1. 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)

2. Introduction to glycobiology
Date
Day
Lecture
Faculty
ONLINE
Saccharides & The Glyco World
Dr. R. Schnaar
Mar 19
Wed
Glycoproteins I
Dr. N. Zachara
Mar 21
Fri
Glycoproteins II
Mar 25
Tue
Glycolipids and GPI anchors
Mar 27
Thu
Carbohydrate Engineering
Dr. K. Yarema
Mar 31
Mon
O-GlcNAc
Dr. G. Hart
Apr 2
Glycomics (Analytical Glycobiology)
Apr 4
Glycans and Disease
Apr 8
Hyaluronan and Proteoglycans
Apr 10
Protein-Glycan Recognition (Lectins)
Apr 14
Glycan Binding Protein Functions

3. Introduction to glycobiology
Course co-directors:
Ronald Schnaar
Natasha Zachara
Lecturers:
Gerald Hart
Kevin Yarema

4. Introduction to glycobiology
Oxford University Press, New York
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Content freely available at:

5. What are:
Glycobiology?
Glycosciences?
Glycomics?

6. 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).

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

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

9. 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

10. 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 millions* of different glycan structures – functional diversity
Glycans may be larger and are 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 faculty/hougland.html
Cummings RD (2009) Molecular BioSystems 5, 1087
*Glycoproteins and glycolipids may contain ~3000 glycan determinants with an additional ~4000 theoretical pentasaccharide sequences in glycosaminoglycans

11. Glycobiology is … NOT a “decoration”
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"
structuralbiologylaboratory/Pages/kwong.aspx
“Structure” of HIV gp120
Zolla-Pazner (2004)
Nature Reviews Immunology 4, 199

12. 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

13. 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

14. Unique to yeast – NO!
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

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

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

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

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

19. Glycobiology = Language (semiotics)
Cohen & Varki (2010) OMICS 4:455
Rosetta Stone, British Museum

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

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

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

23. glucose
fructose

24. 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.

25. Fischer projection
D-glyceraldehyde

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

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

28. Diasteriomers * * *
Highest numbered asymmetric carbon = reference carbon

29. 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

30. Essentials of Glycobiology*
* epimers: differ in only stereogenic center
Essentials of Glycobiology
Second Edition

31. 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

32. Determining anomeric configuration
[PDB]
Lee & Lee (1999) J Chinese Chem Soc 46:

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

34. 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 configuration except fucose

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

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

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

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

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

40. 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

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

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

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

44. 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

45. 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 …

46. 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 pentasaccharide sequences in glycosaminoglycans

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

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

49. Major Glycans of Eukaryotes
Glycoproteins
Glycolipids
Proteoglycans

50. Major Glycans of Eukaryotes
Glycoproteins
Glycolipids
Proteoglycans

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

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

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

54. Families of N-linked glycan structures

55. 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

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

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

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

62. GPI (glycosylphosphatidylinositol) anchored protein

63. 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

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

65. Glycoproteins N-Linked O-Linked GPI Anchor O-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
Glycoproteins
Glycolipids
Proteoglycans

68. Major Glycans of Eukaryotes
Glycoproteins
Glycolipids
Proteoglycans

69. Galactosylceramide (GalCer)
A Glycosphingolipid
Galactosylceramide (GalCer)

70. A Ganglioside
“GD1a”

71. Major Glycans of Eukaryotes
Glycoproteins
Glycolipids
Proteoglycans

72. Major Glycans of Eukaryotes
Glycoproteins
Glycolipids
Proteoglycans

73. 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

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

75. Post-polymerization variations in glycosaminoglycans

76. Cartilage proteoglycan

77. Cartilage proteoglycan

78. 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:

79. 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