Contribution of heavy chain junctional amino acid diversity to antibody affinity among p-azophenylarsonate-specific antibodies.
We showed previously that heavy chain gene junctional amino acid differences among unmutated p-azophenylarsonate (Ars) Abs that share a unique gene segment combination encoding these V regions, termed "canonical," alter affinity. To determine the contribution of junctional amino acid differences to binding, we introduced, by site-directed mutagenesis, various amino acids at position 100 and/or 107 (sequential numbering) into the unmutated Ab 36-65. Among 22 mutant Abs, 15 preserved or showed increased Ars binding (1-to 12.9-fold increase) relative to Ab 36-65, while 7 Abs exhibited lower affinity (< or = 0.5-fold). As much as a 150-fold difference in Ars binding was observed between 2 Abs with different sets of junctions (Asn100/Tyr107 and Val100/Lys107). Thus, amino acid replacements at D gene junctions can produce changes in affinity greater than those for any V region somatic mutation observed thus far in vivo among anti-Ars Abs and, potentially, can result in preferential selection of Abs containing certain junctions during affinity maturation. We combined five different junctional residue pairs with mutations at H chain positions 58 and 59 that are known to be recurrent in vivo and are associated with increased Ars affinity. The mutant Abs all showed increased affinity, indicating that despite variation in D gene junctions of Ars-binding canonical Abs, the combined mutations are additive for enhancement of Ars affinity. These additive effects reflect the "adaptability" of the canonical gene segment combination in sustaining somatic mutations leading to affinity maturation.
Parhami-Seren
B
B
Department of Surgery, Massachusetts General Hospital, Boston 02114, USA.
Margolies
M N
MN
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics / Q000276:immunology*; D000818:Animals; D000915:Antibody Affinity / Q000235:genetics; D000916:Antibody Diversity / Q000235:genetics; D000918:Antibody Specificity / Q000235:genetics; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry* / Q000235:genetics; D007133:Immunoglobulin Joining Region / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics; D051379:Mice; D008805:Mice, Inbred A; D016297:Mutagenesis, Site-Directed; D010132:p-Azobenzenearsonate / Q000235:genetics / Q000276:immunology*
D000596:Amino Acids; D007143:Immunoglobulin Heavy Chains; D007133:Immunoglobulin Joining Region; D007135:Immunoglobulin Variable Region; D010132:p-Azobenzenearsonate