Contribution of the disulfide bond of the A subunit to the action of Escherichia coli heat-labile enterotoxin.
Escherichia coli heat-labile enterotoxin (LT) consists of an A subunit and five B subunits. These subunits oligomerize into an assembled holotoxin within the periplasm. Structural analysis of LT has revealed that the A subunit interacts with the B subunit through its carboxy terminus. This indicates that the carboxy-terminal portion of the protein is required for assembly of holotoxin in the periplasm. However, it is not known whether other regions of the A subunit contribute to the assembly. The A subunit constituting the holotoxin contains a disulfide bond between Cys-187 and Cys-199. It has been observed in many proteins that the intramolecular disulfide bond is deeply involved in the function and tertiary structure of the protein. We speculated that the disulfide bond of the A subunit contributes to the assembly in the periplasm, although the bond is not a structural element of the carboxy-terminal portion of the A subunit. We replaced these cysteine residues of the A subunit by oligonucleotide-directed site-specific mutagenesis and analyzed the LTs produced by cells containing the mutant LT genes. The amount of the mutant holotoxin produced was small compared with that of the wild-type strain, indicating that the disulfide bond of the A subunit contributes to the structure which functions as the site of nucleation in the assembly. A reconstitution experiment in vitro supported the notion. Subsequently, we found that the mutant A subunit constituting holotoxin is easily degraded by trypsin and that in cells incubated with mutant LTs, the lag until the intracellular cyclic AMP begins to accumulate is longer than in cells incubated with native LTs. These results might be useful for the analysis of the interaction of LT with target cells at the molecular level.
Okamoto
K
K
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro, Japan. okamoto@mandala.ne
Nomura
T
T
Fujii
Y
Y
Yamanaka
H
H
D000595:Amino Acid Sequence / Q000235:genetics; D019943:Amino Acid Substitution; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism; D003545:Cysteine / Q000235:genetics; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D004768:Enterotoxins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins; D007525:Isoelectric Focusing; D016297:Mutagenesis, Site-Directed; D019897:Periplasm / Q000378:metabolism; D010957:Plasmids; D011487:Protein Conformation; D014357:Trypsin / Q000494:pharmacology
D001427:Bacterial Toxins; D004220:Disulfides; D004768:Enterotoxins; D029968:Escherichia coli Proteins; C027538:heat-labile enterotoxin, E coli; D000242:Cyclic AMP; D014357:Trypsin; D003545:Cysteine