1. Introduction to glycobiology

2. “What the matter, Sal, not up on your glycobiology? H Ford (2010) Extraordinary Measures, CBS Films, Los Angeles, CA, USA

3. Introduction to glycobiology Learn the basic language of glycobiologyLearn the basic language of glycobiology Monosaccharides (vertebrate) Glycans (oligosaccharide & glycoconjugate basic concepts) Learn basic glycan functionsLearn basic glycan functions Glycans in glycoprotein activity, folding, trafficking O-GlcNAc-mediated regulation Glycolipids in membrane recognition & regulation Proteoglycans in the extracellular matrix Glycan binding proteins Learn the basic concepts of glycan biosynthesisLearn the basic concepts of glycan biosynthesis Learn the basic tools of glycomics (analysis)Learn the basic tools of glycomics (analysis) Learn the basic language of glycobiologyLearn the basic language of glycobiology Monosaccharides (vertebrate) Glycans (oligosaccharide & glycoconjugate basic concepts) Learn basic glycan functionsLearn basic glycan functions Glycans in glycoprotein activity, folding, trafficking O-GlcNAc-mediated regulation Glycolipids in membrane recognition & regulation Proteoglycans in the extracellular matrix Glycan binding proteins Learn the basic concepts of glycan biosynthesisLearn the basic concepts of glycan biosynthesis Learn the basic tools of glycomics (analysis)Learn the basic tools of glycomics (analysis) Course Objectives

4. Introduction to glycobiology DateDayLectureFaculty Mar 16 MonSaccharides & The Glyco WorldDr. R. Schnaar Mar 18 WedGlycoproteins 1Dr. N. Zachara Mar 20 FriGlycoproteins 2Dr. N. Zachara Mar 23 MonHyaluronan and ProteoglycansDr. N. Zachara Mar 25 WedGlycolipids and GPI anchorsDr. R. Schnaar Mar 27 FriO-GlcNAcDr. G. Hart Mar 30 MonCarbohydrate EngineeringDr. K. Yarema Apr 1 WedProtein-Glycan RecognitionDr. R. Schnaar Apr 3 FriGlycan Binding Protein FunctionsDr. R. Schnaar Apr 6 MonGlycomics (Analytical Glycobiology)Dr. G. Hart Apr 8 WedGlycans and DiseaseDr. G. Hart

5. Introduction to glycobiology Course co-directors: Ronald Schnaar (schnaar@jhu.edu) Ronald Schnaar (schnaar@jhu.edu) Natasha Zachara (nzachara@jhmi.edu) Natasha Zachara (nzachara@jhmi.edu)Lecturers: Gerald Hart (gwhart@jhmi.edu) Kevin Yarema (kyarema1@jhu.edu)

6. Introduction to glycobiology Oxford University Press, New York Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Content freely available at: http://www.ncbi.nlm.nih.gov/books/NBK1908

7. What are: Glycobiology?Glycoscience?Glycomics? Glycobiology?Glycoscience?Glycomics?

8. Glycobiology The study of the biological functions, structures, recognition and biosynthesis of glycans (sugar chains, saccharides) in the context of the biological scaffolds to which they are attached (e.g. glycolipids & glycoproteins). Glycobiology

9. Glycoscience Glycobiology & Glycochemistry, including… - Chemical synthesis - Chemoenzymatic synthesis - Metabolic engineering - Glycomimetics - Glycan conformation and structure - Material Sciences - Plant and bacterial polysaccharides - Chemical synthesis - Chemoenzymatic synthesis - Metabolic engineering - Glycomimetics - Glycan conformation and structure - Material Sciences - Plant and bacterial polysaccharidesGlycoscience Glycobiology & Glycochemistry, including… - Chemical synthesis - Chemoenzymatic synthesis - Metabolic engineering - Glycomimetics - Glycan conformation and structure - Material Sciences - Plant and bacterial polysaccharides - Chemical synthesis - Chemoenzymatic synthesis - Metabolic engineering - Glycomimetics - Glycan conformation and structure - Material Sciences - Plant and bacterial polysaccharides

10. Glycomics - Analytical glycosciences - Glycan bioinformatics - Analytical glycosciences - Glycan bioinformatics “Glycome” – The total complement of glycans in a cell or organism “Glycome” – The total complement of glycans in a cell or organismGlycomics - Analytical glycosciences - Glycan bioinformatics - Analytical glycosciences - Glycan bioinformatics “Glycome” – The total complement of glycans in a cell or organism “Glycome” – The total complement of glycans in a cell or organism

11. Glycobiology is … NOT Carbohydrates as food NOT Carbohydrates as food Glycobiology is … NOT Carbohydrates as food NOT Carbohydrates as food Berg, Tymoczko & Stryer (2006) Biochemistry, Sixth Edition, W.H. Freeman, New York “Carbohydrates in food are important sources of energy. Starch found in plant-derived food such as pasta....”

12. Glycobiology is NOT “a” post-translational modification... it is thousands Modified from http://www-che.syr.edu/ faculty/hougland.html Combinatorial linkage position, orientation and branching provide the potential for millions* of different glycan structures – functional diversityCombinatorial linkage position, orientation and branching provide the potential for millions* of different glycan structures – functional diversity Glycans may be larger and are more diverse than their (protein) carriersGlycans may be larger and are more diverse than their (protein) carriers A glycan’s function can supersede that of its (protein) carrierA glycan’s function can supersede that of its (protein) carrier 1-2% of the human genome is devoted to glycosylation1-2% of the human genome is devoted to glycosylation Cummings RD (2009) Molecular BioSystems 5, 1087 *Glycoproteins and glycolipids may contain ~3000 glycan determinants with an additional ~4000 theoretical petasaccharide sequeces in glycosaminoglycans

13. Glycobiology is … NOT a “decoration” “Structure” of HIV gp120 Zolla-Pazner (2004) Nature Reviews Immunology 4, 199 N-linked carbohydrate can form both an immunologically silent face—with carbohydrate masquerading as "self"— and also can protect neighboring epitopes through an "evolving glycan shield" http://www.niaid.nih.gov/labsandresources/labs/aboutlabs/vrc/ structuralbiologylaboratory/Pages/kwong.aspx

14. CD59, a complement defense glycoprotein 20 kDa protein 3 kDa N-linked glycan 1 kDa O-linked glycan 1.5 kDa GPI anchor Proteins typically fold inward, whereas glycans spread out in space “naked” CD59, Huang et al (2007) Acta Crystallographica 63, 714 Rudd et al (1997) J Biol Chem 272, 7229

15. Scanning electron microscopy of C. neoformans yeast cells. Van Duin et al. (2004) Antimicrobial Agents and Chemotherapy 48:2014 Light microscopy micrograph of Cryptococcus neoformans Steenbergen et al. (2003) Microbes and Infection 5:667 What if a MAJOR cell component was invisible by standard microscopy? Light microscopy micrograph of Cryptococcus neoformans capsule delineated by India ink. The inner circle represents the fungal cell, with the wide outer circle being the capsule. Steenbergen et al. (2003) Microbes and Infection 5:667

16. Electron microscopic thin section of Escherichia coli K1 Amako et al. (1988) J Bacteriol 170:4960 Unique to yeast – NO! Electron microscopic thin section of Klebsiella pneumoniae Amako et al. (1988) J Bacteriol 170:4960

17. The Eukaryotic Cell Surface Tiny sugar “decorations”.. added as an afterthought Lodish, et al. (1995) Molecular Cell Biology 3rd ed. ?

18. The “glycocalyx” surrounding a fibroblast. Cell surface carbohydrates are stained black. lipid bilayer fibroblast extracellular intracellular Martinez-Palomo, A., et al. Cancer Res. 29, 925-937, 1969 The Cell Surface -- The Real Picture

19. Tropical Forest Canopy Cohen & Varki (2010) OMICS 4:455 “Evolution has failed to generate a living cell devoid of surface glycosylation” - A. Varki

20. lipid bilayer fibroblastintracellular Martinez-Palomo, A., et al. Cancer Res. 29, 925-937, 1969 The Cell Surface Real image textbook image Among their diverse functions, glycans …Among their diverse functions, glycans … –encode intermolecular and cell-cell recognition –regulate the activities of proteins (enzymes, ion channels, receptors) –Define biophysical space (large, hydrated)

21. Glycobiology = Language* *semiotics: the theory of signs and symbols

22. SaccharidesSaccharides Carbohydrates: The language of glycobiologyCarbohydrates: The language of glycobiology The glycosidic bond and glycan nomenclatureThe glycosidic bond and glycan nomenclature Major vertebrate glycan classesMajor vertebrate glycan classes Carbohydrates: The language of glycobiologyCarbohydrates: The language of glycobiology The glycosidic bond and glycan nomenclatureThe glycosidic bond and glycan nomenclature Major vertebrate glycan classesMajor vertebrate glycan classes

23. SaccharidesSaccharides Carbohydrates: The language of glycobiologyCarbohydrates: The language of glycobiology The glycosidic bond and glycan nomenclatureThe glycosidic bond and glycan nomenclature Major vertebrate glycan classesMajor vertebrate glycan classes Carbohydrates: The language of glycobiologyCarbohydrates: The language of glycobiology The glycosidic bond and glycan nomenclatureThe glycosidic bond and glycan nomenclature Major vertebrate glycan classesMajor vertebrate glycan classes

24. Carbohydrates – the building blocks of Glycobiology (CH 2 O) n = “carbo” “hydrate” Generalized Ketose Generalized Aldose

25. glucose fructose

26. Enantiomers: mirror images of each other that are not superimposable. Diastereomers: stereoisomers that are not enantiomers. Generalized Aldose Monosaccharide structural identity is all about stereochemistry

27. Fischer projection D-glyceraldehyde

28. L-glyceraldehydeD-glyceraldehydeEnantiomers * * * Highest numbered asymmetric carbon = reference carbon

29. Diasteriomers * * *

30. Enantiomers & Diasteriomers * Highest numbered asymmetric carbon = reference carbon * * * * carbons asymmetric carbons diaster- iomers diasteriomers & enantiomers 3102 4224 5348 64816

31. Essentials of Glycobiology Second Edition ** * * * epimers: differ at a single stereogenic center

32. Monosaccharides equilibrate between open chain and ring forms. Two ring forms (anomeric configurations) are possible, α and β Taylor & Drickamer (2011) Introduction to Glycobiology, 3 rd edition D-glucose α-D-glucose β-D-glucose

33. Monosaccharide conformation – Chair configuations Taylor & Drickamer (2006) Introduction to Glycobiology, 2 nd edition

34. Nearly all vertebrate glycans are built from only 9 sugars: 3 hexoses GlucoseGlucose MannoseMannose GalactoseGalactose 2 N-acetylhexosamines N-acetylglucosamineN-acetylglucosamine N-acetylgalactosamineN-acetylgalactosamine xylosexylose glucuronic acidglucuronic acid sialic acidsialic acid L-fucoseL-fucose *all D configuation except fucose

35. Seven eukaryotic sugars and their relationship to glucose Taylor & Drickamer (2011) Introduction to Glycobiology, 3 rd edition

36. Two important terminal eukaryotic sugars Taylor & Drickamer (2011) Introduction to Glycobiology, 3 rd edition

37. Glycan properties for molecular recognition and binding energy Glucose Galactose Sialic Acid Mannose

38. SaccharidesSaccharides Carbohydrates: The language of glycobiologyCarbohydrates: The language of glycobiology The glycosidic bond and glycan nomenclatureThe glycosidic bond and glycan nomenclature Major vertebrate glycan classesMajor vertebrate glycan classes Carbohydrates: The language of glycobiologyCarbohydrates: The language of glycobiology The glycosidic bond and glycan nomenclatureThe glycosidic bond and glycan nomenclature Major vertebrate glycan classesMajor vertebrate glycan classes

39. SaccharidesSaccharides Carbohydrates: The language of glycobiologyCarbohydrates: The language of glycobiology The glycosidic bond and glycan nomenclatureThe glycosidic bond and glycan nomenclature Major vertebrate glycan classesMajor vertebrate glycan classes Carbohydrates: The language of glycobiologyCarbohydrates: The language of glycobiology The glycosidic bond and glycan nomenclatureThe glycosidic bond and glycan nomenclature Major vertebrate glycan classesMajor vertebrate glycan classes

40. When a glycosidic bond is formed the anomeric configuration is “locked” Glycosidic bonds link sugars together Taylor & Drickamer (2011) Introduction to Glycobiology, 3 rd edition (lactose) CCRC

41. Nomenclature: -Name the non-reducing (left-most) sugar (Gal) -Name the anomeric configuration (β) -Name the anomeric carbon number (1) -Name the substituted carbon number (4) -Name the substituted sugar (Glc) -Name the non-reducing (left-most) sugar (Gal) -Name the anomeric configuration (β) -Name the anomeric carbon number (1) -Name the substituted carbon number (4) -Name the substituted sugar (Glc) RESULT: Gal β1-4 Glc Taylor & Drickamer (2011) Introduction to Glycobiology, 3 rd edition

42. NeuAc α2-3 Gal β1-4 (Fuc α1-3) GlcNAc Nomenclature: Branches are placed in parentheses

43. Varki et al. (2009) Proteomics. 9:5398

44. Oligosaccharides: Molecular diversity of glycans PolypeptidesGlycans Building blocks amino acids monosaccharides Number of different monomers 20 common 9 common Linkage sites per monomer 13-4 Possible linkage configurations 12 Possible homodimer structures 16-8 Linkage modes linear linear or branched

45. Three different amino acids (Ala, Ser, Tyr) – 6 structures Ala-Ser-TyrAla-Tyr-SerSer-Ala-TyrSer-Tyr-AlaTyr-Ala-SerTyr-Ser-Ala Three different sugars (Glc, Gal, Man) – 1,056 structures …

46. Gal a1-2 Glc a1-2 Man a1- Gal a1-3 Glc a1-2 Man a1- Gal a1-4 Glc a1-2 Man a1- Gal a1-6 Glc a1-2 Man a1- Gal b1-2 Glc a1-2 Man a1- Gal b1-3 Glc a1-2 Man a1- Gal b1-4 Glc a1-2 Man a1- Gal b1-6 Glc a1-2 Man a1- Gal a1-2 Glc a1-3 Man a1- Gal a1-3 Glc a1-3 Man a1- Gal a1-4 Glc a1-3 Man a1- Gal a1-6 Glc a1-3 Man a1- Gal b1-2 Glc a1-3 Man a1- Gal b1-3 Glc a1-3 Man a1- Gal b1-4 Glc a1-3 Man a1- Gal b1-6 Glc a1-3 Man a1- Gal a1-2 Glc a1-4 Man a1- Gal a1-3 Glc a1-4 Man a1- Gal a1-4 Glc a1-4 Man a1- Gal a1-6 Glc a1-4 Man a1- Gal b1-2 Glc a1-4 Man a1- Gal b1-3 Glc a1-4 Man a1- Gal b1-4 Glc a1-4 Man a1- Gal b1-6 Glc a1-4 Man a1- Gal a1-2 Glc a1-6 Man a1- Gal a1-3 Glc a1-6 Man a1- Gal a1-4 Glc a1-6 Man a1- Gal a1-6 Glc a1-6 Man a1- Gal b1-2 Glc a1-6 Man a1- Gal b1-3 Glc a1-6 Man a1- Gal b1-4 Glc a1-6 Man a1- Gal b1-6 Glc a1-6 Man a1- Gal a1-2 Glc b1-2 Man a1- Gal a1-3 Glc b1-2 Man a1- Gal a1-4 Glc b1-2 Man a1- Gal a1-6 Glc b1-2 Man a1- Gal b1-2 Glc b1-2 Man a1- Gal b1-3 Glc b1-2 Man a1- Gal b1-4 Glc b1-2 Man a1- Gal b1-6 Glc b1-2 Man a1- Gal a1-2 Glc b1-3 Man a1- Gal a1-3 Glc b1-3 Man a1- Gal a1-4 Glc b1-3 Man a1- Gal a1-6 Glc b1-3 Man a1- Gal b1-2 Glc b1-3 Man a1- Gal b1-3 Glc b1-3 Man a1- Gal b1-4 Glc b1-3 Man a1- Gal b1-6 Glc b1-3 Man a1- Gal a1-2 Glc b1-4 Man a1- Gal a1-3 Glc b1-4 Man a1- Gal a1-4 Glc b1-4 Man a1- Gal a1-6 Glc b1-4 Man a1- Gal b1-2 Glc b1-4 Man a1- Gal b1-3 Glc b1-4 Man a1- Gal b1-4 Glc b1-4 Man a1- Gal b1-6 Glc b1-4 Man a1- Gal a1-2 Glc b1-6 Man a1- Gal a1-3 Glc b1-6 Man a1- Gal a1-4 Glc b1-6 Man a1- Gal a1-6 Glc b1-6 Man a1- Gal b1-2 Glc b1-6 Man a1- Gal b1-3 Glc b1-6 Man a1- Gal b1-4 Glc b1-6 Man a1- Gal b1-6 Glc b1-6 Man a1- Gal a1-2 Glc a1-2 Man b1- Gal a1-3 Glc a1-2 Man b1- Gal a1-4 Glc a1-2 Man b1- Gal a1-6 Glc a1-2 Man b1- Gal b1-2 Glc a1-2 Man b1- Gal b1-3 Glc a1-2 Man b1- Gal b1-4 Glc a1-2 Man b1- Gal b1-6 Glc a1-2 Man b1- Gal a1-2 Glc a1-3 Man b1- Gal a1-3 Glc a1-3 Man b1- Gal a1-4 Glc a1-3 Man b1- Gal a1-6 Glc a1-3 Man b1- Gal b1-2 Glc a1-3 Man b1- Gal b1-3 Glc a1-3 Man b1- Gal b1-4 Glc a1-3 Man b1- Gal b1-6 Glc a1-3 Man b1- Gal a1-2 Glc a1-4 Man b1- Gal a1-3 Glc a1-4 Man b1- Gal a1-4 Glc a1-4 Man b1- Gal a1-6 Glc a1-4 Man b1- Gal b1-2 Glc a1-4 Man b1- Gal b1-3 Glc a1-4 Man b1- Gal b1-4 Glc a1-4 Man b1- Gal b1-6 Glc a1-4 Man b1- Gal a1-2 Glc a1-6 Man b1- Gal a1-3 Glc a1-6 Man b1- Gal a1-4 Glc a1-6 Man b1- Gal a1-6 Glc a1-6 Man b1- Gal b1-2 Glc a1-6 Man b1- Gal b1-3 Glc a1-6 Man b1- Gal b1-4 Glc a1-6 Man b1- Gal b1-6 Glc a1-6 Man b1- Gal a1-2 Glc b1-2 Man b1- Gal a1-3 Glc b1-2 Man b1- Gal a1-4 Glc b1-2 Man b1- Gal a1-6 Glc b1-2 Man b1- Gal b1-2 Glc b1-2 Man b1- Gal b1-3 Glc b1-2 Man b1- Gal b1-4 Glc b1-2 Man b1- Gal b1-6 Glc b1-2 Man b1- Gal a1-2 Glc b1-3 Man b1- Gal a1-3 Glc b1-3 Man b1- Gal a1-4 Glc b1-3 Man b1- Gal a1-6 Glc b1-3 Man b1- Gal b1-2 Glc b1-3 Man b1- Gal b1-3 Glc b1-3 Man b1- Gal b1-4 Glc b1-3 Man b1- Gal b1-6 Glc b1-3 Man b1- Gal a1-2 Glc b1-4 Man b1- Gal a1-3 Glc b1-4 Man b1- Gal a1-4 Glc b1-4 Man b1- Gal a1-6 Glc b1-4 Man b1- Gal b1-2 Glc b1-4 Man b1- Gal b1-3 Glc b1-4 Man b1- Gal b1-4 Glc b1-4 Man b1- Gal b1-6 Glc b1-4 Man b1- Gal a1-2 Glc b1-6 Man b1- Gal a1-3 Glc b1-6 Man b1- Gal a1-4 Glc b1-6 Man b1- Gal a1-6 Glc b1-6 Man b1- Gal b1-2 Glc b1-6 Man b1- Gal b1-3 Glc b1-6 Man b1- Gal b1-4 Glc b1-6 Man b1- Gal b1-6 Glc b1-6 Man b1- Gal a1-2 Man a1-2 Glc a1- Gal a1-3 Man a1-2 Glc a1- Gal a1-4 Man a1-2 Glc a1- Gal a1-6 Man a1-2 Glc a1- Gal b1-2 Man a1-2 Glc a1- Gal b1-3 Man a1-2 Glc a1- Gal b1-4 Man a1-2 Glc a1- Gal b1-6 Man a1-2 Glc a1- Gal a1-2 Man a1-3 Glc a1- Gal a1-3 Man a1-3 Glc a1- Gal a1-4 Man a1-3 Glc a1- Gal a1-6 Man a1-3 Glc a1- Gal b1-2 Man a1-3 Glc a1- Gal b1-3 Man a1-3 Glc a1- Gal b1-4 Man a1-3 Glc a1- Gal b1-6 Man a1-3 Glc a1- Gal a1-2 Man a1-4 Glc a1- Gal a1-3 Man a1-4 Glc a1- Gal a1-4 Man a1-4 Glc a1- Gal a1-6 Man a1-4 Glc a1- Gal b1-2 Man a1-4 Glc a1- Gal b1-3 Man a1-4 Glc a1- Gal b1-4 Man a1-4 Glc a1- Gal b1-6 Man a1-4 Glc a1- Gal a1-2 Man a1-6 Glc a1- Gal a1-3 Man a1-6 Glc a1- Gal a1-4 Man a1-6 Glc a1- Gal a1-6 Man a1-6 Glc a1- Gal b1-2 Man a1-6 Glc a1- Gal b1-3 Man a1-6 Glc a1- Gal b1-4 Man a1-6 Glc a1- Gal b1-6 Man a1-6 Glc a1- Gal a1-2 Man b1-2 Glc a1- Gal a1-3 Man b1-2 Glc a1- Gal a1-4 Man b1-2 Glc a1- Gal a1-6 Man b1-2 Glc a1- Gal b1-2 Man b1-2 Glc a1- Gal b1-3 Man b1-2 Glc a1- Gal b1-4 Man b1-2 Glc a1- Gal b1-6 Man b1-2 Glc a1- Gal a1-2 Man b1-3 Glc a1- Gal a1-3 Man b1-3 Glc a1- Gal a1-4 Man b1-3 Glc a1- Gal a1-6 Man b1-3 Glc a1- Gal b1-2 Man b1-3 Glc a1- Gal b1-3 Man b1-3 Glc a1- Gal b1-4 Man b1-3 Glc a1- Gal b1-6 Man b1-3 Glc a1- Gal a1-2 Man b1-4 Glc a1- Gal a1-3 Man b1-4 Glc a1- Gal a1-4 Man b1-4 Glc a1- Gal a1-6 Man b1-4 Glc a1- Gal b1-2 Man b1-4 Glc a1- Gal b1-3 Man b1-4 Glc a1- Gal b1-4 Man b1-4 Glc a1- Gal b1-6 Man b1-4 Glc a1- Gal a1-2 Man b1-6 Glc a1- Gal a1-3 Man b1-6 Glc a1- Gal a1-4 Man b1-6 Glc a1- Gal a1-6 Man b1-6 Glc a1- Gal b1-2 Man b1-6 Glc a1- Gal b1-3 Man b1-6 Glc a1- Gal b1-4 Man b1-6 Glc a1- Gal b1-6 Man b1-6 Glc a1- Gal a1-2 Man a1-2 Glc b1- Gal a1-3 Man a1-2 Glc b1- Gal a1-4 Man a1-2 Glc b1- Gal a1-6 Man a1-2 Glc b1- Gal b1-2 Man a1-2 Glc b1- Gal b1-3 Man a1-2 Glc b1- Gal b1-4 Man a1-2 Glc b1- Gal b1-6 Man a1-2 Glc b1- Gal a1-2 Man a1-3 Glc b1- Gal a1-3 Man a1-3 Glc b1- Gal a1-4 Man a1-3 Glc b1- Gal a1-6 Man a1-3 Glc b1- Gal b1-2 Man a1-3 Glc b1- Gal b1-3 Man a1-3 Glc b1- Gal b1-4 Man a1-3 Glc b1- Gal b1-6 Man a1-3 Glc b1- Gal a1-2 Man a1-4 Glc b1- Gal a1-3 Man a1-4 Glc b1- Gal a1-4 Man a1-4 Glc b1- Gal a1-6 Man a1-4 Glc b1- Gal b1-2 Man a1-4 Glc b1- Gal b1-3 Man a1-4 Glc b1- Gal b1-4 Man a1-4 Glc b1- Gal b1-6 Man a1-4 Glc b1- Gal a1-2 Man a1-6 Glc b1- Gal a1-3 Man a1-6 Glc b1- Gal a1-4 Man a1-6 Glc b1- Gal a1-6 Man a1-6 Glc b1- Gal b1-2 Man a1-6 Glc b1- Gal b1-3 Man a1-6 Glc b1- Gal b1-4 Man a1-6 Glc b1- Gal b1-6 Man a1-6 Glc b1- Gal a1-2 Man b1-2 Glc b1- Gal a1-3 Man b1-2 Glc b1- Gal a1-4 Man b1-2 Glc b1- Gal a1-6 Man b1-2 Glc b1- Gal b1-2 Man b1-2 Glc b1- Gal b1-3 Man b1-2 Glc b1- Gal b1-4 Man b1-2 Glc b1- Gal b1-6 Man b1-2 Glc b1- Gal a1-2 Man b1-3 Glc b1- Gal a1-3 Man b1-3 Glc b1- Gal a1-4 Man b1-3 Glc b1- Gal a1-6 Man b1-3 Glc b1- Gal b1-2 Man b1-3 Glc b1- Gal b1-3 Man b1-3 Glc b1- Gal b1-4 Man b1-3 Glc b1- Gal b1-6 Man b1-3 Glc b1- Gal a1-2 Man b1-4 Glc b1- Gal a1-3 Man b1-4 Glc b1- Gal a1-4 Man b1-4 Glc b1- Gal a1-6 Man b1-4 Glc b1- Gal b1-2 Man b1-4 Glc b1- Gal b1-3 Man b1-4 Glc b1- Gal b1-4 Man b1-4 Glc b1- Gal b1-6 Man b1-4 Glc b1- Gal a1-2 Man b1-6 Glc b1- Gal a1-3 Man b1-6 Glc b1- Gal a1-4 Man b1-6 Glc b1- Gal a1-6 Man b1-6 Glc b1- Gal b1-2 Man b1-6 Glc b1- Gal b1-3 Man b1-6 Glc b1- Gal b1-4 Man b1-6 Glc b1- Gal b1-6 Man b1-6 Glc b1- Man a1-2 Glc a1-2 Gal a1- Man a1-3 Glc a1-2 Gal a1- Man a1-4 Glc a1-2 Gal a1- Man a1-6 Glc a1-2 Gal a1- Man b1-2 Glc a1-2 Gal a1- Man b1-3 Glc a1-2 Gal a1- Man b1-4 Glc a1-2 Gal a1- Man b1-6 Glc a1-2 Gal a1- Man a1-2 Glc a1-3 Gal a1- Man a1-3 Glc a1-3 Gal a1- Man a1-4 Glc a1-3 Gal a1- Man a1-6 Glc a1-3 Gal a1- Man b1-2 Glc a1-3 Gal a1- Man b1-3 Glc a1-3 Gal a1- Man b1-4 Glc a1-3 Gal a1- Man b1-6 Glc a1-3 Gal a1- Man a1-2 Glc a1-4 Gal a1- Man a1-3 Glc a1-4 Gal a1- Man a1-4 Glc a1-4 Gal a1- Man a1-6 Glc a1-4 Gal a1- Man b1-2 Glc a1-4 Gal a1- Man b1-3 Glc a1-4 Gal a1- Man b1-4 Glc a1-4 Gal a1- Man b1-6 Glc a1-4 Gal a1- Man a1-2 Glc a1-6 Gal a1- Man a1-3 Glc a1-6 Gal a1- Man a1-4 Glc a1-6 Gal a1- Man a1-6 Glc a1-6 Gal a1- Man b1-2 Glc a1-6 Gal a1- Man b1-3 Glc a1-6 Gal a1- Man b1-4 Glc a1-6 Gal a1- Man b1-6 Glc a1-6 Gal a1- Man a1-2 Glc b1-2 Gal a1- Man a1-3 Glc b1-2 Gal a1- Man a1-4 Glc b1-2 Gal a1- Man a1-6 Glc b1-2 Gal a1- Man b1-2 Glc b1-2 Gal a1- Man b1-3 Glc b1-2 Gal a1- Man b1-4 Glc b1-2 Gal a1- Man b1-6 Glc b1-2 Gal a1- Man a1-2 Glc b1-3 Gal a1- Man a1-3 Glc b1-3 Gal a1- Man a1-4 Glc b1-3 Gal a1- Man a1-6 Glc b1-3 Gal a1- Man b1-2 Glc b1-3 Gal a1- Man b1-3 Glc b1-3 Gal a1- Man b1-4 Glc b1-3 Gal a1- Man b1-6 Glc b1-3 Gal a1- Man a1-2 Glc b1-4 Gal a1- Man a1-3 Glc b1-4 Gal a1- Man a1-4 Glc b1-4 Gal a1- Man a1-6 Glc b1-4 Gal a1- Man b1-2 Glc b1-4 Gal a1- Man b1-3 Glc b1-4 Gal a1- Man b1-4 Glc b1-4 Gal a1- Man b1-6 Glc b1-4 Gal a1- Man a1-2 Glc b1-6 Gal a1- Man a1-3 Glc b1-6 Gal a1- Man a1-4 Glc b1-6 Gal a1- Man a1-6 Glc b1-6 Gal a1- Man b1-2 Glc b1-6 Gal a1- Man b1-3 Glc b1-6 Gal a1- Man b1-4 Glc b1-6 Gal a1- Man b1-6 Glc b1-6 Gal a1- Man a1-2 Glc a1-2 Gal b1- Man a1-3 Glc a1-2 Gal b1- Man a1-4 Glc a1-2 Gal b1- Man a1-6 Glc a1-2 Gal b1- Man b1-2 Glc a1-2 Gal b1- Man b1-3 Glc a1-2 Gal b1- Man b1-4 Glc a1-2 Gal b1- Man b1-6 Glc a1-2 Gal b1- Man a1-2 Glc a1-3 Gal b1- Man a1-3 Glc a1-3 Gal b1- Man a1-4 Glc a1-3 Gal b1- Man a1-6 Glc a1-3 Gal b1- Man b1-2 Glc a1-3 Gal b1- Man b1-3 Glc a1-3 Gal b1- Man b1-4 Glc a1-3 Gal b1- Man b1-6 Glc a1-3 Gal b1- Man a1-2 Glc a1-4 Gal b1- Man a1-3 Glc a1-4 Gal b1- Man a1-4 Glc a1-4 Gal b1- Man a1-6 Glc a1-4 Gal b1- Man b1-2 Glc a1-4 Gal b1- Man b1-3 Glc a1-4 Gal b1- Man b1-4 Glc a1-4 Gal b1- Man b1-6 Glc a1-4 Gal b1- Man a1-2 Glc a1-6 Gal b1- Man a1-3 Glc a1-6 Gal b1- Man a1-4 Glc a1-6 Gal b1- Man a1-6 Glc a1-6 Gal b1- Man b1-2 Glc a1-6 Gal b1- Man b1-3 Glc a1-6 Gal b1- Man b1-4 Glc a1-6 Gal b1- Man b1-6 Glc a1-6 Gal b1- Man a1-2 Glc b1-2 Gal b1- Man a1-3 Glc b1-2 Gal b1- Man a1-4 Glc b1-2 Gal b1- Man a1-6 Glc b1-2 Gal b1- Man b1-2 Glc b1-2 Gal b1- Man b1-3 Glc b1-2 Gal b1- Man b1-4 Glc b1-2 Gal b1- Man b1-6 Glc b1-2 Gal b1- Man a1-2 Glc b1-3 Gal b1- Man a1-3 Glc b1-3 Gal b1- Man a1-4 Glc b1-3 Gal b1- Man a1-6 Glc b1-3 Gal b1- Man b1-2 Glc b1-3 Gal b1- Man b1-3 Glc b1-3 Gal b1- Man b1-4 Glc b1-3 Gal b1- Man b1-6 Glc b1-3 Gal b1- Man a1-2 Glc b1-4 Gal b1- Man a1-3 Glc b1-4 Gal b1- Man a1-4 Glc b1-4 Gal b1- Man a1-6 Glc b1-4 Gal b1- Man b1-2 Glc b1-4 Gal b1- Man b1-3 Glc b1-4 Gal b1- Man b1-4 Glc b1-4 Gal b1- Man b1-6 Glc b1-4 Gal b1- Man a1-2 Glc b1-6 Gal b1- Man a1-3 Glc b1-6 Gal b1- Man a1-4 Glc b1-6 Gal b1- Man a1-6 Glc b1-6 Gal b1- Man b1-2 Glc b1-6 Gal b1- Man b1-3 Glc b1-6 Gal b1- Man b1-4 Glc b1-6 Gal b1- Man b1-6 Glc b1-6 Gal b1- Man a1-2 Gal a1-2 Glc a1- Man a1-3 Gal a1-2 Glc a1- Man a1-4 Gal a1-2 Glc a1- Man a1-6 Gal a1-2 Glc a1- Man b1-2 Gal a1-2 Glc a1- Man b1-3 Gal a1-2 Glc a1- Man b1-4 Gal a1-2 Glc a1- Man b1-6 Gal a1-2 Glc a1- Man a1-2 Gal a1-3 Glc a1- Man a1-3 Gal a1-3 Glc a1- Man a1-4 Gal a1-3 Glc a1- Man a1-6 Gal a1-3 Glc a1- Man b1-2 Gal a1-3 Glc a1- Man b1-3 Gal a1-3 Glc a1- Man b1-4 Gal a1-3 Glc a1- Man b1-6 Gal a1-3 Glc a1- Man a1-2 Gal a1-4 Glc a1- Man a1-3 Gal a1-4 Glc a1- Man a1-4 Gal a1-4 Glc a1- Man a1-6 Gal a1-4 Glc a1- Man b1-2 Gal a1-4 Glc a1- Man b1-3 Gal a1-4 Glc a1- Man b1-4 Gal a1-4 Glc a1- Man b1-6 Gal a1-4 Glc a1- Man a1-2 Gal a1-6 Glc a1- Man a1-3 Gal a1-6 Glc a1- Man a1-4 Gal a1-6 Glc a1- Man a1-6 Gal a1-6 Glc a1- Man b1-2 Gal a1-6 Glc a1- Man b1-3 Gal a1-6 Glc a1- Man b1-4 Gal a1-6 Glc a1- Man b1-6 Gal a1-6 Glc a1- Man a1-2 Gal b1-2 Glc a1- Man a1-3 Gal b1-2 Glc a1- Man a1-4 Gal b1-2 Glc a1- Man a1-6 Gal b1-2 Glc a1- Man b1-2 Gal b1-2 Glc a1- Man b1-3 Gal b1-2 Glc a1- Man b1-4 Gal b1-2 Glc a1- Man b1-6 Gal b1-2 Glc a1- Man a1-2 Gal b1-3 Glc a1- Man a1-3 Gal b1-3 Glc a1- Man a1-4 Gal b1-3 Glc a1- Man a1-6 Gal b1-3 Glc a1- Man b1-2 Gal b1-3 Glc a1- Man b1-3 Gal b1-3 Glc a1- Man b1-4 Gal b1-3 Glc a1- Man b1-6 Gal b1-3 Glc a1- Man a1-2 Gal b1-4 Glc a1- Man a1-3 Gal b1-4 Glc a1- Man a1-4 Gal b1-4 Glc a1- Man a1-6 Gal b1-4 Glc a1- Man b1-2 Gal b1-4 Glc a1- Man b1-3 Gal b1-4 Glc a1- Man b1-4 Gal b1-4 Glc a1- Man b1-6 Gal b1-4 Glc a1- Man a1-2 Gal b1-6 Glc a1- Man a1-3 Gal b1-6 Glc a1- Man a1-4 Gal b1-6 Glc a1- Man a1-6 Gal b1-6 Glc a1- Man b1-2 Gal b1-6 Glc a1- Man b1-3 Gal b1-6 Glc a1- Man b1-4 Gal b1-6 Glc a1- Man b1-6 Gal b1-6 Glc a1- Man a1-2 Gal a1-2 Glc b1 Man a1-3 Gal a1-2 Glc b1 Man a1-4 Gal a1-2 Glc b1 Man a1-6 Gal a1-2 Glc b1 Man b1-2 Gal a1-2 Glc b1 Man b1-3 Gal a1-2 Glc b1 Man b1-4 Gal a1-2 Glc b1 Man b1-6 Gal a1-2 Glc b1 Man a1-2 Gal a1-3 Glc b1 Man a1-3 Gal a1-3 Glc b1 Man a1-4 Gal a1-3 Glc b1 Man a1-6 Gal a1-3 Glc b1 Man b1-2 Gal a1-3 Glc b1 Man b1-3 Gal a1-3 Glc b1 Man b1-4 Gal a1-3 Glc b1 Man b1-6 Gal a1-3 Glc b1 Man a1-2 Gal a1-4 Glc b1 Man a1-3 Gal a1-4 Glc b1 Man a1-4 Gal a1-4 Glc b1 Man a1-6 Gal a1-4 Glc b1 Man b1-2 Gal a1-4 Glc b1 Man b1-3 Gal a1-4 Glc b1 Man b1-4 Gal a1-4 Glc b1 Man b1-6 Gal a1-4 Glc b1 Man a1-2 Gal a1-6 Glc b1 Man a1-3 Gal a1-6 Glc b1 Man a1-4 Gal a1-6 Glc b1 Man a1-6 Gal a1-6 Glc b1 Man b1-2 Gal a1-6 Glc b1 Man b1-3 Gal a1-6 Glc b1 Man b1-4 Gal a1-6 Glc b1 Man b1-6 Gal a1-6 Glc b1 Man a1-2 Gal b1-2 Glc b1 Man a1-3 Gal b1-2 Glc b1 Man a1-4 Gal b1-2 Glc b1 Man a1-6 Gal b1-2 Glc b1 Man b1-2 Gal b1-2 Glc b1 Man b1-3 Gal b1-2 Glc b1 Man b1-4 Gal b1-2 Glc b1 Man b1-6 Gal b1-2 Glc b1 Man a1-2 Gal b1-3 Glc b1 Man a1-3 Gal b1-3 Glc b1 Man a1-4 Gal b1-3 Glc b1 Man a1-6 Gal b1-3 Glc b1 Man b1-2 Gal b1-3 Glc b1 Man b1-3 Gal b1-3 Glc b1 Man b1-4 Gal b1-3 Glc b1 Man b1-6 Gal b1-3 Glc b1 Man a1-2 Gal b1-4 Glc b1 Man a1-3 Gal b1-4 Glc b1 Man a1-4 Gal b1-4 Glc b1 Man a1-6 Gal b1-4 Glc b1 Man b1-2 Gal b1-4 Glc b1 Man b1-3 Gal b1-4 Glc b1 Man b1-4 Gal b1-4 Glc b1 Man b1-6 Gal b1-4 Glc b1 Man a1-2 Gal b1-6 Glc b1 Man a1-3 Gal b1-6 Glc b1 Man a1-4 Gal b1-6 Glc b1 Man a1-6 Gal b1-6 Glc b1 Man b1-2 Gal b1-6 Glc b1 Man b1-3 Gal b1-6 Glc b1 Man b1-4 Gal b1-6 Glc b1 Man b1-6 Gal b1-6 Glc b1 Glc a1-2 Man a1-2 Gal a1- Glc a1-3 Man a1-2 Gal a1- Glc a1-4 Man a1-2 Gal a1- Glc a1-6 Man a1-2 Gal a1- Glc b1-2 Man a1-2 Gal a1- Glc b1-3 Man a1-2 Gal a1- Glc b1-4 Man a1-2 Gal a1- Glc b1-6 Man a1-2 Gal a1- Glc a1-2 Man a1-3 Gal a1- Glc a1-3 Man a1-3 Gal a1- Glc a1-4 Man a1-3 Gal a1- Glc a1-6 Man a1-3 Gal a1- Glc b1-2 Man a1-3 Gal a1- Glc b1-3 Man a1-3 Gal a1- Glc b1-4 Man a1-3 Gal a1- Glc b1-6 Man a1-3 Gal a1- Glc a1-2 Man a1-4 Gal a1- Glc a1-3 Man a1-4 Gal a1- Glc a1-4 Man a1-4 Gal a1- Glc a1-6 Man a1-4 Gal a1- Glc b1-2 Man a1-4 Gal a1- Glc b1-3 Man a1-4 Gal a1- Glc b1-4 Man a1-4 Gal a1- Glc b1-6 Man a1-4 Gal a1- Glc a1-2 Man a1-6 Gal a1- Glc a1-3 Man a1-6 Gal a1- Glc a1-4 Man a1-6 Gal a1- Glc a1-6 Man a1-6 Gal a1- Glc b1-2 Man a1-6 Gal a1- Glc b1-3 Man a1-6 Gal a1- Glc b1-4 Man a1-6 Gal a1- Glc b1-6 Man a1-6 Gal a1- Glc a1-2 Man b1-2 Gal a1- Glc a1-3 Man b1-2 Gal a1- Glc a1-4 Man b1-2 Gal a1- Glc a1-6 Man b1-2 Gal a1- Glc b1-2 Man b1-2 Gal a1- Glc b1-3 Man b1-2 Gal a1- Glc b1-4 Man b1-2 Gal a1- Glc b1-6 Man b1-2 Gal a1- Glc a1-2 Man b1-3 Gal a1- Glc a1-3 Man b1-3 Gal a1- Glc a1-4 Man b1-3 Gal a1- Glc a1-6 Man b1-3 Gal a1- Glc b1-2 Man b1-3 Gal a1- Glc b1-3 Man b1-3 Gal a1- Glc b1-4 Man b1-3 Gal a1- Glc b1-6 Man b1-3 Gal a1- Glc a1-2 Man b1-4 Gal a1- Glc a1-3 Man b1-4 Gal a1- Glc a1-4 Man b1-4 Gal a1- Glc a1-6 Man b1-4 Gal a1- Glc b1-2 Man b1-4 Gal a1- Glc b1-3 Man b1-4 Gal a1- Glc b1-4 Man b1-4 Gal a1- Glc b1-6 Man b1-4 Gal a1- Glc a1-2 Man b1-6 Gal a1- Glc a1-3 Man b1-6 Gal a1- Glc a1-4 Man b1-6 Gal a1- Glc a1-6 Man b1-6 Gal a1- Glc b1-2 Man b1-6 Gal a1- Glc b1-3 Man b1-6 Gal a1- Glc b1-4 Man b1-6 Gal a1- Glc b1-6 Man b1-6 Gal a1- Glc a1-2 Man a1-2 Gal b1- Glc a1-3 Man a1-2 Gal b1- Glc a1-4 Man a1-2 Gal b1- Glc a1-6 Man a1-2 Gal b1- Glc b1-2 Man a1-2 Gal b1- Glc b1-3 Man a1-2 Gal b1- Glc b1-4 Man a1-2 Gal b1- Glc b1-6 Man a1-2 Gal b1- Glc a1-2 Man a1-3 Gal b1- Glc a1-3 Man a1-3 Gal b1- Glc a1-4 Man a1-3 Gal b1- Glc a1-6 Man a1-3 Gal b1- Glc b1-2 Man a1-3 Gal b1- Glc b1-3 Man a1-3 Gal b1- Glc b1-4 Man a1-3 Gal b1- Glc b1-6 Man a1-3 Gal b1- Glc a1-2 Man a1-4 Gal b1- Glc a1-3 Man a1-4 Gal b1- Glc a1-4 Man a1-4 Gal b1- Glc a1-6 Man a1-4 Gal b1- Glc b1-2 Man a1-4 Gal b1- Glc b1-3 Man a1-4 Gal b1- Glc b1-4 Man a1-4 Gal b1- Glc b1-6 Man a1-4 Gal b1- Glc a1-2 Man a1-6 Gal b1- Glc a1-3 Man a1-6 Gal b1- Glc a1-4 Man a1-6 Gal b1- Glc a1-6 Man a1-6 Gal b1- Glc b1-2 Man a1-6 Gal b1- Glc b1-3 Man a1-6 Gal b1- Glc b1-4 Man a1-6 Gal b1- Glc b1-6 Man a1-6 Gal b1- Glc a1-2 Man b1-2 Gal b1- Glc a1-3 Man b1-2 Gal b1- Glc a1-4 Man b1-2 Gal b1- Glc a1-6 Man b1-2 Gal b1- Glc b1-2 Man b1-2 Gal b1- Glc b1-3 Man b1-2 Gal b1- Glc b1-4 Man b1-2 Gal b1- Glc b1-6 Man b1-2 Gal b1- Glc a1-2 Man b1-3 Gal b1- Glc a1-3 Man b1-3 Gal b1- Glc a1-4 Man b1-3 Gal b1- Glc a1-6 Man b1-3 Gal b1- Glc b1-2 Man b1-3 Gal b1- Glc b1-3 Man b1-3 Gal b1- Glc b1-4 Man b1-3 Gal b1- Glc b1-6 Man b1-3 Gal b1- Glc a1-2 Man b1-4 Gal b1- Glc a1-3 Man b1-4 Gal b1- Glc a1-4 Man b1-4 Gal b1- Glc a1-6 Man b1-4 Gal b1- Glc b1-2 Man b1-4 Gal b1- Glc b1-3 Man b1-4 Gal b1- Glc b1-4 Man b1-4 Gal b1- Glc b1-6 Man b1-4 Gal b1- Glc a1-2 Man b1-6 Gal b1- Glc a1-3 Man b1-6 Gal b1- Glc a1-4 Man b1-6 Gal b1- Glc a1-6 Man b1-6 Gal b1- Glc b1-2 Man b1-6 Gal b1- Glc b1-3 Man b1-6 Gal b1- Glc b1-4 Man b1-6 Gal b1- Glc b1-6 Man b1-6 Gal b1- Glc a1-2 Gal a1-2 Man a1- Glc a1-3 Gal a1-2 Man a1- Glc a1-4 Gal a1-2 Man a1- Glc a1-6 Gal a1-2 Man a1- Glc b1-2 Gal a1-2 Man a1- Glc b1-3 Gal a1-2 Man a1- Glc b1-4 Gal a1-2 Man a1- Glc b1-6 Gal a1-2 Man a1- Glc a1-2 Gal a1-3 Man a1- Glc a1-3 Gal a1-3 Man a1- Glc a1-4 Gal a1-3 Man a1- Glc a1-6 Gal a1-3 Man a1- Glc b1-2 Gal a1-3 Man a1- Glc b1-3 Gal a1-3 Man a1- Glc b1-4 Gal a1-3 Man a1- Glc b1-6 Gal a1-3 Man a1- Glc a1-2 Gal a1-4 Man a1- Glc a1-3 Gal a1-4 Man a1- Glc a1-4 Gal a1-4 Man a1- Glc a1-6 Gal a1-4 Man a1- Glc b1-2 Gal a1-4 Man a1- Glc b1-3 Gal a1-4 Man a1- Glc b1-4 Gal a1-4 Man a1- Glc b1-6 Gal a1-4 Man a1- Glc a1-2 Gal a1-6 Man a1- Glc a1-3 Gal a1-6 Man a1- Glc a1-4 Gal a1-6 Man a1- Glc a1-6 Gal a1-6 Man a1- Glc b1-2 Gal a1-6 Man a1- Glc b1-3 Gal a1-6 Man a1- Glc b1-4 Gal a1-6 Man a1- Glc b1-6 Gal a1-6 Man a1- Glc a1-2 Gal b1-2 Man a1- Glc a1-3 Gal b1-2 Man a1- Glc a1-4 Gal b1-2 Man a1- Glc a1-6 Gal b1-2 Man a1- Glc b1-2 Gal b1-2 Man a1- Glc b1-3 Gal b1-2 Man a1- Glc b1-4 Gal b1-2 Man a1- Glc b1-6 Gal b1-2 Man a1- Glc a1-2 Gal b1-3 Man a1- Glc a1-3 Gal b1-3 Man a1- Glc a1-4 Gal b1-3 Man a1- Glc a1-6 Gal b1-3 Man a1- Glc b1-2 Gal b1-3 Man a1- Glc b1-3 Gal b1-3 Man a1- Glc b1-4 Gal b1-3 Man a1- Glc b1-6 Gal b1-3 Man a1- Glc a1-2 Gal b1-4 Man a1- Glc a1-3 Gal b1-4 Man a1- Glc a1-4 Gal b1-4 Man a1- Glc a1-6 Gal b1-4 Man a1- Glc b1-2 Gal b1-4 Man a1- Glc b1-3 Gal b1-4 Man a1- Glc b1-4 Gal b1-4 Man a1- Glc b1-6 Gal b1-4 Man a1- Glc a1-2 Gal b1-6 Man a1- Glc a1-3 Gal b1-6 Man a1- Glc a1-4 Gal b1-6 Man a1- Glc a1-6 Gal b1-6 Man a1- Glc b1-2 Gal b1-6 Man a1- Glc b1-3 Gal b1-6 Man a1- Glc b1-4 Gal b1-6 Man a1- Glc b1-6 Gal b1-6 Man a1- Glc a1-2 Gal a1-2 Man b1- Glc a1-3 Gal a1-2 Man b1- Glc a1-4 Gal a1-2 Man b1- Glc a1-6 Gal a1-2 Man b1- Glc b1-2 Gal a1-2 Man b1- Glc b1-3 Gal a1-2 Man b1- Glc b1-4 Gal a1-2 Man b1- Glc b1-6 Gal a1-2 Man b1- Glc a1-2 Gal a1-3 Man b1- Glc a1-3 Gal a1-3 Man b1- Glc a1-4 Gal a1-3 Man b1- Glc a1-6 Gal a1-3 Man b1- Glc b1-2 Gal a1-3 Man b1- Glc b1-3 Gal a1-3 Man b1- Glc b1-4 Gal a1-3 Man b1- Glc b1-6 Gal a1-3 Man b1- Glc a1-2 Gal a1-4 Man b1- Glc a1-3 Gal a1-4 Man b1- Glc a1-4 Gal a1-4 Man b1- Glc a1-6 Gal a1-4 Man b1- Glc b1-2 Gal a1-4 Man b1- Glc b1-3 Gal a1-4 Man b1- Glc b1-4 Gal a1-4 Man b1- Glc b1-6 Gal a1-4 Man b1- Glc a1-2 Gal a1-6 Man b1- Glc a1-3 Gal a1-6 Man b1- Glc a1-4 Gal a1-6 Man b1- Glc a1-6 Gal a1-6 Man b1- Glc b1-2 Gal a1-6 Man b1- Glc b1-3 Gal a1-6 Man b1- Glc b1-4 Gal a1-6 Man b1- Glc b1-6 Gal a1-6 Man b1- Glc a1-2 Gal b1-2 Man b1- Glc a1-3 Gal b1-2 Man b1- Glc a1-4 Gal b1-2 Man b1- Glc a1-6 Gal b1-2 Man b1- Glc b1-2 Gal b1-2 Man b1- Glc b1-3 Gal b1-2 Man b1- Glc b1-4 Gal b1-2 Man b1- Glc b1-6 Gal b1-2 Man b1- Glc a1-2 Gal b1-3 Man b1- Glc a1-3 Gal b1-3 Man b1- Glc a1-4 Gal b1-3 Man b1- Glc a1-6 Gal b1-3 Man b1- Glc b1-2 Gal b1-3 Man b1- Glc b1-3 Gal b1-3 Man b1- Glc b1-4 Gal b1-3 Man b1- Glc b1-6 Gal b1-3 Man b1- Glc a1-2 Gal b1-4 Man b1- Glc a1-3 Gal b1-4 Man b1- Glc a1-4 Gal b1-4 Man b1- Glc a1-6 Gal b1-4 Man b1- Glc b1-2 Gal b1-4 Man b1- Glc b1-3 Gal b1-4 Man b1- Glc b1-4 Gal b1-4 Man b1- Glc b1-6 Gal b1-4 Man b1- Glc a1-2 Gal b1-6 Man b1- Glc a1-3 Gal b1-6 Man b1- Glc a1-4 Gal b1-6 Man b1- Glc a1-6 Gal b1-6 Man b1- Glc b1-2 Gal b1-6 Man b1- Glc b1-3 Gal b1-6 Man b1- Glc b1-4 Gal b1-6 Man b1- Glc b1-6 Gal b1-6 Man b1- Gal a1-2 (Glc a1-3) Man a1- Gal a1-2 (Glc a1-4) Man a1- Gal a1-2 (Glc a1-6) Man a1- Gal a1-3 (Glc a1-2) Man a1- Gal a1-3 (Glc a1-4) Man a1- Gal a1-3 (Glc a1-6) Man a1- Gal a1-4 (Glc a1-2) Man a1- Gal a1-4 (Glc a1-3) Man a1- Gal a1-4 (Glc a1-6) Man a1- Gal a1-6 (Glc a1-2) Man a1- Gal a1-6 (Glc a1-3) Man a1- Gal a1-6 (Glc a1-4) Man a1- Gal b1-2 (Glc a1-3) Man a1- Gal b1-2 (Glc a1-4) Man a1- Gal b1-2 (Glc a1-6) Man a1- Gal b1-3 (Glc a1-2) Man a1- Gal b1-3 (Glc a1-4) Man a1- Gal b1-3 (Glc a1-6) Man a1- Gal b1-4 (Glc a1-2) Man a1- Gal b1-4 (Glc a1-3) Man a1- Gal b1-4 (Glc a1-6) Man a1- Gal b1-6 (Glc a1-2) Man a1- Gal b1-6 (Glc a1-3) Man a1- Gal b1-6 (Glc a1-4) Man a1- Gal a1-2 (Glc b1-3) Man a1- Gal a1-2 (Glc b1-4) Man a1- Gal a1-2 (Glc b1-6) Man a1- Gal a1-3 (Glc b1-2) Man a1- Gal a1-3 (Glc b1-4) Man a1- Gal a1-3 (Glc b1-6) Man a1- Gal a1-4 (Glc b1-2) Man a1- Gal a1-4 (Glc b1-3) Man a1- Gal a1-4 (Glc b1-6) Man a1- Gal a1-6 (Glc b1-2) Man a1- Gal a1-6 (Glc b1-3) Man a1- Gal a1-6 (Glc b1-4) Man a1- Gal b1-2 (Glc b1-3) Man a1- Gal b1-2 (Glc b1-4) Man a1- Gal b1-2 (Glc b1-6) Man a1- Gal b1-3 (Glc b1-2) Man a1- Gal b1-3 (Glc b1-4) Man a1- Gal b1-3 (Glc b1-6) Man a1- Gal b1-4 (Glc b1-2) Man a1- Gal b1-4 (Glc b1-3) Man a1- Gal b1-4 (Glc b1-6) Man a1- Gal b1-6 (Glc b1-2) Man a1- Gal b1-6 (Glc b1-3) Man a1- Gal b1-6 (Glc b1-4) Man a1- Gal a1-2 (Glc a1-3) Man b1- Gal a1-2 (Glc a1-4) Man b1- Gal a1-2 (Glc a1-6) Man b1- Gal a1-3 (Glc a1-2) Man b1- Gal a1-3 (Glc a1-4) Man b1- Gal a1-3 (Glc a1-6) Man b1- Gal a1-4 (Glc a1-2) Man b1- Gal a1-4 (Glc a1-3) Man b1- Gal a1-4 (Glc a1-6) Man b1- Gal a1-6 (Glc a1-2) Man b1- Gal a1-6 (Glc a1-3) Man b1- Gal a1-6 (Glc a1-4) Man b1- Gal b1-2 (Glc a1-3) Man b1- Gal b1-2 (Glc a1-4) Man b1- Gal b1-2 (Glc a1-6) Man b1- Gal b1-3 (Glc a1-2) Man b1- Gal b1-3 (Glc a1-4) Man b1- Gal b1-3 (Glc a1-6) Man b1- Gal b1-4 (Glc a1-2) Man b1- Gal b1-4 (Glc a1-3) Man b1- Gal b1-4 (Glc a1-6) Man b1- Gal b1-6 (Glc a1-2) Man b1- Gal b1-6 (Glc a1-3) Man b1- Gal b1-6 (Glc a1-4) Man b1- Gal a1-2 (Glc b1-3) Man b1- Gal a1-2 (Glc b1-4) Man b1- Gal a1-2 (Glc b1-6) Man b1- Gal a1-3 (Glc b1-2) Man b1- Gal a1-3 (Glc b1-4) Man b1- Gal a1-3 (Glc b1-6) Man b1- Gal a1-4 (Glc b1-2) Man b1- Gal a1-4 (Glc b1-3) Man b1- Gal a1-4 (Glc b1-6) Man b1- Gal a1-6 (Glc b1-2) Man b1- Gal a1-6 (Glc b1-3) Man b1- Gal a1-6 (Glc b1-4) Man b1- Gal b1-2 (Glc b1-3) Man b1- Gal b1-2 (Glc b1-4) Man b1- Gal b1-2 (Glc b1-6) Man b1- Gal b1-3 (Glc b1-2) Man b1- Gal b1-3 (Glc b1-4) Man b1- Gal b1-3 (Glc b1-6) Man b1- Gal b1-4 (Glc b1-2) Man b1- Gal b1-4 (Glc b1-3) Man b1- Gal b1-4 (Glc b1-6) Man b1- Gal b1-6 (Glc b1-2) Man b1- Gal b1-6 (Glc b1-3) Man b1- Gal b1-6 (Glc b1-4) Man b1- Gal a1-2 (Man a1-3) Glc a1- Gal a1-2 (Man a1-4) Glc a1- Gal a1-2 (Man a1-6) Glc a1- Gal a1-3 (Man a1-2) Glc a1- Gal a1-3 (Man a1-4) Glc a1- Gal a1-3 (Man a1-6) Glc a1- Gal a1-4 (Man a1-2) Glc a1- Gal a1-4 (Man a1-3) Glc a1- Gal a1-4 (Man a1-6) Glc a1- Gal a1-6 (Man a1-2) Glc a1- Gal a1-6 (Man a1-3) Glc a1- Gal a1-6 (Man a1-4) Glc a1- Gal b1-2 (Man a1-3) Glc a1- Gal b1-2 (Man a1-4) Glc a1- Gal b1-2 (Man a1-6) Glc a1- Gal b1-3 (Man a1-2) Glc a1- Gal b1-3 (Man a1-4) Glc a1- Gal b1-3 (Man a1-6) Glc a1- Gal b1-4 (Man a1-2) Glc a1- Gal b1-4 (Man a1-3) Glc a1- Gal b1-4 (Man a1-6) Glc a1- Gal b1-6 (Man a1-2) Glc a1- Gal b1-6 (Man a1-3) Glc a1- Gal b1-6 (Man a1-4) Glc a1- Gal a1-2 (Man b1-3) Glc a1- Gal a1-2 (Man b1-4) Glc a1- Gal a1-2 (Man b1-6) Glc a1- Gal a1-3 (Man b1-2) Glc a1- Gal a1-3 (Man b1-4) Glc a1- Gal a1-3 (Man b1-6) Glc a1- Gal a1-4 (Man b1-2) Glc a1- Gal a1-4 (Man b1-3) Glc a1- Gal a1-4 (Man b1-6) Glc a1- Gal a1-6 (Man b1-2) Glc a1- Gal a1-6 (Man b1-3) Glc a1- Gal a1-6 (Man b1-4) Glc a1- Gal b1-2 (Man b1-3) Glc a1- Gal b1-2 (Man b1-4) Glc a1- Gal b1-2 (Man b1-6) Glc a1- Gal b1-3 (Man b1-2) Glc a1- Gal b1-3 (Man b1-4) Glc a1- Gal b1-3 (Man b1-6) Glc a1- Gal b1-4 (Man b1-2) Glc a1- Gal b1-4 (Man b1-3) Glc a1- Gal b1-4 (Man b1-6) Glc a1- Gal b1-6 (Man b1-2) Glc a1- Gal b1-6 (Man b1-3) Glc a1- Gal b1-6 (Man b1-4) Glc a1- Gal a1-2 (Man a1-3) Glc b1- Gal a1-2 (Man a1-4) Glc b1- Gal a1-2 (Man a1-6) Glc b1- Gal a1-3 (Man a1-2) Glc b1- Gal a1-3 (Man a1-4) Glc b1- Gal a1-3 (Man a1-6) Glc b1- Gal a1-4 (Man a1-2) Glc b1- Gal a1-4 (Man a1-3) Glc b1- Gal a1-4 (Man a1-6) Glc b1- Gal a1-6 (Man a1-2) Glc b1- Gal a1-6 (Man a1-3) Glc b1- Gal a1-6 (Man a1-4) Glc b1- Gal b1-2 (Man a1-3) Glc b1- Gal b1-2 (Man a1-4) Glc b1- Gal b1-2 (Man a1-6) Glc b1- Gal b1-3 (Man a1-2) Glc b1- Gal b1-3 (Man a1-4) Glc b1- Gal b1-3 (Man a1-6) Glc b1- Gal b1-4 (Man a1-2) Glc b1- Gal b1-4 (Man a1-3) Glc b1- Gal b1-4 (Man a1-6) Glc b1- Gal b1-6 (Man a1-2) Glc b1- Gal b1-6 (Man a1-3) Glc b1- Gal b1-6 (Man a1-4) Glc b1- Gal a1-2 (Man b1-3) Glc b1- Gal a1-2 (Man b1-4) Glc b1- Gal a1-2 (Man b1-6) Glc b1- Gal a1-3 (Man b1-2) Glc b1- Gal a1-3 (Man b1-4) Glc b1- Gal a1-3 (Man b1-6) Glc b1- Gal a1-4 (Man b1-2) Glc b1- Gal a1-4 (Man b1-3) Glc b1- Gal a1-4 (Man b1-6) Glc b1- Gal a1-6 (Man b1-2) Glc b1- Gal a1-6 (Man b1-3) Glc b1- Gal a1-6 (Man b1-4) Glc b1- Gal b1-2 (Man b1-3) Glc b1- Gal b1-2 (Man b1-4) Glc b1- Gal b1-2 (Man b1-6) Glc b1- Gal b1-3 (Man b1-2) Glc b1- Gal b1-3 (Man b1-4) Glc b1- Gal b1-3 (Man b1-6) Glc b1- Gal b1-4 (Man b1-2) Glc b1- Gal b1-4 (Man b1-3) Glc b1- Gal b1-4 (Man b1-6) Glc b1- Gal b1-6 (Man b1-2) Glc b1- Gal b1-6 (Man b1-3) Glc b1- Gal b1-6 (Man b1-4) Glc b1- Glc a1-2 (Man a1-3) Gal a1- Glc a1-2 (Man a1-4) Gal a1- Glc a1-2 (Man a1-6) Gal a1- Glc a1-3 (Man a1-2) Gal a1- Glc a1-3 (Man a1-4) Gal a1- Glc a1-3 (Man a1-6) Gal a1- Glc a1-4 (Man a1-2) Gal a1- Glc a1-4 (Man a1-3) Gal a1- Glc a1-4 (Man a1-6) Gal a1- Glc a1-6 (Man a1-2) Gal a1- Glc a1-6 (Man a1-3) Gal a1- Glc a1-6 (Man a1-4) Gal a1- Glc b1-2 (Man a1-3) Gal a1- Glc b1-2 (Man a1-4) Gal a1- Glc b1-2 (Man a1-6) Gal a1- Glc b1-3 (Man a1-2) Gal a1- Glc b1-3 (Man a1-4) Gal a1- Glc b1-3 (Man a1-6) Gal a1- Glc b1-4 (Man a1-2) Gal a1- Glc b1-4 (Man a1-3) Gal a1- Glc b1-4 (Man a1-6) Gal a1- Glc b1-6 (Man a1-2) Gal a1- Glc b1-6 (Man a1-3) Gal a1- Glc b1-6 (Man a1-4) Gal a1- Glc a1-2 (Man b1-3) Gal a1- Glc a1-2 (Man b1-4) Gal a1- Glc a1-2 (Man b1-6) Gal a1- Glc a1-3 (Man b1-2) Gal a1- Glc a1-3 (Man b1-4) Gal a1- Glc a1-3 (Man b1-6) Gal a1- Glc a1-4 (Man b1-2) Gal a1- Glc a1-4 (Man b1-3) Gal a1- Glc a1-4 (Man b1-6) Gal a1- Glc a1-6 (Man b1-2) Gal a1- Glc a1-6 (Man b1-3) Gal a1- Glc a1-6 (Man b1-4) Gal a1- Glc b1-2 (Man b1-3) Gal a1- Glc b1-2 (Man b1-4) Gal a1- Glc b1-2 (Man b1-6) Gal a1- Glc b1-3 (Man b1-2) Gal a1- Glc b1-3 (Man b1-4) Gal a1- Glc b1-3 (Man b1-6) Gal a1- Glc b1-4 (Man b1-2) Gal a1- Glc b1-4 (Man b1-3) Gal a1- Glc b1-4 (Man b1-6) Gal a1- Glc b1-6 (Man b1-2) Gal a1- Glc b1-6 (Man b1-3) Gal a1- Glc b1-6 (Man b1-4) Gal a1- Glc a1-2 (Man a1-3) Gal b1- Glc a1-2 (Man a1-4) Gal b1- Glc a1-2 (Man a1-6) Gal b1- Glc a1-3 (Man a1-2) Gal b1- Glc a1-3 (Man a1-4) Gal b1- Glc a1-3 (Man a1-6) Gal b1- Glc a1-4 (Man a1-2) Gal b1- Glc a1-4 (Man a1-3) Gal b1- Glc a1-4 (Man a1-6) Gal b1- Glc a1-6 (Man a1-2) Gal b1- Glc a1-6 (Man a1-3) Gal b1- Glc a1-6 (Man a1-4) Gal b1- Glc b1-2 (Man a1-3) Gal b1- Glc b1-2 (Man a1-4) Gal b1- Glc b1-2 (Man a1-6) Gal b1- Glc b1-3 (Man a1-2) Gal b1- Glc b1-3 (Man a1-4) Gal b1- Glc b1-3 (Man a1-6) Gal b1- Glc b1-4 (Man a1-2) Gal b1- Glc b1-4 (Man a1-3) Gal b1- Glc b1-4 (Man a1-6) Gal b1- Glc b1-6 (Man a1-2) Gal b1- Glc b1-6 (Man a1-3) Gal b1- Glc b1-6 (Man a1-4) Gal b1- Glc a1-2 (Man b1-3) Gal b1- Glc a1-2 (Man b1-4) Gal b1- Glc a1-2 (Man b1-6) Gal b1- Glc a1-3 (Man b1-2) Gal b1- Glc a1-3 (Man b1-4) Gal b1- Glc a1-3 (Man b1-6) Gal b1- Glc a1-4 (Man b1-2) Gal b1- Glc a1-4 (Man b1-3) Gal b1- Glc a1-4 (Man b1-6) Gal b1- Glc a1-6 (Man b1-2) Gal b1- Glc a1-6 (Man b1-3) Gal b1- Glc a1-6 (Man b1-4) Gal b1- Glc b1-2 (Man b1-3) Gal b1- Glc b1-2 (Man b1-4) Gal b1- Glc b1-2 (Man b1-6) Gal b1- Glc b1-3 (Man b1-2) Gal b1- Glc b1-3 (Man b1-4) Gal b1- Glc b1-3 (Man b1-6) Gal b1- Glc b1-4 (Man b1-2) Gal b1- Glc b1-4 (Man b1-3) Gal b1- Glc b1-4 (Man b1-6) Gal b1- Glc b1-6 (Man b1-2) Gal b1- Glc b1-6 (Man b1-3) Gal b1- Glc b1-6 (Man b1-4) Gal b1- Man a1-4 Gal a1-3 Glc b1 Man a1-6 Gal a1-3 Glc b1 Man b1-2 Gal a1-3 Glc b1 Man b1-3 Gal a1-3 Glc b1 Man b1-4 Gal a1-3 Glc b1 Man b1-6 Gal a1-3 Glc b1 Man a1-2 Gal a1-4 Glc b1 Man a1-3 Gal a1-4 Glc b1 Man a1-4 Gal a1-4 Glc b1 Man a1-6 Gal a1-4 Glc b1 Man b1-2 Gal a1-4 Glc b1 Man b1-3 Gal a1-4 Glc b1 Man b1-4 Gal a1-4 Glc b1 Man b1-6 Gal a1-4 Glc b1 Man a1-2 Gal a1-6 Glc b1 Man a1-3 Gal a1-6 Glc b1 Cummings RD (2009) Molecular BioSystems 5, 1087 *Glycoproteins and glycolipids may contain ~3000 glycan determinants with an additional ~4000 theoretical petasaccharide sequeces in glycosaminoglycans

47. SaccharidesSaccharides Carbohydrates: The language of glycobiologyCarbohydrates: The language of glycobiology The glycosidic bond and glycan nomenclatureThe glycosidic bond and glycan nomenclature Major vertebrate glycan classesMajor vertebrate glycan classes Carbohydrates: The language of glycobiologyCarbohydrates: The language of glycobiology The glycosidic bond and glycan nomenclatureThe glycosidic bond and glycan nomenclature Major vertebrate glycan classesMajor vertebrate glycan classes

48. SaccharidesSaccharides Carbohydrates: The language of glycobiologyCarbohydrates: The language of glycobiology The glycosidic bond and glycan nomenclatureThe glycosidic bond and glycan nomenclature Major vertebrate glycan classesMajor vertebrate glycan classes Carbohydrates: The language of glycobiologyCarbohydrates: The language of glycobiology The glycosidic bond and glycan nomenclatureThe glycosidic bond and glycan nomenclature Major vertebrate glycan classesMajor vertebrate glycan classes

49. Major Glycans of Eukaryotes GlycoproteinsGlycoproteins GlycolipidsGlycolipids ProteoglycansProteoglycans

50. Major Glycans of Eukaryotes GlycoproteinsGlycoproteins GlycolipidsGlycolipids ProteoglycansProteoglycans

51. Glycoproteins N-LinkedN-Linked O-LinkedO-Linked GPI AnchorGPI Anchor O-GlcNAcO-GlcNAc *A non-inclusive list!

52. Glycoproteins N-LinkedN-Linked O-LinkedO-Linked GPI AnchorGPI Anchor O-GlcNAcO-GlcNAc *A non-inclusive list!

53. “N-linked” glycan linkage to protein asparagine residues

54. Families of N-linked glycan structures

55. 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 = polylactosamine repeats (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

56. Glycoproteins N-LinkedN-Linked O-LinkedO-Linked GPI AnchorGPI Anchor O-GlcNAcO-GlcNAc *A non-inclusive list!

57. Glycoproteins N-LinkedN-Linked O-LinkedO-Linked GPI AnchorGPI Anchor O-GlcNAcO-GlcNAc *A non-inclusive list!

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

59. Examples of variations of O-linked glycans

60. Glycoproteins N-LinkedN-Linked O-LinkedO-Linked GPI AnchorGPI Anchor O-GlcNAcO-GlcNAc *A non-inclusive list!

61. Glycoproteins N-LinkedN-Linked O-LinkedO-Linked GPI AnchorGPI Anchor O-GlcNAcO-GlcNAc *A non-inclusive list!

62. GPI (glycosylphosphatidylinositol) anchored protein

63. 20 kDa protein 3 kDa N-linked glycan 1 kDa O-linked glycan 1.5 kDa GPI anchor plasma membrane CD59, a complement defense glycoprotein

64. Glycoproteins N-LinkedN-Linked O-LinkedO-Linked GPI AnchorGPI Anchor O-GlcNAcO-GlcNAc *A non-inclusive list!

65. Glycoproteins N-LinkedN-Linked O-LinkedO-Linked GPI AnchorGPI Anchor O-GlcNAcO-GlcNAc *A non-inclusive list!

66. O-GlcNAc Note: No further sugar substitutions have been confirmed Highly dynamic post-translational modification!

67. Major Glycans of Eukaryotes GlycoproteinsGlycoproteins GlycolipidsGlycolipids ProteoglycansProteoglycans

68. Major Glycans of Eukaryotes GlycoproteinsGlycoproteins GlycolipidsGlycolipids ProteoglycansProteoglycans

69. A Glycosphingolipid Galactosylceramide (GalCer)

70. A Ganglioside “GD1a”

71. Major Glycans of Eukaryotes GlycoproteinsGlycoproteins GlycolipidsGlycolipids ProteoglycansProteoglycans

72. Major Glycans of Eukaryotes GlycoproteinsGlycoproteins GlycolipidsGlycolipids ProteoglycansProteoglycans

73. Glycosaminoglycans (GAG’s) and Proteoglycans GLYCOSAMINOGLYCANS (GAG’s)GLYCOSAMINOGLYCANS (GAG’s) –long linear glycans made of repeating disaccharides –Hyaluronic acid is the only “stand-alone” GAG, other GAG’s are constituents of … PROTEOGLYCANSPROTEOGLYCANS –GAG’s on proteins –Defined by their repeating disaccharide units –GAG’s on proteoglycans are sulfated

74. Hyaluronic acid -- a “simple” space filling molecule (GlcU β4 GlcNAc) n

75. Post-polymerization variations in glycosaminoglycans

76. Cartilage proteoglycan

78. Glycan Biosynthesis Glycosyltransferase reaction (UDP-Gal:glucose β4 galactosyltransferase) UDP-GlcUDP-GalGDP-ManUDP-GlcNAcUDP-GalNAcUDP-GlcAUDP-XylGDP-FucCMP-NeuAc Activated Sugars:

79. Glycoprotein biosynthesis N-Linked – en-bloc preconstructed core, trimming, terminal elaborationN-Linked – en-bloc preconstructed core, trimming, terminal elaboration O-Linked – stepwise sugar by sugar additionO-Linked – stepwise sugar by sugar addition O-GlcNAc - dynamic transferase/glycosidaseO-GlcNAc - dynamic transferase/glycosidase GPI Anchor – en-bloc preconstructed core, elaborationGPI Anchor – en-bloc preconstructed core, elaboration Glycolipid biosynthesis Stepwise sugar-by-sugar additionStepwise sugar-by-sugar addition 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

80. Introduction to glycobiology “What the matter, Sal, not up on your glycobiology? H Ford (2010) Extraordinary Measures, CBS Films, Los Angeles, CA, USA

81. H Ford and B Fraser (2010) Extraordinary Measures, CBS Films, Los Angeles, CA, USA

82. Hurler (alpha-L iduronidase) Hunter (iduronate-2- sulfatase) I-cell (GlcNAc phosphotransferase) Lysosomal storage diseases are often due to faulty glycan catabolism

83. Lysosomal storage diseases can be studied using patient cells in culture I-cell lyphocytes van der Meer et al (2001) J Clin Pathol 54:724 I-cell fibroblasts Hanai et al (1971) Am J Dis Child 122:34

84. Fibroblasts from Hunter & Hurler patients build up glycosaminoglycans Co-incubation with normal fibroblasts or each other corrects the buildup… Correction is via a soluble factor (direct contact not required). Co-incubation with normal fibroblasts or each other corrects the buildup… Correction is via a soluble factor (direct contact not required). Fratantoni et al (1968) Science 162:170

85. Corrective factor properties Purified lysosomal hydrolases are taken up by fibroblasts – different enzymes correct storage of different diseasesPurified lysosomal hydrolases are taken up by fibroblasts – different enzymes correct storage of different diseases Periodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptakePeriodate pre-treatment of lysosomal hydrolases (cleaves sugar C-C bonds) inhibited enzyme uptake Enzyme uptake was selectively blocked by mannoseEnzyme uptake was selectively blocked by mannose Enzyme uptake was blocked better mannose-6-phosphateEnzyme uptake was blocked better mannose-6-phosphate Phosphatase pre-treatment of the enzyme blocked its uptakePhosphatase pre-treatment of the enzyme blocked its uptake Fibroblasts do not take up I-cell lysosomal hydrolasesFibroblasts do not take up I-cell lysosomal hydrolases Normal lysosomal hydrolases have mannose-6-phosphate residues (unusual!) on their N-linked glycansNormal lysosomal hydrolases have mannose-6-phosphate residues (unusual!) on their N-linked glycans I-cell patients are deficient in addition of 6-linked phosphate to mannose on their N-linked glycansI-cell patients are deficient in addition of 6-linked phosphate to mannose on their N-linked glycans

86. Lysosomal enzyme targeting Dahms et al (1989) J Biol Chem 264:12115

87. Discovery: A unique and specific modification of lysosomal hydrolases for their trafficking to lysosomes Bohnsack et al (2009) J Biol Chem 284:35215

88. Sugar-modifying enzyme lacking in I-cell disease: N-acetylglucosamine-1-phosphate transferase Sugar-modifying enzyme lacking in I-cell disease: N-acetylglucosamine-1-phosphate transferase Reproducing this same Man-6-P modification on expressed therapeutic lysosomal hydrolases effectively targets them to the lysosome in vivo

89. H Ford and B Fraser (2010) Extraordinary Measures, CBS Films, Los Angeles, CA, USA William Canfield and John Crowley, Novazyme (http://blog.newok.com)

90. Introduction to glycobiology