MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 39, Number 4, 2020ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2020.0012

Epitope Mapping of the Anti-Diacylglycerol Kinase
Monoclonal Antibody DhMab-4
for Immunohistochemical Analysis

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Teizo Asano,1 Masato Sano,1 Junko Takei,1 Yusuke Sayama,1 Mika K. Kaneko,1 and Yukinari Kato1,2

Diacylglycerol kinase (DGK) plays a pivotal role in intracellular signaling pathways in mammals. Activated G
protein-coupled receptor activates phospholipase C (PLC) through heterotrimeric G protein, following which
PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol (DG) and inositol 1,4,5trisphosphate (IP3). DGK catalyzes DG phosphorylation to produce phosphatidic acid. DG and phosphatidic
acid function as second messengers and their intracellular concentrations are regulated by DGK; therefore,
DGK plays an important role in regulating many biological processes. There are ten DGK isozymes, of which
DGKZ is classified as a type II DGK. Reports have shown that DGKZ is associated with several diseases; forexample, it is highly expressed in the hippocampus and cerebellum and is a key element in bipolar disorder.
Although a DGKZ-specific monoclonal antibody (mAb) is necessary to reveal the association between theexpression of DGKZ and diseases, an anti-DGKZ mAb for immunohistochemistry has not yet been established.
In this study, we established a specific anti-human DGKZ (hDGKZ) mAb, DhMab-4 (mouse IgG2b, kappa).
DhMab-4 strongly stained Purkinje cells of human cerebellum in immunohistochemistry analysis. For epitope
mapping of DhMab-4, we produced deletion or point mutants of hDGKZ and performed western blotting todetermine the binding epitope of DhMab-4. DhMab-4 reacted with dN745 mutant but not with dN750 mutant,indicating that the N-terminus of the DhMab-4 epitope is located between amino acids 745 and 750. Moredetailed analysis using point mutants demonstrated that five mutants, that is, D747A, P748A, F749A, G750A,
and T752A, were not detected by DhMab-4. These results indicate that Asp747, Pro748, Phe749, Gly750, andThr752 are important for DhMab-4 binding to hDGKZ.
Keywords: DGKh, DhMab-4, monoclonal antibody

Introduction

Diacylglycerol kinase (DGK) plays a key role in the
G protein-mediated signaling pathway, which is an
important signaling cascade in mammalian cells.(1,2) Activated G protein-coupled receptor catalyzes a guanine nucleotide exchange on the alpha subunit of the heterotrimeric
G protein and resulting in its activation. An activated G
protein subsequently activates phospholipase C (PLC).(3,4)
PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2)
into diacylglycerol (DG) and inositol 1,4,5-trisphosphate (IP3),
which functions as a second messenger. IP3 diffuses into thecytosol and increases Ca2+ concentration in the cytoplasm,
whereas DG remains on the plasma membrane and activates
protein kinase C (PKC).(5–8) DGK is an enzyme that phosphorylates DG and produces phosphatidic acid.(1,2) Phosphatidic acid also functions as a second messenger, which
12

mediates intracellular Ca2+ levels and regulates the mTORmediated signaling pathway.(9,10) Therefore, DGK regulates
two signaling pathways to control a variety of biological
functions.(11)
The DGK family consists of 10 isozymes in mammalian
species and each isozyme contains 2 to 3 C1 domains, while
sharing a common catalytic domain.(1) The DGK family isfurther divided into five subtypes according to their subtypespecific functional domains.(12) DGKZ was first cloned fromhamster and is classified as a type II DGK. It exhibits
pleckstrin homology (PH) domain and contains two C1 domains, a catalytic domain and an accessory domain.(13) It is
known that the PH domain is a phosphatidylinositol binding
domain. The PH domain of DGKZ binds to PIP2 strongly andhighly selectively, thereby regulating the subcellular localization of DGKZ.(14) There are two splicing variants of
DGKZ, DGKZ1, and DGKZ2. DGKZ2 has a sterile alphaDepartment 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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motif domain at its carboxyl terminus, while the DGKZ1
variant is devoid of this domain.(15)In this study, we immunized mice with a recombinanthuman DGKZ (hDGKZ) protein and established a novel antihDGKZ monoclonal antibody (mAb), DhMab-4, which may
be used for immunohistochemistry. We also performed epitope mapping to reveal the binding epitope of DhMab-4 using
deletion and point mutations of hDGKZ.
Materials and Methods

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Plasmid preparation

Synthesized DNA (Eurofins Genomics KK, Tokyo, Japan)
encoding hDGKg (Accession No.: AB078967) plus a
C-terminal PA tag (GVAMPGAEDDVV),(16,17) which isrecognized by an anti-PA tag mAb (NZ-1),(18) was subcloned
into the expression vector, pMAL-c2 (New England Biolabs,
Inc., Beverly, MA), using the In-Fusion HD Cloning Kit(Takara Bio, Inc., Shiga, Japan). The resultant construct wasnamed pMAL-c2-hDGKg-PA. The deletion mutants ofhDGKg were produced by PCR and subcloned into pMAL-c2
with a PA tag using the In-Fusion HD Cloning Kit. The
substitution of hDGKg amino acids with alanine at dN705 ofhDGKg was performed using the QuikChange Lightning
Site-Directed Mutagenesis Kit (Agilent Technologies, Inc.,
Santa Clara, CA). These constructs were verified by direct
DNA sequencing.

ASANO ET AL.
Immunohistochemical analyses

Normal human cerebellum tissue was purchased fromProteoGenex, Inc. (Inglewood, CA). Histological sections of
4 lm thickness were directly autoclaved in citrate buffer(pH 6.0; Nichirei Biosciences, Inc., Tokyo, Japan) for 20
minutes. After blocking with SuperBlock T20 (PBS)
Blocking Buffer (Thermo Fisher Scientific, Inc.), the sections
were incubated with DhMab-4 (5 lg/mL) for 1 hour at room
temperature and treated using the EnVision+ Kit (Agilent
Technologies, Inc.) for 30 minutes. Color was developed
using 3,3¢-diaminobenzidine tetrahydrochloride (DAB; Agilent Technologies, Inc.) for 2 minutes, and counterstaining
was performed with hematoxylin (FUJIFILM Wako Pure
Chemical Corporation, Osaka, Japan).
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 polyvinylidene

Production of recombinant DGKg protein

Competent Escherichia coli TOP-10 cells (Thermo Fisher
Scientific, Inc., Waltham, MA) were transformed with theplasmid pMAL-c2-hDGKZ-PA. They were then cultured
overnight at 37C in the LB medium (Thermo Fisher Scientific, Inc.) containing 100 mg/mL ampicillin (SigmaAldrich Corp., St. Louis, MO). Cell pellets were resuspended
in phosphate-buffered saline (PBS) with 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 bed volume) and incubated at 4C for 2 hours undergentle agitation. The resin was then transferred to a column
and washed with 20 mL 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).
Hybridoma production

DhMab-4 was produced using the mouse medial iliac
lymph node method. The Animal Care and Use Committeeof Tohoku University approved all of the animal experiments.
Briefly, B6D2F1/Slc mice ( Japan SLC, Inc., Shizuoka, Japan)
were immunized by injecting 33 lg of pMAL-c2-hDGKg-PA
protein, along with Freund’s complete adjuvant (SigmaAldrich Corp.), into the footpad. Additional immunization
with 60 lg of pMAL-c2-hDGKg-PA protein was performed
using the tailbase. 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 pMAL-c2-hDGKg-PA
protein.

FIG. 1. Immunohistochemical analysis using DhMab-4
against human cerebellum. Tissue sections were incubatedwith DhMab-4 (5 mg/mL) for 1 hour at room temperatureand treated using the EnVision+ kit for 30 minutes. Red
arrows indicate Purkinje cells. Scale bar = 100 mm.

THE EPITOPE OF ANTI-DGKH

difluoride (PVDF) membrane (Merck KGaA, Darmstadt,
Germany). After blocking with 4% skim milk (Nacalai Tesque, Inc.) for 1 hour, the membrane was incubated with
DhMab-4 or NZ-1 for 1 hour, followed by HRP-conjugatedanti-mouse immunoglobulins (1:2000 dilution; Agilent Technologies, Inc.) or HRP-conjugated anti-rat IgG (1:10,000 dilution; Sigma-Aldrich Corp.) for 1 hour. The membrane wasdeveloped with the ImmunoStar LD Chemiluminescence Reagent (FUJIFILM Wako Pure Chemical Corporation) using
the Sayaca-Imager (DRC Co., Ltd., Tokyo, Japan). All western
blot procedures were performed at room temperature.

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Results

In this study, we established DhMab-4 (mouse IgG2b,
kappa), which can recognize hDGKZ, but not other related
isozymes such as DGKa, DGKg, and DGKz in an enzymelinked immunosorbent assay (data not shown). DhMab-4
stained human cerebral Purkinje cells as determined by immunohistochemistry (Fig. 1).
We next performed epitope mapping to characterize thebinding epitope of DhMab-4 using western blot analysis.

119First, we produced a C-terminal deletion mutant (dC583) andan N-terminal deletion mutant (dN584) and investigatedwhether DhMab-4 recognizes either dC583 or dN584 bywestern blot analysis. The results indicated that DhMab-4
interacts with dN584 (Fig. 2A, left panel). We then produced
additional 11 N-terminal deletion mutants (dN600, dN650,
dN700, dN750, dN800, dN850, dN900, dN950, dN1000,
dN1050, and dN1100) of hDGKZ and performed a westernblot. As shown in Figure 2A (left panel), DhMab-4 recognized dN600, dN650, and dN700 but not dN750, dN800,
dN850, dN900, dN950, dN1000, and dN1050. All of thedeletion mutants were detected by an anti-PA tag mAb, NZ-1
(Fig. 2A, right panel). This result indicates that the
N-terminus of the DhMab-4 epitope exists between 700 and
750 amino acids (aa) (Fig. 2B).
We next produced an additional nine N-terminal deletionmutants of hDGKZ (dN705, dN710, dN715, dN720, dN725,
dN730, dN735, dN740, and dN745). The western blot results
showed that DhMab-4 and NZ-1 detected all of the deletionmutants (Fig. 3A), indicating that the N-terminus of the
DhMab-4 binding epitope resides between amino acids 745
and 750 (Fig. 3B).

FIG. 2. Epitope mapping of DhMab-4 using deletion mutants of hDGKZ. (A) Cell lysates of C- or N-terminal deletionmutants of hDGKZ were electrophoresed and transferred onto a PVDF membrane. After blocking, the membrane wasincubated with 1 mg/mL of DhMab-4 or anti-PA tag antibody (NZ-1). (B) Schematic illustration of epitope mapping of
DhMab-4. Black bars, deletion mutants detected by DhMab-4. DGK, diacylglycerol kinase; hDGKZ, human DGKZ; MBP,
maltose-binding protein; PVDF, polyvinylidene difluoride.

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ASANO ET AL.

FIG. 3. Epitope mapping of DhMab-4 using N-terminal deletion mutants of hDGKZ. (A) Cell lysates of 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 DhMab-4 or anti-PA tag antibody (NZ-1). (B) Schematic illustration of epitope mapping of
DhMab-4.

To further investigate the crucial epitope of DhMab-4, we
produced constructs representing 15 point mutations within
hDGKZ (N745A, I746A, D747A, P748A, F749A, G750A,
A751G, T752A, P753A, F754A, I755A, D756A, P757A,
D758A, and L759A). All hDGKZ point mutants were recognized by NZ-1 (Fig. 4A, right panel). In contrast, DhMab-4
did not recognize D747A, P748A, F749A, G750A, or T752A(Fig. 4A, left panel), indicating that DhMab-4 binds to
DGKZ through these five amino acids. These results are
summarized in Figure 4B.
Discussion

DGK plays an important role in signaling pathways byregulating the intracellular level of DG and phosphatidic
acid, and it is considered a potential therapeutic target forsome diseases. Recently, we established anti-DGKa,(19,20)
anti-DGKg,(21) and anti-DGKz(22) mAbs and determined
their respective binding epitopes.(20,23–25) These mAbs arevery useful for immunohistochemical analyses. However, wehave not established anti-DGKZ mAbs for immunohistochemistry.

In this study, we established a novel anti-hDGKZ mAb,
DhMab-4. DhMab-4 can detect hDGKZ in formalin-fixedparaffin-embedded human cerebellum tissue, indicating that
DhMab-4 sensitively interacts with hDGKZ by immunohistochemical analysis (Fig. 1). We further demonstrated thebinding epitope of DhMab-4 by western blot analyses. Fiveamino acids in hDGKZ, Asp747, Pro748, Phe749, Gly750,
and Thr752, are important for DhMab-4 binding to thehDGKZ protein. This epitope is located near the accessory
domain of hDGKZ (Fig. 4C). The amino acid sequence of
this region is highly conserved among vertebrate DGKZ;
hence, DhMab-4 may be useful for detecting DGKZ in otherspecies such as dog, chicken, lizard, and zebrafish.
DGKZ is highly expressed in the brain, especially in the
hippocampus and cerebellum.(26) The expression of DGKZmRNA is increased in patients with bipolar disorder.(27,28)
Furthermore, behavioral tests using mice show that the behavioral profile is similar to human mania in DGKZ knockout
mice.(29) These results indicate that DGKZ plays a key role inbipolar disorder. In addition, DGKZ is also highly expressedin lung cancer containing EGFR mutations, and the knockdown of DGKZ has resulted in impaired growth of EGFRDownloaded by Yukinari Kato from www.liebertpub.com at 08/21/20. For personal use only.

THE EPITOPE OF ANTI-DGKH

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FIG. 4. Epitope mapping of DhMab-4 using hDGKZ point mutants. (A) Cell lysates of point mutants of hDGKZ dN705
were electrophoresed and transferred onto PVDF membranes. After blocking, the membranes were incubated with
1 mg/mL of DhMab-4 or anti-PA tag antibody (NZ-1). (B) Schematic illustration of epitope mapping of DhMab-4 andanti-PA tag antibody (NZ-1). Asp747, Pro748, Phe749, Gly750, and Thr752 are important for DhMab-4 binding tohDGKZ. (C) Schematic illustration of hDGKZ. DhMab-4 epitope is located in between catalytic domain and accessory
domain.

mutant cell lines.(30) To understand the relationship between
the expression of DGKZ and these diseases by immunohistochemical methods, a DGKZ-specific mAb has been required. DhMab-4 can be used to analyze the expression andpathophysiological function of DGKZ and it represents a
useful tool to study the molecular basis for various diseases.
Author Disclosure Statement

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

This research was supported, in part, by AMED underGrant Numbers: JP19am0401013 (Y.K.), JP19am0101078(Y.K.), and JP19ae0101028 (Y.K.) and by JSPS KAKENHI
Grant Number 17K07299 (M.K.K.) and Grant Number
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 15, 2020
Accepted: May 8, 2020