The thrust of this proposal is on carbohydrate binding proteins as probes and reagents for studies of protein-carbohydrate interaction. The models for these studies are lectins of plant and animal origin: their isolation, characterization and interaction with carbohydrates (oligosaccharides and their derivatives, polysaccharides, glycoproteins, etc.) as they occur on cell surfaces and in solution. The molecular basis of these interactions will be studied by X-ray crystallography, NMR spectroscopy, microcalorimetry, equilibrium dialysis, photoaffinity labeling and quantitative precipitation and inhibition. An in-depth study of the slug lectin, Limax flavus will be undertaken. This will include cloning of the gene encoding the lectin with consequent determination of its primary structure, and expression of the lectin in E. coli; NMR studies of its interaction with the methyl alpha-ketoside of N-acetylneuraminic acid and derivatives; and initiation of studies on its crystallization and X-ray analysis. Amaranthin, the lectin present in Amaranthus caudatus has been crystallized and X-ray diffraction studies are well underway. Chemical sequencing of the lectin which is blocked at its N-terminus is also proceeding. A related lectin from the seeds of Amaranthus hypochondriacus which crystallizes in a different form will be compared with A. caudatus by peptide mapping. A microcalorimetric study of amaranthin-T-antigen (Galbeta1,3 GalNAc) interaction will furnish thermodynamic data on the mechanism of binding. Mutants of the adenine binding site of the lima bean lectin will be generated and assayed for their ability to bind adenine. Affinity labeling of the carbohydrate binding sites of concanavalin A and the snowdrop lectin will be conducted employing a high affinity photoaffinity labeling reagent, Man alpha1-3 [Man alpha1-6] manalpha-O-p C/6H/4N/3. A series of new lectins will be isolated and characterized in an effort to identify those with unique carbohydrate binding specificities. As example, a beta-xylosyl binding lectin is being investigated. The effect of lectin valence on the binding of Griffonia simplicifolia I isolectins (A/4, A/3B, A/2B/2, AB/3, B/4) to type A erythrocytes which contain the immunodominant alpha-D-GalNA-p group, the carbohydrate ligand which binds to the A subunit. Ligand peptides that mimic carbohydrates and bind to lectins will identified using the technique of epitope libraries displayed on filamentous phage; the peptides will be synthesized and assayed.