Epitope Mapping of Antihuman Diacylglycerol Kinase Gamma Monoclonal Antibody DgMab-6

Introduction

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-1α, atypical PKCζ, and mammalian target of rapamycin. DGK constitutes an enzyme family comprising 10 isozymes of the mammalian species.^(1,2) Each isozyme has a distinct molecular structure and subcellular localization pattern. DGK gamma (DGKγ) was first isolated in rat⁽⁴⁾ and human,⁽⁵⁾ and is classified into type I DGK, which contains two EF hand motifs (Ca²⁺-biding site), two Zn fingers (DG-binding C1 domain), and a catalytic domain. DGKγ has been reported to be involved in a variety of cell functions including antigen-induced mast cell degranulation,⁽⁶⁾ leukemic cell differentiation,⁽⁷⁾ and membrane traffic between the endoplasmic reticulum and Golgi apparatus.⁽⁸⁾

We previously reported a novel antihuman DGKγ monoclonal antibody (mAb), DgMab-6, which is very useful in immunocytochemical analysis of cultured cells. In this study, we characterize the binding epitope of DgMab-6 using Western blotting.

Materials and Methods

Results

We herein characterized the binding epitope of DgMab-6 using Western blotting. As shown in Figure 1, we produced 19 C-terminal deletion mutants of DGKγ (i.e., dC380, dC360, dC340, dN320, dC300, dC280, dC260, dC240, dC220, dC200, dC180, dC160, dC140, dC120, dC100, dC80, dC60, dC40, and dC20). Western blotting demonstrated that DgMab-6 detected all deletion mutants, which were also detected by an anti-PA tag mAb, NZ-1 (Fig. 1A), indicating that the N-terminus of the DgMab-6 epitope exists between 1st aa and 20th aa.

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FIG. 1.

Production of DGKγ deletion mutants. (A) C-terminal deletion mutants. (B) N-terminal deletion mutants. Cell lysates of deletion mutants or immunoprecipitates of dC80 were electrophoresed and transferred onto PVDF membranes. After blocking, the membranes were incubated with 1 μg/mL of DgMab-6 or anti-PA tag (NZ-1), followed by incubation with peroxidase-conjugated antimouse or antirat IgG. DGKγ, diacylglycerol kinase gamma; PVDF, polyvinylidene difluoride.

Western blotting of the additional deletion mutants of DGKγ—that is, dN6, dN11, dN16, and dN21—demonstrated that DgMab-6 detected dN6, but not dN11, dN16, and dN21, although all deletion mutants were detected by an anti-PA tag mAb, NZ-1 (Fig. 1B), thereby indicating that the N-terminus of the DgMab-6-epitope exists between 6th aa and 20th aa of DGKγ (Fig. 1B). These results are summarized in Figure 2.

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FIG. 2.

Schematic illustration of DgMab-6 epitope. Black bars, deletion mutants detected by DgMab-6. hDGKγ, human DGKγ; MBP, maltose-binding protein.

Accordingly, we produced the following 15 DGKγ point mutants using human DGKγ-dC200: W6A, V7A, S8A, L9A, T10A, P11A, E12A, E13A, F14A, D15A, Q16A, L17A, Q18A, K19A, and Y20A. Western blotting demonstrated that the anti-PA tag mAb, NZ-1, detected all point mutants (Fig. 3A). In contrast, DgMab-6 did not detect E12A. These results are summarized in Figure 3B.

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FIG. 3.

Epitope mapping of DgMab-6 using point mutants of DGKγ. (A) Cell lysates of point mutants were electrophoresed and transferred onto PVDF membranes. After blocking, the membranes were incubated with 1 μg/mL of DgMab-6 and anti-PA tag (NZ-1), followed by incubation with peroxidase-conjugated antimouse IgG and biotin-conjugated antirat IgG, respectively. (B) Schematic illustration of DgMab-6 epitope. A light gray amino acid is a critical epitope of DgMab-6. (C) Schematic illustration of DGKγ. The critical epitope of DgMab-6 is located in the N-terminus of DGKγ.

Discussion

Immunohistochemical examination using anti-DGKγ antibodies on rat tissues demonstrated cytoplasmic localization of DGKγ in several neurons of the brain.^(4,11) In contrast, morphological examination on human cells or tissues has not yet been performed because highly specific and sensitive mAbs against human DGKγ had not been established. Recently, the recombinant MBP-DGKγ-PA was captured using NZ-1-Sepharose and efficiently eluted with PA tag peptide.⁽⁹⁾ Mice were then immunized with MBP-DGKγ-PA to develop novel anti-DGKγ mAbs. Using ELISA, the culture supernatants of hybridomas were screened for binding to recombinant MBP-DGKγ-PA. Finally, DgMab-6 (mouse IgG₁, kappa) was established. Immunocytochemical analysis revealed that immunoreactivity for DgMab-6 is detected as granular pattern in the cytoplasm in HL-60 cells, indicating that a novel anti-DGKγ mAb, DgMab-6, sensitively and specifically reacted with human DGKγ using immunocytochemical analysis. Previously, we further developed DaMab-2 (mouse IgG₁, kappa), a specific mAb against DGKα, which is extremely useful in immunocytochemical analysis.⁽¹²⁾ The binding epitopes of DaMab-2 were determined to be Cys246, Lys249, Pro252, and Cys253 of DGKα using Western blot analysis.⁽¹³⁾ Because many cysteine residues are included in DGKα (24 cysteins in 735 aas; 3.3%), we could not synthesize DGKα peptides, which easily aggregate. Of interest, DaMab-2 epitope was included in Zn finger, and two cysteine residues such as Cys246 and Cys253 are involved in the DaMab-2 epitope. In this study, using Western blotting, we also characterized the binding epitope of DgMab-6 for DGKγ. DgMab-6 did not detect E12A mutant (Fig. 3A), indicating that Glu12 is critical for DgMab-6 binding to DGKγ (Fig. 3B). This epitope exists in the N-terminal domain of DGKγ (Fig. 3C). These findings could be applied for the production of more functional anti-DGKγ mAbs.