1 protein glycosylation adds another layerof structure and specificity to proteins can enhance the...
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Protein glycosylation
Adds another layerof structure and specificity to proteins
Can enhance the function of a protein
Can extend the lifetime of a protein
Can help localize a protein within a cell
Can act as a specific antigen
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Triantennary(also tetra-antennary)
DiantennaryWith bisecting GlcNAcWith fucosylated core
Carbohydrates attached to exterior loops or near termini
Substantial in size
All shown here,N-linked (to amide N of Asnin N-X-S or N-X-T)
Also O-linked, to ser or thr(hydroxyl on side chain); see below
=
=Penta-
saccharidecommon
core
Fucose
Sia= sialic acid (see below)
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1 2
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Examples of O-linked oligosaccharides O-linked oligosaccharides usually consist of only a few carbohydrate residues, which are added one sugar at a time.
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C2
glucose galactose mannose
C4
What’s different from glucose here?
Examples of other hexoses
allose
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Carbohydrate structure specific for:
Cell typePhysiological stateNo. of sites depends on 3-D structure of proteinStructure at that site depends on the site
E.g., transferrin, from different cell types :
Cerebrospinal fluid (made in brain):diantennaryasialo-agalacto-fucosylatedbisecting GlcNAc
Sialic acid structure: see next graphic
Blood (made in liver):diantennary NAcNeu (sialated= sialic acid)
afucosylated
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neuraminic acid – one of the sialic acids = : both terms are used, confusedly
Mannose framework
Carboxyl (acid)
Acetylated amino group
Glycerol moiety
deoxy
NAcNeu:
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Glycosylation pattern affects signaling of proteins used therapeutically, for:Delivery of the soluble glycoprotein drug to the right cell receptor for activityClearance rate
Microheterogeneity:Lots of isoforms typically present
Glycosylation does not seem to represent a bottleneck in high-producing cells:0.1 mg/l (amplify) 200 mg/l = same pattern
Insect cells (Baculovirus, high level transient expression for production):Too simple a pattern compared to human
Mouse and hamster cells: similar to humanHamster: less heterogeneity
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Genetic engineering of glycosylation to:
Modify or enhance activity
E.g.:
Better binding to a receptorMore specific bindingDifferent binding, in theory
Also:
AntigenicityClearance rateDecrease microheterogeneity (for clinical application)
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Modifying glycosylation
1. Add or subtract sites to your favorite protein (cis) 1a. Subtract sites: Easy, change N or S or T to A by site-directed mutagenesis
1b. Add sites: Not so easy. Consensus N-X-S does not work, e.g.:Requires the insertion of a ~12 aa region encompassing a real N-glycosylation site (6 suffices for O-linked)
Place on an end or on a loop (must know protein’s structure)
Works
2. Change the general glycosylation phenotype of the host cell (trans)
E.g., Pam Stanley: lectin-resistant mutants
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2. Clone enzyme genes:Glycosyl transferases, mostlyAlso some synthetases (e.g., NAcNeu synthetase)
Can be complex: e.g., 7 different fucosyl transferases (FTs), with different (overlapping) substrate specificities
Simpler example: Hamster cells do only 2,3 sialylation. Humans do 2,6 as well, via a 2,6-sialyl transferase (ST)Experiment:Over-express cloned human 2,6 ST, along with a substrate protein;produce permanent transfectants of BHK cells (BHK = baby hamster kidney) Get both types of structures now, substantially(although not exactly the same ratio as in human cells).
Modifying glycosylation
1. Add or subtract sites to your favorite protein (cis)2. Change the general glycosylation phenotype of the host cell (trans)
J Biol Chem, Vol. 273, Issue 47, 30985-30994, November 20, 1998 In Vivo Specificity of Human 1,3/4-Fucosyltransferases III-VII in the Biosynthesis of LewisX and Sialyl LewisX Motifs on Complex-type N-Glycans. COEXPRESSION STUDIES FROM BHK-21 CELLS TOGETHER WITH HUMAN -TRACE PROTEIN Eckart Grabenhorst , Manfred Nimtz , Júlia Costa§, and Harald S. Conradt ¶
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Stanley: Isolation of multiply mutated glycosylation mutants by selecting lectin resistanceLectins = carbohydrate-binding proteinsPlant lectins used mostly here (but occur widely in animals as well)
Sequential selections, push - pull on resistance, sensitivity
Resistance: enzyme deficiency failure to add the sugar need for lectin binding
Sensitivity: failure to add a sugar produces greater exposure of underlying sugars A transferase-negative mutant better binding to the exposed sugar
Showed power of selection, usefulness of complementation via cell hybridization
Pam Stanley
Isolate mutant mammalian cell lines deficient in specific glycosylation enzymes
Review: Nature Biotechnology 19, 913 - 917 (2001) , The bittersweet promise of glycobiology. Alan Dove
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Hybrid selection: • All lec-R mutants were: WGA (wheat germ agglutinin) resistant (various degrees) &
pro- (required proline)
• Tester parent was single lec-R + GAT- (req’d glycine, adenine and thymidine) • Select in medium lacking pro and GAT, and with +/- WGA• Complementing hybrids will have regained sensitivity to WGA• Mutants in the same gene will remain WGA resistant (non-complementation)
• Potential: build a production cell line with all glycosyltrasnferases, etc. mutated out. • Could now be used as a tabla rasa (blank slate) introducing a series of enzymes to
build custom tailored glyco-conjugates. Complicated though (order of addition, location in the Golgi, etc. )
Mostly not developed yet.
20Umana, P., Jean-Mairet, J., Moudry, R., Amstutz, H., and Bailey, J.E. 1999.Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity. Nat Biotechnol 17: 176-180.
Target here (bisecting NAcG)
Presence of the bisecting NAcG enhances binding of T-cell receptor to the Fc region of antibodies. Binding is needed for ADCC.Mouse and hamster cell lines used for commercial production lack the glycosyltransferase needed for bisecting NAcG additionA rat myeloma cell line does produce MAb with the bisecting NAcG.
Hypothesis: Expression of the rat enzyme in a CHO cell line will add a bisecting NacG to the anti-neuroblastoma MAb produced by these cells. The modified MAb will be a better mediator of ADCC. Experiment: Clone the cDNA for this enzyme from the rat line and transfer it to CHO cells, driven by an inducible tet promoter. Check sugar structure of Mab (MS) and ADCC efficiency of the Mab (in vitro lysis).
(NAcG = N-acetyl-glucosamine here)
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Genentech(Killer T-cell)
T-cell surface receptor binds Fc region of antibody molecule (Fc gammaR)
Commercial MAb injected as a therapeutic
TARGET CELL
ADCC
22Umana, P., Jean-Mairet, J., Moudry, R., Amstutz, H., and Bailey, J.E. 1999.Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity. Nat Biotechnol 17: 176-180.
Cyt
otox
icity
Anti-neuroblastoma anibody (ng/ml)
High tet, tet-off system, = basal production
Low tet, tet-off system, = higher production
Yet lower tet, tet-off system, = yet higher production
No tet, tet-off system, = highest productionnon-optimalNeuroblastoma
cells + NK T-cells + antibody
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Review:Grabenhorst, E., Schlenke, P., Pohl,., Nimtz, M., and Conradt, H.S. 1999. Genetic engineering of recombinant glycoproteins and the glycosylation pathway in mammalian host cells. Glycoconj J 16: 81-97.
Background:Stanley, P. 1989. Chinese hamster ovary cell mutants with multiple glycosylation defects for production of glycoproteins with minimal carbohydrate heterogeneity. Mol Cell Biol 9: 377-383.
Protein Glycosylation
Naoko Yamane-Ohnuki, et al.. Establishment of FUT8 knockout Chinese hamster ovary cells: an ideal host cell line for producing completely defucosylated antibodies with enhanced antibody-dependent cellular cytotoxicity. Biotechnol Bioeng. 2004 Sep 5;87(5):614-22
Assigned:
Optional Update: Kanda Y, Yamane-Ohnuki N, Sakai N, Yamano K, Nakano R, Inoue M, Misaka H, Iida S, Wakitani M, Konno Y, Yano K, Shitara K, Hosoi S, Satoh M. Comparison of cell lines for stable production of fucose-negative antibodies with enhanced ADCC. Biotechnol Bioeng. 2006 Jul 5;94(4):680-8.
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Biotechnol Bioeng. 2004 Sep 5;87(5):614-22
Hypothesis:Fucose interferes with binding of the T-cell’s Fcgamma3 receptor to the Fc region of an antibody molecule.
Elimination of fucose from produced MAbs will increase ADCC.
Create a mutant CHO cells (starting with amplifiable dhfr- cells) in which the fucose transferase (biosynthesis) genes have been knocked out.
All mAbs produced in these mutant cells will be better at promoting ADCC
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Homology regions
K.O. exon 1 translation start region
DT= diphtheria toxin gene,Kills if integrated via non-homologous recombination
For hemizygote: Select for G418 resistance,Screen by PCR for homologous recomb. 108 cells 45,000 colonies 40 false recombinants (extension-duplications) + 1 true recombinant
Step 2 for homozygote, select for Pur-resistance1.6X10870,000 screened 10 double KO homozygotes.
Remove drug resis. genes bytransient transfection with Cre Recombinase. Exon 1 suffers a 200 nt deletion
Lox sites
Double knock-out strategy for FUT8 an alpha-1,6,fucosyl transferase
Note: 10’s of thousands of PCRs performed to screen for homologous recomb., using 96-well plates
Little sequence data available for Chinese hamsterIsolate CHO cDNA using mouse sequence data for primersUse CHO cDNA to isolate CHO genomic fragments from a commercial lambda library
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Double knockout evidence
mRNA has 200 nt deletion(RT-PCR)
Original KO’d genes have a 1.5 kb insertion(Southern blot)
After Cre treatment
27Use of a fluoresceinated lentil lectin (LCA) that binds fucose oligosaccharides to demonstrate lack of fucosylation in glycosylated proteins in the FUT8 -/- cells
Control background fluorescence(FL-anti avidin)
FUT8 +/+
FUT8 +/-
FUT8 -/-
Surprising: CHO cells do not have excess fucosylation capacity
28Rituxan (retuximab, anti-CD20) produced in FUT -/- cells does not contain fucose(HPLC analysis)
Digestion all the way to monosaccharides
Missing d - g
29Binding to CD20 membranes
FUT8-/- anti CD20 = Rituxan
Complement-mediated cell toxicity is the same for FUT8-/- and Rituxan
In ADCC, FUT8-/- anti-CD20 >> Rituxan
Anti-CD20 from a partially FUT-deficient rat cell line
FUT-/-’sRat line
Rituxan = commercial product, 98% fucosylated
Fc-Receptor protein binding assay
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Very laborious, but apparently a big payoff.
Better selection?:
Why not use the fluorescent LCA to select for the FUT8 KO’s along with G418 resistance (double sequential selection)?
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Hans Henning von Horsten et al., Glycobiology vol. 20 no. 12 pp. 1607–1618, 2010Production of non-fucosylated antibodies by co-expression ofheterologous GDP-6-deoxy-D-lyxo-4-hexulose reductase (RMD)
Clone bacterial RMD cDNAConstruct mam. expn vectorTransfect into CHO cells making Herceptin (anti EGF receptor)Deflects intermediate in fuciose biosynthetic path
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Also absent by MS
No fucose in transfectant glycoproteins
Select for G418 resistance, screen for lack of fucose.
One of 3 clones:WT CHO cells
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Binding assay to Fc receptor (ELISA-type assay)
ELISA = Enzyme-linked immunosorbent assay
About10-fold more effective
WT
3 transfectants
Antibody concentration (ng/ml)