DOUBLE-LECTIN SITE RICIN-B CHAIN MUTANTS EXPRESSED IN INSECT CELLS HAVE RESIDUAL GALACTOSE BINDING - EVIDENCE FOR MORE THAN 2 LECTIN SITES ON THE RICIN TOXIN-B CHAIN

Ricin toxin, the heterodimeric 65 kDa glycoprotein synthesized in cast or 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 R TB by X-ray crystallography, equilibrium dialysis, chemical modificati on, and mutational analysis have yielded conflicting results as to the number, location, and affinity of sugar-combining sites. An accurate assessment of the amino acid residues of RTB involved in galactose bin ding 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 oligonucleoti de-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 cyto toxicity. Two single-site mutants (subdomain 1 alpha or 2 gamma) and t wo double-site mutants (subdomains 1 alpha and 2 gamma) 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 mut ants displayed up to a 1 log decrease in asialofetuin binding avidity, while the double-site mutants showed close to a 2 log decrease in sug ar binding. However, for each of the double-site mutants, residual sug ar binding was demonstrated to both immobilized asialofetuin and cells , and this binding was specifically inhibitable with alpha-lactose. Al l mutants reassociated with RTA, and the mutant heterodimers were cyto toxic to mammalian cells with potencies 1000-fold or more times that o f unreassociated wild-type RTA or RTB. These data support a model for three or more lectin binding subdomains in RTB.