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Glycan Binding Proteins

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.

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Glycan Binding Proteins

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  1. Glycan Binding Proteins

  2. Glycan Binding Proteins – the latest 3D model Y.S. Laurent (2012) Yves Saint Laurent Introduces Glycobiology, www.youtube.com/v/lmyIuUr8Bmk

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

  4. 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

  5. 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

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

  7. There are >80 mammalian lectins in at least a dozen distinct families Second Edition Essentials of Glycobiology

  8. C-type lectins – diverse domain structures for diverse functions Kurt Drickamer, Imperial College Londonhttp://www.imperial.ac.uk/research/animallectins

  9. Animal lectins – structural classes Kurt Drickamer, Imperial College, http://www.imperial.ac.uk/research/animallectins/ctld/lectins.html

  10. Animal lectins – structural classes Kurt Drickamer, Imperial College, http://www.imperial.ac.uk/research/animallectins/ctld/lectins.html

  11. Animal lectins – cellular locations cell membrane intracellular extracellular Dodd, R.B. and Drickamer, K. (2001) Glycobiology 11, 71R-79R

  12. Lectin phylogeny Kurt Drickamer, Imperial College, http://www.imperial.ac.uk/research/animallectins/ctld/lectins.html

  13. 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

  14. 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

  15. Glycan properties for molecular recognition and binding energy Galactose Glucose Mannose Sialic Acid

  16. 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

  17. A “simple” example: GlcNAc binding to wheat germ agglutinin (WGA) Neumann, D., et al. (2004) Adv. Drug Deliv. Rev. 56, 437

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

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

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

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

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

  23. Weis & Drickamer (1994) Structure 2, 1227 • hydrogen bonds • hydrophobic stacking • calcium coordination • - polyvalent binding Drickamer (1997) Structure 5, 465

  24. 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

  25. 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

  26. 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

  27. 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

  28. Method to determine glycan binding specificity by structurally-specific reconstitution Paulson JC and Rogers GN (1987) Methods Enzymol138, 162 Kelm, et al. (1994) Curr. Biol. 4, 965

  29. 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/

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

  31. Microarray decoding of the binding specificity of influenza hemagglutinins Stevens, et al. (2006) Nat Rev Microbiol. 4:857-64

  32. Decoding the binding specificity of recent H7N9 avian/human influenza (bird flu) Yang, et al. (2013) J Virol 87, 12433-46

  33. 6’Su-sialyl-LacNAc 6’Su-sialyl-Lex Siglec-8 glycan microarray binding Bochner et al (2005) J Biol Chem 280:4307& Bochner (2011) http://www.functionalglycomics.org/ Glycan array public resource: http://functionalglycomics.org Influenza A Puerto Rico Cyanovirin-N UlexEuropaeus Agglutinin (UEA-I)

  34. 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

  35. 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

  36. 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

  37. https://www.vectorlabs.com/data/brochures/K4-K7.pdf

  38. “umbrella murder” of GeorgiMarkov by the Bulgarian secret police https://www.vectorlabs.com/data/brochures/K4-K7.pdf

  39. Plant lectin research correlation – seeking diagnostic glycosylation changes in cancer 94 different lectins arrayed for binding tissue extracted proteins Li et al (2011)Anal Chem83:8509

  40. 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

  41. 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

  42. 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 Second Edition Essentials of Glycobiology

  43. 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

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

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

  46. Sialic acid-based influenza inhibitors

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

  48. 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 Second Edition Essentials of Glycobiology

  49. 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

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

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