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
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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DOI：
10.1128/iai.70.3.1260-1271.2002
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发表时间：
2002-03-01
影响因子：
3.1
通讯作者：
Holmes, RK
中科院分区：
文献类型：
--
作者：
Jobling, MG;Holmes, RK
采用亚硫酸盐和寡核苷酸定向诱变方法对霍乱毒素B亚基（CTB）基因进行诱变。通过放射状被动免疫溶血试验（RPIHA）筛选与绵羊红细胞（SRBC）结合缺失的变异。变异CTBs的特点是形成免疫反应性五聚体，体外结合神经节苷脂GM(1)的能力，以及与一组单克隆抗ctb抗体的反应性。在8个位置（即位置22、29、36、45、64、86、93和100）进行替换，极大地降低了免疫反应性CTB的产量。在残基12、33、36、51、52 + 54、91和95上获得了形成免疫反应性五聚体的rpiha阴性取代变异体。酪氨酸-12被鉴定为对GM(1)结合具有重要意义的新残基，因为在所有分离的RPIHA表型改变的新变体中，只有在12号位置用天冬氨酸取代酪氨酸的CTB在体外不能与神经节苷脂GM(1)结合。相比之下，基于CTB的晶体结构和（；M）的寡糖，参与GM(1)结合的其他几个残基（Glu-51、Lys-91和Ala-95）被单次取代的CTB变体，在体外结合GM(1)的能力没有明显改变，尽管它们显示出改变的RPIHA表型，并且不与SRBC结合。将ctxB与相关的大肠杆菌热不稳定肠毒素eltB基因在密码子56处融合而成的杂交B基因产生的CTB变异体分别在单体的氨基或羧基半部分有7个或12个热不稳定肠毒素B残基取代，每个变体也结合GM(1)和野生型CTB。这些103个残基中有47个被替换的变体CTBs被用于绘制9种抗ctb单克隆抗体（mab）的表位。每个单抗与CTB变体具有独特的反应性模式。尽管不同单克隆抗体识别的表位不存在两个相同的表位，但大多数改变CTB免疫反应性的单氨基酸取代影响的不止一个表位。这些抗CTB单克隆抗体表位的三级结构是高度构象的，可能涉及CTB单体内部和单体之间的结构元件。缬氨酸取代第10位和第46位的丙氨酸对CTB的免疫反应性有显著影响，分别影响了8个或6个单克隆抗体识别的表位。
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 GM(1) 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 GM(1) 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 GM(1) in vitro. In contrast, CTB variants with single substitutions for several other residues (Glu-51, Lys-91, and Ala-95) that participate in GM(1) binding, based on the crystal structure of CTB and the oligosaccharide of (;M,, were not appreciably altered in their ability to bind GM(1) 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 GM(1) 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.