1. 1 TSRI/Novartis: 02/04 Glycomics and Bioinformatics What is glycomics? Scope of the informatics problem. Bioinformatics in the glycomics space. Public database resources.

2. 2 TSRI/Novartis: 02/04Glycomics Glycomics: just another omics? - Genes :Genomics - Proteins: Proteomics - Glycans: Glycomics Glycomics: Study of the glycome Glycome : All the glycan structures present in an organism.

3. 3 TSRI/Novartis: 02/04 The 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)?

4. 4 TSRI/Novartis: 02/04 Challenges 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.

5. 5 TSRI/Novartis: 02/04 Functional Glycomics: Systems Biology Focus on Glycan Binding Proteins >30,000 Proteins 50-80% Glycosylated ~100s GBP’s 1000s Glycan structures ~100s GBP Ligands Human and Murine Biology

6. 6 TSRI/Novartis: 02/04 Consortium 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

7. 7 TSRI/Novartis: 02/04 Glycan Binding Proteins in Cell Communication : Three paradigms One Cell Other Cell Glycan Ligand Glycan Binding Protein TRANS Cell Adhesion Selectins CIS Modulate Receptor Signaling CD22 (Siglec-2) Pathogen (HIV-DC-SIGN) Glycoprotein Glycan Targeting / Endocytosis

8. 8 TSRI/Novartis: 02/04 Mammalian Glycan Binding Proteins (GBPs)

9. 9 TSRI/Novartis: 02/04 Specific 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.

10. 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 Transgenics Create & Distribute KO mice TSRI Consortium Databases MIT Steering Committee Analytical Glycan Profiling Imperial College Glycan Specificity GBP ligand specificity U Oklahoma Gene Microarray GBP and GT genes TSRI

11. 11 TSRI/Novartis: 02/04 Consortium for Functional Glycomics : Glycobiology Resources Offered Analysis of your RNAsamples on a highlyannotated custom genemicroarray with 1800human and mouse genes Glyco-gene microarry Glycan specificity of yourcarbohydrate binding proteinon an array with 170 uniqueglycans representingglycoprotein and glycolipidglycans Glycan array screening Over 100 carbohydratecompounds represented onthe glycan array areavailable with azido orbiotin functional groups Carbohydrate compounds 24 mouse strains with genesablated forglycosyltransferase andglycan-binding proteins Mouse knockout strains http://www.functionalglycomics.org

12. 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 Transgenics Create & Distribute KO mice TSRI Consortium Databases MIT Steering Committee Analytical Glycan Profiling Imperial College Glycan Specificity GBP ligand specificity U Oklahoma Gene Microarray GBP and GT genes TSRI

13. 13 TSRI/Novartis: 02/04 Glycan Profiling in Mouse and Human Tissues, Cells and Proteins Imperial College Anne Dell Stuart Haslem Mark Sutton-Smith TSRI/PARC David Goldberg Richard H. Bruce Peter Kuhn TSRI Dan Salomon Mouse Transgenic Core Consortium Database

14. 14 TSRI/Novartis: 02/04 MALDI Screening of WT Kidney

15. 15 TSRI/Novartis: 02/04 PNA Positive Lymphocytes in Germinal Centers of Spleen and Lymph Node G. Cattoretti, Columbia Univ Thorbecke, NY Univ. School of Med Spleen Lymph Node PNA (Gal  1-3GalNAc) PNA (Gal  1-3GalNAc)

16. 16 TSRI/Novartis: 02/04 Protocol for Comparing Resting and Activated Lymphocytes Prepare Mixed Splenocytes (B, CD4+, CD8+) ± Activation in vitro Isolate B, CD4+ or CD8+ lymphocytes using immuno-magnetic beads Left over cells Desired Resting or Activated Cells For Glycan Profiling Extract mRNA for Microarray

17. 17 TSRI/Novartis: 02/04 CD8+ T-cells (IL12 Activated) CD8+ T-cells (Fresh)

18. 18 TSRI/Novartis: 02/04 Gene Microarray: Few Changes in GT Expression 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

19. 19 TSRI/Novartis: 02/04  1-3 Altered Terminal Glycosylation of N-linked Glycans in Activated CD8+ T Cells  2-6 ST6Gal I Key: NeuGc Gal  1- 3GalT ST6Gal I Major structure on resting cells Major structure on activated cells

20. 20 TSRI/Novartis: 02/04 Glycan Synthesis Core - TSRI Synthetic Glycans Construct Library of All Natural Terminal Glycan Sequences Ola Blixt, Nahid Razi-TSRI Nicolai Bovin-Moscow Printed Glycan Microarray Construct Library of All Natural Terminal Glycan Sequences Ola Blixt, Nahid Razi-TSRI Nicolai Bovin-Moscow

21. TSRI/Novartis: 02/04 Glycan-Array: Covalent printing of a glycan library on glass slides Microarray DNA-core facility (Core-E) at Scripps O. Blixt: TSRI Glycan Synthesis Core S. Head: TSRI Microarray-Core C.-H. Wong, M. Bryan, F. Fazio: TSRI N. Bovin: Moscow R. Cummings and R. Alvarez : Univ. Oklahoma

22. 22 TSRI/Novartis: 02/04 Screening Specificity of Human CD22 (Siglec-2) Neu5Ac  2-6Gal(NAc) 4000 spots / slide (ng) Low amount protein consumed (0.5µg / slide) No Blocking required, 1 hr Analysis < 1 hr Re-utilization of slides

23. 23 TSRI/Novartis: 02/04 CD22 is a Negative Regulator of the B Cell Antigen Receptor Complex Antigen (phosphatase- negative regulator) CD22 SHP-1 IgM Ig  src kinase (phosphorylase- positive regulator) ? Does the Ligand Binding Domain Participate In CD22 Function?

24. 24 TSRI/Novartis: 02/04 Public Databases : Glycosyltransferase and Glycan Binding Protein Glycosyltransferase Database - CAZY (extensive genomic portal) CAZY - Consortium for Functional Glycomics (Glycosylation Pathways: menu for CAZY) Consortium for Functional Glycomics - KEGG Glycosylation Pathways http://www.genome.ad.jp/kegg/pathway.html http://www.genome.ad.jp/kegg/pathway.html Glycan Binding Protein Database - Genomics Resource for Animal Lectins Genomics Resource for Animal Lectins

25. 25 TSRI/Novartis: 02/04 Public Databases : Glycan Structure GlycoSuite DB (commercial license req.) GlycoSuite DB Glycominds (commercial license req.) Glycominds Sweet DB (first stop) Sweet DB Consortium for Functional Glycomics (glycan profiles) Consortium for Functional Glycomics GlyCAM -3D (early stage) GlyCAM

26. 26 TSRI/Novartis: 02/04 Glycobiology Resources Glycoforum : http://www.glycoforum.gr.jp/ http://www.glycoforum.gr.jp/ Consortium for Functional Glycomics http://functionalglycomics.org http://functionalglycomics.org

27. 27 TSRI/Novartis: 02/04 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 Lectins http://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

28. 28 TSRI/Novartis: 02/04 End