A single amino acid mutation in CDR3 of the 3-14-9 L chain abolished expression of the IDA 10-defined idiotope and antigen binding.
The ABPC 48 myeloma protein and the 3-14-9 mAb derive their V region genes from the same VH and V kappa gene families. They also share a cross-reactive idiotope defined by the anti-Id mAb IDA 10. Whereas ABPC 48 is specific for bacterial levan, 3-14-9 showed a significant Ag-binding activity to aminophenyl-beta-N-acetylglucosaminide (AZO). In order to define the molecular basis of idiotope expression and Ag-binding activity, we have cloned the genes encoding the 3-14-9 H and L chain V region genes, generated antibodies that carry mutations within the L chain genes, by site-directed mutagenesis, and investigated the effects of those mutations with respect to IDA 10 idiotope expression and binding to AZO. Our findings show that, whereas expression of the IDA 10-defined idiotope requires association of both the H and L chains, a single change (glycine to phenylalanine) at position 91 in the third complementarity-determining region of the L chain abolished both idiotope expression and Ag-binding activity. In addition, a L chain change of alanine to threonine at position 25 allowed idiotope expression to some extent but significantly reduced binding activity to AZO. These data suggest that a single amino acid change can play a crucial role in the functional activity and structural integrity of antibodies.
Hall
B L
BL
Department of Microbiology, Mount Sinai School of Medicine, City University of New York, NY 10029.
Zaghouani
H
H
Daian
C
C
Bona
C A
CA
D000818:Animals; D000941:Antigens / Q000378:metabolism*; D001483:Base Sequence; D000939:Epitopes; D007130:Immunoglobulin Idiotypes / Q000032:analysis*; D007147:Immunoglobulin Light Chains / Q000737:chemistry / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009154:Mutation; D009194:Myeloma Proteins / Q000235:genetics; D013329:Structure-Activity Relationship
D000941:Antigens; D000939:Epitopes; D007130:Immunoglobulin Idiotypes; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D009194:Myeloma Proteins