2. Functional Glycobiology Structure of Glycoconjugates sugar residues, linkage, sequence, conformation types of glycoconjugate, protein and lipid Biosynthesis N- and O-linked Glycosylation, GPIs, PGs, GSLs molecular biology of enzymes Determination of Structure sugar sequencing, HPLC, MS, NMR endo- and exo-glycosidases Function of Glycoconjugates glycoproteins and glycolipids in biology metabolic diseases Course Outline

3. Functional Glycobiology Function of Glycoconjugates Structure of Glycoconjugates Determination of Structure Biosynthesis Revision Topics Carbohydrate/protein interactions Intracellular functions - protein folding pathways Extracellular functions - leukocyte surveillance

4. Lessons from Gene Manipulation or Mutations N-Linked Glycosylation GPT KO in mouse GlcNAcT KO in mouse CDG GSLs GlcCer, NeuAc & GalNAc transferase KO in mouse lysosomal storage disorders PGs Inhibition of CS synthesis using RNAi Hyaluronan synthase disruption GPIs Paroxysmal nocturnal haemoglobinurea

5. P P GlcNAc UDP-GlcNAcUDP GPI-anchored proteins Mutations in Early GPI-anchor Biosynthesis Mutations in the enzyme leads to a loss of all GPI-anchored proteins Paroxysmal nocturnal haemoglobinurea Intravascular haemolysis, cytopenia and thrombosis Loss of GPI-anchored complement inactivating proteins in blood Takeda & Kinoshita (1995), TIBS 20:367

8. Early Embryogenesis in C.elegans Inhibition of chondroitin sulphate (CS) synthesis by RNAi inhibition of cell division inhibition of cytokinesis Sugahara et al (2003) COSB 13:612

9. Targeted Disruption of GSL-Specific Glycosyltransferases in Mice GeneMajor GSLPhenotype CGTnoneLethal at E7.5. Ectodermal apoptosis GM3SLacCerViable. Enhanced insulin sensitivity GalNAcTGM3+GD3Viable. Impaired nerve conductance. Wallerian degeneration and age related motor function defects GD3SGM1+GD1aViable. Impaired nerve regeneration GalNAcTGM3Viable. Peripheral nerve degeneration. and GD3SSpontaneous adult lethal phenotype and sensitivity to audiogenic seizures

10. Effects of GPT Deletion on Pre-implantation Embryos Glc3Man9(GlcNAc) 2 -PP-Dol Man9(GlcNAc) 2 -PP-Dol GlcNAc-PP-Dol (GlcNAc) 2 -PP-Dol PP-Dol P-Dol Asn oligosaccharide transfer Tunicamycin GPT Glc3Man9(GlcNAc) 2

11. n n n n Mannosidase n n n GlcNAcTI mature glycoproteins mannosidase GlcNAc-Transferase I KO in Mouse is Embryonically Lethal

12. Protein N-Glycosylation - why is it so critical? Disruption of early events in lipid cycle (complete absence of N-glycans) ablates early differentiation processes. Disruption of later events (lack of complex N-glycans) is embryonically lethal Points to early events in the ER as having important role Oligosaccharide sequences are recognised by ER resident chaperones calnexin and calreticulin

13. Quality Control in the Protein Folding Pathway ER co-translational addition of N-linked oligosaccharides Trimming with  -glucosidases Recognition of monoglucosylated glycans by chaperones calnexin (membrane bound) careticulin (soluble) Disulphide bond formation and folding Glucosyltransferase ‘senses’ folded state Re-glucosylation and chaperone binding Elimination from the ER or maturation via Golgi

14. P ERp57 (disulphide isomerase) protein folding on/off calnexin glucosyl- transferase  -glucosidase II  -glucosidase I & II P Glc Man Glc other chaperones P G II P cx ER Golgi P EDEM proteasome Sec61 mannosidase ERGIC-53

15. Calnexin - a protein with multifunctional motifs

16.  -Glucosidase II binding Calnexin binding The Glc 1 Man 9 GlcNAc 2 Oligosaccharide Ligand for Calnexin

17. Glycoprotein Biosynthesis

18. MHC Class I Requires Folding Chaperones

19. MHC Class I Molecule peptide

21. P protein folding on/off calnexin glucosyl- transferase  -glucosidase II  -glucosidase I & II P Glc Man Glc other chaperones P G II P cx ER Golgi P NB-DNJ NN-DNJ NB-DNJ NN-DNJ

22. Therapeutic Opportunities Inhibitors of Processing Glucosidases as Antivirals N-alkylated imino sugars are micromolar inhibitors Virus encoded glycoproteins synthesised by host Prevention of calnexin-mediated pathway creates misfolded proteins Aberrant virus envelope assembly leads to non-infectious particles No possibility of ‘escape’ mutations In vitro efficacy for HIV and Hepatitis B

23. Zeng et al.,(1997) Science 277, 339 Lipids are presented to T cells by CD1 Molecules

24. Green = Apolar; Red = Polar CD1b CD1b,d CD1c CD1d CD1 Molecules bind Lipid Ligands

25. nascent CD1 ER GOLGI ? (pH 4.0) LE CD8 CD4 DN IL-4, IFN  Perforin FAS Granzyme B bacteria MR CD1 Pathway of Lipid Presentation

26. Phe144 VAL12 F’channel A’channel C’channel T’tunnel The ligand binding groove of Human CD1b Gadola et al., (2002) Nat Immunol 3, 721

27. Extracellular Protein - Carbohydrate Interactions Transmigration of lymphocytes from lymph to blood HEV cells in lymph nodes Allows antigen-specific B and T cells to survey all possible sites of pathogen entry Leukocyte response to venous endothelial damage mechanical or chemical injury pathogen induced inflammation

29. GROUPS WITHIN THE C-TYPE LECTIN FAMILY

31. Carbohydrate Ligands for Selectins sialyl Lewis x NeuAc  2,3Galß1,4GlcNAcß1-R Fuc  1,3 NeuAc  2,3Galß1,4GlcNAcß1-R Fuc  1,3 SO 3 - 6-sulphated sialyl Lewis x Granulocytes Monocytes Lymphocytes Lymph node HEVs Peyer’s patch HEVs NK cells P-selectin E-selectin L-selectin P-selectin E-selectin

32. E-Selectin binding to Sialyl Lewis X Oligosaccharide

33. Cell Attachment and Rolling is Carbohydrate mediated Requires expression of correct oligosaccharide Terminal carbohydrate sequence found on protein and lipid Weak monomeric affinity but fast K on - and K off -rates Multimeric interactions increase affinity Bonds have a low fractional spring slippage, which means that as the bond is subjected to strain it has a low tendency to break.

34. Flexible Ligands, Flexible Friends

35. Endothelial cell expression of selectins is spatially and temporally restricted

36. Therapeutic Opportunities Carbohydrate based drugs to control inflammation sLeX for P-selectin-dependent acute lung injury (ARDS) carbohydrate based dendrimers (increase valency) peptidemimetics

37. Glycosylation and Disease N-linked Glycosylation Biosynthesis Congenital Disorders of Glycosylation - 8 diseases rare (<500 cases) deficiencies in enzymes or proteins in ER/Golgi pathway multi-organ involvement, psychomotor retardation few therapies Glycolipid biosynthesis GM3 synthase deficiency (Simpson et al., 2004, Nat Gen 36, 1225) Cancer and Metastasis Changes in N-linked glycosylation and GSL expression secondary effects? glycosylation inhibitors reduce metastasis in some forms of disease Lysosomal Storage Disorders deficiencies in catabolic enzymes storage of GSL and glycoprotein relatively rare (1:18000 live births ww) disease can be severe (infantile death) few therapies

38. Further Reading/Sources Control of protein folding Schrag et al., (2003) TIBS 28, 49 Oda et al., (2003) Science 299, 1394 Molinari et al., (2003) Science 299, 1397 Selectins http://hsc.virginia.edu/medicine/basic-sci/biomed/ley/index.html http://lewis.sfsu.edu/glyco/Lsel.html CDG Grunewald et al., (2002) Pediatric Res 52, 618 Lysosomal Storage Diseases Butters et al., (2000) Chem Rev 100, 4683

39. Schwartz, Nancy B (December 1998 ) Proteoglycan. In: Nature Encyclopedia of Life Sciences. London: Nature Publishing Group. http://www.els.net/ Sen–itiroh Hakomori and Ineo Ishizuka (September 2001 ) Glycolipids: Animal. In: Nature Encyclopedia of Life Sciences. London: Nature Publishing Group. http://www.els.net/ Verbert, André ; Cacan, René and (July 2000 ) Cell Surface Glycoconjugates. In: Nature Encyclopedia of Life Sciences. London: Nature Publishing Group. http://www.els.net/ Butters, Terry D (March 2001 ) Glycoproteins. In: Nature Encyclopedia of Life Sciences. London: Nature Publishing Group. http://www.els.net/ Web-based resources