Sompuram S R, Den W, Sharon J
Department of Pathology and Laboratory Medicine, Boston University School of Medicine, MA 02118, USA.
J Immunol. 1996 Feb 1;156(3):1071-81.
We investigated the feasibility and usefulness, for structure-function studies, of removing the side chains of entire complementarity-determining regions (CDRs) of Abs by replacement with polyglycine. The CDRs of a murine Ab specific for p-azophenylarsonate (Ars) were replaced with polyglycine, one CDR at a time and in combinations, by oligonucleotide-directed mutagenesis of the V region genes. Mutant Abs were expressed in transfected hybridoma cells and analyzed for Ars binding and for idiotypic expression. The results suggest that, except for the longest CDRs, polyglycine replacement does not alter the general structure of the Ab molecule. However, for analysis of functional contributions of a CDR, the polyglycine replacement method appears to be most useful for CDRs with extended structures whose replacement by polyglycine does not affect the structure of other parts of the variable regions. In the current studies, such CDRs were CDR1 of the heavy chain (H1) and CDR2 of the light chain (L2). The polyglycine replacement of L2, which does not contain an Ag-contacting residue, allowed the formation of an Ars binding Ab. Furthermore, this mutant Ab revealed previously uncharacterized contributions of L2 to idiotypic expression. Polyglycine replacement of H1 abolished Ars binding as expected, because H1 contains an Ag-contacting residue. However, introduction of the contacting residue (Asn) on the polyglycine-replaced H1 background restored the ability of the Ab to bind Ars. The results suggest that polyglycine replacement of CDRs can provide structural information that complements and extends the information obtained by other methods.
我们研究了通过用聚甘氨酸取代抗体（Ab）整个互补决定区（CDR）的侧链来进行结构 - 功能研究的可行性和实用性。通过对V区基因进行寡核苷酸定向诱变，将对对氨基苯偶氮胂酸（Ars）具有特异性的鼠源Ab的CDR一次一个或组合地用聚甘氨酸取代。突变体Ab在转染的杂交瘤细胞中表达，并分析其对Ars的结合和独特型表达。结果表明，除了最长的CDR外，聚甘氨酸取代不会改变Ab分子的总体结构。然而，对于分析CDR的功能贡献，聚甘氨酸取代方法似乎对具有延伸结构的CDR最有用，用聚甘氨酸取代这些CDR不会影响可变区其他部分的结构。在当前研究中，此类CDR为重链的CDR1（H1）和轻链的CDR2（L2）。不含抗原接触残基的L2的聚甘氨酸取代允许形成结合Ars的Ab。此外，这种突变体Ab揭示了L2对独特型表达以前未被表征的贡献。正如预期的那样，H1的聚甘氨酸取代消除了Ars结合，因为H1含有一个抗原接触残基。然而，在聚甘氨酸取代的H1背景上引入接触残基（Asn）恢复了Ab结合Ars的能力。结果表明，CDR的聚甘氨酸取代可以提供补充并扩展通过其他方法获得的信息的结构信息。
