Lectin leftovers 2/27/07 Lectin leftovers Brief review of

Lectin leftovers 2/27/07 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 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 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 ConA 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 Wash Method Filtration Wash Time Wash Efficiency 20 30 sec High Centrifugation

0 5 min Moderate Inert Phase 10 sec High

Equilibrium Dialysis 0 sec None Ease Easy Problems

Filter binding Moderate Pellet Trapping Moderate Effect of inert phase Difficult Low Signal/Noise

Washing can kill you pKD t0.5 t0.1 11 19 hr 2.9 hr 10 1.9 hr 17 min 9 11.5 min 1.7 min 8 1.1 min 10 sec 7 6.9 sec 1.0 sec

6 0.7 sec 0.1 sec 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

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