Abstract
Diacylglycerol kinase (DGK) converts diacylglycerol (DG) into phosphatidic acid (PA). DGKα, 1 of the 10 DGK isozymes, is involved in T cell function. In the present study, we describe a specific monoclonal antibody DaMab-8 (mouse IgG1, kappa) against DGKα, which is extremely useful for performing immunohistochemical analysis for T cells in oropharyngeal squamous cell carcinomas. Furthermore, we characterized the binding epitope of DaMab-8 using Western blotting and found that the sites Asn610, Leu611, Trp612, Gly613, Asp614, His619, Tyr623, and Gly624 of DGKα are important for facilitating the DaMab-8 binding to the DGKα protein. Thus, DaMab-8 could be advantageous for immunohistochemical analyses toward clarifying the distribution of DGKα-expressing T cells in every pathophysiological tissue.
Highlights
Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DG) to produce phosphatidic acid (PA).(1,2)DG is a neutral lipid derived from various sources, including phosphatidylinositol 4,5-bisphosphate and phosphatidylcholine, and it serves as a second messenger that activates the conventional and novel types of the protein kinase C (PKC)family, RasGRP, Unc-13, and canonical transient receptor potential channels.[2,3] PA functions as a messenger molecule that activates the hypoxia-inducible factor (HIF)-1a, atypical PKCz, and mammalian target of rapamycin
T cell receptor (TCR) signaling via the modulation of the RasGRP activity.[4]. T cells isolated from DGKa-deficient mice demonstrate an altered activity of TCR signaling and hyperproliferation.[5]. DGKa is expressed in T lymphocytes abundantly, in which it facilitates the nonresponsive state known as clonal anergy.[5]. Anergy induction in T cells represents the main mechanism by which advanced tumors avoid immune action.[6]
Several anti-DGKa monoclonal antibodies (mAbs) are commercially available and are reportedly useful in Western blotting and for immunohistochemical analyses.[11] we have developed DaMab-2, a specific mAb against DGKa, which is extremely useful in immunocytochemical analysis.[7]. The binding epitopes of DaMab-2 were determined to be Cys246, Lys249, Pro252, and Cys253 of DGKa.[8]. DaMab-2 was not applicable for immunohistochemical analysis
Summary
Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DG) to produce phosphatidic acid (PA).(1,2)DG is a neutral lipid derived from various sources, including phosphatidylinositol 4,5-bisphosphate and phosphatidylcholine, and it serves as a second messenger that activates the conventional and novel types of the protein kinase C (PKC)family, RasGRP, Unc-13, and canonical transient receptor potential channels.[2,3] PA functions as a messenger molecule that activates the hypoxia-inducible factor (HIF)-1a, atypical PKCz, and mammalian target of rapamycin. We have developed DaMab-2 (mouse IgG1, kappa), a specific mAb against DGKa.[7] DaMab-2 is extremely useful in immunocytochemical analysis using HeLa cells. We further characterized the binding epitope of DaMab-2 using Western blotting and revealed that the Cys246, Lys249, Pro252, and Cys253 sites of DGKa are important for facilitating DaMab-2 binding to the DGKa protein.[8] DaMab-2 was not applicable for immunohistochemical analysis.
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