A multinuclear NMR study of the affinity maturation of anti-NP mouse monoclonal antibodies: comparison of antibody combining sites of primary response antibody N1G9 and secondary response antibody 3B62.
On the basis of multinuclear NMR data, the structures of the antibody combining sites of anti-4-hydroxy-3-nitrophenylacetyl (NP) antibodies were compared for N1G9, which is one of the primary response antibodies with low affinity for NP, and 3B62, which is one of the secondary response antibodies with high affinity for NP. It has been concluded, on the basis of the results of antibody engineering, that in most secondary response antibodies a Trp-->Leu exchange at position 33 of the heavy chain is primarily responsible for the increased affinity for NP [Allen, D., Simon, T., Sablitzky, F., Rajewsky, K., & Cumano, A. (1988) EMBO J. 7, 1995-2001]. Although 3B62 exhibits one of the highest affinities for NP, it lacks the Trp-->Leu exchange at position 33 of the heavy chain. A variety of stable isotope-labeled Fab analogues of N1G9 and 3B62 have been prepared. Chain-specific resonance assignments were made by recombination of the heavy chains and light chains of the Fab fragments. Binding experiments of a spin-labeled hapten and NOESY experiments have demonstrated that, compared with the environment of the antibody combining site of N1G9, the combination of mutations (including one codon deletion) and the particular D-JH rearrangement in the heavy chain of 3B62 affords a more hydrophobic environment, which is formed by one Tyr residue originating from the light chain and two Tyr residues originating from the heavy chain.(ABSTRACT TRUNCATED AT 250 WORDS)
Nakayama
T
T
Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
Arata
Y
Y
Shimada
I
I
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology*; D000915:Antibody Affinity; D001666:Binding Sites, Antibody; D002460:Cell Line; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009154:Mutation; D009596:Nitrophenols / Q000276:immunology*; D010648:Phenylacetates; D017386:Sequence Homology, Amino Acid
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D009596:Nitrophenols; D010648:Phenylacetates; C026242:4-hydroxy-5-nitrophenyl acetic acid