Cytotoxic effects of ricin without an interchain disulfide bond: genetic modification and chemical crosslinking studies.
Ricin is a toxic glycoprotein made of two polypeptide chains (A and B) linked by a disulfide bond. Ricin binds to cells by the B chain and is then internalized. The interchain disulfide bond is believed to be reduced in endosomes, and the A chain is then subsequently translocated to cytoplasm where it inactivates ribosomes. To understand the role of the disulfide bond in ricin toxicity, we prepared two types of ricin molecules. First, cysteine 259 of the A chain was mutated to an alanine residue. The mutant A chain was then reassociated with the native B chain to determine whether ricin is biologically active in the absence of an interchain disulfide bond. Reassociated mutant ricin showed a 40-fold reduction in biological activity. Binding studies using a hydrophobic fluorescence probe indicated that the associated complex was stable only at neutral pH and became highly unstable at a lower pH characteristic of the endosomal milieu. In the second construct, the interchain disulfide bond was replaced with a non-reducible bond by chemical derivatization. Interestingly, the non-reducible ricin molecule was equally cytotoxic as native ricin. These results show: (i) that the interchain disulfide bond is necessary to hold the A chain and the B chain together at endosomal pH, and (ii) that intact ricin may be transported to the cytoplasm where proteolysis or hydrolysis may occur to release the biologically active moiety.
Mohanraj
D
D
Department of Pharmacology, University of Minnesota, Minneapolis 55455, USA.
Ramakrishnan
S
S
D000409:Alanine; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology*; D001483:Base Sequence; D016923:Cell Death; D003432:Cross-Linking Reagents; D004220:Disulfides / Q000737:chemistry*; D004355:Drug Stability; D005260:Female; D005456:Fluorescent Dyes; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010051:Ovarian Neoplasms; D011994:Recombinant Proteins / Q000494:pharmacology; D012276:Ricin / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D003432:Cross-Linking Reagents; D004220:Disulfides; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D012276:Ricin; D000409:Alanine