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-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.
Sompuram
S R
SR
Department of Pathology and Laboratory Medicine, Boston University School of Medicine, MA 02118, USA.
Den
W
W
Sharon
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001666:Binding Sites, Antibody / Q000235:genetics; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics; D007130:Immunoglobulin Idiotypes / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000737:chemistry / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry* / Q000235:genetics
D007143:Immunoglobulin Heavy Chains; D007130:Immunoglobulin Idiotypes; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D010455:Peptides; C011080:polyglycine