MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 36, Number 4, 2017ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2017.0023

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DaMab-2: Anti-Human DGKa Monoclonal
Antibody for Immunocytochemistry
Tomoyuki Nakano,1 Satoshi Ogasawara,2 Toshiaki Tanaka,1 Yasukazu Hozumi,3 Satoru Mizuno,4Eri Satoh,4 Fumio Sakane,4 Naoki Okada,5 Akinobu Taketomi,5 Ryusuke Honma,2 Takuro Nakamura,6
Noriko Saidoh,6 Miyuki Yanaka,6 Shunsuke Itai,6 Saori Handa,6 Yao-Wen Chang,6Shinji Yamada,6 Mika K. Kaneko,6 Yukinari Kato,6,7 and Kaoru Goto1

Diacylglycerol kinase (DGK) is responsible for the enzymatic conversion of diacylglycerol to phosphatidic
acid. Since both diacylglycerol and phosphatidic acid serve as signaling molecules, DGK is regarded as a hub
between diacylglycerol-mediated and phosphatidic acid-mediated signaling. One of the 10 DGK isozymes,
DGKa, is shown to be involved in T cell function. Transfection studies using tagged expression vectors
revealed that DGKa localizes to the cytoplasm and nucleus and translocates to the plasma membrane in
response to T cell receptor stimulation. However, a limited number of studies reported the localization of native
protein of DGKa in tissues and cells. In this study, we immunized mice with recombinant DGKa and developed
several anti-DGKa monoclonal antibodies (mAbs). One of the established anti-DGKa mAbs is a clone DaMab2 (mouse IgG1, kappa). In enzyme-linked immunosorbent assay, DaMab-2 recognized only DGKa, and did notreact with the other isozymes, such as DGKg, DGKz, DGKZ, and DGKd. Importantly, DaMab-2 is very usefulin immunocytochemical analysis of human cultured cells, indicating that DaMab-2 is advantageous to analyze
the localization and function of DGKa.
Keywords: DGKa, monoclonal antibody, immunocytochemical analysis

Introduction

Diacylglycerol kinase (DGK) is an enzyme that
phosphorylates diacylglycerol to produce phosphatidic
acid.(1,2) Diacylglycerol is a neutral lipid derived from various sources, such as phosphatidylinositol 4, 5-bisphosphate
and phosphatidylcholine. Diacylglycerol serves as a second
messenger, which activates conventional and novel types ofthe protein kinase C (PKC) family, RasGRP, Unc-13, andcanonical transient receptor potential channels.(1,3) In addition, phosphatidic acid is also shown to act as a messenger
molecule activating hypoxia-inducible factor 1a, atypical
PKCz, and mammalian target of Rapamycin. Thus, DGK isthought to serve as a signaling hub between diacylglycerolmediated and phosphatidic acid-mediated pathways.
DGK constitutes an enzyme family composed of 10 isozymes in mammalian species.(1–3) Each isozyme has a distinct molecular structure and subcellular localization pattern.

Of DGKs, 80-kDa DGKa is the first identified enzyme, whichcontains EF-hand motif (a Ca2+-binding site), Zn-finger (C1domain, diacylglycerol-binding site), and catalytic domain.
A wide range of cell types and tissues express DGKa, including lymphocytes, glial cells, vascular cells, hepatocytes,
adrenal medullary cells, fibroblasts, sarcoma cells, and melanoma cells.(3–11)
Functional significance of DGKa has been intensely investigated in T lymphocytes. In T cells, DGKa regulates cellproliferation in response to IL-2 stimulation(3) and is involved
in T cell receptor (TCR) signaling through modulating
RasGRP activity.(12) T cells isolated from DGKa-deficient
mice show altered activity of TCR signaling and hyperproliferation.(13) Transfection studies in T cells demonstrated
that DGKa localizes in the cytoplasm and translocates to theplasma membrane in response to the external stimuli.(14)A limited number of studies have been reported about the
localization of native protein using specific monoclonal

1

Department of Anatomy and Cell Biology, Yamagata University Faculty of Medicine, Yamagata, Japan.
Department of Regional Innovation, Tohoku University Graduate School of Medicine, Miyagi, Japan.
Department of Cell Biology and Morphology, Akita University Graduate School of Medicine, Akita, Japan.
4
Department of Chemistry, Graduate School of Science, Chiba University, Chiba, Japan.
5
Department of Gastroenterological Surgery I, Graduate School of Medicine, Hokkaido University, Hokkaido, Japan.
6
Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Miyagi, Japan.
7
New Industry Creation Hatchery Center, Tohoku University, Miyagi, Japan.
2
3

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antibodies (mAbs) because no mAb is available to detecthuman DGKa by immunostaining. Herein, we report a novelanti-human DGKa mAb, DaMab-2, which is very useful inimmunocytochemical analysis of cultured cells.
Materials and Methods

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Cell lines

P3 U1 and HeLa were obtained from the American Type
Culture Collection (Manassas, VA). P3 U1 was cultured in
the RPMI 1640 medium (Nacalai Tesque, Inc., Kyoto, Japan), supplemented with 10% heat-inactivated fetal bovine
serum (FBS) (Thermo Fisher Scientific, Inc., Waltham, MA)
at 37C in a humidified atmosphere of 5% CO2 and 95% air.
HeLa was cultured in DMEM (Wako Pure Chemical Industries Ltd., Osaka, Japan) supplemented with 10% FBS.
Antibiotics, including 100 U/mL of penicillin and 100 mg/mLof streptomycin were added to media.
Plasmid preparation

Human DGKa cDNA(15) was synthesized, and subcloned
into an expression vector, pMAL-c2 (New England Biolabs,
Inc., Beverly, MA), with PA tag (GVAMPGAEDDVV)(16–18)
using the In-Fusion PCR Cloning Kit, named pMAL-c2-DGKaPA. DGKa with PA tag was also subcloned into an expression
vector, pET41a(+) (Novagen; EMD Millipore Corp., Billerica,MA), with PA tag of N-terminus and RAP tag (DMVNPGLEDRIE)(19) of C-terminus, named pET41a-PA-DGKa-RAP. This
construct was verified by direct DNA sequencing.
Production of recombinant protein of DGKa

Competent Escherichia coli TOP-10 cells (Thermo Fisher
Scientific, Inc.) were transformed with the plasmid pMAL-c2DGKa-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 min, 4C). The recombinant PA-DGKa-RAP was expressed in transformed
E. coli BL21 (DE3) cells that were cultured for 3 h at 37C in
the presence of 1 mM of IPTG, and the bacterial pellet from a
400 mL culture was suspended in 10 mL TBS (pH 7.5).
Bacteria were lysed by sonication, and the soluble fraction
was obtained by centrifugation at 15,000 g for 15 min.
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 h under gentle agitation. The resin wasthen transferred to a column and washed with 20 mL TBS(pH 7.5). The bound protein was eluted with PA tag peptide atroom temperature in a stepwise manner (1 mL · 10).

NAKANO ET AL.

an intraperitoneal (i.p.) injection of MBP-DGKa-PA (100 mg)
together with Imject Alum (Thermo Fisher Scientific, Inc.).
After three additional immunizations, a booster injection was
administered through i.p. two days before the spleen cells
were harvested. The spleen cells were fused with P3 U1 cells
using PEG1500 (Roche Diagnostics, Indianapolis, IN). The
fused cells were grown in the RPMI medium with hypoxanthine, aminopterin, and thymidine selection medium supplement (Thermo Fisher Scientific, Inc.). Culture supernatants
were screened using enzyme-linked immunosorbent assay(ELISA) for binding to recombinant MBP-DGKa-PA and
PA-DGKa-RAP.
Enzyme-linked immunosorbent assay

Recombinant MBP-DGKa-PA and PA-DGKa-RAP were
immobilized on Nunc MaxiSorp 96-well immunoplates(Thermo Fisher Scientific, Inc.) at a concentration of 1 mg/mL
for 30 min. After blocking with 1% bovine serum albuminin 0.05% Tween20/PBS, the plates were incubated with culture
supernatant followed by 1:3000-diluted peroxidase-conjugatedanti-mouse IgG (Dako, Glostrup, Denmark). The enzymatic
reaction was performed with a 1-Step Ultra TMB-ELISA(Thermo Fisher Scientific, Inc.). The optical density wasmeasured at 655 nm using an iMark microplate reader (BioRad Laboratories, Inc., Hercules, CA).
Transfection of siRNA against human DGKa

Silencing RNA duplexes directed against human DGKa(siDGKa1, 5¢-UUAGGAGGUUGAACACCUGUCGAGG3¢ and siDGKa2 5¢-CCUGGCCUCUGGACCGGAUCGUAAA-3¢) 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 DGKa orscrambled control using Lipofectamine RNAi MAX (Thermo
Fisher Scientific, Inc.) according to the manufacturer’s instruction. After 48 h of transfection, cells were fixed andstained by DaMab-2 as described above.
Immunocytochemical analysis

HeLa cells were fixed with 2% paraformaldehyde in 0.1 M
phosphate buffer (pH 7.4) for 20 min on ice. Cells were
perforated with 0.3% Triton-X 100/PBS for 20 min at room
temperature, followed by treatment with 5% normal goat
serum in PBS (NGS/PBS) to block nonspecific binding sites,
and were incubated with 20 mg/mL of DaMab-2 or control(NGS/PBS) overnight at room temperature in a moist chamber. They were incubated with goat anti-mouse IgG-Alexa
488 (dilution 1:300; Thermo Fisher Scientific, Inc.) for 30 min
at room temperature. Cells were also treated with DAPI(Thermo Fisher Scientific, Inc.) to stain cell nuclei. They were
examined using confocal laser scanning microscopy (LSM700;
Carl Zeiss, Inc., Jena, Germany).

Hybridoma production

Results

Female BALB/c mice (4 weeks old) were purchased fromSLC Japan (Shizuoka, Japan). Animals were housed underpathogen-free conditions. The Animal Care and Use Committee of Tohoku University approved the animal experiments described herein. BALB/c mice were immunized using

The recombinant MBP-DGKa-PA was captured using NZ1-Sepharose and efficiently eluted with PA tag peptide.(16–18)
Mice were then immunized with MBP-DGKa-PA to develop
novel anti-DGKa mAbs. Using ELISA, the culture supernatantsof hybridomas were screened for binding to recombinant MBP-

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MAB AGAINST DGKALPHA

DGKa-PA or PA-DGKa-RAP that was purified from E. coli. As
a result, DaMab-2 (mouse IgG1, kappa) was established after
limiting dilution. Western blot analysis demonstrated that
DaMab-2 detected MBP-DGKa-PA, but not MBP-IDH1,(20–22)
indicating that DaMab-2 reacts with DGKa, but does not reactwith MBP tag in western blot analysis (Supplementary Fig. S1).
ELISA demonstrated that DaMab-2 reacted only with DGKa,
and did not react with the other isozymes, such as DGKg, DGKz,
DGKZ, and DGKd (data not shown).
Immunocytochemical analyses revealed that DaMab-2
reacted with HeLa cells (Fig. 1). The enlarged view clearly
demonstrated a diffuse staining pattern in both the nucleus
and cytoplasm. This pattern of staining was identical to theprevious studies using rabbit polyclonal antibody on rat aortic
smooth muscle and endothelial cells.(8,9) In addition, DaMab2 reveals faint immunoreactions in the plasma membrane.
Previous cDNA transfection studies described that DGKatranslocates to the plasma membrane in response to TCR
stimulation.(14) Negative control (without primary antibody)
showed an absence of immunoreactions, suggesting thatimages obtained from DaMab-2 antibody were authentic.
To further confirm the accuracy of the immunoreactionsof DaMab-2, we performed siRNA silencing experiments.
As shown in Figure 2, transfection with siDGKa1 significantly reduced the immunoreactivity compared with thetransfected scramble control siRNA (siCont). Similar results were obtained in the experiment using siDGKa2 (datanot shown). These results confirm that DaMab-2 reacts
with DGKa specifically and correctly in human cell line
HeLa cells.

FIG. 1. Immunocytochemical analysis using DaMab-2.
HeLa cells were stained with DaMab-2 at a concentration of
20 mg/mL. Nuclear staining was performed with DAPI. As anegative control, cells were stained without primary antibody (lower right). Enlarged view of the square is shown on
the lower left panel. N; Nucleus, C; Cytoplasm, arrowheads;
plasma membrane. Bar = 10 mm.

183

FIG. 2. Knockdown of DGKa using siRNA. DaMab-2
immunocytochemistry was performed after siRNA treatment for human DGKa (siDGKa1). AllStars Negative
Control siRNA (siCont) was used as a control. Bar = 10 mm.

Discussion

DGKa protein was purified from porcine brain in 1983,(23)followed by cDNA cloning from porcine,(24) human,(15) and rat
brains.(4) In the rat study, a rabbit polyclonal antibody against
rat DGKa was generated to determine DGKa-expressing cells
in the rat brain. Fibrous and dot-like immunoreactions weredetected in white matter by the polyclonal antibody, suggestiveof an oligodendrocyte pattern. Using this polyclonal antibody,
DGKa was shown to localize diffusely in the cytoplasm ofhepatocytes and medullary chromaffin cells,(10,11) and in boththe nucleus and cytoplasm of vascular cells(8,9) of rat tissues.
However, highly specific and sensitive mAbs against human
DGKa remain to be established.
Herein, we immunized mice with recombinant DGKaand successfully developed a clone DaMab-2 (mouse IgG1,
kappa). ELISA demonstrated that DaMab-2 recognized
only DGKa, and did not react with the other isozymes,
such as DGKg, DGKz, DGKZ, and DGKd (data notshown). Furthermore, DaMab-2 is very useful in immunocytochemical analysis of HeLa cells (Fig. 1). Unfortunately, DaMab-2 did not react with endogenous
DGKa of HeLa cells in western blot (data not shown). In
this study, we focused on the development of mAbs against
DGKa for immunocytochemistry. It might often be difficult toestablish mAbs, which are useful for both western blot analysis
and immunocytochemical analysis. The design of immunogenand screening methods to produce specific mAbs are totallydifferent when we aim to develop mAbs for western blot or
immunocytochemical analysis. In particular, it is very difficult to
produce specific and sensitive mAbs to detect endogenous proteins in every experiment. Indeed, DaMab-2 can detect recombinant DaMab-2 in western blot (Supplementary Fig. S1),
although it did not react with endogenous DGKa of HeLa cells inwestern blot. Several anti-DGKa mAbs are commerciallyavailable, and have been reported to be useful in western blot
analysis and immunocytochemical analysis. However, recombinant or overexpressed DGKa are usually used in those studies.
We need to further develop clones for detecting endogenous
DGKa in western blot analysis.
In conclusion, a novel anti-DGKa mAb, DaMab-2,
sensitively and specifically reacted with human DGKa
using immunocytochemical analyses. DaMab-2 could be
advantageous to analyze the localization and function of
DGKa.

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Acknowledgments

This work was supported in part by the Platform for Drug
Discovery, Informatics, and Structural Life Science (PDIS) fromJapan Agency for Medical Research and Development, AMED
(Y.K.), by the Regional Innovation Strategy Support Programfrom the Ministry of Education, Culture, Sports, Science, andTechnology (MEXT) of Japan (Y.K.), and by the Grant-in-Aid
for Challenging Exploratory Research from MEXT (K.G.).

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Author Disclosure Statement

Fumio Sakane, Akinobu Taketomi, Kaoru Goto, and Yukinari Kato received research funding from Ono Pharmaceutical Co., Ltd. The other authors have no conflicts of interest.
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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: March 25, 2017
Accepted: June 6, 2017