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Introduction to glycobiologyIntroduction to glycobiologyIntroduction to glycobiologyIntroduction to glycobiology
• Learn the basic language of glycobiologyLearn the basic language of glycobiology• Monosaccharides (vertebrate)• Glycans (oligosaccharide & glycoconjugate basic concepts)
• Learn basic glycan functionsLearn basic glycan functions• Glycans in glycoprotein activity, folding, trafficking• O-GlcNAc-mediated regulation• Glycolipids in membrane recognition & regulation• Proteoglycans in the extracellular matrix• Glycan binding proteins
• Learn the basic concepts of glycan biosynthesisLearn the basic concepts of glycan biosynthesis• Learn the basic tools of glycomics (analysis)Learn the basic tools of glycomics (analysis)
• Learn the basic language of glycobiologyLearn the basic language of glycobiology• Monosaccharides (vertebrate)• Glycans (oligosaccharide & glycoconjugate basic concepts)
• Learn basic glycan functionsLearn basic glycan functions• Glycans in glycoprotein activity, folding, trafficking• O-GlcNAc-mediated regulation• Glycolipids in membrane recognition & regulation• Proteoglycans in the extracellular matrix• Glycan binding proteins
• Learn the basic concepts of glycan biosynthesisLearn the basic concepts of glycan biosynthesis• Learn the basic tools of glycomics (analysis)Learn the basic tools of glycomics (analysis)
Introduction to glycobiologyIntroduction to glycobiologyIntroduction to glycobiologyIntroduction to glycobiology
Oxford University Press, New YorkOxford University Press, New York Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Content freely available at: Content freely available at: http://www.ncbi.nlm.nih.gov/books/NBK1908http://www.ncbi.nlm.nih.gov/books/NBK1908
GlycobiologyGlycobiologyThe study of the biological functions, structures, The study of the biological functions, structures, recognition and biosynthesis of recognition and biosynthesis of glycansglycans (sugar (sugar chains, saccharides) in the context of the chains, saccharides) in the context of the biological scaffolds to which they are attached biological scaffolds to which they are attached (e.g. glycolipids & glycoproteins).(e.g. glycolipids & glycoproteins).
GlycobiologyGlycobiologyThe study of the biological functions, structures, The study of the biological functions, structures, recognition and biosynthesis of recognition and biosynthesis of glycansglycans (sugar (sugar chains, saccharides) in the context of the chains, saccharides) in the context of the biological scaffolds to which they are attached biological scaffolds to which they are attached (e.g. glycolipids & glycoproteins).(e.g. glycolipids & glycoproteins).
““Glycome” – The total complement of glycansGlycome” – The total complement of glycans in a cell or organism in a cell or organism
Glycobiology is …Glycobiology is … NOT Carbohydrates as foodNOT Carbohydrates as food
Glycobiology is …Glycobiology is … NOT Carbohydrates as foodNOT Carbohydrates as food
Berg, Tymoczko & Stryer (2006) Biochemistry, Sixth Edition, W.H. Freeman, New York
Carbohydrates in food are important sources of energy. Starch found in plant-derived food such as pasta ....
Glycobiology is Glycobiology is NOT “a” post-translational NOT “a” post-translational modification... it is thousandsmodification... it is thousandsGlycobiology is Glycobiology is NOT “a” post-translational NOT “a” post-translational modification... it is thousandsmodification... it is thousands
Modified from http://www-che.syr.edu/ faculty/hougland.html
• Combinatorial linkage position, Combinatorial linkage position, orientation and branching provide orientation and branching provide the potential for millions* of different the potential for millions* of different glycan structures – functional glycan structures – functional diversitydiversity
• Glycans may be larger and are more Glycans may be larger and are more diverse than their (protein) carriersdiverse than their (protein) carriers
• A glycan’s function can supersede A glycan’s function can supersede that of its (protein) carrierthat of its (protein) carrier
• 1-2% of the human genome is 1-2% of the human genome is devoted to glycosylationdevoted to glycosylation
Cummings RD (2009) Molecular BioSystems 5, 1087
*Glycoproteins and glycolipids may contain ~3000 glycan determinants with an *Glycoproteins and glycolipids may contain ~3000 glycan determinants with an additional ~4000 theoretical pentasaccharide sequences in glycosaminoglycansadditional ~4000 theoretical pentasaccharide sequences in glycosaminoglycans
Glycobiology is … Glycobiology is … NOT a “decoration”NOT a “decoration”Glycobiology is … Glycobiology is … NOT a “decoration”NOT a “decoration”
“Structure” of HIV gp120Zolla-Pazner (2004)Nature Reviews Immunology 4, 199
N-linked carbohydrate can form both an immunologically silent face—with carbohydrate masquerading as "self"—and also can protect neighboring epitopes through an "evolving glycan shield"
CD59, a complement CD59, a complement defense glycoproteindefense glycoprotein
20 kDa protein3 kDa N-linked
glycan
1 kDa O-linked
glycan
1.5 kDa GPI
anchor
Proteins typically fold inward, whereas Proteins typically fold inward, whereas glycans spread out in spaceglycans spread out in space
““naked” CD59, Huang et al (2007)naked” CD59, Huang et al (2007)Acta CrystallographicaActa Crystallographica 63, 714 63, 714
Rudd et al (1997) J Biol Chem 272, 7229
Scanning electron microscopy of C. neoformans yeast cells.Van Duin et al. (2004) Antimicrobial Agents and Chemotherapy 48:2014
Light microscopy micrograph of Cryptococcus neoformans
Steenbergen et al. (2003) Microbes and Infection 5:667
What if a MAJOR cell component was invisible by standard microscopy?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
Electron microscopic thin section ofEscherichia coli K1Amako et al. (1988) J Bacteriol 170:4960
Unique to yeast – NO!Unique to yeast – NO!
Electron microscopic thin section ofKlebsiella pneumoniaeAmako et al. (1988) J Bacteriol 170:4960
The Eukaryotic Cell SurfaceThe Eukaryotic Cell Surface
Tiny sugar “decorations”..added as an afterthought
Lodish, et al. (1995) Molecular Cell Biology 3rd ed.Lodish, et al. (1995) Molecular Cell Biology 3rd ed.
??
The “glycocalyx” surrounding a fibroblast. The “glycocalyx” surrounding a fibroblast. Cell surface carbohydrates are stained black.Cell surface carbohydrates are stained black.
lipid bilayerlipid bilayer
fibroblastfibroblast
extracellularextracellular
intracellularintracellular
Martinez-Palomo, A., et al. Cancer Res. 29, 925-937, 1969
The Cell Surface -- The Real PictureThe Cell Surface -- The Real Picture
Tropical Forest
Canopy
Cohen & Varki (2010) OMICS 4:455
“Evolution has failed to generate a living cell devoid of surface glycosylation” - A. Varki
lipid bilayerlipid bilayer
fibroblastfibroblast intracellularintracellular
Martinez-Palomo, A., et al. Cancer Res. 29, 925-937, 1969
The Cell SurfaceThe Cell Surface
Real image textbook imageReal image textbook image
• Among their diverse functions, glycans …Among their diverse functions, glycans …– encode intermolecular and cell-cell recognitionencode intermolecular and cell-cell recognition– regulate the activities of proteins (enzymes, ion channels, regulate the activities of proteins (enzymes, ion channels,
receptors)receptors)– Define biophysical space (large, hydrated)Define biophysical space (large, hydrated)
Rosetta Stone, British MuseumRosetta Stone, British Museum
Cohen & Varki (2010) OMICS 4:455
Glycobiology = Language (semiotics)Glycobiology = Language (semiotics)
SaccharidesSaccharidesSaccharidesSaccharides
• Carbohydrates: The language of glycobiologyCarbohydrates: The language of glycobiology• The glycosidic bond and glycan nomenclatureThe glycosidic bond and glycan nomenclature• Major vertebrate glycan classesMajor vertebrate glycan classes
• Carbohydrates: The language of glycobiologyCarbohydrates: The language of glycobiology• The glycosidic bond and glycan nomenclatureThe glycosidic bond and glycan nomenclature• Major vertebrate glycan classesMajor vertebrate glycan classes
SaccharidesSaccharidesSaccharidesSaccharides
• Carbohydrates: The language of glycobiologyCarbohydrates: The language of glycobiology• The glycosidic bond and glycan nomenclatureThe glycosidic bond and glycan nomenclature• Major vertebrate glycan classesMajor vertebrate glycan classes
• Carbohydrates: The language of glycobiologyCarbohydrates: The language of glycobiology• The glycosidic bond and glycan nomenclatureThe glycosidic bond and glycan nomenclature• Major vertebrate glycan classesMajor vertebrate glycan classes
Carbohydrates – the building blocks of Glycobiology
(CH2O)n = “carbo” “hydrate”
Generalized KetoseGeneralized Ketose
Generalized AldoseGeneralized Aldose
glucoseglucose fructosefructose
Enantiomers: Enantiomers: mirror images of each other that are not superimposable.mirror images of each other that are not superimposable.
Diastereomers: Diastereomers: stereoisomers that are not enantiomers. stereoisomers that are not enantiomers.
Generalized AldoseGeneralized Aldose
Monosaccharide structural identity is all about stereochemistryMonosaccharide structural identity is all about stereochemistry
Monosaccharides equilibrate between open chain and ring forms.Monosaccharides equilibrate between open chain and ring forms.Two ring forms (anomeric configurations) are possible, Two ring forms (anomeric configurations) are possible, αα and and ββ
Taylor & Drickamer (2011) Introduction to Glycobiology, 3rd edition
*all D configuration except fucose*all D configuration except fucose
Seven eukaryotic sugars and their relationship to glucoseSeven eukaryotic sugars and their relationship to glucose
Taylor & Drickamer (2011) Introduction to Glycobiology, 3rd edition
Two important terminal eukaryotic sugarsTwo important terminal eukaryotic sugars
Taylor & Drickamer (2011) Introduction to Glycobiology, 3rd edition
Glycan properties for molecular recognition and binding energyGlycan properties for molecular recognition and binding energyGlycan properties for molecular recognition and binding energyGlycan properties for molecular recognition and binding energy
Glucose Galactose
Sialic Acid
Mannose
SaccharidesSaccharidesSaccharidesSaccharides
• Carbohydrates: The language of glycobiologyCarbohydrates: The language of glycobiology• The glycosidic bond and glycan nomenclatureThe glycosidic bond and glycan nomenclature• Major vertebrate glycan classesMajor vertebrate glycan classes
• Carbohydrates: The language of glycobiologyCarbohydrates: The language of glycobiology• The glycosidic bond and glycan nomenclatureThe glycosidic bond and glycan nomenclature• Major vertebrate glycan classesMajor vertebrate glycan classes
SaccharidesSaccharidesSaccharidesSaccharides
• Carbohydrates: The language of glycobiologyCarbohydrates: The language of glycobiology• The glycosidic bond and glycan nomenclatureThe glycosidic bond and glycan nomenclature• Major vertebrate glycan classesMajor vertebrate glycan classes
• Carbohydrates: The language of glycobiologyCarbohydrates: The language of glycobiology• The glycosidic bond and glycan nomenclatureThe glycosidic bond and glycan nomenclature• Major vertebrate glycan classesMajor vertebrate glycan classes
When a glycosidic bond is formed the anomeric configuration is “locked”When a glycosidic bond is formed the anomeric configuration is “locked”
Glycosidic bonds link sugars togetherGlycosidic bonds link sugars together
Taylor & Drickamer (2011) Introduction to Glycobiology, 3rd edition
(lactose) CCRC
Nomenclature:Nomenclature:
-Name the non-reducing (left-most) sugar (Gal)-Name the non-reducing (left-most) sugar (Gal) -Name the anomeric configuration ( -Name the anomeric configuration (ββ)) -Name the anomeric carbon number (1) -Name the anomeric carbon number (1) -Name the substituted carbon number (4) -Name the substituted carbon number (4) -Name the substituted sugar (Glc) -Name the substituted sugar (Glc)
RESULT: Gal RESULT: Gal ββ1-4 Glc1-4 Glc
Taylor & Drickamer (2011) Introduction to Glycobiology, 3rd edition
NeuAc NeuAc α2-α2-3 Gal 3 Gal β1-β1-4 (Fuc 4 (Fuc α1-α1-3) GlcNAc3) GlcNAc
Nomenclature: Branches are placed in parenthesesNomenclature: Branches are placed in parentheses
Varki et al. (2009) Proteomics. 9:5398
Oligosaccharides: Molecular diversity of glycansOligosaccharides: Molecular diversity of glycans
PolypeptidesPolypeptides GlycansGlycans
Building blocksBuilding blocks amino acidsamino acids monosaccharidesmonosaccharides
Number of different monomersNumber of different monomers 20 common20 common 9 common9 common
Linkage sites per monomerLinkage sites per monomer 11 3-43-4
Possible linkage configurationsPossible linkage configurations 11 22
Possible homodimer structuresPossible homodimer structures 11 6-86-8
Linkage modesLinkage modes linearlinear linear or branchedlinear or branched
HO
HO
O
HO
O
GalHO
OHO
HO
O
HO
O
GalHO
O
H2N C
Ala
OH
OH2N C
Ala
OH
O
Three different amino acids (Ala, Ser, Tyr) – 6 structuresThree different amino acids (Ala, Ser, Tyr) – 6 structures
Three different sugars (Glc, Gal, Man) – 1,056 structures …Three different sugars (Glc, Gal, Man) – 1,056 structures …
Man a1-4 Gal a1-3 Glc b1Man a1-6 Gal a1-3 Glc b1Man b1-2 Gal a1-3 Glc b1Man b1-3 Gal a1-3 Glc b1Man b1-4 Gal a1-3 Glc b1Man b1-6 Gal a1-3 Glc b1Man a1-2 Gal a1-4 Glc b1Man a1-3 Gal a1-4 Glc b1Man a1-4 Gal a1-4 Glc b1Man a1-6 Gal a1-4 Glc b1Man b1-2 Gal a1-4 Glc b1Man b1-3 Gal a1-4 Glc b1Man b1-4 Gal a1-4 Glc b1Man b1-6 Gal a1-4 Glc b1Man a1-2 Gal a1-6 Glc b1Man a1-3 Gal a1-6 Glc b1
SaccharidesSaccharidesSaccharidesSaccharides
• Carbohydrates: The language of glycobiologyCarbohydrates: The language of glycobiology• The glycosidic bond and glycan nomenclatureThe glycosidic bond and glycan nomenclature• Major vertebrate glycan classesMajor vertebrate glycan classes
• Carbohydrates: The language of glycobiologyCarbohydrates: The language of glycobiology• The glycosidic bond and glycan nomenclatureThe glycosidic bond and glycan nomenclature• Major vertebrate glycan classesMajor vertebrate glycan classes
SaccharidesSaccharidesSaccharidesSaccharides
• Carbohydrates: The language of glycobiologyCarbohydrates: The language of glycobiology• The glycosidic bond and glycan nomenclatureThe glycosidic bond and glycan nomenclature• Major vertebrate glycan classesMajor vertebrate glycan classes
• Carbohydrates: The language of glycobiologyCarbohydrates: The language of glycobiology• The glycosidic bond and glycan nomenclatureThe glycosidic bond and glycan nomenclature• Major vertebrate glycan classesMajor vertebrate glycan classes
Major Glycans of EukaryotesMajor Glycans of Eukaryotes
• GlycoproteinsGlycoproteins
• GlycolipidsGlycolipids
• ProteoglycansProteoglycans
Major Glycans of EukaryotesMajor Glycans of Eukaryotes
• GlycoproteinsGlycoproteins
• GlycolipidsGlycolipids
• ProteoglycansProteoglycans
GlycoproteinsGlycoproteins• N-LinkedN-Linked
• O-LinkedO-Linked
• GPI AnchorGPI Anchor
• O-GlcNAcO-GlcNAc
*A non-inclusive list!*A non-inclusive list!
GlycoproteinsGlycoproteins• N-LinkedN-Linked
• O-LinkedO-Linked
• GPI AnchorGPI Anchor
• O-GlcNAcO-GlcNAc
*A non-inclusive list!*A non-inclusive list!
““N-linked” glycan linkage to protein asparagine residuesN-linked” glycan linkage to protein asparagine residues
Families of N-linked glycan structuresFamilies of N-linked glycan structures
Man
4GlcNAc1
2Man
Man
Man
Man
2Man
2Man
2Man
63
Man63 4GlcNAc1 Asn
4GlcNAc1
Man62
Man63 4GlcNAc1 Asn
Man42
4GlcNAc1
4GlcNAc1
4GlcNAc1
4GlcNAc1
6Gal
6Gal
6Gal
6Gal
NeuAc
NeuAc
NeuAcNeuAc
4GlcNAc1
Man
Man63 4GlcNAc1 Asn
2Man4GlcNAc13GalNeuAc
Man
Man
63
core
core
core
High Mannose
Complex
Hybrid
Examples of structures found as termini on the branches of complex Examples of structures found as termini on the branches of complex N-linked glycoprotein glycans….N-linked glycoprotein glycans….
Terminal Saccharide Diversity --- A key to recognitionTerminal Saccharide Diversity --- A key to recognition
NeuAc NeuAc 2-6 Gal 2-6 Gal 1-4 GlcNAc---1-4 GlcNAc---NeuAc NeuAc 2-3 Gal 2-3 Gal 1-4 GlcNAc---1-4 GlcNAc---
Gal Gal 1-3 Gal 1-3 Gal 1-4 GlcNAc---1-4 GlcNAc---4-SO4-SO33-GalNAc -GalNAc 1-4 GlcNAc---1-4 GlcNAc---
(-Gal (-Gal 1-4 GlcNAc 1-4 GlcNAc 1-3-)1-3-)nn = polylactosamine repeats (n may be >6) = polylactosamine repeats (n may be >6)
(-NeuAc (-NeuAc 2-8-) 2-8-) nn = polysialic acid (n may be >50) = polysialic acid (n may be >50)
Major Glycans of EukaryotesMajor Glycans of Eukaryotes
• GlycoproteinsGlycoproteins
• GlycolipidsGlycolipids
• ProteoglycansProteoglycans
Major Glycans of EukaryotesMajor Glycans of Eukaryotes
• GlycoproteinsGlycoproteins
• GlycolipidsGlycolipids
• ProteoglycansProteoglycans
Glycosaminoglycans (GAG’s) Glycosaminoglycans (GAG’s) and Proteoglycansand Proteoglycans
• GLYCOSAMINOGLYCANS (GAG’s)GLYCOSAMINOGLYCANS (GAG’s)– long linear glycans made of repeating long linear glycans made of repeating
disaccharidesdisaccharides– Hyaluronic acid is the only “stand-alone” Hyaluronic acid is the only “stand-alone”
GAG, other GAG’s are constituents of …GAG, other GAG’s are constituents of …
• PROTEOGLYCANSPROTEOGLYCANS– GAG’s on proteinsGAG’s on proteins– Defined by their repeating disaccharide unitsDefined by their repeating disaccharide units– GAG’s on proteoglycans are sulfatedGAG’s on proteoglycans are sulfated
Hyaluronic acid -- a “simple” Hyaluronic acid -- a “simple” space filling moleculespace filling molecule
(GlcU (GlcU β4 GlcNAc)β4 GlcNAc)nn
Post-polymerization variations in glycosaminoglycansPost-polymerization variations in glycosaminoglycans