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

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 IgG2a, 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.


Introduction
Diacylglycerol kinase (DGK) plays a critical role in the regulation of numerous cellular functions by catalyzing the phosphorylation of diacylglycerol to phosphatidic acid [1,2]. Diacylglycerol activates protein kinase C, and the DGK terminates the diacylglycerol-mediated signaling pathway by phosphorylating diacylglycerol [3][4][5][6][7]. Here, the resulting phosphatidic acid functions as a second messenger which regulates the intracellular Ca 2+ level and the mTOR-mediated signaling pathway [8,9].
Ten isozymes of the DGK family have been so far identified in mammals [2]. DGK family is also grouped into five subtypes based on their subtype-specific functional domains. DGKδ is one of the DGK family, and was first cloned from the human testis cDNA library [10].
DGKδ is expressed in several tissues and organs including the stomach, testis, bone marrow, and lymph node [11]. DGKδ is a type II DGK which contains pleckstrin homology (PH) and sterile alpha motif (SAM) domains at the N-and C-terminus of the protein, respectively. The PH domain can bind protein kinase C, the βγ-subunits of heterotrimeric G proteins, and phosphatidylinositol 4,5-bisphosphate [12][13][14]. On the other hand, the SAM domain has been shown to mediate both homo-and hetero-oligomerization, and therefore is a putative protein interaction module [15,16].
DGKδ was previously shown to regulate protein kinase C activity, and thereby control the degradation of epidermal growth factor receptor via modulation of ubiquitin-specific protease 8 expression in cultured human cells [17,18]. Moreover, DGKδ expression and activity levels are reduced in skeletal muscle tissues of Type 2 diabetic patients [19]. Hence, an anti-DGKδ monoclonal antibody (mAb) is required for specific detection of DGKδ in human tissues.
In this study, we established a novel anti-human DGKδ (hDGKδ) mAb, DdMab-1, by immunizing mice with recombinant hDGKδ. We also determined the binding epitope of DdMab-1 using deletion or point mutants of hDGKδ via Western blot analysis.
(New England Biolabs Inc., Beverly, MA) using the In-Fusion HD Cloning Kit (Takara Bio, Inc., Shiga, Japan). The PA tag is recognized by an anti-PA tag mAb (NZ-1) [21]. The resulting construct was named pMAL-c2-hDGKδ-PA. The deletion mutants of hDGKδ DNA were amplified via polymerase chain reaction, and subcloned into the pMAL-c2 with a PA tag using the In-Fusion HD Cloning Kit. The substitution of hDGKδ amino acids with alanine on dN610 of hDGKδ was performed using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Inc., Santa Clara, CA, USA). These constructs were also verified by direct DNA sequencing.

Production of the recombinant DGKδ protein
Competent E. coli TOP-10 cells (Thermo Fisher Scientific Inc., Waltham, MA, USA) were transformed with the pMAL-c2-hDGKδ-PA plasmid. The cells were cultured overnight at 37 • C in LB medium (Thermo Fisher Scientific Inc.) containing 100 μg/ml of ampicillin (Sigma-Aldrich Corp., St. Louis, MO). Cell pellets were resuspended in phosphate-buffered saline (PBS) containing 1% Triton X-100 and 50 μg/ ml aprotinin (Sigma-Aldrich Corp.). After sonication, crude extracts were collected using centrifugation (9000×g, 30 min, 4 • C). The lysates were passed through a 0.45 μm filter to remove trace amounts of insoluble materials. Filtered lysates were then mixed with NZ-1-Sepharose (1 ml of bed volume), and incubated at 4 • C for 2 h under gentle agitation. The resulting resin was then transferred to a column, and washed with 20 ml of Tris-buffered saline (TBS; pH 7.5). The bound protein was eluted with the PA tag peptide at room temperature in a stepwise manner (1 ml × 10 washes).

Hybridoma production
The Animal Care and Use Committee of Tohoku University approved all animal experiments. DdMab-1 was produced using the mouse medial iliac lymph node method. Briefly, three female 8-week old B6D2F1/Slc mice (Japan SLC Inc., Shizuoka, Japan) were immunized by injecting 33 μg of the pMAL-c2-hDGKδ-PA protein and Freund's complete adjuvant (Sigma-Aldrich Corp.) into their footpad. Additional immunization with 50 μg of the pMAL-c2-hDGKδ-PA protein was performed via the tail base. The lymphocytes were fused with mouse myeloma Sp2/0-Ag14 cells using polyethylene glycol (PEG). The culture supernatants were screened using enzyme-linked immunosorbent assay for binding to the pMAL-c2-hDGKδ-PA protein.

Establishment of anti-hDGKδ mAbs
Three B6D2F1/Slc mice were immunized by injecting 33 μg of the pMAL-c2-hDGKδ-PA protein into their footpad. Additional immunization with 50 μg of the pMAL-c2-hDGKδ-PA protein was performed via the tail base. The lymphocytes were fused with mouse myeloma Sp2/0-Ag14 cells using PEG. The culture supernatants were screened using enzyme-linked immunosorbent assay for the binding to the pMAL-c2-hDGKδ-PA protein. After Western blot screening, we established DdMab-1 (mouse IgG 2a , kappa), which is useful for Western blot analysis against hDGKδ (Fig. 1).
Here, we reported a novel anti-hDGKδ mAb, DdMab-1, which is useful for Western blot analysis (Figs. 1B and 2A). We also identified the binding epitope of DdMab-1 by western blotting, and found Arg675, Arg678, Lys679, Val680 and Lys682 to be important for DdMab-1 binding to hDGKδ. The epitope of DdMab-1 is located between catalytic and accessory domains (Fig. 2C). In our next study, we will investigate the utility of this mAb in immunocytochemistry and immunohistochemistry analyses for detection of hDGKδ protein in different tissues/organs including the stomach, testis, bone marrow, and lymph node.