Epitope Mapping of DhMab-1: An Antidiacylglycerol Kinase Monoclonal Antibody

Diacylglycerol kinase (DGK) η is classified as a type II DGK and catalyzes diacylglycerol phosphorylation to produce phosphatidic acid. DGKη has been reported to be highly expressed in the hippocampus and cerebellum. Although a DGKη-specific monoclonal antibody (mAb) is necessary to reveal the association between the expression of DGKη and diseases, an anti-DGKη mAb for immunohistochemistry has not been developed. Recently, we established a specific antihuman DGKη (hDGKη) mAb, DhMab-1 (mouse IgG2a, kappa). For epitope mapping of DhMab-1, here we produced deletion or point mutants of hDGKη and performed Western blotting to determine the binding epitope of DhMab-1. DhMab-1 reacted with the dN755 mutant, but not with the dN760 mutant, indicating that the N-terminus of the DhMab-1 epitope is mainly located between amino acids 755 and 760 of the protein. A more detailed analysis using point mutants demonstrated that seven mutants, that is, A751G, I755A, D756A, P757A, D758A, L759A, and D760A, were not detected by DhMab-1. These results indicate that Ala751, Ile755, Asp756, Pro757, Asp758, Leu759, and Asp760 are important for DhMab-1 binding to hDGKη.