Yamanaka H, Okamoto K
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro, Japan.
Infect Immun. 1996 Jul;64(7):2700-8. doi: 10.1128/iai.64.7.2700-2708.1996.
Escherichia coli heat-stable enterotoxin Ip (STIp), which is a typical extracellular toxin consisting of 18 amino acid residues, is synthesized as a precursor consisting of pre (amino acid residues 1 to 19), pro (amino acid residues 20 to 54), and mature (amino acid residues 55 to 72) regions. Though the pre region functions as a conventional leader peptide that guides the following region to cross the inner membrane, the role of the pro region in the maturation pathway remains to be elucidated. We previously indicated that the sequence from residues 29 to 38 in the pro region increases the efficiency of STI translocation across the inner membrane (H. Yamanaka, Y. Fuke, S. Hitotsubashi, Y. Fujii, and K. Okamoto, Microbiol. Immunol. 37:195-205, 1993). We therefore examined the amino acid residues in the sequence that are responsible for this function. We substituted several amino acid residues in the sequence by means of oligonucleotide-directed site-specific mutagenesis. We then evaluated the effect of the substitution on the efficiency of STI translocation across the inner membrane by determining the enterotoxic activity of the culture supernatant, the amount of a fusion protein consisting of STI and nuclease A released into the periplasm, and the amount of the labeled ST released into the periplasm after pulse-labeling with [35S]cysteine. Substitution of the charged amino acid residues at positions 29 to 31 (K-E-K) with hydrophobic (I-V-L, F-W-F, or F-W-Q) or basic (K-K-K) residues significantly reduced these values in every assay. In contrast, the substitution of these amino acid residues with acidic amino acid residues (E-E-E) increased these values in all assays. This means that the negative charge near position 30 is important for STI to translocate efficiently across the inner membrane. A similar substitution of lysine residues at positions 37 and 38 showed that they are not involved in the translocation of STI across the inner membrane.
大肠杆菌热稳定肠毒素Ip（STIp）是一种典型的细胞外毒素，由18个氨基酸残基组成，它以前体形式合成，前体由前导区（氨基酸残基1至19）、原区（氨基酸残基20至54）和成熟区（氨基酸残基55至72）组成。虽然前导区起着引导后续区域穿过内膜的传统前导肽的作用，但原区在成熟途径中的作用仍有待阐明。我们之前指出，原区中第29至38位的氨基酸序列提高了STI穿过内膜的转运效率（H. Yamanaka、Y. Fuke、S. Hitotsubashi、Y. Fujii和K. Okamoto，《微生物学与免疫学》37:195 - 205，1993）。因此，我们研究了该序列中负责此功能的氨基酸残基。我们通过寡核苷酸定向位点特异性诱变替换了该序列中的几个氨基酸残基。然后，我们通过测定培养上清液的肠毒素活性、释放到周质中的由STI和核酸酶A组成的融合蛋白的量以及用[35S]半胱氨酸脉冲标记后释放到周质中的标记ST的量，评估了这种替换对STI穿过内膜转运效率的影响。将第29至31位带电荷的氨基酸残基（K - E - K）替换为疏水（I - V - L、F - W - F或F - W - Q）或碱性（K - K - K）残基在每次测定中均显著降低了这些值。相反，将这些氨基酸残基替换为酸性氨基酸残基（E - E - E）在所有测定中均提高了这些值。这意味着第30位附近的负电荷对于STI有效穿过内膜很重要。对第37和38位赖氨酸残基进行类似的替换表明，它们不参与STI穿过内膜的转运。
