Effect of alterations of basic amino acid residues of Escherichia coli heat-stable enterotoxin II on enterotoxicity.
Escherichia coli heat-stable enterotoxin II (STII) is composed of 48 amino acid residues. Among these, one histidine, two arginine, and six lysine residues are basic. Isoelectric focusing showed that the isoelectric point of STII is 9.7, indicating that the side chains of some of these basic amino acid residues project outside the molecule. To understand the role that these basic amino acid residues play in toxicity, STII was chemically modified with ethoxyformic anhydride, maleic anhydride, and phenylglyoxal, which alter the side chains of basic amino acid residues in proteins. Maleic anhydride, which modifies the epsilon amino group, caused a significant loss of enterotoxic activity, but the other two modifiers did not. This indicated that lysine residues play an important role in the expression of the enterotoxic activity of STII and that the contribution of the other basic amino acid residues to the toxicity is relatively low. To confirm this hypothesis, we substituted these nine basic amino acid residues by oligonucleotide-directed site-specific mutagenesis and examined the enterotoxicity of these purified mutant STIIs. The enterotoxic activity was reduced when the lysine residues at positions 18, 22, 23, and 46 were substituted. In particular, the substitution at positions 22 and 23 induced a remarkable reduction. These results demonstrate that the lysine residues at positions 22 and 23 are very important in the expression of the enterotoxic activity of STII.
Fujii
Y
Y
Institute of Pharmacognosy, Faculty of Pharmaceutical Sciences, Tosushima Bunri University, Japan.
Okamuro
Y
Y
Hitotsubashi
S
S
Saito
A
A
Akashi
N
N
Okamoto
K
K
D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry / Q000633:toxicity*; D001483:Base Sequence; D004768:Enterotoxins / Q000737:chemistry / Q000633:toxicity*; D004926:Escherichia coli / Q000472:pathogenicity*; D029968:Escherichia coli Proteins; D007422:Intestines / Q000187:drug effects*; D007526:Isoelectric Point; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D013329:Structure-Activity Relationship
D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; C034151:heat stable toxin (E coli)