Eukaryotic cell surface

The “glycocalyx” surrounding a fibroblast – glycans stained black extracellular lipid bilayer fibroblast Martinez-Palomo, A., et al. Cancer Res. 29, 925-937, 1969

• Glycoproteins • Glycolipids • Proteoglycans

intracellular

Modes of glycosylation

Essentials of Glycobiology

Major membrane lipid classes

sphingolipid glycerolipid sterol (ceramide) (diacylglycerol) (cholesterol)

Most vertebrate glycolipids are glycosphingolipids

Glucosylceramide is a key intermediate in skin barrier formation

Holleran et al (2006)

Febs Letters

23:5456 Enzymatic processing of GlcCer generates ceramide, a major lipid required for epidermal barrier function.

galactosylceramide and it’s 3-sulfated from, sulfatide, are major lipids in the brain

Together, GalCer and sulfatide comprise up to 1.9% of brain fresh weight!

Glycosphingolipids are the major glycans of the brain

Glycan ratios in the brain

10 0 40 Myelin total lipids - mole percent Choles terol Phospho lipids 30 Galacto Sulfatide 20

Delcomyn, F. (1998) Foundations of Neurobiology. W.H. Freeman, New York.

Mutant mice lacking GalCer synthase (

Ugt8a

) or GalCer 3-sulfotransferase (

Gal3st1)

display myelin defects and associated behavioral deficits Honke et al (2002)

PNAS

99:4227 Popko (2000)

Glia

29:149 Honke et al (2002)

PNAS

99:4227

-

GlcCer based glycolipid families:

All share a lactosylceramide (Gal β1-4Glcβ1-1Cer) core Biosynthesized stepwise by individual glycosphingolipids, some of which are glycolipid-specific, others of which also make glycoprotein glycans Classified as neutral glycosphingolipids, sulfated glycosphingolipids or gangliosides, which are sialic acid-containing glycosphingolipids Subfamily series Lacto Structure Gal β1-3GlcNAcβ1-3Galβ1-4GlcβCer Neolacto Gal β1-4GlcNAcβ1-3Galβ1-4GlcβCer Ganglio Globo Gal β1-3GalNAcβ1-4Galβ1-4GlcβCer GalNAc β1-3Galα1-4Galβ1-4GlcβCer

Gangliosides – glycosphingolipids with one or more sialic acid residues – are found on

all

vertebrate cells, and are

major

glycans in the brain

Example of a ganglioside – GD1a

Schnaar (2007)

Comprehensive Glycoscience V. 4 ,

323

Cohen & Varki (2010)

OMICS

4:455 Because of their long unsaturated lipid chains, glycolipids cluster together, along with cholesterol, selected other lipids, and selected proteins, in “lipid rafts”

Concepts of glycolipid function:

cis

and

trans

cis

: regulation by lateral association

trans

: regulation by cell-cell recognition Regina Todeschini & Hakomori (2008)

Biochim Biophys Acta

1780:421

Modes of glycosylation

Essentials of Glycobiology

A small but diverse group of proteins are attached to the cell surface by a “glycosylphosphatidylinositol” (GPI) anchor GPI anchors have a minimal (core) structure (black font) with variable additional glycan, fatty acid ester, Thy-1 membrane interaction protein

Essentials of Glycobiology

Second Edition Taylor and Drikamer (2011)

Introduction to Glycobiology

GPI-anchored proteins

*

Mammals Thy-1 CD59 decay accelerating factor (DAF) Alkaline phosphatase 5′-Nucleotidase Renal dipeptidase Trehalase Neural cell adhesion molecule (NCAM-120) cell-cell recognition complement regulation complement regulation cell-surface hydrolase cell-surface hydrolase cell-surface hydrolase cell-surface hydrolase adhesion molecule Neural cell adhesion molecule TAG-1 adhesion molecule CD58 FcγIII receptor Ciliary neurotrophic factor receptor Glial-derived neurotrophic factor receptor Nogo receptor (NgR) CD14 Prion protein (PrP) Glypican family of GPI-anchored proteoglycans adhesion molecule Fc receptor neural receptor neural receptor axon outgrowth regulator LPS receptor unknown extracellular matrix component Parasites Trypanosoma brucei variant surface glycoprotein (VSG) protective coat Leishmania major promastigote surface protease (PSP) bound complement degradation Trypanosoma cruzi GPI anchored mucins host cell invasion Plasmodium falciparum merozoite surface protein 1 (MSP-1) Toxoplasma gondii surface antigen 1 (SAG-1) Entamoeba histolytica GPI proteophosphoglycans erythrocyte invasion host cell invasion virulence factor *plus others in yeast, plants

GPI anchors are complex & diverse • Glucosamine with free amine (rare) • Phosphodiesters at each end • Diverse modifications along the core

Essentials of Glycobiology Second Edition

R3

Variety is the spice of… GPI anchors

Essentials of Glycobiology Second Edition

Like N-linked glycan biosynthesis, the precursor starts assembly on the cytoplasmic face of the ER, then is flipped inside for further processing.

Essentials of Glycobiology Second Edition

Preassembled GPI anchors are covalently transferred

en bloc

via amide linkage to an amino acid near the carboxy terminus of a nacient protein, releasing the C-terminal fragment. This occurs in the ER. Although there is no “consensus sequence,” per se, for GPI transfer, likely sites are identified by their structural features .

Essentials of Glycobiology Second Edition

GPI anchor synthesis has been studied most extensively in parasites, which express millions of GPI-anchored proteins and glycans on the surface of each cell. African sleeping sickness leishmaniasis Chagas disease http://www.lifesci.dundee.ac.uk/people/mike-ferguson

Modes of glycosylation

Essentials of Glycobiology

Glycosaminoglycans (GAG’s) and Proteoglycans

• GLYCOSAMINOGLYCANS (GAG’s)

– 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

Hyaluronic acid - a “simple” space filling molecule (GlcA β1-3 GlcNAc β1-4) n

Essentials of Glycobiology Second Edition

Post-polymerization variations in glycosaminoglycans Heparin / Heparan sulfate GlcA β4 GlcNAc α4 IdoA2S α4 GlcNS6S α4

Glycosaminoglycan repeating disaccharide units

Essentials of Glycobiology Second Edition

Proteoglycans

Name hyaluronic acid chondroitin sulfate dermatan sulfate heparan sulfate heparin keratan sulfate Mol. Wt. (daltons) up to 8 x 10 6 5,000 - 50,000 15,000 - 40,000 5,000 - 12,000 6,000 - 25,000 4,000 - 19,000 disaccharide (A-B) A GlcA β1-3 GlcA β1-3 GlcA β1-3 or IdA α1-3 GlcA β1-4 or IdA α1-4 GlcA β1-4 or IdA α1-4 Gal β1-4 B n GlcNAc β1-4 GalNAc β1-4 GalNAc β1-4 GlcNAc α1-4 GlcNAc α1-4 GlcNAc β1-3 Sulfates per disaccharide 0 0.2 - 2.3 1.0 - 2.0 0.2 - 2.0 2.0 - 3.0 0.9 - 1.8 Protein Linkage None Gal-Gal-Xyl Gal-Gal-Xyl Gal-Gal-Xyl Gal-Gal-Xyl N- and O glycans Tissue distribution connective tissue, skin, vitreous humor, cartilage, synovial fluid cartilage, cornea, bone, skin, arteries skin, blood vessels, heart, heart valves lung, arteries, cell surfaces, basal laminae lung, liver, skin, mast cells cartilage, cornea, intervetebral discs

Proteoglycan/Hyaluronan macromolecular structures

Essentials of Glycobiology Second Edition

Taylor & Drickamer (2011) Introduction to Glycobiology

Cartilage proteoglycan

Glycan-Protein Recognition – Defining terms Carbohydrate binding determinant Glycan Carbohydrate recognition domain (CRD) Lectin = Glycan Binding Protein (GBP) Sharon and Lis (1993) Scientific American

Glycan binding protein diversity – phylogeny & function Sharon and Lis (2004)

Glycobiology

14:53R

Principles of lectin-carbohydrate recognition

• Precisely spaced cooperative hydrogen bonds – direct or through bound water molecules • Hydrophobic stacking • Ionic interactions (for charged glycans) • Calcium coordination (select lectins) • Low site-affinity / high-avidity polyvalent binding

Sialic acids - Enhanced roles in glycan recognition acetyl carboxylate Sialic acid Comb & Roseman,

J Am Chem Soc

80, 497 (1958) glycerol

Diverse sialic acid glycans at the cell surface Essentials of Glycobiology Second Edition sialic acid

Diverse sialic acid linkages at the cell surface

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

Discovery of the “receptor destroying enzyme” of influenza virus Hirst (1942) J. Exp. Med. 76:195

Discovery of sialic acid as the cell surface receptor for influenza virus

• Mucins (e.g. bovine submaxillary mucin… cow drool) inhibited the binding of influenza virus to red blood cells • Pre-treatment of mucin with influenza virus rendered it non-inhibitory • Treatment of highly purified mucin with virus or with an enzyme from

V. cholerae

released the same dialyzable substance • Crystallization of the dialyzable substance identified it as sialic acid (N-acetylneuraminic acid).

Gottschalk (1958) Nature 181:377

Influenza virus lifecycle Laver et al (Jan 1999)

Scientific American

, 78-87 Linda Stannard: http://web.uct.ac.za/depts/mmi /stannard/fluvirus.html

Influenza virus hemagglutinin (side and top views, arrows are sugar binding sites) Weis et al (1988)

Nature

333:426

Sialic acid binding to influenza virus hemagglutinin Weis et al (1988)

Nature

333:426 - hydrogen bonds - hydrophobic stacking - polyvalent binding

Method to determine sialic acid binding specificity Paulson JC and Rogers GN (1987) Methods Enzymol 138, 162

Specificity in influenza virus binding Stevens et al (2006)

Nat Rev Microbiol

4:857

Glycan microarray Stevens et al (2006)

Nat Rev Microbiol

4:857

Specificity in influenza virus binding

Human pandemic

Stevens et al (2006)

Nat Rev Microbiol

4:857

Specificity in influenza virus binding

Bird Flu

Stevens et al (2006)

Nat Rev Microbiol

4:857

Influenza jumps from animals to humans via a slight change in glycan binding specificity Stevens et al (2006)

Nat Rev Microbiol

4:857

Knowledge = opportunity Laver et al (Jan 1999)

Scientific American

, 78-87

Influenza virus neuraminidase Gubareva et al (2000) Lancet 355:827

Rational design of an anti-influenza drug based on sialic acid binding to the viral neuraminidase von Itzstein et al (1993) Nature 363:418

Sialic acid based influenza inhibitors

Influenza virus budding in vitro without (top) and with (bottom) neuraminidase inhibitor Gubareva et al (2000) Lancet 355:827 Peters, P.H., et al. (2001) J Am Geriatr Soc 49, 1025

Bacterial surface lectins

Essentials of Glycobiology Second Edition

E. coli

binding to urinary epithelium in vitro - specific inhibition by Gal α4 Gal (right)

Sharon and Lis (1993)

Scientific American

Jan:82

Bacterial AB

5

toxins

 Cholera toxin •

E coli

enterotoxin • Pertussis toxin • Shiga toxin • Shiga-like verotoxins Merritt et al (1994)

Protein Science

3:166 Fishman and Brady (1976)

Science

194:906

Cholera toxin B-pentamer binding GM1 – extended glycan engagement Merritt et al (1994)

Protein Science

3:166

Essentials of Glycobiology Second Edition

Next: Mammalian glycan binding proteins in health and disease Sharon and Lis (1993) Scientific American