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.
Department of Microbiology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799.
M P
MP
Jackson
E A
EA
Wadolkowski
D L
DL
Weinstein
R K
RK
Holmes
A D
AD
O'Brien
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