title: Evidence that proteolytic separation of Shiga-like toxin type IIv A subunit into A1 and A2 subunits is not required for toxin activity.
abstract: The role for proteolytic activation of Shiga-like toxin type II variant (SLT-IIv) A subunit was examined using site-directed mutagenesis. Processing of the enzymatically active A subunit by trypsin results in cleavage at an arginine residue(s) (Arg247 and/or Arg250) located between two cysteines. After reduction of the disulfide bond, the processed A subunit separates into an enzymatically active A1 and an A2 peptide. Substitution mutations were created in SLT-IIv that replaced each or both of the two arginines with either glutamic acid (R247E, R250E, or R247E/R250E) or histidine (R247H, R250H, or R247H/R250H). The products of all glutamic acid substitution mutations were immunoreactive but were not cytotoxic due to an inability to assemble into holotoxin. The products of all histidine substitution mutations had cytotoxic activities, enzymatic activities, and a lethal dose for mice similar to that of native toxin. R247H and R250H were susceptible to proteolytic cleavage while R247H/R250H was resistant to processing by exogenously added trypsin. Native toxin incubated with Vero cells was completely cleaved while only a fraction of R247H/R250H was cleaved. These results demonstrate that cleavage of SLT-IIv can be mediated by proteases with different specificities and suggest efficient cleavage is not required for toxicity.
authors.affiliation: Microcarb Inc., Gaithersburg, Maryland 20879.
authors.forename: J E
authors.initials: JE
authors.lastname: Samuel
authors.forename: V M
authors.initials: VM
authors.lastname: Gordon
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine; D001427:Bacterial Toxins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000633:toxicity*; D015153:Blotting, Western; D002470:Cell Survival / Q000187:drug effects; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004768:Enterotoxins / Q000096:biosynthesis* / Q000633:toxicity; D004926:Escherichia coli / Q000378:metabolism; D005971:Glutamates; D018698:Glutamic Acid; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000633:toxicity; D022641:Shiga Toxin 2; D014357:Trypsin / Q000378:metabolism*; D014709:Vero Cells
chemical_list: D001427:Bacterial Toxins; D004768:Enterotoxins; D005971:Glutamates; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D022641:Shiga Toxin 2; D018698:Glutamic Acid; D001120:Arginine; D014357:Trypsin