Proteolytic cleavage of the A subunit is essential for maximal cytotoxicity of Escherichia coli O157:H7 Shiga-like toxin-1.
Members of the bacterial Shiga toxin family consist of a single A subunit that is non-covalently associated with a pentamer of B subunits. These toxins bind to receptors on susceptible mammalian cells and enter the cells by endocytic uptake. During cell entry, the 32 kDa A subunit is cleaved by the membrane-anchored protease furin to generate a catalytically active, 27.5 kDa A1 fragment and a 4.5 kDa A2 fragment. Previous studies have shown that mutating the furin site to prevent cleavage did not significantly affect toxin potency, suggesting that cleavage is not required for toxin activity. Here it is confirmed that preventing cleavage at the usual processing site does not prevent proteolytic processing of the Escherichia coli Shiga-like toxin-1 A subunit. However, simultaneous mutation of both the primary furin-recognition site and a nearby putative furin cleavage site did prevent intracellular processing of the A subunit. Comparison of the cytotoxicities of purified recombinant toxins to cultured mammalian cells demonstrated that even on prolonged incubation with toxin, the unprocessed mutant was 60-fold less toxic than the wild-type protein or other mutants still capable of being proteolytically processed during cell entry.
Lea
Nicholas
N
Lord
J Michael
JM
Roberts
Lynne M
LM
D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity*; D002522:Chlorocebus aethiops; D019453:Escherichia coli O157 / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity*; D016297:Mutagenesis, Site-Directed; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism / Q000633:toxicity; D054788:Ribosome Inactivating Proteins; D022622:Shiga Toxin 1; D014709:Vero Cells
D001427:Bacterial Toxins; D011994:Recombinant Proteins; D022622:Shiga Toxin 1; D009699:N-Glycosyl Hydrolases; D054788:Ribosome Inactivating Proteins