Establishment and Epitope Mapping of Anti-Diacylglycerol Kinase α Monoclonal Antibody DaMab-8 for Immunohistochemical Analyses

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.


Introduction
D iacylglycerol 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. DGK constitutes an enzyme family comprising 10 isozymes of the mammalian species. (1,2) Each isozyme possesses a distinct molecular structure and a subcellular localization pattern. DGKa is the first identified enzyme of 80-kDa size that contains an EF-hand motif (Ca 2+ -binding site), a Zn finger (C1 domain, DG-binding site), and a catalytic domain. DGKa regulates cell proliferation in response to IL-2 stimulation in T cells (3) and is involved in 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) Because only few specific anti-DGKa monoclonal antibodies (mAbs) are available to detect human DGKa using immunohistochemistry, the localization of DGKa-expressing cells remains unclear. Recently, we have developed DaMab-2 (mouse IgG 1 , 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) However, DaMab-2 was not applicable for immunohistochemical analysis.
In the present study, we report a novel anti-human DGKa mAb DaMab-8 (mouse IgG 1, kappa) that is extremely useful in immunohistochemical analysis. Furthermore, we have characterized the binding epitope of DaMab-8 using Western blotting.

Plasmid preparation
Human DGKa cDNA (9) was synthesized and subcloned into the expression vector pMAL-c2 (New England Biolabs, Inc., Beverly, MA), along with PA tag (GVAMP-GAEDDVV), (10) using the In-Fusion HD Cloning Kit (Takara Bio, Inc., Shiga, Japan); the resultant construct was named pMAL-c2-DGKa-PA. The deletion mutants of DGKa produced using PCR were subcloned into pMAL-c2 with PA tag using the In-Fusion PCR Cloning Kit. The substitution of DGKa amino acids 605-630 with either alanine or glycine in dN561 of DGKa was performed using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Inc., Santa Clara, CA). These constructs were verified using direct DNA sequencing.

Immunohistochemical analyses
Our study examined a patient with oropharyngeal squamous cell carcinoma who underwent surgery at the Sendai Medical Center. Informed consent for sample procurement and subsequent data analyses was obtained from the patient or the patient's guardian. The tissue samples were processed to produce 4-mm paraffin-embedded tissue sections that were directly autoclaved in citrate buffer (pH 6.0; Nichirei Biosciences, Inc., Tokyo, Japan) for 20 minutes and blocked using the SuperBlock T20 (PBS) Blocking Buffer (Thermo  Fisher Scientific, Inc.,), incubated with DaMab-8 (1 mg/mL) for 1 hour at the room temperature, and then treated using the Envision Kit (Agilent Technologies, Inc.) for 30 minutes. The tissue sections were stained using 3,3¢-diaminobenzidine tetrahydrochloride (DAB; Agilent Technologies, Inc.) for 2 minutes, and counterstaining was performed using hematoxylin (FUJIFILM Wako Pure Chemical Corporation).

Results and Discussion
Several anti-DGKa mAbs are commercially available and are reportedly useful in Western blotting and for immunohistochemical analyses. (11) Furthermore, we have developed DaMab-2 (mouse IgG 1 , kappa), 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) Unfortunately, DaMab-2 was not applicable for immunohisto-chemical analysis. We have previously immunized mice with recombinant DGKa and developed several anti-DGK clones. (7) One of these clones, DaMab-8, recognized only DGKa in ELISA and showed no reaction with other isozymes, such as DGKg, DGKz, DGKZ, and DGKd (data not shown). DGKa was reported to be expressed in T lymphocytes abundantly, in which it facilitates the nonresponsive state known as clonal anergy. (5) Immunohistochemical screening revealed that DaMab-8 was extremely useful in immunohistochemical analysis for T cells in oropharyngeal squamous cell carcinomas (Fig. 1).
In conclusion, Asn610, Leu611, Trp612, Gly613, Asp614, His619, Tyr623, and Gly624 are important for facilitating DaMab-8 binding to the DGKa protein. This epitope exists in the catalytic domain of DGKa, whereas DaMab-2 binds to Zn-finger (Fig. 4C). DaMab-8 could be advantageous for immunohistochemical analyses toward clarifying the distribution of DGKa-expressing T cells in every pathophysiological tissue. Furthermore, these findings could be applied for the production of more functional anti-DGKa mAbs.