Catalytic and cytotoxic activities of recombinant ricin A chain mutants with charged residues added at the carboxyl terminus.
Ricin A chain (RTA) mutants which had been modified by the addition of three lysine residues, three lysines and an alanine, or six histidine residues at the carboxyl terminus were expressed in Escherichia coli. The recombinant proteins were purified to homogeneity by ion-exchange chromatography on CM-Sepharose CL-6B. The 28S ribosomal RNA N-glycosidase activities of the three RTA mutants were indistinguishable from each other and from the activity of wild-type recombinant RTA. The RTA mutants were not impaired, compared with wild-type RTA, in their ability to reassociate with ricin B chain to form ricin holotoxin. Holotoxins containing mutant RTAs were as readily dissociated into subunits under reducing conditions as native holotoxin, and the RTA mutants were indistinguishable from wild-type RTA in the extent of their interaction with biological membranes. Ricin holotoxins containing the RTA mutants were, however, less cytotoxic to Vero cells than ricin containing wild-type RTA. At equivalent concentrations, a time course assay showed that holotoxin containing the mutant RTAs took longer to kill target cells than that containing wild-type recombinant RTA, suggesting that the mutant forms of RTA are less efficiently processed or translocated across an intracellular membrane than is wild-type RTA.
Department of Biological Sciences, University of Warwick, Coventry, United Kingdom.
J C
JC
Simpson
L M
LM
Roberts
J M
JM
Lord
D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D002845:Chromatography / Q000379:methods; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006027:Glycosides / Q000633:toxicity; D009154:Mutation; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D012339:RNA, Ribosomal, 28S / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology; D012276:Ricin / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology*; D014315:Triterpenes / Q000633:toxicity; D014709:Vero Cells
D000596:Amino Acids; D006027:Glycosides; D012339:RNA, Ribosomal, 28S; D011994:Recombinant Proteins; D014315:Triterpenes; C001883:stichoposide; D012276:Ricin