1. Glycoproteins Roles of glycoprotein glycosylationRoles of glycoprotein glycosylation Modes of glycoprotein glycosylationModes of glycoprotein glycosylation N-linked glycansN-linked glycans O-linked glycansO-linked glycans –O-GalNAc – “mucin” type glycosylation –O-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules –O-GlcNAc – intracellular protein regulation Glycan BiosynthesisGlycan Biosynthesis –General Principles –N-linked glycoprotein biosynthesis Roles of glycoprotein glycosylationRoles of glycoprotein glycosylation Modes of glycoprotein glycosylationModes of glycoprotein glycosylation N-linked glycansN-linked glycans O-linked glycansO-linked glycans –O-GalNAc – “mucin” type glycosylation –O-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules –O-GlcNAc – intracellular protein regulation Glycan BiosynthesisGlycan Biosynthesis –General Principles –N-linked glycoprotein biosynthesis

2. Glycoproteins Roles of glycoprotein glycosylationRoles of glycoprotein glycosylation Modes of glycoprotein glycosylationModes of glycoprotein glycosylation N-linked glycansN-linked glycans O-linked glycansO-linked glycans –O-GalNAc – “mucin” type glycosylation –O-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules –O-GlcNAc – intracellular protein regulation Glycan BiosynthesisGlycan Biosynthesis –General Principles –N-linked glycoprotein biosynthesis Roles of glycoprotein glycosylationRoles of glycoprotein glycosylation Modes of glycoprotein glycosylationModes of glycoprotein glycosylation N-linked glycansN-linked glycans O-linked glycansO-linked glycans –O-GalNAc – “mucin” type glycosylation –O-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules –O-GlcNAc – intracellular protein regulation Glycan BiosynthesisGlycan Biosynthesis –General Principles –N-linked glycoprotein biosynthesis

3. The Eukaryotic Cell Surface Lodish, et al. (1995) Molecular Cell Biology 3rd ed. lipid bilayer fibroblast extracellular intracellular Martinez-Palomo, A., et al. Cancer Res. 29, 925-937, 1969

4. HIV gp120 HIV gp120 HIV gp120 HIV gp120 “Structure” of HIV gp120 Zolla-Pazner (2004) Nature Reviews Immunol 4, 199 Burton DR et al (2012) Cell Host & Microbe 4:396

5. HIV gp120 HIV gp120 HIV gp120 HIV gp120 Doores K J et al. (2010) PNAS 107:17107

6. Proteins fold onto themselves… glycans spread out in space Thy-1 cell-cell interaction protein 1.5 kDa GPI anchor 3 kDa N-linked glycans 18 kDa protein membrane Second Edition Essentials of Glycobiology

7. Roles of protein glycosylation Protein folding*Protein folding* Protein conformationProtein conformation Protein trafficking*Protein trafficking* Cell surface glycoprotein lattice*Cell surface glycoprotein lattice* Regulation of signaling pathways*Regulation of signaling pathways* Cell-cell interactions*Cell-cell interactions* Protein folding*Protein folding* Protein conformationProtein conformation Protein trafficking*Protein trafficking* Cell surface glycoprotein lattice*Cell surface glycoprotein lattice* Regulation of signaling pathways*Regulation of signaling pathways* Cell-cell interactions*Cell-cell interactions* * via glycan recognition

8. Protein Folding: N-linked protein glycosylation is essential for chaperone-assisted folding of glycoproteins in the endoplasmic reticulum Helenius, A. and Aebi, M. (2001) Science 291, 2364-2369 Asn

9. http://www.cdgs.com/ Joel Zachary Megan Protein Folding: Children with altered protein N-glycosylation have diverse motor, physiological and cognitive deficits

10. Protein Conformation: Closely spaced short O-linked glycans on mucins

11. Protein Conformation: Conserved glycosylation of IgG is required for interaction with immune effectors (complement, Fc-receptors)

12. Protein Trafficking: Unique glycosylation of lysosomal enzymes “labels” them for trafficking to the lysosome via the Man-P receptor(s) https://wikispaces.psu.edu/v Asn

13. Cell surface glycoprotein lattice: Sugar binding proteins (galectins) bind to and stabilize cell surface glycoproteins Lau et al (2007) Cell 129:123 Altered glycoprotein glycans result in internalization of key glycoproteins Pancreatic β cells stained for Glut-2 (green), insulin (red), and DNA (blue). Ohtsubo et al (2005) Cell 123:1307

14. Regulation of signaling pathways: Notch signaling during development is driven by glycosylation Stanley (2007) Curr Opin Struct Biol 17:530

15. Sharon and Lis (1993) Scientific American Carbohydrate binding determinant Carbohydrate recognition domain (CRD) Glycans in cell-cell interactions Lectin Glycan

16. Roles of protein glycosylation Protein folding*Protein folding* Protein conformationProtein conformation Protein trafficking*Protein trafficking* Cell surface glycoprotein lattice*Cell surface glycoprotein lattice* Regulation of signaling pathways*Regulation of signaling pathways* Cell-cell interactions*Cell-cell interactions* Protein folding*Protein folding* Protein conformationProtein conformation Protein trafficking*Protein trafficking* Cell surface glycoprotein lattice*Cell surface glycoprotein lattice* Regulation of signaling pathways*Regulation of signaling pathways* Cell-cell interactions*Cell-cell interactions* * via glycan recognition

17. Glycoproteins Roles of glycoprotein glycosylationRoles of glycoprotein glycosylation Modes of glycoprotein glycosylationModes of glycoprotein glycosylation N-linked glycansN-linked glycans O-linked glycansO-linked glycans –O-GalNAc – “mucin” type glycosylation –O-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules –O-GlcNAc – intracellular protein regulation Glycan BiosynthesisGlycan Biosynthesis –General Principles –N-linked glycoprotein biosynthesis Roles of glycoprotein glycosylationRoles of glycoprotein glycosylation Modes of glycoprotein glycosylationModes of glycoprotein glycosylation N-linked glycansN-linked glycans O-linked glycansO-linked glycans –O-GalNAc – “mucin” type glycosylation –O-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules –O-GlcNAc – intracellular protein regulation Glycan BiosynthesisGlycan Biosynthesis –General Principles –N-linked glycoprotein biosynthesis

18. Glycoproteins Roles of glycoprotein glycosylationRoles of glycoprotein glycosylation Modes of glycoprotein glycosylationModes of glycoprotein glycosylation N-linked glycansN-linked glycans O-linked glycansO-linked glycans –O-GalNAc – “mucin” type glycosylation –O-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules –O-GlcNAc – intracellular protein regulation Glycan BiosynthesisGlycan Biosynthesis –General Principles –N-linked glycoprotein biosynthesis Roles of glycoprotein glycosylationRoles of glycoprotein glycosylation Modes of glycoprotein glycosylationModes of glycoprotein glycosylation N-linked glycansN-linked glycans O-linked glycansO-linked glycans –O-GalNAc – “mucin” type glycosylation –O-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules –O-GlcNAc – intracellular protein regulation Glycan BiosynthesisGlycan Biosynthesis –General Principles –N-linked glycoprotein biosynthesis

19. Modes of glycosylation

20. Glycoproteins Roles of glycoprotein glycosylationRoles of glycoprotein glycosylation Modes of glycoprotein glycosylationModes of glycoprotein glycosylation N-linked glycansN-linked glycans O-linked glycansO-linked glycans –O-GalNAc – “mucin” type glycosylation –O-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules –O-GlcNAc – intracellular protein regulation Glycan BiosynthesisGlycan Biosynthesis –General Principles –N-linked glycoprotein biosynthesis Roles of glycoprotein glycosylationRoles of glycoprotein glycosylation Modes of glycoprotein glycosylationModes of glycoprotein glycosylation N-linked glycansN-linked glycans O-linked glycansO-linked glycans –O-GalNAc – “mucin” type glycosylation –O-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules –O-GlcNAc – intracellular protein regulation Glycan BiosynthesisGlycan Biosynthesis –General Principles –N-linked glycoprotein biosynthesis

21. Glycoproteins Roles of glycoprotein glycosylationRoles of glycoprotein glycosylation Modes of glycoprotein glycosylationModes of glycoprotein glycosylation N-linked glycansN-linked glycans O-linked glycansO-linked glycans –O-GalNAc – “mucin” type glycosylation –O-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules –O-GlcNAc – intracellular protein regulation Glycan BiosynthesisGlycan Biosynthesis –General Principles –N-linked glycoprotein biosynthesis Roles of glycoprotein glycosylationRoles of glycoprotein glycosylation Modes of glycoprotein glycosylationModes of glycoprotein glycosylation N-linked glycansN-linked glycans O-linked glycansO-linked glycans –O-GalNAc – “mucin” type glycosylation –O-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules –O-GlcNAc – intracellular protein regulation Glycan BiosynthesisGlycan Biosynthesis –General Principles –N-linked glycoprotein biosynthesis

22. “N-linked” glycan linkage to protein asparagine residues N-glycosidic bond

23. N-linked glycan

24. Major classes of N-linked glycans High mannose

25. Examples of structures found as termini on the branches of complex N-linked glycoprotein glycans…. Terminal Saccharide Diversity --- A key to recognition NeuAc  2-6 Gal  1-4 GlcNAc--- NeuAc  2-3 Gal  1-4 GlcNAc--- Gal  1-3 Gal  1-4 GlcNAc--- 4-SO 3 -GalNAc  1-4 GlcNAc--- (-Gal  1-4 GlcNAc  1-3-) n = poly-N-acetyllactosamine (n may be >6) (-NeuAc  2-8-) n = polysialic acid (n may be >50) Fuc residues (e.g. Fuc  1-2Gal; Fuc  1-3 GlcNAc; Fuc  1-4 GlcNAc Fuc residues (e.g. Fuc  1-2 Gal; Fuc  1-3 GlcNAc; Fuc  1-4 GlcNAc

26. Human spleen Human kidney Human spleen Human kidney Human spleen Human kidney

28. N-linked glycosylation consensus sequence The N-linked amino acid consensus sequence is “Asn-[any AA]- [Ser or Thr]” where the middle amino acid is not Pro

29. Glycoforms Whereas a protein is defined by its unique amino acid sequence, the glycan structure at a single protein glycosylation site varies. Although glycan structures vary at each glycosylation site, the profile is reproducible, protein-specific, site- specific, and cell type-specific.

30. Three glycoforms of CD59, a compliment regulatory protein

31. Human erythropoietin glycosylation

32. “Humanization” of yeast to produce complex N-linked glycoproteins Hamilton, et al. Science 313, 1441-1443 (8 September 2006)

33. Glycoproteins Roles of glycoprotein glycosylationRoles of glycoprotein glycosylation Modes of glycoprotein glycosylationModes of glycoprotein glycosylation N-linked glycansN-linked glycans O-linked glycansO-linked glycans –O-GalNAc – “mucin” type glycosylation –O-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules –O-GlcNAc – intracellular protein regulation Glycan BiosynthesisGlycan Biosynthesis –General Principles –N-linked glycoprotein biosynthesis Roles of glycoprotein glycosylationRoles of glycoprotein glycosylation Modes of glycoprotein glycosylationModes of glycoprotein glycosylation N-linked glycansN-linked glycans O-linked glycansO-linked glycans –O-GalNAc – “mucin” type glycosylation –O-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules –O-GlcNAc – intracellular protein regulation Glycan BiosynthesisGlycan Biosynthesis –General Principles –N-linked glycoprotein biosynthesis

34. Glycoproteins Roles of glycoprotein glycosylationRoles of glycoprotein glycosylation Modes of glycoprotein glycosylationModes of glycoprotein glycosylation N-linked glycansN-linked glycans O-linked glycansO-linked glycans –O-GalNAc – “mucin” type glycosylation –O-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules –O-GlcNAc – intracellular protein regulation Glycan BiosynthesisGlycan Biosynthesis –General Principles –N-linked glycoprotein biosynthesis Roles of glycoprotein glycosylationRoles of glycoprotein glycosylation Modes of glycoprotein glycosylationModes of glycoprotein glycosylation N-linked glycansN-linked glycans O-linked glycansO-linked glycans –O-GalNAc – “mucin” type glycosylation –O-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules –O-GlcNAc – intracellular protein regulation Glycan BiosynthesisGlycan Biosynthesis –General Principles –N-linked glycoprotein biosynthesis

35. “O-linked” glycan linkage to protein serine residues

36. O-linked glycan

37. O-linked glycan classes & diversity

38. Human spleen

39. O-GlcNAc Note: No further sugar substitutions have been confirmed

40.  O-GlcNAc is localized to the cytoplasm and nucleus.  O-GlcNAc is present in all higher eukaryotes studied.  O-GlcNAc is as abundant as phosphorylation.  UDP-GlcNAc is as abundant as ATP in some cell types.  O-GlcNAc containing proteins are phosphoproteins.  O-GlcNAc and phosphorylation are often reciprocal.  O-GlcNAc is dynamic, implying a regulatory role. Key Features of O-linked GlcNAc

41. Three-state model: unmodified / O-GlcNAc / phosphate O-GlcNAc Glycoprotein Unmodified protein Unmodified protein Phosphoprotein OPO 3 2- Phosphatase OGT Phosphatase Kinase O-GlcNAcase Natasha Zachara

42. Glycoproteins Roles of glycoprotein glycosylationRoles of glycoprotein glycosylation Modes of glycoprotein glycosylationModes of glycoprotein glycosylation N-linked glycansN-linked glycans O-linked glycansO-linked glycans –O-GalNAc – “mucin” type glycosylation –O-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules –O-GlcNAc – intracellular protein regulation Glycan BiosynthesisGlycan Biosynthesis –General Principles –N-linked glycoprotein biosynthesis Roles of glycoprotein glycosylationRoles of glycoprotein glycosylation Modes of glycoprotein glycosylationModes of glycoprotein glycosylation N-linked glycansN-linked glycans O-linked glycansO-linked glycans –O-GalNAc – “mucin” type glycosylation –O-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules –O-GlcNAc – intracellular protein regulation Glycan BiosynthesisGlycan Biosynthesis –General Principles –N-linked glycoprotein biosynthesis

43. Glycoproteins Roles of glycoprotein glycosylationRoles of glycoprotein glycosylation Modes of glycoprotein glycosylationModes of glycoprotein glycosylation N-linked glycansN-linked glycans O-linked glycansO-linked glycans –O-GalNAc – “mucin” type glycosylation –O-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules –O-GlcNAc – intracellular protein regulation Glycan BiosynthesisGlycan Biosynthesis –General Principles –N-linked glycoprotein biosynthesis Roles of glycoprotein glycosylationRoles of glycoprotein glycosylation Modes of glycoprotein glycosylationModes of glycoprotein glycosylation N-linked glycansN-linked glycans O-linked glycansO-linked glycans –O-GalNAc – “mucin” type glycosylation –O-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules –O-GlcNAc – intracellular protein regulation Glycan BiosynthesisGlycan Biosynthesis –General Principles –N-linked glycoprotein biosynthesis

44. Glycosyltransferase reaction (UDP-Gal:glucose β4 galactosyltransferase) nucleotide sugar donor acceptor glycoside +

45. Sugar donors http://www.cazypedia.org/index.php/Glycosyltransferase

46. http://www.cazypedia.org/index.php/Glycosyltransferase Additional donor intermediates

47. Diversity in glycosyltransferases – Example: 20 human sialyltransfearse genes “St3Gal ” genes (6) “St6GalNAc ” genes (6) “St6Gal ” genes (2) “St8Sia ” genes (6) General rule: at least one distinct enzyme for every linkage

48. Glycoprotein biosynthesis N-Linked – en-bloc preconstructed core, trimming, terminal elaborationN-Linked – en-bloc preconstructed core, trimming, terminal elaboration O-Linked - sugar by sugar additionO-Linked - sugar by sugar addition O-GlcNAc - dynamic transferase/glycosidaseO-GlcNAc - dynamic transferase/glycosidase Glycolipid biosynthesis Stepwise sugar-by-sugar additionStepwise sugar-by-sugar addition GPI Anchor – en-bloc preconstructed core, elaborationGPI Anchor – en-bloc preconstructed core, elaboration Proteoglycan biosynthesis Stepwise sugar-by-sugar addition (core and repeating disaccharide)Stepwise sugar-by-sugar addition (core and repeating disaccharide) Post-polymerization modificationsPost-polymerization modifications

49. N-linked glycosylation starts with a lipid-linked intermediate: Glc 3 Man 9 GlcNAc 2 -P-P-Dol

50. N-linked glycosylation: Core biosynthesis and transfer Freeze & Aebi (2005) Curr Opin Struct Biol 15:490

51. N-linked glycosylation: Oligosaccharyltransferase (OST) Mohorko et al (2011) J Inherit Metab Dis 34:869

52. N-linked glycosylation: Trimming and terminal elaboration Freeze & Aebi (2005) Curr Opin Struct Biol 15:490

53. Courtesy Hudson H. Freeze, Burnham Institute, La Jolla, CA Congenital Disorders of Glycosylation (CDG)

54. Freeze & Aebi (2005) Curr Opin Struct Biol 15:490

55. Congenital Disorders of Glycosylation (CDG) Freeze & Aebi (2005) Curr Opin Struct Biol 15:490

56. Glycoproteins Roles of glycoprotein glycosylationRoles of glycoprotein glycosylation Modes of glycoprotein glycosylationModes of glycoprotein glycosylation N-linked glycansN-linked glycans O-linked glycansO-linked glycans –O-GalNAc – “mucin” type glycosylation –O-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules –O-GlcNAc – intracellular protein regulation Glycan BiosynthesisGlycan Biosynthesis –General Principles –N-linked glycoprotein biosynthesis Roles of glycoprotein glycosylationRoles of glycoprotein glycosylation Modes of glycoprotein glycosylationModes of glycoprotein glycosylation N-linked glycansN-linked glycans O-linked glycansO-linked glycans –O-GalNAc – “mucin” type glycosylation –O-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules –O-GlcNAc – intracellular protein regulation Glycan BiosynthesisGlycan Biosynthesis –General Principles –N-linked glycoprotein biosynthesis