This proposal represents a continuation of our studies on the isolation and characterization of plant lectins, and of their application to biomedical research. The lima bean lectin gene which we have already isolated will be expressed in E. coli (or yeast). Site-directed mutagenesis will be carried out to afford a series of mutant lectins with amino acid substitutions in the carbohydrate and adenine binding sites. These altered lectins will be expressed and examined for their carbohydrate and adenine binding properties. A series of new lectins will be purified and characterized with respect to their physiochemical and carbohydrate binding properties in an effort to discover lectins of unique carbohydrate binding specificity. A number of mannose-specific, monocotyledonous bulb lectins (snow drop, daffodil, amaryllis) will be sequenced, comparing their homologous structures in an effort to account for differences in their fine carbohydrate binding specificity. The snow drop lectin has been crystallized with several carbohydrate binding ligands and its X-ray structure will be solved. Electron spin resonance and photoaffinity labelling probes will be synthesized and employed to investigate the hydrophobic binding sites of the pea, lentil, and Vicia faba lectins which differ in this regard from concanavalin A and related """"""""single chain"""""""" lectins. Focussed studies on the binding of lectins to tumor tissues from colon, breast, and prostate will be carried out in an effort to develop diagnostic reagents for cancer of these organs. Mucus from colon cancer will be fractionated on immobilized amaranthin, the lectin which we isolated and characterized from Amaranthus caudatus seeds. This lectin recognizes the T- or cryptic T-antigen [(NANAalpha2,3)Galbeta1,3GalNAc].
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