Computational 3-D modeling and site-directed mutation of an antibody that binds Neu2en5Ac, a transition state analogue of a sialic acid.
An antibody against a transition state analog (TSA) may share some common features with an enzyme that produces such a transition state. SIC172 antibody binds specifically to Neu2en5Ac, a TSA of Neu5Ac in the sialidase reaction, but has no catalytic activity. To understand how the antibody recognizes Neu2en5Ac and to find out if it is possible to convert it to a catalytic antibody, we made and sequenced the SIC172 ScFv, and constructed a 3-D model of it. The VH-CDR3 contains a unique sequence with Cys at H95. The 3-D model showed that Cys-H95 is exposed inside the antigen-binding cavity. After affinity docking, 4 types emerged. In type I, the carboxyl group of Neu2en5Ac is located near the Cys-H95 and neighboring positively charged residues. The change of Cys-H95 to Asp by site-directed mutation decreased the binding activity, while a change to Arg did not. These and other mutation experiments, and further modeling of mutant Fv, support the 3-D model and docking type I. A comparison with sialidase indicates that SIC172 antibody appears to have some groups of residues that are conserved at the active site of the enzyme. The possibility of Neu2en5Ac-binding antibody being converted to a catalytic antibody is discussed.
Kamei
H
H
Laboratory of Life Science & Biomolecular Engineering, Japan Tobacco Inc., Yokohama, Japan. hiroya.kamei@ims.jti.co.jp
Shimazaki
K
K
Nishi
Y
Y
D019943:Amino Acid Substitution; D000906:Antibodies / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D017151:Antibodies, Catalytic; D001483:Base Sequence; D000939:Epitopes / Q000737:chemistry; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019158:N-Acetylneuraminic Acid / Q000031:analogs & derivatives / Q000737:chemistry / Q000276:immunology*; D009439:Neuraminidase; D017422:Sequence Analysis, DNA
D000906:Antibodies; D017151:Antibodies, Catalytic; D000939:Epitopes; D007135:Immunoglobulin Variable Region; C036137:2-deoxy-2,3-dehydro-N-acetylneuraminic acid; D009439:Neuraminidase; D019158:N-Acetylneuraminic Acid