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 fire B subunits, These subunits oligomerize into an assembled hol otoxin within the periplasm, Structural analysis of LT has revealed th at the A subunit interacts with the B subunit through its carboxy term inus, 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 as sembly, The A subunit constituting the holotoxin contains a disulfide bond bem een Cys-187 and Cys-199, It has been observed in many protein s that the intramolecular disulfide bond is deeply involved in the fun ction and tertiary structure of the protein, We speculated that the di sulfide bond of the A subunit contributes to the assembly in the perip lasm, although the bond is not a structural element of the carboxy-ter minal portion of the A subunit, We replaced these cysteine residues of the A subunit by oligonucleotide-directed site-specific mutagenesis a nd analyzed the LTs produced by cells containing the mutant LT genes, The amount of the mutant holotoxin produced was small compared with th at 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 suppo rted the notion, Subsequently, we found that the mutant A subunit cons tituting holotoxin is easily degraded by trypsin and that in cells inc ubated with mutant LTs, the lag until the intracellular cyclic AMP beg ins to accumulate is longer than in cells incubated with native LTs, T hese results might be useful for the analysis of the interaction of LT with target cells at the molecular level.