Characterization of an anti-digoxin antibody binding site by site-directed in vitro mutagenesis.
Characterization of an anti-digoxin antibody binding site by site-directed in vitro mutagenesis.
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通过定点体外诱变表征抗地高辛抗体结合位点。
DOI：
10.1016/0161-5890(93)90066-k
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发表时间：
1993
影响因子：
3.6
通讯作者：
Ng,SC
中科院分区：
文献类型：
--
作者：
Near,RI;Mudgett-Hunter,M;Novotny,J;Bruccoleri,R;Ng,SC
采用体外诱变和免疫球蛋白基因转染法研究了地高辛单抗2610与地高辛的结合部位。为了选择互补决定区(CDR)中将参与结合的位点，生成了计算机模型。CDR片段中的残基被选择为具有较高的溶剂暴露并位于假定的裂隙中。对克隆的重链可变区和轻链可变区进行体外诱变。然后将突变的M13可变区亚克隆到表达载体中，并导入表达载体。测定了所表达抗体的亲和力和特异性结合特性。许多假定的接触残基的突变体显示出结合性质的显著变化，但不是主要的变化。由于结合位点上的大多数残基是非极性和芳香性的，并且许多突变只导致轻微的结合变化，我们理论上认为大部分高亲和力结合(>；109/M)是许多弱相互作用的累积，产生于口袋中的分散力和疏水效应。初步诱变的两个L链位建议与地高辛的内酯末端结合，具有较大的结合作用。特异性研究表明，突变体更频繁地与地高辛的内酯环结合改变，这改变了与其他地高辛部分的结合。这些数据最让人联想到这样一种模型：内酯位于捆绑袋的底部，紧随其后的是类固醇核，然后是从口袋里挤出的糖份。结合信息可能有助于理解对大的疏水半抗原的免疫反应。
In vitromutagenesis and immunoglobulin gene transfection were used to investigate the binding site of a monoclonal antibody, 2610, that binds to digoxin, a cardiac glycoside. A computer model was generated in order to select sites in the complementarity determining regions (CDR) that would participate in binding. Residues in the CDR segments were chosen that possess high solvent exposure and were located in a putative cleft. The cloned heavy and light chain variable regions were subjected toin vitromutagenesis at these sites. The mutated variable regions in M 13 were then subcloned into expression vectors and transfected. The affinities and specificity binding properties of the resultant expressed antibodies were measured. Many of the mutants of the putative contact residues showed significant but not major alterations of binding properties. Since most of the residues in the binding site are non-polar and aromatic and since many of the mutations resulted in only modest binding changes, we theorize that much of the high affinity binding (> 109/M) is the cumulation of many weak interactions, arising from dispersion forces and hydrophobic effects in the pocket. Preliminary mutagenesis of two L chain positions proposed to bind to the lactone end of digoxin have larger binding effects. Specificity studies show that the mutants more frequently possess altered binding to the lactone ring of digoxin that altered binding to other digoxin moieties. The data are most suggestive of a model in which lactone is at the bottom of a binding pocket, followed by the steroid nucleus and then by the sugar moiety extruding out of the pocket. The binding information may be useful in understanding the immune response to large, hydrophobic haptens.