MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 38, Number 3, 2019ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2019.0011

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

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Masato Sano,1 Mika K. Kaneko,1 and Yukinari Kato1,2

Diacylglycerol kinase (DGK) converts diacylglycerol (DG) into phosphatidic acid (PA). Both DG and PA serveas lipidic second messengers; therefore, DGK plays a critical role in regulating the balance of two signaling
pathways mediated by DG and PA in cellular functions. DGK gamma (DGKg), 1 of the 10 mammalian DGKisozymes, has been reported to be involved in membrane traffic, mast cell function, and leukemic cell differentiation. We previously developed a novel anti-DGKg monoclonal antibody (mAb), DgMab-6, which isvery useful in immunocytochemistry of human cultured cells. In this study, we characterized the binding
epitope of DgMab-6 using Western blotting, and Glu12 is important for facilitating the DgMab-6 binding to theDGKg protein. These results might lead to further development for sensitive and specific mAbs against DGKg.
Keywords: diacylglycerol kinase, DGKg, monoclonal antibody, DgMab-6Introduction

Materials and Methods

DPlasmid of DGKg

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 canonicaltransient receptor potential channels.(2,3) PA functions as amessenger molecule that activates the hypoxia-inducible
factor-1a, atypical PKCz, and mammalian target of rapamycin. DGK constitutes an enzyme family comprising 10
isozymes of the mammalian species.(1,2) Each isozyme has adistinct molecular structure and subcellular localization pattern. DGK gamma (DGKg) was first isolated in rat(4) andhuman,(5) and is classified into type I DGK, which contains
two EF hand motifs (Ca2+-biding site), two Zn fingers (DGbinding C1 domain), and a catalytic domain. DGKg has beenreported to be involved in a variety of cell functions including
antigen-induced mast cell degranulation,(6) leukemic celldifferentiation,(7) and membrane traffic between the endoplasmic reticulum and Golgi apparatus.(8)We previously reported a novel antihuman DGKg monoclonal antibody (mAb), DgMab-6, which is very useful inimmunocytochemical analysis of cultured cells. In this study,
we characterize the binding epitope of DgMab-6 using
Western blotting.

12Human DGKg cDNA was synthesized, and was subcloned
into an expression vector, pMAL-c2 (New England Biolabs,Inc., Beverly, MA), with PA tag (GVAMPGAEDDVV)(9)using the In-Fusion HD Cloning Kit (Takara Bio, Inc., Shiga,Japan); the resultant construct was named pMAL-c2-DGKgPA. The deletion mutants of DGKg produced using PCR
were subcloned into pMAL-c2 with PA tag using the InFusion HD Cloning Kit. The substitution of DGKg aminoacids (aas) 6–20 with alanine in dC200 of DGKg was performed using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Inc., Santa Clara, CA).
These constructs were verified using direct DNA sequencing.
Western blottingCompetent Escherichia coli TOP-10 cells (Thermo FisherScientific, Inc., Waltham, MA) were transformed and cultured overnight at 37C in Luria–Bertani medium (ThermoFisher Scientific, Inc.) containing 100 mg/mL ampicillin(FUJIFILM Wako Pure Chemical Corporation, Osaka,Japan). The cell pellets were resuspended in phosphatebuffered saline containing 1% Triton X-100 and 50 mg/mL
aprotinin (Sigma-Aldrich Corp., St. Louis, MO). Lysates
were boiled in sodium dodecyl sulfate (SDS) sample buffer(Nacalai Tesque, Inc., Kyoto, Japan). About dC80 lysates

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.

120

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EPITOPE OF DGMAB-6

121

were immunoprecipitated using amylose resin (New EnglandBiolabs, Inc.) and boiled in SDS sample buffer (NacalaiTesque, Inc.). The samples were electrophoresed on 5%–20%
polyacrylamide gels (Nacalai Tesque, Inc.) and transferred
onto a polyvinylidene difluoride (PVDF) membrane (MerckKGaA, Darmstadt, Germany). After blocking with 4% skimmilk (Nacalai Tesque, Inc.) for 1 hour, the membrane wasincubated with DgMab-6 for 1 hour, followed by peroxidaseconjugated antimouse IgG (1:2000 dilution; Agilent Technologies, Inc.) for 1 hour. The membrane was also incubatedwith NZ-1 (anti-PA tag)(10) for 1 hour, followed by biotinconjugated antirat IgG (1:1000 dilution; Agilent Technologies, Inc.) for 30 minutes, and further incubated withVECTASTAIN Elite ABC HRP Kit (Vector Laboratories,Inc., Burlingame, CA) for 30 minutes. The membrane wasfinally developed with the ImmunoStar LD Chemiluminescence Reagent (FUJIFILM Wako Pure Chemical Corporation) using the Sayaca-Imager (DRC Co., Ltd., Tokyo,Japan). All procedures of Western blotting were performed atroom temperature.

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), indicatingthat the N-terminus of the DgMab-6 epitope exists between
1st aa and 20th aa.
Western blotting of the additional deletion mutants ofDGKg—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 Nterminus of the DgMab-6-epitope exists between 6th aa and20th aa of DGKg (Fig. 1B). These results are summarized
in Figure 2.
Accordingly, we produced the following 15 DGKg pointmutants using human DGKg-dC200: W6A, V7A, S8A, L9A,T10A, P11A, E12A, E13A, F14A, D15A, Q16A, L17A,
Q18A, K19A, and Y20A. Western blotting demonstrated thatthe 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.

Results

Discussion

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 DGKg (i.e., dC380,dC360, dC340, dN320, dC300, dC280, dC260, dC240,dC220, dC200, dC180, dC160, dC140, dC120, dC100, dC80,

Immunohistochemical examination using anti-DGKg antibodies on rat tissues demonstrated cytoplasmic localizationof DGKg 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

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

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122FIG. 2. Schematic illustration of DgMab-6 epitope. Black
bars, deletion mutants detected by DgMab-6. hDGKg, human DGKg; MBP, maltose-binding protein.

SANO ET AL.

mAbs against human DGKg had not been established. Recently, the recombinant MBP-DGKg-PA was captured using
NZ-1-Sepharose and efficiently eluted with PA tag peptide.(9)Mice were then immunized with MBP-DGKg-PA to develop
novel anti-DGKg mAbs. Using ELISA, the culture supernatants of hybridomas were screened for binding to recombinant MBP-DGKg-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, indicatingthat a novel anti-DGKg mAb, DgMab-6, sensitively andspecifically reacted with human DGKg using immunocytochemical analysis. Previously, we further developed
DaMab-2 (mouse IgG1, kappa), a specific mAb against
DGKa, which is extremely useful in immunocytochemical
analysis.(12) The binding epitopes of DaMab-2 were determined to be Cys246, Lys249, Pro252, and Cys253 of DGKa
using Western blot analysis.(13) Because many cysteine residues are included in DGKa (24 cysteins in 735 aas; 3.3%),
we could not synthesize DGKa 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 epitopeof DgMab-6 for DGKg. DgMab-6 did not detect E12A mutant (Fig. 3A), indicating that Glu12 is critical for DgMab-6binding to DGKg (Fig. 3B). This epitope exists in the Nterminal domain of DGKg (Fig. 3C). These findings could beapplied for the production of more functional anti-DGKg
mAbs.

FIG. 3. Epitope mapping of DgMab-6 using point mutants of DGKg. (A) Cell lysates of point mutants were electrophoresed and transferred onto PVDF membranes. After blocking, the membranes were incubated with 1 mg/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 DGKg. The critical epitope of DgMab-6 is located in the N-terminus of DGKg.

EPITOPE OF DGMAB-6
Acknowledgments

This research was supported, in part, by AMED underGrant numbers JP18am0101te078 (Y.K.), JP18am0301010(Y.K.), and JP18ae0101028 (Y.K.), and by JSPS KAKENHI
Grant number 17K07299 (M.K.K.) and Grant number16K10748 (Y.K.).
Author Disclosure StatementY.K. received research funding from Ono PharmaceuticalCo., Ltd. The other authors have no conflict of interest.

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Address correspondence to:Yukinari Kato
New Industry Creation Hatchery CenterTohoku University
2-1, Seiryo-machi, Aoba-ku
Sendai
Miyagi 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: February 24, 2019
Accepted: May 5, 2019