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The “glycocalyx” surrounding a fibroblast – glycans stained black lipid bilayer fibroblast extracellular intracellular Martinez-Palomo, A., et al. Cancer.

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Presentation on theme: "The “glycocalyx” surrounding a fibroblast – glycans stained black lipid bilayer fibroblast extracellular intracellular Martinez-Palomo, A., et al. Cancer."— Presentation transcript:

1 The “glycocalyx” surrounding a fibroblast – glycans stained black lipid bilayer fibroblast extracellular intracellular Martinez-Palomo, A., et al. Cancer Res. 29, , 1969 Eukaryotic cell surface GlycoproteinsGlycoproteins GlycolipidsGlycolipids ProteoglycansProteoglycans

2 Modes of glycosylation Essentials of Glycobiology

3 Major membrane lipid classes sphingolipid glycerolipid sterol (ceramide) (diacylglycerol) (cholesterol)

4 Most vertebrate glycolipids are glycosphingolipids Most vertebrate glycolipids are glycosphingolipids Most vertebrate glycolipids are glycosphingolipids Most vertebrate glycolipids are glycosphingolipids

5 Glucosylceramide is a key intermediate in skin barrier formation Glucosylceramide is a key intermediate in skin barrier formation Glucosylceramide is a key intermediate in skin barrier formation Glucosylceramide is a key intermediate in skin barrier formation Enzymatic processing of GlcCer generates ceramide, a major lipid required for epidermal barrier function. Holleran et al (2006) Febs Letters 23:5456

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

7 Glycosphingolipids are the major glycans of the brain Glycosphingolipids are the major glycans of the brain Glycosphingolipids are the major glycans of the brain Glycosphingolipids are the major glycans of the brain Glycan ratios in the brain GalCer & Sulfatide

8

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

10 Mutant mice lacking GalCer synthase (Ugt8a) or GalCer 3-sulfotransferase (Gal3st1) display myelin defects and associated behavioral deficits Mutant mice lacking GalCer synthase (Ugt8a) or GalCer 3-sulfotransferase (Gal3st1) display myelin defects and associated behavioral deficits Mutant mice lacking GalCer synthase (Ugt8a) or GalCer 3-sulfotransferase (Gal3st1) display myelin defects and associated behavioral deficits 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

11 GlcCer based glycolipid families: GlcCer based glycolipid families: -All share a lactosylceramide (Galβ1-4Glcβ1-1Cer) core All share a lactosylceramide (Galβ1-4Glcβ1-1Cer) coreAll share a lactosylceramide (Galβ1-4Glcβ1-1Cer) core -Biosynthesized stepwise by individual glycosphingolipids, some of which are glycolipid-specific, others of which also make glycoprotein glycans Biosynthesized stepwise by individual glycosphingolipids, some of which are glycolipid-specific, others of which also make glycoprotein glycansBiosynthesized 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 Classified as neutral glycosphingolipids, sulfated glycosphingolipids or gangliosides, which are sialic acid-containing glycosphingolipidsClassified as neutral glycosphingolipids, sulfated glycosphingolipids or gangliosides, which are sialic acid-containing glycosphingolipids GlcCer based glycolipid families: GlcCer based glycolipid families: -All share a lactosylceramide (Galβ1-4Glcβ1-1Cer) core All share a lactosylceramide (Galβ1-4Glcβ1-1Cer) coreAll share a lactosylceramide (Galβ1-4Glcβ1-1Cer) core -Biosynthesized stepwise by individual glycosphingolipids, some of which are glycolipid-specific, others of which also make glycoprotein glycans Biosynthesized stepwise by individual glycosphingolipids, some of which are glycolipid-specific, others of which also make glycoprotein glycansBiosynthesized 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 Classified as neutral glycosphingolipids, sulfated glycosphingolipids or gangliosides, which are sialic acid-containing glycosphingolipidsClassified as neutral glycosphingolipids, sulfated glycosphingolipids or gangliosides, which are sialic acid-containing glycosphingolipids Subfamily seriesStructure LactoGalβ1-3GlcNAcβ1-3Galβ1-4GlcβCer NeolactoGalβ1-4GlcNAcβ1-3Galβ1-4GlcβCer GanglioGalβ1-3GalNAcβ1-4Galβ1-4GlcβCer GloboGalNAcβ1-3Galα1-4Galβ1-4GlcβCer

12 Gangliosides – glycosphingolipids with one or more sialic acid residues – are found on all vertebrate cells, and are major glycans in the brain Gangliosides – glycosphingolipids with one or more sialic acid residues – are found on all vertebrate cells, and are major glycans in the brain Gangliosides – glycosphingolipids with one or more sialic acid residues – are found on all vertebrate cells, and are major glycans in the brain 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

13 Schnaar (2007) Comprehensive Glycoscience V. 4, 323

14 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”

15 Concepts of glycolipid function: cis and trans Concepts of glycolipid function: cis and trans Concepts of glycolipid function: cis and trans Concepts of glycolipid function: cis and trans cis: regulation by lateral association cis: regulation by lateral association cis: regulation by lateral association cis: regulation by lateral association trans: regulation by cell-cell recognition trans: regulation by cell-cell recognition trans: regulation by cell-cell recognition trans: regulation by cell-cell recognition Regina Todeschini & Hakomori (2008) Biochim Biophys Acta 1780:421

16 Modes of glycosylation Essentials of Glycobiology

17 A small but diverse group of proteins are attached to the cell surface by a “glycosylphosphatidylinositol” (GPI) anchor A small but diverse group of proteins are attached to the cell surface by a “glycosylphosphatidylinositol” (GPI) anchor A small but diverse group of proteins are attached to the cell surface by a “glycosylphosphatidylinositol” (GPI) anchor A small but diverse group of proteins are attached to the cell surface by a “glycosylphosphatidylinositol” (GPI) anchor Thy-1 cell-cell interaction protein membrane Second Edition Essentials of Glycobiology Taylor and Drikamer (2011) Introduction to Glycobiology GPI anchors have a minimal (core) structure (black font) with variable additional glycan, fatty acid ester, and phosphoethanolamine groups (blue font)

18 GPI-anchored proteins * GPI-anchored proteins * GPI-anchored proteins * GPI-anchored proteins *Mammals Thy-1cell-cell recognition CD59complement regulation decay accelerating factor (DAF)complement regulation Alkaline phosphatasecell-surface hydrolase 5′-Nucleotidasecell-surface hydrolase Renal dipeptidasecell-surface hydrolase Trehalasecell-surface hydrolase Neural cell adhesion molecule (NCAM-120) adhesion molecule Neural cell adhesion molecule TAG-1adhesion molecule CD58adhesion molecule FcγIII receptorFc receptor Ciliary neurotrophic factor receptorneural receptor Glial-derived neurotrophic factor receptor neural receptor Nogo receptor (NgR)axon outgrowth regulator CD14LPS receptor Prion protein (PrP)unknown Glypican family of GPI-anchored proteoglycans extracellular matrix component 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) erythrocyte invasion Toxoplasma gondii surface antigen 1 (SAG-1) host cell invasion Entamoeba histolytica GPI proteophosphoglycans virulence factor Parasites *plus others in yeast, plants

19 Essentials of Glycobiology Second Edition GPI anchors are complex & diverse GPI anchors are complex & diverse GPI anchors are complex & diverse GPI anchors are complex & diverse Glucosamine with free amine (rare)Glucosamine with free amine (rare) Phosphodiesters at each endPhosphodiesters at each end Diverse modifications along the coreDiverse modifications along the core R3

20 Variety is the spice of… GPI anchors Variety is the spice of… GPI anchors Variety is the spice of… GPI anchors Variety is the spice of… GPI anchors Essentials of Glycobiology Second Edition

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

22 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. 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. 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. 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

23 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. 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. 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. 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 sicknessChagas diseaseleishmaniasis

24 Modes of glycosylation Essentials of Glycobiology

25 Glycosaminoglycans (GAG’s) and Proteoglycans GLYCOSAMINOGLYCANS (GAG’s)GLYCOSAMINOGLYCANS (GAG’s) –long linear glycans made of repeating disaccharides –Hyaluronic acid is the only “stand-alone” GAG, other GAG’s are constituents of … PROTEOGLYCANSPROTEOGLYCANS –GAG’s on proteins –Defined by their repeating disaccharide units –GAG’s on proteoglycans are sulfated

26 Hyaluronic acid -- a “simple” space filling molecule (GlcA β1-3 GlcNAc β1-4) n Essentials of Glycobiology Second Edition

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

28 Glycosaminoglycan repeating disaccharide units Essentials of Glycobiology Second Edition

29 Proteoglycans

30 Essentials of Glycobiology Second Edition Proteoglycan/Hyaluronan macromolecular structures Taylor & Drickamer (2011) Introduction to Glycobiology

31 Cartilage proteoglycan

32

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

34 Sharon and Lis (2004) Glycobiology 14:53R Glycan binding protein diversity – phylogeny & function Glycan binding protein diversity – phylogeny & function Glycan binding protein diversity – phylogeny & function Glycan binding protein diversity – phylogeny & function

35 Principles of lectin-carbohydrate recognition Precisely spaced cooperative hydrogen bondsPrecisely spaced cooperative hydrogen bonds –direct or through bound water molecules Hydrophobic stackingHydrophobic stacking Ionic interactions (for charged glycans)Ionic interactions (for charged glycans) Calcium coordination (select lectins)Calcium coordination (select lectins) Low site-affinity / high-avidity polyvalent bindingLow site-affinity / high-avidity polyvalent binding

36 Sialic acid glycerol carboxylate acetyl Comb & Roseman, J Am Chem Soc 80, 497 (1958) Sialic acids -- Enhanced roles in glycan recognition

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

38 Diverse sialic acid linkages at the cell surface

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

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

41 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 cellsMucins (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-inhibitoryPre-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 substanceTreatment 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).Crystallization of the dialyzable substance identified it as sialic acid (N-acetylneuraminic acid). Gottschalk (1958) Nature 181:377

42 Influenza virus lifecycle Laver et al (Jan 1999) Scientific American, Linda Stannard: /stannard/fluvirus.html

43 Influenza virus hemagglutinin (side and top views, arrows are sugar binding sites) Weis et al (1988) Nature 333:426

44 Sialic acid binding to influenza virus hemagglutinin Weis et al (1988) Nature 333:426 - hydrogen bonds - hydrophobic stacking - polyvalent binding

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

46 Specificity in influenza virus binding Stevens et al (2006) Nat Rev Microbiol 4:857

47 Glycan microarray Stevens et al (2006) Nat Rev Microbiol 4:857

48 Specificity in influenza virus binding Human pandemic Stevens et al (2006) Nat Rev Microbiol 4:857

49 Specificity in influenza virus binding Bird Flu Stevens et al (2006) Nat Rev Microbiol 4:857

50 Influenza jumps from animals to humans via a slight change in glycan binding specificity Stevens et al (2006) Nat Rev Microbiol 4:857

51 Knowledge = opportunity Laver et al (Jan 1999) Scientific American, 78-87

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

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

54 Sialic acid- based influenza inhibitors

55 Influenza virus budding in vitro without (top) and with (bottom) neuraminidase inhibitor Gubareva et al (2000) Lancet 355:827

56 Bacterial surface lectins Bacterial surface lectins Bacterial surface lectins Bacterial surface lectins Essentials of Glycobiology Second Edition

57 E. coli binding to urinary epithelium in vitro -- specific inhibition by Gal α4 Gal (right) Sharon and Lis (1993) Scientific American Jan:82

58 Bacterial AB 5 toxins Bacterial AB 5 toxins Bacterial AB 5 toxins Bacterial AB 5 toxins Cholera toxin Cholera toxin E coli enterotoxinE coli enterotoxin Pertussis toxinPertussis toxin Shiga toxinShiga toxin Shiga-like verotoxinsShiga-like verotoxins Fishman and Brady (1976) Science 194:906 Merritt et al (1994) Protein Science 3:166

59 Cholera toxin B-pentamer binding GM1 – extended glycan engagement Merritt et al (1994) Protein Science 3:166 Essentials of Glycobiology Second Edition

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


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