Introduction of a disulfide bond into ricin A chain decreases the cytotoxicity of the ricin holotoxin.
Wild type ricin A chain (RTA) contains two cysteine residues (Cys171 and Cys259). Cys259 forms the interchain disulfide bond of ricin holotoxin with Cys4 of ricin B chain (RTB). We have used site-directed mutagenesis of RTA cDNA to convert Cys171 to Ser and to introduce a disulfide bond into RTA by converting Ser215 and Met255 to Cys residues. Mutant RTA was expressed in Escherichia coli and directed to the oxidizing environment of the periplasmic space where the Cys215-Cys255 disulfide bond was formed. The disulfide-containing RTA mutant had an in vitro catalytic activity similar to that of an identical form of recombinant RTA that lacked the S215C and M255C mutations. In the presence of glutathione and protein disulfide isomerase, this RTA variant reassociated with RTB to form ricin holotoxin. Incubation of this holotoxin with increasing concentrations of dithiothreitol showed that the interchain disulfide bond joining RTA and RTB was more readily reduced than the intrachain disulfide bond in RTA. Ricin in which the RTA moiety contained the disulfide bond was 15-18-fold less cytotoxic to HeLa or Vero cells than ricin in which the RTA did not contain the stabilizing disulfide cross-link. Since these ricin molecules had identical RTB cell binding and RTA catalytic activities, we suggest that the observed reduction in cytotoxicity caused by the introduced disulfide bond resulted from a constraint on the unfolding of RTA, indicating that such unfolding is necessary for the membrane translocation of RTA during its entry into the cytosol.
Argent
R H
RH
Department of Biological Sciences, University of Warwick, Coventry, United Kingdom.
Roberts
L M
LM
Wales
R
R
Robertus
J D
JD
Lord
J M
JM
D000818:Animals; D002522:Chlorocebus aethiops; D003545:Cysteine / Q000235:genetics; D003603:Cytotoxins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D004220:Disulfides / Q000737:chemistry / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D037121:Plant Lectins; D010947:Plants, Toxic; D015202:Protein Engineering; D017510:Protein Folding; D012335:RNA, Ribosomal / Q000187:drug effects; D011994:Recombinant Proteins / Q000494:pharmacology; D012276:Ricin / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D012278:Ricinus; D014709:Vero Cells
D003603:Cytotoxins; D004220:Disulfides; D037121:Plant Lectins; D012335:RNA, Ribosomal; D011994:Recombinant Proteins; D012276:Ricin; D003545:Cysteine