Proteolytic cleavage at arginine residues within the hydrophilic disulphide loop of the Escherichia coli Shiga-like toxin I A subunit is not essential for cytotoxicity.
Escherichia coli Shiga-like toxin I is a type II ribosome-inactivating protein composed of an A subunit with RNA-specific N-glycosidase activity, non-covalently associated with a pentamer of B subunits possessing affinity for galabiose-containing glycolipids. The A subunit contains a single intrachain disulphide bond encompassing a hydrophilic sequence containing two trypsin-sensitive arginine residues. By analogy with other bacterial toxins it has been proposed that proteolytic nicking, deemed essential for a cytotoxic effect, occurs within this disulphide-bonded loop to generate the A1 and A2 fragments. Reduced A1 is then believed to translocate an internal membrane to inactivate protein synthesis in the cytosol. In this report, the disulphide-loop arginines of the SLT I A subunit were mutated to block the specific proteolysis presumed to occur. However, the mutant generated remained an effective toxin having similar catalytic activity to wild-type toxin and only a marginally reduced cytotoxicity towards cultured cells. We conclude that the disulphide-loop arginine residues are not the unique and essential processing sites previously assumed, but that processing may occur at alternative accessible sites to compensate for loss of target sites within the loop.
Department of Biological Sciences, University of Warwick, Coventry, UK.
B J
BJ
Burgess
L M
LM
Roberts
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D002522:Chlorocebus aethiops; D010450:Endopeptidases / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000494:pharmacology; D012270:Ribosomes / Q000187:drug effects; D022622:Shiga Toxin 1; D014709:Vero Cells / Q000187:drug effects
D001427:Bacterial Toxins; D011993:Recombinant Fusion Proteins; D022622:Shiga Toxin 1; D001120:Arginine; D010450:Endopeptidases