STRUCTURAL DEFINITION BY ANTIBODY ENGINEERING OF AN IDIOTYPIC DETERMINANT
STRUCTURAL DEFINITION BY ANTIBODY ENGINEERING OF AN IDIOTYPIC DETERMINANT
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DOI：
10.1093/protein/3.6.531
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发表时间：
1990-05-01
期刊：
影响因子：
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通讯作者：
ZANETTI, M
中科院分区：
文献类型：
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作者：
SOLLAZZO, M;CASTIGLIA, D;ZANETTI, M
利用计算机辅助技术预测分子结构，我们构建了小鼠抗甲状腺球蛋白抗体可变结构域的原子模型，该抗体的免疫显性独特型决定因子（Id62）通过定点突变和免疫化学分析绘制。我们之前在实验条件下表明，这种独特型激活抗独特型B细胞和T细胞，并调节小鼠对甲状腺球蛋白的反应。由于独特型相互作用被认为对免疫调节具有重要的生理意义，我们研究了这种独特型，以了解功能和结构之间的关系。为了确定重链和轻链可变结构域对独特型结构的贡献，我们构建了嵌合表达载体，并将其引入（非分泌）P3X63Ag8.653骨髓瘤细胞系。通过定点诱变获得重链可变结构域的突变体，并将其转染到小鼠（.lambda.1）轻链产生者J558L细胞系中。从转染细胞的培养上清液中纯化表达的蛋白并对其进行表征。我们提供的证据表明，重链可变结构域的第三个高变环（D区）是该自身抗体独特型决定因素的结构相关，并且独立于相关轻链的性质。第一和第二互补决定区残基的替换不影响独特型的表达。本文讨论的结果与我们在分子水平上对独特型与B细胞和t细胞区室相互作用的理解有关。
Using computer-aided techniques for predicting molecular structure, we constructed an atomic model of the variable domain of a murine anti-thyroglobulin antibody whose immunodominant idiotypic determinant (Id62) was mapped by site-directed mutagenesis and immunochemical analysis. We previously showed that under experimental conditions this idiotype activates anti-idiotypic B cells and T cells, and modulates the response to thyroglobulin in mice. Because idiotype interactions are considered of physiological importance for immune regulation, we studied this idiotype as a model to understand the relationship between function and structure. To determine the contribution of heavy- and light-chain variable domains to the idiotype structure, we constructed chimeric expression vectors and introduced them into the (non-secreting) P3X63Ag8.653 myeloma cell line. Mutants of the heavy-chain variable domain were obtained by site-directed mutagenesis and transfected into the murine (.lambda.1) light-chain producer J558L cell line. The expressed proteins were purified from culture supernatants of transfected cells and characterized. We provide evidence that the third hypervariable loop (D region) of the heavy-chain variable domain is the structural correlate of the idiotypic determinant of this autoantibody and is independent from the nature of the associated light chain. Substitution of residues of the first and second complementarity-determining regions do not affect idiotype expression. The results described here are discussed in relation to our understanding, at a molecular level, of the interaction of idiotopes with B- and T-cell compartments.