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 A fragment and a 4·5 kDa A 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 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.


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