Glycan Binding Proteins Glycan Binding Proteins
Jan 11, 2016
Glycan Binding ProteinsGlycan Binding ProteinsGlycan Binding ProteinsGlycan Binding Proteins
Y.S. Laurent (2012) Yves Saint Laurent Introduces Glycobiology, www.youtube.com/v/lmyIuUr8Bmk
Glycan Binding Proteins – the latest 3D modelGlycan Binding Proteins – the latest 3D modelGlycan Binding Proteins – the latest 3D modelGlycan Binding Proteins – the latest 3D model
Sharon and Lis (1993) Scientific American
Carbohydrate binding determinant
Carbohydrate recognition domain (CRD)
Glycan-Protein Recognition – Defining termsGlycan-Protein Recognition – Defining terms
Lectin =Lectin =Glycan Binding Glycan Binding Protein (GBP)Protein (GBP)
GlycanGlycan
Glycan Binding ProteinsGlycan Binding ProteinsGlycan Binding ProteinsGlycan Binding Proteins
• Learn the diversity and families of glycan binding Learn the diversity and families of glycan binding proteinsproteins
• Learn the molecular/structural strategies used in Learn the molecular/structural strategies used in protein-glycan binding and recognitionprotein-glycan binding and recognition
• Learn the methods and resources for determining Learn the methods and resources for determining glycan binding protein specificitiesglycan binding protein specificities
• Learn about plant lectins as tools in glycobiology Learn about plant lectins as tools in glycobiology researchresearch
• Learn about glycan binding strategies used by Learn about glycan binding strategies used by pathogenspathogens
• Learn the diversity and families of glycan binding Learn the diversity and families of glycan binding proteinsproteins
• Learn the molecular/structural strategies used in Learn the molecular/structural strategies used in protein-glycan binding and recognitionprotein-glycan binding and recognition
• Learn the methods and resources for determining Learn the methods and resources for determining glycan binding protein specificitiesglycan binding protein specificities
• Learn about plant lectins as tools in glycobiology Learn about plant lectins as tools in glycobiology researchresearch
• Learn about glycan binding strategies used by Learn about glycan binding strategies used by pathogenspathogens
ObjectivesObjectivesObjectivesObjectives
Glycan Binding ProteinsGlycan Binding ProteinsGlycan Binding ProteinsGlycan Binding Proteins
• Learn the diversity and families of glycan binding Learn the diversity and families of glycan binding proteinsproteins
• Learn the molecular/structural strategies used in Learn the molecular/structural strategies used in protein-glycan binding and recognitionprotein-glycan binding and recognition
• Learn the methods and resources for determining Learn the methods and resources for determining glycan binding protein specificitiesglycan binding protein specificities
• Learn about plant lectins as tools in glycobiology Learn about plant lectins as tools in glycobiology researchresearch
• Learn about glycan binding strategies used by Learn about glycan binding strategies used by pathogenspathogens
• Learn the diversity and families of glycan binding Learn the diversity and families of glycan binding proteinsproteins
• Learn the molecular/structural strategies used in Learn the molecular/structural strategies used in protein-glycan binding and recognitionprotein-glycan binding and recognition
• Learn the methods and resources for determining Learn the methods and resources for determining glycan binding protein specificitiesglycan binding protein specificities
• Learn about plant lectins as tools in glycobiology Learn about plant lectins as tools in glycobiology researchresearch
• Learn about glycan binding strategies used by Learn about glycan binding strategies used by pathogenspathogens
ObjectivesObjectivesObjectivesObjectives
Sharon and Lis (2004) Glycobiology 14:53R
Glycan binding protein diversity – phylogeny & functionGlycan binding protein diversity – phylogeny & functionGlycan binding protein diversity – phylogeny & functionGlycan binding protein diversity – phylogeny & function
There are >80 mammalian lectins There are >80 mammalian lectins in at least a dozen distinct families in at least a dozen distinct families
Essentials of Glycobiology Second Edition
C-type lectins – diverse domain structures for diverse functionsC-type lectins – diverse domain structures for diverse functions
Kurt Drickamer, Imperial College LondonKurt Drickamer, Imperial College Londonhttp://www.imperial.ac.uk/research/animallectins
Animal lectins – structural classesAnimal lectins – structural classes
Kurt Drickamer, Imperial College, http://www.imperial.ac.uk/research/animallectins/ctld/lectins.html
Lectin family Lectin family Saccharide ligands Saccharide ligands Subcellular location Subcellular location Examples of functionsExamples of functions
Calnexin Calnexin Glc1Man9 Glc1Man9 ER ER Protein sorting in the ERProtein sorting in the ER
M-type lectins M-type lectins Man8 Man8 ER ER ER -associated degradationER -associated degradation
L-type lectins L-type lectins various various ER, ERGIC, Golgi ER, ERGIC, Golgi Protein sorting in the ERProtein sorting in the ER
P-type lectins P-type lectins Man 6-phosphate, others Man 6-phosphate, others Secretory pathway Secretory pathway post-Golgi glycoprotein traffickingpost-Golgi glycoprotein trafficking
C-type lectins C-type lectins Various, calcium-Various, calcium-dependent dependent
Cell membrane, Cell membrane, extracellular extracellular
Cell adhesion (selectins), Cell adhesion (selectins), glycoprotein clearance, innate glycoprotein clearance, innate
immunity (collectins).immunity (collectins).
Galectins Galectins ββ-galactosides -galactosides Cytoplasm, Cytoplasm, extracellular extracellular
cell surface crosslinking cell surface crosslinking
Siglecs (I-type lectins) Siglecs (I-type lectins) sialic acid sialic acid Cell membrane Cell membrane molecular & cell recognitionmolecular & cell recognition
R-type lectins R-type lectins various various Golgi, Cell membrane Golgi, Cell membrane Enzyme targeting, hormone turnoverEnzyme targeting, hormone turnover
F-box lectins F-box lectins GlcNAc2 GlcNAc2 Cytoplasm Cytoplasm degradation of misfolded proteins.degradation of misfolded proteins.
Ficolins Ficolins GlcNAc, GalNAc GlcNAc, GalNAc cell membrane, cell membrane, extracellular extracellular
Innate immunity.Innate immunity.
Chitinase-like lectins Chitinase-like lectins chito-oligosaccharides chito-oligosaccharides Extracellular Extracellular Collagen metabolismCollagen metabolism
F-type lectins F-type lectins fucose terminifucose termini Extracellular Extracellular Innate immunity.Innate immunity.
Intelectins Intelectins Gal, galactofuranose, pentoses Gal, galactofuranose, pentoses Extracellular/cell membrane Extracellular/cell membrane Innate immunity. Fertilization and Innate immunity. Fertilization and embryogenesis.embryogenesis.
Animal lectins – structural classesAnimal lectins – structural classes
Kurt Drickamer, Imperial College, http://www.imperial.ac.uk/research/animallectins/ctld/lectins.html
Lectin family Lectin family Saccharide ligands Saccharide ligands Subcellular location Subcellular location Examples of functionsExamples of functions
Calnexin Calnexin Glc1Man9 Glc1Man9 ER ER Protein sorting in the ERProtein sorting in the ER
M-type lectins M-type lectins Man8 Man8 ER ER ER -associated degradationER -associated degradation
L-type lectins L-type lectins various various ER, ERGIC, Golgi ER, ERGIC, Golgi Protein sorting in the ERProtein sorting in the ER
P-type lectins P-type lectins Man 6-phosphate, others Man 6-phosphate, others Secretory pathway Secretory pathway post-Golgi glycoprotein traffickingpost-Golgi glycoprotein trafficking
C-type lectins C-type lectins Various, calcium-Various, calcium-dependent dependent
Cell membrane, Cell membrane, extracellular extracellular
Cell adhesion (selectins), Cell adhesion (selectins), glycoprotein clearance, innate glycoprotein clearance, innate
immunity (collectins).immunity (collectins).
Galectins Galectins ββ-galactosides -galactosides Cytoplasm, Cytoplasm, extracellular extracellular
cell surface crosslinking cell surface crosslinking
Siglecs (I-type lectins) Siglecs (I-type lectins) sialic acid sialic acid Cell membrane Cell membrane molecular & cell recognitionmolecular & cell recognition
R-type lectins R-type lectins various various Golgi, Cell membrane Golgi, Cell membrane Enzyme targeting, hormone turnoverEnzyme targeting, hormone turnover
F-box lectins F-box lectins GlcNAc2 GlcNAc2 Cytoplasm Cytoplasm degradation of misfolded proteins.degradation of misfolded proteins.
Ficolins Ficolins GlcNAc, GalNAc GlcNAc, GalNAc cell membrane, cell membrane, extracellular extracellular
Innate immunity.Innate immunity.
Chitinase-like lectins Chitinase-like lectins chito-oligosaccharides chito-oligosaccharides Extracellular Extracellular Collagen metabolismCollagen metabolism
F-type lectins F-type lectins fucose terminifucose termini Extracellular Extracellular Innate immunity.Innate immunity.
Intelectins Intelectins Gal, galactofuranose, pentoses Gal, galactofuranose, pentoses Extracellular/cell membrane Extracellular/cell membrane Innate immunity. Fertilization and Innate immunity. Fertilization and embryogenesis.embryogenesis.
Animal lectins – cellular locationsAnimal lectins – cellular locations
Dodd, R.B. and Drickamer, K. (2001)Dodd, R.B. and Drickamer, K. (2001) Glycobiology Glycobiology 1111, 71R-79R, 71R-79R
cell membrane
intracellular
extracellular
Lectin phylogenyLectin phylogeny
Kurt Drickamer, Imperial College, http://www.imperial.ac.uk/research/animallectins/ctld/lectins.html
Glycan Binding ProteinsGlycan Binding ProteinsGlycan Binding ProteinsGlycan Binding Proteins
• Learn the diversity and families of glycan binding Learn the diversity and families of glycan binding proteinsproteins
• Learn the molecular/structural strategies used in Learn the molecular/structural strategies used in protein-glycan binding and recognitionprotein-glycan binding and recognition
• Learn the methods and resources for determining Learn the methods and resources for determining glycan binding protein specificitiesglycan binding protein specificities
• Learn about plant lectins as tools in glycobiology Learn about plant lectins as tools in glycobiology researchresearch
• Learn about glycan binding strategies used by Learn about glycan binding strategies used by pathogenspathogens
• Learn the diversity and families of glycan binding Learn the diversity and families of glycan binding proteinsproteins
• Learn the molecular/structural strategies used in Learn the molecular/structural strategies used in protein-glycan binding and recognitionprotein-glycan binding and recognition
• Learn the methods and resources for determining Learn the methods and resources for determining glycan binding protein specificitiesglycan binding protein specificities
• Learn about plant lectins as tools in glycobiology Learn about plant lectins as tools in glycobiology researchresearch
• Learn about glycan binding strategies used by Learn about glycan binding strategies used by pathogenspathogens
ObjectivesObjectivesObjectivesObjectives
Glycan Binding ProteinsGlycan Binding ProteinsGlycan Binding ProteinsGlycan Binding Proteins
• Learn the diversity and families of glycan binding Learn the diversity and families of glycan binding proteinsproteins
• Learn the molecular/structural strategies used in Learn the molecular/structural strategies used in protein-glycan binding and recognitionprotein-glycan binding and recognition
• Learn the methods and resources for determining Learn the methods and resources for determining glycan binding protein specificitiesglycan binding protein specificities
• Learn about plant lectins as tools in glycobiology Learn about plant lectins as tools in glycobiology researchresearch
• Learn about glycan binding strategies used by Learn about glycan binding strategies used by pathogenspathogens
• Learn the diversity and families of glycan binding Learn the diversity and families of glycan binding proteinsproteins
• Learn the molecular/structural strategies used in Learn the molecular/structural strategies used in protein-glycan binding and recognitionprotein-glycan binding and recognition
• Learn the methods and resources for determining Learn the methods and resources for determining glycan binding protein specificitiesglycan binding protein specificities
• Learn about plant lectins as tools in glycobiology Learn about plant lectins as tools in glycobiology researchresearch
• Learn about glycan binding strategies used by Learn about glycan binding strategies used by pathogenspathogens
ObjectivesObjectivesObjectivesObjectives
Glycan properties for molecular recognition and binding energyGlycan properties for molecular recognition and binding energyGlycan properties for molecular recognition and binding energyGlycan properties for molecular recognition and binding energy
Glucose Galactose
Sialic Acid
Mannose
Principles of lectin-carbohydrate recognitionPrinciples of lectin-carbohydrate recognition
• Precisely spaced cooperative hydrogen bondsPrecisely spaced cooperative hydrogen bonds– direct or through bound water moleculesdirect 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
A “simple” example: A “simple” example: GlcNAc binding to wheat germ agglutinin (WGA)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)Weis et al (1988) Nature Nature 333:426333:426
Sialic acid binding to influenza virus hemagglutinin
Weis et al (1988)Weis et al (1988) Nature Nature 333:426333:426
- hydrogen bonds - hydrogen bonds - hydrophobic stacking- hydrophobic stacking- polyvalent binding- polyvalent binding
Siglec-1 CRDSiglec-1 CRD
May, A.P., et al. (1998) Mol. Cell 1, 719-728
Principles of glycan-Principles of glycan-lectin binding:lectin binding:
-hydrogen bonds -hydrogen bonds -hydrophobic stacking-hydrophobic stacking-ionic interactions-ionic interactions
May, A.P., et al. (1998) Mol. Cell 1, 719-728
Weis & Drickamer (1994) Structure 2, 1227
Drickamer (1997) Structure 5, 465
- hydrogen bondshydrogen bonds- hydrophobic stackinghydrophobic stacking- calcium coordinationcalcium coordination- polyvalent binding- polyvalent binding
Polyvalency in lectin binding – Polyvalency in lectin binding – low site affinity, high polyvalent aviditylow site affinity, high polyvalent avidity
Lee et al (1983) J Biol Chem 258, 199
The mammalian hepatic C-type lectin is a A2B trimer, each subunit having a single galactose binding site Glycan added (M)
Inhibition of radiolabeled galactoside binding to intact rabbit hepatocytes
Glycan Binding ProteinsGlycan Binding ProteinsGlycan Binding ProteinsGlycan Binding Proteins
• Learn the diversity and families of glycan binding Learn the diversity and families of glycan binding proteinsproteins
• Learn the molecular/structural strategies used in Learn the molecular/structural strategies used in protein-glycan binding and recognitionprotein-glycan binding and recognition
• Learn the methods and resources for determining Learn the methods and resources for determining glycan binding protein specificitiesglycan binding protein specificities
• Learn about plant lectins as tools in glycobiology Learn about plant lectins as tools in glycobiology researchresearch
• Learn about glycan binding strategies used by Learn about glycan binding strategies used by pathogenspathogens
• Learn the diversity and families of glycan binding Learn the diversity and families of glycan binding proteinsproteins
• Learn the molecular/structural strategies used in Learn the molecular/structural strategies used in protein-glycan binding and recognitionprotein-glycan binding and recognition
• Learn the methods and resources for determining Learn the methods and resources for determining glycan binding protein specificitiesglycan binding protein specificities
• Learn about plant lectins as tools in glycobiology Learn about plant lectins as tools in glycobiology researchresearch
• Learn about glycan binding strategies used by Learn about glycan binding strategies used by pathogenspathogens
ObjectivesObjectivesObjectivesObjectives
Glycan Binding ProteinsGlycan Binding ProteinsGlycan Binding ProteinsGlycan Binding Proteins
• Learn the diversity and families of glycan binding Learn the diversity and families of glycan binding proteinsproteins
• Learn the molecular/structural strategies used in Learn the molecular/structural strategies used in protein-glycan binding and recognitionprotein-glycan binding and recognition
• Learn the methods and resources for determining Learn the methods and resources for determining glycan binding protein specificitiesglycan binding protein specificities
• Learn about plant lectins as tools in glycobiology Learn about plant lectins as tools in glycobiology researchresearch
• Learn about glycan binding strategies used by Learn about glycan binding strategies used by pathogenspathogens
• Learn the diversity and families of glycan binding Learn the diversity and families of glycan binding proteinsproteins
• Learn the molecular/structural strategies used in Learn the molecular/structural strategies used in protein-glycan binding and recognitionprotein-glycan binding and recognition
• Learn the methods and resources for determining Learn the methods and resources for determining glycan binding protein specificitiesglycan binding protein specificities
• Learn about plant lectins as tools in glycobiology Learn about plant lectins as tools in glycobiology researchresearch
• Learn about glycan binding strategies used by Learn about glycan binding strategies used by pathogenspathogens
ObjectivesObjectivesObjectivesObjectives
Lectins were originally identified as “hemagglutinins”Lectins were originally identified as “hemagglutinins”
Influenza “hemagglutinin”…Influenza “hemagglutinin”…red cells fall to the bottom red cells fall to the bottom unlessunless they are they are crosslinked. Samples A, D, and E are activecrosslinked. Samples A, D, and E are activehttp://www.virology.ws/2009/05/27/influenza-hemagglutination-http://www.virology.ws/2009/05/27/influenza-hemagglutination-inhibition-assay/inhibition-assay/
Sugar binding specificity - Sugar binding specificity - inhibition of galectin-inhibition of galectin-induced hemagglutination induced hemagglutination by lactoseby lactoseOla et al (2007) Ola et al (2007) Cell Biology International Cell Biology International 31, 57831, 578
Method to determine glycan Method to determine glycan binding specificity by binding specificity by structurally-specific reconstitutionstructurally-specific reconstitution
Paulson JC and Rogers GN (1987) Paulson JC and Rogers GN (1987) Methods Enzymol Methods Enzymol 138, 162138, 162
Kelm, et al. (1994) Curr. Biol. 4, 965Kelm, et al. (1994) Curr. Biol. 4, 965
Latest technology – glycan microarrays Latest technology – glycan microarrays
Ola Blixt, Copenhagen Center for GlycomicsOla Blixt, Copenhagen Center for Glycomicshttp://glycomics.ku.dk/research/glyco-biomarkers/microarray/http://glycomics.ku.dk/research/glyco-biomarkers/microarray/
Stamp derivatized glycans onto glass slidesStamp derivatized glycans onto glass slides
Overlay with labeled GBP, wash, detectOverlay with labeled GBP, wash, detect
Glycan microarrayGlycan microarray
Stevens et al (2006) Nat Rev Microbiol 4:857
Microarray decoding of the binding specificity Microarray decoding of the binding specificity of influenza hemagglutininsof influenza hemagglutinins
Stevens, et al. (2006) Nat Rev Microbiol. 4:857-64
Decoding the Decoding the binding specificity binding specificity
of recent H7N9 of recent H7N9 avian/human avian/human
influenza (bird flu)influenza (bird flu)
Yang, et al. (2013) J Virol 87, 12433-46
Siglec-8 glycan microarray bindingSiglec-8 glycan microarray binding
Bochner et al (2005) J Biol Chem 280:4307& Bochner (2011) http://www.functionalglycomics.org/
0 100 200 300 400 500 600
2000
4000
6000
8000
100006’Su-sialyl-LacNAc 6’Su-sialyl-Lex
Glycan array public resource: Glycan array public resource: http://functionalglycomics.org
Cyanovirin-N Ulex Europaeus Agglutinin (UEA-I) Influenza A Puerto Rico
Glycan Binding ProteinsGlycan Binding ProteinsGlycan Binding ProteinsGlycan Binding Proteins
• Learn the diversity and families of glycan binding Learn the diversity and families of glycan binding proteinsproteins
• Learn the molecular/structural strategies used in Learn the molecular/structural strategies used in protein-glycan binding and recognitionprotein-glycan binding and recognition
• Learn the methods and resources for determining Learn the methods and resources for determining glycan binding protein specificitiesglycan binding protein specificities
• Learn about plant lectins as tools in glycobiology Learn about plant lectins as tools in glycobiology researchresearch
• Learn about glycan binding strategies used by Learn about glycan binding strategies used by pathogenspathogens
• Learn the diversity and families of glycan binding Learn the diversity and families of glycan binding proteinsproteins
• Learn the molecular/structural strategies used in Learn the molecular/structural strategies used in protein-glycan binding and recognitionprotein-glycan binding and recognition
• Learn the methods and resources for determining Learn the methods and resources for determining glycan binding protein specificitiesglycan binding protein specificities
• Learn about plant lectins as tools in glycobiology Learn about plant lectins as tools in glycobiology researchresearch
• Learn about glycan binding strategies used by Learn about glycan binding strategies used by pathogenspathogens
ObjectivesObjectivesObjectivesObjectives
Glycan Binding ProteinsGlycan Binding ProteinsGlycan Binding ProteinsGlycan Binding Proteins
• Learn the diversity and families of glycan binding Learn the diversity and families of glycan binding proteinsproteins
• Learn the molecular/structural strategies used in Learn the molecular/structural strategies used in protein-glycan binding and recognitionprotein-glycan binding and recognition
• Learn the methods and resources for determining Learn the methods and resources for determining glycan binding protein specificitiesglycan binding protein specificities
• Learn about plant lectins as tools in glycobiology Learn about plant lectins as tools in glycobiology researchresearch
• Learn about glycan binding strategies used by Learn about glycan binding strategies used by pathogenspathogens
• Learn the diversity and families of glycan binding Learn the diversity and families of glycan binding proteinsproteins
• Learn the molecular/structural strategies used in Learn the molecular/structural strategies used in protein-glycan binding and recognitionprotein-glycan binding and recognition
• Learn the methods and resources for determining Learn the methods and resources for determining glycan binding protein specificitiesglycan binding protein specificities
• Learn about plant lectins as tools in glycobiology Learn about plant lectins as tools in glycobiology researchresearch
• Learn about glycan binding strategies used by Learn about glycan binding strategies used by pathogenspathogens
ObjectivesObjectivesObjectivesObjectives
Plant lectinsPlant lectins
• Found in a variety of legumes and other plants, Found in a variety of legumes and other plants, often in seedsoften in seeds
• Discovered by their hemagglutinating activitiesDiscovered by their hemagglutinating activities• Proposed to function in plant pathogen or Proposed to function in plant pathogen or
symbiont recognition but data is not compellingsymbiont recognition but data is not compelling• Broad variety of glycan specificities - many Broad variety of glycan specificities - many
have yet to be defined in detailhave yet to be defined in detail• Often sturdy proteins suitable for research, Often sturdy proteins suitable for research,
biotechnology and diagnostic applicationsbiotechnology and diagnostic applications
https://https://www.vectorlabs.com/data/www.vectorlabs.com/data/brochures/K4-K7.pdfbrochures/K4-K7.pdf
https://https://www.vectorlabs.com/data/www.vectorlabs.com/data/brochures/K4-K7.pdfbrochures/K4-K7.pdf
““umbrella murder” of umbrella murder” of Georgi Markov by the Georgi Markov by the Bulgarian secret policeBulgarian secret police
Plant lectin research correlation – seeking diagnostic Plant lectin research correlation – seeking diagnostic glycosylation changes in cancerglycosylation changes in cancer
94 different lectins arrayed for 94 different lectins arrayed for binding tissue extracted proteinsbinding tissue extracted proteins
Li et al (2011)Li et al (2011)Anal ChemAnal Chem 83:8509 83:8509
Glycan Binding ProteinsGlycan Binding ProteinsGlycan Binding ProteinsGlycan Binding Proteins
• Learn the diversity and families of glycan binding Learn the diversity and families of glycan binding proteinsproteins
• Learn the molecular/structural strategies used in Learn the molecular/structural strategies used in protein-glycan binding and recognitionprotein-glycan binding and recognition
• Learn the methods and resources for determining Learn the methods and resources for determining glycan binding protein specificitiesglycan binding protein specificities
• Learn about plant lectins as tools in glycobiology Learn about plant lectins as tools in glycobiology researchresearch
• Learn about glycan binding strategies used by Learn about glycan binding strategies used by pathogenspathogens
• Learn the diversity and families of glycan binding Learn the diversity and families of glycan binding proteinsproteins
• Learn the molecular/structural strategies used in Learn the molecular/structural strategies used in protein-glycan binding and recognitionprotein-glycan binding and recognition
• Learn the methods and resources for determining Learn the methods and resources for determining glycan binding protein specificitiesglycan binding protein specificities
• Learn about plant lectins as tools in glycobiology Learn about plant lectins as tools in glycobiology researchresearch
• Learn about glycan binding strategies used by Learn about glycan binding strategies used by pathogenspathogens
ObjectivesObjectivesObjectivesObjectives
Glycan Binding ProteinsGlycan Binding ProteinsGlycan Binding ProteinsGlycan Binding Proteins
• Learn the diversity and families of glycan binding Learn the diversity and families of glycan binding proteinsproteins
• Learn the molecular/structural strategies used in Learn the molecular/structural strategies used in protein-glycan binding and recognitionprotein-glycan binding and recognition
• Learn the methods and resources for determining Learn the methods and resources for determining glycan binding protein specificitiesglycan binding protein specificities
• Learn about plant lectins as tools in glycobiology Learn about plant lectins as tools in glycobiology researchresearch
• Learn about glycan binding strategies used by Learn about glycan binding strategies used by pathogenspathogens
• Learn the diversity and families of glycan binding Learn the diversity and families of glycan binding proteinsproteins
• Learn the molecular/structural strategies used in Learn the molecular/structural strategies used in protein-glycan binding and recognitionprotein-glycan binding and recognition
• Learn the methods and resources for determining Learn the methods and resources for determining glycan binding protein specificitiesglycan binding protein specificities
• Learn about plant lectins as tools in glycobiology Learn about plant lectins as tools in glycobiology researchresearch
• Learn about glycan binding strategies used by Learn about glycan binding strategies used by pathogenspathogens
ObjectivesObjectivesObjectivesObjectives
Pathogen glycan binding proteinsPathogen glycan binding proteinsPathogen glycan binding proteinsPathogen glycan binding proteins
Viruses use glycan binding proteins (viral Viruses use glycan binding proteins (viral hemagglutinins) to infect cellshemagglutinins) to infect cells
• Many bacterial toxins are lectinsMany bacterial toxins are lectins• Bacteria use surface lectins to adhere to Bacteria use surface lectins to adhere to
target cellstarget cells• Eukaryotic microbes use lectins to adhere to Eukaryotic microbes use lectins to adhere to
target cellstarget cells
Essentials of Glycobiology Second Edition
Influenza virus lifecycleInfluenza virus lifecycle
Laver et al (1999)Laver et al (1999) Scientific American Scientific American, Jan issue, pp78-87, Jan issue, pp78-87
Linda Stannard: Linda Stannard: http://web.uct.ac.za/depts/mmhttp://web.uct.ac.za/depts/mmi/stannard/fluvirus.htmli/stannard/fluvirus.html
Influenza virus Influenza virus neuraminidaseneuraminidase
Gubareva et al (2000) Lancet 355:827
Rational design of an anti-influenza drug based on Rational design of an anti-influenza drug based on
sialic acid binding to the viral neuraminidasesialic acid binding to the viral neuraminidase
von Itzstein et al (1993) Nature 363:418
Sialic acid-Sialic acid-based influenza based influenza
inhibitorsinhibitors
Influenza virus budding in Influenza virus budding in vitro without (top) and with vitro without (top) and with
(bottom) neuraminidase (bottom) neuraminidase inhibitorinhibitor
Gubareva et al (2000) Lancet 355:827
Pathogen glycan binding proteinsPathogen glycan binding proteinsPathogen glycan binding proteinsPathogen glycan binding proteins
• Viruses use glycan binding proteins (viral Viruses use glycan binding proteins (viral hemagglutinins) to infect cellshemagglutinins) to infect cells
Many bacterial toxins are lectinsMany bacterial toxins are lectins• Bacteria use surface lectins to adhere to Bacteria use surface lectins to adhere to
target cellstarget cells• Eukaryotic microbes use lectins to adhere to Eukaryotic microbes use lectins to adhere to
target cellstarget cells
Essentials of Glycobiology Second Edition
Bacterial ABBacterial AB55 toxins toxinsBacterial ABBacterial AB55 toxins toxins Cholera toxinCholera toxin• E coliE coli enterotoxin 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
Cholera toxin B-pentamer binding GM1 – extended glycan engagementCholera toxin B-pentamer binding GM1 – extended glycan engagement
Merritt et al (1994) Protein Science 3:166Essentials of Glycobiology
Second Edition
Pathogen glycan binding proteinsPathogen glycan binding proteinsPathogen glycan binding proteinsPathogen glycan binding proteins
• Viruses use glycan binding proteins (viral Viruses use glycan binding proteins (viral hemagglutinins) to infect cellshemagglutinins) to infect cells
• Many bacterial toxins are lectinsMany bacterial toxins are lectins Bacteria use surface lectins to adhere to Bacteria use surface lectins to adhere to
target cellstarget cells• Eukaryotic microbes use lectins to adhere to Eukaryotic microbes use lectins to adhere to
target cellstarget cells
Pathogen glycan binding proteinsPathogen glycan binding proteinsPathogen glycan binding proteinsPathogen glycan binding proteins Bacteria use surface lectins to adhere to Bacteria use surface lectins to adhere to
target cellstarget cells• Eukaryotic microbes use lectins to adhere to Eukaryotic microbes use lectins to adhere to
target cellstarget cells
Essentials of Glycobiology Second Edition
Bacterial surface lectinsBacterial surface lectinsBacterial surface lectinsBacterial surface lectins
Essentials of Glycobiology Second Edition
E. coli binding to urinary epithelium in vitro -- E. coli binding to urinary epithelium in vitro -- specific inhibition by Gal specific inhibition by Gal αα1-4 Gal (right)1-4 Gal (right)E. coli binding to urinary epithelium in vitro -- E. coli binding to urinary epithelium in vitro -- specific inhibition by Gal specific inhibition by Gal αα1-4 Gal (right)1-4 Gal (right)
Sharon and Lis (1993) Scientific American Jan:82
Inhibition of Helicobacter Inhibition of Helicobacter pylori binding to human pylori binding to human gastric epithelium by gastric epithelium by multivalent carbohydratesmultivalent carbohydrates
Inhibition of Helicobacter Inhibition of Helicobacter pylori binding to human pylori binding to human gastric epithelium by gastric epithelium by multivalent carbohydratesmultivalent carbohydrates
Boren et al (1993) Science 262:1892
Pathogen glycan binding proteinsPathogen glycan binding proteinsPathogen glycan binding proteinsPathogen glycan binding proteins
• Viruses use glycan binding proteins (viral Viruses use glycan binding proteins (viral hemagglutinins) to infect cellshemagglutinins) to infect cells
• Many bacterial toxins are lectinsMany bacterial toxins are lectins• Bacteria use surface lectins to adhere to Bacteria use surface lectins to adhere to
target cellstarget cells Eukaryotic microbes use lectins to adhere to Eukaryotic microbes use lectins to adhere to
target cellstarget cells
Binding of Binding of Candida glabrata Candida glabrata to human epithelial cellsto human epithelial cellsBinding of Binding of Candida glabrata Candida glabrata to human epithelial cellsto human epithelial cells
Cormack et al (1999) Science 285:578
LINK: EPA7 glycan array screening
Glycan Binding ProteinsGlycan Binding ProteinsGlycan Binding ProteinsGlycan Binding Proteins
• Learn the diversity and families of glycan binding Learn the diversity and families of glycan binding proteinsproteins
• Learn the molecular/structural strategies used in Learn the molecular/structural strategies used in protein-glycan binding and recognitionprotein-glycan binding and recognition
• Learn the methods and resources for determining Learn the methods and resources for determining glycan binding protein specificitiesglycan binding protein specificities
• Learn about plant lectins as tools in glycobiology Learn about plant lectins as tools in glycobiology researchresearch
• Learn about glycan binding strategies used by Learn about glycan binding strategies used by pathogenspathogens
• Learn the diversity and families of glycan binding Learn the diversity and families of glycan binding proteinsproteins
• Learn the molecular/structural strategies used in Learn the molecular/structural strategies used in protein-glycan binding and recognitionprotein-glycan binding and recognition
• Learn the methods and resources for determining Learn the methods and resources for determining glycan binding protein specificitiesglycan binding protein specificities
• Learn about plant lectins as tools in glycobiology Learn about plant lectins as tools in glycobiology researchresearch
• Learn about glycan binding strategies used by Learn about glycan binding strategies used by pathogenspathogens
ObjectivesObjectivesObjectivesObjectives
(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.
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.
Sadat et al (2014) NEJM, yesterday