 0.3 µM of RSA, 50 µM of H2O2 in caspase-8 and -9, and 0.01% of DMSO in
caspase-7 assay as positive control 24 h, untreated cells were used as negative control. Caspase-Glo-
3/7, -8 and -9 kits were used to measure caspase activities. Data are presented as mean relative
luminescence units (RLU) ± SD after 1 h reaction with caspase-3/7, -8 and 9 reagents. Values are
followed by a different letter (a-c) are significantly different (post hoc Tukey-Kramer test with
p=0.05).
In contrast, caspase-7, -8 and -9 activities were detected when the midgut CF-203 cells were
treated with 0.3 µM of RSA. Caspase-7 was induced significantly (p<0.0001) in RSA-treated
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cells and its activity was increased 2-fold over untreated cells (control) (Fig. 5.7B). Besides,
RSA induced caspase-8 and -9 activities significantly (p<0.0001) in CF-203 cells. The
activity of these caspases achieved a 2.2 and 2.3-fold increase, respectively, in treated cells
over the control (Fig. 5.7C,D).
5.4.8 Proteomic analysis of soluble and membrane proteins of midgut cells bound to
RSA column
Extracts containing both soluble and membrane proteins from the midgut CF-203 cells were
analyzed by chromatography on immobilized RSA followed by LC-MS/MS. So 4941 and
5454 unique peptides were identified from membrane and soluble fractions of CF-203 cells,
respectively. From these peptides, 1115 and 1183 proteins were identified (Appendix 1 and
2), and of these proteins about 80 and 77% were found to have putative N-glycosylation
site(s), respectively. A search for proteins known to be involved in apoptosis yielded 55
proteins from the membrane fraction and 65 proteins from the soluble fraction. Among the
RSA-binding proteins identified from the membrane fraction, only 4 proteins are known to be
located in the plasma membrane, and two of them are predicted to have N- or O-glycosylation
site(s) on the exterior surface of the cells. These proteins are Neuroglian and
Q9VZ34_DROME (Fig. 5.8 and 5.9), and could be possible interacting partners for RSA.
Finally, in the soluble fraction two proteins were found that are involved in apoptosis
induction via caspases, namely the Fas-associated protein factor-1 (FAF-1) and the
Apoptosis-linked gene-2 (ALG-2).
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Figure 5.8. Neuroglian. (A) N-Glycosylation sites were predicted by NetNGlyc 1.0 Server. The graph
illustrates predicted N-glycosylation sites across the protein chain (x-axis represents protein length
from N- to C-terminal). A position with a potential (vertical lines) crossing the threshold (horizontal
line at 0.5) is predicted to be glycosylated. (B) O-Glycosylation sites were predicted by NetOGlyc 3.1
Server. A position with a potential (vertical lines) crossing the threshold (horizontal line at 0.5) is
predicted to be glycosylated. (C) Prediction of the glycosylation sites position. The analysis was done
using TMHMM 2.0 Server. Red bars and peaks represent putative transmembrane domains. Blue lines
represent putative intracellular portions of the protein; pink lines represent putative extracellular
portions of the protein.
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Figure 5.9. Q9VZ34_DROME. (A) N-Glycosylation sites were predicted by NetNGlyc 1.0 Server.
The graph illustrates predicted N-glycosylation sites across the protein chain (x-axis represents protein
length from N- to C-terminal). A position with a potential (vertical lines) crossing the threshold
(horizontal line at 0.5) is predicted to be glycosylated. (B) O-Glycosylation sites were predicted by
NetOGlyc 3.1 Server. A position with a potential (vertical lines) crossing the threshold (horizontal line
at 0.5) is predicted to be glycosylated. No O-glycan was predicted. (C) Prediction of the glycosylation
sites position. The analysis was done using TMHMM 2.0 Server. Red bars and peaks represent
putative transmembrane domains. Blue lines represent putative intracellular portions of the protein;
pink lines represent putative extracellular portions of the protein.
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5.5 DISCUSSION
Many reports have suggested that lectins can be used as tools to control insects belonging to
different orders (Vasconcelos and Oliveira, 2004; Hussain et al., 2008; Lagarda-Diaz et al.,
2009). Moreover, genetically modified crops expressing insecticidal lectins showed enhanced
resistance against pest insects (Bell et al., 2001; Saha et al., 2006; Sengupta et al., 2010).
However, the exact mechanism of action of these lectins with insecticidal properties still
remains enigmatic and needs more in-depth studies.
Here we demonstrated that feeding of larvae of S. littoralis on an artificial diet containing
different concentrations of RSA resulted in a significant reduction of larval weight which was
accompanied with effects on larval development and survival. Machuka et al. (1999)
investigated the activity of eight Gal/GalNAc-specific plant lectins towards the legume pod
borer (Maruca vitrata) and reported insecticidal activity for five of them. Interestingly, the
activity of RSA towards S. littoralis was considerably higher than for these lectins. For
example the highest mortality and larval weight reduction of M. vitrata were 31 ± 6% and 59
± 9%, respectively, after 10 days feeding on diet containing 2% of Bauhinia purpurea
agglutinin (BPA) or Iris hybrid agglutinin (IRA), respectively (Machuka et al., 1999). While
these respective values were 48 ± 5% mortality and 84 ± 2% larval weight reduction when S.
littoralis larvae were fed on diet containing 1% (10 mg/g) of RSA for 8 days.
In addition, because some proteins have been reported to be toxic towards some insect orders
without any toxicity effects for other orders, as reported for Bt toxin which has high toxicity
against Lepidoptera but no effect on the performance of aphids (Lawo et al., 2009), we
expanded the toxicity assay to the pea aphid A. pisum, a sap-sucking insect. Interestingly, high
mortality rates were detected in A. pisum nymphs fed on a liquid artificial diet containing
different concentrations of RSA, with an LC50 value of 9 µM. Recently, we reported that the
fungal lectin SSA from S. sclerotiorum also has strong toxicity towards A. pisum. The LC50
for RSA was about 4-fold higher than for SSA (2 µM, 95% CL: 1.5-2.5 µM) (Chapter 4).
Moreover, Trigueros et al. (2003) showed lower toxicity of XCL towards A. pisum; the LC50
was 15 µM for nymphs fed on artificial diet containing different concentrations of XCL for 7
days, while RSA was tested only for 3 days.
Cross sections through the body of S. littoralis larvae and A. pisum nymphs fed on an artificial
diet containing FITC-labeled RSA distinctly showed that RSA bound to the surface of the
epithelial cells of both insects, but did not internalize in the cytoplasm. Similar results were
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reported recently when pea aphids were fed on a diet containing SSA labeled with FITC. SSA
bound to the surface of the epithelial cells and was also not seen in the cytoplasm (Chapter
4). Sauvion et al. (2004) reported also similar observations in A. pisum when the aphid
nymphs were treated with the plant lectin ConA which has a different carbohydrate binding
specificity (namely mannose/glucose binding lectin) compared to RSA and SSA.
Further studies at the cell level revealed a significant toxicity of RSA towards midgut CF-203
cells (LC50=0.3 µM) after 4 days of incubation with different concentrations of purified RSA.
The lectin toxicity in these midgut cells was inhibited by specific carbohydrates or
glycoproteins which demonstrated the importance of carbohydrate-binding for RSA toxicity.
Interestingly, more in-depth analysis by using FITC-labeled RSA showed that RSA bound to
the cell surface but was not internalized in the CF-203 cells. Very similar results were also
observed when RSA binding was analyzed to primary midgut cell cultures from S. littoralis.
The lectin only attached clearly to the apical microvilli of columnar cells and this without
internalization.
The results obtained agree with previous reports where it was shown that SSA exerted
significant toxicity in CF-203 cells and bound to cell surface but did not get internalized in the
cells (Chapter 4). These finding together with the results of a histofluorescence study on A.
pisum nymphs and S. littoralis demonstrate the midgut as a primary target for RSA. Some
studies have shown that lectins can interact with a specific receptor in the midgut of
lepidopteran insects. For instance, ferritin can act as a target site for the snowdrop lectin
(GNA) in the midgut of the cotton leafworm S. littoralis (Sadeghi et al., 2009b). Harper et al.
(1995) also demonstrated binding of some lectins to Ostrinia nubilalis brush border
membrane proteins. Furthermore, Fitches and Gatehouse (1998) observed that GNA and
ConA can bind to the soluble and brush border membrane enzymes in the midgut of
Lacanobia oleracea, which affected the activities of soluble and brush border membrane
enzymes. In addition it is possible that the binding of lectins to the midgut epithelium may
damage the epithelial cells and as such can disrupt nutrient assimilation (Czapla and Lang,
1990; Zhu-Salzman et al., 1998; Michiels et al., 2010) or it could inhibit plasma membrane
repair (Miyake et al., 2007).
Apoptosis, also known as programmed cell death (PCD), is a mechanism by which cells
undergo death to control cell proliferation or in response to DNA damage. Apoptotic cells can
be characterized by typical morphological changes such as blebbing or budding, cell
shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA
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fragmentation (Lawen, 2003; Ma et al., 2005). Interestingly, clear evidence of apoptosis was
seen in midgut CF-203 cells treated with RSA as judged by DNA fragmentation, nuclear
condensation, and apoptotic bodies in addition to caspase activity. Caspases, consisting a
family of cysteine proteases, are one of the main executors of the apoptotic process that are
activated in proteolytic cascades during cell death (Kumar and Harvey, 1995; Thornberry and
Lazebnik, 1998; Taylor et al., 2008). Caspases exist as an inactive form in the cells and are
activated upon exposure to apoptotic stimuli. Upstream initiator caspases (caspase-8, -9, -10)
are first activated, and then as a consequence the downstream effector caspases (caspase-3, -6,
-7) are activated that is inducing apoptosis (Alenzi et al., 2010).
Fas (also called Apo1 or CD95) is a death receptor on the cell surface which is considered as
the major cell surface receptor involved in the induction of apoptosis (Li et al., 2007b). Fas
triggers apoptosis by recruiting the apoptosis initiator caspase-8 through the adaptor Fas-
associated death domain (FADD) which binds to the death domain of Fas and the death
effector domain of caspase-8 (Krammer, 2000). After activation of caspase-8 by Fas receptor,
apoptosis could be induced by two different signaling pathways according to the amount of
activated caspase-8 (Scaffidi et al., 1998; Shatnyeva et al., 2011). The direct pathway
occurs when there are high levels of active caspase-8 which results in direct activation of
downstream effector caspases such as caspase-7. In contrast, when caspase-8 is activated in
low concentrations, it induces apoptosis indirectly by releasing cytochrome C from
mitochondria into cytosol which is reported to be an important event during apoptosis in
Lepidoptera (Kumarswamy et al., 2009). The release of cytochrome C from the mitochondria
results in activation of caspase-9 which induces apoptosis via activation of downstream
effector caspases (Shatnyeva et al., 2011).
In the current study, similar pathways as reported for Fas signaling pathway could be
responsible to induce apoptosis in midgut CF-203 cells upon RSA treatment, especially given
that Fas was reported to have two N-glycosylation sites as well as a Thr-rich region that may
possibly have O-glycosylated sites on the cell surface (Li et al., 2007b) which suggests that
Fas could be a suitable partner for RSA. Interestingly, the involvement of the Fas signaling
pathway in RSA activity was suggested by proteomic analysis of the soluble and membrane
proteins of CF-203 cells. Indeed, although the Fas protein itself was not found among the
proteins eluted from the RSA affinity column, we identified two proteins which link to Fas.
The first protein is Fas-associated protein factor-1 (FAF-1) and was reported by Chu et al.
(1995). FAF1 binds to the death domain of Fas and to the death effector domains of FADD
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and caspase-8 (Ryu and Kim, 2001; Ryu et al., 2003). The second protein that is known to
link to Fas is the Apoptosis-linked gene-2 (ALG-2) which also binds to the Death Domain of
Fas (Jung et al., 2001). This protein translocates from the plasma membrane to cytosol upon
Fas activation (Maki and Shibata, 2007). Based on these data we have set out a working
model that needs to be studied in more detail in future experiments. Binding of RSA to the
glycans of Fas receptor ---for instance by GalNAc--- could lead to activation of caspase-8
which in turn could activate caspase-7 (but not caspase-3) directly or indirectly via activation
of caspase-9. Finally, activation of caspase-7 could then induce apoptosis. It is
worth mentioning that RSA activated caspase-8, -9 and -7 in CF-203 cells. The respective
homolog of caspase-8 and -9 in insects is Dredd and DRONC, and that of caspase-7 is Drice,
Dcp-1, Decay or/and Damm (Cooper et al., 2009).
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Chapter 6
Parts of this chapter are published in:
Hamshou M, Van Damme EJM, Vandenborre G, Ghesquière B, Trooskens G, Gevaert K,
Smagghe G (2012) GalNAc/Gal-binding Rhizoctonia solani agglutinin has antiproliferative
activity in Drosophila melanogaster S2 cells via MAPK and JAK/STAT signaling pathways.
PLoS ONE 7(4):e33680.
GalNAc/Gal-binding Rhizoctonia solani agglutinin
has antiproliferative activity in Drosophila
melanogaster S2 cells via MAPK and JAK/STAT
signaling pathways
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6.1 ABSTRACT
Rhizoctonia solani agglutinin, further referred to as RSA, is a lectin isolated from the plant
pathogenic fungus Rhizoctonia solani. Previously, we reported a high entomotoxic activity of
RSA towards the cotton leafworm Spodoptera littoralis. To better understand the mechanism
of action of RSA, Drosophila melanogaster Schneider S2 cells were treated with different
concentrations of the lectin and FITC-labeled RSA binding was examined using confocal
fluorescence microscopy. RSA has antiproliferative activity with a median effect
concentration (EC50) of 0.35 µM. In addition, the lectin was typically bound to the cell surface
but not internalized. In contrast, the N-acetylglucosamine-binding lectin WGA and the
galactose-binding lectin PNA, that were both also inhibitory for S2 cell proliferation, were
internalized whereas the mannose-binding lectin GNA did not show any activity on these
cells, although it was internalized. Extracted DNA and nuclei from S2 cells treated with RSA
were not different from untreated cells, which confirms inhibition of proliferation without
apoptosis. Pre-incubation of RSA with N-acetylgalactosamine clearly inhibited the
antiproliferative activity by RSA in S2 cells, demonstrating the importance of carbohydrate
binding. Similarly, the use of MEK and JAK inhibitors reduced the activity of RSA. Finally,
RSA affinity chromatography of membrane proteins from S2 cells allowed the identification
of several cell surface receptors involved in both signaling transduction pathways.
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6.2 INTRODUCTION
Lectins are a diverse group of proteins or glycoproteins that can bind to carbohydrates on the
cell surface and induce various biological effects. They are omnipresent in nature, and are
found in plants, animals and microorganisms. In addition to the extensive studies on plant
lectins, a number of carbohydrate-binding proteins has been isolated from fungi, especially
mushrooms, and studied for their physiological functions. Fungal lectins probably play an
important role in some biological processes such as dormancy, growth and morphogenesis
(Guillot and Konska, 1997; Konska, 2006). At present very few fungal lectins have been
studied with regard to their insecticidal activity.
The Rhizoctonia solani agglutinin (RSA) is a fungal lectin which was purified from the
sclerotes of the soil plant pathogenic fungus Rhizoctonia solani (Vranken et al., 1987). RSA is
a homodimeric protein consisting of two 15.5 kDa subunits with specificity towards N-
acetylgalactosamine (GalNAc) and galactose (Candy et al., 2001), and has been proposed to
play a role as a storage protein in the sclerotes of the fungus (Kellens and Peumans, 1990;
Chapter 2). Previously, we reported that RSA has toxic effects on the growth, development
and survival of the cotton leafworm Spodoptera littoralis which is an important pest in
agriculture (Chapter 3).
Many factors can influence the biological activity of lectins on cells such as their binding on
the cell surface or internalization in the cell and the availability of suitable targets. It was
shown that the fungal lectin from Xerocomus chrysenteron (XCL), which exerts high toxicity
in several insect species from different orders (Wang et al., 2002b; Trigueros et al., 2003), is
internalized by clathrin-dependent endocytosis and is then delivered to late
endosome/lysosome compartments in insect (SF9) or mammalian (NIH-3T3 and Hela) cell
lines (Francis et al., 2003). The internalization of the Sambucus nigra agglutinin (SNA-I)
which induces (cyto)toxicity by caspase-dependent apoptosis, occurs via clathrin and
caveolae-mediated endocytosis in insect midgut CF-203 cells (Shahidi-Noghabi et al., 2010a,
2011). In contrast, other cytotoxic lectins bind to the cell surface and cause cell death without
internalization of the lectin into the cytoplasm. For example, the fungal lectin from Sclerotinia
sclerotiorum (SSA) with a carbohydrate specificity for galactose (Gal) and N-
acetylgalactosamine (GalNAc), was found to be toxic to midgut CF-203 cells, although it was
not taken up in the cells but only bound to the cell surface (Chapter 4). SSA was proposed to
kill the cells by the induction of apoptosis via a caspase-3 independent pathway. The
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Cucumaria echinata lectin (CEL-I) also bound to the cell surface and exerted high toxicity
towards mammalian cells (Kuramoto et al., 2005), but the effect was apoptosis-independent
by causing changes in the plasma membrane integrity.
In the present study, the mode of action of RSA was investigated in the Drosophila
melanogaster Schneider S2 cell line. This cell line was originally derived from primary
cultures of late-stage of D. melanogaster embryos (Schneider, 1972). These cells are typically
round with a diameter of 15-20 µm and many features of the S2 cell line suggest that it is
derived from a macrophage-like lineage. For this study the S2 cells were chosen because D.
melanogaster represents an important model insect and because of the availability of the
Drosophila genome and proteome database (Adams et al, 2000). In addition, a comparative
analysis was made of the activity of RSA and selected plant lectins in S2 cells, and we
investigated to what extent the FITC-labeled lectins were bound and/or taken up by these
insect cells. For RSA the importance of its binding to carbohydrates on the cell surface was
shown using an excess of GalNAc in the culture medium. In addition, nuclear morphological
changes and DNA fragmentation were evaluated in RSA-treated S2 cells to study whether
RSA activity relates to apoptosis. Different kinase inhibitors were used on S2 cells to block
specific signaling transduction pathways, and highlighted those that were involved in the RSA
signal transduction pathway leading to inhibition of cell proliferation. Finally, potential target
proteins for RSA in the cell membrane of S2 cells were identified using RSA affinity
chromatography and LC-MS/MS.
6.3 MATERIALS AND METHODS
6.3.1 Isolation of lectins and labeling with FITC
RSA was isolated from the sclerotes of the plant pathogenic fungus R. solani using affinity
chromatography on galactose-Sepharose 4B and ion exchange chromatography on Q Fast
Flow column (GE Healthcare, Uppsala, Sweden), as described previously (Chapter 3). Other
plant lectins used in this study were peanut (Arachis hypogaea) agglutinin (PNA), wheat germ
(Triticum aestivum) agglutinin (WGA) and snowdrop (Galanthus nivalis) lectin (GNA)
recognizing Gal, N-acetylglucosamine (GlcNAc) and mannose, respectively. All these lectins
were purified in the laboratory as described previously (Van Damme et al., 1998).
Lectins were labeled with fluorescein isothiocyanate (FITC) as described previously in
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Chapter 4.
6.3.2 Cell proliferation assay
The cytotoxic action of RSA was investigated in S2 cells, a cell line derived from D.
melanogaster embryos (originally from The Drosophila Genomics Resource Center, Indiana
University, Bloomington, IN) which was cultured in HYQ SFX-Insect medium (Perbio
Science, Erembodegem, Belgium) (Soin et al., 2008). 100 µl of a cell suspension containing 1
x 106 cells per ml was incubated in wells of a 96-well microtiter plate for 4 days at 27°C with
different concentrations of RSA or an equal amount of PBS in the control treatment. Four
replicates were performed for each concentration, and the overall experiment was repeated
twice. After incubation, cell proliferation was monitored using the 3-(4,5)dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described previously in Chapter 4.
In addition, the effect of three plant lectins PNA, WGA and GNA on S2 cells was
investigated and compared with that of RSA to check whether there is a correlation between
carbohydrate specificity of the lectins and their antiproliferative activity on S2 cells. For each
lectin, S2 cells were treated with a 0.7 µM solution of these lectins.
Detailed studies of the carbohydrate-binding properties of RSA using glycan array analyses
from the Consortium for Functional Glycomics
(http://www.functionalglycomics.org/glycomics/publicdata/primaryscreen.jsp) have shown
that RSA interacts with GalNAc α1,3 Gal and has a clear preference for GalNAc residues
over Gal. In contrast, PNA interacts well with Gal β1,3 GalNAc, and clearly prefers Gal over
GalNAc, whereas WGA interacts preferentially with GlcNAc oligomers and GNA with
terminal mannose residues.
Significant differences between treatments were determined by one-way analysis of variance
(ANOVA) using a post hoc Tukey-Kramer test which was performed using SPSS v17.0
(SPSS Inc., Chicago, IL). In addition, a concentration-response curve, a 50%-effect
concentration (EC50) and the corresponding 95% confidence limits (95% CL) were estimated
with Prism v4 (GraphPad, La Jolla, CA); the accuracy of data fitting to the sigmoid curve
model was evaluated through examination of R2 values and the comparison of EC50 was done
using the overlapping of 95% CL as a criterion.
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6.3.3 Effect of carbohydrates on RSA antiproliferative activity on S2 cells
The effect of sugars on the antiproliferative activity induced by RSA in S2 cells was
investigated. Therefore, 0.7 µM of RSA was pre-incubated for one hour with the specific
sugar GalNAc at 100 mM, while the non-specific sugar mannose at 100 mM was used as a
negative control. Afterwards, the mixture was added to S2 cells and incubated for 24 h at
27°C. The MTT assay was used to determine cell proliferation parameters.
6.3.4 RSA activity in S2 cells following pre-incubation with kinase inhibitors
To study the effect of kinase inhibitors on the antiproliferative activity of RSA, S2 cells were
pre-incubated with different inhibitors: 10 µM of SB203580 (p38 MAP kinase inhibitor), 50
µM of PD98059 (MAP kinase (MEK) inhibitor) and 50 µM of AG490 (JAK inhibitor). All
inhibitors were purchased from Calbiochem (Darmstadt, Germany). The inhibitors were used
at the highest possible concentration that did not affect growth of S2 cells as determined in
preliminary experiments using MTT cell viability bioassays (data not shown). After
incubation in the presence or absence of the individual inhibitors for 1 h, cells were treated
with 0.3 µM RSA and incubated for 3 h at 27°C. Similar volumes of solvent (DMSO for the
inhibitors, and PBS for RSA) were used in all treatments as well in control cells. For every
treatment, three replicates were prepared and the experiment was repeated twice. After
incubation, the cell proliferation was determined using an MTT assay.
6.3.5 Internalization assay
Uptake of RSA in S2 cells was investigated as described previously (Chapter 4). The uptake
of RSA in S2 cells was also compared to that of the plant lectins PNA, WGA and GNA. Cells
were examined with a Nikon A1R confocal fluorescence microscope (Nikon, France).
6.3.6 DNA fragmentation analysis in S2 cells
DNA was extracted from embryonic S2 cells after 24 h of incubation in the presence or
absence of RSA using a phenol/chloroform/isoamyl alcohol method as described in (Chapter
4). Then 10 µg DNA was analyzed on a 2% agarose gel and DNA was visualized by ethidium
bromide staining and subsequent UV illumination.
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6.3.7 Nuclear staining with Hoechst dyes
A similar experimental setup was used as described in Chapter 5.
6.3.8 Proteomic analysis of the RSA binding proteins in the membrane of S2 cells
S2 cells grown in HYQ SFX-Insect medium were collected and washed with PBS. The cells
were suspended in 2 mM of phenylmethylsulphonyl fluoride (PMSF), vortexed and frozen at -
80°C overnight. The next day, cells were thawed at 4°C and the resulting solution was
homogenized in an Eppendorf tube using a pestle. After vortexing for 1-2 min, the extract was
centrifuged at 16,000g for 2 h at 4°C. The supernatant was removed and the pellet was
resuspended in 10 mM HEPES buffer (pH 7.4), containing 1.5% Triton X-100 and incubated
at 4°C for 1 h. In between the sample was vortexed a few times. After that, the supernatant
containing the membrane proteins was collected by centrifugation at 20,000g for 30 min at
4°C and frozen at -80°C.
RSA affinity chromatography was performed as described previously (Vandenborre et al.,
2010, 2011a). Briefly, membrane protein fractions were loaded on the RSA column and the
captured proteins were eluted using 20 mM unbuffered 1,3-diaminopropane. The membrane
glycoproteins captured by RSA were dried and re-dissolved in 50 mM
triethylammoniumbicarbonate (TEAB, Sigma-Aldrich, Steinheim, Germany) pH 7.8.
Following a denaturing step at 95°C for 10 min and cooling down on ice, trypsin (ultragrade,
Promega, Madison, WI) was added in a 1:100 ratio (w/w) and digestion of the protein
samples was carried out overnight at 37°C. Following digestion, each protein digestion
mixture (equivalent to approximately 300 µg of proteins) was acidified with 10%
trifluoroacetic acid to a final concentration of 0.5%, and loaded for RP-HPLC separation on a
2.1 mm internal diameter x 150 mm 300SB-C18 column (Zorbax®, Agilent technologies,
Waldbronn, Germany) using an Agilent 1100 Series HPLC system. Briefly, following a 10
min wash with 0.1% TFA in water/acetonitrile (98/2 (v/v), both Baker HPLC analysed,
Mallinckrodt Baker B.V., Deventer, the Netherlands), a linear gradient to 0.1% TFA in
water/acetonitrile (30/70, v/v) was applied over 100 min at a constant flow rate of 80 µl/min.
48 fractions of eluting peptides were collected between 20 and 68 min, and fractions separated
by 16 min were pooled, dried and stored at -20°C until LC-MS/MS analysis.
These dried fractions (16 fraction per sample) were re-dissolved in 80 µl of 2.5% acetonitrile
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and 8 µl was used for LC-MS/MS analysis using an Ultimate 3000 HPLC system (Dionex,
Amsterdam, the Netherlands) in-line connected to a LTQ Orbitrap XL mass spectrometer
(Thermo Electron, Bremen, Germany). Peptides were first trapped on a trapping column
(PepMap™ C18 column, 0.3 mm I.D. x 5 mm (Dionex)) and, following back-flushing from
this trapping column, peptides were loaded on a 75 m I.D. x 150 mm reverse-phase column
(PepMap™ C18, Dionex). Bound peptides were eluted with a linear gradient of 1.8% solvent
B (0.05% formic acid in water/acetonitrile (2/8, v/v)) increase per minute at a constant flow
rate of 300 nl/min.
The mass spectrometer was operated in data-dependent mode, and automatically switched
between MS and MS/MS acquisition for the six most abundant ion peaks per MS spectrum.
Full scan MS spectra were acquired at a target value of 1E6 with a resolution of 30,000. The
six most intense ions were then isolated for fragmentation in the linear ion trap. In this LTQ,
MS/MS scans were recorded in profile mode at a target value of 5,000. Peptides were
fragmented after filling the ion trap with a maximum ion time of 10 ms and a maximum of
1E4 ion counts. From the MS/MS data in each LC-run, Mascot generic files (mgf) were
created using the Mascot Distiller software (version 2.2.1.0, Matrix Science). When
generating these peak lists, grouping of spectra was performed with a maximum intermediate
retention time of 30 s and maximum intermediate scan count of 5, where possible. Grouping
was done with 0.1 Da tolerance on the precursor ion. A peak list was only generated when the
MS/MS spectrum contained more than 10 peaks, no de-isotoping was performed and the
relative S/N (signal/noise) limit was set at 2.
The generated peak lists were searched with Mascot using the Mascot Daemon interface
(version 2.3.01, Matrix Science). Spectra were searched against the UniProt database
(http://www.uniprot.org/) with taxonomy restricted to Drosophila melanogaster. Variable
modifications were set to methionine oxidation, pyro-glutamate formation of amino terminal
glutamine and acetylation of the N-terminus. Mass tolerance of the precursor ions was set to
10 ppm and for fragment ions to 0.5 Da. The peptide charge was set to 1+, 2+ or 3+ and one
missed tryptic cleavage site was allowed. Also, Mascot’s C13 setting was set to 1. Only
peptides that were ranked one and scored above the threshold score set at 99% confidence
were withheld. For extraction and storage of peptide identifications, the ms_lims platform was
used (ref: PMID: 20058248). All data were submitted to PRIDE.
The identified proteins were annotated by performing a BLAST search
(http://blast.ncbi.nlm.nih.gov) against GenBank. In addition, the number of predicted N-
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glycosylation sites present on the polypeptide backbone was calculated using the NetNGlyc
1.0 server (http://www.cbs.dtu.dk/services/NetNGlyc). Only Asn-X-Ser/Thr sequences (where
X is any amino acid except proline) with a prediction score >0.5 were withheld as potential
N-glycosylation sites. The potential O-glycosylation sites were predicted using NetOGlyc 3.1
Server (http://www.cbs.dtu.dk/services/NetOGlyc-3.1/) and only sequences with a prediction
score >0.5 were withheld as potential O-glycosylation sites. Finally, the location or
orientation of the predicted glycoproteins in the cell membrane was determined using the
TMHMM Server v. 2.0 server (http://www.cbs.dtu.dk/services/TMHMM/).
6.4 RESULTS
6.4.1 RSA causes inhibition of cell proliferation in S2 cells
Exposure of S2 cells to different concentrations of RSA for 4 days caused an inhibitory effect
on cell proliferation. The numbers of S2 cells increased 5.2 fold in the control series, but only
2.8 fold in cells exposed to 0.7 µM RSA (Fig. 6.1C). The latter cells also showed typical
clumping (Fig. 6.1B). As depicted in Fig. 6.1D, sigmoid curve analysis estimated a 50%-
response concentration (EC50) for RSA of 0.35 µM (95% CL: 0.32-0.41; R2=0.9).
In a separate experiment S2 cells were treated with RSA or with selected plant lectins with
different carbohydrate binding specificities. A comparative analysis was made for S2 cells
exposed to 0.7 µM of RSA, PNA, WGA and GNA for 24 h. While the cellular proliferation
inhibitory effects by RSA (56±1%) and WGA (59±5%) were very similar (p=0.84), PNA
showed a higher inhibition (71±1%; p=0.03) whereas GNA caused no effect (Fig. 6.2A).
6.4.2 Importance of carbohydrate binding for antiproliferative activity of RSA
As shown in Fig. 6.2B, pre-incubation of RSA with 100 mM GalNAc reduced the
antiproliferative activity of the lectin with about 70%, indicating that GalNAc competes with
binding of RSA to the cell. In contrast, 100 mM mannose did not affect the RSA activity.
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Figure 6.1. Effect of RSA on S2 cells. (A) Control, (B) Treated cells with 0.7 µM RSA, (C) S2 cell
number at time zero (at the beginning of the assay) and after 4 days incubation in the presence and
absence of 0.7 µM RSA. Cell numbers increased 5.2 and 2.8 fold in control and treated cells,
respectively, which showed clearly that RSA inhibited cell proliferation. (D) Concentration-response
curves of S2 cells challenged with RSA for 4 days after sigmoid curve fitting in Prism v4. Cell number
was measured using an MTT assay.
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Figure 6.2. (A) Effect of RSA, GNA, PNA and WGA on cell proliferation of S2 cells. Cells were
treated with 0.7 µM of different lectins for 24 h at 27°C. (B) Inhibitory effect of sugars on the activity
of RSA on S2 cells. 0.7 µM RSA was pre-incubated with 100 µM of the specific sugar GalNAc or the
non-specific sugar mannose (negative control) and PBS in the control treatments for 1 h. Then the
mixtures of RSA and sugars were added to S2 cells and incubated for 24 h at 27°C. Cell proliferation
was measured using an MTT assay. Data are presented as mean percentages of cell proliferation
inhibition ± SE compared to the control, and based on four repeats. Values are followed by a different
letter (a-c) are significantly different (post hoc Tukey-Kramer test with p=0.05)
6.4.3 Binding and internalization of RSA compared to plant lectins
Confocal microscopy analysis of S2 cells exposed to FITC-labeled RSA demonstrated that the
fungal lectin bound to the cell surface but was not internalized (Fig. 6.3A). In contrast, the
plant lectins GNA, WGA and PNA were clearly taken up by the S2 cells as shown in Fig.
6.3B, C and D.
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Figure 6.3. Confocal microscopy S2 cells incubated with different lectins: S2 cells were incubated
with 0.7 µM FITC-lectin for 1 h. (A) RSA- FITC (B) GNA-FITC (C) WGA-FITC and (D) PNA-
FITC. Scale bars are 2.5 µM.
6.4.4 RSA treatment does not induce apoptosis
When S2 cells were incubated with RSA at 0.7 µM for 24 h, no DNA fragmentation was
observed (Fig. 6.4A). In addition, there were no signs of apoptosis such as condensed and
fragmented nuclei or apoptotic bodies in the RSA-treated cells (Fig. 6.4C).
6.4.5 Effect of kinase inhibitors on RSA activity
Pre-incubation of S2 cells with different MEK and JAK inhibitors for 1 h significantly
(p<0.0001) reduced the antiproliferative activity of RSA (Fig. 6.5). The inhibitors for MEK
and JAK caused a respective reduction of 49±4% and 80±5%. In contrast, the p38 MAP
kinase inhibitor had no effect.
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Figure 6.4. (A) DNA fragmentation in S2 cells. Cells were treated with 0.7 µM RSA compared to
control (untreated) cells. Ten micrograms of extracted DNA was loaded on the 2% agarose gel. (B,C)
Nuclear condensation assay: Upon treatment with 0.7 µM RSA, the nuclei of the S2 cells were stained
with Hoechst. Typically, treated cells (C) showed a normal, non-fragmented nucleus similar to the
untreated control cells (B).
6.4.6 Proteomic analysis of membrane proteins of S2 cells retained on RSA affinity
column
Chromatography on immobilized RSA was used to capture surface glycoproteins from S2
cells. Following LC-MS/MS analysis, 4127 peptides were sequenced, leading to the
identification of 216 proteins (Appendix 3). Of these only 34 proteins were found to be cell
membrane proteins and most of them (32 proteins) have putative N- and/or O-glycosylation
site(s) as determined by the NetNGlyc 1.0 and NetOGlyc 3.1 algorithms. Finally, with the use
of TMHMM Server, 17 proteins were predicted to have N- and/or O-glycosylation site(s)
oriented towards the cell surface (Fig. 6.6-6.9), suggesting that these particular proteins could
be possible binding partners for RSA.
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Figure 6.5. Inhibition of RSA activity after pre-incubation of the S2 cells with different kinase
inhibitors. Cells were pre-incubated with three inhibitors (individually): 10 µM of SB203580 (p38
MAP kinase inhibitor), 50 µM of PD98059 [MAP kinase, (MEK) inhibitor] and 50 µM of
AG490 (JAK inhibitor) for 1 h before exposure to 0.3 µM RSA for 24 h. Values are given as means ±
SEM based on two independent repetitions. Values are followed by a different letter (a-c) are
significantly different (post hoc Tukey-Kramer test with p=0.05).
6.5 DISCUSSION
Previously, we have investigated the insecticidal activity of RSA towards the cotton leafworm
S. littoralis (Chapter 3). We demonstrated that RSA has high entomotoxic activity on the
development and survival of this economically important caterpillar insect. Therefore RSA
has been reported as an insecticidal protein that could possibly be used in crop protection
(Chapter 3). At present, the mechanism behind the toxic effect of RSA is not known. In this
paper, an attempt was made to elucidate the mode of action of this lectin with the use of S2
cells derived from embryos of D. melanogaster.
The exposure of S2 cells to RSA resulted in a significant reduction of cell proliferation. Cell
trafficking with FITC-labeled RSA under a confocal microscope demonstrated that the lectin
was not taken up by the S2 cells, but bound to their cell surface. Since RSA preferentially
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binds to GalNAc (and to a lesser extent Gal), which effectively inhibited the RSA activity in
S2 cells, it appears that the binding of RSA to specific carbohydrate moieties on the cell
surface is a prerequisite for its activity which initiated a cascade of signaling process(es)
inside the cells leading to inhibition of cellular proliferation. The interaction of RSA in S2
cells agrees with previous work in which we reported that SSA, another fungal lectin purified
from the fungus S. sclerotiorum, exerted a dramatic toxicity in the insect midgut cell line CF-
203, and similar to RSA, SSA too was only bound to the cell surface without internalization
in the cell (Chapter 4). The toxicity of SSA towards CF-203 cells was accompanied with
DNA fragmentation which most likely indicates that the effect of SSA is apoptosis-
dependent. Moreover, Shahidi-Noghabi et al. (2010a) reported induction of apoptosis when
CF-203 cells were incubated with the NeuAc(α-2,6)Gal/GalNAc specific lectin SNA-I from
elderberry Sambucus nigra. The involvement of apoptosis in the mechanism of cell death,
induced by SSA and SNA-I, in the midgut CF-203 cells raised the question whether a similar
mechanism is responsible for the activity of RSA in S2 cells, but our experiments yielded
different results. Indeed, no DNA fragmentation, no nuclear condensation and no apoptotic
bodies were detected in S2 cells upon incubation with RSA, and the treated cells were similar
in appearance to untreated cells, indicating that a different mechanism should be involved in
the activity of RSA.
In an attempt to unravel the mode of action of RSA in S2 cells, various kinase inhibitors were
used in an attempt to block the antiproliferative activity of the lectin. Interestingly, the activity
of RSA in S2 cells was inhibited by pre-incubation of the cells with MEK inhibitor
(PD98059) and JAK inhibitor (AG490). These results provide evidence that multiple
pathways could be involved in the activity of RSA.
MEK (also called MAP kinase and abbreviated MAPKK) is an upstream activator of the
MAP kinase (MAPK) (Xu et al., 1997). MAPK is a family of serine/threonine kinases that
can transfer various extracellular signals such as growth factors, mitogens and stress-inducing
agents to the nucleus (Davis, 1993). This pathway includes several kinase proteins
Raf/MEK/ERK which are mainly activated by receptor tyrosine kinases (RTKs) (McKay and
Morrison, 2007), but evidence suggests that it could also be activated by other classes of
membrane receptors such as integrins (Ojaniemi and Vuori, 1997) or G-protein-coupled
receptors (GPCRs) (Goldsmith and Dhanasekaran, 2007). The MAPK signaling pathway has
been reported to play a role in regulating cell proliferation and cell differentiation in
Drosophila as demonstrated genetically (Wassarman and Therrien, 1997). In Drosophila, a
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small GTPase Ras oncogene at 85D (Ras85D) is activated by different signals on the cell
surface receptors (Simon et al., 1991). The activated Ras85D initiates phosphorylation of the
three MAPKs within the cascade sequentially; phl (Raf1 homologue) which phosphorylates
Dsor1 (MEK homologue) which then activates Rolled (Rl) (ERK homologue) (Tsuda et al.,
1993; Brunner et al., 1994; Biggs et al., 1994; Ragab at al., 2011).
Interestingly, the proteomic analysis of this project helped to identify some membrane
proteins that could play a role as cell surface receptors for RSA and that could be involved in
MAPK signaling pathway. Two of these RSA-binding proteins are integrins (Integrin α-PS3
and Integrin β-nu). Figures 6.6 and 6.7 show the putative N- and O-glycosylation sites and
their position in both integrins. The third RSA-binding protein is a GPCR (Latrophilin Cirl)
(Fig. 6.8). It seems that binding of RSA to one or more of these proteins might be responsible
to activate the Ras85D/phl/Dsor1/Rl signaling pathway, resulting in inhibition of cellular
proliferation. This hypothesis was confirmed by pre-incubation of S2 cells with the MEK
(Dsor1) inhibitor which reduced the inhibition of cell proliferation of RSA for approximately
80% (Fig. 6.5).
The second pathway which could be involved in the activity of RSA in S2 cells involves the
Janus kinase/signal transducer and activator of transcription (JAK/STAT). Involvement of
this pathway in RSA activity was confirmed by using the JAK inhibitor. This pathway is
reported to be activated by different membrane receptors such as the cytokine receptors, and
as demonstrated in mammals and in Drosophila, it mediates many biological effects, for
example immune response, cell survival, proliferation, differentiation, and oncogenesis
(Rawlings et al., 2004). Based on the structure and the activities of these receptors, they have
been divided into several families, including cytokine receptors (type I and II), TNF receptor
family, chemokine receptors, TGF-β receptors and members of the immunoglobulin
superfamily (Howard et al., 1993; Ihle, 1995; Leonard and Lin, 2000). The JAK/STAT
signaling pathway mechanism starts by binding of a ligand (such as a cytokine) to a cell
surface receptor, which activates JAK. Afterwards, JAK phosphorylates STAT, which
translocates into the cell nucleus and regulates the expression of specific target genes
(Patterson, 2002). In Drosophila, the ligand is encoded by unpaired (upd), the receptor by
Domeless (DOME), JAK by hopscotch (hop), and STAT by Stat92E (also known as marelle)
(Arbouzova and Zeidler, 2006).
Proteomic analysis also identified a membrane protein which could be a cell surface receptor
for RSA and that could interact with the JAK signaling pathway. This protein is Neuroglian,
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and its putative N- and O-glycosylation sites and their position are given in Fig. 6.9. This
membrane protein belongs to the immunoglobulin (Ig) superfamily receptors which are
considered as a receptor for the JAK/STAT pathway as mentioned above. RSA may bind to
Neuroglian and in turn activates hop (JAK) which phosphorylates Stat92E or marelle (STAT).
Stat92E then moves to the nucleus and inhibits the cell proliferation. Interestingly, it has
recently been reported that the binding of a fungal lectin from Rhizoctonia bataticola lectin
(RBL) to complex sugars on the cell surface of human PBMC cells also affected cell
proliferation via the MAPK (but through p38 and not MEK) and JAK/STAT signaling
pathways (Pujari et al., 2010). However and in contrast to RSA in S2 cells, the RBL activity
was not inhibited by GalNAc, but by some glycoproteins such as mucin, fetuin and
asialofetuin. Moreover, multiple MAPK signaling pathways were reported to be activated by
binding of the Gal/GalNAc-binding lectin of Entamoeba histolytica with the cell membrane
receptor of human intestinal epithelial cell line (Henle-407) (Rawal et al., 2005). These
multiple MAPK signaling pathways are known to affect the cell physiology by acting on
different nuclear substrates or by binding to different transcription factors.
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Figure 6.6. Integrin α-PS3 (A) N-glycosylation sites were predicted by NetNGlyc 1.0 Server. The
graph illustrates predicted N-glycosylation sites across the protein chain (x-axis represents protein
length from N- to C-terminal). A position with a potential (vertical lines) crossing the threshold
(horizontal line at 0.5) is predicted to be glycosylated. (B) O-glycosylation sites were predicted by
NetOGlyc 3.1 Server. A position with a potential (vertical lines) crossing the threshold (horizontal line
at 0.5) is predicted to be glycosylated. (B) Prediction of the orientation in the cell membrane. The
analysis was done using TMHMM 2.0 Server. Red bars and peaks represent putative transmembrane
domains. Blue lines represent putative intracellular portions of the protein; pink lines represent
putative extracellular portions of the protein.
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Figure 6.7. Integrin β-nu (A) N-glycosylation sites were predicted by NetNGlyc 1.0 Server. The graph
illustrates predicted N-glycosylation sites across the protein chain (x-axis represents protein length
from N- to C-terminal). A position with a potential (vertical lines) crossing the threshold (horizontal
line at 0.5) is predicted to be glycosylated. (B) O-Glycosylation sites were predicted by NetOGlyc 3.1
Server. A position with a potential (vertical lines) crossing the threshold (horizontal line at 0.5) is
predicted to be glycosylated. (C) Prediction of the orientation in the cell membrane. The analysis was
done using TMHMM 2.0 Server. Red bars and peaks represent putative transmembrane domains. Blue
lines represent putative intracellular portions of the protein; pink lines represent putative extracellular
portions of the protein.
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Figure 6.8. Latrophilin Cirl (A) N-glycosylation sites were predicted by NetNGlyc 1.0 Server. The
graph illustrates predicted N-glycosylation sites across the protein chain (x-axis represents protein
length from N- to C-terminal). A position with a potential (vertical lines) crossing the threshold
(horizontal line at 0.5) is predicted to be glycosylated. (B) O-glycosylation sites were predicted by
NetOGlyc 3.1 Server. A position with a potential (vertical lines) crossing the threshold (horizontal line
at 0.5) is predicted to be glycosylated. (C) Prediction of the orientation in the cell membrane. The
analysis was done using TMHMM 2.0 Server. Red bars and peaks represent putative transmembrane
domains. Blue lines represent putative intracellular portions of the protein; pink lines represent
putative extracellular portions of the protein.
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Figure 6.9. Neuroglian (A) N-glycosylation sites were predicted by NetNGlyc 1.0 Server. The graph
illustrates predicted N-glycosylation sites across the protein chain (x-axis represents protein length
from N- to C-terminal). A position with a potential (vertical lines) crossing the threshold (horizontal
line at 0.5) is predicted to be glycosylated. (B) O-glycosylation sites were predicted by NetOGlyc 3.1
Server. A position with a potential (vertical lines) crossing the threshold (horizontal line at 0.5) is
predicted to be glycosylated. (C) Prediction of the orientation in the cell membrane. The analysis was
done using TMHMM 2.0 Server. Red bars and peaks represent putative transmembrane domains. Blue
lines represent putative intracellular portions of the protein; pink lines represent putative extracellular
portions of the protein.
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DISCUSSION, CONCLUSIONS & PERSPECTIVES
Chapter 7
GENERAL DISCUSSION, CONCLUSIONS AND
PERSPECTIVES FOR FUTURE RESEARCH
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DISCUSSION, CONCLUSIONS & PERSPECTIVES
7.1 GENERAL DISCUSSION
7.1.1 Fungi as a source for bioactive compounds
Fungi are an important source of natural bioactive compounds which can be useful in
agriculture, medicine and food industry. Recently, many valuable bioactive compounds with
antimicrobial, insecticidal, cytotoxic and anticancer activities have been successfully
identified in mushrooms and other fungi including plant endophytic fungi (Zhou et al., 2010).
Several compounds found in mushrooms have been reported to possess antitumor, antifungal
and antibacterial activities (Ferreira at al., 2010; Muhsin at al., 2011). For instance,
panepoxydone isolated from three mushrooms (Panus conchatus, Panus rudis, and Lentinus
crinitus) was found to possess antitumor properties (Erkel at al., 1996). Moreover, plant
endophytic fungi have been reported to have antifungal and antibacterial activities (Park et al.,
2003; Ding et al., 2010). In addition, other fungal compounds have the ability to prevent
several diseases such as hypertension, atherosclerosis and cancer (Ribeiro et al., 2006;
Wasser, 2011). For instance, CAPE (caffeic acid phenethyl ester), which is produced by
different mushrooms such as Agaricus bisporus and Marasmius oreades, inhibited human
cancer cells (Wasser, 2011) and many molecules with antioxidant properties were found in
mushrooms (Chirinang and Intarapichet, 2009; Ferreira et al., 2009; Vidovic et al., 2010).
When insects were reared on a diet containing powdered fungi, a few species were reported to
possess insecticidal activity against Drosophila melanogaster and Spodoptera littoralis (Mier
et al., 1996). These findings indicate that fungi possibly contain bioactive proteins with
insecticidal activity. One group of potentially interesting proteins belongs to the class of
lectins. Unfortunately, only a few fungal lectins have been investigated in detail for their
biological activities against insects, although many fungal lectins have been identified and
characterized in some detail.
7.1.2 Fungal lectins as bio-insecticidal proteins
The use of chemical pesticides to control agricultural pests, especially insects, has resulted in
many problems since several of these chemicals cause detrimental effects on non-target
organisms, development of insecticide resistance, environmental pollution and toxicity to
humans. These problems have prompted researchers and the industrial community to spend
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DISCUSSION, CONCLUSIONS & PERSPECTIVES
more efforts on the development of new technologies and durable strategies for crop
protection. In fact, plants resistant against pests offer a good strategy for pest control to reduce
pesticide usage. Interestingly, recent advances in molecular biology and genetic engineering
have provided the opportunity to produce genetically modified plants with high levels of
resistance to pests such as the European corn borer Ostrinia nubilalis and the cotton bollworm
Helicoverpa armigera (Kos et al., 2009). The well-known and most studied example in this
area is the Bacillus thuringiensis toxin (Bt) which was used to genetically engineer several
crops such as corn, cotton and potato (De Maagd et al., 1999; Bravo et al., 2011). Over the
years some insects have developed resistance against Bt toxin (Ferré and Van Rie, 2002)
which urged to look for other proteins with insecticidal activity. Many lectins, especially plant
lectins, have been reported to possess insecticidal properties. Moreover, several of these plant
lectins have been expressed in transgenic plants (Vandenborre et al., 2009). At present, there
is insufficient information about the insecticidal activity of fungal lectins. For this purpose,
this PhD research project provides a detailed study about the entomotoxic activities of the
Rhizoctonia solani agglutinin (RSA) and the Sclerotinia sclerotiorum agglutinin (SSA), two
fungal lectins isolated from the phytopathogenic fungi Rhizoctonia solani and Sclerotinia
sclerotiorum, respectively. In addition to the insecticidal properties of these lectins, their
mechanism of action was also investigated. Studies were performed both in vitro with insect
cells and in vivo with whole insects.
In this PhD project, the insecticidal activity of RSA and SSA was first investigated against the
sap-sucking pea aphid Acyrthosiphon pisum (Chapters 4 and 5). The study of the toxicity of
these fungal lectins was done using a liquid artificial diet which was considered an easy and
fast method to investigate the efficacy of different components towards insects.
In Chapter 4 rearing of A. pisum on an artificial diet containing different concentrations of
SSA, revealed high toxicity of this lectin against the pea aphid and the effect was time and
dose-dependent. In Chapter 5 a similar assay method was used to determine the entomotoxic
activity of RSA against A. pisum. The results demonstrated that SSA had about 4-fold more
toxicity than RSA. These results can be compared with the results from Trigueros et al.
(2003), where the fungal lectin (XCL) from the edible mushroom Xerocomus chrysenteron
was tested against A. pisum in a similar experimental setup. The toxicity of XCL towards A.
pisum was 7.5 and 1.6-fold lower compared to SSA and RSA, respectively. In addition, it is
worth mentioning here that the activity was calculated after 7 days of feeding on different
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DISCUSSION, CONCLUSIONS & PERSPECTIVES
concentrations of XCL, whereas in the case of SSA and RSA, calculations were done after a
much shorter period of 3 days of feeding on the lectins.
Moreover, in Chapters 3 and 5 the insecticidal activities of RSA on the growth, development
and survival of an economically important caterpillar in agriculture and horticulture, the
cotton leafworm Spodoptera littoralis, were investigated using a solid artificial diet. Rearing
larvae of S. littoralis on this diet containing 5 mg/g RSA for 11 days resulted in a significant
reduction in larval weight which ranged up to 74%. This value increased to approximately
90% when the concentration of RSA was increased to 10 mg/g. Since larvae have to reach a
minimum critical fresh weight before entering the pupal stage, the reduction in the larval
weight induced by RSA caused retardation in larval development. For example, the larvae
which were fed on 10 mg/g RSA took 11 extra days to reach the pupal stage, compared with
the control treatment. In addition, these pupae were smaller in size and weight, and the
emerged adults from treated larvae were also smaller than the adults that emerged from non-
treated insects. Moreover, a high larval mortality rate of 81% was scored with 10 mg/g RSA.
To our knowledge, RSA is the first and the only lectin which was tested against S. littoralis in
the artificial diet, but some plant lectins expressed in transgenic plants have been reported
before to affect growth and survival of this insect. For instance, rearing S. littoralis larvae on
tobacco leaves expressing 0.7% APA, a mannose-binding lectin from leek Allium porrum,
resulted in 21% reduction of the larval weight and 28% larval mortality (Sadeghi et al.,
2009a). Although these results were obtained using a lectin with a different carbohydrate
specificity than RSA and using transgenic plants, the results of this project demonstrated a
high potential of the fungal lectin RSA in the control of an important pest within the
Lepidoptera.
Based on the idea that genetically engineered plants that express multiple genes encoding
insecticidal proteins, provide a better and a more effective insect control and also reduce the
potential for development of resistant insect pests, we investigated the additive effects of RSA
with the Bt toxin in Chapter 3. Interestingly, the results showed that RSA can be combined
with Bt toxin to give a greater effect than each protein alone. Similarly, Maqbool et al. (2001)
and Zhang et al. (2007) reported before that transgenic lines expressing both GNA and Bt
toxin showed a higher resistance to insect pests, compared with the transgenic lines
expressing a single gene.
In conclusion, the insecticidal activities of RSA and SSA against different insects and the
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DISCUSSION, CONCLUSIONS & PERSPECTIVES
ability to combine at least RSA with other proteins such as Bt toxin, clearly indicate that these
fungal lectins could be used in bioengineering insect resistance in crops of agronomic
importance.
7.1.3 The midgut as primary target for RSA and SSA
The insect midgut has been considered as one of the most important targets for insecticidal
proteins and one of the main entrances for pathogens, toxins and insecticides (Hakim et al.,
2010). Determining the primary target of any toxic compound in the insect body is one of the
important steps to study this compound in more detail. Therefore, the primary target of the
fungal lectins under study was investigated.
In Chapters 4 and 5, 4th
-instar nymphs of the pea aphid A. pisum were fed on an artificial diet
containing RSA or SSA labeled with FITC. The insects were embedded in paraffin and serial
sections of 10 µm thickness were cut using a microtome. Later, the location of FITC-labeled
RSA in the insect tissues was analyzed under a fluorescence microscope. The obtained results,
demonstrate that both lectins were bound to the microvilli (brush border zone) of the
epithelial midgut cells, and typically there was no internalization in the cytoplasm of these
cells. In a similar experimental setup, RSA was found to bind strongly to the brush border
zone (apical microvillar side of the epithelium) in the midgut of S. littoralis larvae without
further internalization into the cytoplasm. This indicates the importance of the interaction of
these fungal lectins with the cell surface of the insect midgut. Furthermore, these results were
confirmed at (the) cellular level by studies of the interaction of RSA and SSA with the insect
midgut cell line CF-203. Interestingly, exposure of CF-203 cells to RSA or SSA for 4 days
showed a strong cytotoxic effect towards these cells with an LC50 of 0.3 and 0.12 µM,
respectively. In addition, RSA showed a stronger effect towards the midgut lepidopteran CF-
203 cells than to the embryonic S2 cells from the dipteran Drosophila melanogaster (Chapter
6). The latter result may indicate that binding of RSA and SSA to the midgut epithelial cells
plays an important role in the lectin toxicity. Interaction of some lectins with the insect
midgut has been reported before: ferritin in the midgut of S. littoralis is considered a target
site for the snowdrop lectin (GNA) (Sadeghi et al., 2009b). The brush border membrane
proteins of Ostrinia nubilalis were also shown to be a target for some lectins such as wheat
germ agglutinin and Bauhinia purpurea lectin (Harper et al., 1995). Furthermore, the cell
surface of the midgut epithelium cells of A. pisum was also targeted by the plant lectins from
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DISCUSSION, CONCLUSIONS & PERSPECTIVES
Jack bean (Concanavalin A, ConA) and garlic leaves (ASAL) (Sauvion et al., 2004;
Majumder et al., 2004). The fungal lectin called CGL2 from Coprinopsis cinerea was
reported to bind carbohydrates on the intestinal epithelium of the nematode Caenorhabditis
elegans and to damage the microvilli of the epithelium (Butschi et al., 2010).
7.1.4 Study of RSA and SSA binding at cellular level
The use of lectins as a biological tool needs a better understanding of the targeting, binding,
uptake, intracellular routing and delivery in the cell. Since RSA and SSA show clear binding
to the surface of the midgut cells of insects, it will be good to confirm this result at cellular
level using the insect midgut cell line CF-203 and to make a detailed study for a better
understanding of the lectin-midgut cell interaction. In Chapters 4 and 5, fluorescence
confocal microscopy was used to demonstrate that FITC-labeled SSA and RSA were not
internalized in the insect midgut CF-203 cells, but only bound to the cell surface. These
findings confirm the results of the histofluorescence study on the whole body of A. pisum and
S. littoralis. However, the current results are in contrast with other lectins as investigated in
previous studies. Good examples are the Sambucus nigra agglutinins (SNA-I and SNA-II)
that were internalized into the cytoplasm of CF-203 cells (Shahidi-Noghabi et al., 2011), and
also the fungal lectin XCL was found to internalize in insect SF9 cells via a clathrin-
dependent pathway (Francis et al., 2003).
It is well known that the activity of the lectins depends on their carbohydrate-binding domain.
Therefore the carbohydrate-binding dependency of RSA and SSA to the cell surface was
investigated by preincubation of these lectins with their specific sugar, compared with a non-
specific sugar. The results showed that the specific sugar (GalNAc) significantly inhibited the
toxicity of both lectins in the insect midgut CF-203 cells, while a non-specific sugar
(mannose) did not show any effect on the activity of these lectins. This clearly indicates that
RSA and SSA bind with specific carbohydrate moieties on the surface of the midgut cells.
Furthermore, we tried to force RSA and SSA to internalize into the midgut cells by
preincubation of the cells with saponin, which is known to increase the permeability of the
cell membrane prior to lectin treatment. The results showed that saponin allowed partial
internalization of the lectins into CF-203 cells. However, this internalization did not increase
the cell toxicity of both lectins which revealed again the importance of the binding of RSA
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DISCUSSION, CONCLUSIONS & PERSPECTIVES
and SSA to the cell surface to induce their toxicity.
7.1.5 Investigation of the mode of action of RSA and SSA at cellular level
Understanding the mode of action of insecticidal proteins is a very important factor to
determine the ability and efficiency of using a specific protein in genetically modified plants
for insect control. Here in this project different techniques were used to elucidate the mode of
action of the fungal lectins under study such as FITC-labeling, addition of inhibitors, DNA
fragmentation analysis and a proteomics approach. In Chapter 4 an attempt was made to
clarify the mode of action of SSA in the insect midgut CF-203 cells. The activity of SSA was
accompanied with DNA fragmentation, a hallmark of apoptosis (programmed cell death)
which indicated that the activity of SSA in CF-203 cells is apoptosis-dependent. Moreover,
the induction of cell death by SSA was found to be caspase-3 independent. In Chapter 5
further investigation on the mode of action of RSA was performed. Similar to SSA, the
activity of RSA was also found to be apoptosis-dependent as evidenced by DNA
fragmentation, nuclear condensation, apoptotic bodies and caspase activation (caspase-7, 8
and 9 dependent, but not caspase-3 dependent). In a recent study, the Gal/GalNAc-specific
plant lectins SNA-I and SNA-II from elderberry also showed a high cytotoxicity to the same
midgut cells of CF-203 via apoptosis induction, but in contrast to the effect of RSA and SSA,
the effect of the elderberry lectins was caspase-3-dependent (Shahidi-Noghabi et al. 2010a).
The fact that some fungal lectins induce apoptosis in human cells was also documented. For
example, the activity of mushroom lectins from Boletopsis leucomelas and Agrocybe aegerita
was found to be apoptosis-dependent when they were incubated with human monoblastic
leukemia U937 and HeLa cells (Koyama et al., 2002; Yang et al., 2005a).
To confirm whether or not the activity of RSA is similar for different cells, the mode of action
of RSA was investigated using S2 cells derived from fruit fly embryos of D. melanogaster in
Chapter 6. First, the cytotoxic activity of RSA was checked on S2 cells. The results showed
that RSA inhibited the cellular proliferation of the cells significantly. In contrast to the midgut
CF-203 cells, no signs of apoptosis such as DNA fragmentation, nuclear condensation and
apoptotic bodies were detected in the embryonic S2 cells treated with RSA, indicating that a
different mechanism should be involved to explain the activity of RSA in these cells. Various
inhibitors were used in order to block the antiproliferative activity of the lectin. Two of these
inhibitors, MAPK or MEK inhibitor and JAK inhibitor, significantly reduced the lectin effect
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DISCUSSION, CONCLUSIONS & PERSPECTIVES
indicating that multiple pathways could be involved in the activity of RSA in S2 cells.
Actually, several fungal lectins were reported to inhibit the cellular proliferation of various
mammalian cells. For instance, the proliferation activity of HepG2 and MCF-7 tumor cells
was inhibited by different lectins from mushrooms such as Agaricus arvensis (Zhao et al.,
2011), Inocybe umbrinella (Zhao et al., 2009b) and Pholiota adipose (Zhang et al., 2009). In
addition, XCL, A. aegerita lectin and Armillaria luteo-virens lectin induced antiproliferative
activity in Hela cells (Marty-Detraves et al., 2004; Zhao et al., 2003; Feng et al., 2006).
In 2011 Pujari et al. have shown that cellular proliferation of the human PBMC cells was
affected by binding of the fungal lectin RBL from Rhizoctonia bataticola to the cell surface.
Interaction of RBl with the plasmamembrane also involved activation of the p38MAPK and
STAT5 signaling pathways (Pujari et al., 2010). It is worth mentioning that in a very recent
study the same authors have reported the involvement of CD45, a receptor-like protein
tyrosine phosphatase, in RBL-induced PBMC proliferation (Pujari et al., 2012).
Potential binding partners for RSA were identified using RSA affinity chromatography of
soluble membrane extracts of CF203 and S2 cells. In Chapter 5 it is shown that although the
proteins identified by the proteomics analysis from CF-203 cells did not include any protein
that could be a suitable receptor for RSA and could be involved directly in apoptosis
induction, the list of proteins interacting with RSA contained some interesting proteins. We
identified two proteins, namely Fas-associated protein factor-1 (FAF-1) and the Apoptosis-
linked gene-2 (ALG-2), which are linked to an important receptor on the cell surface called
Fas. The Fas receptor is a death receptor on the cell membrane and it is considered one of the
major cell surface receptors involved in the induction of apoptosis (Li et al., 2007b). In
addition, Fas has N- and O-glycosylation sites on the cell surface which can explain why Fas
was bound to RSA. All the above results allowed us to draw a working hypothesis for the
mode of action of RSA, as exemplified in Fig. 7.1.
Binding of RSA to the glycans of the Fas receptor, for instance by GalNAc, could activate
caspase-8 which in turn could activate caspase-7 directly or indirectly via activation of
caspase-9. Subsequently, activation of caspase-7 could then induce apoptosis. It is worth
mentioning that the respective homologs of caspase-7 in insects are Drice, Dcp-1, Decay
or/and Damm, and that the homologs for caspase-8 and -9 are Dredd and DRONC,
respectively (Cooper et al., 2009).
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DISCUSSION, CONCLUSIONS & PERSPECTIVES
Figure 7.1. Schematic representation of the working hypothesis for the mechanism of activity of RSA
on insect midgut lepidopteran CF-203 cells.
Furthermore, in Chapter 6 the proteomics analysis for embryonic S2 cells of D. melanogaster
enabled to identify some glycosylated membrane proteins that could play a role as cell surface
receptors for RSA. Three proteins (Latrophilin Cirl, Integrin α-PS3, and Integrin β-nu) could
be involved in the MAPK signaling pathway. In addition, a protein called Neuroglian could
be involved in the signaling pathway. Our working hypothesis to explain the mechanism of
RSA activity on S2 cells is shown in Fig. 7.2. Binding of RSA to Latrophilin Cirl or/and
Integrins could activate the Ras85D/phl (Raf1 homologue) /Dsor1 (MEK homologue) /Rl
(ERK homologue) signaling pathway which then results in inhibition of the proliferation of
S2 cells. Binding of RSA to Neuroglian can lead to an activation of hop (JAK) which in turn
can phosphorylate Stat92E or marelle (STAT). Stat92E can then move to the nucleus and
inhibit the cell proliferation.
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DISCUSSION, CONCLUSIONS & PERSPECTIVES
Figure 7.2. Schematic representation of the working hypothesis for the mechanism of activity of RSA
on embryonic S2 cells from Drosophila melanogaster.
Actually the most important factor for the insecticidal activity of RSA is the interaction
between the lectin and the receptor. This interaction depends mainly on the ability of lectins
to recognize specific carbohydrates on the cell surface according to the lectin specificity.
Since the lectins in this study have a high affinity for Gal/GalNAc this clearly indicates the
importance of the complex glycans containing Gal/GalNAc residues on the cell surface. In
fact, only little is known about targeting Gal/GalNAc on the cell surface. For instance, the
binding of the Gal/GalNAc-specific lectin from peanut (PNA) to the Gal/GalNAc resides on
the surface of colon cancer cells affected the cellular proliferation via activation of the MAPK
signaling pathway (Singh et al., 2006). Another example is the Gal/GalNAc-binding lectin
from Entamoeba histolytica that was reported to bind to the cell membrane of the human
intestinal epithelial cell line (Henle-407) and so it activated the MAPK signaling pathway
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DISCUSSION, CONCLUSIONS & PERSPECTIVES
(Rawal et al., 2005). These findings as well as ours show the importance of Gal/GalNAc
residing on the cell surface for the activity of proteins which can recognize these residues. In
addition, it was reported that binding of the Bt toxin to GalNAc moieties on the midgut
epithelial cells plays a role in the activity of this toxin (Rodrigo-Simon et al., 2008).
Moreover, binding of ricin toxin to Gal/GalNAc sites on the cell surface was found to be the
first important step in the toxicity of ricin (Doan, 2004).
Interestingly, several reports suggested that the O-glycosylation in insects was restricted to
GalNAc-α-Ser/Thr and Galβ1–3GalNAc-α-Ser/Th in several lepidopteran cell lines (Thomsen
et al., 1990; Kramerov et al., 1996; Lopez et al., 1999; Maes et al., 2005; Garenaux et al.,
2011). In addition, the most abundant O-glycan structure in Drosophila is the mucin type O-
glycosylation which involves the addition of GalNAc to serine or threonine residues in protein
substrates, often extended with galactose (Gal) (Tian and Hagen, 2009). This type of
glycosylation is initiated in Drosophila by PGANTs (UDP-GalNAc, a polypeptide N-acetyl
galactosaminyl transferase family of enzymes) (Tran et al., 2012). Interestingly, different
members of this family were found to be essential during the life of Drosophila. For instance,
pgant35A was reported to be essential for viability (Schwientek et al., 2002; Ten Hagen and
Tran 2002). Another good example is pgant3 that has was reported to disrupt integrin-
mediated cell adhesion during Drosophila development by affecting the secretion of an
extracellular matrix protein (Zhang et al., 2010a). Recently, 4 additional pgant genes were
identified and found to be required for viability of Drosophila cells (Tran et al., 2012).
Moreover, a recent study showed that among 148 proteins identified from Drosophila by
RSA-affinity chromatography followed by LC-MS/MS, about 82% of these proteins were
found to possess putative N-glycosylation sites indicating that complex N-glycans with
terminal Gal/GalNAc could be present on these proteins (Vandenborre et al., 2010). As a
consequence, it can be envisaged that RSA and SSA could bind to (some of) these
glycosylated structures and this binding could explain the insecticidal activities of the lectins.
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DISCUSSION, CONCLUSIONS & PERSPECTIVES
7.2 GENERAL CONCLUSIONS
RSA is present in different R. solani strains belonging to different anastomosis groups
but some strains clearly contain a higher lectin concentration than others. In addition,
the amount of RSA in the sclerotia is higher than in the mycelium of the same strain.
RSA has significant effects on the weight, development and survival of the caterpillar
cotton leafworm S. littoralis.
RSA can be combined successfully with other entomotoxic proteins such as Bt toxin to
give greater effects than the individual treatment.
RSA has strong entomotoxic effects against nymphal stages of the pea aphid A. pisum.
Upon feeding to insects (S. littoralis and A. pisum), RSA was bound to the
apical/luminal side of the midgut epithelium with the brush border zone, confirming
the insect midgut as a primary target for RSA.
In vitro assays revealed that RSA has strong effects on the insect midgut CF-203 and
embryonic S2 cells.
Confocal microscopic analysis showed that RSA was bound to the cell surface of both
CF-203 and S2 cells but was not internalized.
Activity of RSA depends on its carbohydrate binding specificity mainly towards
GalNAc in both insect cell lines.
The activity of RSA in the midgut CF-203 cells was found to be apoptosis-dependent
as evidenced by different aspects including DNA fragmentation, nuclear condensation,
apoptotic bodies, and induction of caspases (-7 , 8 and 9) but not caspase-3.
The activity of RSA in the embryonic S2 cells was found to be antiproliferative via
interaction with the MAPK and JAK/STAT signaling pathways.
Proteomics analysis of CF-203 and S2 cells helped to identify some proteins as
potential binding partners for RSA. These proteins are Fas receptor in CF-203 cells
and Latrophilin Cirl, Integrin α-PS3, and Integrin β-nu in S2 cells.
A working model to explain the toxic effects of RSA in lepidopteran midgut cells
(CF203) was made; binding of RSA to the Fas receptor could activate caspase-8 which
in turn could activate caspase-7 directly or indirectly via activation of caspase-9.
Subsequently, activation of caspase-7 could then induce apoptosis.
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DISCUSSION, CONCLUSIONS & PERSPECTIVES
A working model to explain the antiproliferative effects of RSA in dipteran embryonic
cells (S2) was made; binding of RSA to Latrophilin Cirl or/and Integrins could
activate the phl /Dsor1/Rl signaling pathway which then can inhibit the cellular
proliferation. Furthermore binding of RSA to Neuroglian can activate hop which in
turn can phosphorylate Stat92E that can translocate to the nucleus and inhibit the cell
proliferation.
Some experiments in this thesis were performed in parallel with the fungal lectin from
Sclerotinia sclerotiorum. The results suggest a similar insecticidal activity and mode
of action as observed for RSA.
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DISCUSSION, CONCLUSIONS & PERSPECTIVES
7.3 PERSPECTIVES FOR FUTURE RESEARCH
During the recent decades, lots of efforts have been made to develop new technologies and
strategies for crop protection instead of using chemical pesticides. Many reports suggested
lectins, especially plant lectins, as insecticidal proteins that could be used as part of the IPM
program.
In this PhD thesis, the insecticidal activities of two different fungal lectins RSA (isolated from
Rhizoctonia solani) and SSA (isolated from Sclerotinia sclerotiorum) were investigated
towards two economically important insect pests the cotton leafworm, Spodoptera littoralis
and the pea aphids Acyrthosiphon pisum. The results showed that both lectins possess strong
insecticidal activity. Furthermore, the mode of action of these lectins has been investigated at
the cellular level using different insect cell lines from different insects and tissues. According
to the obtained results, we can propose some important points for future investigation in these
fields.
Since only very few fungal lectins (less than 2% of total number of fungal lectins
reported) have been studied for their insecticidal activities, more investigations of
fungal lectins could reveal other interesting insecticidal lectins which could be useful
to control some economic important insect pests.
It will be interesting to confirm the toxicity of RSA and SSA in other insect pests
belonging to the same order as well as other orders such as the Colorado potato beetle,
the brown planthopper and the cotton bollworm to check whether these lectins could
be used to control a wide range of insect pests.
Since many proteins, some of them lectins, have been expressed in transgenic plants
and these genetically modified plants showed good resistance against different insects,
it can be envisaged to express these fungal lectins (RSA/SSA) in some important crops
such as cotton, rice and potato.
Up to date, very little is known about the resistance of insect pests to lectins.
Therefore, more efforts will be necessary to elucidate this point.
More information is required about the indirect effects of RSA and SSA towards non-
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DISCUSSION, CONCLUSIONS & PERSPECTIVES
target insects such as pollinators like honeybees or bumblebees, predators and
parasitoids. These could be tested by direct exposure of these insects to the lectins or
indirectly by rearing predators and parasitoids on insects which were fed on the lectins
or on treated prey/food (food-chain contamination).
In addition, the effect of SSA and RSA towards other organisms including humans
should be investigated to check whether these lectins can be used safely in the IPM
program. It is worth mentioning that until now these lectins have never been tested
against vertebrates or vertebrate cells. This point is very important especially when
these lectins will be expressed in some plants which can be used human consumption,
such as the food crops rice and potato.
In vivo and in vitro assays will be necessary to study the synergistic effects of these
lectins with other insecticidal proteins such as protease inhibitors, ribosome
inactivating proteins, α-amylase inhibitors as well as other lectins with different
carbohydrate specificities, allowing to increase the number of target sites in the insect
body.
The mode of action of RSA was investigated in two cell lines derived from different
insects and tissues: S2 and CF-203 cells. The obtained results showed that multiple
signaling pathways could be involved in the activity of RSA. However, these findings
still need more confirmation. For example, here we identified some glycosylated
proteins on the cell surface as receptors for RSA. Some new technologies such as
RNA interference (RNAi) could be helpful to inhibit these receptors and study if they
are responsible for the RSA activities observed.
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DISCUSSION, CONCLUSIONS & PERSPECTIVES
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Summary / Samenvatting
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Summary / Samenvatting
Summary
The urgent need for more and safer agricultural products, especially food, is rapidly
increasing due to an increase of the global human population. Agriculture has suffered from
multiple problems, a major threat being insects. These insects have been controlled with
different methods mainly by using chemical insecticides. But many problems are associated
with the use of these insecticides, such as for example the harmful effects of insecticides to
our environment and non-target organisms including humans. In addition, many insects have
developed resistance to these insecticides. These problems pushed researchers and
entomologists to develop alternative methods for these chemical insecticides. Many plant
lectins have been reported to possess insecticidal properties but very little is known about the
entomotoxic effects of fungal lectins.
The main objective of this PhD thesis is to study the insecticidal activity of fungal lectins
isolated from two basidiomycetes namely Rhizoctonia solani and Sclerotinia sclerotiorum
towards different pest insects and insect cell lines and to investigate the mode of action of
these lectins.
Chapter 1 consists of a literature review about insects and lectins. The first part presents a
survey on the control of pest insects, the insect midgut, glycosylation in insects, regulation of
cell death (apoptosis) in insects and the pest insects used in this project. In the second part of
this chapter, a general introduction is presented dealing with lectins in general and fungal
lectins in particular with discussion on the insecticidal activity of lectins and the possible
mechanisms involved in insecticidal activity of the fungal lectins towards different insects.
In Chapter 2, the Rhizoctonia solani agglutinin (RSA) has been investigated in several strains
of the phytopathogenic basidiomycete R. solani belonging to different anastomosis groups
using agglutination assays to detect and determine lectin activity. Both the mycelium and
sclerotia of all isolates have been evaluated for their lectin content. This investigation showed
that the amount of lectin in the sclerotia was higher than in the mycelium of the same strain
and some strains clearly contained a higher lectin concentration than others. One strain of R.
solani namely AG 1-1B was selected for cultivation, extraction and purification of the lectin
because this strain contained the highest amount of lectin and showed very good growth.
Rearing of R. solani AG 1-1B on autoclaved wheat grains enabled to produce large quantities
of sclerotia which allowed the purification of large amounts of the pure lectin for feeding
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Summary / Samenvatting
experiments with the cotton leafworm, Spodoptera littoralis, which was tested in Chapter 3.
The insecticidal effect of RSA was investigated on the growth, development and survival of
this economically important caterpillar. The larvae of S. littoralis were fed on a diet
containing different concentrations of RSA and then mortality, fresh weight of larvae, weight
of pupae, weight of adults, adult emergence and developmental stages were scored. RSA
feeding resulted in a significant reduction in the larval weight gain and the effect was
concentration and time dependent. This effect on larval weight was accompanied with a high
mortality in the treated larvae. Moreover, the developmental stages of pupation and adult
formation were also affected. In the second part of this chapter, the combinatory effects of
RSA with Bt toxin towards larvae of S. littoralis were investigated. The results showed higher
entomotoxic effects on the larval weight and mortality than with RSA or Bacillus
thuringiensis endotoxin alone.
In Chapter 4, we expanded the assays to another fungal lectin, the Sclerotinia sclerotiorum
agglutinin (SSA). First the insecticidal activity of SSA was evaluated towards an
economically important insect pest belonging to the orders of Hemiptera, the pea aphid
(Acyrthosiphon pisum). A high mortality of pea aphid nymphs was demonstrated by feeding
these nymphs on an artificial diet containing different concentrations of SSA. Binding of the
SSA with pea aphid tissues was investigated by feeding these aphids on a diet containing
FITC-labeled SSA and making cross sections of the aphids. The results indicated that the
insect midgut with its brush border zone was the primary target for SSA. Further toxicity
evaluation was done with use of the insect midgut FPMI-CF-203/2.5 cells. The MTT assay
has been used to determine the cell toxicity of SSA after 4 days of incubation with CF-203
cells and the results showed a total loss of cell viability. More in depth investigations using
CF-203 cells were performed in an attempt to understand the mode of action of SSA.
Analyses of the internalization of SSA in CF-203 cells using FITC-labeled SSA showed that
SSA was bound to the cell surface but not internalized in the insect midgut cells. In
continuation, DNA fragmentation and caspase-3 activity were studied and the results showed
that cell death was associated with DNA fragmentation, but the effect was not caspase-3
dependent. Interestingly, when saponin was used to improve the cell membrane permeation
SSA internalization in the insect midgut cells increased, but was not accompanied with an
increase in SSA toxicity for the insect midgut CF-203 cells.
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In Chapter 5 the toxicity of RSA towards the cotton leafworm S. littoralis and pea aphids A.
pisum was studied using artificial diet. RSA showed high entomotoxic effects in both insects.
Similar to SSA in previous chapter, the surface of the insect midgut epithelium was found to
be the primary target as revealed by fluorescence microscopy. Furthermore, RSA also exerted
a high cytotoxicity towards the insect midgut CF203 cells. Dependency of RSA toxicity on its
carbohydrate specificity was demonstrated by preincubation of RSA with GalNAc which
inhibited the RSA activity significantly. Interestingly, the cytotoxic action of RSA was found
to be apoptosis-dependent as evidenced by DNA fragmentation, nuclear condensation,
apoptotic bodies and caspase activation. Finally, RSA affinity chromatography of soluble and
membrane extracts of CF203 cells followed by LC-MS/MS allowed to determine the FAS
receptor as a potential binding partner for RSA.
To confirm if the activity and the mechanism of RSA toxicity is similar in all cells or insects,
in Chapter 6 the activity and mode of action of RSA were investigated in S2 cells derived
from embryos of the fruit fly Drosophila melanogaster. Incubation of S2 cells with RSA
resulted in a significant inhibition of the cellular proliferation of these cells. The lectin was
bound to the cell surface of S2 cells, but did not internalize in the cytoplasm. In contrast to the
activity of RSA in CF-203 cells as investigated in Chapter 5, the cytotoxic activity of RSA in
S2 cells was apoptosis-independent. These findings indicated that a different pathway(s)
should be involved in the activity of RSA on S2 cells compared to CF203 cells. Various
inhibitors were used in order to block the antiproliferative activity of RSA. Among these
inhibitors, two inhibitors (MAPK or MEK inhibitor and JAK inhibitor) reduced the activity of
RSA significantly, indicating the involvement of multiple pathways in the activity of RSA in
S2 cells. Interestingly, a proteomic analysis on S2 cell proteins enabled to identify a selection
of glycosylated membrane proteins (such as Neuroglian, Latrophilin Cirl, Integrin α-PS3 and
Integrin β-nu) being involved in both pathways that could play a role as cell surface receptors
for binding of RSA.
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Samenvatting
Er is een steeds toenemende druk op de globale landbouwsector om meer en veiligere
voedingsgewassen te produceren, teneinde een steeds sneller groeiende wereldpopulatie te
kunnen voeden. Echter, de broodnodige hogere productie in de land- en tuinbouw heeft op
zijn beurt geleid tot een waaier aan nieuwe problemen, waarvan insectenplagen de
voornaamste zijn. De meest voorkomende bestrijdingsmethode hiertegen was -en is nog
steeds- het gebruik van diverse insecticiden. Maar er zijn vele problemen geassocieerd met
het gebruik van zulke stoffen, met name dat vele insecten snel resistentie ontwikkelen en dat
er schadelijke neveneffecten optreden, specifiek bij niet-doelorganismen maar ook algemeen
bij de mens en zijn leefmilieu. In die context, tracht(t)en onderzoekers alternatieven te
ontwikkelen voor insecticiden. Een mogelijk piste is het gebruik van lectines afkomstig uit
planten, waarvan intussen algemeen aanvaard is dat ze insecticidale eigenschappen bezitten.
Echter, over het potentieel entomotoxisch effect van lectines afkomstig van andere bronnen
dan planten, zoals schimmels en bacteriën, is nog té weinig gekend.
Het doel van dit doctoraatsproject is dan ook het onderzoeken van de insecticidale
eigenschappen van lectines geïsoleerd uit twee Basidiomycota (of steeltjeszwammen):
namelijk Rhizoctonia solani (J.G. Kühn, 1858) en Sclerotinia sclerotiorum ((Lib.) de Bary,
1884). Er werden in vivo en in vitro biotoetsen uitgevoerd met verscheidene soorten van
plaaginsecten en insectencellijnen om zo de moleculaire werking van deze lectines te kunnen
achterhalen.
Hoofdstuk 1 bestaat uit een literatuuronderzoek omtrent insecten en lectines. In het eerste
deel wordt een overzicht gegeven over de bestrijding van plaaginsecten, de insectendarm,
glycosylatie bij insecten, de regulatie van celdood (apoptose) bij insecten en de gebruikte
plaaginsecten. In het tweede deel een overzicht van lectines met de nadruk op lectines
gewonnen uit schimmels. Hierbij wordt vooral hun insecticidale activiteit tegen verschillende
plaaginsecten alsook hun respectievelijke werkingsmechanismen besproken.
Hoofdstuk 2 focust op het lectine Rhizoctonia solani agglutinine (RSA), dat reeds onderzocht
werd bij verschillende stammen van R. solani, die behoren tot verschillende
anastomosegroepen. Met behulp van agglutinatietesten (om lectines te detecteren en lectine
activiteit te bepalen) werden het mycelium en de scleroten van elk van deze stammen
onderzocht. Daaruit bleek dat het gehalte aan lectine in de scleroten hoger was dan in het
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Summary / Samenvatting
mycelium, en dat de gehaltes duidelijk verschilden tussen de stammen onderling. Uiteindelijk
werd de stam R. solani AG 1-1B geselecteerd als lectinebron omdat deze het hoogste gehalte
aan RSA had en ook zeer goed groeide. R. solani AG 1-1B werd gecultiveerd op
geautoclaveerde tarwekorrels om zo veel mogelijk sclerotia te produceren, waaruit dan grote
hoeveelheden zuiver lectine kon gewonnen worden voor voederexperimenten met de
katoenbladrups, Spodoptera littoralis (Boisduval, 1833). Dit plaaginsect werd gekozen omdat
het moeilijk te bestrijden is en vele economisch belangrijke gewassen beschadigt.
In Hoofdstuk 3 werd het effect van RSA getest op de groei, ontwikkeling en overleving van
rupsen van S. littoralis. Verschillende hoeveelheden van RSA werden toegevoegd aan de
vaste kunstmatige voeding waarna de mortaliteit, het gewicht van de larven, poppen en
adulten, het uitkomen van de adulten en de verschillende ontwikkelingsstadia werden
gescoord. De toevoeging van RSA aan de voeding leidde, op een concentratie- en
tijdsafhankelijke manier, tot een significante vermindering in gewichtstoename,
gecombineerd met een sterke verhoging van de mortaliteit, gedurende de larvale periode. Ook
de ontwikkelingsstadia van de poppen en het ontluiken van de adulten werd door RSA
negatief beïnvloed. In het tweede deel van dit hoofdstuk werd het effect van een combinatie
van RSA en Bacillus thuringiensis endotoxine onderzocht. Er bleek een duidelijke synergie,
waarbij de combinatie van beide een hogere entomotoxiciteit vertoonde (reductie in larvaal
gewicht en verhoogde mortaliteit) dan wanneer enkel RSA of Bt toxine getest werden.
In Hoofdstuk 4 werden gelijkaardige testen uitgevoerd als in hoofdstuk 3, maar op een ander
belangrijk plaaginsect, namelijk de erwtenbladluis, Acyrthosiphon pisum (Hemiptera, Harris,
1776). Hier werden verschillende concentraties van het schimmellectine Sclerotinia
sclerotiorum agglutinine (SSA) toegevoegd aan een vloeibare kunstmatige voeding. De
behandelde bladluisnimfen vertoonden een sterk verhoogde mortaliteit. Bijkomend werd
FITC-gemerkt SSA toegevoegd aan de voeding en kon de binding van SSA met de weefsels
van de bladluis worden bevestigd onder de fluorescentiemicroscoop. Uit deze coupes bleek
dat de meeste hoeveelheid FITC-gemerkte SSA gebonden was aan de borstelzoom
(microvillaire zone) van de middendarm. Daarna werd een reeks van cellulaire
toxiciteitsproeven uitgevoerd met FPMI-CF-203/2.5 cellen, die origineel afkomstig zijn van
de middendarm van rupsen. Daaruit bleek dat na een incubatie van 4 dagen met SSA, de
cellen zich niet meer deelden. Incubatie van CF-203 cellen met FITC-gemerkte SSA toonde
aan dat het lectine voornamelijk aan het celoppervlak bond en niet opgenomen werd door de
167
Summary / Samenvatting
cellen. Verder bleek de incubatie ook een fragmentatie van het DNA van de cellen te
veroorzaken, maar de inductie van celdood (apoptose) ging niet gepaard met een stijging van
de activiteit van de caspase-3 enzymen. Tot slot werd saponine toegevoegd aan de cellen met
als doel een verhoogde permeabiliteit te realiseren. De verhoogde opname van SSA in de
cellen leidde echter niet tot een wijziging van de toxiciteit van SSA voor de CF-203 cellen.
In Hoofdstuk 5 werd de toxiciteit van RSA bepaald na toevoeging aan de kunstmatige
voeding van S. littoralis en A pisum. Daaruit bleek dat RSA sterk entomotoxisch was voor
beide insectensoorten. Net zoals voor SSA in hoofdstuk 4, situeerde de binding van FITC-
gemerkt RSA zich voornamelijk ter hoogte van de microvillaire zone van het
middendarmepithelium. Verder bleek RSA ook sterk cytotoxisch te zijn voor de CF-203
cellen. De inductie van celdood (apoptose) door RSA ging gepaard met het optreden van o.a.
DNA-fragmentatie, condensatie van de celkern en inductie van verschillende caspase
enzymen. Bijkomend bleek de werking van RSA afhankelijk te zijn om bepaalde suikers te
binden en deze stelling werd bevestigd in proeven waarbij een pre-incubatie van N-
acetylgalactosamine (GalNAc) de activiteit van RSA sterk deed dalen. Uiteindelijk werd een
RSA-affiniteitschromatografie uitgevoerd en werden de bekomen de fracties van oplosbare en
membraaneiwitten afkomstig van de CF-203 cellen onderzocht met behulp van LC-MS/MS.
Deze proeven toonden aan dat de FAS-receptor waarschijnlijk een bindingspartner is van
RSA.
Om te bevestigen of de eerder gevonden activiteit en het werkingsmechanisme van RSA
algemeen is voor insecten(cellen), werden in hoofdstuk 6 gelijkaardige tests gedaan met S2-
cellen, die afkomstig zijn van de embryo's van de fruitvlieg, Drosophila melanogaster
(Meigen, 1830). Incubatie van S2-cellen met RSA resulteerde in een significante reductie in
celproliferatie en het lectine bleek ook nu weer gebonden te zijn aan het celoppervlak en dit
zonder opname in het cytoplasma. In tegenstelling tot de activiteit van RSA in de
middendarmcellen van CF-203 in hoofdstuk 5, bleek het cytotoxisch effect van RSA in S2-
cellen apoptose-onafhankelijk te zijn. Deze waarnemingen impliceren dat de activiteit van
RSA bij S2-cellen via een andere pathway verloopt. Om het mechanisme achter de anti-
proliferatieve effecten van RSA te achterhalen werden verschillende inhibitoren getest. Twee
hiervan, namelijk de MAPK- of MEK-inhibitor en de JAK-inhibitor, reduceerden de activiteit
van RSA. Tot slot kon een proteoomanalyse van de S2 eiwitten helpen om een selectie te
maken van geglycosyleerde membraaneiwitten, namelijk Neuroglian, Latrophilin Cirl,
168
Summary / Samenvatting
Integrin α-PS3, en Integrin β-nu, en het was opmerkelijk dat deze eiwitten in beide
bovengenoemde pathways voorkomen. Deze data suggereren dat deze drie receptoren,
aanwezig aan de oppervlakte van de S2-cellen, kunnen fungeren als bindingpartners van RSA.
169
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Zhao JK, Zhao YC, Li SH, Wang HX, Ng TB (2011) Isolation and characterization of a
novel thermostable lectin from the wild edible mushroom Agaricus arvensis. Journal of Basic
Microbiology 51, 304-311.
Zhao S, Guo YX, Liu QH, Wang HX, Ng TB (2009a) Lectins but not antifungal proteins
exhibit anti-nematode activity. Environmental Toxicology and Pharmacology 28, 265-268.
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Zheng SY, Li CX, Ng TB, Wang HX (2007) A lectin with mitogenic activity from the edible
wild mushroom Boletus edulis. Process Biochemistry 42, 1620-1624.
Zhou X, Zhu H, Liu L, Lin J, Tang K (2010) A review: recent advances and future
prospects of taxol-producing endophytic fungi. Applied Microbiology and Biotechnology 86,
1707-1717.
Zhu-Salzman K, Shade RE, Koiwa H, Salzman RA, Narasimhan M, Bressan RA,
Hasegawa PM, Murdock LL (1998) Carbohydrate binding and resistance to proteolysis
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Zuzarte-Luis V, Hurle JM (2002) Programmed cell death in the developing limb.
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205
CURRICULUM VITAE
Curriculum vitae
206
CURRICULUM VITAE
1. PERSONAL INFORMATION
Surname: Hamshou
First name: Mohamad
Place of birth: Hama/Halfaya
Date of birth: 15 august 1977
Nationality: Syrian
Marital status: Married
E-mail address: [email protected]
2. EDUCATION
SEPT 1995-JULY 1999:
B.Sc Plant Protection, Faculty of agriculture, Aleppo university, Aleppo, Syria. The degree
was obtained with good average.
DURING THE ACADAMIC YEAR 2000/2001.
Diploma in Plant Protection, Faculty of Agriculture, Aleppo University, Aleppo, Syria. The
degree was obtained with very good average.
SEPT 2006- TILL NOW:
Ph.D. student at Ghent University, Ghent, Belgium. Title of thesis: "Toxicity and mode of
action of fungal lectins in pest insects important in agriculture", Promoters: Prof. Dr. ir. Guy
Smagghe and Prof. Dr. Els J.M. Van Damme.
3. PROFESSIONAL CAREER
MAY 2001 - SEPT 2006
Lecturer in the Department of Plant Protection, Faculty of Agriculture, Aleppo University,
Aleppo, Syria.
SEPT 2006 - MAY 2012:
Doctoral research supported by a Ph.D. scholarship awarded from the General Commission
for Scientific Agricultural Research in Syria.
Supervision of students during their thesis:
Silke Jacques 2010, "Toxicity and working mechanism of AtSerpin1 towards pea aphid
Acyrthosiphon pisum".
207
CURRICULUM VITAE
4. SCIENTIFIC PUBLICATIONS:
Publications in international journals with peer reviewing and impact factor:
Hamshou M, Smagghe G, Van Damme EJM (2010) Entomotoxic effects of fungal
lectin from Rhizoctonia solani towards Spodoptera littoralis. Fungal Biology 114, 34-
40.
Hamshou M, Smagghe G, Shahidi-Noghabi S, De Geyter E, Lannoo N, Van Damme
EJM (2010) Insecticidal properties of Sclerotinia sclerotiorum agglutinin and its
interaction with insect tissues and cells. Insect Biochemistry and Molecular Biology
40, 883-890.
Vandenborre G, Van Damme E, Ghesquière B, Menschaert G, Hamshou M, Rao R,
Gevaert K, Smagghe G (2010) Glycosylation signatures in Drosophila: fishing with
lectins. Journal of Proteome Research 9, 3235-3242.
Nachman RJ, Hamshou M, Kaczmarek K, Zabrocki J, Smagghe G (2012) Biostable
and PEG polymer-conjugated insect pyrokinin analogs demonstrate antifeedant
activity and induce high mortality in the pea aphid Acyrthosiphon pisum (Hemiptera:
Aphidae). Peptides 34, 266-273.
Hamshou M, Van Damme EJM, Vandenborre G, Ghesquière B, Trooskens G,
Gevaert K, Smagghe G (2012) GalNAc/Gal-binding Rhizoctonia solani agglutinin has
antiproliferative activity in Drosophila melanogaster S2 cells via MAPK and
JAK/STAT signaling pathways. PLoS ONE 7(4):e33680..
Hamshou M, Van Damme EJM, Caccia S, Vandenborre G, Ghesquière B, Gevaert K,
Smagghe G. High entomotoxic activity of the GalNAc/Gal-specific Rhizoctonia
solani lectin in pest insects relies on caspase 3-independent midgut cell apoptosis.
Insect Biochemistry and Molecular Biology. Submitted.
Publications in local journals:
Hamshou M, Smagghe G, Van Damme EJM (2007) Analysis of lectin concentrations
in different Rhizoctonia solani strains. Communications in Agricultural and Applied
Biological Sciences, Ghent University 72, 639-44.
208
CURRICULUM VITAE
5. PRESENTATION AT SYMPOSIA AND CONFERENCES:
Contribution with oral presentation:
Hamshou M, Van Damme EJM, Smagghe G. Insecticidal activity of the fungal lectin
from Sclerotinia sclerotiorum. 62nd
International Symposium on Crop Protection.
Ghent, Belgium, 18 May 2010.
Hamshou M, Van Damme EJM, Smagghe G. The midgut brush border membrane in
insects is the primary target of the fungal lectin (RSA) from Rhizoctonia solani. VII-
TH International Conference on Arthropods. Chemical, Physiological,
Biotechnological and Environmental Aspects. Białka Tatrzanska near Zakopane,
Poland, 18-23 September 2011.
Hamshou M, Van Damme EJM, Vandenborre G, Ghesquière B, Trooskens G,
Gevaert K, Smagghe G. The Gal/GalNAc-specific lectin from Rhizoctonia solani
induces antiproliferative activity in Drosophila melanogaster S2 cells via MAPK and
JAK/STAT signalling pathways. 22nd
Joint Glycobiology Meeting, Lille, France, 27-
29 November 2011.
Contribution with poster presentation:
Hamshou M, Smagghe G, Van Damme EJM. Analysis of lectin concentrations in
different Rhizoctonia solani strains. 59th International Symposium on Crop
Protection. Ghent, Belgium, 21 May 2007.
Smagghe G, Vandenborre G, Sadeghi A, Shahidi-Noghabi S, Hamshou M, Rao N,
Michiels K, Kabera A, Vaeyens L, Van Damme EJM. Plant lectins as tools for
controlling pest insects. 6th International Integrated Pest Management Symposium.
Portland, OR (USA), 24-26 March 2009.
Smagghe G, Vandenborre G, Shahidi-Noghabi S, Hamshou M, Lannoo N, Van
Damme EJM (2010). Plant lectins as tools in pest control. Chemical Entomology,
Gembloux Agro-Bio Tech, University of Liege, 5 May 2010.
Hamshou M, Smagghe G, Lannoo N, Van Damme. Insecticidal properties of
Sclerotinia sclerotiorum agglutinin and interaction with insect tissues and cells.
International Applied Plant Biotechnology & International Congress on In Vitro
Biology Sustainability through agricultural biotechnology: Food, biomaterials,
209
CURRICULUM VITAE
energy, and environment, St. Louis, MO (USA), 6-11 June 2010.
Hamshou M, Smagghe G, Lannoo N, Van Damme. Insecticidal properties of
Sclerotinia sclerotiorum agglutinin. 21st
Joint Glycobiology Meeting, Ghent, Belgium,
7-9 November 2010.
Hamshou M, Smagghe G, Van Damme EJM. Interactions of Rhizoctonia solani
agglutinin with insect midgut CF-203 cells. 63rd
International Symposium on Crop
Protection. Ghent, Belgium, 24 May 2011.
Hamshou M, Van Damme EJM, Smagghe G. The Fungal Lectin Rhizoctonia solani
Agglutinin as a Biological Insecticide. 17th Symposium on Applied Biological
Sciences, Campus Arenberg, Leuven, Belgium, 10 February 2012.
210
CURRICULUM VITAE
211
APPENDIX
Appendix 1 ….. Page 212 Appendix 2 ….. Page 224 Appendix 3 ….. Page 237
APPENDIX
212
APPENDIX
Accession (BGIBMGA-)
Protein name
000064-PA Peroxiredoxin 5037
000066-PA Ribosomal protein L32
000074-PA Fibrillarin
000086-PA CG18812
000097-PA Calpain-A
000103-PA CG30122
000104-PA delta-coatomer protein
000106-PA Tim10
000114-PA CG5913
000120-PA CG14804
000132-PA CG14476
000201-PA Proteasome 26kD subunit
000230-PA Phospholipase A2 activator protein
000236-PA CG6638
000292-PA CG7430
000295-PA Rab-protein 3
000321-PA CG6664
000381-PA nudC
000382-PA nudC
000390-PA Adenosine 2
000398-PA CG1140
000415-PA claret
000425-PA CG4729
000447-PA comatose
000469-PA CG10103
000475-PA Calreticulin
000481-PA CG4670
000508-PA putative sperm associated antigen 17
000511-PA scully
000516-PA IGF-II mRNA-binding protein
000526-PA Ypsilons chachtel
000542-PA belphegor
000552-PA Failed axon connections
000559-PA Triosephosphate isomerase
000581-PA CG8531
000596-PA Vacuolar H[+]-ATPase SFD subunit
000617-PA Syntaxin 7
Accession (BGIBMGA-)
Protein name
000647-PA NUCB1
000672-PA knockdown
000701-PA Catalase
000710-PA Vap-33-1
000714-PA pathetic
000715-PA CG12262
000743-PA flotillin
000767-PA atlastin
000793-PA CG5484
000804-PA mitochondrial ATP synthase coupling factor 6
000806-PA CG32626
000809-PA CG10354
000828-PA Minichromosome maintenance 6
000829-PA CG13900
000858-PA Drop dead
000867-PA String of pearls
000897-PA CG6020
000903-PA Transport and Golgi organization 7
000918-PA skpA
000926-PA Tal
000937-PA Updo
000941-PA Sema-2a
000943-PA n-synaptobrevin
000959-PA Ribosomal protein L31
001011-PA Edem1
001019-PA CG10158
001035-PA raspberry
001043-PA Ribosomal protein L27A
001044-PA Apoptosis-linked gene-2
001064-PA Polypeptide GalNAc transferase 6
001070-PA Arflike at 72A
001097-PA Calnexin 99A
001106-PA Ribosomal protein L21
001107-PA super coiling factor
001118-PA CG3362
001119-PA GDP dissociation inhibitor
001126-PA Rab escort protein
001136-PA Spase 22/23-subunit
001165-PA Dynamin related protein 1
001168-PA CG18811
001206-PA CG7033
001209-PA COP9 complex homolog subunit 3
001218-PA Hsc70Cb
Appendix 1. List of proteins form membrane fractions of CF-203 cells identified after RSA affinity
chromatography and LC-MS/MS analysis. (Chapter 5).
213
APPENDIX
001223-PA Dead box protein 80
001236-PA CG14757
001241-PA Trap1
001243-PA Ribosomal protein S10b
001258-PA CG1599
001265-PA eIF5B
001318-PA Calmodulin
001319-PA Calmodulin
001323-PA CG31548
001335-PA CG10463
001336-PA CG10077
001352-PA ergic53
001353-PA ergic53
001363-PA overgrown hematopoietic organs at 23B
001385-PA CG7460
001386-PA CG6767
001395-PA Pros beta 3
001396-PA CG9267
001402-PA Aldehyde dehydrogenase
001406-PA Cct gamma
001415-PA Ubiquitin-63E
001458-PA Downstream of raf1
001471-PA Ribosomal protein S15
001483-PA Ribosomal protein S17
001490-PA FK506-binding protein 1
001501-PA Ribosomal protein S6
001525-PA Tyrosyl-tRNA synthetase
001540-PA Upf1
001542-PA rumpelstiltskin
001549-PA Ubiquitin-63E
001551-PA Ubiquilin
001580-PA Cap binding protein 80
001584-PA Tropomyosin 1
001638-PA Aminopeptidase P
001644-PA La related protein
001648-PA ferrochelatase
001670-PA Specifically Rac1-associated protein 1
001683-PA beta-Tubulin at 56D
001707-PA beta-Tubulin at 56D
001725-PA Iron regulatory protein 1B
001726-PA Iron regulatory protein 1B
001751-PA CG17271
001797-PA CG8031
001800-PA Ribosomal protein L4
001801-PA reptin
001806-PA CG3689
001811-PA CG5077
001825-PA CG17768
001847-PA CG4972
001849-PA Threonyl-tRNA synthetase
001853-PA bellwether
001870-PA Srp54
001873-PA vesicle amine transport protein
001874-PA CG3523
001876-PA Amylase proximal
001915-PA Proteasome 29kD subunit
001918-PA CG4406
001926-PA Farnesyl pyrophosphate synthase
001928-PA Rab-protein 2
001929-PA CG3415
001943-PA CG1598
001950-PA Pabp2
001953-PA bancal
001954-PA Heterogeneous nuclear ribonucleoprotein K
001966-PA Aldehyde dehydrogenase type III
001991-PA Ribosomal protein L36
002004-PA CG9281
002005-PA stress-sensitive B
002012-PA moleskin
002013-PA moleskin
002052-PA CG3731
002103-PA alpha-Tubulin at 84B
002149-PA CG32649
002186-PA thioredoxin peroxidase 1
002203-PA Rab-related protein 4
002209-PA Rab-protein 11
002215-PA mitochondrial import inner membrane translocase
002222-PA Glutathione S transferase D1
002237-PA Protein phosphatase 2A at 29B
002241-PA Vacuolar H[+]-ATPase 55kD B subunit
002295-PA growl
002330-PA CG1983
002346-PA C-terminal Binding protein
002361-PA CG3909
002381-PA Heat shock protein cognate 4
002393-PA Aralar 1
002405-PA Ribosomal protein S14b
002406-PA thioredoxin peroxidase 2
002423-PA CG1458
002429-PA Cyclophilin 1
214
APPENDIX
002432-PA Vha100-2
002449-PA RabX4
002457-PA Aldehyde dehydrogenase
002462-PA CG11089
002470-PA CG6259
002481-PA CG6905
002493-PA CG10576
002504-PA sar1
002508-PA Pyruvate kinase
002538-PA Vha100-1
002539-PA Vha100-1
002542-PA alpha-Tubulin at 84B
002543-PA Moesin
002544-PA Moesin
002559-PA bendless
002569-PA CaBP1
002570-PA CG33303
002572-PA Ribosomal protein L19
002582-PA CG3731
002592-PA CG5355
002593-PA CG5355
002594-PA Adenylate kinase-2
002604-PA CG10527
002636-PA Na[+]/H[+] hydrogen exchanger 3
002639-PA Aspartyl beta-hydroxylase
002640-PA Ubiquitin carboxy-terminal hydrolase
002642-PA CG8707
002644-PA 14-3-3 zeta
002649-PA CG5366
002651-PA Sialic acid phosphate synthase
002709-PA CG8026
002711-PA lightoid
002750-PA CG11876
002759-PA CG5384
002779-PA dre4
002790-PA COP9 complex homolog subunit 5
002811-PA Qm
002814-PA Oligosaccharyl transferase 48kD subunit
002818-PA Thioredoxin reductase-1
002820-PA mitochondrial assembly regulatory factor
002829-PA mitochondrial Ribosomal protein L21
002837-PA GTP-binding-protein
002840-PA CG17514
002886-PA sniffer
002914-PA AP-1 gamma
002953-PA Diphenoloxidase A2
002967-PA CG4389
002972-PA Female sterile (2) Ketel
002978-PA CG7364
002981-PA chickadee
002984-PA Lysyl-tRNA synthetase
003004-PA small nuclear ribonucleoprotein polypeptide
003018-PA RNA polymerase II 140kD subunit
003028-PA Proteasome 28kD subunit 1
003042-PA CG5168
003049-PA Glycerophosphate oxidase-1
003070-PA CG34132
003073-PA small nuclear ribonucleoprotein polypeptide
003086-PA tartan
003116-PA Glutamate oxaloacetate transaminase 2
003126-PA CG11092
003129-PA CG5958
003143-PA CG12125
003153-PA CG4164
003165-PA CG2076
003169-PA CG7382
003184-PA Mo25
003186-PA Eukaryotic initiation factor 4a
003196-PA VhaAC39
003197-PA Ribosomal protein S18
003212-PA Rpn7
003221-PA CG8801
003243-PA CG5323
003258-PA Pyruvate dehydrogenase kinase
003267-PA CG12918
003279-PA CG3107
003296-PA beta-Tubulin at 56D
003305-PA Transcription factor II F alpha
003309-PA Ribosomal protein LP0
003319-PA Glutamate oxaloacetate transaminase 1
003335-PA lethal (2) 05070
003337-PA Ribosomal protein L10Ab
003355-PA CG11968
003361-PA Karyopherin beta 3
003362-PA hypothetical protein KGM_20868
003390-PA CG11577
003391-PA CG7394
003397-PA Ribosomal protein S8
003412-PA Ribosomal protein LP1
003428-PA CG1518
215
APPENDIX
003442-PA beta-Tubulin at 56D
003457-PA Ras-associated protein 2-like
003464-PA CG12773
003466-PA CG5676
003467-PA Leucyl-tRNA synthetase
003469-PA polyA-binding protein
003474-PA Casein kinase II alpha subunit
003490-PA lethal (2) s5379
003515-PA capulet
003519-PA CG5174
003539-PA lethal (2) 35Df
003555-PA CG33096
003562-PA CG6463
003584-PA PRL-1
003587-PA ERp60
003597-PA Rab-protein 6
003601-PA kurtz
003607-PA frayed
003608-PA Elongation factor 1 alpha 48D
003627-PA ADP ribosylation factor 51F
003632-PA CG3402
003635-PA Transportin
003652-PA Lamin
003656-PA Eip55E
003669-PA Int6 homologue
003671-PA CG13349
003702-PA CG10286
003717-PA N-myristoyl transferase
003725-PA N-myristoyl transferase
003726-PA Ribosomal protein L13
003744-PA peanut
003758-PA Karyopherin alpha 3
003805-PA CG10298
003806-PA Glued
003815-PA CG5214
003820-PA Rpb8
003829-PA stress-sensitive B
003833-PA Nucleolar protein at 60B
003892-PA beta'-coatomer protein
003901-PA ATP synthase-beta
003923-PA Ribosomal protein S24
003924-PA NADH dehydrogenase ubiquinone
003961-PA CG4673
003972-PA Selenide water dikinase
003978-PA Ribosomal protein L12
003984-PA TER94
003985-PA TER94
004021-PA G protein o alpha 47A
004038-PA Eb1
004078-PA mitochondrial Ribosomal protein S9
004096-PA cAMP-dependent protein kinase R2
004103-PA lethal (2) essential for life
004109-PA spenito
004112-PA CG10602
004136-PA Tumor suppressor protein 101
004165-PA Elongation factor 2b
004166-PA Dak1
004175-PA Adam
004218-PA CG10166
004221-PA Phosphoglucose isomerase
004241-PA CAS/CSE1segregation protein
004267-PA CG14480
004302-PA eIF-2alpha
004315-PA P-element somatic inhibitor
004356-PA Ribosomal protein S7
004374-PA Ribosomal protein S12
004375-PA Ribosomal protein L7
004376-PA Pros beta 5
004408-PA CG6133
004447-PA CG4752
004501-PA Elongin C
004531-PA CG42314
004533-PA CG42314
004534-PA CG42314
004535-PA CG42314
004540-PA lethal (2) essential for life
004541-PA lethal (2) essential for life
004557-PA Purine-rich binding protein -alpha
004603-PA beta-Tubulin at 56D
004608-PA Fimbrin
004612-PA Heat shock protein 83
004614-PA Heat-shock-protein-70Ba
004626-PA G protein beta-subunit 13F
004646-PA Bub3
004647-PA sec71
004655-PA p270
004657-PA Proteasome alpha 6 subunit
004661-PA CG18591
004666-PA Rab-protein 10
004687-PA auxillin
216
APPENDIX
004688-PA Dipeptidyl aminopeptidase III
004703-PA Rheb
004706-PA Sh3beta
004711-PA pontin
004739-PA CG9413
004740-PA DDB1
004776-PA Arginine methyltransferase 1
004782-PA Tat-binding protein -1
004785-PA CG10221
004819-PA CG2185
004822-PA eIF4AIII
004824-PA CG7834
004838-PA mitochondrial Ribosomal protein S5
004846-PA Rab-protein 8
004847-PA sec13
004850-PA CG5823
004852-PA sec23
004873-PA CG11337
004891-PA baiser
004892-PA Rox8
004898-PA Oligosaccharyl transferase 3
004904-PA CG5919
004905-PA CG11919
004908-PA Proteasome 26S subunit 4 ATPase
004909-PA Presenilin
004919-PA CG7789
004939-PA CG1646
004940-PA Septin interacting protein 3
004942-PA ATP synthase subunit d
004980-PA widerborst
004993-PA Signal recognition particle protein 54k
005058-PA Na pump alpha subunit
005062-PA Brf
005064-PA CG17446
005089-PA CG5045
005115-PA Rab-protein 1
005126-PA Tryptophanyl-tRNA synthetase
005151-PA Minichromosome maintenance 5
005161-PA CG1969
005186-PA CG11591
005270-PA Translocation protein 1
005306-PA CG17494
005315-PA Rpn1
005319-PA CG9099
005323-PA Aly
005328-PA CG5885
005347-PA ADP ribosylation factor 79F
005372-PA boca
005395-PA loquacious
005425-PA lesswright
005434-PA Replication protein A2
005439-PA CG9172
005466-PA alpha-Adaptin
005473-PA CG4225
005493-PA Enolase
005494-PA hypothetical protein KGM_20175
005523-PA Spt6
005535-PA zipper
005536-PA zipper
005550-PA Histone H3,3A
005559-PA ARP-like
005570-PA lethal (2) giant larvae
005576-PA Actin 5C
005592-PA eukaryotic translation initiation factor 3 subunit D
005656-PA CG6543
005701-PA singed
005793-PA Cytochrome c oxidase subunit Va
005794-PA COQ7
005812-PA Arginine kinase
005816-PA CG7791
005853-PA Myosin light chain cytoplasmic
005928-PA Ribosomal protein L35
005940-PA Glass bottom boat
005965-PA CG3107
005966-PA Zwischen ferment
005969-PA CG14939
005994-PA Mov34
005999-PA CG6621
006011-PA CG9140
006022-PA La auto antigen-like
006043-PA stathmin
006045-PA Rab-protein 5
006056-PA CG4038
006085-PA nop5
006134-PA lethal (2) k09022
006141-PA Eukaryotic translation initiation factor 3 subunit H
006144-PA CG4567
006158-PA lethal (2) 37Cc
006173-PA RNA polymerase II Elongation factor
006178-PA hook-like
217
APPENDIX
006185-PA fatty acid transport protein
006200-PA CG10470
006209-PA Sec61 beta
006221-PA Rab-protein 14
006243-PA CG6842
006245-PA CG2021
006246-PA Mdh
006257-PA Roughened
006263-PA Tcp1-like
006405-PA heterogeneous nuclear ribonucleo protein
006414-PA Ribosomal protein L15
006419-PA Malic enzyme
006424-PA CG5706
006462-PA eIF2B-alpha
006474-PA CG7770
006476-PA cleavage stimulation factor 64-kDa subunit
006507-PA Glutamate dehydrogenase
006539-PA Ance-3
006590-PA Syntaxin 1A
006603-PA Calcium ATPase at 60A
006620-PA CG8003
006704-PA Vasa intronic gene
006709-PA clot
006735-PA CG8549
006749-PA Elongation factor Tu mitochondrial
006751-PA ran
006771-PA CG17266
006777-PA cdc2-related-kinase
006779-PA CG10417
006789-PA CG9257
006809-PA Kinesin heavy chain
006816-PA rudimentary
006819-PA Rad23
006823-PA CG7433
006828-PA CG9987
006835-PA Ribosomal protein L23A
006837-PA Cytochrome P450 reductase
006840-PA Eukaryotic initiation factor 1A
006850-PA Calcineurin A at 14F
006867-PA Ribosomal protein S15Aa
006878-PA myospheroid
006897-PA CG6876
006907-PA Isocitrate dehydrogenase
006932-PA CG9132
006945-PA CG1354
006975-PA mitochondrial Ribosomal protein L12
006976-PA Starving
006986-PA Ribosomal protein L22
007025-PA CG7860
007035-PA hypothetical protein KGM_17753
007043-PA Spase12-subunit
007079-PA CG3011
007082-PA U2 small nuclear ribonucleo protein auxiliary factor 2
007092-PA Chd64
007098-PA NADP-dependent oxidoreductase
007103-PA CG4365
007110-PA Rac1
007114-PA eIF3-S9
007118-PA CG5590
007119-PA CG3590
007121-PA Aconitase
007131-PA CG6195
007141-PA tweety
007160-PA lethal (1) G0156
007169-PA Ras oncogene at 85D
007172-PA hypothetical protein
007174-PA CG8915
007194-PA CG8798
007210-PA CG4538
007224-PA CG11444
007230-PA Ef1 alpha-like factor
007248-PA CG7564
007258-PA CG7322
007266-PA CG5189
007267-PA Pdsw
007268-PA Rpn5
007311-PA stubarista
007315-PA ubiquitin-specific protease 7
007332-PA Rpt1
007334-PA Nedd8
007349-PA Heat shock protein 60
007360-PA 3-hydroxyacyl-CoA dehydrogenase
007363-PA Ribosomal protein S26
007389-PA olfactory receptor 35
007409-PA 3-hydroxyacyl-CoA dehydrogenase
007410-PA lark
007425-PA Phosphogluconate mutase
007436-PA burgundy
007438-PA Fumarylacetoacetase
007441-PA Inos
218
APPENDIX
007460-PA Microtubule star
007469-PA eIF-5A
007476-PA graves disease carrier protein homolog
007477-PA receptor for activated protein kinase C
007482-PA CG17593
007490-PA glyceraldehyde-3-phosphate dehydrogenase
007497-PA CG2118
007502-PA putative nadp transhydrogenase
007503-PA CG6643
007509-PA CG11964
007512-PA glycoprotein glucosyltransferase-like
007519-PA Hyper plastic discs
007522-PA COP9 complex homolog subunit 6
007523-PA Adenylate kinase-3
007577-PA PAK-kinase
007582-PA putative glutamate cysteine ligase isoform 1
007586-PA Isocitrate dehydrogenase
007594-PA belle
007637-PA Glycyl-tRNA synthetase
007639-PA Surfeit 4
007645-PA Ribosomal protein S23
007656-PA Rbp1-like
007661-PA Ribosomal protein L7A
007665-PA Elp3
007681-PA Phosphoglycerate kinase
007701-PA Abnormal wing discs
007702-PA Multiprotein bridging factor 1
007710-PA Ribosomal protein S5a
007712-PA Rab-protein 7
007719-PA Hostce ll factor
007728-PA cytosolic non-specific dipeptidase-like
007735-PA CG5789
007743-PA Ribosomal protein L8
007770-PA Sac1
007783-PA CG11963
007785-PA CG5789
007824-PA Sucb
007862-PA CG7375
007879-PA Ribosomal protein L5
007889-PA Trip1
007919-PA CG3603
007935-PA ade5
007948-PA Alpha Spectrin
007949-PA Porphobilinogen synthase
007950-PA Heat shock protein cognate 3
007954-PA CG4849
007988-PA Nucleosome remodeling factor-38kD
008020-PA icarus
008095-PA CG31548
008117-PA CG2093
008154-PA Opticatrophy 1-like
008214-PA CG4095
008226-PA eIF5
008228-PA CG11779
008238-PA Plexin A
008239-PA 1-acylglycerol-3-phosphate acyltransferase
008284-PA RNA-binding protein 9
008291-PA CG4447
008293-PA CG1637
008295-PA Vha68-2
008302-PA Ef1 gamma
008304-PA Akt1
008312-PA lethal (2) 03709
008342-PA CG7828
008364-PA CoRest
008392-PA GTP-binding protein
008415-PA CG5254
008425-PA Rpn2
008426-PA Rpn2
008433-PA GDP-mannose 4,6-dehydratase
008442-PA CG1516
008447-PA Small bristles
008498-PA lethal (1) G0155
008501-PA Flap wing
008511-PA putative semaphorin 2a
008542-PA Vha36
008551-PA CG2918
008554-PA hypothetical protein KGM_11293
008595-PA Rpn12
008620-PA Black pearl
008625-PA Minichromosome maintenance 2
008670-PA ATP synthase subunit b
008682-PA Extra bases
008706-PA flare
008714-PA mitochondrial Ribosomal protein L45
008723-PA Ras oncogene at 64B
008725-PA UGP
008726-PA Kinesin-like protein at 61F
008768-PA Ferritin 1 heavy chain homologue
008776-PA CG40084
219
APPENDIX
008846-PA CG32500
008865-PA Ribosomal protein L13A
008867-PA CG10641
008874-PA Fas-associated factor
008881-PA Ribosomal protein L11
008894-PA ADP ribosylation factor 102F
008899-PA CG13277
008904-PA Polypeptide GalNAc transferase 5
008914-PA CG6227
008921-PA Elongation factor 1 beta
008922-PA COP9 complex homolog subunit 4
008924-PA Target of rapamycin
008928-PA CG31688
008930-PA Arginyl-tRNA synthetase
008955-PA porin
008956-PA CG9642
008957-PA hypothetical protein DAPPUDRAFT_192333
008983-PA CG5261
008998-PA CG6984
009000-PA hypothetical protein KGM_04050
009010-PA Rpn11
009012-PA Cytochrome c proximal
009018-PA CG11208
009043-PA CG31674
009045-PA Chloride intracellular channel
009048-PA congested-like trachea
009057-PA CG1907
009101-PA CG13868
009103-PA sterol carrier protein x
009121-PA coracle
009130-PA Ribosomal protein S20
009131-PA beta-Tubulin at 56D
009132-PA beta-Tubulin at 56D
009138-PA Granny smith
009139-PA 26-29kD-proteinase
009208-PA Ribosomal protein L28
009218-PA lethal (2) k09913
009234-PA Proteasome 35kD subunit
009254-PA Stromal interaction molecule
009255-PA Stromal interaction molecule
009276-PA vermilion
009319-PA Ribosomal protein S3
009321-PA CG6796
009359-PA tamas
009375-PA Kruppel homolog 2
009380-PA Septin-2
009404-PA CG42347
009408-PA CG12267
009409-PA CG7185
009411-PA Ribosomal protein S16
009420-PA CG12140
009504-PA ubiquitin-fold modifier 1
009515-PA lethal (1) G0334
009554-PA CG5641
009562-PA Proteasome 25kD subunit
009563-PA CG40084
009578-PA CG11107
009647-PA His4:CG33907
009671-PA CG3663
009674-PA lin-19-like
009698-PA nascent polypeptide associated complex protein alpha subunit
009699-PA T-complex Chaperonin 5
009734-PA CG8814
009751-PA Ribosomal protein L23
009794-PA Aspartyl-tRNA synthetase
009795-PA Aspartyl-tRNA synthetase
009805-PA Brahma associated protein 55kD
009816-PA prp8
009821-PA Actin 88F
009830-PA eIF6
009841-PA CG6459
009851-PA Valyl-tRNA synthetase
009857-PA Myosin 61F
009863-PA Ribosomal protein L27
009881-PA CG32138
009885-PA CG9253
009888-PA Transformer 2
009895-PA 6-phosphofructo-2-kinase
009900-PA Annexin X
009911-PA Ubiquitin activating enzyme 1
009916-PA CG8372
009918-PA Ras-related protein
009933-PA CG31439
009941-PA CG12082
009945-PA c12,2
009963-PA uninitiated
009967-PA sec63
009974-PA Neural conserved at 73EF
009981-PA CG8963
009986-PA CG12567
220
APPENDIX
009992-PA Putative Achaete Scute Target 1
010000-PA putative secreted protein
010067-PA like-AP180
010068-PA Soluble NSF attachment protein
010088-PA CG6512
010090-PA heartless
010120-PA putative succinyl-CoA synthetase small subunit
010129-PA pixie
010130-PA Annexin IX
010139-PA Ribosomal protein S27
010154-PA Deoxyuridine triphosphatase
010178-PA B52
010193-PA short-chain dehydrogenease/reductase
010200-PA CG1291
010246-PA Vacuolar H[+]-ATPase 26kD E subunit
010247-PA Vacuolar H[+]-ATPase 26kD E subunit
010330-PA Multidrug-Resistance like Protein 1
010331-PA Multidrug-Resistance like Protein 1
010333-PA putative U2 snrnp auxiliary factor
010361-PA Glycogen phosphorylase
010376-PA lethal (3) 87Df
010381-PA SLY-1 homologous
010396-PA xl6
010423-PA CG5362
010446-PA CG8209
010449-PA CG11490
010471-PA Muscle-specific protein 300
010472-PA Muscle-specific protein 300
010475-PA Dynein heavy chain at 36C
010487-PA Ribosomal protein L36A
010523-PA putative n-acetylgalactosaminyl transferase
010527-PA CG6766
010538-PA Bicoid stability factor
010559-PA CG2107
010562-PA Connector of kinase to AP-1
010571-PA Ribosomal protein L9
010595-PA CG2947
010621-PA hypothetical protein KGM_04264
010635-PA Translocase of outermembrane 70
010644-PA Aubergine
010657-PA embargoed
010658-PA CG3702
010666-PA CG5525
010673-PA maternal expression at 31B
010723-PA snRNP2
010725-PA alpha-coatomer protein
010739-PA Nop56
010751-PA bicaudal
010790-PA Succinate dehydrogenase B
010794-PA Rpt4
010829-PA CG5508
010840-PA maleless
010851-PA CG9536
010867-PA Ribosomal protein S4
010906-PA mutagen-sensitive 209
010941-PA Srp72
010943-PA CG7891
010959-PA eclair
010970-PA Ribosomal protein L26
010978-PA goldengoal
010982-PA hypothetical protein KGM_17152
011029-PA CG10932
011087-PA CG9000
011117-PA CG8386
011131-PA CG1970
011168-PA Adenosyl homocysteinase at 13
011173-PA Infertile crescent
011180-PA gamma-coatomer protein
011186-PA Snx6
011190-PA MAP kinase activated protein-kinase-2
011218-PA CG10175
011237-PA Rpn9
011268-PA Coproporphyrinogen oxidase
011280-PA Rab39
011282-PA Ribosomal protein S13
011294-PA CG6812
011308-PA CG3902
011317-PA CG6359
011334-PA ciboulot
011344-PA cathD
011386-PA Lachesin
011387-PA Lachesin
011412-PA CG5028
011416-PA Ribosomal protein S3A
011426-PA CG4589
011446-PA Ribosomal protein S30
011467-PA Ribosomal protein L18A
011468-PA Juvenile hormone epoxide hydrolase 1
011477-PA Hexokinase A
011499-PA Suppressor of variegation 3-9
221
APPENDIX
011500-PA putative heterochromatin protein isoform 2
011507-PA CG8258
011508-PA CG8258
011521-PA Asparagine synthetase
011572-PA CG8165
011581-PA smt3
011599-PA lethal (1) G0320
011619-PA Trailer hitch
011620-PA Ribosomal protein L18
011631-PA CG32479
011641-PA CG3529
011650-PA Neosin
011674-PA Puromycin sensitive aminopeptidase
011695-PA Megator
011696-PA CG17233
011710-PA ubiquinol-cytochrome c reductase core protein II
011746-PA Rm62
011747-PA Rpd3
011754-PA CG10777
011779-PA CG10635
011803-PA Uev1A
011821-PA CG17081
011824-PA Ard1
011844-PA Protein disulfide isomerase
011862-PA Arginine methyltransferase 7
011870-PA CG5703
011922-PA FormAldehyde dehydrogenase
011936-PA ATP synthase subunit d
011948-PA Ribosomal protein S28b
011965-PA CG10077
011966-PA CG6550
011973-PA Aps
011983-PA CG1640
012029-PA CG8993
012032-PA CG11267
012038-PA CG4933
012046-PA lethal (1) G0431
012072-PA CG8743
012075-PA pumpless
012078-PA putative sorting nexin 14
012093-PA Mitochondrial carrier homolog 1
012110-PA CG10083
012116-PA Smt3 activating enzyme 2
012126-PA CG5642
012142-PA CG1597
012151-PA Actin-related protein 87C
012152-PA CG6084
012159-PA hulitaishao
012182-PA Tcp-1eta
012223-PA CG2658
012230-PA Not1
012282-PA Neurocalcin
012295-PA Cdc42
012298-PA Phosphogluconate dehydrogenase
012304-PA Peroxiredoxin 6005
012309-PA CG6287
012336-PA Ecdysone-inducible gene L3
012388-PA Zn72D
012406-PA Tinytim 50
012414-PA Ribosomal protein L17
012428-PA hnRNPA/B-like 28
012449-PA Groucho
012474-PA CG9922
012485-PA CG11899
012489-PA coro
012492-PA G protein alpha 49B
012507-PA CG12065
012541-PA DNAJ-like-2
012546-PA Ribosomal protein L24
012549-PA ATP synthase-beta
012550-PA CG13343
012560-PA Glycerol 3 phosphate dehydrogenase
012626-PA Ribosomal protein S9
012665-PA Cappin G protein alpha
012687-PA Sec61 alpha
012690-PA CG7338
012701-PA CG9674
012743-PA CG7955
012753-PA Glycoprotein 93
012772-PA Helicase at 25E
012851-PA eIF3-S8
012852-PA Ubiquitin conjugating enzyme 10
012859-PA Nipsnap
012860-PA HP1b
012870-PA CG9947
012904-PA Rab35
012905-PA Nup358
012922-PA Beta Adaptin
012931-PA windbeutel
012935-PA Clathrin heavy chain
222
APPENDIX
012960-PA vibrator
012961-PA spaghetti squash
012966-PA Septin-1
012977-PA CG41099
012983-PA Dead-box-1
012998-PA CG9518
013010-PA Protein phosphatase 19C
013021-PA Aldolase
013025-PA eIF3-S10
013063-PA CG7998
013085-PA CG4572
013087-PA CG11679
013096-PA SF2
013100-PA Small ribonucleo protein SmD3
013116-PA T-cp1zeta
013133-PA Asparaginyl-tRNA synthetase
013140-PA Arc42
013142-PA Oxysterol binding protein
013168-PA CG6782
013201-PA 14-3-3 epsilon
013220-PA 3-dehydroecdysone 3 alpha-reductase
013230-PA Protein phosphatase 1at 87B
013244-PA Phosphoglycero mutase
013317-PA putative carnitine o-acyltransferase
013327-PA Tudor-SN
013340-PA Dihydrofolate reductase
013348-PA Translocase of outermembrane 40
013369-PA Vinculin
013413-PA alpha-Tubulin at 84B
013447-PA rasputin
013449-PA Histone H2 Avariant
013464-PA squid
013473-PA glorund
013498-PA CG6370
013499-PA alien
013510-PA ribonucleoside diphosphate reductase
013536-PA CG5001
013537-PA CG1749
013545-PA lethal (2) essential for life
013548-PA guftagu
013567-PA Ribosomal protein L3
013587-PA CG5629
013590-PA Minichromosome maintenance 3
013610-PA CG13827
013631-PA CG5854
013653-PA CG3033
013713-PA CG8860
013717-PA beta-coatomer protein
013765-PA mitochondrial ribosomal protein S16
013792-PA Ribosomal protein S11
013801-PA shrub
013898-PA Proteasome alpha subunit
013916-PA CG17333
013963-PA Vacuolar H[+] ATPase 44kD C subunit
013964-PA Vacuolar H[+] ATPase 44kD C subunit
013965-PA ATP synthase-gamma chain
013996-PA exuperantia
013998-PA Hemeoxygenase
014035-PA CG42400
014065-PA Neuroglian
014083-PA CG9393
014094-PA Ribosomal protein L14
014136-PA Proteasome p44,5 subunit
014155-PA Quaking related 54B
014177-PA Pros45
014181-PA Thiolase
014211-PA CG8036
014217-PA CG7920
014218-PA CG7920
014339-PA CG2093
014340-PA CG11526
014417-PA CG7461
014442-PA CG2246
014453-PA aldo-keto reductase
014470-PA pawn
014483-PA CG12079
014548-PA CG5235
014607-PA Tim8
223
APPENDIX
Accession (BGIBMGA-)
Protein name
000029-PA NHP2
000064-PA Peroxiredoxin 5037
000066-PA Ribosomal protein L32
000074-PA Fibrillarin
000081-PA Chrac-14
000084-PA Hiiragi
000097-PA Calpain-A
000103-PA CG30122
000104-PA delta-coatomer protein
000114-PA CG5913
000120-PA CG14804
000129-PA twinstar
000132-PA CG14476
000201-PA Proteasome 26kD subunit
000204-PA Sin3A
000230-PA Phospholipase A2 activator protein
000235-PA PAPS synthetase
000292-PA CG7430
000295-PA Rab-protein 3
000312-PA SRm160
000321-PA CG6664
000381-PA nudC
000382-PA nudC
000390-PA Adenosine 2
000397-PA kermit
000415-PA claret
000447-PA comatose
000469-PA CG10103
000473-PA pacman
000475-PA Calreticulin
000508-PA putative sperm associated antigen 17
000516-PA IGF-II mRNA-binding protein
000526-PA Ypsilon schachtel
000528-PA hypothetical protein KGM_01293
000552-PA Failed axon connections
000559-PA Triosephosphate isomerase
000596-PA Vacuolar H[+]-ATPase SFD subunit
Accession (BGIBMGA-)
Protein name
000617-PA Syntaxin 7
000647-PA NUCB1
000672-PA knockdown
000680-PA Glutamine synthetase 2
000683-PA CG32528
000701-PA Catalase
000710-PA Vap-33-1
000715-PA CG12262
000743-PA flotillin
000791-PA Protein Kinase D
000803-PA Viral IAP-associated factor
000804-PA mitochondrial ATP synthase coupling factor 6
000806-PA CG32626
000809-PA CG10354
000828-PA Minichromosome maintenance 6
000829-PA CG13900
000865-PA CG12259
000867-PA String of pearls
000893-PA CG7766
000898-PA CG10754
000903-PA Transport and Golgi organization 7
000918-PA skpA
000926-PA Tal
000930-PA Imitation SWI
000931-PA Pop2
000937-PA Updo
000944-PA CG14207
000959-PA Ribosomal protein L31
000991-PA Histidyl-tRNA synthetase
001032-PA SH3PX1
001035-PA raspberry
001037-PA CG33123
001038-PA CG10191
001043-PA Ribosomal protein L27A
001044-PA Apoptosis-linked gene-2
001070-PA Arflike at 72A
001100-PA corkscrew
001106-PA Ribosomal protein L21
001107-PA super coiling factor
001118-PA CG3362
001119-PA GDP dissociation inhibitor
001123-PA Ranbp21
001124-PA Recombination repair protein 1
Appendix 2. List of proteins form soluble fractions of CF-203 cells identified after RSA affinity
chromatography and LC-MS/MS analysis (chapter 5).
224
APPENDIX
001126-PA Rab escort protein
001136-PA Spase 22/23-subunit
001159-PA Vps36
001160-PA CG9940
001165-PA Dynamin related protein 1
001168-PA CG18811
001175-PA Aac11
001206-PA CG7033
001209-PA COP9 complex homolog subunit 3
001218-PA Hsc70Cb
001223-PA Dead box protein 80
001236-PA CG14757
001238-PA Cysteinyl-tRNA synthetase
001241-PA Trap 1
001243-PA Ribosomal protein S10b
001249-PA CG8435
001265-PA eIF5B
001318-PA Calmodulin
001319-PA Calmodulin
001335-PA CG10463
001336-PA CG10077
001363-PA overgrown hematopoietic organs at 23B
001372-PA rhea
001385-PA CG7460
001386-PA CG6767
001395-PA Pros beta 3
001406-PA Cct gamma
001415-PA Ubiquitin-63E
001423-PA crinkled
001437-PA Eukaryotic initiation factor 2 beta
001458-PA Downstream of raf 1
001471-PA Ribosomal protein S15
001483-PA Ribosomal protein S17
001490-PA FK506-binding protein 1
001501-PA Ribosomal protein S6
001525-PA Tyrosyl-tRNA synthetase
001536-PA pyd3
001540-PA Upf1
001542-PA rumpelstiltskin
001546-PA Dynein heavy chain 64C
001547-PA Dynein heavy chain 64C
001549-PA Ubiquitin-63E
001550-PA Cyclin-dependent kinase 7
001551-PA Ubiquilin
001558-PA Isoleucyl-tRNA synthetase
001579-PA Cap binding protein 80
001581-PA MRG15
001584-PA Tropomyosin 1
001585-PA tropomyosin-2 isoform 3
001644-PA La related protein
001670-PA Specifically Rac1-associated protein 1
001683-PA beta-Tubulin at 56D
001707-PA beta-Tubulin at 56D
001725-PA Iron regulatory protein 1B
001726-PA Iron regulatory protein 1B
001799-PA abstrakt
001800-PA Ribosomal protein L4
001801-PA reptin
001806-PA CG3689
001811-PA CG5077
001825-PA CG17768
001849-PA Threonyl-tRNA synthetase
001853-PA bellwether
001870-PA Srp54
001873-PA vesicle amine transport protein
001874-PA CG3523
001876-PA Amylase proximal
001887-PA TXBP181-like
001915-PA Proteasome 29kD subunit
001921-PA Actin-related protein 66B
001926-PA Farnesyl pyrophosphate synthase
001928-PA Rab-protein 2
001943-PA CG1598
001950-PA Pabp2
001953-PA bancal
001954-PA Heterogeneous nuclear ribonucleo protein K
001966-PA Aldehyde dehydrogenase type III
001991-PA Ribosomal protein L36
002004-PA CG9281
002005-PA stress-sensitive B
002008-PA Myosin binding subunit
002012-PA moleskin
002013-PA moleskin
002103-PA alpha-Tubulin at 84B
002105-PA CG9135
002151-PA Cyclophilin-like
002186-PA thioredoxin peroxidase 1
002209-PA Rab-protein 11
002222-PA Glutathione S transferase D1
002235-PA CG5871
225
APPENDIX
002237-PA Protein phosphatase 2A at 29B
002241-PA Vacuolar H[+]-ATPase 55kD B subunit
002295-PA Growl
002330-PA CG1983
002346-PA C-terminal Binding protein
002353-PA CG18815
002361-PA CG3909
002381-PA Heat shock protein cognate 4
002394-PA CG13630
002405-PA Ribosomal protein S14b
002406-PA thioredoxin peroxidase 2
002423-PA CG1458
002428-PA HEM-protein
002429-PA Cyclophilin1
002457-PA Aldehyde dehydrogenase
002462-PA CG11089
002470-PA CG6259
002481-PA CG6905
002493-PA CG10576
002504-PA sar1
002508-PA Pyruvate kinase
002542-PA alpha-Tubulin at 84B
002543-PA Moesin
002544-PA Moesin
002559-PA bendless
002569-PA CaBP1
002570-PA CG33303
002572-PA Ribosomal protein L19
002592-PA CG5355
002593-PA CG5355
002594-PA Adenylate kinase-2
002605-PA CG10527
002640-PA Ubiquitin carboxy-terminal hydrolase
002644-PA 14-3-3 zeta
002649-PA CG5366
002651-PA Sialic acid phosphate synthase
002678-PA SHC-adaptor protein
002711-PA Lightoid
002750-PA CG11876
002759-PA CG5384
002763-PA Disc proliferation abnormal
002779-PA dre4
002790-PA COP9 complex homolog subunit 5
002811-PA Qm
002814-PA Oligosaccharyl transferase 48kD subunit
002818-PA Thioredoxin reductase-1
002837-PA GTP-binding-protein
002840-PA CG17514
002886-PA sniffer
002914-PA AP-1gamma
002931-PA Mi-2
002953-PA Diphenoloxidase A2
002967-PA CG4389
002970-PA CG9317
002972-PA Female sterile (2) Ketel
002981-PA Chickadee
002984-PA Lysyl-tRNA synthetase
003004-PA small nuclear ribonucleo protein polypeptide
003018-PA RNA polymerase II 140kD subunit
003028-PA Proteasome 28kD subunit 1
003038-PA Rho-kinase
003042-PA CG5168
003049-PA Glycerophosphateoxidase-1
003070-PA CG34132
003073-PA small nuclear ribonucleo protein polypeptide
003116-PA Glutamate oxaloacetate transaminase 2
003126-PA CG11092
003129-PA CG5958
003184-PA Mo25
003186-PA Eukaryotic initiation factor 4a
003197-PA Ribosomal protein S18
003212-PA Rpn7
003221-PA CG8801
003243-PA CG5323
003279-PA CG3107
003296-PA beta-Tubulin at 56D
003300-PA CG7427
003305-PA Transcription factor II F alpha
003309-PA Ribosomal protein LP0
003319-PA Glutamate oxaloacetate transaminase 1
003335-PA lethal (2) 05070
003337-PA Ribosomal protein L10Ab
003342-PA hypothetical protein KGM_10216
003351-PA Minichromosome maintenance 7
003361-PA Karyopherin beta 3
003397-PA Ribosomal protein S8
003402-PA CG6353
003403-PA E2F transcription factor
003412-PA Ribosomal protein LP1
003429-PA CG10333
226
APPENDIX
003442-PA beta-Tubulin at 56D
003462-PA Spf45
003465-PA TH1
003466-PA CG5676
003469-PA polyA-binding protein
003470-PA CG8920
003472-PA tango
003474-PA Casein kinase II alpha subunit
003475-PA eEF1delta
003480-PA CG7843
003484-PA RNA polymerase II 215kD subunit
003513-PA CG3714
003515-PA capulet
003519-PA CG5174
003539-PA lethal (2) 35Df
003587-PA ERp60
003594-PA Klp31E
003597-PA Rab-protein 6
003601-PA kurtz
003607-PA frayed
003608-PA Elongation factor 1 alpha 48D
003632-PA CG3402
003635-PA Transportin
003652-PA Lamin
003656-PA Eip55E
003669-PA Int6 homologue
003670-PA Argonaute-1
003671-PA CG13349
003700-PA Muscle protein 20
003702-PA CG10286
003717-PA N-myristoyl transferase
003725-PA N-myristoyl transferase
003726-PA Ribosomal protein L13
003744-PA peanut
003758-PA Karyopherin alpha 3
003759-PA Spt5
003805-PA CG10298
003806-PA Glued
003820-PA Rpb8
003822-PA CG10080
003829-PA stress-sensitive B
003833-PA Nucleolar protein at 60B
003892-PA beta'-coatomer protein
003901-PA ATP synthase-beta
003910-PA CG1486
003923-PA Ribosomal protein S24
003961-PA CG4673
003972-PA Selenide, water dikinase
003977-PA CG17259
003978-PA Ribosomal protein L12
003984-PA TER94
003985-PA TER94
003987-PA Dihydropteridine reductase
003997-PA Suppressor of variegation 2-10
004021-PA G protein o alpha 47A
004038-PA Eb1
004090-PA CG13185
004096-PA cAMP-dependent protein kinase R2
004103-PA lethal (2) essential for life
004109-PA Spenito
004112-PA CG10602
004136-PA Tumor suppressor protein 101
004165-PA Elongation factor 2b
004166-PA Dak1
004175-PA Adam
004177-PA CG1513
004178-PA CG1513
004221-PA Phosphoglucose isomerase
004241-PA CAS/CSE1segregation protein
004267-PA CG14480
004302-PA eIF-2 alpha
004309-PA Dek
004315-PA P-element somatic inhibitor
004331-PA FK506-binding protein 2
004337-PA CG8243
004356-PA Ribosomal protein S7
004374-PA Ribosomal protein S12
004375-PA Ribosomal protein L7
004376-PA Pros beta 5
004379-PA hypothetical protein KGM_02143
004409-PA Short stop
004447-PA CG4752
004482-PA CG8207
004501-PA Elongin C
004540-PA lethal (2) essential for life
004541-PA lethal (2) essential for life
004557-PA Purine-rich binding protein alpha
004573-PA Small ribonucleo protein G
004603-PA beta-Tubulin at 56D
004608-PA Fimbrin
227
APPENDIX
004612-PA Heat shock protein 83
004614-PA Heat-shock-protein-70Ba
004626-PA G protein beta-subunit 13F
004646-PA Bub3
004647-PA sec71
004655-PA p270
004657-PA Proteasome alpha 6 subunit
004661-PA CG18591
004666-PA Rab-protein 10
004674-PA CG8858
004687-PA auxillin
004688-PA Dipeptidyl aminopeptidase III
004691-PA Poly-(ADP-ribose) polymerase
004706-PA Sh3 beta
004711-PA pontin
004739-PA CG9413
004740-PA DDB1
004776-PA Arginine methyltransferase 1
004782-PA Tat-binding protein -1
004807-PA Protein phosphatase D3
004822-PA eIF4AIII
004823-PA CG3605
004824-PA CG7834
004852-PA sec23
004857-PA CG17273
004858-PA CG17273
004891-PA baiser
004905-PA CG11919
004908-PA Proteasome 26S subunit 4 ATPase
004919-PA CG7789
004939-PA CG1646
004963-PA CG31368
004971-PA sec10
004980-PA widerborst
004993-PA Signal recognition particle protein 54k
004994-PA RNA polymerase II 33kD subunit
005014-PA hypothetical protein KGM_14676
005022-PA falafel
005035-PA Neurochondrin
005062-PA Brf
005064-PA CG17446
005071-PA Bruce
005102-PA Kinesin-like protein at 3A
005115-PA Rab-protein 1
005116-PA Glutamyl-prolyl-tRNA synthetase
005117-PA putative aminoacyl-tRNA synthetase
005126-PA Tryptophanyl-tRNA synthetase
005151-PA Minichromosome maintenance 5
005181-PA Trehalose-6-phosphatesynthase 1
005182-PA Trehalose-6-phosphatesynthase 1
005186-PA CG11591
005249-PA Upstream of RpIII 128
005250-PA GTP-binding protein 128 up-like
005267-PA COP9 complex homolog subunit 7
005288-PA Structure specific recognition protein
005315-PA Rpn1
005319-PA CG9099
005323-PA Aly
005328-PA CG5885
005333-PA G protein-coupled receptor kinase 1
005339-PA hypothetical protein KGM_20357
005347-PA ADP ribosylation factor 79F
005372-PA boca
005395-PA loquacious
005425-PA lesswright
005428-PA hypothetical protein KGM_18067
005434-PA Replication protein A2
005448-PA Replication protein A70
005466-PA alpha-Adaptin
005488-PA replication factor C
005493-PA Enolase
005494-PA hypothetical protein KGM_20175
005497-PA RhoGAP1A
005522-PA Spt6
005523-PA Spt6
005535-PA zipper
005536-PA zipper
005550-PA Histone H3,3A
005559-PA ARP-like
005560-PA Proteasome 54kD subunit
005564-PA CG32409
005570-PA lethal (2) giant larvae
005576-PA Actin5C
005587-PA CG10254
005588-PA hypothetical protein KGM_13479
005592-PA eukaryotic translation initiation factor 3 subunit D
005593-PA Srp68
005641-PA licorne
005656-PA CG6543
005701-PA singed
228
APPENDIX
005758-PA cullin-4
005777-PA Klp10A
005812-PA Arginine kinase
005853-PA Myosin light chain cytoplasmic
005930-PA CG17293
005965-PA CG3107
005966-PA Zwischen ferment
005969-PA CG14939
005994-PA Mov34
005999-PA CG6621
006014-PA Transcription factor II B
006022-PA La auto antigen-like
006043-PA stathmin
006045-PA Rab-protein 5
006056-PA CG4038
006085-PA nop5
006109-PA HP1b
006121-PA Dlic2
006141-PA Eukaryotic translation initiation factor 3 subunit H
006158-PA lethal (2) 37Cc
006173-PA RNA polymerase II Elongation factor
006178-PA hook-like
006208-PA CG11266
006218-PA Kinesin associated protein 3
006221-PA Rab-protein 14
006243-PA CG6842
006245-PA CG2021
006246-PA Mdh
006257-PA Roughened
006263-PA Tcp1-like
006328-PA putative Ankyrin repeat domain-containing protein 17
006357-PA pitchoune
006358-PA noisette
006376-PA CG16817
006401-PA CG6523
006405-PA heterogeneous nuclear ribonucleo protein
006414-PA Ribosomal protein L15
006419-PA Malic enzyme
006424-PA CG5706
006426-PA Modifier of mdg4
006462-PA eIF2B-alpha
006463-PA Eukaryotic initiation factor 4B
006474-PA CG7770
006476-PA cleavage stimulation factor 64-kDa subunit
006507-PA Glutamate dehydrogenase
006537-PA CG17639
006567-PA CG2258
006590-PA Syntaxin 1A
006603-PA Calcium ATPase at 60A
006613-PA tenectin
006620-PA CG8003
006683-PA CG9109
006704-PA Vasa intronic gene
006705-PA CG5290
006709-PA Clot
006724-PA rolled
006725-PA rolled
006728-PA Bre1
006735-PA CG8549
006749-PA Elongation factor Tu mitochondrial
006751-PA Ran
006752-PA Rpb11
006770-PA Ect4
006771-PA CG17266
006772-PA CG8545
006777-PA cdc2-related-kinase
006779-PA CG10417
006780-PA CG13531
006783-PA CG4045
006784-PA putative rotatin
006789-PA CG9257
006807-PA RPS6-p70-protein kinase
006809-PA Kinesin heavy chain
006816-PA rudimentary
006818-PA Enhancer of bithorax
006819-PA Rad23
006823-PA CG7433
006828-PA CG9987
006830-PA Nipped-B
006835-PA Ribosomal protein L23A
006840-PA Eukaryotic initiation factor 1A
006850-PA Calcineurin A at 14F
006851-PA CG2807
006867-PA Ribosomal protein S15Aa
006897-PA CG6876
006907-PA Isocitrate dehydrogenase
006939-PA DNA-polymerase-delta
006945-PA CG1354
006964-PA CG9330
006986-PA Ribosomal protein L22
229
APPENDIX
007000-PA CG8223
007012-PA CG31145
007035-PA hypothetical protein KGM_17753
007043-PA Spase12-subunit
007079-PA CG3011
007082-PA U2 small nuclear ribonucleo protein auxiliary factor 2
007092-PA Chd64
007114-PA eIF3-S9
007118-PA CG5590
007119-PA CG3590
007121-PA Aconitase
007131-PA CG6195
007169-PA Ras oncogene at 85D
007171-PA CG6388
007194-PA CG8798
007208-PA futsch
007224-PA CG11444
007230-PA Ef1 alpha-like factor
007258-PA CG7322
007266-PA CG5189
007268-PA Rpn5
007310-PA Downstream of receptor kinase
007311-PA stubarista
007315-PA ubiquitin-specific protease 7
007318-PA CG2446
007332-PA Rpt1
007334-PA Nedd8
007349-PA Heat shock protein 60
007360-PA 3-hydroxyacyl-CoA dehydrogenase
007363-PA Ribosomal protein S26
007389-PA olfactory receptor 35
007409-PA 3-hydroxyacyl-CoA dehydrogenase
007410-PA lark
007418-PA CG11255
007425-PA Phosphogluconate mutase
007436-PA burgundy
007441-PA Inos
007460-PA Microtubule star
007468-PA Cutlet
007469-PA eIF-5A
007477-PA receptor for activated protein kinase C
007490-PA glyceraldehyde-3-phosphate dehydrogenase
007507-PA fatfacets
007509-PA CG11964
007512-PA glycoprotein glucosyltransferase-like
007518-PA TBPH
007519-PA Hyper plastic discs
007522-PA COP9 complex homolog subunit 6
007543-PA CG11858
007577-PA PAK-kinase
007582-PA putative glutamate cysteine ligase isoform 1
007586-PA Isocitrate dehydrogenase
007594-PA belle
007637-PA Glycyl-tRNA synthetase
007645-PA Ribosomal protein S23
007656-PA Rbp1-like
007661-PA Ribosomal protein L7A
007665-PA Elp3
007681-PA Phosphoglycerate kinase
007694-PA Mediator complex subunit 22
007695-PA brahma
007701-PA Abnormal wing discs
007702-PA Multiprotein bridging factor 1
007710-PA Ribosomal protein S5a
007712-PA Rab-protein 7
007719-PA Host cell factor
007728-PA cytosolic non-specific dipeptidase-like
007730-PA misshapen
007734-PA byS6
007743-PA Ribosomal protein L8
007783-PA CG11963
007821-PA hyrax
007822-PA heterogeneous nuclear ribonucleo protein A1
007824-PA Sucb
007862-PA CG7375
007869-PA CG1703
007879-PA Ribosomal protein L5
007889-PA Trip1
007895-PA Will die slowly
007906-PA Purity of essence
007935-PA ade5
007942-PA Casein kinase II beta subunit
007946-PA Extra denticle
007948-PA Alpha Spectrin
007949-PA Porphobilinogen synthase
007950-PA Heat shock protein cognate 3
007954-PA CG4849
007971-PA CG7359
007988-PA Nucleosome remodelling factor-38kD
008020-PA icarus
230
APPENDIX
008051-PA jaguar
008088-PA snRNP-U1
008095-PA CG31548
008111-PA CG10375
008116-PA CG6854
008117-PA CG2093
008214-PA CG4095
008226-PA eIF5
008228-PA CG11779
008284-PA RNA-binding protein 9
008291-PA CG4447
008294-PA Dead-box-1
008295-PA Vha68-2
008302-PA Ef1gamma
008312-PA lethal (2) 03709
008314-PA XNP
008364-PA CoRest
008392-PA GTP-binding protein
008399-PA CG2097
008408-PA Kinesin light chain
008425-PA Rpn2
008426-PA Rpn2
008433-PA GDP-mannose 4,6-dehydratase
008442-PA CG1516
008447-PA small bristles
008498-PA lethal (1) G0155
008501-PA Flap wing
008532-PA Separation anxiety
008542-PA Vha36
008544-PA CG12214
008545-PA putative tubulin-specific chaperone e
008554-PA hypothetical protein KGM_11293
008563-PA Calcineurin B
008595-PA Rpn12
008596-PA Small minded
008625-PA Minichromosome maintenance 2
008633-PA CG5068
008682-PA Extra bases
008706-PA Flare
008723-PA Ras oncogene at 64B
008725-PA UGP
008726-PA Kinesin-like protein at 61F
008779-PA CG5931
008864-PA CG31715
008865-PA Ribosomal protein L13A
008867-PA CG10641
008881-PA Ribosomal protein L11
008894-PA ADP ribosylation factor 102F
008899-PA CG13277
008911-PA CG6841
008914-PA CG6227
008921-PA Elongation factor 1 beta
008922-PA COP9 complex homolog subunit 4
008924-PA Target of rapamycin
008927-PA Arc-p34
008928-PA CG31688
008930-PA Arginyl-tRNA synthetase
008955-PA porin
008966-PA CG31184
008983-PA CG5261
008998-PA CG6984
009000-PA hypothetical protein KGM_04050
009010-PA Rpn11
009012-PA Cytochrome c proximal
009021-PA CG3226
009037-PA Argonaute 3
009043-PA CG31674
009045-PA Chloride intracellular channel
009118-PA vihar
009121-PA coracle
009130-PA Ribosomal protein S20
009131-PA beta-Tubulin at 56D
009132-PA beta-Tubulin at 56D
009133-PA beta-Tubulin at 60D
009138-PA granny smith
009207-PA lethal (1) G0020
009208-PA Ribosomal protein L28
009218-PA lethal (2) k09913
009228-PA dalao
009234-PA Proteasome 35kD subunit
009240-PA Nat1
009248-PA CG16721
009276-PA vermilion
009319-PA Ribosomal protein S3
009355-PA CG4646
009359-PA tamas
009372-PA Pcf11
009380-PA Septin-2
009409-PA CG7185
009411-PA Ribosomal protein S16
231
APPENDIX
009419-PA CG5941
009430-PA Capping protein beta
009463-PA hypothetical protein KGM_06507
009495-PA Replication-factor-C40kD subunit
009515-PA lethal (1) G0334
009554-PA CG5641
009562-PA Proteasome 25kD subunit
009563-PA CG40084
009565-PA hypothetical protein KGM_21100
009578-PA CG11107
009595-PA CstF-50
009618-PA Roe1
009647-PA His4: CG33907
009668-PA CG7946
009674-PA lin-19-like
009698-PA nascent polypeptide associated complex protein alpha subunit
009699-PA T-complex Chaperonin 5
009726-PA translin
009740-PA CG10907
009751-PA Ribosomal protein L23
009778-PA Lasp
009791-PA Start 1
009794-PA Aspartyl-tRNA synthetase
009795-PA Aspartyl-tRNA synthetase
009805-PA Brahma associated protein 55kD
009816-PA prp8
009821-PA Actin 88F
009830-PA eIF6
009835-PA SMC1
009841-PA CG6459
009851-PA Valyl-tRNA synthetase
009854-PA lethal (2) NC136
009857-PA Myosin 61F
009863-PA Ribosomal protein L27
009885-PA CG9253
009895-PA 6-phosphofructo-2-kinase
009899-PA CG8841
009900-PA Annexin X
009903-PA CG6418
009911-PA Ubiquitin activating enzyme 1
009916-PA CG8372
009918-PA Ras-related protein
009933-PA CG31439
009941-PA CG12082
009963-PA uninitiated
009967-PA sec63
009974-PA Neural conserved at 73EF
009983-PA CG11583
009986-PA CG12567
009992-PA Putative Achaete Scute Target 1
009999-PA Arp11
010000-PA putative secreted protein
010068-PA Soluble NSF attachment protein
010072-PA Cortactin
010088-PA CG6512
010120-PA putative succinyl-CoA synthetase small subunit
010129-PA pixie
010130-PA Annexin IX
010139-PA Ribosomal protein S27
010147-PA CG11984
010154-PA Deoxyuridine triphosphatase
010171-PA CG5205
010178-PA B52
010180-PA without children
010193-PA short-chain dehydrogenease/reductase
010197-PA CG5934
010200-PA CG1291
010209-PA CG14213
010220-PA CG7379
010222-PA Ubiquitin conjugating enzyme
010245-PA Cdc37
010246-PA Vacuolar H[+]-ATPase 26kD E subunit
010247-PA Vacuolar H[+]-ATPase 26kD E subunit
010249-PA moira
010260-PA CG1129
010333-PA putative U2 snrnp auxiliary factor
010361-PA Glycogen phosphorylase
010381-PA SLY-1 homologous
010396-PA xl6
010403-PA CG31075
010423-PA CG5362
010446-PA CG8209
010471-PA Muscle-specific protein 300
010475-PA Dynein heavy chain at 36C
010487-PA Ribosomal protein L36A
010525-PA CG1620
010538-PA Bicoid stability factor
010541-PA Effete
010571-PA Ribosomal protein L9
010572-PA calcium/calmodulin-dependent protein kinase type 1
232
APPENDIX
010595-PA CG2947
010621-PA hypothetical protein KGM_04264
010626-PA sec6
010644-PA Aubergine
010655-PA Menin 1
010657-PA Embargoed
010666-PA CG5525
010673-PA maternal expression at 31B
010723-PA snRNP2
010725-PA alpha-coatomer protein
010739-PA Nop56
010744-PA Protein on ecdysone puffs
010751-PA Bicaudal
010790-PA Succinate dehydrogenase B
010792-PA lethal (3) 07882
010794-PA Rpt4
010831-PA twins
010840-PA maleless
010867-PA Ribosomal protein S4
010871-PA CG32242
010906-PA mutagen-sensitive 209
010941-PA Srp72
010970-PA Ribosomal protein L26
010978-PA Golden goal
010991-PA CG4266
011029-PA CG10932
011033-PA O-glycosyl transferase
011039-PA Chromosome-associated protein
011082-PA Toll-6
011117-PA CG8386
011133-PA Suppressor of forked
011168-PA Adenosylhomocysteinase at 13
011180-PA gamma-coatomer protein
011186-PA Snx6
011190-PA MAP kinase activated protein-kinase-2
011218-PA CG10175
011222-PA CG11198
011223-PA acetyl-CoA carboxylase-like
011246-PA Enhancer of rudimentary
011272-PA Intronic Protein 259
011281-PA CG10153
011282-PA Ribosomal protein S13
011297-PA CG1416
011308-PA CG3902
011317-PA CG6359
011334-PA ciboulot
011344-PA cathD
011360-PA CG6904
011416-PA Ribosomal protein S3A
011426-PA CG4589
011429-PA cAMP-dependent protein kinase 1
011438-PA PHGPx
011446-PA Ribosomal protein S30
011461-PA Replication factor C 38kD subunit
011467-PA Ribosomal protein L18A
011468-PA Juvenile hormone epoxidehydrolase 1
011477-PA Hexokinase A
011479-PA CG32549
011490-PA Spell checker 1
011499-PA Suppressor of variegation 3-9
011500-PA putative heterochromatin protein isoform 2
011507-PA CG8258
011508-PA CG8258
011511-PA cullin-5
011521-PA Asparagine synthetase
011572-PA CG8165
011581-PA smt3
011615-PA Topo isomerase 2
011619-PA Trailer hitch
011620-PA Ribosomal protein L18
011631-PA CG32479
011641-PA CG3529
011650-PA Neosin
011659-PA CG11489
011674-PA Puromycin sensitive aminopeptidase
011695-PA Megator
011696-PA CG17233
011747-PA Rpd3
011754-PA CG10777
011770-PA COP9 complex homolog subunit 1b
011779-PA CG10635
011803-PA Uev1A
011824-PA Ard1
011841-PA putative ubiquitin-binding protein
011844-PA Protein disulphide isomerase
011862-PA Arginine methyltransferase 7
011922-PA Formaldehyde dehydrogenase
011948-PA Ribosomal protein S28b
011965-PA CG10077
011973-PA Aps
233
APPENDIX
011983-PA CG1640
012029-PA CG8993
012032-PA CG11267
012034-PA CG2685
012038-PA CG4933
012045-PA Developmental embryonic B
012046-PA lethal (1) G0431
012075-PA pumpless
012110-PA CG10083
012116-PA Smt3 activating enzyme 2
012118-PA sansfille
012119-PA Integrin linked kinase
012126-PA CG5642
012151-PA Actin-related protein 87C
012152-PA CG6084
012159-PA hulitaishao
012163-PA CG7757
012171-PA karst
012182-PA Tcp-1eta
012230-PA Not1
012282-PA Neurocalcin
012295-PA Cdc42
012298-PA Phosphogluconate dehydrogenase
012304-PA Peroxiredoxin 6005
012323-PA CG4420
012336-PA Ecdysone-inducible gene L3
012388-PA Zn72D
012414-PA Ribosomal protein L17
012428-PA hnRNPA/B-like 28
012449-PA groucho
012470-PA putative Ccar1 protein
012474-PA CG9922
012485-PA CG11899
012489-PA coro
012492-PA G protein alpha 49B
012507-PA CG12065
012515-PA Minute (2) 21AB
012541-PA DNAJ-like-2
012546-PA Ribosomal protein L24
012549-PA ATP synthase-beta
012550-PA CG13343
012560-PA Glycerol 3 phosphate dehydrogenase
012577-PA CG9153
012626-PA Ribosomal protein S9
012665-PA cappinG protein alpha
012674-PA Eukaryotic initiation factor 4E
012687-PA Sec61 alpha
012701-PA CG9674
012753-PA Glycoprotein 93
012762-PA Cytoplasmic dynein light chain 2
012772-PA Helicase at 25E
012774-PA CG15027
012827-PA Aldolase
012851-PA eIF3-S8
012852-PA Ubiquitin conjugating enzyme 10
012859-PA Nipsnap
012860-PA HP1b
012893-PA CG3911
012904-PA Rab35
012905-PA Nup358
012922-PA Beta Adaptin
012931-PA wind beutel
012934-PA Arc-p20
012935-PA Clathrin heavy chain
012960-PA vibrator
012961-PA Spaghetti squash
012966-PA Septin-1
012977-PA CG41099
012983-PA Dead-box-1
012995-PA CG6428
012998-PA CG9518
013010-PA Protein phosphatase 19C
013021-PA Aldolase
013023-PA CG1440
013025-PA eIF3-S10
013061-PA CG31120
013063-PA CG7998
013079-PA strawberry notch
013085-PA CG4572
013096-PA SF2
013100-PA Small ribonucleo protein SmD3
013112-PA CG40045
013116-PA T-cp1zeta
013117-PA hypothetical protein KGM_14452
013133-PA Asparaginyl-tRNA synthetase
013134-PA cdc2
013140-PA Arc42
013142-PA Oxysterol binding protein
013148-PA CG11652
013171-PA Phosphorylase kinase gamma
234
APPENDIX
013190-PA CG4764
013201-PA 14-3-3 epsilon
013230-PA Protein phosphatase 1 at 87B
013244-PA Phosphoglycero mutase
013255-PA Snf5-related 1
013327-PA Tudor-SN
013340-PA Dihydrofolate reductase
013348-PA Translocase of outermembrane 40
013365-PA Ubc-E2H
013369-PA Vinculin
013413-PA alpha-Tubulin at 84B
013423-PA RhoGAPp190
013447-PA Rasputin
013449-PA Histone H2 Avariant
013450-PA Splicing factor 1
013463-PA Mobas tumor suppressor
013464-PA Squid
013473-PA Glorund
013478-PA Hippo
013499-PA Alien
013500-PA gamma-Tubulin at 23C
013508-PA Endosulfine
013510-PA ribonucleoside diphosphate reductase
013536-PA CG5001
013543-PA CG5343
013548-PA Guftagu
013567-PA Ribosomal protein L3
013587-PA CG5629
013590-PA Minichromosome maintenance 3
013594-PA CG16941
013631-PA CG5854
013678-PA Vacuolar H[+] ATPase G-subunit
013704-PA CG9776
013717-PA beta-coatomer protein
013792-PA Ribosomal protein S11
013801-PA shrub
013898-PA Proteasome alpha subunit
013916-PA CG17333
013963-PA Vacuolar H[+] ATPase 44kD C subunit
013964-PA Vacuolar H[+] ATPase 44kD C subunit
013976-PA homer
013996-PA exuperantia
014035-PA CG42400
014063-PA CG32495
014074-PA CG9667
014087-PA CG7217
014094-PA Ribosomal protein L14
014118-PA Adenosine 3
014121-PA Decapping protein 1
014136-PA Proteasome p44,5 subunit
014155-PA Quaking related 54B
014177-PA Pros45
014181-PA Thiolase
014194-PA CG5625
014211-PA CG8036
014217-PA CG7920
014294-PA ATP citrate lyase
014335-PA CG2246
014339-PA CG2093
014398-PA Eukaryotic release factor 1
014423-PA Mnf
014442-PA CG2246
014446-PA ATP citrate lyase
014453-PA aldo-keto reductase
014474-PA CG17904
014483-PA CG12079
014499-PA His2A: CG33859
014548-PA CG5235
014558-PA CG30291
014563-PA CG2982
014579-PA His2A: CG33823
014605-PA CG31249
014607-PA Tim8
235
APPENDIX
Appendix 3. List of proteins form membrane fractions of S2 cells identified after RSA
affinity chromatography and LC-MS/MS analysis (chapter 6).
Accession Protein name
A1Z7G7 Latrophilin Cirl
A1ZA92 Polynucleotide 5'-hydroxyl-kinase NOL9
B3LVG7 Serine protease HTRA2, mitochondrial
B3LVQ1 Cell cycle regulator Mat89Bb
B3M7W0 Eukaryotic translation initiation factor 3 subunit L
B3MC02 Ubiquitin-fold modifier-conjugating enzyme 1
B3NLK7 Ribosome biogenesis protein BOP1 homolog
B3NRC6 Eukaryotic translation initiation factor 3 subunit M
B3NXQ7 tRNA (guanine-N(7)-)-methyltransferase subunit wuho
B3NY03 Neuropathy target esterase sws
B3P045 DDRGK domain-containing protein 1
B3P239 Eukaryotic translation initiation factor 3 subunit F-1
B3P7K6 Zinc finger protein-like 1 homolog
B3P8M3 Ribosome-releasing factor 2, mitochondrial
B4GDM7 Probable cytolic iron-sulfur protein assembly protein Ciao1
B4I100 Lysine-specific demethylase NO66
B4I7U3 Eukaryotic translation initiation factor 3 subunit K
B4I9J6 Serine/threonine-protein phosphatase Pgam5, mitochondrial
B4IL64 Neuropathy target esterase sws
O01367 Protein held out wings
O16068 Heat shock protein 83 (Fragment)
O44386 Integrin alpha-PS3
O46106 Splicing factor 3A subunit 3
O61491 Flotillin-1
O61492 Flotillin-2
O76511 Thymidylate synthase
O77237 Protein pellino
O77286 Protein anon-73B1
O77459 Transcription factor Ken
O96539 Arginyl-tRNA--protein transferase 1
P02828 Heat shock protein 83
P04282 Retrovirus-related Gag polyprotein from copia-like transposable element 17.6
P08181 Casein kinase II subunit alpha
P08970 Protein suppressor of hairy wing
P10040 Protein crumbs
P11146 Heat shock 70 kDa protein cognate 2
P12982 Serine/threonine-protein phosphatase alpha-2 isoform
P17917 Proliferating cell nuclear antigen
P20241 Neuroglian
P20354 Guanine nucleotide-binding protein G(s) subunit alpha
236
APPENDIX
P23257 Tubulin gamma-1 chain
P23380 V-type proton ATPase 16 kDa proteolipid subunit
P23696 Serine/threonine-protein phosphatase PP2A
P25007 Peptidyl-prolyl cis-trans isomerase
P25160 ADP-ribosylation factor-like protein 1
P25171 Regulator of chromosome condensation
P26019 DNA polymerase alpha catalytic subunit
P28750 Maternal protein exuperantia
P32234 GTP-binding protein 128up
P32392 Actin-related protein 3
P35128 Ubiquitin-conjugating enzyme E2 N
P36975 Synaptosomal-Associated Protein 25
P40304 Proteasome subunit beta type-1
P40320 S-adenylmethionine synthase
P40417 Mitogen-activated protein kinase ERK-A
P42787 Carboxypeptidase D
P48461 Serine/threonine-protein phosphatase alpha-1 isoform
P48462 Serine/threonine-protein phosphatase beta isoform
P49071 MAP kinase-activated protein kinase 2
P49415 Syndecan
P49735 DNA replication licensing factor Mcm2
P49963 Signal recognition particle 19 kDa protein
P50245 Putative adenylhomocysteinase 2
P51592 E3 ubiquitin-protein ligase hyd
P54362 AP-3 complex subunit delta
P56079 Phphatidate cytidylyltransferase, photoreceptor-specific
P91660 Probable multidrug resistance-associated protein lethal (2) 03659
Q01989 Myin heavy chain 95F
Q23979 Myin-IB
Q24169 Origin recognition complex subunit 5
Q24192 Ras-like GTP-binding protein RhoL
Q24323 Semaphorin-2A
Q24331 Protein tumorous imaginal discs, mitochondrial
Q24338 Polycomb protein esc
Q24547 Syntaxin-1A
Q26454 DNA replication licensing factor MCM4
Q27294 RNA-binding protein cabeza
Q27333 Lethal (2) neighbour of tid protein
Q27591 Integrin beta-nu
Q27869 Protein-L-isoaspartate(D-aspartate) O-methyltransferase
Q28ZX3 Polyadenylate-binding protein 2
Q293C2 Dymeclin
Q295Y7 Manne-1-phosphate guanyltransferase beta
Q297K8 Protein jagunal
Q29BL9 LMBR1 domain-containing protein 2 homolog
Q29IM3 60S ribosomal protein L17
237
APPENDIX
Q29L43 Protein MON2 homolog
Q29PG4 39S ribosomal protein L51, mitochondrial
Q2M146 Protein ST7 homolog
Q6WV17 Polypeptide N-acetylgalactaminyltransferase 5
Q6XJ13 60S ribosomal protein L17
Q7JW12 Thioredoxin-related transmembrane protein 2 homolog
Q7JZM8 39S ribosomal protein L41, mitochondrial
Q7K0L4 WD repeat-containing protein 26 homolog
Q7K2B0 Ribosomal RNA-processing protein 8
Q7KN79 Protein LTV1 homolog
Q7KNF2 Polyadenylate-binding protein 2
Q7KQM6 PERQ amino acid-rich with GYF domain-containing protein CG11148
Q7KRI2 Longitudinals lacking protein-like
Q7KRW1 Protein TRC8 homolog
Q7PLI2 Nipped-B protein
Q7YU24 Transmembrane GTPase Marf
Q86B79 RING finger protein unkempt
Q86BN8 Protein-tyrine phosphatase mitochondrial 1-like protein
Q8I8V0 Transcriptional adapter 2B
Q8IH00 Nucleolar protein 6
Q8INQ7 Protein KRTCAP2 homolog
Q8MLV1 Lamin-B receptor
Q8MLZ7 Chitinase-like protein Idgf3
Q8MSU3 Putative ferric-chelate reductase 1 homolog
Q8MV48 N-acetylgalactaminyltransferase 7
Q8SWR8 Ataxin-2 homolog
Q8SYG2 COP9 signalome complex subunit 3
Q8SZ63 Golgin-84
Q8T4E1 Putative GPI-anchor transamidase
Q94524 Dynein light chain Tctex-type
Q94981 Protein ariadne-1
Q95SP2 E3 ubiquitin-protein ligase HRD1
Q95SS8 Transmembrane protein 70 homolog, mitochondrial
Q95TU8 Netrin receptor unc-5
Q9GQQ0 Protein spinster
Q9NEF6 Probable V-type proton ATPase subunit D 2
Q9NHD5 Probable N-acetyltransferase san
Q9NIV1 Eukaryotic translation initiation factor 2-alpha kinase
Q9U4L6 Mitochondrial import receptor subunit TOM40 homolog 1
Q9V359 Vacuolar protein sorting-associated protein 28 homolog
Q9V3G7 26S proteasome non-ATPase regulatory subunit 6
Q9V3J8 Protein will die slowly
Q9V3R8 UbiA prenyltransferase domain-containing protein 1 homolog
Q9V407 Axin
Q9V4A7 Plexin-B
Q9V4P1 Actin-binding protein anillin
238
APPENDIX
Q9V4S8 COP9 signalosome complex subunit 7
Q9V564 Conserved oligomeric Golgi complex subunit 6
Q9V6U9 Probable trans-2-enoyl-CoA reductase, mitochondrial
Q9V6Y3 Probable 28S ribosomal protein S16, mitochondrial
Q9V7D2 V-type proton ATPase subunit D 1
Q9V7H4 Transmembrane protein 131 homolog
Q9V9Z1 39S ribosomal protein L32, mitochondrial
Q9VAD6 Conserved oligomeric Golgi complex subunit 7
Q9VAF0 Uncharacterized protein CG7816
Q9VAI1 Probable complex I intermediate-associated protein 30, mitochondrial
Q9VAL0 Signal peptidase complex subunit 1
Q9VB14 40S ribosomal protein S10a
Q9VBG6 UPF0570 protein CG6073
Q9VC45 Protein abnormal spindle
Q9VC57 Atlastin
Q9VCA8 Ankyrin repeat and KH domain-containing protein mask
Q9VCR7 Cystinin homolog
Q9VCX3 Probable 39S ribosomal protein L45, mitochondrial
Q9VDD1 DDRGK domain-containing protein 1
Q9VE50 Probable 28 kDa Golgi SNARE protein
Q9VF87 Cytoplasmic FMR1-interacting protein
Q9VF89 39S ribosomal protein L9, mitochondrial
Q9VFB2 28S ribosomal protein S10, mitochondrial
Q9VFL5 Methionyl-tRNA synthetase, mitochondrial
Q9VGC3 Conserved oligomeric Golgi complex subunit 1
Q9VH38 Dimethyladenine transferase 2, mitochondrial
Q9VHB6 Metaxin-1 homolog
Q9VI55 E3 UFM1-protein ligase 1 homolog
Q9VIF0 Nucleolar complex protein 2 homolog
Q9VIL0 NIK- and IKBKB-binding protein homolog
Q9VIU7 Probable dolichol-phosphate mannyltransferase
Q9VJ87 Pre-mRNA-splicing factor CWC22 homolog
Q9VKH0 Conserved oligomeric Golgi complex subunit 8
Q9VL00 Ubiquitin thioesterase otubain-like
Q9VLM5 Dolichyl-diphosphooligaccharide--protein glycyltransferase subunit DAD1
Q9VLU0 Barrier-to-autointegration factor
Q9VMX0 39S ribosomal protein L28, mitochondrial
Q9VNH1 Probable methyltransferase CG1239
Q9VNH6 Exocyst complex component 4
Q9VNI3 UPF0609 protein CG1218
Q9VPH2 DNA primase large subunit
Q9VQ57 Derlin-1
Q9VQH2 Dual oxidase
Q9VQZ6 Probable elongator complex protein 3
Q9VRH6 Translation factor waclaw, mitochondrial
Q9VRJ2 FIT family protein CG10671
239
APPENDIX
Q9VRJ8 UPF0139 membrane protein CG10674
Q9VRL2 Probable Golgi SNAP receptor complex member 2
Q9VS46 RINT1-like protein
Q9VS60 Sphingomyelin synthase-related 1
Q9VSD7 Lariat debranching enzyme
Q9VSF3 NEDD8-conjugating enzyme Ubc12
Q9VSU7 Vesicle transport protein USE1
Q9VTC4 MIP18 family protein CG7949
Q9VU65 POC1 centriolar protein homolog
Q9VUJ0 39S ribosomal protein L39, mitochondrial
Q9VUV9 Putative U5 small nuclear ribonucleoprotein 200 kDa helicase
Q9VVI3 E3 ubiquitin-protein ligase Nedd-4
Q9VVT2 Protein I'm not dead yet
Q9VW97 Psible lysine-specific histone demethylase 1
Q9VWE6 Protein ELYS homolog
Q9VXE5 Serine/threonine-protein kinase PAK mbt
Q9VXF1 Serine/threonine-protein phosphatase 2B catalytic subunit 3
Q9VYI3 Probable 39S ribosomal protein L49, mitochondrial
Q9VYS3 Regulator of nonsense transcripts 1 homolog
Q9VYS5 UPF0551 protein CG15738 homolog, mitochondrial
Q9VYX1 Enhancer of yellow 2 transcription factor
Q9W021 39S ribosomal protein L23, mitochondrial
Q9W032 Protein SGT1 homolog ecdysoneless
Q9W092 Probable chitinase 2
Q9W0Y1 Troponin C-akin-1 protein
Q9W130 Cytochrome P450 9c1
Q9W1I2 Benign gonial cell neoplasm protein
Q9W1X9 OCIA domain-containing protein 1
Q9W376 Neuferricin homolog
Q9W391 Probable phosphorylase b kinase regulatory subunit alpha
Q9W3J5 Probable phenylalanyl-tRNA synthetase alpha chain
Q9W3W8 60S ribosomal protein L17
Q9W429 UDP-xyle and UDP-N-acetylglucosamine transporter-like
Q9W4J5 Probable Ribosome biogenesis protein NEP1
Q9W4L1 39S ribosomal protein L33, mitochondrial
Q9XY35 Cytochrome b-c1 complex subunit 9
Q9XYP7 Gamma-tubulin complex component 2 homolog
Q9Y140 Dehydrogenase/reductase SDR family protein 7-like