Double-site ricin B chain mutants retain galactose binding.
Three distinct double-site and two single-site ricin B chain (RTB) mutants were expressed in Spodoptera frugiperda insect cells and purified from infected cell supernatants. The yields of recombinant proteins were 0.01-0.2 mg/l. The purity after monoclonal antibody affinity chromatography was 1-20%. The mutant proteins were soluble, immunoreactive with monoclonal antibodies and polyclonal antibodies to RTB and demonstrated molecular weights of 32 kDa, similar to plant RTB. All three double-site and both single-site mutants bound asialofetuin and mammalian cell surfaces based on an asialofetuin ELISA and cell binding immunofluorescence assay. While one double-site mutant, W37S/Y248S, had a 1 log drop in sugar binding, the other two double-site mutants W37S/Y248H and D22E/D234E had 2 log reductions in sugar binding. Each mutant reassociated efficiently (25-75%) with plant ricin A chain (RTA) to form cytotoxic heterodimers. The concentration of protein required to reduce protein synthesis 50% (ID50) was 1 log higher than plant ricin for W37S/Y248S-RTA and the single-site mutant heterodimers, Q35N-RTA and D22E-RTA and 2 logs higher than plant ricin for the other two double-site mutant heterodimers. The results suggest amino acid residues in both the 1 alpha and 2 gamma subdomains of RTB participate in sugar binding. However, other subdomains must contribute to the avidity of ricin for cell surface oligosaccharides.
Frankel
A
A
Department of Medicine, Medical University of South Carolina, Charleston 29425, USA.
Tagge
E
E
Chandler
J
J
Burbage
C
C
Willingham
M
M
D000818:Animals; D001212:Asialoglycoproteins / Q000378:metabolism; D016367:Baculoviridae / Q000235:genetics; D001667:Binding, Competitive; D004305:Dose-Response Relationship, Drug; D060748:Fetuins; D005690:Galactose / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D012276:Ricin / Q000235:genetics* / Q000378:metabolism* / Q000633:toxicity; D018411:Spodoptera / Q000166:cytology; D018675:Toxicity Tests; D014407:Tumor Cells, Cultured; D000509:alpha-Fetoproteins / Q000378:metabolism
D001212:Asialoglycoproteins; D060748:Fetuins; D011994:Recombinant Proteins; D000509:alpha-Fetoproteins; C017029:asialofetuin; D012276:Ricin; D005690:Galactose