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

Bioconjugate Chemistry
T FuA Frankel

Abstract

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 amino 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 1 alpha or 2 gamma) and two double-site mutants (subdomains 1 alpha 2 gamma) were ...Continue Reading

References

Sep 26, 1978·Biochimica Et Biophysica Acta·C ZentzR Bourrillon
Apr 10, 1991·European Journal of Biochemistry·A Rivera-SagredoM Martín-Lomas
Nov 1, 1994·The Biochemical Journal·A FrankelM Willingham
Jan 1, 1996·Bioconjugate Chemistry·A FrankelM Willingham

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