Essentials of Glycobiology

March 30th, 2004

Ajit Varki

Lecture 1 Course Overview & General Introduction

Online at NCBI http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Books

Lecture 1 - Key Points

• What is Glycobiology?

• Central Dogma

• Basic definitions

• Monosaccharides - the structural units of glycans

• Oligoaccharides consist of monosaccharides linked together

• Nomenclature and symbolic representations

• Major classes of glycoconjugates and oligosaccharides

• Clustered oligosaccharides

• Proteins can bind at either the ends of glycans or within the chains

DNA RNA PROTEIN CELL ORGANISM

DNA

ORGANISM

? ?

All Cells Are Coated with “Glycans”

Electron micrograph of a human lymphocyte (Ruthenium Red staining)

LIPIDS

DNA RNA PROTEINS

MATRIXCELL

GLYCOPROTEINSPROTEOGLYCANSGLYCOLIPIDS

SIGNALLINGMOLECULES

TISSUES & ORGANS

TRANSCRIPTION FACTORS

PHYSICAL ENVIRONMENT

MICROBESPARASITES

ENZYMES

DIET

DNA

SUGARS

ORGANISM

DNA

Macromolecules

Macromolecule Building Block AproximateMass

PossibleVariations in a

TrimerProtein Amino acids 125 ’ 104-105 6

Nucleic Acid Nucleotides 330 ’ 103-109 6

Lipid Fatty acids 250 ’ 103 NA

Carbohydrate Monosaccharides 200 ’ 102-106 1,056 to 27,648!

What is Glycobiology?

¶ Study of the structure, biosynthesis and biology of saccharides (sugar chains or “glycans”) in nature.

¶ Glycobiology is an integrative science, crossing all subfields of chemistry, biology and medicine.

¶ Chemical analysis and biochemistry dominated the field in the early part of the century.

¶ In the 1960’s, glycans in the extracellular matrix, on the cell surface, and in the interior of cells were found to have biological properties independent of the underlying protein or lipid.

¶ The term “Glycobiology” was coined in 1988 by Rademacher, Parekh, and Dwek: Annu Rev Biochem. 57:785-838.

“Central Dogma”

Glycans occur in patterns that are characteristic of the cell-type, tissue and organism

Glycan patterning is not template driven

Instead, the glycan composition and pattern depends on expression of biosynthetic enzyme (transferases), substrate specificity, and the availability of precursors (i.e., more like “assembly-line driven)

Lecture 1 - Key Points

• What is Glycobiology?

• Central Dogma

• Basic definitions

• Monosaccharides - the structural units of glycans

• Oligoaccharides consist of monosaccharides linked together

• Nomenclature and symbolic representations

• Major classes of glycoconjugates and oligosaccharides

• Clustered oligosaccharides

• Proteins can bind at either the ends of glycans or within the chains

Basic Definitions

• Monosaccharide: A carbohydrate that cannot be hydrolyzed into a simpler carbohydrate. The building block of oligosaccharides and polysaccharides.

• Oligosaccharide: Linear or branched chain of monosaccharides attached to one another via glycosidic linkages. The number of monosaccharide units can vary.

• Polysaccharide: Glycan composed of repeating monosaccharides, generally greater than ten monosaccharide units in length.

• Carbohydrate, glycan, saccharide, sugar: Generic terms used interchangeably. Includes monosaccharides, oligosaccharides, polysaccharides, and derivatives of these compounds. Carbohydrates consist of “hydrated carbon”, [CH2O]n

• Preferred generic term is “Glycan”

Monosaccharides - the basic structural unit

• Carbonyl group at the end of the carbon chain (aldoses) or at an inner carbon (ketoses) has potential reducing power. This end is called the reducing terminus, or reducing end

• The ring form of a monosaccharide generates a chiral (anomeric) center (at C-1 for aldo sugars or at C-2 for keto sugars). Notice that other positions are chiral, which therefore imparts stereochemical information

HO

CHO

H

HHO

OHH

OHH

CH2OH

HOO

H

H

HO

OH

OHHH

H

OH

H O

OH

H

OH

OH

HH

OH

CH2OH

H

1

2

3

4

5

6

1

23

4

5

6

123

4

5

6

Fischer Hayworth Stereochemical

Oligosaccharides and Polysaccharides

-linkage -linkage

OH O

H

H

H

OHH

OH

CH2OH

H1

23

4

5

6

H O

H

H

OHH

OH

CH2OH

H1

23

4

5

6

O

H O H

OH

OH

HH

OH

CH2OH

H1

23

4

5

6

O

Glycosidic linkage can be or The convention is to draw the reducing end to the right and the non-reducing end to the left. Once in glycosidic linkage, ring can no longer open Reducing end is still called the same even when it is covelantly bound to another macromolecule (tying up its reducing power)

Common Monosaccharides

NeutralSugars

AminoSugars

AcidicSugars

OOH

OH

OH

HO

CH2OH

Galactose(Gal)

OOH

OHOHHO

CH2OH

Mannose(Man)

OOH

OH

OHHO

Xylose(Xyl)

OOH

OH

OH

HO

CH3

Fucose(Fuc)

OOH

OH

OHHO

CH2OH

Glucose(Glc)

CHOHCH2OH

CHOHO

COOH

OH

AcN

OH

Sialic acids(Sia)

OOH

OH

OHHO

COOH

Glucuronic acid(GlcA)

OOH

OH

OHHO

COOH

Iduronic acid(IdoA)

OOH

NAC

OH

HOCH2OH

N-acetyl Galactosamine(GalNAc)

OOH

NAC

OHHO

CH2OH

N-acetyl Glucosamine(GlcNAc)

Lecture 1 - Key Points

• What is Glycobiology?

• Central Dogma

• Basic definitions

• Monosaccharides - the structural units of glycans

• Oligoaccharides consist of monosaccharides linked together

• Nomenclature and symbolic representations

• Major classes of glycoconjugates and oligosaccharides

• Clustered oligosaccharides

• Proteins can bind at either the ends of glycans or within the chains

Symbolic Representation of Common Monosaccharides

To simplify the structural complexity of glycans, we will generally use a standard set of symbols to represent sugars.

= Glucose (Glc)= Mannose (Man)= Galactose (Gal)= N-acetylglucosamine (GlcNAc)= N-acetylgalactosamine (GalNAc)= N-acetylhexosamine, unspecified (HexNAc)

= Fucose (Fuc)= Xylose (Xyl)= Sialic acid, unspecified (Sia)= Glucuronic acid (GlcA)= Iduronic acid (IdoA)= Uronic acid, unspecified (HexA)

Hexose,unspecified (Hex)

Symbol set is being modified for second edition of “Essentials”, in a consensus agreement with NCBI, Consortium for Functional Glycomics, KEGG and other national and international bodies

Saccharide Modifications increase the diversity and functionality of glycans

= GlcNAc= GlcA= Gal= Xyl = IdoA

NS NSNS NSNS NSNS2S 2S2S 2S6S 6S 6S 6S 6S6S

3S

Ac = O-acetyl P = Phosphate S = O-Sulfate NS = N-Sulfate NH2 = free amino group

Lecture 1 - Key Points

• What is Glycobiology?

• Central Dogma

• Basic definitions

• Monosaccharides - the structural units of glycans

• Oligoaccharides consist of monosaccharides linked together

• Nomenclature and symbolic representations

• Major classes of glycoconjugates and oligosaccharides

• Clustered oligosaccharides

• Proteins can bind at either the ends of glycans or within the chains

Glycoconjugates

Glycoconjugate: A compound in which one or more glycans (the glycone) are covalently linked to a non-carbohydrate moiety (the aglycone).

Glycoproteins: A protein with one or more covalently bound glycans.

Glycolipids: A molecule containing a saccharide linked to a lipid.

Proteoglycans: Any glycoprotein with one or more covalently attached glycosaminoglycan chains.

N-glycan N-glycanGPI-glycan

GPI-acyl chains

Polypeptide

Schematic representation of the Thy-1 glycoprotein

Major Classes of Animal Glyconconjugates

N-GlcNAc linkedoligosaccharideN-linked chain

N-glycan

O-GalNAc linkedoligosaccharideO-linked chain

O-glycan

O-Xyl linkedoligosaccharide

GlycosaminoglycanGAG chain

Ceramide-linkedoligosaccharides

GlycosphingolipidsGlycolipids

Glycophospholipidanchor

GPI anchorPI-glycan

O--linked GlcNAcO-GlcNAc

OSer/Thr

4

NAsn

4

63

OSer

4

3

3

4

NH2

6

4

2

OSer/Thr

OUTSIDE

INSIDE

*

*Several additional kinds of O-glycans have emerged recently

Major Glycan

Classes in Animal Cells

OSer

OSer/Thr

NAsn

Ser-O-

OUTSIDE

INSIDE

NAsn

S S S

-O-SerS SSS S

EtnP

INOSITOL

P

NH

Ac

P

NS NS

Ac

S

2

P

GlycoproteinGlycoprotein

ProteoglycanProteoglycan

GLYCOPHOSPHO-GLYCOPHOSPHO-LIPIDLIPID

ANCHORANCHOR

N-LINKED CHAINSN-LINKED CHAINS

O-LINKED O-LINKED CHAINCHAIN

HYALURONANHYALURONAN

GLYCOSAMINO-GLYCOSAMINO-GLYCANSGLYCANS HEPARAN SULFATEHEPARAN SULFATE

CHONDROITINCHONDROITIN SULFATESULFATE

Sialic AcidsSialic Acids

GLYCOSPHINGOLIPIDGLYCOSPHINGOLIPID

O-LINKED GlcNAcO-LINKED GlcNAc

Symbolic Representation of Oligosaccharides

4β4

β 4 β2

9Ac

α3α3

α6 β 4 β2

α 6

3α

α69Acβ4 -β4

β4 β2

9Ac

α3

β4 β2 α3

α3α6α6

α3

Symbolic Representation

Fuc 3 Si3Gl4GlcNAc2Mn Fuc 6 6 Mn4GlcNAc4GlcNAc∼ 39Ac-Si6Gl4GlcNAc2Mn

Simplified Traditional

Fucp1 3 Sip2-3Glp1-4GlcNAcp1-2Mnp1 Fucp1 6 6 Mnp1-4GlcNAcp1-4GlcNAcp∼ 39OAc-Sip2-6Glp1-4GlcNAcp1-2Mnp1

Full Traditional

Clustered O-linked Oligosaccharides

• Electron micrograph of a cartilage proteoglycan shows the classical “bottle-brush” appearance

• Many mucins contain clustered O-GalNAc linked oligosaccharides

• These highly hydrated conjugates fill space, provide lubrication, and create clustered glycan ligands for binding receptors

Lecture 1 - Key Points

• What is Glycobiology?

• Central Dogma

• Basic definitions

• Monosaccharides - the structural units of glycans

• Oligoaccharides consist of monosaccharides linked together

• Nomenclature and symbolic representations

• Major classes of glycoconjugates and oligosaccharides

• Clustered oligosaccharides

• Proteins can bind at either the ends of glycans or within the chains

Outer Chains Shared by Different Classes of Glycans can be recognized by Specific

Proteins

OSer/Thr

NAsn

N-LINKED CHAINN-LINKED CHAIN

O-LINKED CHAINO-LINKED CHAIN

GLYCOSPHINGOLIPIDGLYCOSPHINGOLIPID

OUTSIDE

INSIDE

S

CELLMEMBRANE

Membrane ProteinMembrane Protein

OSer/Thr

NAsn

S

= Sialic acid

Secreted ProteinSecreted Protein

Glycosaminoglycan Chains Contain Multiple Protein Binding Sites

� Proteins can bind at the ends of N-linked and O-linked chains, and typically have shallow binding pockets

� Other proteins bind to internal sugar sequences, and have binding clefts

� Valency is a major factor in determining overall affinity

NS NSNS NSNS NSNS2S 2S2S 2S6S 6S 6S 6S 6S6S

3SAntithrombinFGF-1

Essentials of Glycobiology

March 30th, 2004

Ajit Varki

Lecture 1 Course Overview & General Introduction