Double-lectin site ricin B chain mutants expressed in insect cells have residual galactose binding: Evidence for more than two lectin sites on the ricin toxin B chain

Ricin toxin, the heterodimeric 65 kDa glycoprotein synthesized in castor bean seeds, contains a cell binding lectin subunit (RTB) disulfide linked to an RNA N-glycosidase protein synthesis-inactivating subunit (RTA). Investigations of the molecular nature of the lectin sites in RTB by X-ray crystallography, equilibrium dialysis, chemical modification, and mutational analysis have yielded conflicting results as to the number, location, and affinity of sugar-combining sites. An accurate assessment of the amine acid residues of RTB involved in galactose binding is needed both for correlating structure-function of a number of plant lectins and for the design and synthesis of targeted toxins for cancer and autoimmune disease therapy. We have performed oligonucleotide-directed mutagenesis on cDNA encoding RTB and expressed the mutant RTBs in insect cells. Partially purified recombinant proteins obtained from infected cell supernatants and cell extracts were characterized as to yields, immunoreactivities, asialofetuin binding, cell binding, ability to reassociate with RTA, and recombinant heterodimer cell cytotoxicity. Two single-site mutants (subdomain 1α or 2γ) and two double- site mutants (subdomains 1α and 2γ) were produced and studied. Yields varied by two logs with lower recoveries of double-site mutants. All the mutants showed immunoreactivity with a panel of anti-RTB monoclonal and polyclonal antibodies. Single-lectin site mutants displayed up to a 1 log decrease in asialofetuin binding avidity, while the double-site mutants showed close to a 2 log decrease in sugar binding. However, for each of the double-site mutants, residual sugar binding was demonstrated to both immobilized asialofetuin and cells, and this binding was specifically inhibitable with α-lactose. All mutants reassociated with RTA, and the mutant heterodimers were cytotoxic to mammalian cells with potencies 1000- fold or more times that of unreassociated wild-type RTA or RTB. These data support a model for three or more lectin binding subdomains in RTB.