Introduction to glycobiology

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)

Course ObjectivesCourse ObjectivesCourse ObjectivesCourse Objectives

http://www.youtube.com/watch?v=v9j-g0qspdk

http://www.youtube.com/watch?v=v9j-g0qspdk

Introduction to glycobiologyIntroduction to glycobiologyIntroduction to glycobiologyIntroduction to glycobiologyDate Day Lecture Faculty

ONLINE Saccharides & The Glyco World Dr. R. Schnaar

Mar 19 Wed Glycoproteins I Dr. N. Zachara

Mar 21 Fri Glycoproteins II Dr. N. Zachara

Mar 25 Tue Glycolipids and GPI anchors Dr. R. Schnaar

Mar 27 Thu Carbohydrate Engineering Dr. K. Yarema

Mar 31 Mon O-GlcNAc Dr. G. Hart

Apr 2 Wed Glycomics (Analytical Glycobiology) Dr. G. Hart

Apr 4 Fri Glycans and Disease Dr. G. Hart

Apr 8 Tue Hyaluronan and Proteoglycans Dr. N. Zachara

Apr 10 Thu Protein-Glycan Recognition (Lectins) Dr. R. Schnaar

Apr 14 Mon Glycan Binding Protein Functions Dr. R. Schnaar


Course co-directors: Course co-directors: Ronald Schnaar ([email protected])Ronald Schnaar ([email protected]) Natasha Zachara ([email protected])Natasha Zachara ([email protected])Lecturers: Lecturers:

Gerald Hart ([email protected])Gerald Hart ([email protected])Kevin Yarema ([email protected])Kevin Yarema ([email protected])


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

What are:What are:

Glycobiology?Glycobiology?Glycosciences?Glycosciences?

Glycomics?Glycomics?

What are:What are:

Glycobiology?Glycobiology?Glycosciences?Glycosciences?

Glycomics?Glycomics?

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).

GlycosciencesGlycosciencesGlycobiology & Glycochemistry, including…Glycobiology & Glycochemistry, including…

-Chemical synthesis-Chemical synthesis -Chemoenzymatic synthesis -Chemoenzymatic synthesis -Metabolic engineering -Metabolic engineering -Glycomimetics -Glycomimetics -Glycan conformation and structure -Glycan conformation and structure -Material Sciences -Material Sciences -Plant and bacterial polysaccharides -Plant and bacterial polysaccharides

GlycosciencesGlycosciencesGlycobiology & Glycochemistry, including…Glycobiology & Glycochemistry, including…

-Chemical synthesis-Chemical synthesis -Chemoenzymatic synthesis -Chemoenzymatic synthesis -Metabolic engineering -Metabolic engineering -Glycomimetics -Glycomimetics -Glycan conformation and structure -Glycan conformation and structure -Material Sciences -Material Sciences -Plant and bacterial polysaccharides -Plant and bacterial polysaccharides

GlycomicsGlycomics- Analytical glycosciences- Analytical glycosciences -Glycan bioinformatics -Glycan bioinformatics

““Glycome” – The total complement of glycansGlycome” – The total complement of glycans in a cell or organism in a cell or organism

GlycomicsGlycomics- Analytical glycosciences- Analytical glycosciences -Glycan bioinformatics -Glycan bioinformatics

““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"

http://www.niaid.nih.gov/labsandresources/labs/aboutlabs/vrc/structuralbiologylaboratory/Pages/kwong.aspx

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 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

Fischer projectionFischer projection

D-glyceraldehyde

L-glyceraldehydeD-glyceraldehyde

EnantiomersEnantiomers

* *

* Highest numbered asymmetric carbon = reference carbon

L-glyceraldehydeD-glyceraldehyde

EnantiomersEnantiomers

* *


DiasteriomersDiasteriomers


* *

Enantiomers & DiasteriomersEnantiomers & Diasteriomers


* *

**

carbonsasymmetric

carbons

diaster-

iomers

diasteriomers &

enantiomers

3 1 0 2

4 2 2 4

5 3 4 8

6 4 8 16

Essentials of Glycobiology Second Edition

** * *

* epimers: differ in only stereogenic center

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

D-glucose

α-D-glucose

β-D-glucose

Lee & Lee (1999) J Chinese Chem Soc 46:283-291

C

C

C

C

H

HH

H

O

O O

O

O

CH OH

C

O

CHO H

C

Anomeric

carbon

Ring oxygen

Determining anomeric configurationDetermining anomeric configuration

[PDB]

Monosaccharide conformation – Chair configurationsMonosaccharide conformation – Chair configurations

Taylor & Drickamer (2006) Introduction to Glycobiology, 2nd edition

Nearly all vertebrate glycans Nearly all vertebrate glycans are built from only 9 sugars:are built from only 9 sugars:

3 hexoses*3 hexoses*• GlucoseGlucose• MannoseMannose• GalactoseGalactose

2 N-acetylhexosamines2 N-acetylhexosamines• N-acetylglucosamineN-acetylglucosamine• N-acetylgalactosamineN-acetylgalactosamine

•xylosexylose•glucuronic acidglucuronic acid•sialic acidsialic acid•L-fucoseL-fucose

*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


Two important terminal eukaryotic sugarsTwo important terminal eukaryotic sugars


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







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


(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


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

Ala-Ser-TyrAla-Ser-TyrAla-Tyr-SerAla-Tyr-SerSer-Ala-TyrSer-Ala-TyrSer-Tyr-AlaSer-Tyr-AlaTyr-Ala-SerTyr-Ala-SerTyr-Ser-AlaTyr-Ser-Ala

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







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!






““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)

Fuc residues (e.g. Fuc Fuc residues (e.g. Fuc 1-21-2 Gal; Fuc Gal; Fuc 1-3 GlcNAc; Fuc 1-3 GlcNAc; Fuc 1-4 GlcNAc1-4 GlcNAc











“O-linked” glycan linkage to protein serine residues

Examples of variations of O-linked glycans

GalNAc Serine

GalNAc Serine

NeuAc 26

GalNAc Serine

Gal 1

NeuAc 263

GalNAc SerineNeuAc 2-3 Gal 1

NeuAc 263

Gal 1-4 GlcNAc 1

Fuc 1

GalNAc Serine

GlcNAc 1

63

4NeuAc 2-3 Gal 1

3











GPI (glycosylphosphatidylinositol) anchored proteinGPI (glycosylphosphatidylinositol) anchored protein

Gal 2Gal62Gal

Gal

Man 2Man

Man63 4GlcNH2

1 6myo Inositol1

P=O

O

O

O

O

CH2

CH2

NHC

O

Asp

[Phosphoethanolamine]

6

P=O

O

O

O

O

CH2CH CH2

[Phosphatidylinositol]

20 kDa protein3 kDa N-linked

glycan

1 kDa O-linked

glycan

1.5 kDa GPI

anchorplasma membrane

CD59, a complement defense glycoproteinCD59, a complement defense glycoprotein











O-GlcNAc

Note: No further sugar substitutions have been confirmedHighly dynamic post-translational modification!









A GlycosphingolipidA Glycosphingolipid

Galactosylceramide (GalCer)Galactosylceramide (GalCer)

HO

HO

O

HO

O

NH

HO

Gal Sphingosine

Ceramide

HO

A GangliosideA Ganglioside

““GD1a”GD1a”

HO

OO

HO

HOOHO

O

HO

HOHO

O

HO

NH

HOHO

O

H3COH3C

NH

H3CNH

OO

HO

O

NH

HO

GlcGalGalGalNAcNeuAc

NeuAc

Sphingosine

Ceramide









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

Cartilage proteoglycanCartilage proteoglycan

Cartilage proteoglycanCartilage proteoglycan

Glycan BiosynthesisGlycan Biosynthesis

Glycosyltransferase reaction Glycosyltransferase reaction (UDP-Gal:glucose (UDP-Gal:glucose β4 β4 galactosyltransferase)galactosyltransferase)

UDP-GlcUDP-GlcUDP-GalUDP-GalGDP-ManGDP-ManUDP-GlcNAcUDP-GlcNAcUDP-GalNAcUDP-GalNAc

UDP-GlcAUDP-GlcAUDP-XylUDP-XylGDP-FucGDP-FucCMP-NeuAcCMP-NeuAc

Activated Sugars:Activated Sugars:

Glycoprotein biosynthesisGlycoprotein biosynthesis

• N-Linked – en-bloc preconstructed core, trimming, N-Linked – en-bloc preconstructed core, trimming, terminal elaborationterminal elaboration

• O-Linked – stepwise sugar by sugar additionO-Linked – stepwise sugar by sugar addition• O-GlcNAc - dynamic transferase/glycosidaseO-GlcNAc - dynamic transferase/glycosidase• GPI Anchor – en-bloc preconstructed core, elaborationGPI Anchor – en-bloc preconstructed core, elaboration

Glycolipid biosynthesisGlycolipid biosynthesis

• Stepwise sugar-by-sugar additionStepwise sugar-by-sugar addition

Proteoglycan biosynthesisProteoglycan biosynthesis

• Stepwise sugar-by-sugar addition (core and repeating Stepwise sugar-by-sugar addition (core and repeating disaccharide)disaccharide)

• Post-polymerization modificationsPost-polymerization modifications

Documents

Introduction to glycobiology