Lectin leftovers

1. Lectin leftovers 2/27/07

2. Lectin leftovers • Brief review of animal lectins • Brief overview of plant lectins • Determining/detecting lectin activities

3. 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 After Alvarez-Manilla

4. Animal lectins are everywhere After Schnaar, R.L.

5. After Schnaar, R.L.

6. Common structural features of animal lectins After Schnaar, R.L.

7. 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 After Alvarez-Manilla

8. 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 After Alvarez-Manilla

9. 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 After Alvarez-Manilla

10. Structure of ConA After Alvarez-Manilla

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 After Alvarez-Manilla

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 After Alvarez-Manilla

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 After Alvarez-Manilla

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 After Alvarez-Manilla

15. Affinity/Avidity/Multivalency

16. Affinity determines approach

17. Washing can kill you

18. Characterizing lectin binding • Equilibrium dialysis against labeled hapten • Equilibrium binding, stop by PEG with centrifugation (solubilized receptor) • Equilibrium binding, stop by filtration (membranes) • Multivalent ligands • Multivalent receptor probes • Biacore realtime kinetics • Cell adhesion, flow under shear to immobilized glycan or receptor • Cell adhesion, static adhesion to immobilized glycan • X-ray co-crystallography, NMR, and MS mapping of relevant contacts and protein dynamics

19. Static cell adhesion, controlled force Blackburn, C., 1982

20. Adhesive specificity determined by controlled detachment Blackburn, C., 1985

21. Adhesive strength by controlled detachment Blackburn, C., 1985

22. Binding specificity to resolved glycoconjugates Tiemeyer, M., 1991

23. General Principles • Lectins generally bind with low affinity but achieve high avidity through multi-valency • A relatively small set of protein motifs have been identified as lectins • Lectin motifs comprise distributed sequence similarities and structural homologies; extended primary amino acid sequence conservation is not generally associated with lectin-like activities • Methods for lectin characterization must consider affinity, valency, and avidity • The development of multivalent probes, as well as methods for determining static and dynamic adhesion have been instrumental for defining lectin binding specificity and lectin function