DgMab-6: Antihuman DGKγ Monoclonal Antibody for Immunocytochemistry

Nakano, Tomoyuki; Ogasawara, Satoshi; Tanaka, Toshiaki; Hozumi, Yasukazu; Yamaki, Atsumi; Sakane, Fumio; Shirai, Yasuhito; Nakamura, Takuro; Yanaka, Miyuki; Yamada, Shinji; Kaneko, Mika K.; Kato, Yukinari; Goto, Kaoru

doi:10.1089/mab.2018.0026

Cited by 4 publications

(3 citation statements)

References 16 publications

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“…DGK plays an important role in signaling pathways by regulating the intracellular level of DG and phosphatidic acid, and it is considered a potential therapeutic target for some diseases. Recently, we established anti-DGKa, (19,20) anti-DGKg, (21) and anti-DGKz (22) mAbs and determined their respective binding epitopes. (20,(23)(24)(25) These mAbs are very useful for immunohistochemical analyses.…”

Section: Discussionmentioning

confidence: 99%

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

Asano

Sano

Takei

et al. 2020

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy

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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.

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Section: Discussionmentioning

confidence: 99%

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

Asano

Sano

Takei

et al. 2020

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy

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“…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.…”

Section: Discussionmentioning

confidence: 99%

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

Sano

Asano

Kaneko

et al. 2020

Biochemistry and Biophysics Reports

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Diacylglycerol kinase δ (DGKδ) is a type II DGK, which catalyzes diacylglycerol phosphorylation to produce phosphatidic acid. DGKδ is expressed in several types of tissues and organs including the stomach, testis, bone marrow, and lymph node. Here, we established an anti-human DGKδ (hDGKδ) mAb, DdMab-1 (mouse IgG 2a , kappa), which is useful for Western blot analysis. We also introduced deletion or point mutations to hDGKδ, and performed western blotting to determine the binding epitope of DdMab-1. DdMab-1 reacted with the dN670 mutant, but not with the dN680 mutant, indicating that the N-terminus of the DdMab-1 epitope is mainly located between amino acids 670 and 680 of the protein. Further analysis using point mutants demonstrated that R675A, R678A, K679A, and K682A mutants were not detected, and V680A was only weakly detected by DdMab-1, indicating that Arg675, Arg678, Lys679, Val680 and Lys682 are important for binding of DdMab-1 to hDGKδ.

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“…We have developed anti-DGKa (clone: DaMab-2) (12) and anti-DGKg (clone: DgMab-6) (13) mAbs, both of which are extremely useful for immunocytochemical analysis. We characterized the binding epitope of DaMab-2 using Western blots and revealed that the Cys246, Lys249, Pro252, and Cys253 residues of DGKa are important for binding of DaMab-2 to DGKa.…”

Section: Discussionmentioning

confidence: 99%

Epitope Mapping of Anti-Diacylglycerol Kinase ζ Monoclonal Antibody for the Detection of T Cells by Immunohistochemical Analyses

Kato

Itai

Yamada

et al. 2019

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy

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The diacylglycerol kinases (DGKs) are a family of proteins that catalyze the phosphorylation of the cell membrane lipid diacylglycerol (DG), a cellular component that is important in lipid biochemistry and signal transduction, into phosphatidic acid. DG-mediated signal transduction downstream of the T cell receptor has previously been reported to be terminated in most cases by one of 10 DGK isoforms, DGKz. In this study, we performed immunohistochemical analysis using a rabbit anti-DGKz monoclonal antibody (mAb) (clone EPR22040-80) against tissues from the tonsils of a patient with oropharyngeal squamous cell carcinoma. We demonstrated that many DGKz-expressing T cells are localized in the tonsils. We further characterized the binding epitope using an enzyme-linked immunosorbent assay and found that Pro790, Gln791, Gly792, and Leu795 residues of DGKz are important for facilitating anti-DGKz mAb binding to DGKz. This anti-DGKz mAb could be valuable in immunohistochemical analyses in determining the distribution of DGKz-expressing T cells in pathophysiological tissues.

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DgMab-6: Antihuman DGKγ Monoclonal Antibody for Immunocytochemistry

Cited by 4 publications

References 16 publications

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

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

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

Epitope Mapping of Anti-Diacylglycerol Kinase ζ Monoclonal Antibody for the Detection of T Cells by Immunohistochemical Analyses

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