Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
Monoclon Antib Immunodiagn Immunother. 2020 Aug;39(4):117-122. doi: 10.1089/mab.2020.0012. Epub 2020 Jun 2.
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) into diacylglycerol (DG) and inositol 1,4,5-trisphosphate (IP). 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 DGKη is classified as a type II DGK. Reports have shown that DGKη 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 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 yet been established. In this study, we established a specific anti-human DGKη (hDGKη) mAb, DhMab-4 (mouse IgG, 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 hDGKη 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 hDGKη.
二酰基甘油激酶（DGK）在哺乳动物的细胞内信号通路中发挥着关键作用。激活的 G 蛋白偶联受体通过异三聚体 G 蛋白激活磷脂酶 C（PLC），随后 PLC 将磷脂酰肌醇 4,5-二磷酸（PIP）水解为二酰基甘油（DG）和肌醇 1,4,5-三磷酸（IP）。DGK 催化 DG 磷酸化生成磷酸脂酸。DG 和磷酸脂酸作为第二信使，其细胞内浓度受 DGK 调节；因此，DGK 在调节许多生物学过程中起着重要作用。有十种 DGK 同工酶，其中 DGKη 被归类为 II 型 DGK。有报道表明，DGKη 与几种疾病有关；例如，它在海马体和小脑高表达，是双相情感障碍的关键因素。尽管需要 DGKη 特异性单克隆抗体（mAb）来揭示 DGKη 表达与疾病之间的关联，但尚未建立用于免疫组织化学的抗-DGKη mAb。在这项研究中，我们建立了一种特异性抗人 DGKη（hDGKη）mAb，DhMab-4（鼠 IgG，kappa）。在免疫组织化学分析中，DhMab-4 强烈染色人小脑的浦肯野细胞。为了对 DhMab-4 的表位进行作图，我们产生了 hDGKη 的缺失或点突变体，并进行了 Western blot 以确定 DhMab-4 的结合表位。DhMab-4 与 dN745 突变体反应，但不与 dN750 突变体反应，表明 DhMab-4 表位的 N 端位于氨基酸 745 和 750 之间。使用点突变体的更详细分析表明，五个突变体，即 D747A、P748A、F749A、G750A 和 T752A，未被 DhMab-4 检测到。这些结果表明，Asp747、Pro748、Phe749、Gly750 和 Thr752 对 DhMab-4 与 hDGKη 的结合很重要。
