glycobiology introduction to glycobiologyintroduction to glycobiology glycan structuresglycan...

GlycobiologyGlycobiology

• Introduction to GlycobiologyIntroduction to Glycobiology

• Glycan structuresGlycan structures

• Glycan functionsGlycan functions

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

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?

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 – glycans stained blackThe “glycocalyx” surrounding a fibroblast – glycans 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

““Evolution has failed to generate a living cell Evolution has failed to generate a living cell devoid of surface glycosylation” devoid of surface glycosylation” - A. Varki- A. Varki

““Every eukaryotic cell is covered with a dense and complex array Every eukaryotic cell is covered with a dense and complex array of glycans, which also feature prominently in extracellular matrix of glycans, which also feature prominently in extracellular matrix and secreted soluble molecules.” … in fact…and secreted soluble molecules.” … in fact…

Tropical Forest

Canopy

Ajit Varki, “Perspectives on the Future of Glycobiology,” Athens, GA, 14-Mar-2011

Tropical Forest

Canopy

Cohen & Varki (2010) OMICS 4:455

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

Sharon and Lis (1993) Scientific American

Cell-cell recognition is one important functionCell-cell recognition is one important function

Lectin

Glycan

Life cycle of the influenza virus - the role of glycansLife cycle of the influenza virus - the role of glycans

Influenza jumps from animals to humans via a Influenza jumps from animals to humans via a slight change in glycan binding specificityslight change in glycan binding specificity

Stevens, et al. (2006) Nat Rev Microbiol. 4:857-64

Inflammation: How we fight bacterial infection

Leukocyte cell surface glycan ligand Leukocyte cell surface glycan ligand required for endothelial recognitionrequired for endothelial recognition

R

Marquardt, et al. (1999) J. Pediatr. 134, 681

You fight bacterial infection using glycans. This You fight bacterial infection using glycans. This child could not …child could not …

Marquardt, et al. (1999) Blood 94, 3976

……until he was fed FUCOSE (a sugar), after which…until he was fed FUCOSE (a sugar), after which…

“…“…neutrophil levels returned to the normal range .... neutrophil levels returned to the normal range .... there were no further infections”there were no further infections”

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 onto themselves, Proteins typically fold onto themselves, whereas glycans spread out in spacewhereas glycans spread out in space

plasma membrane

plasma membrane

Glycoprotein Glycolipid

Monosaccharides equilibrate between open chain and ring forms.Monosaccharides equilibrate between open chain and ring forms.For each monosaccharide, two ring forms (anomeric For each monosaccharide, two ring forms (anomeric

configurations) are possible, configurations) are possible, αα and and ββ

Seven sugars and their structural relationship to glucoseSeven sugars and their structural relationship to glucose

Two important terminal sugarsTwo important terminal sugars

When a glycosidic bond is formed,When a glycosidic bond is formed,the anomeric configuration is “locked”the anomeric configuration is “locked”

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 α2-3 Gal β1-4 (Fuc α1-3) GlcNAcNeuAc α2-3 Gal β1-4 (Fuc α1-3) GlcNAc

Nomenclature: Branches are placed in parenthesesNomenclature: Branches are placed in parentheses

NeuAc α2-3 Gal β1-4 (Fuc α1-3) GlcNAcNeuAc α2-3 Gal β1-4 (Fuc α1-3) GlcNAc

Molecular diversity glycansMolecular diversity 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 …

Major Glycoconjugates of EukaryotesMajor Glycoconjugates 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

Diversity in complex N-linked glycan structureDiversity in complex N-linked glycan structure

4GlcNAc1

Man642

Man643

4GlcNAc1 Asn

Man42

4GlcNAc1

4GlcNAc1

6Gal

6Gal

NeuAc

4GlcNAc16GalNeuAc

4GlcNAc16GalNeuAc

NeuAc4GlcNAc16GalNeuAc

GlcNAc1

Fuc

6

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

Glycoforms add yet more variation-- Glycoforms add yet more variation--

Whereas a protein is defined by its unique amino acid Whereas a protein is defined by its unique amino acid sequence, sequence, the glycan structure at a single protein the glycan structure at a single protein glycosylation site variesglycosylation site varies. .

Although glycan structures vary at each glycosylation Although glycan structures vary at each glycosylation site, the profile is reproducible, protein-specific, site-site, the profile is reproducible, protein-specific, site-specific, and cell type-specific.specific, and cell type-specific.

Three glycoforms of CD59Three glycoforms of CD59

“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 confirmed

O-GlcNAc is localized to the cytoplasm and nucleus.O-GlcNAc is localized to the cytoplasm and nucleus.

O-GlcNAc is present in all higher eukaryotes studied.O-GlcNAc is present in all higher eukaryotes studied.

O-GlcNAc is as abundant as phosphorylation.O-GlcNAc is as abundant as phosphorylation.

UDP-GlcNAc is as abundant as ATP in some cell types. UDP-GlcNAc is as abundant as ATP in some cell types.

O-GlcNAc containing proteins are phosphoproteins.O-GlcNAc containing proteins are phosphoproteins.

O-GlcNAc and phosphorylation are often reciprocal.O-GlcNAc and phosphorylation are often reciprocal.

O-GlcNAc is dynamic, implying a regulatory role.O-GlcNAc is dynamic, implying a regulatory role.

Key Features of O-linked GlcNAc

Three-state model: unmodified / O-GlcNAc / phosphate

O-GlcNAc

GlycoproteinGlycoproteinUnmodified protein

Unmodified proteinPhosphoprotein

OPO32-

PhosphatasePhosphatase

OGTOGT

OGTOGT

PhosphatasePhosphatase

KinaseKinase

KinaseKinase

O-GlcNAcaseO-GlcNAcase

O-GlcNAcaseO-GlcNAcase

Natasha Zachara

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

Glycolipids are the major glycoconjugates in the brainGlycolipids are the major glycoconjugates in the brain

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 β4 GlcNAc)(GlcU β4 GlcNAc)nn

Post-polymerization variations in glycosaminoglycansPost-polymerization variations in glycosaminoglycans

Proteoglycans

disaccharide (A-B)n

NameMol. Wt.(daltons) A B

Sulfates perdisaccharide

Linked toprotein? Core Tissue distribution

hyaluronicacid

up to 8 x 106 GlcA GlcNAc 0 N NAconnective tissue,skin, vitreoushumor, cartilage,synovial fluid

chondroitinsulfate

5,000 -50,000 GlcA GalNAc 0.2 - 2.3 Y Gal-Gal-Xyl

cartilage, cornea,bone, skin, arteries

dermatansulfate

15,000 -40,000

GlcA orIdA

GalNAc 1.0 - 2.0 Y Gal-Gal-Xylskin, bloodvessels, heart,heart valves

heparansulfate

5,000 -12,000

GlcA orIdA

GlcNAc 0.2 - 2.0 Y Gal-Gal-Xyllung, arteries, cellsurfaces, basallaminae

heparin6,000 -25,000

GlcA orIdA GlcNAc 2.0 - 3.0 Y Gal-Gal-Xyl

lung, liver, skin,mast cells

keratansulfate

4,000 -19,000 Gal GlcNAc 0.9 - 1.8 Y

N- and O-linkedglycoproteins

cartilage, cornea,intervetebral discs

Cartilage proteoglycanCartilage proteoglycan

fibroblast

extracellular

intracellular

Squamus Epithelium

Glycoproteins and glycolipids are found prominently on extracellular leaflet of the plasma membrane and on the intralumenal leaflet of lysosomes. They also are found in the ER and Golgi apparatus, where they are synthesized. GAG’s and proteoglycans are found on the cell surface and as prominent structures of the extracelllular matrix.




• Glycan functions Glycan functions – RecognitionRecognition– Biophysical effectsBiophysical effects– Biochemical effectsBiochemical effects




• Glycan functions Glycan functions – RecognitionRecognition

• General principlesGeneral principles• Influenza virusInfluenza virus• InflammationInflammation

– Biophysical effectsBiophysical effects– Biochemical effectsBiochemical effects

Sharon and Lis (1993) Scientific American

Carbohydrate binding determinant

Carbohydrate recognition domain (CRD)

Glycans in cell-cell recognitionGlycans in cell-cell recognition

Lectin

Glycan

Sialoadhesin CRDSialoadhesin CRD

May, A.P., et al. (1998) Mol. Cell 1, 719-728

Principles of glycan-Principles of glycan-lectin binding:lectin binding:

-hydrogen bonds -hydrogen bonds -hydrophobic stacking-hydrophobic stacking-multivalent binding-multivalent binding-(salt bridges)-(salt bridges)-(calcium coordination)-(calcium coordination)

May, A.P., et al. (1998) Mol. Cell 1, 719-728

Sialic acid

glycerol

carboxylate

acetyl

Comb & Roseman, J Am Chem Soc 80, 497 (1958)

Sialic acids --Sialic acids -- Enhanced roles in glycan recognition Enhanced roles in glycan recognition

Diverse sialic acid glycans at the cell surfaceDiverse sialic acid glycans at the cell surface

Essentials of GlycobiologySecond Edition

sialic acid

Diverse sialic acid linkages at the cell surfaceDiverse sialic acid linkages at the cell surface

NeuAc-α2,3-Gal-β1,4-GlcNAc NeuAc-α2,6-Gal-β1,4-GlcNAc

Life cycle of the influenza virus - the role of sialic acidsLife cycle of the influenza virus - the role of sialic acids

Influenza virus hemagglutinin (side and top views, arrows are sugar binding sites)

Sialoglycan structure drives biological recognition:Sialoglycan structure drives biological recognition:Influenza pandemics are determined by sialic acid linkage specificityInfluenza pandemics are determined by sialic acid linkage specificity

Stevens et al (2006) Nat Rev Microbiol. 4:857-64

Influenza virus Influenza virus neuraminidaseneuraminidase

(sialidase)(sialidase)

Rational design of an anti-influenza drug based on Rational design of an anti-influenza drug based on sialic acid binding to the viral neuraminidasesialic acid binding to the viral neuraminidase

1

2

345

67

89

OH

HO

HO

OOR

NH

OH

COOH

NH

NH

NH2

OH

HO

ONH

OH

COOH

O

O

NH2

NH

O

COOR

O

Sialic Acid Relenza Tamiflu

Influenza virus Influenza virus budding in vitro budding in vitro

without (top) and without (top) and with (bottom) with (bottom)

neuraminidase neuraminidase inhibitorinhibitor

Prophylactic efficacy of a sialic acid-based Prophylactic efficacy of a sialic acid-based influenza neuraminidase inhibitorinfluenza neuraminidase inhibitor

Peters, P.H., et al. (2001) J Am Geriatr Soc 49, 1025

Inflammation: How we fight bacterial infection

Leukocyte adhesion to E-selectin transfected COS cells

Selectin

glycoconjugate

Endothelium

Neutrophil

Leukocyte cell surface glycan ligand Leukocyte cell surface glycan ligand required for endothelial recognitionrequired for endothelial recognition

R

Marquardt, et al. (1999) J. Pediatr. 134, 681

Leukocyte adhesion deficiency (LAD) Type IILeukocyte adhesion deficiency (LAD) Type II

Human blood group antigens and Selectin ligandsHuman blood group antigens and Selectin ligands

Fuc 1-2 Gal 1- O (H) Blood Type

Fuc 1-2 Gal 1- A Blood Type |

GalNAc 1-3

Fuc 1-2 Gal 1- B Blood Type |

Gal 1-3

Gal 1- Bombay Phenotype

NeuAc 2-3 Gal 1-4 GlcNAc- Selectin ligand |

Fuc 1-3

NeuAc 2-3 Gal 1-4 GlcNAc- LAD Type II

““Within days of starting fucose therapy, neutrophil levels returned to the Within days of starting fucose therapy, neutrophil levels returned to the normal range .... there were no further infections and antibiotic normal range .... there were no further infections and antibiotic prophylaxis was discontinued.”prophylaxis was discontinued.”

Marquardt, et al. (1999) Blood 94, 3976

Hyaluronic acid -- a “simple” Hyaluronic acid -- a “simple” space filling moleculespace filling molecule

(GlcU β4 GlcNAc)(GlcU β4 GlcNAc)nn

Anticoagulant carbohydrate determinantAnticoagulant carbohydrate determinant

Reference textsReference textsReference textsReference texts

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

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

Introduction to GlycobiologyIntroduction to GlycobiologyME:330.712ME:330.712

Mar-April 2013Mar-April 2013

Introduction to GlycobiologyIntroduction to GlycobiologyME:330.712ME:330.712

Mar-April 2013Mar-April 2013

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

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

glycobiology introduction to glycobiologyintroduction to glycobiology glycan structuresglycan...

Documents

eukaryotic cell surfacelodish

varkievery eukaryotic

molecular cell biology

major cell component

sugar gal

anomeric carbon number

slight change

substituted carbon number