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Glycan Binding Proteins Glycan Binding Proteins – the latest 3D model Y.S. Laurent (2012) Yves Saint Laurent Introduces Glycobiology, www.youtube.com/v/lmyIuUr8Bmk Glycan-Protein Recognition – Defining terms Carbohydrate binding determinant Carbohydrate recognition domain (CRD) Lectin = Glycan Binding Protein (GBP) Glycan Sharon and Lis (1993) Scientific American Glycan Binding Proteins Objectives • Learn the diversity and families of glycan binding proteins • Learn the molecular/structural strategies used in protein-glycan binding and recognition • Learn the methods and resources for determining glycan binding protein specificities • Learn about plant lectins as tools in glycobiology research • Learn about glycan binding strategies used by pathogens Glycan Binding Proteins Objectives • Learn the diversity and families of glycan binding proteins • Learn the molecular/structural strategies used in protein-glycan binding and recognition • Learn the methods and resources for determining glycan binding protein specificities • Learn about plant lectins as tools in glycobiology research • Learn about glycan binding strategies used by pathogens Glycan binding protein diversity – phylogeny & function Sharon and Lis (2004) Glycobiology 14:53R There are >80 mammalian lectins in at least a dozen distinct families Essentials of Glycobiology Second Edition C-type lectins – diverse domain structures for diverse functions Kurt Drickamer, Imperial College London http://www.imperial.ac.uk/research/animallectins Animal lectins – structural classes Lectin family Saccharide ligands Subcellular location Examples of functions Calnexin Glc1Man9 ER Protein sorting in the ER M-type lectins Man8 ER ER -associated degradation L-type lectins various ER, ERGIC, Golgi Protein sorting in the ER P-type lectins Man 6-phosphate, others Secretory pathway post-Golgi glycoprotein trafficking C-type lectins Various, calcium-dependent Cell membrane, extracellular Cell adhesion (selectins), glycoprotein clearance, innate immunity (collectins). Galectins β-galactosides Cytoplasm, extracellular cell surface crosslinking Siglecs (I-type lectins) sialic acid Cell membrane molecular & cell recognition R-type lectins various Golgi, Cell membrane Enzyme targeting, hormone turnover F-box lectins GlcNAc2 Cytoplasm degradation of misfolded proteins. Ficolins GlcNAc, GalNAc cell membrane, extracellular Innate immunity. Chitinase-like lectins chito-oligosaccharides Extracellular Collagen metabolism F-type lectins fucose termini Extracellular Innate immunity. Intelectins Gal, galactofuranose, pentoses Extracellular/cell membrane Innate immunity. Fertilization and embryogenesis. Kurt Drickamer, Imperial College, http://www.imperial.ac.uk/research/animallectins/ctld/lectins.html Animal lectins – structural classes Lectin family Saccharide ligands Subcellular location Examples of functions Calnexin Glc1Man9 ER Protein sorting in the ER M-type lectins Man8 ER ER -associated degradation L-type lectins various ER, ERGIC, Golgi Protein sorting in the ER P-type lectins Man 6-phosphate, others Secretory pathway post-Golgi glycoprotein trafficking C-type lectins Various, calcium-dependent Cell membrane, extracellular Cell adhesion (selectins), glycoprotein clearance, innate immunity (collectins). Galectins β-galactosides Cytoplasm, extracellular cell surface crosslinking Siglecs (I-type lectins) sialic acid Cell membrane molecular & cell recognition R-type lectins various Golgi, Cell membrane Enzyme targeting, hormone turnover F-box lectins GlcNAc2 Cytoplasm degradation of misfolded proteins. Ficolins GlcNAc, GalNAc cell membrane, extracellular Innate immunity. Chitinase-like lectins chito-oligosaccharides Extracellular Collagen metabolism F-type lectins fucose termini Extracellular Innate immunity. Intelectins Gal, galactofuranose, pentoses Extracellular/cell membrane Innate immunity. Fertilization and embryogenesis. Kurt Drickamer, Imperial College, http://www.imperial.ac.uk/research/animallectins/ctld/lectins.html Animal lectins – cellular locations cell membrane Dodd, R.B. and Drickamer, K. (2001) Glycobiology 11, 71R-79R Lectin phylogeny Kurt Drickamer, Imperial College, http://www.imperial.ac.uk/research/animallectins/ctld/lectins.html Glycan Binding Proteins Objectives • Learn the diversity and families of glycan binding proteins • Learn the molecular/structural strategies used in protein-glycan binding and recognition • Learn the methods and resources for determining glycan binding protein specificities • Learn about plant lectins as tools in glycobiology research • Learn about glycan binding strategies used by pathogens Glycan Binding Proteins Objectives • Learn the diversity and families of glycan binding proteins • Learn the molecular/structural strategies used in protein-glycan binding and recognition • Learn the methods and resources for determining glycan binding protein specificities • Learn about plant lectins as tools in glycobiology research • Learn about glycan binding strategies used by pathogens Glycan properties for molecular recognition and binding energy Glucose Galactose Sialic Acid Mannose 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 A “simple” example: GlcNAc binding to wheat germ agglutinin (WGA) Neumann, D., et al. (2004) Adv. Drug Deliv. Rev. 56, 437 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 Siglec-1 CRD May, A.P., et al. (1998) Mol. Cell 1, 719-728 Principles of glycanlectin binding: -hydrogen bonds -hydrophobic stacking -ionic interactions May, A.P., et al. (1998) Mol. Cell 1, 719-728 Weis & Drickamer (1994) Structure 2, 1227 - hydrogen bonds hydrophobic stacking calcium coordination polyvalent binding Drickamer (1997) Structure 5, 465 Polyvalency in lectin binding – low site affinity, high polyvalent avidity Inhibition of radiolabeled galactoside binding to intact rabbit hepatocytes The mammalian hepatic C-type lectin is a A2B trimer, each subunit having a single galactose binding site Glycan added (M) Lee et al (1983) J Biol Chem 258, 199 Glycan Binding Proteins Objectives • Learn the diversity and families of glycan binding proteins • Learn the molecular/structural strategies used in protein-glycan binding and recognition • Learn the methods and resources for determining glycan binding protein specificities • Learn about plant lectins as tools in glycobiology research • Learn about glycan binding strategies used by pathogens Glycan Binding Proteins Objectives • Learn the diversity and families of glycan binding proteins • Learn the molecular/structural strategies used in protein-glycan binding and recognition • Learn the methods and resources for determining glycan binding protein specificities • Learn about plant lectins as tools in glycobiology research • Learn about glycan binding strategies used by pathogens Lectins were originally identified as “hemagglutinins” Influenza “hemagglutinin”… red cells fall to the bottom unless they are crosslinked. Samples A, D, and E are active http://www.virology.ws/2009/05/27/influenza-hemagglutinationinhibition-assay/ Sugar binding specificity inhibition of galectininduced hemagglutination by lactose Ola et al (2007) Cell Biology International 31, 578 Method to determine glycan binding specificity by structurally-specific reconstitution Paulson JC and Rogers GN (1987) Methods Enzymol 138, 162 Kelm, et al. (1994) Curr. Biol. 4, 965 Latest technology – glycan microarrays Overlay with labeled GBP, wash, detect Stamp derivatized glycans onto glass slides Ola Blixt, Copenhagen Center for Glycomics http://glycomics.ku.dk/research/glyco-biomarkers/microarray/ Glycan microarray Stevens et al (2006) Nat Rev Microbiol 4:857 Microarray decoding of the binding specificity of influenza hemagglutinins Stevens, et al. (2006) Nat Rev Microbiol. 4:857-64 Decoding the binding specificity of recent H7N9 avian/human influenza (bird flu) Yang, et al. (2013) J Virol 87, 12433-46 Siglec-8 glycan microarray binding 10000 6’Su-sialyl-LacNAc 6’Su-sialyl-Lex 8000 6000 4000 2000 0 100 200 300 400 500 600 Bochner et al (2005) J Biol Chem 280:4307 & Bochner (2011) http://www.functionalglycomics.org/ Glycan array public resource: http://functionalglycomics.org Cyanovirin-N Ulex Europaeus Agglutinin (UEA-I) Influenza A Puerto Rico Glycan Binding Proteins Objectives • Learn the diversity and families of glycan binding proteins • Learn the molecular/structural strategies used in protein-glycan binding and recognition • Learn the methods and resources for determining glycan binding protein specificities • Learn about plant lectins as tools in glycobiology research • Learn about glycan binding strategies used by pathogens Glycan Binding Proteins Objectives • Learn the diversity and families of glycan binding proteins • Learn the molecular/structural strategies used in protein-glycan binding and recognition • Learn the methods and resources for determining glycan binding protein specificities • Learn about plant lectins as tools in glycobiology research • Learn about glycan binding strategies used by pathogens Plant lectins • Found in a variety of legumes and other plants, often in seeds • Discovered by their hemagglutinating activities • Proposed to function in plant pathogen or symbiont recognition but data is not compelling • Broad variety of glycan specificities - many have yet to be defined in detail • Often sturdy proteins suitable for research, biotechnology and diagnostic applications https://www.vectorlabs.com/ data/brochures/K4-K7.pdf “umbrella murder” of Georgi Markov by the Bulgarian secret police https://www.vectorlabs.com/ data/brochures/K4-K7.pdf Plant lectin research correlation – seeking diagnostic glycosylation changes in cancer 94 different lectins arrayed for binding tissue extracted proteins Li et al (2011) Anal Chem 83:8509 Glycan Binding Proteins Objectives • Learn the diversity and families of glycan binding proteins • Learn the molecular/structural strategies used in protein-glycan binding and recognition • Learn the methods and resources for determining glycan binding protein specificities • Learn about plant lectins as tools in glycobiology research • Learn about glycan binding strategies used by pathogens Glycan Binding Proteins Objectives • Learn the diversity and families of glycan binding proteins • Learn the molecular/structural strategies used in protein-glycan binding and recognition • Learn the methods and resources for determining glycan binding protein specificities • Learn about plant lectins as tools in glycobiology research • Learn about glycan binding strategies used by pathogens Pathogen glycan binding proteins Viruses use glycan binding proteins (viral hemagglutinins) to infect cells • Many bacterial toxins are lectins • Bacteria use surface lectins to adhere to target cells • Eukaryotic microbes use lectins to adhere to target cells Essentials of Glycobiology Second Edition Influenza virus lifecycle Linda Stannard: http://web.uct.ac.za/depts/mmi /stannard/fluvirus.html Laver et al (1999) Scientific American, Jan issue, pp78-87 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 acidbased 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 Pathogen glycan binding proteins • Viruses use glycan binding proteins (viral hemagglutinins) to infect cells Many bacterial toxins are lectins • Bacteria use surface lectins to adhere to target cells • Eukaryotic microbes use lectins to adhere to target cells Essentials of Glycobiology Second Edition Bacterial AB5 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 Pathogen glycan binding proteins • Viruses use glycan binding proteins (viral hemagglutinins) to infect cells • Many bacterial toxins are lectins Bacteria use surface lectins to adhere to target cells • Eukaryotic microbes use lectins to adhere to target cells Pathogen glycan binding proteins Bacteria use surface lectins to adhere to target cells • Eukaryotic microbes use lectins to adhere to target cells Essentials of Glycobiology Second Edition Bacterial surface lectins Essentials of Glycobiology Second Edition E. coli binding to urinary epithelium in vitro -specific inhibition by Gal α1-4 Gal (right) Sharon and Lis (1993) Scientific American Jan:82 Inhibition of Helicobacter pylori binding to human gastric epithelium by multivalent carbohydrates Boren et al (1993) Science 262:1892 Pathogen glycan binding proteins • Viruses use glycan binding proteins (viral hemagglutinins) to infect cells • Many bacterial toxins are lectins • Bacteria use surface lectins to adhere to target cells Eukaryotic microbes use lectins to adhere to target cells Binding of Candida glabrata to human epithelial cells Cormack et al (1999) Science 285:578 LINK: EPA7 glycan array screening Glycan Binding Proteins Objectives • Learn the diversity and families of glycan binding proteins • Learn the molecular/structural strategies used in protein-glycan binding and recognition • Learn the methods and resources for determining glycan binding protein specificities • Learn about plant lectins as tools in glycobiology research • Learn about glycan binding strategies used by pathogens Influenza virus fails to proliferate in CDG-IIb patient cells CDG-IIb patient glycosylation (red) of a blood protein (IgG) compared to normal control. These patients lack glucosidase I. (Upper) Viral titers assessed by hemagglutination (HA) and 50% tissue-culture infective dose (TCID50) of virus produced by monocyte-derived macrophages (MDM) from the patients and the healthy control. (Lower) HA titers and cytopathic effect (CPE) in MDCK cells that infected with equivalent amounts of influenza virus produced by MDM cultures from Patient 1 and a control. Sadat et al (2014) NEJM, yesterday
