1 tsri/novartis: 02/04 glycomics and bioinformatics what is glycomics? scope of the informatics...
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TSRI/Novartis: 02/04 1
Glycomics and BioinformaticsGlycomics and Bioinformatics
• What is glycomics?• Scope of the informatics
problem.• Bioinformatics in the glycomics
space. • Public database resources.
TSRI/Novartis: 02/04 2
GlycomicsGlycomics
• Glycomics: just another omics?- Genes :Genomics
- Proteins: Proteomics
- Glycans: Glycomics
• Glycomics: Study of the glycome
• Glycome : All the glycan structures present in an organism.
TSRI/Novartis: 02/04 3
The Glycome : How should it be definedThe Glycome : How should it be defined
• Glycosylation is the most common post-translational modification of proteins, with 50-80% of all glycoproteins being glycosylated
• Glycan structure at a single site is often heterogenous, with single glycoproteins having 20-50 glycan structures.
• Glycosylation is cell type specific, so a single glycoprotein can have a different spectrum of glycan structures when expressed in different cells.
• Should the glycome be defined as the library of glycans expressed in an organism (1000s), or the glycans associated with every protein in every cell (1,000,000s)?
TSRI/Novartis: 02/04 4
Challenges in GlycomicsChallenges in Glycomics
• Glycan structure analysis is low throughput. Technical hurdles for obtaining information is high.
• The goals of glycomics and proteo-glycomics are not clear.
• Will the information be useful if it is obtained?• The resources needed to do glycomics far
exceed the need for the information.• Solution: Develop a focused approach to
glycomics based on function and information content.
TSRI/Novartis: 02/04 5
Functional Glycomics: Systems Biology Functional Glycomics: Systems Biology Focus on Glycan Binding Proteins Focus on Glycan Binding Proteins
>30,000 Proteins 50-80%
Glycosylated~100sGBP’s
1000s Glycan structures
~100sGBP
Ligands
Human and Murine Biology
TSRI/Novartis: 02/04 6
Consortium for Functional GlycomicsConsortium for Functional Glycomics
Goal: Define paradigms by which
protein-carbohydrate interactions mediate cell communication
http://www.functionalglycomics.org
Define paradigms by which protein-carbohydrate interactions mediate
cell communication
• NIGMS sponsored 'glue grant' to TSRI (~$8 MM/yr).
• Over 110 participating investigators worldwide
• Focus in innate and acquired immunity
TSRI/Novartis: 02/04 7
Glycan Binding Proteins in Cell Glycan Binding Proteins in Cell Communication : Three paradigmsCommunication : Three paradigms
One Cell
Other Cell
Glycan Ligand
Glycan Binding Protein
TRANSTRANS
Cell Adhesion
Selectins
CISCIS
ModulateReceptor Signaling
CD22(Siglec-2)
•Pathogen (HIV-DC-SIGN)
•Glycoprotein
Glycan Targeting /
Endocytosis
TSRI/Novartis: 02/04 8
CBP Family Ligands Functions
C-type lectins Various• Cell Adhesion• Glycoprotein clearance• Inn ate Immunity
Siglecs( -I ty pe lectins) Sialosides
• Cell signaling• Cell adhesion
Galectins Galactosides • Extr -a cellula r matrixcros -s linking
CD1/TCRGlycolipids/
Glycopeptides• Inn ate immunity• Acquire d immunity
M-type lectins Hig h Man N-linked • Qualit y contro l inprotei n fold ing i n ER
L-typ e lectins Various • Protei n sorting inER
P-typ e lectins Man-6-phosphate • Lysosom al enzymesortin (g Post-Gol )gi
R-typ e lectins Various • Enzym e targeting• Hormon e turnover
Mammalian Glycan Binding Proteins (GBPs)Mammalian Glycan Binding Proteins (GBPs)
CBP Family Ligands Functions
C-type lectins Various• Cell Adhesion• Glycoprotein clearance• Inn ate Immunity
Siglecs( -I ty pe lectins) Sialosides
• Cell signaling• Cell adhesion
Galectins Galactosides • Extr -a cellula r matrixcros -s linking
CD1/TCRGlycolipids/
Glycopeptides• Inn ate immunity• Acquire d immunity
M-type lectins Hig h Man N-linked • Qualit y contro l inprotei n fold ing i n ER
L-typ e lectins Various • Protei n sorting inER
P-typ e lectins Man-6-phosphate • Lysosom al enzymesortin (g Post-Gol )gi
R-typ e lectins Various • Enzym e targeting• Hormon e turnover
TSRI/Novartis: 02/04 9
Specific AimsSpecific Aims
For selected glycan binding proteins (GBPs):
• Define the specificity for binding sugar structures.
• Identify the macromolecular-ligands on biologically relevant cell types.
• Determine how GBP-ligand interactions mediate cell communication.
• Establish the extent to which regulation of glycosylation regulates their function.
• Determine the crystal structure.
Part. Investigators•Explore Biology using tools
•Contribute data> 80 Institutions
Functional Glycomics : Systems Biology Perspective
Glycan Library Tools for specificity analysis and biology
TSRI
Mouse Phenotype UC San Diego
Mouse TransgenicsCreate & Distribute
KO mice
TSRI
Consortium DatabasesMIT
Steering Committee
AnalyticalGlycan Profiling
Imperial College
Glycan SpecificityGBP ligand specificity
U Oklahoma
Gene MicroarrayGBP and GT genes
TSRI
TSRI/Novartis: 02/04 11
Consortium for Functional Glycomics : Consortium for Functional Glycomics : Glycobiology Resources Offered Glycobiology Resources Offered
Analysis of your RNA samples on a highly annotated custom gene microarray with 1800 human and mouse genes
Glyco-gene microarry
Glycan specificity of your carbohydrate binding protein on an array with 170 unique glycans representing glycoprotein and glycolipid glycans
Glycan array screening
Over 100 carbohydrate compounds represented on the glycan array are available with azido or biotin functional groups
Carbohydrate compounds
24 mouse strains with genes ablated for glycosyltransferase and glycan-binding proteins
Mouse knockout strains
http://www.functionalglycomics.org
Part. Investigators•Explore Biology using tools
•Contribute data> 80 Institutions
Functional Glycomics : Systems Biology Perspective
Glycan Library Tools for specificity analysis and biology
TSRI
Mouse Phenotype UC San Diego
Mouse TransgenicsCreate & Distribute
KO mice
TSRI
Consortium DatabasesMIT
Steering Committee
AnalyticalGlycan Profiling
Imperial College
Glycan SpecificityGBP ligand specificity
U Oklahoma
Gene MicroarrayGBP and GT genes
TSRI
TSRI/Novartis: 02/04 13
Glycan Profiling in Mouse and Human Tissues, Cells and Proteins
Imperial CollegeAnne DellStuart HaslemMark Sutton-Smith
TSRI/PARCDavid GoldbergRichard H. BrucePeter Kuhn
TSRIDan SalomonMouse Transgenic Core
Consortium Database
TSRI/Novartis: 02/04 14MALDI Screening of WT Kidney
KEY:
Fucose
NeuAc
GalNAc
Galactose
GlcNAc
Mannose
NeuGc
TSRI/Novartis: 02/04 15
PNA Positive Lymphocytes in Germinal PNA Positive Lymphocytes in Germinal Centers of Spleen and Lymph NodeCenters of Spleen and Lymph Node
G. Cattoretti, Columbia Univ
Thorbecke, NY Univ. School of Med
Spleen Lymph Node
PNA(Gal1-3GalNAc) PNA
(Gal1-3GalNAc)
TSRI/Novartis: 02/04 16
Protocol for Comparing Resting and Protocol for Comparing Resting and Activated LymphocytesActivated Lymphocytes
Prepare Mixed Splenocytes (B, CD4+, CD8+)
± Activation in vitro
Isolate B, CD4+ or CD8+ lymphocytes using
immuno-magnetic beads
Left over cellsDesired Resting or Activated Cells
For Glycan Profiling
Extract mRNA for Microarray
TSRI/Novartis: 02/04 172500 2700 2900 3100 3300 3500
Mass (m/z)
0
10
20
30
40
50
60
70
80
90
100
% Intensity
Voyager Spec #1=>BC=>NF0.7[BP = 1133.6, 51713]
2653.2455
2655.2445
3306.5343
2601.2317
3308.52613102.44522519.1766 2928.37832723.2846 3104.42852603.2231
2898.33732696.2586 3310.46543134.44102521.2049 2926.37412827.29902625.2310 3027.4188 3379.59612720.2697 3221.53873123.51662541.1002 2789.7348
2500 2700 2900 3100 3300 3500
Mass (m/z)
0
10
20
30
40
50
60
70
80
90
100
% Intensity
Voyager Spec #1=>BC=>NF0.7[BP = 2397.2, 48908]
3027.4080
2601.2399
3029.34772519.2553
2636.2781
2723.37092603.21972504.2313 2840.31312693.3151
2533.2539 2927.43973085.46952663.3107 2811.29532737.3507 2883.33932665.3168 3131.40092522.2347 2594.2482 3289.53742968.3952 3042.41492793.3733 2899.3482 3116.45522622.24982508.1122 3336.23762690.3760 3041.30142806.2946 2931.2588 3260.43083165.6332 3408.43442616.2495 3339.32332537.9715 2703.0636
CD8+ T-cells(IL12 Activated)
CD8+ T-cells (Fresh)
TSRI/Novartis: 02/04 18
Gene Microarray: Few Changes in GT Gene Microarray: Few Changes in GT Expression After Activation of CD8+ T cellsExpression After Activation of CD8+ T cells
CD8+T Cells
Fold Change (Activated/Resting)
1.91
1.60
- 6.22
- 7.71
- 2.33
ST6GalNAc IV
1,3Gal GT
CMP-NeuAc Hydroxylase
ST6Gal I
ST8Sia IV
0
Relevant to Changes in N-
Linked Glycosylation
TSRI/Novartis: 02/04 19
1-31-3
Altered Terminal Glycosylation of N-linked Altered Terminal Glycosylation of N-linked Glycans in Activated CD8+ T CellsGlycans in Activated CD8+ T Cells
2-62-6
ST6Gal I
Key:
NeuGc
Gal1-
3GalT1-
3GalT
ST6Gal I
Major structure on resting cells
Major structure on activated
cells
TSRI/Novartis: 02/04 20
Glycan Synthesis Core - TSRIGlycan Synthesis Core - TSRI
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
Synthetic GlycansConstruct Library of All
Natural Terminal Glycan Sequences
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
Ola Blixt, Nahid Razi-TSRINicolai Bovin-Moscow
Printed Glycan
Microarray
Construct Library of All Natural Terminal
Glycan Sequences
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
Ola Blixt, Nahid Razi-TSRINicolai Bovin-Moscow
TSRI/Novartis: 02/04
Glycan-Array: Covalent printing of a glycan library on glass slides
Glycan-Array: Covalent printing of a glycan library on glass slides
Microarray DNA-core facility(Core-E) at Scripps
OO
NH2
OO
NH2
OO
NH2
N
O
O
O
O
OO
NH
O
O HN
NH2
O
CO2H
Glycoprotein Glycans
multistep chemoenzymatic synthesis
One-PotChemical Synthesis
PNGNase cleavageand purification
+
Stable amide bond
NHS-activated surface
Making a diverse carbohydrate library
Library with a selectivefunctional amino group
"High CarbChips"
O. Blixt: TSRI Glycan Synthesis CoreS. Head: TSRI Microarray-CoreC.-H. Wong, M. Bryan, F. Fazio: TSRIN. Bovin: MoscowR. Cummings and R. Alvarez : Univ.
Oklahoma
TSRI/Novartis: 02/04 22
Screening Specificity of Human CD22 (Siglec-2)Screening Specificity of Human CD22 (Siglec-2)
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
hCD22
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
11000
12000
13000
14000
15000
16000
17000
18000
19000
20000
21000
22000
Neu5,9NAc2,6LacNAc
5Neu Gc2,6LacNAc
5Neu Gc2,6Tn5Neu Ac2,6-
Tn
5Neu Ac2,6 -LacNAc LacNAc
5Neu Ac2,6 2LacNAc
5Neu Ac2,6[6 ]Su LacNAc
5Neu Ac2,6 1LacNAc
Ligands
Neu5Ac2-6Gal(NAc)
• 4000 spots / slide (ng)• Low amount protein consumed
(0.5µg / slide)• No Blocking required, 1 hr
Analysis < 1 hr • Re-utilization of slides
TSRI/Novartis: 02/04 23
CD22 is a Negative Regulator of theCD22 is a Negative Regulator of the B Cell Antigen Receptor Complex B Cell Antigen Receptor Complex
Antigen
(phosphatase-negative regulator)
CD22
SHP-1
IgM
IgIg IgIg
src kinase
(phosphorylase-positive regulator)
?
Does theLigand Binding
Domain ParticipateIn CD22 Function?
TSRI/Novartis: 02/04 24
Public Databases : Glycosyltransferase and Public Databases : Glycosyltransferase and Glycan Binding ProteinGlycan Binding Protein
• Glycosyltransferase Database- CAZY (extensive genomic portal)
- Consortium for Functional Glycomics (Glycosylation Pathways: menu for CAZY)
- KEGG Glycosylation Pathways http://www.genome.ad.jp/kegg/pathway.html
• Glycan Binding Protein Database- Genomics Resource for Animal Lectins
TSRI/Novartis: 02/04 25
Public Databases : Glycan StructurePublic Databases : Glycan Structure
• GlycoSuite DB (commercial license req.)
• Glycominds (commercial license req.)
• Sweet DB (first stop)
• Consortium for Functional Glycomics (glycan profiles)
• GlyCAM -3D (early stage)
TSRI/Novartis: 02/04 26
Glycobiology ResourcesGlycobiology Resources
• Glycoforum : http://www.glycoforum.gr.jp/
• Consortium for Functional Glycomics http://functionalglycomics.org
TSRI/Novartis: 02/04 27
URL Addresses:URL Addresses:
- CAZY (extensive genomic portal)http://afmb.cnrs-mrs.fr/CAZY/GT.html
- Consortium for Functional Glycomics http://www.functionalglycomics.org/
- KEGG Glycosylation Pathways http://www.genome.ad.jp/kegg/pathway.html
- Genomics Resource for Animal Lectinshttp://ctld.glycob.ox.ac.uk/
- GlycoSuite DB (commercial license req.)http://www.proteomesystems.com/glycosuite/
- Glycominds (commercial license req.)http://www.glycominds.com/GlycoInfo.asp?HighLight=LinearCode
- Sweet DB (first stop)http://www.dkfz-heidelberg.de/spec2/sweetdb/
- Consortium for Functional Glycomics (glycan profiles)http://www.functionalglycomics.org/
- GlyCAM -3D (early stage)http://glycam.ccrc.uga.edu/index.html
TSRI/Novartis: 02/04 28
EndEnd