A single engineered amino acid substitution changes antibody fine specificity.
During the acquisition of humoral immunity, the process of somatic hypermutation introduces nucleotide substitutions into expressed antibody (Ab) V region genes. Studies employing in vitro mutagenesis have shown that recurrent mutations observed in vivo often enhance the affinity of the target Ab for Ag. Here we show that a single amino acid replacement at position 35 in the H chain of an unmutated Ab with specificity for p-azophenylarsonate (Ars) confers specificity for the structurally related hapten p-azophenylsulfonate (Sulf) while abolishing specificity for Ars. The mutant Ab binds Sulf with an affinity characteristic of Ab produced by memory B cells. The same mutation in the somatically mutated anti-Ars Ab 36-71, for which the Fab crystal structure is known, resulted in a significant shift in fine specificity from Ars to Sulf. Examination of the crystal structure suggests that the specificity change is caused by a decrease in binding site size and/or new hydrogen bond geometry. Because the mutation at position 35 had been observed in somatically mutated Ab elicited by immunization with Ars followed by Sulf, the results confirm that somatic mutation in vivo can alter Ab specificity. The results also support the potential of Ab engineering to alter antigenic specificity.
Kussie
P H
PH
Department of Surgery, Massachusetts General Hospital, Boston.
Parhami-Seren
B
B
Wysocki
L J
LJ
Margolies
M N
MN
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001391:Azo Compounds / Q000276:immunology; D001557:Benzenesulfonates / Q000276:immunology; D002460:Cell Line; D006241:Haptens / Q000276:immunology; D006825:Hybridomas; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics; D051379:Mice; D008805:Mice, Inbred A; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D010132:p-Azobenzenearsonate / Q000276:immunology
D001391:Azo Compounds; D001557:Benzenesulfonates; D006241:Haptens; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; C061256:4,4'-azobis(benzenesulfonic acid); D010132:p-Azobenzenearsonate