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Marshall UniversityMarshall Digital Scholar
Chemistry Faculty Research Chemistry
Winter 12-2012
Designing Tools for Studying the DynamicGlycomeJohn F. RakusMarshall University, [email protected]
Follow this and additional works at: http://mds.marshall.edu/chemistry_facultyPart of the Organic Chemistry Commons
This Presentation is brought to you for free and open access by the Chemistry at Marshall Digital Scholar. It has been accepted for inclusion inChemistry Faculty Research by an authorized administrator of Marshall Digital Scholar. For more information, please contact [email protected].
Recommended CitationRakus, J. F. (2012, December). Designing tools for studying the dynamic glycome. Invited Lecture at Sonoma State University,Rohnert Park, CA.
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NYU Cover
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Cells are primarily compose of three types of biomolecules
Protein (50% dry weight)
Nucleic acid (25% dry weight)
Carbohydrate (10% dry weight)
HeLa cell
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Carbohydrates are pervasive and involved in many cellular interactions
Holgersson et al, Immuno Cell Biol, 2005Laughlin et al, Science, 2008
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Nucleic acids and proteins are synthesized with a defined template and dedicated polymerases
Macromolecule: Nucleic acid
Polymerase: DNA Pol or RNA Pol
Template: DNA strand
Macromolecule: polypeptide
Polymerase: Ribosome
Template: mRNA strand
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Glycan biosynthesis lacks a dedicated polymerase and genetic template
Formation of Glc3Man9GlcNAc2-DolPP,an intermediate in the N-linked glycosylation pathway, requires 12 separate enzymes
Essentially, each linkage in an oligosaccharide is formed by a specific transferase enzyme with specific expression control and subcellular localization.
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Consortium for Functional Glycomics (CFG) Notation
Rakus and Mahal, Ann Rev Anal Chem, 2011
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N-linked glycosylation occurs in the ER andGolgi and involves construction of a lipid-linked 14-mer precursor before beingtransferred to an Asn residue and furthermodified to form the final structure.Modified proteins have N-x-S/T consensussequence
O-linked glycosylation occurs in theGolgi apparatus and involves transfer ofa monosaccharide directly to a Ser/Thrresidue by a specific ppGalNacTfollowed by further elaboration. Noknown consensus sequence
Essentials of Glycobiology
There are two primary glycosylation pathways
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Carbohydrate synthetic regulation
• Synthesizing a glycome requires a large commitment of cellular resources
• Many glycosylation enzymes (glycosyltransferases and glycosidases), sugar transporter and metabolic proteins, and regulation elements (over 120 identified as of 2011)
N-linked glycosylation network (Reactome)
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There are three types of N-linked glycans
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Glycosylation is complicated
• The structures are hard to discern
• The pathways are often redundant and overlapping
• The effects are subtle and, occasionally, conflicting
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Systems Biology
“Systems biology” is a strategy to look at complex phenomena and try to break it down into discernible pathways
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The experiment
1. Look at the cell surface (glycome)
2. See if different cells express different carbohydrates
3. If so, try to explain why
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Model System: The NCI-60 Cell Panel
• NCI-60: 60 cell lines for screening of potential cancer therapeutics
• Vary in tissue type, metastasis, individual of origin
• CellMiner.org: open source database containing mRNA, miRNA and protein array data, genetic mapping, pharmacological and mutational analysis
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Generation of Lectin Microarrays
• Lectins are printed on NHS-ester coated glass slides in high spatial density at 10°C and ambient humidity
• Protein lysine residues react with esters to form amide-bound conjugates
• Unreacted esters are blocked with ethanolamine
• Slides can be stored for up to two months
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Ratiometric lectin microarray analysis for semi-quantitative analysis of the dynamic glycome
Pilobello et al, PNAS, 2007Rakus and Mahal, Ann Rev Anal Chem, 2011
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Interpreting a two-color experimentProbe Sample
FluorescenceReferenceFluorescence
Sample/Reference Log2(S/R)
A 500.00 500.00 1 0.00
B 2497.0 525.00 4.76 2.25
C 5500.0 12485 0.44 -1.18
D 125.20 137.23 0.91 -0.13
E 2545.0 1928.0 1.32 0.40
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Interpreting a two-color experiment
When you have many samples and many probes, the data gets represented as a heat map
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Experimental Strategy
Isolate, labelmembranes
Combine and Integrate
Culture NCI-60 lines
GLYCOMICS
Analyze lectinmicroarray
Confirm with whole cell labeling
GENOMICS
Isolate total mRNA
Analyze genemicroarray
SVD
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Lectin microarray analysis shows meaningful patterns
• 90 lectins
• 56 cell lines
• Biological replicates of two cell lines
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Lectin microarray analysis shows meaningful patterns
• Several clusters of lectins by carbohydrate specificity
• Cell lines cluster by tissue of origin
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Cell lines cluster based on their glycosylation
COLON (5/6, p=0.39)LEUKEMIA (4/6, p=0.39)
CNS (4/5, p=0.44)LUNG (4/7, p=0.44)
MELANOMA (8/9, p=0.60)
RENAL (6/8, p=0.39)
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Glycosylation signatures: Mannose
Mannose (high)GNA, NPA, CVN, RPA
Mannose (complex)UDA, SVN, Calsepa, HHL, LcH, PHA-E, MAL-I, PSA
RENAL
MELANOMALUNG,COLON
COLON, LUNG
MELANOMA, RENAL
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Maturation of N-linked glycans
• Glc3Man9GlcNAc2 precursor is transferred to nascent polypeptide in ER
• Upon proper folding, glycan is trimmed to high-mannose and, potentially further modified to hybrid/complex
• α-mannosidase I controls all hybrid/complex maturation steps
Essentials of Glycobiology
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Conclusions (some)!
• Human cells express different, specific carbohydrate patterns on their surfaces, based on their histological function
• What controls the glycome variation? (what does the interior of the house look like?)
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Experimental Strategy
Isolate, labelmembranes
Combine and Integrate
Culture NCI-60 lines
GLYCOMICS
Analyze lectinmicroarray
Confirm with whole cell labeling
GENOMICS
Isolate total mRNA
Analyze genemicroarray
SVD
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Patterns of NCI-60 Gene Expression Regulation
• mRNA and miRNA expression patterns investigated across entire NCI-60 (Liu et al, Mol Biol Cell, 2010)
OBSERVATION OF HIGHLY EXPRESSED AND DIVERSE PROBES-Melanoma (8/9 mRNA, 9/9 miRNA) and leukemia (6/6 mRNA and miRNA) lines cluster together based on expression patterns
-Renal (8/8 mRNA, 6/8 miRNA) and colon (6/7 mRNA and miRNA) also show strong correlation
-CNS and lung cancer show little correlation
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microRNAs (miRNAs) regulate gene expression
• miRNA are genomicallyencoded short (~22 nt) molecules involved in repressing expression of mRNA
• Bind target miRNAs in a 6-10 bp seed region
• Upon processing, miRNAs recruit a post-transcriptional silencing complex to inhibit expression
Thamilarasan et al, Autoimmun Rev, 2012
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How we make a cluster
𝒓𝒓 =𝟏𝟏𝒏𝒏�𝒊𝒊=𝟏𝟏
𝒏𝒏
(𝒙𝒙𝒊𝒊 − �𝒙𝒙σ𝒙𝒙
)(𝒚𝒚𝒊𝒊 − �𝒚𝒚𝝈𝝈𝒚𝒚
)
�𝒙𝒙 = average across all set of numbersσ = standard deviation against all set of
numbers
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• This is a fancy way of saying I am working with vectors
• Probes vs cell line (horizontal) and cell line vsprobe (vertical)
• The intensity for each point on a heat map has a distance from every other data point
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Singular Value Decomposition
• Method to decompose a matrix into an orthogonally transformed set of variables
• Used to identify significant, co-varying patterns in large data sets. I.E. What signals account for the variation?
• Can be applied to any array individually or combined for SYSTEMS BIOLOGY examination of glycosylation pathways
M = U ΣVT
SVD Matrix
Eigenarrays vs genes/lectins
Arrays vs eigengenes/eigenlectins
U Σ VT
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Lectin plus miRNA SVD Results• The first six principal
components (six most signif-icant) are shown
• All miRNA and lectin probes (two separate experiments)
With Lani Pilobello
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We now have a hypothesis
• miRNA’s regulate the N-linked maturation pathway
• miRNA expression controls N-linked glycome by repressing (or activating) α-mannosidase I
• High mannose cells have high miRNAexpression which means low α-mannosidase I
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We need evidence
Use high mannose (SN12C, SK-MEL-5) and complex (HCT-116, HT29) lines
Modulate miRNA expression in cells
Isolate total RNA
Isolate total
proteinAnalyze
mannosidase gene expression
(RT-PCR)
Analyze mannosidase
protein expression (Western blot)
Analyze glycome (lectin microarray)
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Can the miRNAs bind the mannosidase transcript?
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Luciferase read-through assay
Promega.com
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miR-30c and -361 bind the MAN1A2 3’ UTRMAN1A2.30cMut.stk< CCTGTGTTTCGCTCATATGGACCACTACAGAAATTAGTTTGAAGGGGCGGCTTTTGAAAAMAN1A2.3UTR< CCTGTGTTTTGTTTACATGGACCACTACAGAAATTAGTTTGAAGGGGCGGCTTTTGAAAA
********* * * * ********************************************MAN1A2.361Mut.stk< AAATTTCTTGGTTAAGTTTAATTTTCTATAGAGACATGATCTCACAAAGAATACTCAAGT MAN1A2.3UTR< AAATTTCTTGGTTAAGTTTAATTTTCTCTGGGGGCATGATCTCACAAAGAATACTCAAGT
***************************.*.*.*.**************************
0
5000
10000
15000
20000
25000
30000
35000
1
Fluo
resc
ence
(AFU
)
MAN1A2 +Scramble
MAN1A2 + 361 mimic
MAN1A2 + 30c mimic
MAN1A2-30c mut +ScrambleMAN1A2-30c mut + 30cmimicMAN1A2-361 mut +scrambleMAN1A2-361 mut + 361mimicMAN1A2
MAN1A2 + 361 mimic
MAN1A2-361 mut
+ 361 mimic,
MAN1A2 + 30c
mimic,
MAN1A2-30c mut
+ 30c mimic, -2
-1.5
-1
-0.5
0
0.5
1
Log 2
ratio
(mim
ic/s
cram
ble)
With Praveen Agrawal
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Do the miRNAs affect mannosidase transcription?
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Transfection of cell lines with miRNA repress MAN1A2
0
0.002
0.004
0.006
0.008
0.01
scramble mimic hsa-miR-30c hsa-miR-181a hsa-miR-181b hsa-miR-361-5p
MAN1A2 2^ΔCt
24 h post- transfection
Rela
tive
quan
titat
ion
0
5E-09
1E-08
1.5E-08
2E-08
2.5E-08
3E-08
scramble mimic hsa-miR-30c hsa-miR-181a hsa-miR-181b hsa-miR-361-5p
MAN1A2 2^ΔCt
72 h post- transfection
Rela
tive
quan
titat
ion
With Praveen Agrawal
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Do the miRNAs affect mannosidase protein expression?
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00.20.40.60.8
11.2
MAN1A2/GAPDH
72 h post- transfection
anti-MAN1A2
anti-GAPDH
Rela
tive
quan
titat
ion
Western Blot of MAN1A2 72 hrs after transfection with miRNA
MAN1A2 expression decreases ~50%
With Praveen Agrawal
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anti-MAN1A2
anti-GAPDH
72 h post- transfectionRe
lativ
e qu
antit
atio
n
00.20.40.60.8
11.2
MAN1A2/GAPDH
Western Blot of MAN1A2 72 hrs after transfection with miRNA inhibitor
MAN1A2 expression increases ~50%
With Praveen Agrawal
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Seeing inside the house
• miRNA-30c and -361 affect expression of the enzyme α-mannosidase I (maybe -181b as well)
• Expression of α-mannosidase I determines the type of N-linked glycan expresses
• Cells differentially express miRNAs resulting in different glycomes
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A model for cell-type specific carbohydrate expression?
α-mannosidase I
miR-30cmiR-361
Expression of hybrid/complex glycans (renal)
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A model for cell-type specific carbohydrate expression?
α-mannosidase I
miR-30cmiR-361
Expression of hybrid/complex glycans (renal)
Expression of high mannose (colon)
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N-linked glycosylation occurs in the ER andGolgi and involves construction of a lipid-linked 14-mer precursor before beingtransferred to an Asn residue and furthermodified to form the final structure.Modified proteins have N-x-S/T consensussequence
O-linked glycosylation occurs in theGolgi apparatus and involves transfer ofa monosaccharide directly to a Ser/Thrresidue by a specific ppGalNacTfollowed by further elaboration. Noknown consensus sequence
Essentials of Glycobiology
There are two primary glycosylation pathways
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N-linked glycosylation O-linked glycosylation
Glycolipids C-linked glycosylation
Functions: protein folding and trafficking; signaling ligand
Functions: protein folding and trafficking; ECM composition
Functions: Cell surface receptors; membrane composition
Functions: Unknown (stabilization?)Cer Cer
AsnAsnSer/Thr Ser/Thr
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Background: C-linked glycosylation• First identified in human RNaseB by Edman degradation, NMR and MS
(Hofsteenge et al, Biochemistry, 1994)
• Mannose is transferred from dolichyl-phosphate-mannose (not GDP-mannose) by an endoplasmic reticulum-associated protein (Doucey et al, Mol Biol Cell, 1998)
• Several dozen human proteins confirmed to be C-mannosylated; over 2500 candidates contain W-x-x-W consensus sequence (Julenius, Glycobiology, 2007)
• Present in insects and C. elegans, not in yeast or E. coli (Krieg et al, J BiolChem, 1997)
• The glycosyltransferase responsible for this modification is not known
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Non-Leloir Transferases utilize dolichyl-phosphate mannose
Non-Leloir transferases share a common ancestor and are evolutionarily related in two distinct sequence regions
(Oriol et al, Mol BiolEvol, 2002)
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Topology of Non-Leloir Mannosyltransferases
O-mannosyltransferases N-linked and GPI anchor
1 1E D E D
+H3N +H3N
CO2-
CO2-
2 2
Loop 1: Contains N-terminal acidic domain (DE/EE/DD). Work on POMT1/2 family suggests this is the catalytic domain
Loop 2: Homologous throughout dolichyl-phosphate-carbohydrate utilizing enzymes
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OHO
HO
OH
ODolP
OH
+ HO-R
OHO
HO
OH
OR
OH
H2O + DolP
OHO
HO
OH
ODolP
OH
OHO
HO
OH
OH
H2O
+ DolP
HN
HN
+H
Transfer of α–Mannose from Dolichyl-Phosphate-Mannose to Hydroxyl Acceptor
Transfer of α–Mannose from Dolichyl-Phosphate-Mannose to Tryptophan
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PHI-BLAST Method to Identify Potential C-MT SequencesStep 1: Query human non-redundant protein database with Alg3 sequence, PHI-BLAST loop 2
+H3N CO2-
+H3N CO2-
+H3N CO2-
+H3N CO2-
Step 2: Iterate PHI-BLAST search to find “loop 2” sequences in distantly-related sequences+H3N CO2-
+H3N CO2-
+H3N CO2-
+H3N
+H3N+H3N
CO2-
CO2-CO2-
Step 3: a.) Repeat search with 7 other human DPM transferase sequencesb.) Pool and align search resultsc.) Query C. elegans and S. cerevisiae for presence of candidate
Search Term
Result sequences
Sequences containingPHI-BLAST term
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Alg3PigUPigMPigBAlg12Alg9PomT1
PomT2
PigV
Q7Z602
Q86U75Q9HCN8
Two unique candidate sequences emerge from the pool which are functionally unannotated, present in C. elegans, and not present in S. cerevisiae
I propose to express these candidates and screen them for C-MT activity
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C-MT, what are my goals?
• Identify C-MT
• Determine if acidic motif is responsible for catalysis
• Identify C-MT in other organisms
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Proposal: in vivo identification of C-mannosylatedproteins
• Over 2500 mammalian proteins may be C-mannosylated
• Nearly all remain unconfirmed
• What stimuli enhance C-mannosylation?
• Innate immune system activation results in several C-mannosylated proteins
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Proposal: Role for C-linked glycosylation in lipo-polysaccharide-induced TNFα activation
KNOWN: Stimulation of macrophages with LPS and C-mannosylatedpeptides induces TNFα signaling (Muroi et al, Glycobiology, 2007) via interaction with Hsc70 (Ihara et al, Glycobiology, 2010)
Question 1: What role does C-mannosylation have in co-activating this response?
Question 2: What proteins are involved?
Proposal: Utilize glycomic strategy to identify response of C-mannosylated proteins.
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Glycoproteomics
• Galanthus nivalis agglutinin (GNA) binds terminally exposed mannose, included mannosyl-tryptophan (Perez-Villar et al, Glycobiology, 2004)
• PNGaseF can remove mannose-containing N-linked glycans
• Cellular proteins can be deglycosylated with PNGaseF, the only remaining terminal mannoses will be mannosyl-tryptophan, which can be recognized by GNA-pulldown
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Strategy to identify C-mannosylatedproteins in vivo
Isolated C-linkedglycoproteome
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Goals
• Get from 150 to 2500 C-mannosylated proteins
• Standardized proteomic method, identification of C-mannosylation under stimulated conditions (LPS-induced macrophage response)
• Identification of protein-protein interactions and activation pathways
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Acknowledgements