MOLECULAR GENETIC-ANALYSIS OF GANGLIOSIDE GD1B-BINDING ACTIVITY OF ESCHERICHIA-COLI TYPE-IIA HEAT-LABILE ENTEROTOXIN BY USE OF RANDOM AND SITE-DIRECTED MUTAGENESIS
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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DOI：
10.1128/iai.60.1.63-70.1992
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发表时间：
1992-01-01
影响因子：
3.1
通讯作者：
HOLMES, RK
中科院分区：
文献类型：
--
作者：
CONNELL, TD;HOLMES, RK
用亚硫酸氢钠对大肠杆菌不耐热肠毒素LT-Ⅱ a B亚单位基因进行了体外诱变。通过放射状被动免疫溶血测定法初步筛选突变体与红细胞结合的丧失。突变体B多肽的特征在于免疫反应性;与神经节苷脂GD 1 B、GD 1 a和GM 1的结合;全毒素的形成;以及生物活性。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编码LT-Ⅱ a成熟B多肽中第13、14和34位残基的Thr-至-Ile取代。每种突变蛋白都不能与神经节苷脂GD 1b结合，尽管Ile-14突变体保留了与神经节苷脂GM 1结合的能力。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位的丝氨酸取代了苏氨酸的突变蛋白质保留了与神经节苷脂GD 1b结合的能力，从而表明了苏氨酸或丝氨酸的羟基在神经节苷脂GD 1b结合中的作用。
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.