Epitope Mapping of the Anti-Diacylglycerol Kinase Monoclonal Antibody DhMab-4 for Immunohistochemical Analysis

Abstract

Diacylglycerol kinase (DGK) plays a pivotal role in intracellular signaling pathways in mammals. Activated G protein-coupled receptor activates phospholipase C (PLC) through heterotrimeric G protein, following which PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP 2 ) into diacylglycerol (DG) and inositol 1,4,5trisphosphate (IP 3 ). DGK catalyzes DG phosphorylation to produce phosphatidic acid. DG and phosphatidic acid function as second messengers and their intracellular concentrations are regulated by DGK; therefore, DGK plays an important role in regulating many biological processes. There are ten DGK isozymes, of which DGKZ is classified as a type II DGK. Reports have shown that DGKZ is associated with several diseases; for example, it is highly expressed in the hippocampus and cerebellum and is a key element in bipolar disorder. Although a DGKZ-specific monoclonal antibody (mAb) is necessary to reveal the association between the expression of DGKZ and diseases, an anti-DGKZ mAb for immunohistochemistry has not yet been established. In this study, we established a specific anti-human DGKZ (hDGKZ) mAb, DhMab-4 (mouse IgG 2b , kappa). DhMab-4 strongly stained Purkinje cells of human cerebellum in immunohistochemistry analysis. For epitope mapping of DhMab-4, we produced deletion or point mutants of hDGKZ and performed western blotting to determine the binding epitope of DhMab-4. DhMab-4 reacted with dN745 mutant but not with dN750 mutant, indicating that the N-terminus of the DhMab-4 epitope is located between amino acids 745 and 750. More detailed analysis using point mutants demonstrated that five mutants, that is, D747A, P748A, F749A, G750A, and T752A, were not detected by DhMab-4. These results indicate that Asp747, Pro748, Phe749, Gly750, and Thr752 are important for DhMab-4 binding to hDGKZ.

Epitope mapping of an anti-diacylglycerol kinase delta monoclonal antibody DdMab-1

“…Previously, we established DaMab-2 as an anti-DGKα mAb [ 22 ], DgMab-6 as an anti-DGKγ mAb [ 23 ], and DzMab-1 as an anti-DGKζ mAb [ 24 ] for immunocytochemistry. We further developed DaMab-8 as an anti-DGKα mAb [ 25 ] and DhMab-1 [ 26 ]/DhMab-4 [ 27 ] as anti-DGKη mAbs for immunohistochemistry. We determined their respective binding epitopes [ [25] , [26] , [27] , [28] , [29] , [30] ].…”

“…We further developed DaMab-8 as an anti-DGKα mAb [ 25 ] and DhMab-1 [ 26 ]/DhMab-4 [ 27 ] as anti-DGKη mAbs for immunohistochemistry. We determined their respective binding epitopes [ [25] , [26] , [27] , [28] , [29] , [30] ]. Accordingly, DaMab-2 and DaMab-8 was found to bind to the Zn-finger and catalytic domains of DGKα, respectively [ 25 , 28 ].…”

“…DgMab-6 and DzMab-1 were shown to bind to the N-termini of DGKγ and DGKζ, respectively [ 29 , 30 ]. DhMab-1/DhMab-4 epitope was found to be located near the accessory domain of hDGKη [ 26 , 27 ]. These epitope analyses revealed that each sensitive and specific mAb for use in immunocytochemistry or immunohistochemistry against different DGK isotypes included different epitope regions.…”