Functional analysis of the Shiga toxin and Shiga-like toxin type II variant binding subunits by using site-directed mutagenesis.
The B subunit of Shiga toxin and the Shiga-like toxins (SLTs) mediates receptor binding, cytotoxic specificity, and extracellular localization of the holotoxin. While the functional receptor for Shiga toxin, SLT type I (SLT-I), and SLT-II is the glycolipid designated Gb3, SLT-II variant (SLT-IIv) may use a different glycolipid receptor. To identify the domains responsible for receptor binding, localization in Escherichia coli, and recognition by neutralizing monoclonal antibodies, oligonucleotide-directed site-specific mutagenesis was used to alter amino acid residues in the B subunits of Shiga toxin and SLT-IIv. Mutagenesis of a well-conserved hydrophilic region near the amino terminus of the Shiga toxin B subunit rendered the molecule nontoxic but did not affect immunoreactivity or holotoxin assembly. In addition, elimination of one cysteine residue, as well as truncation of the B polypeptide by 5 amino acids, caused a total loss of activity. Changing a glutamate to a glutamine at the carboxyl terminus of the Shiga toxin B subunit resulted in the loss of receptor binding and immunoreactivity. However, the corresponding mutation in the SLT-IIv B subunit (glutamine to glutamate) did not reduce the levels of cytotoxicity but did affect extracellular localization of the holotoxin in E. coli.
Jackson
M P
MP
Department of Microbiology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799.
Wadolkowski
E A
EA
Weinstein
D L
DL
Holmes
R K
RK
O'Brien
A D
AD
D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D014644:Genetic Variation; D006367:HeLa Cells / Q000166:cytology / Q000187:drug effects; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D009876:Operon; D010957:Plasmids; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic / Q000378:metabolism*; D015183:Restriction Mapping; D022603:Shiga Toxins; D012762:Shigella dysenteriae / Q000235:genetics* / Q000276:immunology
D001427:Bacterial Toxins; D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic; D022603:Shiga Toxins; C045390:shigella toxin receptor