ANALYSIS OF ANTIGEN-BINDING AND IDIOTYPIC EXPRESSION BY ANTIBODIES WITH POLYGLYCINE-REPLACED COMPLEMENTARITY-DETERMINING REGIONS

We investigated the feasibility and usefulness, for structure-function studies, of removing the side chains of entire complementarity-determ ining regions (CDRs) of Abs by replacement with polyglycine, The CDRs of a murine Ab specific for p-azophenylarsonate (Ars) were replaced wi th polyglycine, one CDR at a time and in combinations, by oligonucleot ide-directed mutagenesis of the V region genes, Mutant Abs were expres sed in transfected hybridoma cells and analyzed for Ars binding and fo r idiotypic expression, The results suggest that, except for the longe st 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 do es 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 a n Ars binding Ab. Furthermore, this mutant Ab revealed previously unch aracterized contributions of L2 to idiotypic expression, Polyglycine r eplacement of H1 abolished Ars binding as expected, because H1 contain s an Ag-contacting residue. However, introduction of the contacting re sidue (Asn) on the polyglycine-replaced H1 background restored the abi lity of the Ab to bind Ars. The results suggest that polyglycine repla cement of CDRs can provide structural information that complements and extends the information obtained by other methods.