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首页> 外文期刊>Infection and immunity >Molecular genetic analysis of ganglioside GD1b-binding activity of Escherichia coli type IIa heat-labile enterotoxin by use of random and site-directed mutagenesis.
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Molecular genetic analysis of ganglioside GD1b-binding activity of Escherichia coli type IIa heat-labile enterotoxin by use of random and site-directed mutagenesis.

机译：利用随机和定点诱变技术对IIa型热不稳定肠毒素的神经节苷脂GD1b结合活性进行分子遗传学分析。

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摘要

Mutagenesis of the B-subunit gene of Escherichia coli heat-labile enterotoxin LT-IIa was performed in vitro with sodium bisulfite. Mutants were screened initially by radial passive immune hemolysis assays for loss of binding to erythrocytes. Mutant B polypeptides were characterized for immunoreactivity; for binding to gangliosides GD1b, GD1a, and GM1; for formation of holotoxin; and for biological activity. Mutant alleles that determined altered binding specificities were sequenced. Three such mutant alleles encoded Thr-to-Ile substitutions at residues 13, 14, and 34 in the mature B polypeptide of LT-IIa. Each mutant protein failed to bind to ganglioside GD1b, although the Ile-14 mutant retained the ability to bind to ganglioside GM1. Site-specific mutagenesis was used to construct mutants with various amino acid substitutions at residue 13, 14, or 34. Only those mutant proteins with Ser substituted for Thr at position 13, 14, or 34 retained the ability to bind to ganglioside GD1b, thereby suggesting a role for the hydroxyl group of Thr or Ser in ganglioside GD1b binding.
机译：用亚硫酸氢钠体外诱变大肠杆菌热不稳定肠毒素LT-IIa的B亚基基因。最初通过放射状被动免疫溶血测定法筛选突变体，以检测与红细胞的结合丧失。突变的B多肽具有免疫反应性。用于结合神经节苷脂GD1b，GD1a和GM1;用于形成全毒素;并具有生物活性。对确定改变的结合特异性的突变等位基因进行测序。三个这样的突变等位基因在LT-IIa的成熟B多肽的残基13、14和34处编码Thr-Ile取代。尽管Ile-14突变体保留了与神经节苷脂GM1结合的能力，但每个突变蛋白均未能与神经节苷脂GD1b结合。使用位点特异性诱变来构建在残基13、14或34处具有各种氨基酸取代的突变体。只有那些在13、14或34位用Ser取代Thr的突变蛋白保留了与神经节苷脂GD1b结合的能力，从而提示Thr或Ser的羟基在神经节苷脂GD1b结合中的作用。

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