Lectin leftovers 22707 Lectin leftovers Brief review of

Lectin leftovers 2/27/07

Lectin leftovers – Brief review of animal lectins – Brief overview of plant lectins – Determining/detecting lectin activities

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

Animal lectins are everywhere After Schnaar, R. L.

After Schnaar, R. L.

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

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

Vegetable Lectins • Leguminosae – Con. A (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

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

Structure of Con. A After Alvarez-Manilla

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

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

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

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

Affinity/Avidity/Multivalency

Affinity determines approach

Washing can kill you

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

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

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

Adhesive strength by controlled detachment Blackburn, C. , 1985

Binding specificity to resolved glycoconjugates Tiemeyer, M. , 1991

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