Near R I, Mudgett-Hunter M, Novotny J, Bruccoleri R, Ng S C
Cellular and Molecular Research Laboratory, Massachusetts General Hospital, Boston 02114.
Mol Immunol. 1993 Mar;30(4):369-77. doi: 10.1016/0161-5890(93)90066-k.
In vitro mutagenesis 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 to in vitro mutagenesis at these sites. The mutated variable regions in M13 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 (> 10(9)/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.
利用体外诱变和免疫球蛋白基因转染来研究一种与强心苷地高辛结合的单克隆抗体2610的结合位点。生成了一个计算机模型，以便在互补决定区（CDR）中选择参与结合的位点。选择了CDR片段中具有高溶剂暴露且位于假定裂隙中的残基。在这些位点对克隆的重链和轻链可变区进行体外诱变。然后将M13中突变的可变区亚克隆到表达载体中并进行转染。测量所得表达抗体的亲和力和特异性结合特性。许多假定接触残基的突变体显示出结合特性有显著但非主要的改变。由于结合位点中的大多数残基是非极性和芳香性的，并且由于许多突变仅导致适度的结合变化，我们推测许多高亲和力结合（>10⁹/M）是由口袋中的色散力和疏水效应产生的许多弱相互作用的累积。对两个提议与地高辛内酯末端结合的轻链位置进行的初步诱变具有更大的结合效应。特异性研究表明，突变体更频繁地出现与地高辛内酯环结合的改变，而不是与地高辛其他部分结合的改变。这些数据最能说明一种模型，即内酯位于结合口袋的底部，其次是甾体核，然后是从口袋中伸出的糖部分。该结合信息可能有助于理解对大的疏水性半抗原的免疫反应。
