Ricin toxin contains at least three galactose-binding sites located in B chain subdomains 1 alpha, 1 beta, and 2 gamma.

Ricin toxin, the heterodimeric 65 kDa glycoprotein synthesized in castor bean seeds, consists of a cell binding lectin subunit (RTB) disulfide linked to an rRNA N-glycosidase protein synthesis inactivating subunit (RTA). While X-ray crystallography and equilibrium dialysis suggested two sugar-combining sites located in subdomains 1 alpha and 2 gamma, biochemical and mutational analyses suggested the existence of a third lectin site. We performed oligonucleotide-directed mutagenesis on RTB cDNA to create mutants with modifications in subdomains 1 alpha, 2 gamma, and either 1 beta or 2 alpha. The triple-site mutant RTBs were expressed in insect cells. Partially purified recombinant proteins obtained from infected cell extracts and cell supernatants were characterized for asialofetuin and cell binding, immunoreactivites, ability to reassociate with RTA, and recombinant heterodimer cell cytotoxicity. Yields of both triple-site mutants were similar to the parent double-site mutant. Both mutants showed immunoreactivity with a panel of anti-RTB monoclonal and polyclonal antibodies. The triple-site mutant with modification of amino acid residues in subdomains 1 alpha, 2 alpha, and 2 gamma bound asialofetuin and cells similarly to the parent 1 alpha, 2 gamma, subdomain mutant. In contrast, the 1 alpha, 1 beta, 2 gamma subdomain triple-site mutant had a one and one-half log decrease in asialofetuin and cell binding relative to the parent double-site mutant. The 1 alpha, 2 alpha, 2 gamma triple-site mutant and 1 alpha, 2 gamma parent protein had sugar binding which was inhibited by 3-27-fold by lactose and asialofetuin. Both triple-site mutants reassociated well with RTA. The 1 alpha, 2 alpha, 2 gamma triple-site mutant-RTA was equally cytotoxic to mammalian cells as the double-site mutant-RTA heterodimer. In contrast, the 1 alpha, 1 beta, 2 gamma triple-site mutant-RTA was 25 times less toxic than the double mutant and 20 times more toxic than RTA alone. These data support a model for at least three lectin-binding subdomains in RTB.