Effect of substitution for arginine residues near position 146 of the A subunit of Escherichia coli heat-labile enterotoxin on the holotoxin assembly.
Escherichia coli heat-labile enterotoxin (LT) is a holotoxin which consists of one A and five B subunits. Although B subunit monomers released into periplasm can associate into pentameric structures in the absence of the A subunit, the A subunit accelerates the assembly. To express the function, A subunit constructs the proper spatial structure. However, the regions involved in the construction are unknown. To identify the regions, we substituted arginine residues near position 146 of the A subunit with glycine by oligonucleotide-directed site-specific mutagenesis and obtained the mutants expressing LT(R141G), LT(R143G), LT(R146G), LT(R143G, R146G), LT(R141G, R143G, R146G) and LT(R143G, R146G, R148G). We purified these mutant LTs by using an immobilized D-galactose column and analyzed the purified mutant LTs by SDS-PAGE to examine the amount of A subunit associated with B-subunit oligomer. The substitution of an arginine residue at any position did not induce a significant alteration in the amount of A subunit associated with B-subunit oligomer. However, the substitution of more than two arginine residues induced a significant decrease in the amount of A subunits associated with the B-subunit oligomer. Subsequently, we measured the level of the intracellular B-subunit oligomer of these mutant strains. The measurement revealed that the amount of B-subunit oligomer in cells decreased as the number of substituted arginine residues increased. These results show that all arginine residues near position 146 are important for the construction of the functional A subunit, and thus for holotoxin formation, although each individual arginine residue is not an absolute requirement.
Okamoto
K
K
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Japan.
Takatori
R
R
Okamoto
K
K
D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial; D001120:Arginine; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004768:Enterotoxins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005998:Glycine; D005779:Immunodiffusion; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D009154:Mutation; D011817:Rabbits; D013329:Structure-Activity Relationship
D000907:Antibodies, Bacterial; D001427:Bacterial Toxins; D017931:DNA Primers; D004768:Enterotoxins; D000939:Epitopes; D029968:Escherichia coli Proteins; D001120:Arginine; C027538:heat-labile enterotoxin, E coli; D005998:Glycine