| Abstract
| - Ricin toxin, the heterodimeric 65 kDa glycoproteinsynthesized in castor bean seeds, consistsof a cell binding lectin subunit (RTB) disulfide linked to an rRNAN-glycosidase protein synthesisinactivating subunit (RTA). While X-ray crystallography andequilibrium dialysis suggested two sugar-combining sites located in subdomains 1α and 2γ, biochemical andmutational analyses suggested theexistence of a third lectin site. We performedoligonucleotide-directed mutagenesis on RTB cDNA tocreate mutants with modifications in subdomains 1α, 2γ, and either1β or 2α. The triple-site mutantRTBs were expressed in insect cells. Partially purifiedrecombinant proteins obtained from infected cellextracts and cell supernatants were characterized for asialofetuin andcell binding, immunoreactivites,ability to reassociate with RTA, and recombinant heterodimer cellcytotoxicity. Yields of both triple-sitemutants were similar to the parent double-site mutant. Bothmutants showed immunoreactivity with apanel of anti-RTB monoclonal and polyclonal antibodies. Thetriple-site mutant with modification ofamino acid residues in subdomains 1α, 2α, and 2γ boundasialofetuin and cells similarly to the parent1α, 2γ, subdomain mutant. In contrast, the 1α, 1β, 2γsubdomain triple-site mutant had a one and one-half log decrease in asialofetuin and cell binding relative to theparent double-site mutant. The 1α, 2α,2γ triple-site mutant and 1α, 2γ parent protein had sugar bindingwhich was inhibited by 3−27-fold bylactose and asialofetuin. Both triple-site mutants reassociatedwell with RTA. The 1α, 2α, 2γ triple-sitemutant−RTA was equally cytotoxic to mammalian cells as thedouble-site mutant−RTA heterodimer.In contrast, the 1α, 1β, 2γ triple-site mutant−RTA was 25times less toxic than the double mutant and20 times more toxic than RTA alone. These data support a model forat least three lectin-bindingsubdomains in RTB.
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