Introduction to glycobiology Course Objectives Learn the basic language of glycobiology Monosaccharides (vertebrate) Glycans (oligosaccharide & glycoconjugate basic concepts) Learn 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 biosynthesis Learn the basic tools of glycomics (analysis)

Introduction to glycobiology Date Day Lecture Faculty Mar 27 Mon Saccharides & The Glyco World Dr. R. Schnaar Mar 28 Tue Glycoproteins 1 Dr. N. Zachara Mar 29 Wed Glycoproteins 2 Mar 30 Thu O-GlcNAc Dr. G. Hart Mar 31 Fri Glycolipids and GPI anchors Apr 3 Hyaluronan and Proteoglycans Apr 4 Carbohydrate Engineering Dr. K. Yarema Apr 5 Protein-Glycan Recognition Apr 5* Glycan Binding Protein Functions Apr 6 Glycomics (Analytical Glycobiology) Apr 6* Glycans and Disease

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

Introduction to glycobiology Oxford University Press, 2011 Cold Spring Harbor Laboratory Press, 2008 Content freely available: http://www.ncbi.nlm.nih.gov/books/NBK1908 NOTE: 3rd Edition release date June 2017 https://www.ncbi.nlm.nih.gov/books/NBK310274/

What are: Glycobiology? Glycoscience? Glycomics?

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).

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

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

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

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

Glycobiology is … NOT a “decoration” Zolla-Pazner (2004) Nature Reviews Immunology 4, 199 Guillaume et al (2016) Cell 165:813

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

The mammalian cell surface ? Martinez-Palomo, A., et al. Cancer Res. 29, 925-937, 1969 The “glycocalyx” revealed (glycans stained with ruthinium red) intracellular fibroblast extracellular lipid bilayer intracellular fibroblast extracellular lipid bilayer https://en.wikipedia.org// wiki/Plasma_membrane Krijnse Locker J. & Schmid S.L. PLoS Biol 11, e1001639, 2013 “Every eukaryotic cell is covered with a dense and complex array of glycans.” … in fact… “Evolution has failed to generate a [single] living cell devoid of surface glycosylation” - A. Varki

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 Light microscopy micrograph of Cryptococcus neoformans Steenbergen et al. (2003) Microbes and Infection 5:667 Scanning electron microscopy of C. neoformans yeast cells. Van Duin et al. (2004) Antimicrobial Agents and Chemotherapy 48:2014

Each prokaryote carries a surface glycan coat Electron microscopic thin section of Escherichia coli K1 Amako et al. (1988) J Bacteriol 170:4960 Electron microscopic thin section of Klebsiella pneumoniae Amako et al. (1988) J Bacteriol 170:4960

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

Real image textbook image The Cell Surface Real image textbook image lipid bilayer fibroblast intracellular Martinez-Palomo, A., et al. Cancer Res. 29, 925-937, 1969 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)

Glycobiology = Language* *semiotics: the theory of signs and symbols Rosetta Stone, British Museum

Saccharides Carbohydrates: The language of glycobiology The glycosidic bond and glycan nomenclature Major vertebrate glycan classes

Saccharides Carbohydrates: The language of glycobiology The glycosidic bond and glycan nomenclature Major vertebrate glycan classes

(CH2O)n = “carbo” “hydrate” Carbohydrates – the building blocks of Glycobiology (CH2O)n = “carbo” “hydrate” Generalized Ketose Generalized Aldose

glucose fructose

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

Fischer projection D-glyceraldehyde

Enantiomers * * D-glyceraldehyde L-glyceraldehyde * Highest numbered asymmetric carbon = reference carbon

Diasteriomers * * * Highest numbered asymmetric carbon = reference carbon

diasteriomers & enantiomers Enantiomers & Diasteriomers * * carbons asymmetric carbons diaster- iomers diasteriomers & enantiomers 3 1 2 4 5 8 6 16 * * * Highest numbered asymmetric carbon = reference carbon

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

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

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

Nearly all vertebrate glycans are built from only 9 sugars: 3 hexoses Glucose Mannose Galactose 2 N-acetylhexosamines N-acetylglucosamine N-acetylgalactosamine xylose glucuronic acid sialic acid L-fucose *all D configuation except fucose

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

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

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

Saccharides Carbohydrates: The language of glycobiology The glycosidic bond and glycan nomenclature Major vertebrate glycan classes

Saccharides Carbohydrates: The language of glycobiology The glycosidic bond and glycan nomenclature Major vertebrate glycan classes

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

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) RESULT: Gal β1-4 Glc Taylor & Drickamer (2011) Introduction to Glycobiology, 3rd edition

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

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

Oligosaccharides: Molecular diversity of glycans Polypeptides Glycans Building blocks amino acids monosaccharides Number of different monomers 20 common 9 common Linkage sites per monomer 1 3-4 Possible linkage configurations 2 Possible homodimer structures 6-8 Linkage modes linear linear or branched

Three different amino acids (Ala, Ser, Tyr) – 6 structures Ala-Ser-Tyr    Ala-Ser-Tyr Ala-Tyr-Ser Ser-Ala-Tyr Ser-Tyr-Ala Tyr-Ala-Ser Tyr-Ser-Ala Three different sugars (Glc, Gal, Man) – 1,056 structures …

Man a1-4 Gal a1-3 Glc b1 Man a1-6 Gal a1-3 Glc b1  Gal a1-2 Glc a1-2 Man a1- Gal a1-3 Glc a1-6 Man b1- Gal a1-4 Man b1-4 Glc a1- Man a1-6 Glc a1-3 Gal a1- Man b1-2 Glc b1-2 Gal b1- Man b1-3 Gal b1-6 Glc a1- Glc b1-4 Man a1-4 Gal a1- Glc b1-6 Man b1-3 Gal b1- Glc a1-2 Gal a1-3 Man b1- Gal a1-6 (Glc b1-2) Man a1- Gal a1-2 (Man b1-6) Glc a1- Glc b1-4 (Man a1-3) Gal a1- Gal a1-3 Glc a1-2 Man a1- Gal a1-4 Glc a1-6 Man b1- Gal a1-6 Man b1-4 Glc a1- Man b1-2 Glc a1-3 Gal a1- Man b1-3 Glc b1-2 Gal b1- Man b1-4 Gal b1-6 Glc a1- Glc b1-6 Man a1-4 Gal a1- Glc a1-2 Man b1-4 Gal b1- Glc a1-3 Gal a1-3 Man b1- Gal a1-6 (Glc b1-3) Man a1- Gal a1-3 (Man b1-2) Glc a1- Glc b1-4 (Man a1-6) Gal a1- Gal a1-4 Glc a1-2 Man a1- Gal a1-6 Glc a1-6 Man b1- Gal b1-2 Man b1-4 Glc a1- Man b1-3 Glc a1-3 Gal a1- Man b1-4 Glc b1-2 Gal b1- Man b1-6 Gal b1-6 Glc a1- Glc a1-2 Man a1-6 Gal a1- Glc a1-3 Man b1-4 Gal b1- Glc a1-4 Gal a1-3 Man b1- Gal a1-6 (Glc b1-4) Man a1- Gal a1-3 (Man b1-4) Glc a1- Glc b1-6 (Man a1-2) Gal a1- Gal a1-6 Glc a1-2 Man a1- Gal b1-2 Glc a1-6 Man b1- Gal b1-3 Man b1-4 Glc a1- Man b1-4 Glc a1-3 Gal a1- Man b1-6 Glc b1-2 Gal b1- Man a1-2 Gal a1-2 Glc b1 Glc a1-3 Man a1-6 Gal a1- Glc a1-4 Man b1-4 Gal b1- Glc a1-6 Gal a1-3 Man b1- Gal b1-2 (Glc b1-3) Man a1- Gal a1-3 (Man b1-6) Glc a1- Glc b1-6 (Man a1-3) Gal a1- Gal b1-2 Glc a1-2 Man a1- Gal b1-3 Glc a1-6 Man b1- Gal b1-4 Man b1-4 Glc a1- Man b1-6 Glc a1-3 Gal a1- Man a1-2 Glc b1-3 Gal b1- Man a1-3 Gal a1-2 Glc b1 Glc a1-4 Man a1-6 Gal a1- Glc a1-6 Man b1-4 Gal b1- Glc b1-2 Gal a1-3 Man b1- Gal b1-2 (Glc b1-4) Man a1- Gal a1-4 (Man b1-2) Glc a1- Glc b1-6 (Man a1-4) Gal a1- Gal b1-3 Glc a1-2 Man a1- Gal b1-4 Glc a1-6 Man b1- Gal b1-6 Man b1-4 Glc a1- Man a1-2 Glc a1-4 Gal a1- Man a1-3 Glc b1-3 Gal b1- Man a1-4 Gal a1-2 Glc b1 Glc a1-6 Man a1-6 Gal a1- Glc b1-2 Man b1-4 Gal b1- Glc b1-3 Gal a1-3 Man b1- Gal b1-2 (Glc b1-6) Man a1- Gal a1-4 (Man b1-3) Glc a1- Glc a1-2 (Man b1-3) Gal a1- Gal b1-4 Glc a1-2 Man a1- Gal b1-6 Glc a1-6 Man b1- Gal a1-2 Man b1-6 Glc a1- Man a1-3 Glc a1-4 Gal a1- Man a1-4 Glc b1-3 Gal b1- Man a1-6 Gal a1-2 Glc b1 Glc b1-2 Man a1-6 Gal a1- Glc b1-3 Man b1-4 Gal b1- Glc b1-4 Gal a1-3 Man b1- Gal b1-3 (Glc b1-2) Man a1- Gal a1-4 (Man b1-6) Glc a1- Glc a1-2 (Man b1-4) Gal a1- Gal b1-6 Glc a1-2 Man a1- Gal a1-2 Glc b1-2 Man b1- Gal a1-3 Man b1-6 Glc a1- Man a1-4 Glc a1-4 Gal a1- Man a1-6 Glc b1-3 Gal b1- Man b1-2 Gal a1-2 Glc b1 Glc b1-3 Man a1-6 Gal a1- Glc b1-4 Man b1-4 Gal b1- Glc b1-6 Gal a1-3 Man b1- Gal b1-3 (Glc b1-4) Man a1- Gal a1-6 (Man b1-2) Glc a1- Glc a1-2 (Man b1-6) Gal a1- Gal a1-2 Glc a1-3 Man a1- Gal a1-3 Glc b1-2 Man b1- Gal a1-4 Man b1-6 Glc a1- Man a1-6 Glc a1-4 Gal a1- Man b1-2 Glc b1-3 Gal b1- Man b1-3 Gal a1-2 Glc b1 Glc b1-4 Man a1-6 Gal a1- Glc b1-6 Man b1-4 Gal b1- Glc a1-2 Gal a1-4 Man b1- Gal b1-3 (Glc b1-6) Man a1- Gal a1-6 (Man b1-3) Glc a1- Glc a1-3 (Man b1-2) Gal a1- Gal a1-3 Glc a1-3 Man a1- Gal a1-4 Glc b1-2 Man b1- Gal a1-6 Man b1-6 Glc a1- Man b1-2 Glc a1-4 Gal a1- Man b1-3 Glc b1-3 Gal b1- Man b1-4 Gal a1-2 Glc b1 Glc b1-6 Man a1-6 Gal a1- Glc a1-2 Man b1-6 Gal b1- Glc a1-3 Gal a1-4 Man b1- Gal b1-4 (Glc b1-2) Man a1- Gal a1-6 (Man b1-4) Glc a1- Glc a1-3 (Man b1-4) Gal a1- Gal a1-4 Glc a1-3 Man a1- Gal a1-6 Glc b1-2 Man b1- Gal b1-2 Man b1-6 Glc a1- Man b1-3 Glc a1-4 Gal a1- Man b1-4 Glc b1-3 Gal b1- Man b1-6 Gal a1-2 Glc b1 Glc a1-2 Man b1-2 Gal a1- Glc a1-3 Man b1-6 Gal b1- Glc a1-4 Gal a1-4 Man b1- Gal b1-4 (Glc b1-3) Man a1- Gal b1-2 (Man b1-3) Glc a1- Glc a1-3 (Man b1-6) Gal a1- Gal a1-6 Glc a1-3 Man a1- Gal b1-2 Glc b1-2 Man b1- Gal b1-3 Man b1-6 Glc a1- Man b1-4 Glc a1-4 Gal a1- Man b1-6 Glc b1-3 Gal b1- Man a1-2 Gal a1-3 Glc b1 Glc a1-3 Man b1-2 Gal a1- Glc a1-4 Man b1-6 Gal b1- Glc a1-6 Gal a1-4 Man b1- Gal b1-4 (Glc b1-6) Man a1- Gal b1-2 (Man b1-4) Glc a1- Glc a1-4 (Man b1-2) Gal a1- Gal b1-2 Glc a1-3 Man a1- Gal b1-3 Glc b1-2 Man b1- Gal b1-4 Man b1-6 Glc a1- Man b1-6 Glc a1-4 Gal a1- Man a1-2 Glc b1-4 Gal b1- Man a1-3 Gal a1-3 Glc b1 Glc a1-4 Man b1-2 Gal a1- Glc a1-6 Man b1-6 Gal b1- Glc b1-2 Gal a1-4 Man b1- Gal b1-6 (Glc b1-2) Man a1- Gal b1-2 (Man b1-6) Glc a1- Glc a1-4 (Man b1-3) Gal a1- Gal b1-3 Glc a1-3 Man a1- Gal b1-4 Glc b1-2 Man b1- Gal b1-6 Man b1-6 Glc a1- Man a1-2 Glc a1-6 Gal a1- Man a1-3 Glc b1-4 Gal b1- Man a1-4 Gal a1-3 Glc b1 Glc a1-6 Man b1-2 Gal a1- Glc b1-2 Man b1-6 Gal b1- Glc b1-3 Gal a1-4 Man b1- Gal b1-6 (Glc b1-3) Man a1- Gal b1-3 (Man b1-2) Glc a1- Glc a1-4 (Man b1-6) Gal a1- Gal b1-4 Glc a1-3 Man a1- Gal b1-6 Glc b1-2 Man b1- Gal a1-2 Man a1-2 Glc b1- Man a1-3 Glc a1-6 Gal a1- Man a1-4 Glc b1-4 Gal b1- Man a1-6 Gal a1-3 Glc b1 Glc b1-2 Man b1-2 Gal a1- Glc b1-3 Man b1-6 Gal b1- Glc b1-4 Gal a1-4 Man b1- Gal b1-6 (Glc b1-4) Man a1- Gal b1-3 (Man b1-4) Glc a1- Glc a1-6 (Man b1-2) Gal a1- Gal b1-6 Glc a1-3 Man a1- Gal a1-2 Glc b1-3 Man b1- Gal a1-3 Man a1-2 Glc b1- Man a1-4 Glc a1-6 Gal a1- Man a1-6 Glc b1-4 Gal b1- Man b1-2 Gal a1-3 Glc b1 Glc b1-3 Man b1-2 Gal a1- Glc b1-4 Man b1-6 Gal b1- Glc b1-6 Gal a1-4 Man b1- Gal a1-2 (Glc a1-3) Man b1- Gal b1-3 (Man b1-6) Glc a1- Glc a1-6 (Man b1-3) Gal a1- Gal a1-2 Glc a1-4 Man a1- Gal a1-3 Glc b1-3 Man b1- Gal a1-4 Man a1-2 Glc b1- Man a1-6 Glc a1-6 Gal a1- Man b1-2 Glc b1-4 Gal b1- Man b1-3 Gal a1-3 Glc b1 Glc b1-4 Man b1-2 Gal a1- Glc b1-6 Man b1-6 Gal b1- Glc a1-2 Gal a1-6 Man b1- Gal a1-2 (Glc a1-4) Man b1- Gal b1-4 (Man b1-2) Glc a1- Glc a1-6 (Man b1-4) Gal a1- Gal a1-3 Glc a1-4 Man a1- Gal a1-4 Glc b1-3 Man b1- Gal a1-6 Man a1-2 Glc b1- Man b1-2 Glc a1-6 Gal a1- Man b1-3 Glc b1-4 Gal b1- Man b1-4 Gal a1-3 Glc b1 Glc b1-6 Man b1-2 Gal a1- Glc a1-2 Gal a1-2 Man a1- Glc a1-3 Gal a1-6 Man b1- Gal a1-2 (Glc a1-6) Man b1- Gal b1-4 (Man b1-3) Glc a1- Glc b1-2 (Man b1-3) Gal a1- Gal a1-4 Glc a1-4 Man a1- Gal a1-6 Glc b1-3 Man b1- Gal b1-2 Man a1-2 Glc b1- Man b1-3 Glc a1-6 Gal a1- Man b1-4 Glc b1-4 Gal b1- Man b1-6 Gal a1-3 Glc b1 Glc a1-2 Man b1-3 Gal a1- Glc a1-3 Gal a1-2 Man a1- Glc a1-4 Gal a1-6 Man b1- Gal a1-3 (Glc a1-2) Man b1- Gal b1-4 (Man b1-6) Glc a1- Glc b1-2 (Man b1-4) Gal a1- Gal a1-6 Glc a1-4 Man a1- Gal b1-2 Glc b1-3 Man b1- Gal b1-3 Man a1-2 Glc b1- Man b1-4 Glc a1-6 Gal a1- Man b1-6 Glc b1-4 Gal b1- Man a1-2 Gal a1-4 Glc b1 Glc a1-3 Man b1-3 Gal a1- Glc a1-4 Gal a1-2 Man a1- Glc a1-6 Gal a1-6 Man b1- Gal a1-3 (Glc a1-4) Man b1- Gal b1-6 (Man b1-2) Glc a1- Glc b1-2 (Man b1-6) Gal a1- Gal b1-2 Glc a1-4 Man a1- Gal b1-3 Glc b1-3 Man b1- Gal b1-4 Man a1-2 Glc b1- Man b1-6 Glc a1-6 Gal a1- Man a1-2 Glc b1-6 Gal b1- Man a1-3 Gal a1-4 Glc b1 Glc a1-4 Man b1-3 Gal a1- Glc a1-6 Gal a1-2 Man a1- Glc b1-2 Gal a1-6 Man b1- Gal a1-3 (Glc a1-6) Man b1- Gal b1-6 (Man b1-3) Glc a1- Glc b1-3 (Man b1-2) Gal a1- Gal b1-3 Glc a1-4 Man a1- Gal b1-4 Glc b1-3 Man b1- Gal b1-6 Man a1-2 Glc b1- Man a1-2 Glc b1-2 Gal a1- Man a1-3 Glc b1-6 Gal b1- Man a1-4 Gal a1-4 Glc b1 Glc a1-6 Man b1-3 Gal a1- Glc b1-2 Gal a1-2 Man a1- Glc b1-3 Gal a1-6 Man b1- Gal a1-4 (Glc a1-2) Man b1- Gal b1-6 (Man b1-4) Glc a1- Glc b1-3 (Man b1-4) Gal a1- Gal b1-4 Glc a1-4 Man a1- Gal b1-6 Glc b1-3 Man b1- Gal a1-2 Man a1-3 Glc b1- Man a1-3 Glc b1-2 Gal a1- Man a1-4 Glc b1-6 Gal b1- Man a1-6 Gal a1-4 Glc b1 Glc b1-2 Man b1-3 Gal a1- Glc b1-3 Gal a1-2 Man a1- Glc b1-4 Gal a1-6 Man b1- Gal a1-4 (Glc a1-3) Man b1- Gal a1-2 (Man a1-3) Glc b1- Glc b1-3 (Man b1-6) Gal a1- Gal b1-6 Glc a1-4 Man a1- Gal a1-2 Glc b1-4 Man b1- Gal a1-3 Man a1-3 Glc b1- Man a1-4 Glc b1-2 Gal a1- Man a1-6 Glc b1-6 Gal b1- Man b1-2 Gal a1-4 Glc b1 Glc b1-3 Man b1-3 Gal a1- Glc b1-4 Gal a1-2 Man a1- Glc b1-6 Gal a1-6 Man b1- Gal a1-4 (Glc a1-6) Man b1- Gal a1-2 (Man a1-4) Glc b1- Glc b1-4 (Man b1-2) Gal a1- Gal a1-2 Glc a1-6 Man a1- Gal a1-3 Glc b1-4 Man b1- Gal a1-4 Man a1-3 Glc b1- Man a1-6 Glc b1-2 Gal a1- Man b1-2 Glc b1-6 Gal b1- Man b1-3 Gal a1-4 Glc b1 Glc b1-4 Man b1-3 Gal a1- Glc b1-6 Gal a1-2 Man a1- Glc a1-2 Gal b1-2 Man b1- Gal a1-6 (Glc a1-2) Man b1- Gal a1-2 (Man a1-6) Glc b1- Glc b1-4 (Man b1-3) Gal a1- Gal a1-3 Glc a1-6 Man a1- Gal a1-4 Glc b1-4 Man b1- Gal a1-6 Man a1-3 Glc b1- Man b1-2 Glc b1-2 Gal a1- Man b1-3 Glc b1-6 Gal b1- Man b1-4 Gal a1-4 Glc b1 Glc b1-6 Man b1-3 Gal a1- Glc a1-2 Gal a1-3 Man a1- Glc a1-3 Gal b1-2 Man b1- Gal a1-6 (Glc a1-3) Man b1- Gal a1-3 (Man a1-2) Glc b1- Glc b1-4 (Man b1-6) Gal a1- Gal a1-4 Glc a1-6 Man a1- Gal a1-6 Glc b1-4 Man b1- Gal b1-2 Man a1-3 Glc b1- Man b1-3 Glc b1-2 Gal a1- Man b1-4 Glc b1-6 Gal b1- Man b1-6 Gal a1-4 Glc b1 Glc a1-2 Man b1-4 Gal a1- Glc a1-3 Gal a1-3 Man a1- Glc a1-4 Gal b1-2 Man b1- Gal a1-6 (Glc a1-4) Man b1- Gal a1-3 (Man a1-4) Glc b1- Glc b1-6 (Man b1-2) Gal a1- Gal a1-6 Glc a1-6 Man a1- Gal b1-2 Glc b1-4 Man b1- Gal b1-3 Man a1-3 Glc b1- Man b1-4 Glc b1-2 Gal a1- Man b1-6 Glc b1-6 Gal b1- Man a1-2 Gal a1-6 Glc b1 Glc a1-3 Man b1-4 Gal a1- Glc a1-4 Gal a1-3 Man a1- Glc a1-6 Gal b1-2 Man b1- Gal b1-2 (Glc a1-3) Man b1- Gal a1-3 (Man a1-6) Glc b1- Glc b1-6 (Man b1-3) Gal a1- Gal b1-2 Glc a1-6 Man a1- Gal b1-3 Glc b1-4 Man b1- Gal b1-4 Man a1-3 Glc b1- Man b1-6 Glc b1-2 Gal a1- Man a1-2 Gal a1-2 Glc a1- Man a1-3 Gal a1-6 Glc b1 Glc a1-4 Man b1-4 Gal a1- Glc a1-6 Gal a1-3 Man a1- Glc b1-2 Gal b1-2 Man b1- Gal b1-2 (Glc a1-4) Man b1- Gal a1-4 (Man a1-2) Glc b1- Glc b1-6 (Man b1-4) Gal a1- Gal b1-3 Glc a1-6 Man a1- Gal b1-4 Glc b1-4 Man b1- Gal b1-6 Man a1-3 Glc b1- Man a1-2 Glc b1-3 Gal a1- Man a1-3 Gal a1-2 Glc a1- Man a1-4 Gal a1-6 Glc b1 Glc a1-6 Man b1-4 Gal a1- Glc b1-2 Gal a1-3 Man a1- Glc b1-3 Gal b1-2 Man b1- Gal b1-2 (Glc a1-6) Man b1- Gal a1-4 (Man a1-3) Glc b1- Glc a1-2 (Man a1-3) Gal b1- Gal b1-4 Glc a1-6 Man a1- Gal b1-6 Glc b1-4 Man b1- Gal a1-2 Man a1-4 Glc b1- Man a1-3 Glc b1-3 Gal a1- Man a1-4 Gal a1-2 Glc a1- Man a1-6 Gal a1-6 Glc b1 Glc b1-2 Man b1-4 Gal a1- Glc b1-3 Gal a1-3 Man a1- Glc b1-4 Gal b1-2 Man b1- Gal b1-3 (Glc a1-2) Man b1- Gal a1-4 (Man a1-6) Glc b1- Glc a1-2 (Man a1-4) Gal b1- Gal b1-6 Glc a1-6 Man a1- Gal a1-2 Glc b1-6 Man b1- Gal a1-3 Man a1-4 Glc b1- Man a1-4 Glc b1-3 Gal a1- Man a1-6 Gal a1-2 Glc a1- Man b1-2 Gal a1-6 Glc b1 Glc b1-3 Man b1-4 Gal a1- Glc b1-4 Gal a1-3 Man a1- Glc b1-6 Gal b1-2 Man b1- Gal b1-3 (Glc a1-4) Man b1- Gal a1-6 (Man a1-2) Glc b1- Glc a1-2 (Man a1-6) Gal b1- Gal a1-2 Glc b1-2 Man a1- Gal a1-3 Glc b1-6 Man b1- Gal a1-4 Man a1-4 Glc b1- Man a1-6 Glc b1-3 Gal a1- Man b1-2 Gal a1-2 Glc a1- Man b1-3 Gal a1-6 Glc b1 Glc b1-4 Man b1-4 Gal a1- Glc b1-6 Gal a1-3 Man a1- Glc a1-2 Gal b1-3 Man b1- Gal b1-3 (Glc a1-6) Man b1- Gal a1-6 (Man a1-3) Glc b1- Glc a1-3 (Man a1-2) Gal b1- Gal a1-3 Glc b1-2 Man a1- Gal a1-4 Glc b1-6 Man b1- Gal a1-6 Man a1-4 Glc b1- Man b1-2 Glc b1-3 Gal a1- Man b1-3 Gal a1-2 Glc a1- Man b1-4 Gal a1-6 Glc b1 Glc b1-6 Man b1-4 Gal a1- Glc a1-2 Gal a1-4 Man a1- Glc a1-3 Gal b1-3 Man b1- Gal b1-4 (Glc a1-2) Man b1- Gal a1-6 (Man a1-4) Glc b1- Glc a1-3 (Man a1-4) Gal b1- Gal a1-4 Glc b1-2 Man a1- Gal a1-6 Glc b1-6 Man b1- Gal b1-2 Man a1-4 Glc b1- Man b1-3 Glc b1-3 Gal a1- Man b1-4 Gal a1-2 Glc a1- Man b1-6 Gal a1-6 Glc b1 Glc a1-2 Man b1-6 Gal a1- Glc a1-3 Gal a1-4 Man a1- Glc a1-4 Gal b1-3 Man b1- Gal b1-4 (Glc a1-3) Man b1- Gal b1-2 (Man a1-3) Glc b1- Glc a1-3 (Man a1-6) Gal b1- Gal a1-6 Glc b1-2 Man a1- Gal b1-2 Glc b1-6 Man b1- Gal b1-3 Man a1-4 Glc b1- Man b1-4 Glc b1-3 Gal a1- Man b1-6 Gal a1-2 Glc a1- Man a1-2 Gal b1-2 Glc b1 Glc a1-3 Man b1-6 Gal a1- Glc a1-4 Gal a1-4 Man a1- Glc a1-6 Gal b1-3 Man b1- Gal b1-4 (Glc a1-6) Man b1- Gal b1-2 (Man a1-4) Glc b1- Glc a1-4 (Man a1-2) Gal b1- Gal b1-2 Glc b1-2 Man a1- Gal b1-3 Glc b1-6 Man b1- Gal b1-4 Man a1-4 Glc b1- Man b1-6 Glc b1-3 Gal a1- Man a1-2 Gal a1-3 Glc a1- Man a1-3 Gal b1-2 Glc b1 Glc a1-4 Man b1-6 Gal a1- Glc a1-6 Gal a1-4 Man a1- Glc b1-2 Gal b1-3 Man b1- Gal b1-6 (Glc a1-2) Man b1- Gal b1-2 (Man a1-6) Glc b1- Glc a1-4 (Man a1-3) Gal b1- Gal b1-3 Glc b1-2 Man a1- Gal b1-4 Glc b1-6 Man b1- Gal b1-6 Man a1-4 Glc b1- Man a1-2 Glc b1-4 Gal a1- Man a1-3 Gal a1-3 Glc a1- Man a1-4 Gal b1-2 Glc b1 Glc a1-6 Man b1-6 Gal a1- Glc b1-2 Gal a1-4 Man a1- Glc b1-3 Gal b1-3 Man b1- Gal b1-6 (Glc a1-3) Man b1- Gal b1-3 (Man a1-2) Glc b1- Glc a1-4 (Man a1-6) Gal b1- Gal b1-4 Glc b1-2 Man a1- Gal b1-6 Glc b1-6 Man b1- Gal a1-2 Man a1-6 Glc b1- Man a1-3 Glc b1-4 Gal a1- Man a1-4 Gal a1-3 Glc a1- Man a1-6 Gal b1-2 Glc b1 Glc b1-2 Man b1-6 Gal a1- Glc b1-3 Gal a1-4 Man a1- Glc b1-4 Gal b1-3 Man b1- Gal b1-6 (Glc a1-4) Man b1- Gal b1-3 (Man a1-4) Glc b1- Glc a1-6 (Man a1-2) Gal b1- Gal b1-6 Glc b1-2 Man a1- Gal a1-2 Man a1-2 Glc a1- Gal a1-3 Man a1-6 Glc b1- Man a1-4 Glc b1-4 Gal a1- Man a1-6 Gal a1-3 Glc a1- Man b1-2 Gal b1-2 Glc b1 Glc b1-3 Man b1-6 Gal a1- Glc b1-4 Gal a1-4 Man a1- Glc b1-6 Gal b1-3 Man b1- Gal a1-2 (Glc b1-3) Man b1- Gal b1-3 (Man a1-6) Glc b1- Glc a1-6 (Man a1-3) Gal b1- Gal a1-2 Glc b1-3 Man a1- Gal a1-3 Man a1-2 Glc a1- Gal a1-4 Man a1-6 Glc b1- Man a1-6 Glc b1-4 Gal a1- Man b1-2 Gal a1-3 Glc a1- Man b1-3 Gal b1-2 Glc b1 Glc b1-4 Man b1-6 Gal a1- Glc b1-6 Gal a1-4 Man a1- Glc a1-2 Gal b1-4 Man b1- Gal a1-2 (Glc b1-4) Man b1- Gal b1-4 (Man a1-2) Glc b1- Glc a1-6 (Man a1-4) Gal b1- Gal a1-3 Glc b1-3 Man a1- Gal a1-4 Man a1-2 Glc a1- Gal a1-6 Man a1-6 Glc b1- Man b1-2 Glc b1-4 Gal a1- Man b1-3 Gal a1-3 Glc a1- Man b1-4 Gal b1-2 Glc b1 Glc b1-6 Man b1-6 Gal a1- Glc a1-2 Gal a1-6 Man a1- Glc a1-3 Gal b1-4 Man b1- Gal a1-2 (Glc b1-6) Man b1- Gal b1-4 (Man a1-3) Glc b1- Glc b1-2 (Man a1-3) Gal b1- Gal a1-4 Glc b1-3 Man a1- Gal a1-6 Man a1-2 Glc a1- Gal b1-2 Man a1-6 Glc b1- Man b1-3 Glc b1-4 Gal a1- Man b1-4 Gal a1-3 Glc a1- Man b1-6 Gal b1-2 Glc b1 Glc a1-2 Man a1-2 Gal b1- Glc a1-3 Gal a1-6 Man a1- Glc a1-4 Gal b1-4 Man b1- Gal a1-3 (Glc b1-2) Man b1- Gal b1-4 (Man a1-6) Glc b1- Glc b1-2 (Man a1-4) Gal b1- Gal a1-6 Glc b1-3 Man a1- Gal b1-2 Man a1-2 Glc a1- Gal b1-3 Man a1-6 Glc b1- Man b1-4 Glc b1-4 Gal a1- Man b1-6 Gal a1-3 Glc a1- Man a1-2 Gal b1-3 Glc b1 Glc a1-3 Man a1-2 Gal b1- Glc a1-4 Gal a1-6 Man a1- Glc a1-6 Gal b1-4 Man b1- Gal a1-3 (Glc b1-4) Man b1- Gal b1-6 (Man a1-2) Glc b1- Glc b1-2 (Man a1-6) Gal b1- Gal b1-2 Glc b1-3 Man a1- Gal b1-3 Man a1-2 Glc a1- Gal b1-4 Man a1-6 Glc b1- Man b1-6 Glc b1-4 Gal a1- Man a1-2 Gal a1-4 Glc a1- Man a1-3 Gal b1-3 Glc b1 Glc a1-4 Man a1-2 Gal b1- Glc a1-6 Gal a1-6 Man a1- Glc b1-2 Gal b1-4 Man b1- Gal a1-3 (Glc b1-6) Man b1- Gal b1-6 (Man a1-3) Glc b1- Glc b1-3 (Man a1-2) Gal b1- Gal b1-3 Glc b1-3 Man a1- Gal b1-4 Man a1-2 Glc a1- Gal b1-6 Man a1-6 Glc b1- Man a1-2 Glc b1-6 Gal a1- Man a1-3 Gal a1-4 Glc a1- Man a1-4 Gal b1-3 Glc b1 Glc a1-6 Man a1-2 Gal b1- Glc b1-2 Gal a1-6 Man a1- Glc b1-3 Gal b1-4 Man b1- Gal a1-4 (Glc b1-2) Man b1- Gal b1-6 (Man a1-4) Glc b1- Glc b1-3 (Man a1-4) Gal b1- Gal b1-4 Glc b1-3 Man a1- Gal b1-6 Man a1-2 Glc a1- Gal a1-2 Man b1-2 Glc b1- Man a1-3 Glc b1-6 Gal a1- Man a1-4 Gal a1-4 Glc a1- Man a1-6 Gal b1-3 Glc b1 Glc b1-2 Man a1-2 Gal b1- Glc b1-3 Gal a1-6 Man a1- Glc b1-4 Gal b1-4 Man b1- Gal a1-4 (Glc b1-3) Man b1- Gal a1-2 (Man b1-3) Glc b1- Glc b1-3 (Man a1-6) Gal b1- Gal b1-6 Glc b1-3 Man a1- Gal a1-2 Man a1-3 Glc a1- Gal a1-3 Man b1-2 Glc b1- Man a1-4 Glc b1-6 Gal a1- Man a1-6 Gal a1-4 Glc a1- Man b1-2 Gal b1-3 Glc b1 Glc b1-3 Man a1-2 Gal b1- Glc b1-4 Gal a1-6 Man a1- Glc b1-6 Gal b1-4 Man b1- Gal a1-4 (Glc b1-6) Man b1- Gal a1-2 (Man b1-4) Glc b1- Glc b1-4 (Man a1-2) Gal b1- Gal a1-2 Glc b1-4 Man a1- Gal a1-3 Man a1-3 Glc a1- Gal a1-4 Man b1-2 Glc b1- Man a1-6 Glc b1-6 Gal a1- Man b1-2 Gal a1-4 Glc a1- Man b1-3 Gal b1-3 Glc b1 Glc b1-4 Man a1-2 Gal b1- Glc b1-6 Gal a1-6 Man a1- Glc a1-2 Gal b1-6 Man b1- Gal a1-6 (Glc b1-2) Man b1- Gal a1-2 (Man b1-6) Glc b1- Glc b1-4 (Man a1-3) Gal b1- Gal a1-3 Glc b1-4 Man a1- Gal a1-4 Man a1-3 Glc a1- Gal a1-6 Man b1-2 Glc b1- Man b1-2 Glc b1-6 Gal a1- Man b1-3 Gal a1-4 Glc a1- Man b1-4 Gal b1-3 Glc b1 Glc b1-6 Man a1-2 Gal b1- Glc a1-2 Gal b1-2 Man a1- Glc a1-3 Gal b1-6 Man b1- Gal a1-6 (Glc b1-3) Man b1- Gal a1-3 (Man b1-2) Glc b1- Glc b1-4 (Man a1-6) Gal b1- Gal a1-4 Glc b1-4 Man a1- Gal a1-6 Man a1-3 Glc a1- Gal b1-2 Man b1-2 Glc b1- Man b1-3 Glc b1-6 Gal a1- Man b1-4 Gal a1-4 Glc a1- Man b1-6 Gal b1-3 Glc b1 Glc a1-2 Man a1-3 Gal b1- Glc a1-3 Gal b1-2 Man a1- Glc a1-4 Gal b1-6 Man b1- Gal a1-6 (Glc b1-4) Man b1- Gal a1-3 (Man b1-4) Glc b1- Glc b1-6 (Man a1-2) Gal b1- Gal a1-6 Glc b1-4 Man a1- Gal b1-2 Man a1-3 Glc a1- Gal b1-3 Man b1-2 Glc b1- Man b1-4 Glc b1-6 Gal a1- Man b1-6 Gal a1-4 Glc a1- Man a1-2 Gal b1-4 Glc b1 Glc a1-3 Man a1-3 Gal b1- Glc a1-4 Gal b1-2 Man a1- Glc a1-6 Gal b1-6 Man b1- Gal b1-2 (Glc b1-3) Man b1- Gal a1-3 (Man b1-6) Glc b1- Glc b1-6 (Man a1-3) Gal b1- Gal b1-2 Glc b1-4 Man a1- Gal b1-3 Man a1-3 Glc a1- Gal b1-4 Man b1-2 Glc b1- Man b1-6 Glc b1-6 Gal a1- Man a1-2 Gal a1-6 Glc a1- Man a1-3 Gal b1-4 Glc b1 Glc a1-4 Man a1-3 Gal b1- Glc a1-6 Gal b1-2 Man a1- Glc b1-2 Gal b1-6 Man b1- Gal b1-2 (Glc b1-4) Man b1- Gal a1-4 (Man b1-2) Glc b1- Glc b1-6 (Man a1-4) Gal b1- Gal b1-3 Glc b1-4 Man a1- Gal b1-4 Man a1-3 Glc a1- Gal b1-6 Man b1-2 Glc b1- Man a1-2 Glc a1-2 Gal b1- Man a1-3 Gal a1-6 Glc a1- Man a1-4 Gal b1-4 Glc b1 Glc a1-6 Man a1-3 Gal b1- Glc b1-2 Gal b1-2 Man a1- Glc b1-3 Gal b1-6 Man b1- Gal b1-2 (Glc b1-6) Man b1- Gal a1-4 (Man b1-3) Glc b1- Glc a1-2 (Man b1-3) Gal b1- Gal b1-4 Glc b1-4 Man a1- Gal b1-6 Man a1-3 Glc a1- Gal a1-2 Man b1-3 Glc b1- Man a1-3 Glc a1-2 Gal b1- Man a1-4 Gal a1-6 Glc a1- Man a1-6 Gal b1-4 Glc b1 Glc b1-2 Man a1-3 Gal b1- Glc b1-3 Gal b1-2 Man a1- Glc b1-4 Gal b1-6 Man b1- Gal b1-3 (Glc b1-2) Man b1- Gal a1-4 (Man b1-6) Glc b1- Glc a1-2 (Man b1-4) Gal b1- Gal b1-6 Glc b1-4 Man a1- Gal a1-2 Man a1-4 Glc a1- Gal a1-3 Man b1-3 Glc b1- Man a1-4 Glc a1-2 Gal b1- Man a1-6 Gal a1-6 Glc a1- Man b1-2 Gal b1-4 Glc b1 Glc b1-3 Man a1-3 Gal b1- Glc b1-4 Gal b1-2 Man a1- Glc b1-6 Gal b1-6 Man b1- Gal b1-3 (Glc b1-4) Man b1- Gal a1-6 (Man b1-2) Glc b1- Glc a1-2 (Man b1-6) Gal b1- Gal a1-2 Glc b1-6 Man a1- Gal a1-3 Man a1-4 Glc a1- Gal a1-4 Man b1-3 Glc b1- Man a1-6 Glc a1-2 Gal b1- Man b1-2 Gal a1-6 Glc a1- Man b1-3 Gal b1-4 Glc b1 Glc b1-4 Man a1-3 Gal b1- Glc b1-6 Gal b1-2 Man a1- Gal a1-2 (Glc a1-3) Man a1- Gal b1-3 (Glc b1-6) Man b1- Gal a1-6 (Man b1-3) Glc b1- Glc a1-3 (Man b1-2) Gal b1- Gal a1-3 Glc b1-6 Man a1- Gal a1-4 Man a1-4 Glc a1- Gal a1-6 Man b1-3 Glc b1- Man b1-2 Glc a1-2 Gal b1- Man b1-3 Gal a1-6 Glc a1- Man b1-4 Gal b1-4 Glc b1 Glc b1-6 Man a1-3 Gal b1- Glc a1-2 Gal b1-3 Man a1- Gal a1-2 (Glc a1-4) Man a1- Gal b1-4 (Glc b1-2) Man b1- Gal a1-6 (Man b1-4) Glc b1- Glc a1-3 (Man b1-4) Gal b1- Gal a1-4 Glc b1-6 Man a1- Gal a1-6 Man a1-4 Glc a1- Gal b1-2 Man b1-3 Glc b1- Man b1-3 Glc a1-2 Gal b1- Man b1-4 Gal a1-6 Glc a1- Man b1-6 Gal b1-4 Glc b1 Glc a1-2 Man a1-4 Gal b1- Glc a1-3 Gal b1-3 Man a1- Gal a1-2 (Glc a1-6) Man a1- Gal b1-4 (Glc b1-3) Man b1- Gal b1-2 (Man b1-3) Glc b1- Glc a1-3 (Man b1-6) Gal b1- Gal a1-6 Glc b1-6 Man a1- Gal b1-2 Man a1-4 Glc a1- Gal b1-3 Man b1-3 Glc b1- Man b1-4 Glc a1-2 Gal b1- Man b1-6 Gal a1-6 Glc a1- Man a1-2 Gal b1-6 Glc b1 Glc a1-3 Man a1-4 Gal b1- Glc a1-4 Gal b1-3 Man a1- Gal a1-3 (Glc a1-2) Man a1- Gal b1-4 (Glc b1-6) Man b1- Gal b1-2 (Man b1-4) Glc b1- Glc a1-4 (Man b1-2) Gal b1- Gal b1-2 Glc b1-6 Man a1- Gal b1-3 Man a1-4 Glc a1- Gal b1-4 Man b1-3 Glc b1- Man b1-6 Glc a1-2 Gal b1- Man a1-2 Gal b1-2 Glc a1- Man a1-3 Gal b1-6 Glc b1 Glc a1-4 Man a1-4 Gal b1- Glc a1-6 Gal b1-3 Man a1- Gal a1-3 (Glc a1-4) Man a1- Gal b1-6 (Glc b1-2) Man b1- Gal b1-2 (Man b1-6) Glc b1- Glc a1-4 (Man b1-3) Gal b1- Gal b1-3 Glc b1-6 Man a1- Gal b1-4 Man a1-4 Glc a1- Gal b1-6 Man b1-3 Glc b1- Man a1-2 Glc a1-3 Gal b1- Man a1-3 Gal b1-2 Glc a1- Man a1-4 Gal b1-6 Glc b1 Glc a1-6 Man a1-4 Gal b1- Glc b1-2 Gal b1-3 Man a1- Gal a1-3 (Glc a1-6) Man a1- Gal b1-6 (Glc b1-3) Man b1- Gal b1-3 (Man b1-2) Glc b1- Glc a1-4 (Man b1-6) Gal b1- Gal b1-4 Glc b1-6 Man a1- Gal b1-6 Man a1-4 Glc a1- Gal a1-2 Man b1-4 Glc b1- Man a1-3 Glc a1-3 Gal b1- Man a1-4 Gal b1-2 Glc a1- Man a1-6 Gal b1-6 Glc b1 Glc b1-2 Man a1-4 Gal b1- Glc b1-3 Gal b1-3 Man a1- Gal a1-4 (Glc a1-2) Man a1- Gal b1-6 (Glc b1-4) Man b1- Gal b1-3 (Man b1-4) Glc b1- Glc a1-6 (Man b1-2) Gal b1- Gal b1-6 Glc b1-6 Man a1- Gal a1-2 Man a1-6 Glc a1- Gal a1-3 Man b1-4 Glc b1- Man a1-4 Glc a1-3 Gal b1- Man a1-6 Gal b1-2 Glc a1- Man b1-2 Gal b1-6 Glc b1 Glc b1-3 Man a1-4 Gal b1- Glc b1-4 Gal b1-3 Man a1- Gal a1-4 (Glc a1-3) Man a1- Gal a1-2 (Man a1-3) Glc a1- Gal b1-3 (Man b1-6) Glc b1- Glc a1-6 (Man b1-3) Gal b1- Gal a1-2 Glc a1-2 Man b1- Gal a1-3 Man a1-6 Glc a1- Gal a1-4 Man b1-4 Glc b1- Man a1-6 Glc a1-3 Gal b1- Man b1-2 Gal b1-2 Glc a1- Man b1-3 Gal b1-6 Glc b1 Glc b1-4 Man a1-4 Gal b1- Glc b1-6 Gal b1-3 Man a1- Gal a1-4 (Glc a1-6) Man a1- Gal a1-2 (Man a1-4) Glc a1- Gal b1-4 (Man b1-2) Glc b1- Glc a1-6 (Man b1-4) Gal b1- Gal a1-3 Glc a1-2 Man b1- Gal a1-4 Man a1-6 Glc a1- Gal a1-6 Man b1-4 Glc b1- Man b1-2 Glc a1-3 Gal b1- Man b1-3 Gal b1-2 Glc a1- Man b1-4 Gal b1-6 Glc b1 Glc b1-6 Man a1-4 Gal b1- Glc a1-2 Gal b1-4 Man a1- Gal a1-6 (Glc a1-2) Man a1- Gal a1-2 (Man a1-6) Glc a1- Gal b1-4 (Man b1-3) Glc b1- Glc b1-2 (Man b1-3) Gal b1- Gal a1-4 Glc a1-2 Man b1- Gal a1-6 Man a1-6 Glc a1- Gal b1-2 Man b1-4 Glc b1- Man b1-3 Glc a1-3 Gal b1- Man b1-4 Gal b1-2 Glc a1- Man b1-6 Gal b1-6 Glc b1 Glc a1-2 Man a1-6 Gal b1- Glc a1-3 Gal b1-4 Man a1- Gal a1-6 (Glc a1-3) Man a1- Gal a1-3 (Man a1-2) Glc a1- Gal b1-4 (Man b1-6) Glc b1- Glc b1-2 (Man b1-4) Gal b1- Gal a1-6 Glc a1-2 Man b1- Gal b1-2 Man a1-6 Glc a1- Gal b1-3 Man b1-4 Glc b1- Man b1-4 Glc a1-3 Gal b1- Man b1-6 Gal b1-2 Glc a1- Glc a1-2 Man a1-2 Gal a1- Glc a1-3 Man a1-6 Gal b1- Glc a1-4 Gal b1-4 Man a1- Gal a1-6 (Glc a1-4) Man a1- Gal a1-3 (Man a1-4) Glc a1- Gal b1-6 (Man b1-2) Glc b1- Glc b1-2 (Man b1-6) Gal b1- Gal b1-2 Glc a1-2 Man b1- Gal b1-3 Man a1-6 Glc a1- Gal b1-4 Man b1-4 Glc b1- Man b1-6 Glc a1-3 Gal b1- Man a1-2 Gal b1-3 Glc a1- Glc a1-3 Man a1-2 Gal a1- Glc a1-4 Man a1-6 Gal b1- Glc a1-6 Gal b1-4 Man a1- Gal b1-2 (Glc a1-3) Man a1- Gal a1-3 (Man a1-6) Glc a1- Gal b1-6 (Man b1-3) Glc b1- Glc b1-3 (Man b1-2) Gal b1- Gal b1-3 Glc a1-2 Man b1- Gal b1-4 Man a1-6 Glc a1- Gal b1-6 Man b1-4 Glc b1- Man a1-2 Glc a1-4 Gal b1- Man a1-3 Gal b1-3 Glc a1- Glc a1-4 Man a1-2 Gal a1- Glc a1-6 Man a1-6 Gal b1- Glc b1-2 Gal b1-4 Man a1- Gal b1-2 (Glc a1-4) Man a1- Gal a1-4 (Man a1-2) Glc a1- Gal b1-6 (Man b1-4) Glc b1- Glc b1-3 (Man b1-4) Gal b1- Gal b1-4 Glc a1-2 Man b1- Gal b1-6 Man a1-6 Glc a1- Gal a1-2 Man b1-6 Glc b1- Man a1-3 Glc a1-4 Gal b1- Man a1-4 Gal b1-3 Glc a1- Glc a1-6 Man a1-2 Gal a1- Glc b1-2 Man a1-6 Gal b1- Glc b1-3 Gal b1-4 Man a1- Gal b1-2 (Glc a1-6) Man a1- Gal a1-4 (Man a1-3) Glc a1- Glc a1-2 (Man a1-3) Gal a1- Glc b1-3 (Man b1-6) Gal b1- Gal b1-6 Glc a1-2 Man b1- Gal a1-2 Man b1-2 Glc a1- Gal a1-3 Man b1-6 Glc b1- Man a1-4 Glc a1-4 Gal b1- Man a1-6 Gal b1-3 Glc a1- Glc b1-2 Man a1-2 Gal a1- Glc b1-3 Man a1-6 Gal b1- Glc b1-4 Gal b1-4 Man a1- Gal b1-3 (Glc a1-2) Man a1- Gal a1-4 (Man a1-6) Glc a1- Glc a1-2 (Man a1-4) Gal a1- Glc b1-4 (Man b1-2) Gal b1- Gal a1-2 Glc a1-3 Man b1- Gal a1-3 Man b1-2 Glc a1- Gal a1-4 Man b1-6 Glc b1- Man a1-6 Glc a1-4 Gal b1- Man b1-2 Gal b1-3 Glc a1- Glc b1-3 Man a1-2 Gal a1- Glc b1-4 Man a1-6 Gal b1- Glc b1-6 Gal b1-4 Man a1- Gal b1-3 (Glc a1-4) Man a1- Gal a1-6 (Man a1-2) Glc a1- Glc a1-2 (Man a1-6) Gal a1- Glc b1-4 (Man b1-3) Gal b1- Gal a1-3 Glc a1-3 Man b1- Gal a1-4 Man b1-2 Glc a1- Gal a1-6 Man b1-6 Glc b1- Man b1-2 Glc a1-4 Gal b1- Man b1-3 Gal b1-3 Glc a1- Glc b1-4 Man a1-2 Gal a1- Glc b1-6 Man a1-6 Gal b1- Glc a1-2 Gal b1-6 Man a1- Gal b1-3 (Glc a1-6) Man a1- Gal a1-6 (Man a1-3) Glc a1- Glc a1-3 (Man a1-2) Gal a1- Glc b1-4 (Man b1-6) Gal b1- Gal a1-4 Glc a1-3 Man b1- Gal a1-6 Man b1-2 Glc a1- Gal b1-2 Man b1-6 Glc b1- Man b1-3 Glc a1-4 Gal b1- Man b1-4 Gal b1-3 Glc a1- Glc b1-6 Man a1-2 Gal a1- Glc a1-2 Man b1-2 Gal b1- Glc a1-3 Gal b1-6 Man a1- Gal b1-4 (Glc a1-2) Man a1- Gal a1-6 (Man a1-4) Glc a1- Glc a1-3 (Man a1-4) Gal a1- Glc b1-6 (Man b1-2) Gal b1- Gal a1-6 Glc a1-3 Man b1- Gal b1-2 Man b1-2 Glc a1- Gal b1-3 Man b1-6 Glc b1- Man b1-4 Glc a1-4 Gal b1- Man b1-6 Gal b1-3 Glc a1- Glc a1-2 Man a1-3 Gal a1- Glc a1-3 Man b1-2 Gal b1- Glc a1-4 Gal b1-6 Man a1- Gal b1-4 (Glc a1-3) Man a1- Gal b1-2 (Man a1-3) Glc a1- Glc a1-3 (Man a1-6) Gal a1- Glc b1-6 (Man b1-3) Gal b1- Gal b1-2 Glc a1-3 Man b1- Gal b1-3 Man b1-2 Glc a1- Gal b1-4 Man b1-6 Glc b1- Man b1-6 Glc a1-4 Gal b1- Man a1-2 Gal b1-4 Glc a1- Glc a1-3 Man a1-3 Gal a1- Glc a1-4 Man b1-2 Gal b1- Glc a1-6 Gal b1-6 Man a1- Gal b1-4 (Glc a1-6) Man a1- Gal b1-2 (Man a1-4) Glc a1- Glc a1-4 (Man a1-2) Gal a1- Glc b1-6 (Man b1-4) Gal b1- Gal b1-3 Glc a1-3 Man b1- Gal b1-4 Man b1-2 Glc a1- Gal b1-6 Man b1-6 Glc b1- Man a1-2 Glc a1-6 Gal b1- Man a1-3 Gal b1-4 Glc a1- Glc a1-4 Man a1-3 Gal a1- Glc a1-6 Man b1-2 Gal b1- Glc b1-2 Gal b1-6 Man a1- Gal b1-6 (Glc a1-2) Man a1- Gal b1-2 (Man a1-6) Glc a1- Glc a1-4 (Man a1-3) Gal a1- Gal b1-4 Glc a1-3 Man b1- Gal b1-6 Man b1-2 Glc a1- Man a1-2 Glc a1-2 Gal a1- Man a1-3 Glc a1-6 Gal b1- Man a1-4 Gal b1-4 Glc a1- Glc a1-6 Man a1-3 Gal a1- Glc b1-2 Man b1-2 Gal b1- Glc b1-3 Gal b1-6 Man a1- Gal b1-6 (Glc a1-3) Man a1- Gal b1-3 (Man a1-2) Glc a1- Glc a1-4 (Man a1-6) Gal a1- Gal b1-6 Glc a1-3 Man b1- Gal a1-2 Man b1-3 Glc a1- Man a1-3 Glc a1-2 Gal a1- Man a1-4 Glc a1-6 Gal b1- Man a1-6 Gal b1-4 Glc a1- Glc b1-2 Man a1-3 Gal a1- Glc b1-3 Man b1-2 Gal b1- Glc b1-4 Gal b1-6 Man a1- Gal b1-6 (Glc a1-4) Man a1- Gal b1-3 (Man a1-4) Glc a1- Glc a1-6 (Man a1-2) Gal a1- Gal a1-2 Glc a1-4 Man b1- Gal a1-3 Man b1-3 Glc a1- Man a1-4 Glc a1-2 Gal a1- Man a1-6 Glc a1-6 Gal b1- Man b1-2 Gal b1-4 Glc a1- Glc b1-3 Man a1-3 Gal a1- Glc b1-4 Man b1-2 Gal b1- Glc b1-6 Gal b1-6 Man a1- Gal a1-2 (Glc b1-3) Man a1- Gal b1-3 (Man a1-6) Glc a1- Glc a1-6 (Man a1-3) Gal a1- Gal a1-3 Glc a1-4 Man b1- Gal a1-4 Man b1-3 Glc a1- Man a1-6 Glc a1-2 Gal a1- Man b1-2 Glc a1-6 Gal b1- Man b1-3 Gal b1-4 Glc a1- Glc b1-4 Man a1-3 Gal a1- Glc b1-6 Man b1-2 Gal b1- Glc a1-2 Gal a1-2 Man b1- Gal a1-2 (Glc b1-4) Man a1- Gal b1-4 (Man a1-2) Glc a1- Glc a1-6 (Man a1-4) Gal a1- Gal a1-4 Glc a1-4 Man b1- Gal a1-6 Man b1-3 Glc a1- Man b1-2 Glc a1-2 Gal a1- Man b1-3 Glc a1-6 Gal b1- Man b1-4 Gal b1-4 Glc a1- Glc b1-6 Man a1-3 Gal a1- Glc a1-2 Man b1-3 Gal b1- Glc a1-3 Gal a1-2 Man b1- Gal a1-2 (Glc b1-6) Man a1- Gal b1-4 (Man a1-3) Glc a1- Glc b1-2 (Man a1-3) Gal a1- Gal a1-6 Glc a1-4 Man b1- Gal b1-2 Man b1-3 Glc a1- Man b1-3 Glc a1-2 Gal a1- Man b1-4 Glc a1-6 Gal b1- Man b1-6 Gal b1-4 Glc a1- Glc a1-2 Man a1-4 Gal a1- Glc a1-3 Man b1-3 Gal b1- Glc a1-4 Gal a1-2 Man b1- Gal a1-3 (Glc b1-2) Man a1- Gal b1-4 (Man a1-6) Glc a1- Glc b1-2 (Man a1-4) Gal a1- Gal b1-2 Glc a1-4 Man b1- Gal b1-3 Man b1-3 Glc a1- Man b1-4 Glc a1-2 Gal a1- Man b1-6 Glc a1-6 Gal b1- Man a1-2 Gal b1-6 Glc a1- Glc a1-3 Man a1-4 Gal a1- Glc a1-4 Man b1-3 Gal b1- Glc a1-6 Gal a1-2 Man b1- Gal a1-3 (Glc b1-4) Man a1- Gal b1-6 (Man a1-2) Glc a1- Glc b1-2 (Man a1-6) Gal a1- Gal b1-3 Glc a1-4 Man b1- Gal b1-4 Man b1-3 Glc a1- Man b1-6 Glc a1-2 Gal a1- Man a1-2 Glc b1-2 Gal b1- Man a1-3 Gal b1-6 Glc a1- Glc a1-4 Man a1-4 Gal a1- Glc a1-6 Man b1-3 Gal b1- Glc b1-2 Gal a1-2 Man b1- Gal a1-3 (Glc b1-6) Man a1- Gal b1-6 (Man a1-3) Glc a1- Glc b1-3 (Man a1-2) Gal a1- Gal b1-4 Glc a1-4 Man b1- Gal b1-6 Man b1-3 Glc a1- Man a1-2 Glc a1-3 Gal a1- Man a1-3 Glc b1-2 Gal b1- Man a1-4 Gal b1-6 Glc a1- Glc a1-6 Man a1-4 Gal a1- Glc b1-2 Man b1-3 Gal b1- Glc b1-3 Gal a1-2 Man b1- Gal a1-4 (Glc b1-2) Man a1- Gal b1-6 (Man a1-4) Glc a1- Glc b1-3 (Man a1-4) Gal a1- Gal b1-6 Glc a1-4 Man b1- Gal a1-2 Man b1-4 Glc a1- Man a1-3 Glc a1-3 Gal a1- Man a1-4 Glc b1-2 Gal b1- Man a1-6 Gal b1-6 Glc a1- Glc b1-2 Man a1-4 Gal a1- Glc b1-3 Man b1-3 Gal b1- Glc b1-4 Gal a1-2 Man b1- Gal a1-4 (Glc b1-3) Man a1- Gal a1-2 (Man b1-3) Glc a1- Glc b1-3 (Man a1-6) Gal a1- Gal a1-2 Glc a1-6 Man b1- Gal a1-3 Man b1-4 Glc a1- Man a1-4 Glc a1-3 Gal a1- Man a1-6 Glc b1-2 Gal b1- Man b1-2 Gal b1-6 Glc a1- Glc b1-3 Man a1-4 Gal a1- Glc b1-4 Man b1-3 Gal b1- Glc b1-6 Gal a1-2 Man b1- Gal a1-4 (Glc b1-6) Man a1- Gal a1-2 (Man b1-4) Glc a1- Glc b1-4 (Man a1-2) Gal a1- 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

Saccharides Carbohydrates: The language of glycobiology The glycosidic bond and glycan nomenclature Major vertebrate glycan classes

Saccharides Carbohydrates: The language of glycobiology The glycosidic bond and glycan nomenclature Major vertebrate glycan classes

Major Glycans of Eukaryotes Glycoproteins Glycolipids Proteoglycans

Major Glycans of Eukaryotes Glycoproteins Glycolipids Proteoglycans

Glycoproteins N-Linked O-Linked GPI Anchor O-GlcNAc *A non-inclusive list!

Glycoproteins N-Linked O-Linked GPI Anchor O-GlcNAc *A non-inclusive list!

“N-linked” glycan linkage to protein asparagine residues

Families of N-linked glycan structures

Terminal Saccharide Diversity --- A key to recognition Examples of structures found as termini on the branches of complex N-linked glycoprotein glycans…. NeuAc 2-6 Gal 1-4 GlcNAc--- NeuAc 2-3 Gal 1-4 GlcNAc--- Gal 1-3 Gal 1-4 GlcNAc--- 4-SO3-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 1-2 Gal; Fuc 1-3 GlcNAc; Fuc 1-4 GlcNAc

Glycoproteins N-Linked O-Linked GPI Anchor O-GlcNAc *A non-inclusive list!

Glycoproteins N-Linked O-Linked GPI Anchor O-GlcNAc *A non-inclusive list!

“O-linked” glycan linkage to protein serine residues

Examples of variations of O-linked glycans

Glycoproteins N-Linked O-Linked GPI Anchor O-GlcNAc *A non-inclusive list!

Glycoproteins N-Linked O-Linked GPI Anchor O-GlcNAc *A non-inclusive list!

GPI (glycosylphosphatidylinositol) anchored protein

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

Glycoproteins N-Linked O-Linked GPI Anchor O-GlcNAc *A non-inclusive list!

Glycoproteins N-Linked O-Linked GPI Anchor O-GlcNAc *A non-inclusive list!

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

Major Glycans of Eukaryotes Glycoproteins Glycolipids Proteoglycans

Major Glycans of Eukaryotes Glycoproteins Glycolipids Proteoglycans

Galactosylceramide (GalCer) A Glycosphingolipid Galactosylceramide (GalCer)

A Ganglioside “GD1a”

Major Glycans of Eukaryotes Glycoproteins Glycolipids Proteoglycans

Major Glycans of Eukaryotes Glycoproteins Glycolipids Proteoglycans

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

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

Post-polymerization variations in glycosaminoglycans

Cartilage proteoglycan

Cartilage proteoglycan

Glycan Biosynthesis Glycosyltransferase reaction (UDP-Gal:glucose β4 galactosyltransferase) UDP-Glc UDP-Gal GDP-Man UDP-GlcNAc UDP-GalNAc UDP-GlcA UDP-Xyl GDP-Fuc CMP-NeuAc Activated Sugars:

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

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

Harrison Ford and Brendon Fraser (2010) Extraordinary Measures

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

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

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). Fratantoni et al (1968) Science 162:170

Corrective factor properties Purified 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 uptake Enzyme uptake was selectively blocked by mannose Enzyme uptake was blocked better mannose-6-phosphate Phosphatase pre-treatment of the enzyme blocked its uptake Fibroblasts do not take up I-cell lysosomal hydrolases Normal 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 glycans

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

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

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

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

Introduction to glycobiology Date Day Lecture Faculty Mar 27 Mon Saccharides & The Glyco World Dr. R. Schnaar Mar 28 Tue Glycoproteins 1 Dr. N. Zachara Mar 29 Wed Glycoproteins 2 Mar 30 Thu O-GlcNAc Dr. G. Hart Mar 31 Fri Glycolipids and GPI anchors Apr 3 Hyaluronan and Proteoglycans Apr 4 Carbohydrate Engineering Dr. K. Yarema Apr 5 Protein-Glycan Recognition Apr 5* Glycan Binding Protein Functions Apr 6 Glycomics (Analytical Glycobiology) Apr 6* Glycans and Disease