Glycan Binding Proteins

Glycan Binding ProteinsGlycan Binding ProteinsGlycan Binding ProteinsGlycan Binding Proteins

http://www.youtube.com/watch?v=v9j-g0qspdk

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


• 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

http://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.

http://www.imperial.ac.uk/research/animallectins/ctld/lectins.html

Animal lectins – structural classesAnimal lectins – structural classes


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































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

C:\RLS\03.rls\Teach\Intro_Glycobiology\ITG_2013\GBP2013\d_glucose.pdb


C:\RLS\03.rls\Teach\Intro_Glycobiology\ITG_2013\GBP2013\RW_NeuAc.pdb

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 stackinghydrophobic 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





























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

http://www.functionalglycomics.org/

http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_3000

http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_PA_v2_728_09262005

http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_3000

























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

























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


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


• 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




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



• Many bacterial toxins are lectinsMany bacterial toxins are lectins Bacteria use surface lectins to adhere to Bacteria use surface lectins to adhere to



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




Bacterial surface lectinsBacterial surface lectinsBacterial surface lectinsBacterial surface lectins


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



• 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


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













(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

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Glycan Binding Proteins