MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 39, Number 4, 2020ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2020.0014

Epitope Mapping of DhMab-1:
An Antidiacylglycerol Kinase Monoclonal Antibody

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

Diacylglycerol kinase (DGK) Z is classified as a type II DGK and catalyzes diacylglycerol phosphorylation toproduce phosphatidic acid. DGKZ has been reported to be highly expressed in the hippocampus and cerebellum. Although a DGKZ-specific monoclonal antibody (mAb) is necessary to reveal the association between
the expression of DGKZ and diseases, an anti-DGKZ mAb for immunohistochemistry has not been developed.
Recently, we established a specific antihuman DGKZ (hDGKZ) mAb, DhMab-1 (mouse IgG2a, kappa). For
epitope mapping of DhMab-1, here we produced deletion or point mutants of hDGKZ and performed Western
blotting to determine the binding epitope of DhMab-1. DhMab-1 reacted with the dN755 mutant, but not withthe dN760 mutant, indicating that the N-terminus of the DhMab-1 epitope is mainly located between aminoacids 755 and 760 of the protein. A more detailed analysis using point mutants demonstrated that seven mutants,that is, A751G, I755A, D756A, P757A, D758A, L759A, and D760A, were not detected by DhMab-1. These
results indicate that Ala751, Ile755, Asp756, Pro757, Asp758, Leu759, and Asp760 are important for DhMab-1
binding to hDGKZ.
Keywords: DGKh, DhMab-1, monoclonal antibodyIntroductionDMaterials and Methods

iacylglycerol kinase (DGK) plays a key role in theG-protein–mediated signaling pathway, which is an
important signaling cascade in mammalian cells.(1,2) DGK isan enzyme that phosphorylates diacylglycerol to produce
phosphatidic acid. In turn, phosphatidic acid functions as asecond messenger that mediates intracellular Ca2+ levels andregulates the mTOR-mediated signaling pathway.(3,4) The
DGK family consists of 10 isozymes in mammalian species,
with each isozyme containing two to three C1 domains, whilesharing a common catalytic domain.(1) The DGK family isfurther divided into five subtypes according to their subtypespecific functional domains.(5) DGKZ is classified as a type IIDGK. It contains a pleckstrin homology (PH) domain and
two C1 domains, a catalytic domain, and an accessory domain.(6) The PH domain is a phosphatidylinositol-binding
domain. In particular, the PH domain of DGKZ binds tophosphatidylinositol 4,5-bisphosphate and regulates thesubcellular localization of DGKZ.(7)Recently, we established a specific antihuman DGKZ(hDGKZ) monoclonal antibody (mAb), DhMab-1 (mouseIgG2a, kappa). In this study, we performed epitope mapping
to identify the binding epitope of DhMab-1 using deletionand point mutations of hDGKZ.
12Plasmid preparation and productionof recombinant DGKg protein

Synthesized DNA (Eurofins Genomics KK, Tokyo,Japan) encoding hDGKg (accession No. AB078967) plus a
C-terminal PA tag (GVAMPGAEDDVV)(8,9) was subcloned into the expression vector pMAL-c2 (New England
Biolabs, Inc., Beverly, MA) using the In-Fusion HDCloning Kit (Takara Bio, Inc., Shiga, Japan). The PA tag isrecognized by an anti-PA tag mAb (NZ-1).(10) The resulting
construct was named pMAL-c2-hDGKg-PA. The deletionmutants of hDGKg were produced by polymerase chain
reaction and were subcloned into pMAL-c2 with a PA tag
using the In-Fusion HD Cloning Kit. The substitution of theamino acids of hDGKg with alanine at dN705 of hDGKg
was performed using the QuikChange Lightning SiteDirected Mutagenesis Kit (Agilent Technologies, Inc.,
Santa Clara, CA). These constructs were verified by direct
DNA sequencing.
Competent Escherichia coli TOP-10 cells (Thermo FisherScientific, Inc., Waltham, MA) were transformed with thepMAL-c2-hDGKZ-PA plasmid. They were cultured overnight at 37C in Luria-Bertani broth (Thermo Fisher Scientific, Inc.) containing 100 mg/mL ampicillin (Sigma-Aldrich

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

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EPITOPE MAPPING OF AN ANTI-DGKH MAB

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Corp., St. Louis, MO). Cell pellets were resuspended inphosphate-buffered saline containing 1% Triton X-100 and
50 mg/mL aprotinin (Sigma-Aldrich Corp.). After sonication,
the crude extracts were collected by centrifugation (9000 g,30 minutes, 4C). The lysates were passed through a 0.45 mm
filter to remove any trace amounts of insoluble materials.
Cleared lysates were mixed with NZ-1-Sepharose (1 mL bedvolume) and incubated at 4C for 2 hours under gentle agitation. The resin was then transferred to a column and washedwith 20 mL Tris-buffered saline (pH 7.5). The bound protein
was eluted with the PA tag peptide at room temperature in astepwise manner (1 mL · 10 washes).

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Hybridoma production

The Animal Care and Use Committee of Tohoku University approved all animal experiments. DhMab-1 wasproduced using the mouse medial iliac lymph node method.
In brief, B6D2F1/Slc mice ( Japan SLC, Inc., Shizuoka,Japan) were immunized by injecting 33 lg of the pMAL-c2hDGKg-PA protein together with Freund’s complete adjuvant (Sigma-Aldrich Corp.) into their footpad. Additionalimmunization with 60 lg of the pMAL-c2-hDGKg-PA protein was performed through the tail base. The lymphocytes
were fused with mouse myeloma Sp2/0-Ag14 cells using
polyethylene glycol. The culture supernatants were screenedusing enzyme-linked immunosorbent assay of the binding to
the pMAL-c2-hDGKg-PA protein.
Western blot analyses

Lysates were boiled in sodium dodecyl sulfate sample
buffer (Nacalai Tesque, Inc., Kyoto, Japan). The samples
were electrophoresed on 5%–20% polyacrylamide gels
(Nacalai Tesque, Inc.) and transferred onto a polyvinylidenedifluoride membrane (Merck KGaA, Darmstadt, Germany).
After blocking with 4% skim milk (Nacalai Tesque, Inc.)for 1 hour, the membrane was incubated with DhMab-1(1 lg/mL) or NZ-1 (1 lg/mL) for 1 hour, followed by incubation with hydrogen peroxidase-conjugated antimouse immunoglobulins (1:2000 dilution; Agilent Technologies, Inc.)
or HRP-conjugated antirat IgG (1:10,000 dilution; SigmaAldrich Corp.) for 1 hour. The membrane was developed with
the ImmunoStar LD Chemiluminescence Reagent (FUJIFILM Wako Pure Chemical Corporation) using the SayacaImager (DRC Co., Ltd., Tokyo, Japan). All Western blot
procedures were performed at room temperature.
Results

Previously, we established DhMab-1 (mouse IgG2a,kappa), which can recognize hDGKZ but not other related
isozymes, such as DGKa, DGKg, and DGKz, in an enzymelinked immunosorbent assay (data not shown). In this study,
we performed epitope mapping to characterize the binding
epitope of DhMab-1 using Western blot analysis.
We produced a C-terminal deletion mutant (dC583) andan N-terminal deletion mutant (dN584), as shown inFigure 1. First, we investigated whether DhMab-1 recognizes either dC583 or dN584 by Western blotting. The results of this experiment indicated that DhMab-1 interacts
with dN584 (Fig. 2A).

FIG. 1. Schematic illustration of the hDGKZ deletionmutants. Black bars, deletion mutants detected by DhMab-1.
DGKZ, diacylglycerol kinase Z; hDGKZ, human DGKZ;
MBP, maltose-binding protein.

We then produced an additional 11 N-terminal deletionmutants (dN600, dN650, dN700, dN750, dN800, dN850,dN900, dN950, dN1000, dN1050, and dN1100) of hDGKZ(Fig. 1) and performed Western blotting. As shown in
Figure 2A, DhMab-1 recognized dN600, dN650, dN700, anddN750, but not dN800, dN850, dN900, dN950, dN1000,dN1050, and dN1100. All of the deletion mutants were detected by the anti-PA tag mAb, NZ-1 (Fig. 2A). This result
indicates that the DhMab-1 epitope is located between aminoacids 750 and 800.
Next, we produced an additional nine N-terminal deletionmutants (dN755, dN760, dN765, dN770, dN775, dN780,dN785, dN790, and dN795) of hDGKZ (Fig. 1). Western
blotting demonstrated that DhMab-1 recognized dN755, but
not dN760, dN765, dN770, dN775, dN780, dN785, dN790,
and dN795, indicating that the DhMab-1 epitope is locatedbetween amino acids 755 and 760. All of the deletion mutants
were detected by the anti-PA tag mAb, NZ-1 (Fig. 2B).
To investigate further the critical epitope of DhMab-1, we
produced constructs representing 16 point mutants within
hDGKZ (P748A, F749A, G750A, A751G, T752A, P753A,F754A, I755A, D756A, P757A, D758A, L759A, D760A,S761A, V762A, and D763A). All hDGKZ point mutants
were recognized by NZ-1 (Fig. 2C). In contrast, DhMab-1 did
not recognize A751G, I755A, D756A, P757A, D758A,L759A, and D760A (Fig. 2C), indicating that DhMab-1 binds
to DGKZ through the following seven amino acids: Ala751,Ile755, Asp756, Pro757, Asp758, Leu759, and Asp760.
These results are summarized in Figure 2D.

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SANO ET AL.

FIG. 2. Epitope mapping of DhMab-1 using deletion mutants and point mutants of hDGKZ. (A) Cell lysates of the C- or
N-terminal deletion mutants of hDGKZ were electrophoresed and transferred onto a PVDF membrane. After blocking, themembrane was incubated with 1 mg/mL of the DhMab-1 or anti-PA tag antibody (NZ-1). (B) Cell lysates of the N-terminal
deletion mutants of hDGKZ were electrophoresed and transferred onto a PVDF membrane. After blocking, the membrane
was incubated with 1 mg/mL of the DhMab-1 or anti-PA tag antibody (NZ-1). (C) Cell lysates of the dN705 point mutants ofhDGKZ were electrophoresed and transferred onto PVDF membranes. After blocking, the membranes were incubated with1 mg/mL of the DhMab-1 or anti-PA tag antibody (NZ-1). (D) Schematic illustration of hDGKZ. The DhMab-1 epitope islocated between the catalytic domain and the accessory domain. The amino acids shown in gray are important for DhMab-1
binding to hDGKZ. PVDF, polyvinylidene difluoride.
Discussion

Previously, we established the anti-DGKa mAbs DaMab-2
and DaMab-8,(11,12) an anti-DGKg mAb, DgMab-6,(13) andan anti-DGKz mAb, DzMab-1.(14) We further determined
their respective binding epitopes.(12,15–17) DaMab-2 andDaMab-8 bind to the Zn-finger domain and catalytic domain
of DGKa, respectively.(12,15) DgMab-6 and DzMab-1 bind to
the N-terminus of DGKg and DGKz, respectively.(16,17) Recently, we established a novel anti-hDGKZ mAb, DhMab-1,

which is useful for immunohistochemical analysis (data notshown). In this study, we identified the binding epitope ofDhMab-1 by Western blotting. Seven amino acids ofhDGKZ, that is, Ala751, Ile755, Asp756, Pro757, Asp758,
Leu759, and Asp760, are critical for DhMab-1 binding to thehDGKZ protein. This epitope is located near the accessory
domain of hDGKZ (Fig. 2D).
DGKZ is highly expressed in the hippocampus and cerebellum areas of the brain.(18) The DGKZ mRNA is upregulated in patients with bipolar disorder.(19,20) DGKZ is also

EPITOPE MAPPING OF AN ANTI-DGKH MAB

highly expressed in lung cancer containing EGFR mutations,and knockdown of DGKZ resulted in impaired growth of
EGFR-mutant cell lines.(21) DhMab-1 can be used to analyze
the expression and pathophysiological function of DGKZ
and represents a useful tool to study the molecular basis ofvarious diseases.
Author Disclosure Statement

Y.K. received research funding from Ono PharmaceuticalCo., Ltd. The other authors have no conflict of interest.
Funding Information

Downloaded by Yukinari Kato from www.liebertpub.com at 08/21/20. For personal use only.

This research was supported, in part, by AMED underGrant Nos. JP19am0401013 (Y.K.), JP19am0101078 (Y.K.),
and JP19ae0101028 (Y.K.), and by JSPS KAKENHI Grant
No. 17K07299 (M.K.K.) and Grant No. 19K07705 (Y.K.).
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Address correspondence to:
Yukinari Kato
New Industry Creation Hatchery Center
Tohoku University
2-1, Seiryo-machi, Aoba-ku
Sendai
Miyagi 980-8575
JapanE-mail: yukinari-k@bea.hi-ho.ne.jp
Received: March 18, 2020
Accepted: June 14, 2020