Structural characterization of H chain-associated idiotopes of anti-p-azophenylarsonate monoclonal antibodies.
The majority of antibodies directed against p-azophenylarsonate (Ars) protein conjugates elicited during secondary immune responses of A/J mice bear a heritable cross-reactive Id (CRIa or IdCR) which corresponds to the utilization of a unique combination of variable region gene segments that can differ by somatic mutations. One such monoclonal anti-Ars antibody, 44-10, bears IdCR as defined by rabbit antisera but does not react with two anti-idiotypic mAb, 5Ci and AD8, which react with all primary (unmutated) IdCR+ antibodies and some secondary response IdCR+ antibodies. We therefore determined the complete sequence of antibody 44-10, which differs from the germline encoded (unmutated) IdCR+ antibody 36-65 at four positions in the H chain V region (VH): position 55 in the second complementarity determining region, 100 and 107 (D-gene junctions) and 110 (in JH2). The 44-10 L chain is unmutated. Sequence analyses of five other secondary immune response anti-Ars IdCR+ antibodies chosen on the basis of sharing one or more of the amino acid substitutions found in 44-10, were correlated with idiotypic expression of this set of antibodies. The results suggest that the mutation at VH position 55 (Asn----Lys) is responsible for loss of the 5Ci idiotope. To substantiate this hypothesis, oligonucleotide-directed mutagenesis of the germline encoded (unmutated) IdCR+ antibody was used to produce two mutants, one with VH Lys 55 and the other containing residues at positions 100, 107 and 110 identical to those found in 44-10. Id binding studies on these mutants confirm that 5Ci idiotope loss is due to conformational changes resulting from a mutation at VH position 55. This mutation also results in loss of the AD8 idiotope in the structural context of antibody 44-10.
Parhami-Seren
B
B
Department of Medicine, Massachusetts General Hospital, Boston 02114.
Sharon
J
J
Margolies
M N
MN
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology*; D000915:Antibody Affinity; D000916:Antibody Diversity; D001391:Azo Compounds / Q000276:immunology*; D001483:Base Sequence; D004252:DNA Mutational Analysis; D005803:Genes, Immunoglobulin; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007130:Immunoglobulin Idiotypes / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics; D007156:Immunologic Memory; D051379:Mice; D008969:Molecular Sequence Data; D010132:p-Azobenzenearsonate / Q000276:immunology*
D000911:Antibodies, Monoclonal; D001391:Azo Compounds; D007143:Immunoglobulin Heavy Chains; D007130:Immunoglobulin Idiotypes; D007135:Immunoglobulin Variable Region; D010132:p-Azobenzenearsonate