Ricin is the highly toxic heterodimeric protein from castor bean plants which has been used for producing targeted therapeutics for cancer. However, minimal information is known about the structure-function relationships of this molecule other than the presence of enzyme activity in the A subunit and binding activity in the B subunit. Five experiments will be conducted to define structural requirements for different ricin intoxication functions. The first experiment will attempt to define the mechanisms of catalysis of the N-glycosidic bond hydrolysis by ricin. Ricin toxin A chain (RTA) will be expressed in Escherichia coli (E. coli) and site-specific mutagenesis will be used to produce mutant RTAs which maintain protein immunologic determinants but have altered enzyme activity. Enzyme kinetics will be measured with 32P labeled ribosomes. The second experiments should define amino acid residues critical for RTB lectin function. Ricin toxin B chain (RTB) will also be expressed in E. coli and site-specific mutagenesis will be used to produce mutants which maintain immunological determinants but have reduced galactosyl binding. The third experiment will identify RTA structural properties needed for membrane translocation. Site-specific mutagenesis will be used to produce RTA molecules with tertiary structure stabilized by an intrachain disulfide bond. If this molecule retains enzymatic and RTB association functions but fails to produce fully toxic heterodimers it suggests protein unfolding is required for translocation. The fourth experiment will identify RTB translocation functions unrelated to sugar binding. RTB molecules with or without galactosyl binding will be associated with native plant RTA and the dimers will be coupled to an antibody and tested for cytotoxicity. In the final experiment the location, linkage and role of the ligand in ricin cytotoxicity will be studied. MSH will be inserted at either the N- terminus or C-terminus of RTA with and without disulfide bridges. These genetically engineered toxins will be tested for selective in vitro cytotoxicity and whole animal toxicity and pharmacology.
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