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首页> 外文期刊>Infection and immunity >Mutational Analysis of Ganglioside GM1-Binding Ability, Pentamer Formation, and Epitopes of Cholera Toxin B (CTB) Subunits and CTB/Heat-Labile Enterotoxin B Subunit Chimeras
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Mutational Analysis of Ganglioside GM1-Binding Ability, Pentamer Formation, and Epitopes of Cholera Toxin B (CTB) Subunits and CTB/Heat-Labile Enterotoxin B Subunit Chimeras

机译：神经节苷脂GM1结合能力，五聚体形成和霍乱毒素B（CTB）亚基和CTB /不耐热肠毒素B亚基嵌合体表位的突变分析

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Variants of cholera toxin B subunit (CTB) were made by bisulfite- and oligonucleotide-directed mutagenesis of the ctxB gene. Variants were screened by a radial passive immune hemolysis assay (RPIHA) for loss of binding to sheep erythrocytes (SRBC). Variant CTBs were characterized for the formation of immunoreactive pentamers, the ability to bind ganglioside GM1 in vitro, and reactivity with a panel of monoclonal anti-CTB antibodies. Substitutions at eight positions (i.e., positions 22, 29, 36, 45, 64, 86, 93, and 100) greatly reduced the yield of immunoreactive CTB. RPIHA-negative substitution variants that formed immunoreactive pentamers were obtained for residues 12, 33, 36, 51, 52 + 54, 91, and 95. Tyrosine-12 was identified as a novel residue important for GM1 binding since, among all of the novel variants isolated with altered RPIHA phenotypes, only CTB with aspartate substituted for tyrosine at position 12 failed to bind significantly to ganglioside GM1 in vitro. In contrast, CTB variants with single substitutions for several other residues (Glu-51, Lys-91, and Ala-95) that participate in GM1 binding, based on the crystal structure of CTB and the oligosaccharide of GM1, were not appreciably altered in their ability to bind GM1 in vitro, even though they showed altered RPIHA phenotypes and did not bind to SRBC. Hybrid B genes made by fusing ctxB and the related Escherichia coli heat-labile enterotoxin eltB genes at codon 56 produced CTB variants that had 7 or 12 heat-labile enterotoxin B residue substitutions in the amino or carboxyl halves of the monomer, respectively, each of which which also bound GM1 as well as wild-type CTB. This collection of variant CTBs in which 47 of the 103 residues were substituted was used to map the epitopes of nine anti-CTB monoclonal antibodies (MAbs). Each MAb had a unique pattern of reactivity with the panel of CTB variants. Although no two of the epitopes recognized by different MAbs were identical, most of the single amino acid substitutions that altered the immunoreactivity of CTB affected more that one epitope. The tertiary structures of the epitopes of these anti-CTB MAbs are highly conformational and may involve structural elements both within and between CTB monomers. Substitution of valine for alanine at positions 10 and 46 had dramatic effects on the immunoreactivity of CTB, affecting epitopes recognized by eight or six MAbs, respectively.
机译：通过 ctxB 基因的亚硫酸氢盐和寡核苷酸定向诱变制备霍乱毒素B亚基（CTB）。通过放射状被动免疫溶血测定法（RPIHA）筛选变异体，以测定与绵羊红细胞（SRBC）结合的丧失。变异CTBs具有免疫反应性五聚体的形成，体外结合神经节苷脂GM 1 的能力以及与一系列单克隆抗CTB抗体的反应性。八个位置（即22、29、36、45、64、86、93和100位）的取代大大降低了免疫反应性CTB的产量。对于残基12、33、36、51、52、54、91和95，获得了形成免疫反应性五聚体的RPIHA负替代变体。酪氨酸12被鉴定为对GM 1 重要的新残基因为在所有具有改变的RPIHA表型的新颖变体中，只有12位用天冬氨酸替代酪氨酸的CTB不能在体外与神经节苷脂GM 1 显着结合。相反，基于CTB和寡糖的晶体结构，具有几个其他残基（Glu-51，Lys-91和Ala-95）的单取代取代的CTB变体会参与GM 1 的结合GM 1 的体外结合GM 1 的能力没有明显改变，即使它们表现出改变的RPIHA表型并且不与SRBC结合。通过融合 ctxB 和相关的 Escherichia coli 不耐热肠毒素 eltB 基因制成的杂种B基因在56位密码子上产生的CTB变异体具有7或12热不稳定的肠毒素B残基分别在单体的氨基或羧基半部分取代，每个残基也与GM 1 和野生型CTB结合。用103个残基中的47个被取代的变异CTB集合来绘制九种抗CTB单克隆抗体（MAb）的表位。每个单克隆抗体与CTB变异体都有独特的反应模式。尽管没有被不同的单克隆抗体识别的两个表位是相同的，但是大多数改变CTB免疫反应性的单个氨基酸取代对一个表位的影响更大。这些抗CTB MAb的表位的三级结构是高度构象的，可能涉及CTB单体内部和之间的结构元素。缬氨酸取代10和46位的丙氨酸对CTB的免疫反应性有显着影响，分别影响被8个或6个MAb识别的表位。

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