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Carbohydrate Binding Proteins

This article provides an overview of carbohydrate binding proteins, including lectins, their functions, structures, and various uses in cell biology and research.

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Carbohydrate Binding Proteins

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  1. Carbohydrate Binding Proteins BCH 8130 Gerardo Alvarez-Manilla

  2. Carbohydrates are good recognition sites

  3. Carbohydrate Binding proteins • Enzymes • Glycosidases, glycosyltransferases • Antibodies • Blood groups • Lectins

  4. LECTINS • Carbohydrate binding proteins that are not antibodies or enzymes • Bind with high specificity • Latin: lectus, meaning to gather or select • Relatively high dissociation constants (ca 100 M) • Carbohydrate recognition domains are small • Most lectins are multivalent

  5. Carbohydraye Recognition domains

  6. Lectins are present in all organisms • Virus----- Influenza • Bacteria ----- binding to hosts during pathogenesis • Vegetable • Many have been purified and characterized • Physiological function is unknown • Animal • Several proteins with a wide variety of functions

  7. Vegetable Lectins • Leguminosae • ConA (Concanavalin A from Jack bean) • Phaseolus Vulgaris (PHA-L and PHE) • Soy bean agglutinin • Graminae • Wheat germ agglutinin • Solanaceae • Tomato lectin • Potato lectin

  8. Structure of Vegetable lectins • Compact -barrel, no alpha helices • Antiparalell beta-sheets • Many require metals (leguminosae) • Ca and Mn • Metals do not participate directly in the binding but are required

  9. Structure of ConA

  10. Structure of PHA-L

  11. Functions of Plant lectins • Little is known • In legume seeds can comprise up to 30% of the total protein • They are expressed in other parts of the plant • Nodulation factor in roots

  12. Functions of Plant lectins (cont) • May function as defense against pathogens • Some lectins posses other activities besides carbohydrate binding • RCAII (Ricin) RNA-N-glycosidase • DBA has an adenine binding site in addition to CRD

  13. Uses of Plant lectins • Agglutination of cells and blood typing • Cell separation and analysis • Bacterial typing • Identification and selection of mutated cells with altered glycosylation • Toxic conjugates for tumor cell killing • Cytochemical characterization/staining of cells and tissues

  14. Uses of Plant lectins (cont) • Mitogenesis of cells • Mapping neuronal pathways • Purification of glycoconjugates • Assays of glycosyltransferases and glycosidases • Defining glycosylation status of target glycoconjugates

  15. Animal Lectins • Very heterogenous group • Many physiological functions • Little sequence homology • However most of the are multivalent • Current classification is based on amino acid sequence homology and evolutionary relatedness

  16. Classification of animal lectins • C-type lectins • Require calcium • S-type • Disulfide bonds, bind galactosides • P-type • Man 6-P receptors • I-Type • Immunoglobulin like

  17. Classification of animal lectins (cont) • Calnexin and calreticulin • Protein folding • Hyaluronan binding proteins • CD 44, verscan, aggrecan • Frog egg lectins

  18. Classification of animal lectins (cont.) Varki et. al. Essentials of glycobiology

  19. Animal Lectins Varki et. al. Essentials of glycobiology

  20. P-Type lectins, Man-6-P receptors • Calcium Independent (CIMPR) • 275 kDa • Highest afffinity for Glycans with two phosphomonoesters > one phosphomonoester > phosphodiester • Calcium dependent (CDMPR) • 45 kDa • Binds only to glycans with one phosphomonoester

  21. Varki et. al. Essentials of glycobiology

  22. lysosomal enzyme traficking Varki et. al. 1999 Essentials of glycobiology

  23. Lysosomal enzyme trafficking Kornfeld, 1987

  24. Lysosomal enzyme deficiencies • I Cell disease • Pseudo-Hurler polydistrophy • AKA Mucolipidosis II and IIIC • Deficiency in GlcNAc-1-Phosphotransferase

  25. C-Type lectins • Characteristic CRD is structurally preserved • Calcium is involved directly in glycan recognition and stabilization of CRD

  26. Types of C-type lectins, soluble • Lecticans (versican, aggregan,neurocan) • Proteoglycans • organizers of the brain extracellular matrix forming complexes with tenascin and other glycoproteins • Collectins • Host defense • Mannose binding proteins, Conglutinnin, etc

  27. C-type lectins, membrane bound • Selectins • Neutrophil and leukocyte trafficking • Type II membrane receptors • Asialoglycoprotein receptor • Type I transmembrane protein with tandem extracellular CRDs. • Molecular uptake into the cells • surface mannose receptors on macrophages • DEC-205, (cell surface molecule of dendritic cells)

  28. Selectins • molecules that facilitate the recirculation of lymphoid cells from the intravascular compartment to the secondary lymphoid organs • Type I membrane proteins with sequence and structural homology

  29. Selectins Varki et. al. 1999 Essentials of glycobiology

  30. L-Selectin • CD 62L • Discovered as the antigen recognized by MEL14 • Inhibited vitro adhesion of lymphocytes to HEVs and in vivo homing of lymphocytes to peripheral lymph nodes. • L-selectin also has a role in adhesion of neutrophils, eosinophils, and monocytes to nonlymphoid vascular endothelium • Leukocyte-endothelial cell and Leukocyte-Leukocyte interactions have been reported

  31. L-selectin (cont) • Molecular mechanisms are not yet understood completely • Some of the described receptors are GlyCAM-1, CD34, Sgp200 and MAdCAM-1 • Heparan sulfate GAGS are involved • Sialylated sulfated and fucosylated O-linked structures are recognized • L-Selectin can also bind to PSGL-1

  32. E-Selectin • CD 62E • Endothelial Leukocyte Adhesion Molecule I (ELAM-1) • Induced in Endothelial cells via cytokine stimulation • Ligand are ESL-1 • N-linked glycans with sialyl-lewis-X • Can also bind PSGL in vitro

  33. N-Acetyllactosamine chains

  34. Lewis a and b are on type1 N-Acetyllactosamines

  35. Lewis X and Y are on type 2 lactosamines

  36. Carbohydrate ligands for E-Selectin Varki et. al. 1999 Essentials of glycobiology

  37. Structure of E-selectin Somers et al. 2000 Cell103(3):467-79

  38. Crystal structure of recombinant E-selectin

  39. P-Selectin • 140 kDa protein • expressed in cortical granules in platelets • Stored in Weibel-Palade bodies in Endothelial cells • Expressed in the surface upon stimulation with cytokines • Principal ligand is PSGL-1 • Sialyl-Lewis-X and tyrosine sulfation

  40. Crystal structure of P-selectin

  41. Leukocyte Blood stream Leukocyte Leukocyte ligand TNF Leukocyte selectin Leukocyte Role of Selectins in inflammation Endothelial cells Inflammation triggers release Of cytokines (TNF)

  42. Galectins • Galactose binding proteins • Share substantial homology in their CRD • All are soluble • Expressed as cytoplasmic proteins • Translocated through membrane to extracellular space via unknown mechanism

  43. Carbohydrate ligands • B-linked Galactose • Lactose • N-Acetyllactosamine • Poly-N-Acetyllactosamine

  44. Galectins (cont) • Do not require cations for binding • require reducing conditions to maintain activity in the absence of ligands • -sheet barrel “jelly roll” structure • Wide distyribution among the animal kingdom

  45. Structural motifs

  46. Structure of galectin1

  47. Functions of galectins • Cell-cell adhesion • Cell matrix interaction • Cell signaling • Growth arrest • Mitogenesis • apoptosis

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