Functional Glycobiology

1. Functional Glycobiology Course Outline • 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

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

3. 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

4. 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 UDP-GlcNAc UDP GPI-anchored proteins GlcNAc P P Takeda & Kinoshita (1995), TIBS 20:367

7. 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

8. Targeted Disruption of GSL-Specific Glycosyltransferases in Mice Gene Major GSL Phenotype CGT none Lethal at E7.5. Ectodermal apoptosis GM3S LacCer Viable. Enhanced insulin sensitivity GalNAcT GM3+GD3 Viable. Impaired nerve conductance. Wallerian degeneration and age related motor function defects GD3S GM1+GD1a Viable. Impaired nerve regeneration GalNAcT GM3 Viable. Peripheral nerve degeneration. and GD3S Spontaneous adult lethal phenotype and sensitivity to audiogenic seizures

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

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

11. 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

12. Quality Control in the Protein Folding Pathway • ER co-translational addition of N-linked oligosaccharides • Trimming with a-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

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

14. Calnexin - a protein with multifunctional motifs

15. The Glc1Man9GlcNAc2 Oligosaccharide Ligand for Calnexin a-Glucosidase II binding Calnexin binding

16. Glycoprotein Biosynthesis

17. MHC Class I Requires Folding Chaperones

18. MHC Class I Molecule peptide

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

21. 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

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

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

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

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

26. 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

28. GROUPS WITHIN THE C-TYPE LECTIN FAMILY

30. Carbohydrate Ligands for Selectins sialyl Lewis x P-selectin E-selectin Granulocytes Monocytes Lymphocytes NeuAca2,3Galß1,4GlcNAcß1-R Fuca1,3 6-sulphated sialyl Lewis x L-selectin P-selectin E-selectin Lymph node HEVs Peyer’s patch HEVs NK cells SO3- NeuAca2,3Galß1,4GlcNAcß1-R Fuca1,3

31. E-Selectin binding to Sialyl Lewis X Oligosaccharide

32. 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 Kon- and Koff-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.

33. Flexible Ligands, Flexible Friends

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

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

36. 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

37. 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

38. Web-based resources 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/