MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 40, Number 6, 2021
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2021.0034
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Immunocytochemical Analysis of DGKZ in Cultured Cells
Using a Monoclonal Antibody DhMab-4

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Tomoyuki Nakano,1 Toshiaki Tanaka,1 Fumio Sakane,2 Mika K. Kaneko,3 Yukinari Kato,3,4,i and Kaoru Goto1

Diacylglycerol kinase (DGK) is a lipid kinase that converts diacylglycerol (DG) to phosphatidic acid (PA).
Since both DG and PA serve as intracellular second messenger molecules, DGK plays a pivotal role inbalancing these two signaling pathways. Of the DGK family, DGKZ is classified as a type II DGK. Reportedly,
DGKZ is expressed ubiquitously through mammalian tissues and cells. Previous studies using cDNA transfection methods reported cytoplasmic localization of DGKZ in cultured human cells. However, subcellularlocalization of native protein is still unknown. Recently, we established a human DGKZ-specific monoclonalantibody, DhMab-4. In this study, we examined subcellular localization of native protein of DGKZ using
DhMab-4 by immunocytochemistry in human cultured cells.
Keywords: DGKZ, monoclonal antibody, immunocytochemical analysis

Introduction

Diacylglycerol (DG) is a unique lipid, which represents as an intermediate product of phospholipid
metabolism and also serves as an intracellular signaling
molecule. DG activates several proteins including conventional and novel types of protein kinase C (PKC), RasGRP,
Unc-13, and canonical transient receptor potential channels.(1,2)
DG is produced through several enzymatic pathways, such as(1) hydrolysis of phosphatidylinositol 4,5-bisphosphate byphospholipase C, (2) hydrolysis of phosphatidylcholine bysequential action of phospholipase D and phosphatidic acid(PA) phosphatase, and (3) acylation of monoacylglycerol(MG) by MG acyltransferase during triglyceride synthesis.(2–5)
Among DG-converting enzymes, diacylglycerol kinase(DGK) phosphorylates DG to produce PA.(1,3) Since PA, aproduct of DGK, also serves as a second messenger activating various proteins such as hypoxia inducible factor 1a,
atypical PKCz, and mammalian target of rapamycin, DGK isthought to balance DG-mediated and PA-mediated pathways.
DGK family consists of 10 isozymes in mammalian species.(1–3) Each isozyme has distinct molecular structures andsubcellular localizations.(1,6) Of the DGKs, DGKZ is classi-

fied as a type II DGK, which contains pleckstrin homology
domain, two Zn -fingers (DG-binding C1 domain), separated
catalytic domain, and sterile a-motif domain.(2,7) Reportedly,
DGKZ is expressed ubiquitously in mammalian organs, including the brain, liver, lung, kidney, thymus, spleen, and reproductive organ.(7–9)
Previous studies using cDNA transfection technique disclosed that DGKZ localizes to the cytoplasm under normal conditions.(7,8) However, detailed localization of native
DGKZ protein remains undetermined, because no specificantibody is available for immunostaining. Herein, we reporta novel anti-human DGKZ (hDGKZ) monoclonal antibody,DhMab-4, which is very useful in immunocytochemical analysis of cultured human cells.
Materials and Methods
Cell lines

HeLa and HepG2 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.,Kyoto, Japan), supplemented with 10% heat-inactivated fetal
bovine serum (Thermo Fisher Scientific, Inc., Waltham, MA)

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.
Departments of 3Antibody Drug Development and 4Molecular Pharmacology, Tohoku University Graduate School of Medicine,Sendai, Japani
ORCID ID (https://orcid.org/0000-0001-5385-8201).
2261

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cells were transfected with siRNAs against hDGKg orscrambled control using Lipofectamine RNAi MAX (ThermoFisher Scientific, Inc.) according to manufacturer’s instruction.
Immunocytochemical analysis

HeLa and HepG2 cells were fixed with 3% glyoxal fixative(10) for 30 minutes on ice, followed by at room temperature for additional 30 minutes. After fixation, cells wereDownloaded by Yukinari Kato from www.liebertpub.com at 12/27/21. For personal use only.

at 37C in a humidified atmosphere of 5% CO2 and 95% air.
Antibiotics, including 100 units/mL of penicillin, 100 lg/mL ofstreptomycin, were added to media.
Silencing RNA duplexes directed against hDGKg(siDGKg1, 5¢- CCAAGACGCUAUGUGAAACUGUAAA-3¢
and siDGKg2, 5¢- CAAGGGAAAUCAUGUUGCGGGCA
AA-3¢) were purchased from Thermo Fisher Scientific,
Inc. Scrambled siRNA duplexes (AllStars Negative Control
siRNA; Qiagen, Valencia, CA) were used as control. HeLa

NAKANO ET AL.

FIG. 1. Immunocytochemistry of HeLa cells using DhMab-4 in the presence (A) or absence (B) of the antibody. Inset in(A) is an enlarged view of squared area. Nuclear staining was performed with DAPI. Arrows indicate perinuclear staining.
Scale bar = 10 mm. (C) Immunocytochemistry using DhMab-4 in siRNA-treated HeLa cells. Cells were transfected withsiDGKZ1 or siDGKZ2 at an amount of 60 pmol/60 mm dish. After 48 hours, immunocytochemistry was performed using
DhMab-4. Nuclear staining was performed with DAPI. AllStar Negative Control siRNA (siControl) was used as a control.
Scale bar = 20 mm.

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They were examined using confocal laser-scanning microscopy (LSM700; Carl Zeiss, Inc., Jena, Germany).
Results and Discussion

Previous studies revealed that DGKZ is targeted to thecytoplasm in transfected cultured cells including COS-7,HeLa, and NEC8 (human embryonal carcinoma cells derived
from testis) cells.(7,8,11) In this study we aimed to investigate
detailed subcellular localization of native DGKZ protein inhuman cells. To this end, we established a specific monoclonal antibody against hDGKZ (DhMab-4).(12)In our previous study, DhMab-4 strongly stained Purkinjecells of human cerebellum in immunohistochemical analysis.(12) However, since it is hard to obtain fresh human brain
specimen for morphological examination at subcellular level,
we used in this study human cervical cancer cell line HeLa

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perforated with 0.3% Triton-X 100/phosphate-bufferedsaline (PBS) for 15 minutes at room temperature, followed bytreatment with 5% normal goat serum (NGS) in PBS to block
nonspecific binding sites, and were incubated with 0.5 lg/mLof DhMab-4 or control (NGS/PBS) for overnight at room
temperature in a moist chamber. In some cases, cells weredouble stained with cytochrome oxidase subunit 4 (COX IV)(dilution 1:200; Cell Signaling Technology, Inc., Danvers,MA, USA), calreticulin (dilution 1:200; Novus Biological,Centennial, CO, USA) or Golgin 97 (dilution 1:200; Thermo
Fisher Scientific, Inc.) antibodies as organelle markers.
Immunoreactions for DhMab-4 and organelle markers
were visualized with anti-mouse IgG-Alexa 488 (dilution1:300; Thermo Fisher Scientific, Inc.) and anti-rabbit IgGAlexa 594 (Thermo Fisher Scientific, Inc.), respectively, for
30 minutes at room temperature. Cells were also reacted withDAPI (Thermo Fisher Scientific, Inc.) to stain the cell nuclei.

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FIG. 2. Double immunostaining of DhMab-4 with the organelle markers in HeLa cells. (A) Anti-calreticulin antibody(endoplasmic reticulum), (B) anti-Golgin 97 antibody (trans-Golgi network), (C) COX IV antibody (mitochondria). Nuclear
staining was performed with DAPI. Scale bar = 10 mm. COX IV, cytochrome oxidase subunit 4.

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264

cells, in which DGKZ protein expression is reported.(11)
Immunocytochemical examination showed the immunoreactivity for DhMab-4 is predominantly detected in a granularmanner in the cytoplasm (Fig. 1 A). On closer examination,
the immunoreactivity was rather intensely recognized in theperinuclear region (arrows in Fig. 1A). The same stainingpattern was obtained in another human cell line HepG2 cells(data not shown).
As a control experiment, immunostaining without primary
antibody abolished the immunoreaction (Fig. 1B), showing
no artifact in our antigen–antibody detection method. To
verify the specificity of DhMab-4 in immunocytochemistry,
we performed siRNA silencing experiment. As shown inFigure 1C, transfection with siRNAs for DGKZ (siDGKZ1and -Z2) significantly reduced DhMab-4 immunoreactivity,
whereas transfection with scramble control siRNA (siControl) gave no effect. These results confirm that DhMab-4
stains native DGKZ protein in human culture cells.
We next performed double immunostaining to determine
whether native DGKZ protein specifically localizes to someorganelle in the cytoplasm. We used anti-calreticulin antibody(a marker for the endoplasmic reticulum(13)), anti-Golgin
97 (a marker for trans-Golgi network(14)), and anti-COX. IV(a marker for mitochondria). None of these marker antibodies was colocalized with DhMab-4 (Fig. 2). These double
staining results demonstrate that DGKZ native protein localizes to the cytoplasm but is not associated with distinct organelles such as endoplasmic reticulum (ER) Golgi complex,and mitochondria.
Reportedly, DGKZ translocates to the endosomes by theosmotic stress in cDNA-transfection studies.(7,8) Considering
our present data, granular pattern of DGKZ protein in thecytoplasm might represent the endosomes, if not all. Endosomes are membrane-bound cytosolic vesicles, which include early, late, and recycling endosomal compartments.
These small membrane-bound structures play some roles inendocytosis or cargo between the ER and Golgi complex.(15)
From the functional point of view, DGKZ is identified
as a bipolar disorder susceptibility gene.(16–18) Furthermore,
DGKZ is also implicated in oncogenic properties of lungcancers and DGKZ knockdown leads to growth impairment ofepidermal growth factor receptor pathway.(19) These results
suggest that DGKZ plays a pivotal role in higher brain function
and tumorigenesis. Further study is needed to elucidate thesepoints. This study showed that a novel anti-DGKZ mAb,
DhMab-4, specifically reacts with native hDGKZ protein in
immunocytochemistry and localizes to the cytoplasm of humancultured cells. To gain an insight understanding the relationshipbetween these disorders and morphological aspects of DGKZ,
a specific antibody DhMab-4 would be a useful tool.
Author Disclosure Statement

K.G. and Y.K. received research funding from Ono Pharmaceutical Co., Ltd. The other authors have no conflict ofinterest.
Funding Information

This study was supported in part by AMED under Grant
No. JP21am0101078 (Y.K.) and by the grant-in-aid fromMEXT (T.N. and K.G.).

NAKANO ET AL.
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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: July 13, 2021
Accepted: November 17, 2021