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 recognition domain (CRD) Carbohydrate binding determinant 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

Essentials of Glycobiology 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 -associated degradation L-type lectins various ER, ERGIC, Golgi 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 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 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 -associated degradation L-type lectins various ER, ERGIC, Golgi 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 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 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 intracellular extracellular 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 Mannose Sialic Acid

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 glycan-lectin binding: -hydrogen bonds -hydrophobic stacking -ionic interactions May, A.P., et al. (1998) Mol. Cell 1, 719-728

-hydrophobic stacking -ionic interactions Structural basis for sulfation-dependent self-glycan recognition by the human immune-inhibitory receptor Siglec-8 -hydrogen bonds -hydrophobic stacking -ionic interactions Pröpster et al. (2016) PNAS 113:E4170

hydrogen bonds hydrophobic stacking calcium coordination 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-hemagglutination- inhibition-assay/ Sugar binding specificity - inhibition of galectin-induced hemagglutination by lactose Ola et al (2007) Cell Biology International 31, 578

Method to determine glycan binding specificity by structurally-specific reconstitution Kelm, et al. (1994) Curr. Biol. 4, 965 Paulson JC and Rogers GN (1987) Methods Enzymol 138, 162

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 6’Su-sialyl-LacNAc 6’Su-sialyl-Lex 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

Essentials of Glycobiology 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 Laver et al (1999) Scientific American, Jan issue, pp78-87 Linda Stannard: http://web.uct.ac.za/depts/mmi/stannard/fluvirus.html

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 Peters, P.H., et al. (2001) J Am Geriatr Soc 49, 1025 Gubareva et al (2000) Lancet 355:827

Essentials of Glycobiology 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 FIGURE 34.6. Crystal structure of cholera toxin B-subunit pentamer with bound GM1 pentasaccharide shown from the bottom (a) and from the side (b). (Redrawn, with permission, from Merritt E.A., Sarfaty S., van den Akker F., L’Hoir C., Martial J.A., and Hol W.G. 1994. Protein Sci. 3: 166–175.) (c) Shiga toxin pentamer with an artificial pentavalent ligand, a powerful inhibitor of the toxin. The carbohydrate ligands are shown in a ball-and-stick representation. (Dashed magenta lines) Possible conformation for the linker. (Redrawn, with permission of Macmillan Publisher Ltd., from Kitov P.I., Sadowska J.M., Mulvey G., et al. 2000. Nature 403: 669–672.) Essentials of Glycobiology Second Edition Merritt et al (1994) Protein Science 3:166

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 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

Essentials of Glycobiology 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