MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 38, Number 4, 2019ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2019.0024

DzMab-1: Anti-Human Diacylglycerol Kinasez Monoclonal
Antibody for Immunocytochemistry

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Tomoyuki Nakano,1 Satoshi Ogasawara,2 Toshiaki Tanaka,1 Yasukazu Hozumi,3 Masato Sano,4Yusuke Sayama,4 Shinji Yamada,4 Mika K. Kaneko,4 Yukinari Kato,4,5 and Kaoru Goto1Diacylglycerol kinase (DGK) is an enzyme that converts diacylglycerol (DG) to phosphatidic acid (PA). As both
DG and PA serve as lipidic second messengers, DGK plays a pivotal role in controlling the balance of two signaling
pathways mediated by DG and PA in cellular functions. DGKz, one member of the mammalian DGK family, isreported to contain a nuclear localization signal, which suggests its functional role in the nucleus. Previously,morphological studies using tagged expression vectors and immunostaining of rat tissues or cells have revealed thatDGKz localizes mainly to the nucleus. However, a limited number of studies reported the detailed localizationof native protein of DGKz in human tissues and cells. In this study, we developed a novel anti-human DGKz
monoclonal antibody, DzMab-1, which is very advantageous in immunocytochemistry of human cultured cells.
Keywords: DGKz, monoclonal antibody, immunocytochemical analysis

Introduction

Diacylglycerol (DG) is a neutral lipid that is produced from various sources including phosphatidylinositol 4,5-bisphosphate, phosphatidylcholine, and triglyceride.
Functionally, DG is not just an intermediate of lipid metabolism, but serves as a signaling molecule, which activates
RasGRP, conventional and novel types of protein kinase C(PKC), Unc-13, and canonical transient receptor potentialchannels.(1,2) Within the cell, DG is distributed to the membranous structures, such as the plasma membrane, nuclear
envelope, and endoplasmic reticulum membrane, and also to
the nuclear matrix.(3) Therefore, DG metabolism should betightly regulated to keep its levels within physiological range
in the biological membranes and nucleus.(4)One mechanism responsible for this regulation is an action of DG kinase (DGK), an enzyme that phosphorylates DG
to produce phosphatidic acid (PA).(4,5) Because PA, a product of DGK, also serves as a second messenger activating
hypoxia-inducible factor 1a, atypical PKCz and mammaliantarget of Rapamycin, DGK is thought to control the switch
between DG-mediated and PA-mediated pathways.
DGK constitutes an enzyme family composed of 10 isozymes in mammalian species.(2,5,6) Each isozyme has distinct
properties of enzymatic activity, molecular structure, andsubcellular localization.(6) Of the DGKs, DGKz is classified

into type IV DGK, which contains two EF-hand motifs(Ca2+ binding site), two Zn-fingers (DG-binding C1 domain),nuclear localization signal, and a catalytic domain.(7,8) It isreported that DGKz is expressed ubiquitously in mammalianorgans, including the brain, lung, liver, muscle, and pituitary
gland.(9) Subcellular localization of DGKz has been examined by cDNA transfection technique and immunohistochemistry on rat tissues by polyclonal antibody raised against
rat DGKz.(6,10) These studies show that DGKz predominantly
localizes to the nucleus, but in some occasions such as transient brain ischemia or kainite-induced seizure, DGKztranslocates from the nucleus to the cytoplasm in hippocampal neurons.(11,12) However, detailed morphologicalanalysis of native DGKz protein on human tissues or cells has
not been reported yet, because no specific monoclonal antibody (mAb) is available to detect human DGKz by immunostaining. In this study, we report a novel anti-human
DGKz mAb, DzMab-1, which is a very advantageous tool in
immunocytochemical analysis of cultured human cells.
Materials and Methods
Cell lines

HeLa cells were obtained from the American Type Culture
Collection (Manassas, VA) and were cultured in the Dulbecco’s modified Eagle’s medium (Nacalai Tesque, Inc.,

1

Department of Anatomy and Cell Biology, Yamagata University Faculty of Medicine, Yamagata, Japan.
Department of Chemistry, Graduate School of Science, Chiba University, Chiba, Japan.
3
Department of Cell Biology and Morphology, Akita University Graduate School of Medicine, Akita, Japan.
4
Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
5
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
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180Kyoto, Japan), supplemented with 10% heat-inactivated fetalbovine serum (Thermo Fisher Scientific, Inc., Waltham, MA)
at 37C in a humidified atmosphere of 5% CO2 and 95% air.
Antibiotics, including 100 U/mL of penicillin and 100 mg/mLof streptomycin were added to media.
Silencing RNA duplexes directed against human DGKz(siDGKz1, 5¢-CCAGUCUUUCCUCUGGUAUCUCAAU-3¢
and siDGKz2 5¢-GCCGCUUUCGGAAUAAGAUGUUCUA3¢) were purchased from Thermo Fisher Scientific, Inc.
Scrambled siRNA duplexes (AllStars Negative Control siRNA)(Qiagen, Valencia, CA) were used as control. HeLa cells
were transfected with siRNAs against human DGKz or
scrambled control using Lipofectamine RNAi MAX (ThermoFisher Scientific, Inc.) according to manufacturer’s instruction.

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Plasmid preparation

Mouse DGKz (mDGKz) cDNA and human DGKz (hDGKz)
cDNA were synthesized and subcloned into an expression
vector, pMAL-c2 (New England Biolabs, Inc., Beverly,MA), with PA tag (GVAMPGAEDDVV)(13) using the InFusion HD Cloning Kit (Takara Bio, Inc., Shiga, Japan); theresultant construct was named pMAL-c2-mDGKz-PA andpMAL-c2-hDGKz-PA, respectively. The deletion mutants ofhDGKz produced using PCR were subcloned into pMAL-c2with PA tag using the In-Fusion HD Cloning Kit. The substitution of hDGKz amino acids 1–10 with alanine in dC200of hDGKz was performed using the QuikChange LightningSite-Directed Mutagenesis Kit (Agilent Technologies, Inc.,
Santa Clara, CA). These constructs were verified using direct
DNA sequencing.
Production of recombinant protein of DGKz

Competent Escherichia coli TOP-10 cells (Thermo FisherScientific, Inc.) were transformed with the plasmid pMALc2-DGKz-PA. They were then cultured overnight at 37C in
the LB medium (Thermo Fisher Scientific, Inc.) containing
100 mg/mL ampicillin (Sigma-Aldrich Corp., St. Louis, MO).
Cell pellets were resuspended in phosphate-buffered saline(PBS) with 1% Triton X-100 and 50 mg/mL aprotinin (SigmaAldrich Corp.). After sonication, the crude extracts werecollected 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 weremixed with NZ-1-Sepharose (1 mL bed volume) and incubated at 4C for 2 hours under gentle agitation. The resin wasthen transferred to a column and washed with 20 mL Trisbuffered saline (pH 7.5). The bound protein was eluted with
PA tag peptide at room temperature in a stepwise manner(1 mL · 10).
Hybridoma production

DzMab-1 was produced using rat medial iliac lymph nodemethods. The Animal Care and Use Committee of Tohoku
University approved all animal experiments. In brief,WKY/Izm rats ( Japan SLC, Inc., Shizuoka, Japan) wereimmunized by injecting 50 mg of pMAL-c2-mDGKz-PA
proteins together with Freund’s complete adjuvant (SigmaAldrich Corp.) into the footpad. Additional immunization of
74 mg of pMAL-c2-mDGKz-PA proteins were performed
into the tailbase. The lymphocytes were fused with mouse

NAKANO ET AL.

myeloma Sp2/0-Ag14 cells using polyethylene glycol. The
culture supernatants were screened using enzyme-linked
immunosorbent assay (ELISA) for the binding to pMAL-c2mDGKz-PA proteins. Then, second screening was performed
using pMAL-c2-hDGKz-PA.
Western blotting

Lysates were immunoprecipitated using a NZ-1-Sepharoseand boiled in sodium dodecyl sulfate sample buffer (NacalaiTesque, Inc.). The samples were electrophoresed on 5%–20%
polyacrylamide gels (Nacalai Tesque, Inc.) and transferred
onto a polyvinylidene difluoride membrane (Merck KGaA,Darmstadt, Germany). After blocking with 4% skim milk
(Nacalai Tesque, Inc.) for 1 hour, the membrane was incubated with DzMab-1 or NZ-1 for 1 hour, followed by biotinconjugated anti-rat immunoglobulin G (IgG) (1:1000 dilution;Agilent Technologies, Inc.) for 1 hour, and further incubatedwith VECTASTAIN Elite ABC HRP Kit (Vector Laboratories, Inc., Burlingame, CA) for 30 minutes. The membrane
was finally developed with the ImmunoStar LD Chemiluminescence Reagent (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) using the Sayaca-Imager (DRC Co.,Ltd., Tokyo, Japan). All procedures of western blotting were
performed at the room temperature.
Immunocytochemical analysis

HeLa cells were fixed with 2% paraformaldehyde in 0.1 M
phosphate buffer (pH 7.4) for 20 minutes on ice. After fixation, cells were perforated with 0.3% Triton-X 100/PBS for
30 minutes at room temperature, followed by treatment with
5% bovine serum albumin (BSA) in PBS (BSA/PBS) to block
nonspecific binding sites, and were incubated with 10 mg/mLof DzMab-1 or control (BSA/PBS) for overnight at room
temperature in a moist chamber. They were incubated withgoat anti-rat IgG-Alexa 488 (dilution 1:300; Thermo FisherScientific, Inc.) for 30 minutes at room temperature. Cells
were also treated with DAPI (Thermo Fisher Scientific, Inc.)
to stain the cell nuclei. They were examined using confocal
laser scanning microscopy (LSM700; Carl Zeiss, Inc., Jena,
Germany).
Results and Discussion

Previously, immunohistochemical studies using rabbit
polyclonal DGKz antibody on rat tissues showed predominant localization of the immunoreactivity in the nuclei ofvarious cell types, including neurons, hepatocytes, alveolar
epithelial cells, and macrophages.(6) Nuclear localization ofDGKz immunoreactivity is consistent with cDNA tranfection
studies in COS-7 cells and primary cultured neurons.(10,14)
As this antibody was not useful in immunohistochemical
study on human tissues and cells, we aimed at obtaining a
specific DGKz antibody that is suitable for immunostainingof human cells. The recombinant maltose-binding protein(MBP)-mDGKz-PA was captured using NZ-1-Sepharose andefficiently eluted with PA tag peptide. Rat was then immunized with MBP-mDGKz-PA to develop novel anti-mDGKzmAbs. Using ELISA, the culture supernatants of hybridomas
were screened for the binding to MBP-mDGKz-PA that waspurified from E. coli. Furthermore, MBP-hDGKz-PA wasused as a second ELISA screening. As a result, DzMab-1 (ratMAB AGAINST DGKf

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FIG. 1. Specificity of DzMab-1 for DGKz. Immunoprecipitates of hDGKa, hDGKg, and hDGKz by anti-PA tag(NZ-1) were electrophoresed and transferred onto membranes. After blocking, the membranes were incubated with
10 mg/mL of DzMab-1 and 1 mg/mL of NZ-1, respectively.
hDGK, human diacylglycerol kinase; PA, phosphatidic acid.

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Immunostaining without primary antibody abolished theimmunoreactions (data not shown). Furthermore, we performed siRNA silencing experiment. As given in Figure 3,transfection with 20 pmol of siRNA for DGKz (siDGKz)
significantly reduced the immunoreactivity for DzMab-1
compared with the transfection with scramble control siRNA(siControl). These results confirm that DzMab-1 reacts withnative DGKz protein specifically in human HeLa cells andthat the images obtained with this antibody is authentic.
In addition, it should be mentioned that many of the nuclei
in cells transfected with 40 pmol of siDGKz are shrunk orirregular (Fig. 3, lower panels). Nuclear deformation is moreevident in cells transfected with 60 pmol of siDGKz (datanot shown). No changes in the nuclear morphology wereIgG1, kappa) that can react with both MBP-mDGKz-PA
and MBP-hDGKz-PA was established after limiting dilution. Western blot analysis demonstrated that DzMab-1
detected MBP-hDGKz-PA but not MBP-hDGKa-PA or
MBP-hDGKg-PA (Fig. 1), indicating that DzMab-1 specifically reacts with hDGKz.
In this study, we used HeLa cells, a human cervical cancer cell line, in which expression of DGKz protein is reported.(15,16) Immunocytochemical analysis revealed that
immunoreactivity for DzMab-1 is predominantly detectedin the nucleus with a granular pattern of labeling and lessintensely in the cytoplasm with a diffuse pattern (Fig. 2).
Immunoreactivity observed in this study is similar to the one
in previous studies using rabbit polyclonal antibody.(17,18)

FIG. 2. Immunocytochemistry using DzMab-1 in HeLa
cells. HeLa cells were stained with DzMab-1 antibody at aconcentration of 10 mg/mL. Nuclear staining was performedwith DAPI. Enlarged view (lower left panel) clearly shows
granular staining in the nucleus (N) and diffuse staining inthe cytoplasm. Bar = 10 mm.

FIG. 3. Knockdown of DGKz using siRNA in HeLa
cells. Cells were transfected with siDGKz at an amount of20 pmol (upper panels) and 40 pmol/60 mm dish (lowerpanels). After 48 hours, immunocytochemistry was performed using DzMab-1. Note that shrunk or irregular nuclei
are frequently observed in 40 pmol siDGKz-treated cells(asterisks). AllStar Negative Control siRNA (siCont) wasused as a control. Bar = 10 mm.

182

observed in cells transfected with siControl. These findings
raise a possibility that reduced levels of DGKz could induce
nuclear deformability.
In this regard, we previously reported that DGKz knockdown induces cleavage of PARP, an apoptotic marker, inHeLa cells after treatment with doxorubicin,(15) suggestingthat DGKz downregulation renders cells vulnerable to cell
death in response to genotoxic stress. This seems to be correlated with the present findings that DGKz knockdown induces nuclear deformation, showing that DGKz is deeply
involved in the regulation of nuclear morphology and function.
In conclusion, a novel anti-DGKz mAb, DzMab-1, specifically reacts with native human DGKz protein of HeLa
cells in immunocytochemistry.

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Acknowledgments

This work was supported in part by AMED under Grant
Number JP18am0101078 (Y.K.) and by the Grant-in-Aid for
Challenging Exploratory Research from MEXT (T.N., K.G.).
Author Disclosure Statement

K.G. and Y.K. received research funding from Ono Pharmaceutical Co., Ltd. The other authors have no conflicts ofinterest.
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Address correspondence to:
Kaoru Goto
Department of Anatomy and Cell Biology
Yamagata University Faculty of Medicine
2-2-2 Iida-nishi
Yamagata 990-9585
JapanE-mail: kgoto@med.id.yamagata-u.ac.jp
Received: May 14, 2019
Accepted: June 24, 2019