MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 37, Number 5, 2018ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2018.0026

Short Communications

DgMab-6: Antihuman DGKg Monoclonal Antibodyfor Immunocytochemistry

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Tomoyuki Nakano,1 Satoshi Ogasawara,2 Toshiaki Tanaka,1 Yasukazu Hozumi,3 Atsumi Yamaki,2Fumio Sakane,2 Yasuhito Shirai,4 Takuro Nakamura,5 Miyuki Yanaka,5 Shinji Yamada,5Mika K. Kaneko,5 Yukinari Kato,5,6 and Kaoru Goto1

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DG) to produce phosphatidic acid (PA). Sinceboth DG and PA serve as lipidic second messengers, DGK plays a pivotal role in regulating the balance of two
signaling pathways mediated by DG and PA in cellular functions. Reportedly, DGKg, one of the 10 mammalian
DGK isozymes, is involved in leukemic cell differentiation, mast cell function, and membrane traffic. Transfection studies using tagged expression vectors and immunohistochemistry on rat tissues revealed that DGKg
localizes to the cytoplasm, plasma membrane, and Golgi apparatus. However, a limited number of studies
reported the detailed localization of native protein of DGKg in human tissues and cells. In this study, wedeveloped a novel anti-DGKg monoclonal antibody, DgMab-6, which is very useful in immunocytochemistryof human cultured cells.
Keywords: DGKg, monoclonal antibody, immunocytochemical analysis

Introduction

Diacylglycerol (DG) serves as a lipidic second messenger that activates various proteins containing DGsensitive C1 domain, including conventional and novel typesof protein kinase C (PKC), RasGRP, Unc-13, and canonicaltransient receptor potential channels.(1,2) Therefore, DG
metabolism should be tightly regulated to keep its levels
within physiological range. Of the DG metabolic pathways,
diacylglycerol kinase (DGK) phosphorylates DG to produce
phosphatidic acid (PA).(3,4) In addition to DG, PA is also
revealed to serve as a messenger molecule activating
hypoxia-inducible factor 1a, atypical PKCz, and mammaliantarget of rapamycin. Thus, DGK is thought to control theswitch between DG-mediated and PA-mediated pathways.
In mammalian species, DGK constitutes an enzyme family
composed of 10 isozymes.(2,4,5) Each isozyme has a distinct
molecular structure and subcellular localization pattern. Of
DGKs, DGKg cDNA was isolated in rat(6) and human(7) as
the third DGK isozyme. DGKg is classified into type I DGK,
which contains two EF-hand motifs (Ca2+-binding site), twoZn fingers (DG-binding C1 domain), and a catalytic domain.

Reportedly, DGKg is involved in a variety of cell functions
including leukemic cell differentiation,(8) antigen-induced
mast cell degranulation,(9) and membrane traffic between theendoplasmic reticulum and Golgi apparatus.(10) Analysis of
DGKg subcellular localization was performed mainly by
cDNA transfection, which suggests that the subcellular localization of DGKg is differentially regulated in cell speciesdependent manner. In NIE-115 (a neuroblastoma cell line),
DGKg localizes to the cytoplasm and plasma membrane.(11)In U937 (a human promonocytic leukemic cell line), DGKglocalizes to the cytoplasm and translocates to the plasma
membrane in response to TPA (12-O-tetradecanoyl phorbol13 acetate), a membrane-permeant DG analogue typicallyused in cell-based studies.(8) In COS-7 (an African greenmonkey kidney fibroblast-like cell line), DGKg is targeted to
the Golgi apparatus, which is confirmed by double staining
with a Golgi marker.(12)Immunohistochemical examination using specific antibody
against DGKg is reported solely on rat tissues, including
the brain, vascular endothelium, and adrenal cortex.(6,10,13)
However, detailed morphological analysis of DGKg nativeprotein on human cells or tissues has not been reported yet,because no specific monoclonal antibody (mAb) is available

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 Applied Chemistry in Bioscience, Graduate School of Agricultural Science, Kobe University, Kobe, Japan.
5
Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan.
6
New Industry Creation Hatchery Center, Tohoku University, Sendai, Miyagi, Japan.
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to detect human DGKg by immunostaining. Herein, we report a novel antihuman DGKg mAb, DgMab-6, which is very
useful in immunocytochemical analysis of cultured cells.
Materials and Methods

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Cell lines

P3U1 and HL-60 cells were obtained from the American
Type Culture Collection (Manassas, VA) and were cultured
in the RPMI 1640 medium (Nacalai Tesque, Inc., Kyoto,Japan), supplemented with 10% heat-inactivated fetal bovineserum (Thermo Fisher Scientific, Inc., Waltham, MA) at37C in a humidified atmosphere of 5% CO2 and 95% air.
Antibiotics including 100 units/mL of penicillin and 100 mg/mLof streptomycin were added to medium.
Silencing RNA duplexes directed against human DGKg
(siDGKg1, 5¢-CCACAUCUUACUGCCCACCUCCAUA-3¢
and siDGKg2 5¢-CCACAGAGCUGAGGCCUAUAUUGAA-3¢) were purchased from Thermo Fisher Scientific, Inc.
Scrambled siRNA duplexes (AllStars Negative Control siRNA)(Qiagen, Valencia, CA) were used as control. HL-60 cells weretransfected with siRNAs against human DGKg or scrambledcontrol using Lipofectamine RNAi MAX (Thermo Fisher Scientific Inc.) according to manufacturer’s instruction.
Plasmid and recombinant protein of DGKg

Human DGKg cDNA was synthesized and was subcloned
into an expression vector, pMAL-c2 (New England Biolabs,Inc., Beverly, MA), with PA tag (GVAMPGAEDDVV)(14)
using the In-Fusion PCR cloning kit, named pMAL-c2DGKg-PA. This construct was verified by direct DNA sequencing.
Competent Escherichia coli TOP-10 cells (Thermo FisherScientific, Inc.) were transformed with the plasmid pMAL-c2DGKg-PA. They were then cultured overnight at 37C in theLuria broth 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 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) andincubated at 4C for 2 hours under gentle agitation. The resin
was then transferred to a column and washed with 20 mL
PBS. The bound protein was eluted with PA tag peptide atroom temperature in a step-wise manner (1 mL · 10).

NAKANO ET AL.

dianapolis, IN). The fused cells were grown in the RPMI
medium with hypoxanthine, aminopterin, and thymidine selection medium supplement (Thermo Fisher Scientific, Inc.).
Recombinant MBP-DGKg-PA was immobilized on Nunc
Maxisorp 96-well immunoplates (Thermo Fisher Scientific,Inc.) at a concentration of 1 mg/mL for 30 minutes. Afterblocking with 1% bovine serum albumin in 0.05% Tween20/PBS, the plates were incubated with culture supernatantfollowed by 1:3000-diluted peroxidase-conjugated antimouse IgG (Agilent Technologies, Inc., Santa Clara, CA).
The enzymatic reaction was performed with a 1-Step Ultra
TMB-ELISA (Thermo Fisher Scientific, Inc.). Optical density was measured at 655 nm using an iMark microplate
reader (Bio-Rad Laboratories, Inc., Berkeley, CA).
Immunocytochemical analysis

HL-60 cells were fixed with 2% paraformaldehyde in
0.1 M phosphate buffer (pH 7.4) in a test tube for 20 minutes
on ice. After fixation, cells were washed with PBS and wereput on a slide glass as cell smear specimen. After brief airdrying, cells were perforated with 0.3% Triton-X 100/PBS
for 20 minutes at room temperature, followed by treatment
with 5% normal goat serum in PBS (NGS/PBS) to block
nonspecific binding sites, and were incubated with 5 mg/mLof DgMab-6 or control (NGS/PBS) for overnight at room
temperature in a moist chamber. They were incubated withgoat antimouse IgG-Alexa 488 (dilution 1:300; Thermo
Fisher Scientific, 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).

Hybridoma production

Female Balb/c mice (4 weeks old) were purchased fromSLC Japan (Shizuoka, Japan). Animals were housed underspecific pathogen-free conditions. The ‘‘Animal Care andUse Committee of Tohoku University’’ approved the animal
experiments described herein. Balb/c mice were immunizedusing an intraperitoneal (i.p.) injection of MBP-DGKg-PA(100 mg) together with Imject Alum (Thermo Fisher Scientific, Inc.). After three additional immunizations, a booster
injection was administered through i.p. 2 days before thespleen cells were harvested. The spleen cells were fused withP3U1 cells using PEG1500 (Roche Diagnostics, In-FIG. 1. Immunocytochemical analysis of human HL-60
cells using DgMab-6. Cells were stained with DgMab-6 at aconcentration of 5 mg/mL. DAPI was used for nuclear
staining. Arrowheads indicate immunoreactive site in thecytoplasm. As a negative control, cells were stained without
primary antibody (lower panels). Scale bar = 10 mm.

MAB AGAINST DGKc

Densitometry and statistical analysis

Fluorescent intensity was measured by ImageJ (NationalInstitutes of Health, Bethesda, MD). Intensity per cell wasexpressed as mean – standard error of the mean. Significance was determined by Welch’s t-test at significanceof P < 0.001.

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Results and Discussion

In earlier studies, immunohistochemical examination using DGKg antibody on rat tissues showed cytoplasmic localization of this isozyme in several neurons of the brain,including cerebellar Purkinje cells.(6,15) Subsequently, another polyclonal antibody against rat DGKg was generated toexamine subcellular localization of DGKg on rat aortic endothelial cells.(10) By double immunostaining with organelle
makers, immunoreactive site for this antibody was localized
to the Golgi apparatus. A subsequent study revealed that theimmunoreactivity is detected in the Golgi apparatus of rat adrenal cortex cells.(13) These ‘‘Golgi patterns’’ of localization are
consistent with the data on transfected COS-7 cells.(12) However, morphological examination has not yet been performed onhuman cells or tissues since highly specific and sensitive mAbs
against human DGKg remained to be established.
In this study, the recombinant MBP-DGKg-PA was captured using NZ-1-Sepharose and efficiently eluted with PA231

tag peptide.(14) Mice were then immunized with MBPDGKg-PA to develop novel anti-DGKg mAbs. Using ELISA, the culture supernatants of hybridomas were screened forbinding to recombinant MBP-DGKg-PA that was purified
from E. coli. As a result, DgMab-6 (mouse IgG1, kappa) wasestablished after limiting dilution. Immunoblot analysis
demonstrated that DgMab-6 detected MBP-DGKg-PA but
not MBP-IDH1(16) (data not shown), indicating that DgMab6 is specific against DGKg in immunoblot analysis.
In this study, we used a human promonocytic leukemic cellline HL-60 cells in which mRNA expression of DGKg wasreported.(8) Immunocytochemical analysis revealed that immunoreactivity for DgMab-6 is detected as granular patternin the cytoplasm (Fig. 1). This pattern of staining is similar
to that in previous studies using rabbit polyclonal antibody,(10,13) suggesting Golgi pattern of distribution. At thismoment, we failed to identify the immunoreactive structure
as Golgi apparatus because of very narrow cytoplasmic spaceof HL-60 leukemic cells.
As a negative control, immunostaining without primary
antibody abolished the immunoreactions, suggesting thatthe images obtained from DgMab-6 antibody are authentic.
To further confirm the accuracy of the immunoreactionsof DgMab-6, we performed siRNA silencing experiment. As
shown in Figure 2, transfection with siRNA for DGKg1(siDGKg1) significantly reduced the immunoreactivity
compared with the transfection with scramble control siRNA (siControl). Similar results were obtained in the experiment using another siDGKg2 (data not shown). Theseresults confirm that DgMab-6 reacts with DGKg specificallyand correctly in human leukemic cell line HL-60 cells.
In conclusion, a novel anti-DGKg mAb, DgMab-6, sensitively and specifically reacted with human DGKg using
immunocytochemical analysis. We could not observe specific bands of DGKg using DgMab-6 in HL-60 cells by immunoblot analysis. We should further develop more usefulanti-DGKg mAbs for immunoblot or immunohistochemical
analyses.
Acknowledgments

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

K.G., Y.S., F.S., and Y.K. received research funding fromOno Pharmaceutical Co., Ltd. The other authors have noconflict of interest.
References

FIG. 2. Knockdown of DGKg using siRNA. (A) Cells
were transfected with human DGKg siRNA (siDGKg1).
After 48 hours, immunocytochemistry was performed withDgMab-6. AllStars Negative Control siRNA (siControl) wasused as a control. Scale bar = 10 mm. (B) Densitometry of theimmunoreactivity on a single cell was carried out by NIHimage J. Data (intensity/cell) are represented as mean – SE(n = 30). *P < 0.001. DGK, diacylglycerol kinase.

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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: June 28, 2018
Accepted: September 22, 2018