Identification and characterization of an anti-isoaspartic acid monoclonal antibody.
The deamidation and rearrangement of protein-bound asparagine residues occurs when peptides and proteins are exposed to acidic or alkaline aqueous media. Asn99 of bovine growth hormone (bGH) is readily modified via these mechanisms. We have generated a monoclonal antibody (MAb) that interacts with a bGH fragment that contains an isoaspartyl residue. To obtain this antibody, CAF1/J mice were immunized with [isoaspartyl99]-bGH(96-112) conjugated to BSA. Using a competitive ELISA assay, the interaction of this MAb to [isoaspartyl99]-bGH(96-112) has been observed to have an apparent Km of 150 nM. The corresponding native peptide and other bGH fragments do not bind to this antibody with high affinity. For example, the binding affinities of [Asp99]-bGH(96-112) and [Glu99]-bGH(96-112) to this antibody are 54- and 78-fold lower than the corresponding isoaspartyl peptide. The antibody also binds to bGH that is enriched in isoaspartic acid at position 99, but not to the unmodified protein. The binding epitope of the peptide has been further characterized by comparing the binding of bGH(96-112) analogues to the MAb. Alanine substitution at residues 99, 100, 101, and 103 reduce binding affinity to the antibody by more than 10(3)-fold. Replacement of valine with alanine at position 102 has much less impact on antibody affinity. Further experiments suggest that the relative insensitivity to this substitution is due to the structural similarity of these sidechains. Other isoaspartic acid-containing peptides not derived from the bGH sequence do not bind to the antibody. We conclude that the epitope binding site of this MAb is highly specific for 99-103 of [isoaspartyl99]-bGH (96-112).(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000915:Antibody Affinity; D000918:Antibody Specificity; D000941:Antigens / Q000276:immunology; D001224:Aspartic Acid / Q000276:immunology*; D001666:Binding Sites, Antibody; D000939:Epitopes / Q000378:metabolism; D013006:Growth Hormone / Q000276:immunology*; D006863:Hydrogen-Ion Concentration; D007114:Immunization; D051379:Mice; D010446:Peptide Fragments / Q000276:immunology
D000911:Antibodies, Monoclonal; D000941:Antigens; D000939:Epitopes; D010446:Peptide Fragments; D001224:Aspartic Acid; D013006:Growth Hormone