Glycobiology resources Concise clear introduction Oxford University Press
Glycobiology resources “Concise, clear introduction” Oxford University Press, New York “Thorough, detailed, comprehensive” Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. FREE – http: //www. ncbi. nlm. nih. gov/books/NBK 1908 FREE – http & coming soon: https: //www. ncbi. nlm. nih. gov/books/NBK 310274 Schnaar RL “Glycobiology Simplified” J Leuk Biol 99: 825
The Glyco World saccharides, glycosides & glycans • • Glycans: the "missing mass" of the biological universe Carbohydrates: The language of glycoscience The glycosidic bond and glycan nomenclature Major carbohydrate classes: sugars, polysaccharides, oligosaccharides and glycans • Major glycans of eukaryotes
The Glyco World saccharides, glycosides & glycans • • Glycans: the "missing mass" of the biological universe Carbohydrates: The language of glycoscience The glycosidic bond and glycan nomenclature Major carbohydrate classes: sugars, polysaccharides, oligosaccharides and glycans • Major glycans of eukaryotes
The mammalian cell surface ? The “glycocalyx” revealed (glycans stained with ruthinium red) extracellular https: //en. wikipedia. org// wiki/Plasma_membrane fibroblast lipid bilayer intracellular Krijnse Locker J. & Schmid S. L. PLo. S Biol 11, e 1001639, 2013 Martinez-Palomo, A. , et al. Cancer Res. 29, 925 -937, 1969 “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
A MAJOR cell component invisible to standard phase microscopy Light (phase) micrograph of neoformans capsule Cryptococcus neoformans 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 Scanning electron micrograph of C. neoformans yeast cells. Van Duin et al. (2004) Antimicrobial Agents and Chemotherapy 48: 2014
Evolution has failed to generate a living cell devoid of surface glycosylation Electron microscopic thin section of Escherichia coli K 1 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 Cohen & Varki (2010) OMICS 4: 455 Ajit Varki, “Perspectives on the Future of Glycobiology, ” Athens, GA, 14 -Mar-2011
The Cell Surface Textbook image Real image fibroblast Martinez-Palomo, A. , et al. Cancer Res. 29, 925 -937, 1969
Glycobiology = Language (semiotics) Cohen & Varki (2010) OMICS 4: 455 Rosetta Stone, British Museum
The Glyco World saccharides, glycosides & glycans • • Glycans: the "missing mass" of the biological universe Carbohydrates: The language of glycoscience The glycosidic bond and glycan nomenclature Major carbohydrate classes: sugars, polysaccharides, oligosaccharides and glycans • Major glycans of eukaryotes
The Glyco World saccharides, glycosides & glycans • • Glycans: the "missing mass" of the biological universe Carbohydrates: The language of glycoscience The glycosidic bond and glycan nomenclature Major carbohydrate classes: sugars, polysaccharides, oligosaccharides and glycans • Major glycans of eukaryotes
Carbohydrates – the building blocks of Glycobiology (CH 2 O)n = “carbo” “hydrate” Generalized Aldose Generalized Ketose
D-glucose D-fructose
Sugar diversity – structurally and functionally – is all about STEREOCHEMISTRY Enantiomers: mirror images of each other that are not superimposable. In sugar nomenclature, enantiomers get a letter “D” or “L” Diastereomers: stereoisomers that are not enantiomers. In sugar nomenclature, diastereomers get different names
Enantiomers Mirror images of each other that are not superimposable. In sugar nomenclature, enantiomers get a letter “D” or “L” * D-glyceraldehyde * L-glyceraldehyde * Highest numbered asymmetric carbon = reference carbon
Fischer projection D-glyceraldehyde
Diasteriomers Stereoisomers that are not enantiomers. In sugar nomenclature, diastereomers get different names * * * Highest numbered asymmetric carbon = reference carbon
Enantiomers & Diasteriomers * * * Highest numbered asymmetric carbon = reference carbons asymmetric carbons diasteriomers & enantiomers 3 1 0 2 4 2 2 4 5 3 4 8 6 4 8 16
* * * * epimers: differ in one stereogenic center Essentials of Glycobiology Second Edition
• Monosaccharides are predominantly in RING FORM at equilibrium. • Free sugars 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
Determining anomeric configuration Step 1: D or L? Highest numbered asymeric carbon Lee & Lee (1999) J Chinese Chem Soc 46: 283 -291
Monosaccharide conformation – Chair configurations Taylor & Drickamer (2006) Introduction to Glycobiology, 2 nd edition
• Just nine monosaccharides are responsible for building nearly all mammalian glycans. • glucose, galactose, mannose • N-acetylglucosamine, N-acetylgalactosamine • xylose • glucuronic acid • fucose • sialic acid • All are six-membered rings (5 carbons, 1 oxygen) varying in the substituents at each carbon (mostly hydroxyls). • Seven are closely structurally related to glucose.
Seven eukaryotic sugars and their relationship to glucose Taylor & Drickamer (2011) Introduction to Glycobiology, 3 rd edition
Two important terminal eukaryotic sugars Taylor & Drickamer (2011) Introduction to Glycobiology, 3 rd edition
Glycan properties for molecular recognition and binding energy Glucose Galactose Sialic Acid Mannose
The Glyco World saccharides, glycosides & glycans • • Glycans: the "missing mass" of the biological universe Carbohydrates: The language of glycoscience The glycosidic bond and glycan nomenclature Major carbohydrate classes: sugars, polysaccharides, oligosaccharides and glycans • Major glycans of eukaryotes
The Glyco World saccharides, glycosides & glycans • • Glycans: the "missing mass" of the biological universe Carbohydrates: The language of glycoscience The glycosidic bond and glycan nomenclature Major carbohydrate classes: sugars, polysaccharides, oligosaccharides and glycans • Major glycans of eukaryotes
Glycosidic bonds link sugars together (lactose) When a glycosidic bond is formed the anomeric configuration is “locked” Taylor & Drickamer (2011) Introduction to Glycobiology, 3 rd 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 β 1 -4 Glc Taylor & Drickamer (2011) Introduction to Glycobiology, 3 rd edition
Nomenclature: Branches are placed in parentheses Neu. Ac α 2 -3 Gal β 1 -4 (Fuc α 1 -3) Glc. NAc
Varki et al. (2009) Proteomics. 9: 5398
The Glyco World saccharides, glycosides & glycans • • Glycans: the "missing mass" of the biological universe Carbohydrates: The language of glycoscience The glycosidic bond and glycan nomenclature Major carbohydrate classes: sugars, polysaccharides, oligosaccharides and glycans • Major glycans of eukaryotes
The Glyco World saccharides, glycosides & glycans • • Glycans: the "missing mass" of the biological universe Carbohydrates: The language of glycoscience The glycosidic bond and glycan nomenclature Major carbohydrate classes: sugars, polysaccharides, oligosaccharides and glycans • Major glycans of eukaryotes
Sugars, polysaccharides, oligosaccharides and glycans • Sugars: often from plants or breakdown products of plant polysaccharides. – Examples: glucose, sucrose (glucose α 1 -2 fructose), maltose (Glc α 1 -4 Glc), lactose (Gal β 1 -4 Glc). • Polysaccharides: long polymers (linear or branched) of a single monosaccharide – Examples: Cellulose, amylose (starch), glycogen, chitin • Oligosaccharides: groups of monosaccharides (typically 3 or more) in linear or branched glycosidic linkage – Seldom found as free structures – components of glycans • Glycans: Oligosaccharides on proteins or lipids – Examples: Glycoproteins, glycolipids
Examples of polysaccharides: Cellulose Most abundant organic compound on Earth. ~90% of cotton fiber, 50% of wood, 33% of all plant matter. (β 1 -4 Glc)n Intra- and inter-strand hydrogen bonding provides strength Material sciences http: //en. wikipedia. org/wiki/Cellulose
Examples of polysaccharides: Amylose Linear (amylose) and branched (amylopectin) forms are the carbohydrates of starch. This major food source is enzymatically broken down to glucose during digestion… (α 1 -4 Glc)n …and during ethanol production α 1 -6 Glc branches http: //en. wikipedia. org/wiki/Amylopectin
Examples of polysaccharides: Glycogen α 1 -6 Glc branches Tissue storage form of glucose (primarily in the liver & muscle). “Animal Starch” – same general structure as plant starch, but more branched & compact. Broken down to glucose at need to quickly increase blood glucose levels. (α 1 -4 Glc)n http: //en. wikipedia. org/wiki/Glycogen
Examples of polysaccharides: Chitin (β 1 -4 Glc. NAc)n Among the most abundant of organic molecules on Earth, it is the main component of the cell walls of fungi, the exoskeletons of arthropods such as insects and crustaceans (e. g. , crabs, lobsters and shrimps). http: //en. wikipedia. org/wiki/Chitin
oligosaccharides as molecular language Cohen & Varki (2010) OMICS 4: 455 Rosetta Stone, British Museum
Oligosaccharides: Molecular diversity of glycans Building blocks Polypeptides amino acids Glycans monosaccharides Number of different monomers 20 common 9 common Linkage sites per monomer 1 3 -4 Possible linkage configurations 1 2 Possible homodimer structures 1 6 -8 linear or branched Linkage modes
Three different amino acids (Ala, Ser, Tyr) – 6 structures Ala-Ser-Tyr Ala-Tyr-Ser Ser-Ala-Tyr Ser-Tyr-Ala-Ser Tyr-Ser-Ala Three different sugars (Glc, Gal, Man) – 1, 056 structures …
Gal a 1 -2 Glc a 1 -2 Man a 1 Gal a 1 -3 Glc a 1 -2 Man a 1 Gal a 1 -4 Glc a 1 -2 Man a 1 Gal a 1 -6 Glc a 1 -2 Man a 1 Gal b 1 -2 Glc a 1 -2 Man a 1 Gal b 1 -3 Glc a 1 -2 Man a 1 Gal b 1 -4 Glc a 1 -2 Man a 1 Gal b 1 -6 Glc a 1 -2 Man a 1 Gal a 1 -2 Glc a 1 -3 Man a 1 Gal a 1 -3 Glc a 1 -3 Man a 1 Gal a 1 -4 Glc a 1 -3 Man a 1 Gal a 1 -6 Glc a 1 -3 Man a 1 Gal b 1 -2 Glc a 1 -3 Man a 1 Gal b 1 -3 Glc a 1 -3 Man a 1 Gal b 1 -4 Glc a 1 -3 Man a 1 Gal b 1 -6 Glc a 1 -3 Man a 1 Gal a 1 -2 Glc a 1 -4 Man a 1 Gal a 1 -3 Glc a 1 -4 Man a 1 Gal a 1 -4 Glc a 1 -4 Man a 1 Gal a 1 -6 Glc a 1 -4 Man a 1 Gal b 1 -2 Glc a 1 -4 Man a 1 Gal b 1 -3 Glc a 1 -4 Man a 1 Gal b 1 -4 Glc a 1 -4 Man a 1 Gal b 1 -6 Glc a 1 -4 Man a 1 Gal a 1 -2 Glc a 1 -6 Man a 1 Gal a 1 -3 Glc a 1 -6 Man a 1 Gal a 1 -4 Glc a 1 -6 Man a 1 Gal a 1 -6 Glc a 1 -6 Man a 1 Gal b 1 -2 Glc a 1 -6 Man a 1 Gal b 1 -3 Glc a 1 -6 Man a 1 Gal b 1 -4 Glc a 1 -6 Man a 1 Gal b 1 -6 Glc a 1 -6 Man a 1 Gal a 1 -2 Glc b 1 -2 Man a 1 Gal a 1 -3 Glc b 1 -2 Man a 1 Gal a 1 -4 Glc b 1 -2 Man a 1 Gal a 1 -6 Glc b 1 -2 Man a 1 Gal b 1 -2 Glc b 1 -2 Man a 1 Gal b 1 -3 Glc b 1 -2 Man a 1 Gal b 1 -4 Glc b 1 -2 Man a 1 Gal b 1 -6 Glc b 1 -2 Man a 1 Gal a 1 -2 Glc b 1 -3 Man a 1 Gal a 1 -3 Glc b 1 -3 Man a 1 Gal a 1 -4 Glc b 1 -3 Man a 1 Gal a 1 -6 Glc b 1 -3 Man a 1 Gal b 1 -2 Glc b 1 -3 Man a 1 Gal b 1 -3 Glc b 1 -3 Man a 1 Gal b 1 -4 Glc b 1 -3 Man a 1 Gal b 1 -6 Glc b 1 -3 Man a 1 Gal a 1 -2 Glc b 1 -4 Man a 1 Gal a 1 -3 Glc b 1 -4 Man a 1 Gal a 1 -4 Glc b 1 -4 Man a 1 Gal a 1 -6 Glc b 1 -4 Man a 1 Gal b 1 -2 Glc b 1 -4 Man a 1 Gal b 1 -3 Glc b 1 -4 Man a 1 Gal b 1 -4 Glc b 1 -4 Man a 1 Gal b 1 -6 Glc b 1 -4 Man a 1 Gal a 1 -2 Glc b 1 -6 Man a 1 Gal a 1 -3 Glc b 1 -6 Man a 1 Gal a 1 -4 Glc b 1 -6 Man a 1 Gal a 1 -6 Glc b 1 -6 Man a 1 Gal b 1 -2 Glc b 1 -6 Man a 1 Gal b 1 -3 Glc b 1 -6 Man a 1 Gal b 1 -4 Glc b 1 -6 Man a 1 Gal b 1 -6 Glc b 1 -6 Man a 1 Gal a 1 -2 Glc a 1 -2 Man b 1 Gal a 1 -3 Glc a 1 -2 Man b 1 Gal a 1 -4 Glc a 1 -2 Man b 1 Gal a 1 -6 Glc a 1 -2 Man b 1 Gal b 1 -2 Glc a 1 -2 Man b 1 Gal b 1 -3 Glc a 1 -2 Man b 1 Gal b 1 -4 Glc a 1 -2 Man b 1 Gal b 1 -6 Glc a 1 -2 Man b 1 Gal a 1 -2 Glc a 1 -3 Man b 1 Gal a 1 -3 Glc a 1 -3 Man b 1 Gal a 1 -4 Glc a 1 -3 Man b 1 Gal a 1 -6 Glc a 1 -3 Man b 1 Gal b 1 -2 Glc a 1 -3 Man b 1 Gal b 1 -3 Glc a 1 -3 Man b 1 Gal b 1 -4 Glc a 1 -3 Man b 1 Gal b 1 -6 Glc a 1 -3 Man b 1 Gal a 1 -2 Glc a 1 -4 Man b 1 Gal a 1 -3 Glc a 1 -4 Man b 1 Gal a 1 -4 Glc a 1 -4 Man b 1 Gal a 1 -6 Glc a 1 -4 Man b 1 Gal b 1 -2 Glc a 1 -4 Man b 1 Gal b 1 -3 Glc a 1 -4 Man b 1 Gal b 1 -4 Glc a 1 -4 Man b 1 Gal b 1 -6 Glc a 1 -4 Man b 1 Gal a 1 -2 Glc a 1 -6 Man b 1 - Gal a 1 -3 Glc a 1 -6 Man b 1 Gal a 1 -4 Glc a 1 -6 Man b 1 Gal a 1 -6 Glc a 1 -6 Man b 1 Gal b 1 -2 Glc a 1 -6 Man b 1 Gal b 1 -3 Glc a 1 -6 Man b 1 Gal b 1 -4 Glc a 1 -6 Man b 1 Gal b 1 -6 Glc a 1 -6 Man b 1 Gal a 1 -2 Glc b 1 -2 Man b 1 Gal a 1 -3 Glc b 1 -2 Man b 1 Gal a 1 -4 Glc b 1 -2 Man b 1 Gal a 1 -6 Glc b 1 -2 Man b 1 Gal b 1 -2 Glc b 1 -2 Man b 1 Gal b 1 -3 Glc b 1 -2 Man b 1 Gal b 1 -4 Glc b 1 -2 Man b 1 Gal b 1 -6 Glc b 1 -2 Man b 1 Gal a 1 -2 Glc b 1 -3 Man b 1 Gal a 1 -3 Glc b 1 -3 Man b 1 Gal a 1 -4 Glc b 1 -3 Man b 1 Gal a 1 -6 Glc b 1 -3 Man b 1 Gal b 1 -2 Glc b 1 -3 Man b 1 Gal b 1 -3 Glc b 1 -3 Man b 1 Gal b 1 -4 Glc b 1 -3 Man b 1 Gal b 1 -6 Glc b 1 -3 Man b 1 Gal a 1 -2 Glc b 1 -4 Man b 1 Gal a 1 -3 Glc b 1 -4 Man b 1 Gal a 1 -4 Glc b 1 -4 Man b 1 Gal a 1 -6 Glc b 1 -4 Man b 1 Gal b 1 -2 Glc b 1 -4 Man b 1 Gal b 1 -3 Glc b 1 -4 Man b 1 Gal b 1 -4 Glc b 1 -4 Man b 1 Gal b 1 -6 Glc b 1 -4 Man b 1 Gal a 1 -2 Glc b 1 -6 Man b 1 Gal a 1 -3 Glc b 1 -6 Man b 1 Gal a 1 -4 Glc b 1 -6 Man b 1 Gal a 1 -6 Glc b 1 -6 Man b 1 Gal b 1 -2 Glc b 1 -6 Man b 1 Gal b 1 -3 Glc b 1 -6 Man b 1 Gal b 1 -4 Glc b 1 -6 Man b 1 Gal b 1 -6 Glc b 1 -6 Man b 1 Gal a 1 -2 Man a 1 -2 Glc a 1 Gal a 1 -3 Man a 1 -2 Glc a 1 Gal a 1 -4 Man a 1 -2 Glc a 1 Gal a 1 -6 Man a 1 -2 Glc a 1 Gal b 1 -2 Man a 1 -2 Glc a 1 Gal b 1 -3 Man a 1 -2 Glc a 1 Gal b 1 -4 Man a 1 -2 Glc a 1 Gal b 1 -6 Man a 1 -2 Glc a 1 Gal a 1 -2 Man a 1 -3 Glc a 1 Gal a 1 -3 Man a 1 -3 Glc a 1 Gal a 1 -4 Man a 1 -3 Glc a 1 Gal a 1 -6 Man a 1 -3 Glc a 1 Gal b 1 -2 Man a 1 -3 Glc a 1 Gal b 1 -3 Man a 1 -3 Glc a 1 Gal b 1 -4 Man a 1 -3 Glc a 1 Gal b 1 -6 Man a 1 -3 Glc a 1 Gal a 1 -2 Man a 1 -4 Glc a 1 Gal a 1 -3 Man a 1 -4 Glc a 1 Gal a 1 -4 Man a 1 -4 Glc a 1 Gal a 1 -6 Man a 1 -4 Glc a 1 Gal b 1 -2 Man a 1 -4 Glc a 1 Gal b 1 -3 Man a 1 -4 Glc a 1 Gal b 1 -4 Man a 1 -4 Glc a 1 Gal b 1 -6 Man a 1 -4 Glc a 1 Gal a 1 -2 Man a 1 -6 Glc a 1 Gal a 1 -3 Man a 1 -6 Glc a 1 Gal a 1 -4 Man a 1 -6 Glc a 1 Gal a 1 -6 Man a 1 -6 Glc a 1 Gal b 1 -2 Man a 1 -6 Glc a 1 Gal b 1 -3 Man a 1 -6 Glc a 1 Gal b 1 -4 Man a 1 -6 Glc a 1 Gal b 1 -6 Man a 1 -6 Glc a 1 Gal a 1 -2 Man b 1 -2 Glc a 1 Gal a 1 -3 Man b 1 -2 Glc a 1 Gal a 1 -4 Man b 1 -2 Glc a 1 Gal a 1 -6 Man b 1 -2 Glc a 1 Gal b 1 -2 Man b 1 -2 Glc a 1 Gal b 1 -3 Man b 1 -2 Glc a 1 Gal b 1 -4 Man b 1 -2 Glc a 1 Gal b 1 -6 Man b 1 -2 Glc a 1 Gal a 1 -2 Man b 1 -3 Glc a 1 Gal a 1 -3 Man b 1 -3 Glc a 1 Gal a 1 -4 Man b 1 -3 Glc a 1 Gal a 1 -6 Man b 1 -3 Glc a 1 Gal b 1 -2 Man b 1 -3 Glc a 1 Gal b 1 -3 Man b 1 -3 Glc a 1 Gal b 1 -4 Man b 1 -3 Glc a 1 Gal b 1 -6 Man b 1 -3 Glc a 1 Gal a 1 -2 Man b 1 -4 Glc a 1 Gal a 1 -3 Man b 1 -4 Glc a 1 - Gal a 1 -4 Man b 1 -4 Glc a 1 Gal a 1 -6 Man b 1 -4 Glc a 1 Gal b 1 -2 Man b 1 -4 Glc a 1 Gal b 1 -3 Man b 1 -4 Glc a 1 Gal b 1 -4 Man b 1 -4 Glc a 1 Gal b 1 -6 Man b 1 -4 Glc a 1 Gal a 1 -2 Man b 1 -6 Glc a 1 Gal a 1 -3 Man b 1 -6 Glc a 1 Gal a 1 -4 Man b 1 -6 Glc a 1 Gal a 1 -6 Man b 1 -6 Glc a 1 Gal b 1 -2 Man b 1 -6 Glc a 1 Gal b 1 -3 Man b 1 -6 Glc a 1 Gal b 1 -4 Man b 1 -6 Glc a 1 Gal b 1 -6 Man b 1 -6 Glc a 1 Gal a 1 -2 Man a 1 -2 Glc b 1 Gal a 1 -3 Man a 1 -2 Glc b 1 Gal a 1 -4 Man a 1 -2 Glc b 1 Gal a 1 -6 Man a 1 -2 Glc b 1 Gal b 1 -2 Man a 1 -2 Glc b 1 Gal b 1 -3 Man a 1 -2 Glc b 1 Gal b 1 -4 Man a 1 -2 Glc b 1 Gal b 1 -6 Man a 1 -2 Glc b 1 Gal a 1 -2 Man a 1 -3 Glc b 1 Gal a 1 -3 Man a 1 -3 Glc b 1 Gal a 1 -4 Man a 1 -3 Glc b 1 Gal a 1 -6 Man a 1 -3 Glc b 1 Gal b 1 -2 Man a 1 -3 Glc b 1 Gal b 1 -3 Man a 1 -3 Glc b 1 Gal b 1 -4 Man a 1 -3 Glc b 1 Gal b 1 -6 Man a 1 -3 Glc b 1 Gal a 1 -2 Man a 1 -4 Glc b 1 Gal a 1 -3 Man a 1 -4 Glc b 1 Gal a 1 -4 Man a 1 -4 Glc b 1 Gal a 1 -6 Man a 1 -4 Glc b 1 Gal b 1 -2 Man a 1 -4 Glc b 1 Gal b 1 -3 Man a 1 -4 Glc b 1 Gal b 1 -4 Man a 1 -4 Glc b 1 Gal b 1 -6 Man a 1 -4 Glc b 1 Gal a 1 -2 Man a 1 -6 Glc b 1 Gal a 1 -3 Man a 1 -6 Glc b 1 Gal a 1 -4 Man a 1 -6 Glc b 1 Gal a 1 -6 Man a 1 -6 Glc b 1 Gal b 1 -2 Man a 1 -6 Glc b 1 Gal b 1 -3 Man a 1 -6 Glc b 1 Gal b 1 -4 Man a 1 -6 Glc b 1 Gal b 1 -6 Man a 1 -6 Glc b 1 Gal a 1 -2 Man b 1 -2 Glc b 1 Gal a 1 -3 Man b 1 -2 Glc b 1 Gal a 1 -4 Man b 1 -2 Glc b 1 Gal a 1 -6 Man b 1 -2 Glc b 1 Gal b 1 -2 Man b 1 -2 Glc b 1 Gal b 1 -3 Man b 1 -2 Glc b 1 Gal b 1 -4 Man b 1 -2 Glc b 1 Gal b 1 -6 Man b 1 -2 Glc b 1 Gal a 1 -2 Man b 1 -3 Glc b 1 Gal a 1 -3 Man b 1 -3 Glc b 1 Gal a 1 -4 Man b 1 -3 Glc b 1 Gal a 1 -6 Man b 1 -3 Glc b 1 Gal b 1 -2 Man b 1 -3 Glc b 1 Gal b 1 -3 Man b 1 -3 Glc b 1 Gal b 1 -4 Man b 1 -3 Glc b 1 Gal b 1 -6 Man b 1 -3 Glc b 1 Gal a 1 -2 Man b 1 -4 Glc b 1 Gal a 1 -3 Man b 1 -4 Glc b 1 Gal a 1 -4 Man b 1 -4 Glc b 1 Gal a 1 -6 Man b 1 -4 Glc b 1 Gal b 1 -2 Man b 1 -4 Glc b 1 Gal b 1 -3 Man b 1 -4 Glc b 1 Gal b 1 -4 Man b 1 -4 Glc b 1 Gal b 1 -6 Man b 1 -4 Glc b 1 Gal a 1 -2 Man b 1 -6 Glc b 1 Gal a 1 -3 Man b 1 -6 Glc b 1 Gal a 1 -4 Man b 1 -6 Glc b 1 Gal a 1 -6 Man b 1 -6 Glc b 1 Gal b 1 -2 Man b 1 -6 Glc b 1 Gal b 1 -3 Man b 1 -6 Glc b 1 Gal b 1 -4 Man b 1 -6 Glc b 1 Gal b 1 -6 Man b 1 -6 Glc b 1 Man a 1 -2 Glc a 1 -2 Gal a 1 Man a 1 -3 Glc a 1 -2 Gal a 1 Man a 1 -4 Glc a 1 -2 Gal a 1 Man a 1 -6 Glc a 1 -2 Gal a 1 Man b 1 -2 Glc a 1 -2 Gal a 1 Man b 1 -3 Glc a 1 -2 Gal a 1 Man b 1 -4 Glc a 1 -2 Gal a 1 Man b 1 -6 Glc a 1 -2 Gal a 1 Man a 1 -2 Glc a 1 -3 Gal a 1 Man a 1 -3 Glc a 1 -3 Gal a 1 Man a 1 -4 Glc a 1 -3 Gal a 1 - Man a 1 -6 Glc a 1 -3 Gal a 1 Man b 1 -2 Glc a 1 -3 Gal a 1 Man b 1 -3 Glc a 1 -3 Gal a 1 Man b 1 -4 Glc a 1 -3 Gal a 1 Man b 1 -6 Glc a 1 -3 Gal a 1 Man a 1 -2 Glc a 1 -4 Gal a 1 Man a 1 -3 Glc a 1 -4 Gal a 1 Man a 1 -4 Glc a 1 -4 Gal a 1 Man a 1 -6 Glc a 1 -4 Gal a 1 Man b 1 -2 Glc a 1 -4 Gal a 1 Man b 1 -3 Glc a 1 -4 Gal a 1 Man b 1 -4 Glc a 1 -4 Gal a 1 Man b 1 -6 Glc a 1 -4 Gal a 1 Man a 1 -2 Glc a 1 -6 Gal a 1 Man a 1 -3 Glc a 1 -6 Gal a 1 Man a 1 -4 Glc a 1 -6 Gal a 1 Man a 1 -6 Glc a 1 -6 Gal a 1 Man b 1 -2 Glc a 1 -6 Gal a 1 Man b 1 -3 Glc a 1 -6 Gal a 1 Man b 1 -4 Glc a 1 -6 Gal a 1 Man b 1 -6 Glc a 1 -6 Gal a 1 Man a 1 -2 Glc b 1 -2 Gal a 1 Man a 1 -3 Glc b 1 -2 Gal a 1 Man a 1 -4 Glc b 1 -2 Gal a 1 Man a 1 -6 Glc b 1 -2 Gal a 1 Man b 1 -2 Glc b 1 -2 Gal a 1 Man b 1 -3 Glc b 1 -2 Gal a 1 Man b 1 -4 Glc b 1 -2 Gal a 1 Man b 1 -6 Glc b 1 -2 Gal a 1 Man a 1 -2 Glc b 1 -3 Gal a 1 Man a 1 -3 Glc b 1 -3 Gal a 1 Man a 1 -4 Glc b 1 -3 Gal a 1 Man a 1 -6 Glc b 1 -3 Gal a 1 Man b 1 -2 Glc b 1 -3 Gal a 1 Man b 1 -3 Glc b 1 -3 Gal a 1 Man b 1 -4 Glc b 1 -3 Gal a 1 Man b 1 -6 Glc b 1 -3 Gal a 1 Man a 1 -2 Glc b 1 -4 Gal a 1 Man a 1 -3 Glc b 1 -4 Gal a 1 Man a 1 -4 Glc b 1 -4 Gal a 1 Man a 1 -6 Glc b 1 -4 Gal a 1 Man b 1 -2 Glc b 1 -4 Gal a 1 Man b 1 -3 Glc b 1 -4 Gal a 1 Man b 1 -4 Glc b 1 -4 Gal a 1 Man b 1 -6 Glc b 1 -4 Gal a 1 Man a 1 -2 Glc b 1 -6 Gal a 1 Man a 1 -3 Glc b 1 -6 Gal a 1 Man a 1 -4 Glc b 1 -6 Gal a 1 Man a 1 -6 Glc b 1 -6 Gal a 1 Man b 1 -2 Glc b 1 -6 Gal a 1 Man b 1 -3 Glc b 1 -6 Gal a 1 Man b 1 -4 Glc b 1 -6 Gal a 1 Man b 1 -6 Glc b 1 -6 Gal a 1 Man a 1 -2 Glc a 1 -2 Gal b 1 Man a 1 -3 Glc a 1 -2 Gal b 1 Man a 1 -4 Glc a 1 -2 Gal b 1 Man a 1 -6 Glc a 1 -2 Gal b 1 Man b 1 -2 Glc a 1 -2 Gal b 1 Man b 1 -3 Glc a 1 -2 Gal b 1 Man b 1 -4 Glc a 1 -2 Gal b 1 Man b 1 -6 Glc a 1 -2 Gal b 1 Man a 1 -2 Glc a 1 -3 Gal b 1 Man a 1 -3 Glc a 1 -3 Gal b 1 Man a 1 -4 Glc a 1 -3 Gal b 1 Man a 1 -6 Glc a 1 -3 Gal b 1 Man b 1 -2 Glc a 1 -3 Gal b 1 Man b 1 -3 Glc a 1 -3 Gal b 1 Man b 1 -4 Glc a 1 -3 Gal b 1 Man b 1 -6 Glc a 1 -3 Gal b 1 Man a 1 -2 Glc a 1 -4 Gal b 1 Man a 1 -3 Glc a 1 -4 Gal b 1 Man a 1 -4 Glc a 1 -4 Gal b 1 Man a 1 -6 Glc a 1 -4 Gal b 1 Man b 1 -2 Glc a 1 -4 Gal b 1 Man b 1 -3 Glc a 1 -4 Gal b 1 Man b 1 -4 Glc a 1 -4 Gal b 1 Man b 1 -6 Glc a 1 -4 Gal b 1 Man a 1 -2 Glc a 1 -6 Gal b 1 Man a 1 -3 Glc a 1 -6 Gal b 1 Man a 1 -4 Glc a 1 -6 Gal b 1 Man a 1 -6 Glc a 1 -6 Gal b 1 Man b 1 -2 Glc a 1 -6 Gal b 1 Man b 1 -3 Glc a 1 -6 Gal b 1 Man b 1 -4 Glc a 1 -6 Gal b 1 Man b 1 -6 Glc a 1 -6 Gal b 1 Man a 1 -2 Glc b 1 -2 Gal b 1 Man a 1 -3 Glc b 1 -2 Gal b 1 Man a 1 -4 Glc b 1 -2 Gal b 1 Man a 1 -6 Glc b 1 -2 Gal b 1 - Man b 1 -2 Glc b 1 -2 Gal b 1 Man b 1 -3 Glc b 1 -2 Gal b 1 Man b 1 -4 Glc b 1 -2 Gal b 1 Man b 1 -6 Glc b 1 -2 Gal b 1 Man a 1 -2 Glc b 1 -3 Gal b 1 Man a 1 -3 Glc b 1 -3 Gal b 1 Man a 1 -4 Glc b 1 -3 Gal b 1 Man a 1 -6 Glc b 1 -3 Gal b 1 Man b 1 -2 Glc b 1 -3 Gal b 1 Man b 1 -3 Glc b 1 -3 Gal b 1 Man b 1 -4 Glc b 1 -3 Gal b 1 Man b 1 -6 Glc b 1 -3 Gal b 1 Man a 1 -2 Glc b 1 -4 Gal b 1 Man a 1 -3 Glc b 1 -4 Gal b 1 Man a 1 -4 Glc b 1 -4 Gal b 1 Man a 1 -6 Glc b 1 -4 Gal b 1 Man b 1 -2 Glc b 1 -4 Gal b 1 Man b 1 -3 Glc b 1 -4 Gal b 1 Man b 1 -4 Glc b 1 -4 Gal b 1 Man b 1 -6 Glc b 1 -4 Gal b 1 Man a 1 -2 Glc b 1 -6 Gal b 1 Man a 1 -3 Glc b 1 -6 Gal b 1 Man a 1 -4 Glc b 1 -6 Gal b 1 Man a 1 -6 Glc b 1 -6 Gal b 1 Man b 1 -2 Glc b 1 -6 Gal b 1 Man b 1 -3 Glc b 1 -6 Gal b 1 Man b 1 -4 Glc b 1 -6 Gal b 1 Man b 1 -6 Glc b 1 -6 Gal b 1 Man a 1 -2 Gal a 1 -2 Glc a 1 Man a 1 -3 Gal a 1 -2 Glc a 1 Man a 1 -4 Gal a 1 -2 Glc a 1 Man a 1 -6 Gal a 1 -2 Glc a 1 Man b 1 -2 Gal a 1 -2 Glc a 1 Man b 1 -3 Gal a 1 -2 Glc a 1 Man b 1 -4 Gal a 1 -2 Glc a 1 Man b 1 -6 Gal a 1 -2 Glc a 1 Man a 1 -2 Gal a 1 -3 Glc a 1 Man a 1 -3 Gal a 1 -3 Glc a 1 Man a 1 -4 Gal a 1 -3 Glc a 1 Man a 1 -6 Gal a 1 -3 Glc a 1 Man b 1 -2 Gal a 1 -3 Glc a 1 Man b 1 -3 Gal a 1 -3 Glc a 1 Man b 1 -4 Gal a 1 -3 Glc a 1 Man b 1 -6 Gal a 1 -3 Glc a 1 Man a 1 -2 Gal a 1 -4 Glc a 1 Man a 1 -3 Gal a 1 -4 Glc a 1 Man a 1 -4 Gal a 1 -4 Glc a 1 Man a 1 -6 Gal a 1 -4 Glc a 1 Man b 1 -2 Gal a 1 -4 Glc a 1 Man b 1 -3 Gal a 1 -4 Glc a 1 Man b 1 -4 Gal a 1 -4 Glc a 1 Man b 1 -6 Gal a 1 -4 Glc a 1 Man a 1 -2 Gal a 1 -6 Glc a 1 Man a 1 -3 Gal a 1 -6 Glc a 1 Man a 1 -4 Gal a 1 -6 Glc a 1 Man a 1 -6 Gal a 1 -6 Glc a 1 Man b 1 -2 Gal a 1 -6 Glc a 1 Man b 1 -3 Gal a 1 -6 Glc a 1 Man b 1 -4 Gal a 1 -6 Glc a 1 Man b 1 -6 Gal a 1 -6 Glc a 1 Man a 1 -2 Gal b 1 -2 Glc a 1 Man a 1 -3 Gal b 1 -2 Glc a 1 Man a 1 -4 Gal b 1 -2 Glc a 1 Man a 1 -6 Gal b 1 -2 Glc a 1 Man b 1 -2 Gal b 1 -2 Glc a 1 Man b 1 -3 Gal b 1 -2 Glc a 1 Man b 1 -4 Gal b 1 -2 Glc a 1 Man b 1 -6 Gal b 1 -2 Glc a 1 Man a 1 -2 Gal b 1 -3 Glc a 1 Man a 1 -3 Gal b 1 -3 Glc a 1 Man a 1 -4 Gal b 1 -3 Glc a 1 Man a 1 -6 Gal b 1 -3 Glc a 1 Man b 1 -2 Gal b 1 -3 Glc a 1 Man b 1 -3 Gal b 1 -3 Glc a 1 Man b 1 -4 Gal b 1 -3 Glc a 1 Man b 1 -6 Gal b 1 -3 Glc a 1 Man a 1 -2 Gal b 1 -4 Glc a 1 Man a 1 -3 Gal b 1 -4 Glc a 1 Man a 1 -4 Gal b 1 -4 Glc a 1 Man a 1 -6 Gal b 1 -4 Glc a 1 Man b 1 -2 Gal b 1 -4 Glc a 1 Man b 1 -3 Gal b 1 -4 Glc a 1 Man b 1 -4 Gal b 1 -4 Glc a 1 Man b 1 -6 Gal b 1 -4 Glc a 1 Man a 1 -2 Gal b 1 -6 Glc a 1 Man a 1 -3 Gal b 1 -6 Glc a 1 Man a 1 -4 Gal b 1 -6 Glc a 1 Man a 1 -6 Gal b 1 -6 Glc a 1 Man b 1 -2 Gal b 1 -6 Glc a 1 - Man b 1 -3 Gal b 1 -6 Glc a 1 Man b 1 -4 Gal b 1 -6 Glc a 1 Man b 1 -6 Gal b 1 -6 Glc a 1 Man a 1 -2 Gal a 1 -2 Glc b 1 Man a 1 -3 Gal a 1 -2 Glc b 1 Man a 1 -4 Gal a 1 -2 Glc b 1 Man a 1 -6 Gal a 1 -2 Glc b 1 Man b 1 -2 Gal a 1 -2 Glc b 1 Man b 1 -3 Gal a 1 -2 Glc b 1 Man b 1 -4 Gal a 1 -2 Glc b 1 Man b 1 -6 Gal a 1 -2 Glc b 1 Man a 1 -2 Gal a 1 -3 Glc b 1 Man a 1 -3 Gal a 1 -3 Glc b 1 Man a 1 -4 Gal a 1 -3 Glc b 1 Man a 1 -6 Gal a 1 -3 Glc b 1 Man b 1 -2 Gal a 1 -3 Glc b 1 Man b 1 -3 Gal a 1 -3 Glc b 1 Man b 1 -4 Gal a 1 -3 Glc b 1 Man b 1 -6 Gal a 1 -3 Glc b 1 Man a 1 -2 Gal a 1 -4 Glc b 1 Man a 1 -3 Gal a 1 -4 Glc b 1 Man a 1 -4 Gal a 1 -4 Glc b 1 Man a 1 -6 Gal a 1 -4 Glc b 1 Man b 1 -2 Gal a 1 -4 Glc b 1 Man b 1 -3 Gal a 1 -4 Glc b 1 Man b 1 -4 Gal a 1 -4 Glc b 1 Man b 1 -6 Gal a 1 -4 Glc b 1 Man a 1 -2 Gal a 1 -6 Glc b 1 Man a 1 -3 Gal a 1 -6 Glc b 1 Man a 1 -4 Gal a 1 -6 Glc b 1 Man a 1 -6 Gal a 1 -6 Glc b 1 Man b 1 -2 Gal a 1 -6 Glc b 1 Man b 1 -3 Gal a 1 -6 Glc b 1 Man b 1 -4 Gal a 1 -6 Glc b 1 Man b 1 -6 Gal a 1 -6 Glc b 1 Man a 1 -2 Gal b 1 -2 Glc b 1 Man a 1 -3 Gal b 1 -2 Glc b 1 Man a 1 -4 Gal b 1 -2 Glc b 1 Man a 1 -6 Gal b 1 -2 Glc b 1 Man b 1 -2 Gal b 1 -2 Glc b 1 Man b 1 -3 Gal b 1 -2 Glc b 1 Man b 1 -4 Gal b 1 -2 Glc b 1 Man b 1 -6 Gal b 1 -2 Glc b 1 Man a 1 -2 Gal b 1 -3 Glc b 1 Man a 1 -3 Gal b 1 -3 Glc b 1 Man a 1 -4 Gal b 1 -3 Glc b 1 Man a 1 -6 Gal b 1 -3 Glc b 1 Man b 1 -2 Gal b 1 -3 Glc b 1 Man b 1 -3 Gal b 1 -3 Glc b 1 Man b 1 -4 Gal b 1 -3 Glc b 1 Man b 1 -6 Gal b 1 -3 Glc b 1 Man a 1 -2 Gal b 1 -4 Glc b 1 Man a 1 -3 Gal b 1 -4 Glc b 1 Man a 1 -4 Gal b 1 -4 Glc b 1 Man a 1 -6 Gal b 1 -4 Glc b 1 Man b 1 -2 Gal b 1 -4 Glc b 1 Man b 1 -3 Gal b 1 -4 Glc b 1 Man b 1 -4 Gal b 1 -4 Glc b 1 Man b 1 -6 Gal b 1 -4 Glc b 1 Man a 1 -2 Gal b 1 -6 Glc b 1 Man a 1 -3 Gal b 1 -6 Glc b 1 Man a 1 -4 Gal b 1 -6 Glc b 1 Man a 1 -6 Gal b 1 -6 Glc b 1 Man b 1 -2 Gal b 1 -6 Glc b 1 Man b 1 -3 Gal b 1 -6 Glc b 1 Man b 1 -4 Gal b 1 -6 Glc b 1 Man b 1 -6 Gal b 1 -6 Glc b 1 Glc a 1 -2 Man a 1 -2 Gal a 1 Glc a 1 -3 Man a 1 -2 Gal a 1 Glc a 1 -4 Man a 1 -2 Gal a 1 Glc a 1 -6 Man a 1 -2 Gal a 1 Glc b 1 -2 Man a 1 -2 Gal a 1 Glc b 1 -3 Man a 1 -2 Gal a 1 Glc b 1 -4 Man a 1 -2 Gal a 1 Glc b 1 -6 Man a 1 -2 Gal a 1 Glc a 1 -2 Man a 1 -3 Gal a 1 Glc a 1 -3 Man a 1 -3 Gal a 1 Glc a 1 -4 Man a 1 -3 Gal a 1 Glc a 1 -6 Man a 1 -3 Gal a 1 Glc b 1 -2 Man a 1 -3 Gal a 1 Glc b 1 -3 Man a 1 -3 Gal a 1 Glc b 1 -4 Man a 1 -3 Gal a 1 Glc b 1 -6 Man a 1 -3 Gal a 1 Glc a 1 -2 Man a 1 -4 Gal a 1 Glc a 1 -3 Man a 1 -4 Gal a 1 Glc a 1 -4 Man a 1 -4 Gal a 1 Glc a 1 -6 Man a 1 -4 Gal a 1 Glc b 1 -2 Man a 1 -4 Gal a 1 Glc b 1 -3 Man a 1 -4 Gal a 1 - Glc b 1 -4 Man a 1 -4 Gal a 1 Glc b 1 -6 Man a 1 -4 Gal a 1 Glc a 1 -2 Man a 1 -6 Gal a 1 Glc a 1 -3 Man a 1 -6 Gal a 1 Glc a 1 -4 Man a 1 -6 Gal a 1 Glc a 1 -6 Man a 1 -6 Gal a 1 Glc b 1 -2 Man a 1 -6 Gal a 1 Glc b 1 -3 Man a 1 -6 Gal a 1 Glc b 1 -4 Man a 1 -6 Gal a 1 Glc b 1 -6 Man a 1 -6 Gal a 1 Glc a 1 -2 Man b 1 -2 Gal a 1 Glc a 1 -3 Man b 1 -2 Gal a 1 Glc a 1 -4 Man b 1 -2 Gal a 1 Glc a 1 -6 Man b 1 -2 Gal a 1 Glc b 1 -2 Man b 1 -2 Gal a 1 Glc b 1 -3 Man b 1 -2 Gal a 1 Glc b 1 -4 Man b 1 -2 Gal a 1 Glc b 1 -6 Man b 1 -2 Gal a 1 Glc a 1 -2 Man b 1 -3 Gal a 1 Glc a 1 -3 Man b 1 -3 Gal a 1 Glc a 1 -4 Man b 1 -3 Gal a 1 Glc a 1 -6 Man b 1 -3 Gal a 1 Glc b 1 -2 Man b 1 -3 Gal a 1 Glc b 1 -3 Man b 1 -3 Gal a 1 Glc b 1 -4 Man b 1 -3 Gal a 1 Glc b 1 -6 Man b 1 -3 Gal a 1 Glc a 1 -2 Man b 1 -4 Gal a 1 Glc a 1 -3 Man b 1 -4 Gal a 1 Glc a 1 -4 Man b 1 -4 Gal a 1 Glc a 1 -6 Man b 1 -4 Gal a 1 Glc b 1 -2 Man b 1 -4 Gal a 1 Glc b 1 -3 Man b 1 -4 Gal a 1 Glc b 1 -4 Man b 1 -4 Gal a 1 Glc b 1 -6 Man b 1 -4 Gal a 1 Glc a 1 -2 Man b 1 -6 Gal a 1 Glc a 1 -3 Man b 1 -6 Gal a 1 Glc a 1 -4 Man b 1 -6 Gal a 1 Glc a 1 -6 Man b 1 -6 Gal a 1 Glc b 1 -2 Man b 1 -6 Gal a 1 Glc b 1 -3 Man b 1 -6 Gal a 1 Glc b 1 -4 Man b 1 -6 Gal a 1 Glc b 1 -6 Man b 1 -6 Gal a 1 Glc a 1 -2 Man a 1 -2 Gal b 1 Glc a 1 -3 Man a 1 -2 Gal b 1 Glc a 1 -4 Man a 1 -2 Gal b 1 Glc a 1 -6 Man a 1 -2 Gal b 1 Glc b 1 -2 Man a 1 -2 Gal b 1 Glc b 1 -3 Man a 1 -2 Gal b 1 Glc b 1 -4 Man a 1 -2 Gal b 1 Glc b 1 -6 Man a 1 -2 Gal b 1 Glc a 1 -2 Man a 1 -3 Gal b 1 Glc a 1 -3 Man a 1 -3 Gal b 1 Glc a 1 -4 Man a 1 -3 Gal b 1 Glc a 1 -6 Man a 1 -3 Gal b 1 Glc b 1 -2 Man a 1 -3 Gal b 1 Glc b 1 -3 Man a 1 -3 Gal b 1 Glc b 1 -4 Man a 1 -3 Gal b 1 Glc b 1 -6 Man a 1 -3 Gal b 1 Glc a 1 -2 Man a 1 -4 Gal b 1 Glc a 1 -3 Man a 1 -4 Gal b 1 Glc a 1 -4 Man a 1 -4 Gal b 1 Glc a 1 -6 Man a 1 -4 Gal b 1 Glc b 1 -2 Man a 1 -4 Gal b 1 Glc b 1 -3 Man a 1 -4 Gal b 1 Glc b 1 -4 Man a 1 -4 Gal b 1 Glc b 1 -6 Man a 1 -4 Gal b 1 Glc a 1 -2 Man a 1 -6 Gal b 1 Glc a 1 -3 Man a 1 -6 Gal b 1 Glc a 1 -4 Man a 1 -6 Gal b 1 Glc a 1 -6 Man a 1 -6 Gal b 1 Glc b 1 -2 Man a 1 -6 Gal b 1 Glc b 1 -3 Man a 1 -6 Gal b 1 Glc b 1 -4 Man a 1 -6 Gal b 1 Glc b 1 -6 Man a 1 -6 Gal b 1 Glc a 1 -2 Man b 1 -2 Gal b 1 Glc a 1 -3 Man b 1 -2 Gal b 1 Glc a 1 -4 Man b 1 -2 Gal b 1 Glc a 1 -6 Man b 1 -2 Gal b 1 Glc b 1 -2 Man b 1 -2 Gal b 1 Glc b 1 -3 Man b 1 -2 Gal b 1 Glc b 1 -4 Man b 1 -2 Gal b 1 Glc b 1 -6 Man b 1 -2 Gal b 1 Glc a 1 -2 Man b 1 -3 Gal b 1 Glc a 1 -3 Man b 1 -3 Gal b 1 Glc a 1 -4 Man b 1 -3 Gal b 1 Glc a 1 -6 Man b 1 -3 Gal b 1 Glc b 1 -2 Man b 1 -3 Gal b 1 Glc b 1 -3 Man b 1 -3 Gal b 1 Glc b 1 -4 Man b 1 -3 Gal b 1 - Glc b 1 -6 Man b 1 -3 Gal b 1 Glc a 1 -2 Man b 1 -4 Gal b 1 Glc a 1 -3 Man b 1 -4 Gal b 1 Glc a 1 -4 Man b 1 -4 Gal b 1 Glc a 1 -6 Man b 1 -4 Gal b 1 Glc b 1 -2 Man b 1 -4 Gal b 1 Glc b 1 -3 Man b 1 -4 Gal b 1 Glc b 1 -4 Man b 1 -4 Gal b 1 Glc b 1 -6 Man b 1 -4 Gal b 1 Glc a 1 -2 Man b 1 -6 Gal b 1 Glc a 1 -3 Man b 1 -6 Gal b 1 Glc a 1 -4 Man b 1 -6 Gal b 1 Glc a 1 -6 Man b 1 -6 Gal b 1 Glc b 1 -2 Man b 1 -6 Gal b 1 Glc b 1 -3 Man b 1 -6 Gal b 1 Glc b 1 -4 Man b 1 -6 Gal b 1 Glc b 1 -6 Man b 1 -6 Gal b 1 Glc a 1 -2 Gal a 1 -2 Man a 1 Glc a 1 -3 Gal a 1 -2 Man a 1 Glc a 1 -4 Gal a 1 -2 Man a 1 Glc a 1 -6 Gal a 1 -2 Man a 1 Glc b 1 -2 Gal a 1 -2 Man a 1 Glc b 1 -3 Gal a 1 -2 Man a 1 Glc b 1 -4 Gal a 1 -2 Man a 1 Glc b 1 -6 Gal a 1 -2 Man a 1 Glc a 1 -2 Gal a 1 -3 Man a 1 Glc a 1 -3 Gal a 1 -3 Man a 1 Glc a 1 -4 Gal a 1 -3 Man a 1 Glc a 1 -6 Gal a 1 -3 Man a 1 Glc b 1 -2 Gal a 1 -3 Man a 1 Glc b 1 -3 Gal a 1 -3 Man a 1 Glc b 1 -4 Gal a 1 -3 Man a 1 Glc b 1 -6 Gal a 1 -3 Man a 1 Glc a 1 -2 Gal a 1 -4 Man a 1 Glc a 1 -3 Gal a 1 -4 Man a 1 Glc a 1 -4 Gal a 1 -4 Man a 1 Glc a 1 -6 Gal a 1 -4 Man a 1 Glc b 1 -2 Gal a 1 -4 Man a 1 Glc b 1 -3 Gal a 1 -4 Man a 1 Glc b 1 -4 Gal a 1 -4 Man a 1 Glc b 1 -6 Gal a 1 -4 Man a 1 Glc a 1 -2 Gal a 1 -6 Man a 1 Glc a 1 -3 Gal a 1 -6 Man a 1 Glc a 1 -4 Gal a 1 -6 Man a 1 Glc a 1 -6 Gal a 1 -6 Man a 1 Glc b 1 -2 Gal a 1 -6 Man a 1 Glc b 1 -3 Gal a 1 -6 Man a 1 Glc b 1 -4 Gal a 1 -6 Man a 1 Glc b 1 -6 Gal a 1 -6 Man a 1 Glc a 1 -2 Gal b 1 -2 Man a 1 Glc a 1 -3 Gal b 1 -2 Man a 1 Glc a 1 -4 Gal b 1 -2 Man a 1 Glc a 1 -6 Gal b 1 -2 Man a 1 Glc b 1 -2 Gal b 1 -2 Man a 1 Glc b 1 -3 Gal b 1 -2 Man a 1 Glc b 1 -4 Gal b 1 -2 Man a 1 Glc b 1 -6 Gal b 1 -2 Man a 1 Glc a 1 -2 Gal b 1 -3 Man a 1 Glc a 1 -3 Gal b 1 -3 Man a 1 Glc a 1 -4 Gal b 1 -3 Man a 1 Glc a 1 -6 Gal b 1 -3 Man a 1 Glc b 1 -2 Gal b 1 -3 Man a 1 Glc b 1 -3 Gal b 1 -3 Man a 1 Glc b 1 -4 Gal b 1 -3 Man a 1 Glc b 1 -6 Gal b 1 -3 Man a 1 Glc a 1 -2 Gal b 1 -4 Man a 1 Glc a 1 -3 Gal b 1 -4 Man a 1 Glc a 1 -4 Gal b 1 -4 Man a 1 Glc a 1 -6 Gal b 1 -4 Man a 1 Glc b 1 -2 Gal b 1 -4 Man a 1 Glc b 1 -3 Gal b 1 -4 Man a 1 Glc b 1 -4 Gal b 1 -4 Man a 1 Glc b 1 -6 Gal b 1 -4 Man a 1 Glc a 1 -2 Gal b 1 -6 Man a 1 Glc a 1 -3 Gal b 1 -6 Man a 1 Glc a 1 -4 Gal b 1 -6 Man a 1 Glc a 1 -6 Gal b 1 -6 Man a 1 Glc b 1 -2 Gal b 1 -6 Man a 1 Glc b 1 -3 Gal b 1 -6 Man a 1 Glc b 1 -4 Gal b 1 -6 Man a 1 Glc b 1 -6 Gal b 1 -6 Man a 1 Glc a 1 -2 Gal a 1 -2 Man b 1 Glc a 1 -3 Gal a 1 -2 Man b 1 Glc a 1 -4 Gal a 1 -2 Man b 1 Glc a 1 -6 Gal a 1 -2 Man b 1 Glc b 1 -2 Gal a 1 -2 Man b 1 Glc b 1 -3 Gal a 1 -2 Man b 1 Glc b 1 -4 Gal a 1 -2 Man b 1 Glc b 1 -6 Gal a 1 -2 Man b 1 - Man a 1 -4 Gal a 1 -3 Glc b 1 Man a 1 -6 Gal a 1 -3 Glc b 1 Man b 1 -2 Gal a 1 -3 Glc b 1 Man b 1 -3 Gal a 1 -3 Glc b 1 Man b 1 -4 Gal a 1 -3 Glc b 1 Man b 1 -6 Gal a 1 -3 Glc b 1 Man a 1 -2 Gal a 1 -4 Glc b 1 Man a 1 -3 Gal a 1 -4 Glc b 1 Man a 1 -4 Gal a 1 -4 Glc b 1 Man a 1 -6 Gal a 1 -4 Glc b 1 Man b 1 -2 Gal a 1 -4 Glc b 1 Man b 1 -3 Gal a 1 -4 Glc b 1 Man b 1 -4 Gal a 1 -4 Glc b 1 Man b 1 -6 Gal a 1 -4 Glc b 1 Man a 1 -2 Gal a 1 -6 Glc b 1 Man a 1 -3 Gal a 1 -6 Glc b 1 Glc a 1 -2 Gal a 1 -3 Man b 1 - Gal a 1 -6 (Glc b 1 -2) Man a 1 - Gal a 1 -2 (Man b 1 -6) Glc a 1 - Glc b 1 -4 (Man a 1 -3) Gal a 1 Glc a 1 -3 Gal a 1 -3 Man b 1 - Gal a 1 -6 (Glc b 1 -3) Man a 1 - Gal a 1 -3 (Man b 1 -2) Glc a 1 - Glc b 1 -4 (Man a 1 -6) Gal a 1 Glc a 1 -4 Gal a 1 -3 Man b 1 - Gal a 1 -6 (Glc b 1 -4) Man a 1 - Gal a 1 -3 (Man b 1 -4) Glc a 1 - Glc b 1 -6 (Man a 1 -2) Gal a 1 Glc a 1 -6 Gal a 1 -3 Man b 1 - Gal b 1 -2 (Glc b 1 -3) Man a 1 - Gal a 1 -3 (Man b 1 -6) Glc a 1 - Glc b 1 -6 (Man a 1 -3) Gal a 1 Glc b 1 -2 Gal a 1 -3 Man b 1 - Gal b 1 -2 (Glc b 1 -4) Man a 1 - Gal a 1 -4 (Man b 1 -2) Glc a 1 - Glc b 1 -6 (Man a 1 -4) Gal a 1 Glc b 1 -3 Gal a 1 -3 Man b 1 - Gal b 1 -2 (Glc b 1 -6) Man a 1 - Gal a 1 -4 (Man b 1 -3) Glc a 1 -2 (Man b 1 -3) Gal a 1 Glc b 1 -4 Gal a 1 -3 Man b 1 - Gal b 1 -3 (Glc b 1 -2) Man a 1 - Gal a 1 -4 (Man b 1 -6) Glc a 1 -2 (Man b 1 -4) Gal a 1 Glc b 1 -6 Gal a 1 -3 Man b 1 - Gal b 1 -3 (Glc b 1 -4) Man a 1 - Gal a 1 -6 (Man b 1 -2) Glc a 1 -2 (Man b 1 -6) Gal a 1 Glc a 1 -2 Gal a 1 -4 Man b 1 - Gal b 1 -3 (Glc b 1 -6) Man a 1 - Gal a 1 -6 (Man b 1 -3) Glc a 1 -3 (Man b 1 -2) Gal a 1 Glc a 1 -3 Gal a 1 -4 Man b 1 - Gal b 1 -4 (Glc b 1 -2) Man a 1 - Gal a 1 -6 (Man b 1 -4) Glc a 1 -3 (Man b 1 -4) Gal a 1 Glc a 1 -4 Gal a 1 -4 Man b 1 - Gal b 1 -4 (Glc b 1 -3) Man a 1 - Gal b 1 -2 (Man b 1 -3) Glc a 1 -3 (Man b 1 -6) Gal a 1 Glc a 1 -6 Gal a 1 -4 Man b 1 - Gal b 1 -4 (Glc b 1 -6) Man a 1 - Gal b 1 -2 (Man b 1 -4) Glc a 1 -4 (Man b 1 -2) Gal a 1 Glc b 1 -2 Gal a 1 -4 Man b 1 - Gal b 1 -6 (Glc b 1 -2) Man a 1 - Gal b 1 -2 (Man b 1 -6) Glc a 1 -4 (Man b 1 -3) Gal a 1 Glc b 1 -3 Gal a 1 -4 Man b 1 - Gal b 1 -6 (Glc b 1 -3) Man a 1 - Gal b 1 -3 (Man b 1 -2) Glc a 1 -4 (Man b 1 -6) Gal a 1 Glc b 1 -4 Gal a 1 -4 Man b 1 - Gal b 1 -6 (Glc b 1 -4) Man a 1 - Gal b 1 -3 (Man b 1 -4) Glc a 1 -6 (Man b 1 -2) Gal a 1 Glc b 1 -6 Gal a 1 -4 Man b 1 - Gal a 1 -2 (Glc a 1 -3) Man b 1 - Gal b 1 -3 (Man b 1 -6) Glc a 1 -6 (Man b 1 -3) Gal a 1 Glc a 1 -2 Gal a 1 -6 Man b 1 - Gal a 1 -2 (Glc a 1 -4) Man b 1 - Gal b 1 -4 (Man b 1 -2) Glc a 1 -6 (Man b 1 -4) Gal a 1 Glc a 1 -3 Gal a 1 -6 Man b 1 - Gal a 1 -2 (Glc a 1 -6) Man b 1 - Gal b 1 -4 (Man b 1 -3) Glc a 1 - Glc b 1 -2 (Man b 1 -3) Gal a 1 Glc a 1 -4 Gal a 1 -6 Man b 1 - Gal a 1 -3 (Glc a 1 -2) Man b 1 - Gal b 1 -4 (Man b 1 -6) Glc a 1 - Glc b 1 -2 (Man b 1 -4) Gal a 1 Glc a 1 -6 Gal a 1 -6 Man b 1 - Gal a 1 -3 (Glc a 1 -4) Man b 1 - Gal b 1 -6 (Man b 1 -2) Glc a 1 - Glc b 1 -2 (Man b 1 -6) Gal a 1 Glc b 1 -2 Gal a 1 -6 Man b 1 - Gal a 1 -3 (Glc a 1 -6) Man b 1 - Gal b 1 -6 (Man b 1 -3) Glc a 1 - Glc b 1 -3 (Man b 1 -2) Gal a 1 Glc b 1 -3 Gal a 1 -6 Man b 1 - Gal a 1 -4 (Glc a 1 -2) Man b 1 - Gal b 1 -6 (Man b 1 -4) Glc a 1 - Glc b 1 -3 (Man b 1 -4) Gal a 1 Glc b 1 -4 Gal a 1 -6 Man b 1 - Gal a 1 -4 (Glc a 1 -3) Man b 1 - Gal a 1 -2 (Man a 1 -3) Glc b 1 -3 (Man b 1 -6) Gal a 1 Glc b 1 -6 Gal a 1 -6 Man b 1 - Gal a 1 -4 (Glc a 1 -6) Man b 1 - Gal a 1 -2 (Man a 1 -4) Glc b 1 -4 (Man b 1 -2) Gal a 1 Glc a 1 -2 Gal b 1 -2 Man b 1 - Gal a 1 -6 (Glc a 1 -2) Man b 1 - Gal a 1 -2 (Man a 1 -6) Glc b 1 -4 (Man b 1 -3) Gal a 1 Glc a 1 -3 Gal b 1 -2 Man b 1 - Gal a 1 -6 (Glc a 1 -3) Man b 1 - Gal a 1 -3 (Man a 1 -2) Glc b 1 -4 (Man b 1 -6) Gal a 1 Glc a 1 -4 Gal b 1 -2 Man b 1 - Gal a 1 -6 (Glc a 1 -4) Man b 1 - Gal a 1 -3 (Man a 1 -4) Glc b 1 -6 (Man b 1 -2) Gal a 1 Glc a 1 -6 Gal b 1 -2 Man b 1 - Gal b 1 -2 (Glc a 1 -3) Man b 1 - Gal a 1 -3 (Man a 1 -6) Glc b 1 -6 (Man b 1 -3) Gal a 1 Glc b 1 -2 Gal b 1 -2 Man b 1 - Gal b 1 -2 (Glc a 1 -4) Man b 1 - Gal a 1 -4 (Man a 1 -2) Glc b 1 -6 (Man b 1 -4) Gal a 1 Glc b 1 -3 Gal b 1 -2 Man b 1 - Gal b 1 -2 (Glc a 1 -6) Man b 1 - Gal a 1 -4 (Man a 1 -3) Glc b 1 - Glc a 1 -2 (Man a 1 -3) Gal b 1 Glc b 1 -4 Gal b 1 -2 Man b 1 - Gal b 1 -3 (Glc a 1 -2) Man b 1 - Gal a 1 -4 (Man a 1 -6) Glc b 1 - Glc a 1 -2 (Man a 1 -4) Gal b 1 Glc b 1 -6 Gal b 1 -2 Man b 1 - Gal b 1 -3 (Glc a 1 -4) Man b 1 - Gal a 1 -6 (Man a 1 -2) Glc b 1 - Glc a 1 -2 (Man a 1 -6) Gal b 1 Glc a 1 -2 Gal b 1 -3 Man b 1 - Gal b 1 -3 (Glc a 1 -6) Man b 1 - Gal a 1 -6 (Man a 1 -3) Glc b 1 - Glc a 1 -3 (Man a 1 -2) Gal b 1 Glc a 1 -3 Gal b 1 -3 Man b 1 - Gal b 1 -4 (Glc a 1 -2) Man b 1 - Gal a 1 -6 (Man a 1 -4) Glc b 1 - Glc a 1 -3 (Man a 1 -4) Gal b 1 Glc a 1 -4 Gal b 1 -3 Man b 1 - Gal b 1 -4 (Glc a 1 -3) Man b 1 - Gal b 1 -2 (Man a 1 -3) Glc b 1 - Glc a 1 -3 (Man a 1 -6) Gal b 1 Glc a 1 -6 Gal b 1 -3 Man b 1 - Gal b 1 -4 (Glc a 1 -6) Man b 1 - Gal b 1 -2 (Man a 1 -4) Glc b 1 - Glc a 1 -4 (Man a 1 -2) Gal b 1 Glc b 1 -2 Gal b 1 -3 Man b 1 - Gal b 1 -6 (Glc a 1 -2) Man b 1 - Gal b 1 -2 (Man a 1 -6) Glc b 1 - Glc a 1 -4 (Man a 1 -3) Gal b 1 Glc b 1 -3 Gal b 1 -3 Man b 1 - Gal b 1 -6 (Glc a 1 -3) Man b 1 - Gal b 1 -3 (Man a 1 -2) Glc b 1 - Glc a 1 -4 (Man a 1 -6) Gal b 1 Glc b 1 -4 Gal b 1 -3 Man b 1 - Gal b 1 -6 (Glc a 1 -4) Man b 1 - Gal b 1 -3 (Man a 1 -4) Glc b 1 - Glc a 1 -6 (Man a 1 -2) Gal b 1 Glc b 1 -6 Gal b 1 -3 Man b 1 - Gal a 1 -2 (Glc b 1 -3) Man b 1 - Gal b 1 -3 (Man a 1 -6) Glc b 1 - Glc a 1 -6 (Man a 1 -3) Gal b 1 Glc a 1 -2 Gal b 1 -4 Man b 1 - Gal a 1 -2 (Glc b 1 -4) Man b 1 - Gal b 1 -4 (Man a 1 -2) Glc b 1 - Glc a 1 -6 (Man a 1 -4) Gal b 1 Glc a 1 -3 Gal b 1 -4 Man b 1 - Gal a 1 -2 (Glc b 1 -6) Man b 1 - Gal b 1 -4 (Man a 1 -3) Glc b 1 -2 (Man a 1 -3) Gal b 1 Glc a 1 -4 Gal b 1 -4 Man b 1 - Gal a 1 -3 (Glc b 1 -2) Man b 1 - Gal b 1 -4 (Man a 1 -6) Glc b 1 -2 (Man a 1 -4) Gal b 1 Glc a 1 -6 Gal b 1 -4 Man b 1 - Gal a 1 -3 (Glc b 1 -4) Man b 1 - Gal b 1 -6 (Man a 1 -2) Glc b 1 -2 (Man a 1 -6) Gal b 1 Glc b 1 -2 Gal b 1 -4 Man b 1 - Gal a 1 -3 (Glc b 1 -6) Man b 1 - Gal b 1 -6 (Man a 1 -3) Glc b 1 -3 (Man a 1 -2) Gal b 1 Glc b 1 -3 Gal b 1 -4 Man b 1 - Gal a 1 -4 (Glc b 1 -2) Man b 1 - Gal b 1 -6 (Man a 1 -4) Glc b 1 -3 (Man a 1 -4) Gal b 1 Glc b 1 -4 Gal b 1 -4 Man b 1 - Gal a 1 -4 (Glc b 1 -3) Man b 1 - Gal a 1 -2 (Man b 1 -3) Glc b 1 -3 (Man a 1 -6) Gal b 1 Glc b 1 -6 Gal b 1 -4 Man b 1 - Gal a 1 -4 (Glc b 1 -6) Man b 1 - Gal a 1 -2 (Man b 1 -4) Glc b 1 -4 (Man a 1 -2) Gal b 1 Glc a 1 -2 Gal b 1 -6 Man b 1 - Gal a 1 -6 (Glc b 1 -2) Man b 1 - Gal a 1 -2 (Man b 1 -6) Glc b 1 -4 (Man a 1 -3) Gal b 1 Glc a 1 -3 Gal b 1 -6 Man b 1 - Gal a 1 -6 (Glc b 1 -3) Man b 1 - Gal a 1 -3 (Man b 1 -2) Glc b 1 -4 (Man a 1 -6) Gal b 1 Glc a 1 -4 Gal b 1 -6 Man b 1 - Gal a 1 -6 (Glc b 1 -4) Man b 1 - Gal a 1 -3 (Man b 1 -4) Glc b 1 -6 (Man a 1 -2) Gal b 1 Glc a 1 -6 Gal b 1 -6 Man b 1 - Gal b 1 -2 (Glc b 1 -3) Man b 1 - Gal a 1 -3 (Man b 1 -6) Glc b 1 -6 (Man a 1 -3) Gal b 1 Glc b 1 -2 Gal b 1 -6 Man b 1 - Gal b 1 -2 (Glc b 1 -4) Man b 1 - Gal a 1 -4 (Man b 1 -2) Glc b 1 -6 (Man a 1 -4) Gal b 1 Glc b 1 -3 Gal b 1 -6 Man b 1 - Gal b 1 -2 (Glc b 1 -6) Man b 1 - Gal a 1 -4 (Man b 1 -3) Glc b 1 - Glc a 1 -2 (Man b 1 -3) Gal b 1 Glc b 1 -4 Gal b 1 -6 Man b 1 - Gal b 1 -3 (Glc b 1 -2) Man b 1 - Gal a 1 -4 (Man b 1 -6) Glc b 1 - Glc a 1 -2 (Man b 1 -4) Gal b 1 Glc b 1 -6 Gal b 1 -6 Man b 1 - Gal b 1 -3 (Glc b 1 -4) Man b 1 - Gal a 1 -6 (Man b 1 -2) Glc b 1 - Glc a 1 -2 (Man b 1 -6) Gal b 1 Gal a 1 -2 (Glc a 1 -3) Man a 1 - Gal b 1 -3 (Glc b 1 -6) Man b 1 - Gal a 1 -6 (Man b 1 -3) Glc b 1 - Glc a 1 -3 (Man b 1 -2) Gal b 1 Gal a 1 -2 (Glc a 1 -4) Man a 1 - Gal b 1 -4 (Glc b 1 -2) Man b 1 - Gal a 1 -6 (Man b 1 -4) Glc b 1 - Glc a 1 -3 (Man b 1 -4) Gal b 1 Gal a 1 -2 (Glc a 1 -6) Man a 1 - Gal b 1 -4 (Glc b 1 -3) Man b 1 - Gal b 1 -2 (Man b 1 -3) Glc b 1 - Glc a 1 -3 (Man b 1 -6) Gal b 1 Gal a 1 -3 (Glc a 1 -2) Man a 1 - Gal b 1 -4 (Glc b 1 -6) Man b 1 - Gal b 1 -2 (Man b 1 -4) Glc b 1 - Glc a 1 -4 (Man b 1 -2) Gal b 1 Gal a 1 -3 (Glc a 1 -4) Man a 1 - Gal b 1 -6 (Glc b 1 -2) Man b 1 - Gal b 1 -2 (Man b 1 -6) Glc b 1 - Glc a 1 -4 (Man b 1 -3) Gal b 1 Gal a 1 -3 (Glc a 1 -6) Man a 1 - Gal b 1 -6 (Glc b 1 -3) Man b 1 - Gal b 1 -3 (Man b 1 -2) Glc b 1 - Glc a 1 -4 (Man b 1 -6) Gal b 1 Gal a 1 -4 (Glc a 1 -2) Man a 1 - Gal b 1 -6 (Glc b 1 -4) Man b 1 - Gal b 1 -3 (Man b 1 -4) Glc b 1 - Glc a 1 -6 (Man b 1 -2) Gal b 1 Gal a 1 -4 (Glc a 1 -3) Man a 1 - Gal a 1 -2 (Man a 1 -3) Glc a 1 - Gal b 1 -3 (Man b 1 -6) Glc b 1 - Glc a 1 -6 (Man b 1 -3) Gal b 1 Gal a 1 -4 (Glc a 1 -6) Man a 1 - Gal a 1 -2 (Man a 1 -4) Glc a 1 - Gal b 1 -4 (Man b 1 -2) Glc b 1 - Glc a 1 -6 (Man b 1 -4) Gal b 1 Gal a 1 -6 (Glc a 1 -2) Man a 1 - Gal a 1 -2 (Man a 1 -6) Glc a 1 - Gal b 1 -4 (Man b 1 -3) Glc b 1 -2 (Man b 1 -3) Gal b 1 Gal a 1 -6 (Glc a 1 -3) Man a 1 - Gal a 1 -3 (Man a 1 -2) Glc a 1 - Gal b 1 -4 (Man b 1 -6) Glc b 1 -2 (Man b 1 -4) Gal b 1 Gal a 1 -6 (Glc a 1 -4) Man a 1 - Gal a 1 -3 (Man a 1 -4) Glc a 1 - Gal b 1 -6 (Man b 1 -2) Glc b 1 -2 (Man b 1 -6) Gal b 1 -2 (Glc a 1 -3) Man a 1 - Gal a 1 -3 (Man a 1 -6) Glc a 1 - Gal b 1 -6 (Man b 1 -3) Glc b 1 -3 (Man b 1 -2) Gal b 1 -2 (Glc a 1 -4) Man a 1 - Gal a 1 -4 (Man a 1 -2) Glc a 1 - Gal b 1 -6 (Man b 1 -4) Glc b 1 -3 (Man b 1 -4) Gal b 1 -2 (Glc a 1 -6) Man a 1 - Gal a 1 -4 (Man a 1 -3) Glc a 1 -2 (Man a 1 -3) Gal a 1 - Glc b 1 -3 (Man b 1 -6) Gal b 1 -3 (Glc a 1 -2) Man a 1 - Gal a 1 -4 (Man a 1 -6) Glc a 1 -2 (Man a 1 -4) Gal a 1 - Glc b 1 -4 (Man b 1 -2) Gal b 1 -3 (Glc a 1 -4) Man a 1 - Gal a 1 -6 (Man a 1 -2) Glc a 1 -2 (Man a 1 -6) Gal a 1 - Glc b 1 -4 (Man b 1 -3) Gal b 1 -3 (Glc a 1 -6) Man a 1 - Gal a 1 -6 (Man a 1 -3) Glc a 1 -3 (Man a 1 -2) Gal a 1 - Glc b 1 -4 (Man b 1 -6) Gal b 1 -4 (Glc a 1 -2) Man a 1 - Gal a 1 -6 (Man a 1 -4) Glc a 1 -3 (Man a 1 -4) Gal a 1 - Glc b 1 -6 (Man b 1 -2) Gal b 1 -4 (Glc a 1 -3) Man a 1 - Gal b 1 -2 (Man a 1 -3) Glc a 1 -3 (Man a 1 -6) Gal a 1 - Glc b 1 -6 (Man b 1 -3) Gal b 1 -4 (Glc a 1 -6) Man a 1 - Gal b 1 -2 (Man a 1 -4) Glc a 1 -4 (Man a 1 -2) Gal a 1 - Glc b 1 -6 (Man b 1 -4) Gal b 1 -6 (Glc a 1 -2) Man a 1 - Gal b 1 -2 (Man a 1 -6) Glc a 1 -4 (Man a 1 -3) Gal a 1 Gal b 1 -6 (Glc a 1 -3) Man a 1 - Gal b 1 -3 (Man a 1 -2) Glc a 1 -4 (Man a 1 -6) Gal a 1 Gal b 1 -6 (Glc a 1 -4) Man a 1 - Gal b 1 -3 (Man a 1 -4) Glc a 1 -6 (Man a 1 -2) Gal a 1 -2 (Glc b 1 -3) Man a 1 - Gal b 1 -3 (Man a 1 -6) Glc a 1 -6 (Man a 1 -3) Gal a 1 -2 (Glc b 1 -4) Man a 1 - Gal b 1 -4 (Man a 1 -2) Glc a 1 -6 (Man a 1 -4) Gal a 1 -2 (Glc b 1 -6) Man a 1 - Gal b 1 -4 (Man a 1 -3) Glc a 1 - Glc b 1 -2 (Man a 1 -3) Gal a 1 -3 (Glc b 1 -2) Man a 1 - Gal b 1 -4 (Man a 1 -6) Glc a 1 - Glc b 1 -2 (Man a 1 -4) Gal a 1 -3 (Glc b 1 -4) Man a 1 - Gal b 1 -6 (Man a 1 -2) Glc a 1 - Glc b 1 -2 (Man a 1 -6) Gal a 1 -3 (Glc b 1 -6) Man a 1 - Gal b 1 -6 (Man a 1 -3) Glc a 1 - Glc b 1 -3 (Man a 1 -2) Gal a 1 -4 (Glc b 1 -2) Man a 1 - Gal b 1 -6 (Man a 1 -4) Glc a 1 - Glc b 1 -3 (Man a 1 -4) Gal a 1 -4 (Glc b 1 -3) Man a 1 - Gal a 1 -2 (Man b 1 -3) Glc a 1 - Glc b 1 -3 (Man a 1 -6) Gal a 1 -4 (Glc b 1 -6) Man a 1 - Gal a 1 -2 (Man b 1 -4) Glc a 1 - Glc b 1 -4 (Man a 1 -2) Gal a 1 -
The Glyco World saccharides, glycosides & glycans • • Glycans: the "missing mass" of the biological universe Carbohydrates: The language of glycoscience The glycosidic bond and glycan nomenclature Major carbohydrate classes: sugars, polysaccharides, oligosaccharides and glycans • Major glycans of eukaryotes
The Glyco World saccharides, glycosides & glycans • • Glycans: the "missing mass" of the biological universe Carbohydrates: The language of glycoscience The glycosidic bond and glycan nomenclature Major carbohydrate classes: sugars, polysaccharides, oligosaccharides and glycans • Major glycans of eukaryotes
Major Glycans of Eukaryotes • Glycoproteins • Glycolipids • Proteoglycans
Major Glycans of Eukaryotes • Glycoproteins • Glycolipids • Proteoglycans
Glycans are large hydrated surface components of glycoproteins HIV gp 120 Burton DR et al (2012) Cell Host & Microbe 4: 396
HIV gp 120 Doores K J et al. (2010) PNAS 107: 17107
Proteins fold onto themselves… glycans spread out in space 3 k. Da N-linked glycans 1 k. Da N-linked glycan 1. 5 k. Da GPI anchor 18 k. Da protein membrane Thy-1 cell-cell interaction protein Essentials of Glycobiology Second Edition
Glycoproteins • • Roles of glycoprotein glycosylation Modes of glycoprotein glycosylation N-linked glycans O-linked glycans – O-Gal. NAc – “mucin” type glycosylation – O-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules – O-Glc. NAc – intracellular protein regulation • Glycan Biosynthesis – General Principles – N-linked glycoprotein biosynthesis
Glycoproteins • • Roles of glycoprotein glycosylation Modes of glycoprotein glycosylation N-linked glycans O-linked glycans – O-Gal. NAc – “mucin” type glycosylation – O-Man, O-Fuc, O-Glc, O-Xyl – signaling molecules – O-Glc. NAc – intracellular protein regulation • Glycan Biosynthesis – General Principles – N-linked glycoprotein biosynthesis
Roles of protein glycosylation • • • Protein conformation Protein folding* Protein trafficking* Cell surface glycoprotein lattice* Regulation of signaling pathways* Cell-cell interactions* * via glycan recognition
Protein Conformation: Closely spaced short O-linked glycans on mucins
Protein Folding: N-linked protein glycosylation is essential for chaperone-assisted folding of glycoproteins in the endoplasmic reticulum Asn Helenius, A. and Aebi, M. (2001) Science 291, 2364 -2369
Protein Conformation: Conserved glycosylation of Ig. G is required for interaction with immune effectors (complement, Fc-receptors)
Protein Trafficking: Unique glycosylation of lysosomal enzymes “labels” them for trafficking to the lysosome via the Man-P receptor(s) https: //wikispaces. psu. edu/v Asn
Cell surface glycoprotein lattice: Sugar binding proteins (galectins) bind to and stabilize cell surface glycoproteins Altered glycoprotein glycans result in internalization of key glycoproteins Wild type Glycosyaltion mutant Lau et al (2007) Cell 129: 123 Pancreatic β cells stained for Glut-2 (green), insulin (red), and DNA (blue). Ohtsubo et al (2005) Cell 123: 1307
Regulation of signaling pathways: Notch signaling during development is driven by glycosylation Stanley (2007) Curr Opin Struct Biol 17: 530
Glycans in cell-cell interactions Carbohydrate binding determinant Carbohydrate recognition domain (CRD) Lectin Glycan Sharon and Lis (1993) Scientific American
Roles of protein glycosylation • • • Protein conformation Protein folding* Protein trafficking* Cell surface glycoprotein lattice* Regulation of signaling pathways* Cell-cell interactions* * via glycan recognition
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