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INTRODUCTION OF A DISULFIDE BOND INTO RICIN-A CHAIN DECREASES THE CYTOTOXICITY OF THE RICIN HOLOTOXIN
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UNSPECIFIED (1994) INTRODUCTION OF A DISULFIDE BOND INTO RICIN-A CHAIN DECREASES THE CYTOTOXICITY OF THE RICIN HOLOTOXIN. JOURNAL OF BIOLOGICAL CHEMISTRY, 269 (43). pp. 26705-26710. ISSN 0021-9258.
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Abstract
Wild type ricin A chain (RTA) contains two cysteine residues (Cys(171) and Cys(259)). Cys(259) forms the interchain disulfide bond of ricin holotoxin with Cys(4) of ricin B chain (RTB). We have used site-directed mutagenesis of RTA cDNA to convert Cys(171) to Ser and to introduce a disulfide bond into RTA by converting Ser(215) and Met(255) to Cys residues. Mutant RTA was expressed in Escherichia coil and directed to the oxidizing environment of the periplasmic space where the Cys(215)-Cys(255) 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 crosslink. 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.
Item Type: | Journal Article | ||||
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Subjects: | Q Science > QD Chemistry | ||||
Journal or Publication Title: | JOURNAL OF BIOLOGICAL CHEMISTRY | ||||
Publisher: | AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC | ||||
ISSN: | 0021-9258 | ||||
Official Date: | 28 October 1994 | ||||
Dates: |
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Volume: | 269 | ||||
Number: | 43 | ||||
Number of Pages: | 6 | ||||
Page Range: | pp. 26705-26710 | ||||
Publication Status: | Published |
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