MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 39, Number 1, 2020
Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2019.0004

Original Articles

Establishment and Epitope Mapping of Anti-Diacylglycerol
Kinase a Monoclonal Antibody DaMab-8
for Immunohistochemical Analyses
Masato Sano,1 Mika K. Kaneko,1 Hiroyoshi Suzuki,2 and Yukinari Kato1,3

Diacylglycerol kinase (DGK) converts diacylglycerol (DG) into phosphatidic acid (PA). DGKa, 1 of the 10
DGK isozymes, is involved in T cell function. In the present study, we describe a specific monoclonal antibody
DaMab-8 (mouse IgG1, kappa) against DGKa, which is extremely useful for performing immunohistochemical
analysis for T cells in oropharyngeal squamous cell carcinomas. Furthermore, we characterized the binding
epitope of DaMab-8 using Western blotting and found that the sites Asn610, Leu611, Trp612, Gly613, Asp614,His619, Tyr623, and Gly624 of DGKa are important for facilitating the DaMab-8 binding to the DGKa protein.
Thus, DaMab-8 could be advantageous for immunohistochemical analyses toward clarifying the distribution ofDGKa-expressing T cells in every pathophysiological tissue.
Keywords: diacylglycerol kinase, DGKa, monoclonal antibody, epitope mapping
Because only few specific anti-DGKa monoclonal antibodies(mAbs) are available to detect human DGKa using immunohistochemistry, the localization of DGKa-expressing cells remains unclear. Recently, we have developed DaMab-2 (mouseIgG1, kappa), a specific mAb against DGKa.(7) DaMab-2 isextremely useful in immunocytochemical analysis using HeLacells. We further characterized the binding epitope of DaMab-2
using Western blotting and revealed that the Cys246, Lys249,
Pro252, and Cys253 sites of DGKa are important for facilitating
DaMab-2 binding to the DGKa protein.(8) However, DaMab-2
was not applicable for immunohistochemical analysis.
In the present study, we report a novel anti-human DGKa
mAb DaMab-8 (mouse IgG1, kappa) that is extremely useful inimmunohistochemical analysis. Furthermore, we have characterized the binding epitope of DaMab-8 using Western blotting.

IntroductionDiacylglycerol kinase (DGK) phosphorylates diacylglycerol (DG) to produce phosphatidic acid (PA).(1,2)
DG is a neutral lipid derived from various sources, including
phosphatidylinositol 4,5-bisphosphate and phosphatidylcholine, and it serves as a second messenger that activates theconventional and novel types of the protein kinase C (PKC)
family, RasGRP, Unc-13, and canonical transient receptor
potential channels.(2,3) PA functions as a messenger moleculethat activates the hypoxia-inducible factor (HIF)-1a, atypical
PKCz, and mammalian target of rapamycin. DGK constitutes
an enzyme family comprising 10 isozymes of the mammalianspecies.(1,2) Each isozyme possesses a distinct molecularstructure and a subcellular localization pattern. DGKa is thefirst identified enzyme of 80-kDa size that contains an EF-handmotif (Ca2+-binding site), a Zn finger (C1 domain, DG-bindingsite), and a catalytic domain. DGKa regulates cell proliferationin response to IL-2 stimulation in T cells(3) and is involved inT cell receptor (TCR) signaling via the modulation of theRasGRP activity.(4) T cells isolated from DGKa-deficient micedemonstrate an altered activity of TCR signaling and hyperproliferation.(5) DGKa is expressed in T lymphocytes abundantly, in which it facilitates the nonresponsive state known as
clonal anergy.(5) Anergy induction in T cells represents the main
mechanism by which advanced tumors avoid immune action.(6)Materials and Methods
Plasmid preparation

Human DGKa cDNA(9) was synthesized and subcloned
into the expression vector pMAL-c2 (New England Biolabs,Inc., Beverly, MA), along with PA tag (GVAMPGAEDDVV),(10) using the In-Fusion HD Cloning Kit (TakaraBio, Inc., Shiga, Japan); the resultant construct was namedpMAL-c2-DGKa-PA. The deletion mutants of DGKa

1Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
Department of Pathology and Laboratory Medicine, Sendai Medical Center, Sendai, Japan.
3
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.
2ª Masato Sano et al. 2019; Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the terms of the CreativeCommons License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium,provided the original work is properly cited.

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produced using PCR were subcloned into pMAL-c2 with PA
tag using the In-Fusion PCR Cloning Kit. The substitution ofDGKa amino acids 605–630 with either alanine or glycine in
dN561 of DGKa was performed using the QuikChangeLightning Site-Directed Mutagenesis Kit (Agilent Technologies, Inc., Santa Clara, CA). These constructs were verified
using direct DNA sequencing.
Western blottingCompetent Escherichia coli TOP-10 cells (Thermo Fisher
Scientific, Inc., Waltham, MA) were transformed and cultured overnight at 37C in Luria–Bertani medium (ThermoFisher Scientific, Inc.,) containing 100 mg/mL ampicillin(FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan).
The cell pellets were resuspended in phosphate-buffered solution containing 1% Triton X-100 and 50 mg/mL aprotinin(Sigma-Aldrich). Lysates were immunoprecipitated using
amylose resin (New England Biolabs, Inc.) and boiled in sodium dodecyl sulfate (SDS) sample buffer (Nacalai Tesque,Inc., Kyoto, Japan). The samples were electrophoresed on
5%–20% polyacrylamide gels (FUJIFILM Wako Pure Chemical Corporation) and transferred onto a polyvinylidene
difluoride membrane (Merck KGaA, Darmstadt, Germany).
After blocking with 4% skim milk (Nacalai Tesque, Inc.) forSANO ET AL.

1 hour, the membrane was incubated with DaMab-8 for 1hour, followed by peroxidase-conjugated anti-mouse IgG(1:2000 dilution; Agilent Technologies, Inc.) for 1 hour. Themembrane was also incubated with NZ-1 (anti-PA tag) for 1hour, followed by biotin-conjugated anti-rat IgG (1:1000dilution; Agilent Technologies, Inc.) for 30 minutes, andfurther incubated with the avidin–biotin complex (Vectorlaboratories, Inc., Burlingame, CA) for 30 minutes. The
membrane was finally developed with the ImmunoStar LD
Chemiluminescence Reagent (FUJIFILM Wako Pure Chemical Corporation) using the Sayaca-Imager (DRC Co., Ltd.,Tokyo, Japan). All procedures of Western blotting wereperformed at room temperature.
Immunohistochemical analyses

Our study examined a patient with oropharyngeal squamous cell carcinoma who underwent surgery at the Sendai
Medical Center. Informed consent for sample procurement
and subsequent data analyses was obtained from the patientor the patient’s guardian. The tissue samples were processedto produce 4-mm paraffin-embedded tissue sections that weredirectly autoclaved in citrate buffer (pH 6.0; Nichirei Biosciences, Inc., Tokyo, Japan) for 20 minutes and blocked
using the SuperBlock T20 (PBS) Blocking Buffer (ThermoFIG. 1. Immunohistochemical analysis using DaMab-8 against oropharyngeal squamous cell carcinomas. Tissue sections
were incubated with DaMab-8 (1 mg/mL; A, B) or blocking buffer (C, D) for 1 hour at room temperature and treated using
the Envision Kit (Agilent Technologies, Inc.) for 30 minutes. Scale bar = 100 mm. (E, F) Hematoxylin and eosin staining.

FIG. 2. Production of DGKa deletion mutants. (A) Immunoprecipitates of deletion mutants were electrophoresed andtransferred onto PVDF membranes. After blocking, the membranes were incubated with 1 mg/mL of DaMab-8 or anti-PA tag(NZ-1), followed by incubation with peroxidase-conjugated anti-mouse or anti-rat IgG. (B) Schematic illustration of DaMab-8
epitope. Black bars, deletion mutants detected by DaMab-8; white bars, deletion mutants not detected by DaMab-8. hDGKa,human diacylglycerol kinase a; MBP, maltose-binding protein; PVDF, polyvinylidene difluoride.

FIG. 3. Production of additional deletion mutants of DGKa. (A) Immunoprecipitates of deletion mutants were electrophoresed and transferred onto PVDF membranes. After blocking, the membranes were incubated with 1 mg/mL of DaMab-8
and anti-PA tag (NZ-1), followed by incubation with peroxidase-conjugated anti-mouse IgG and biotin-conjugated anti-rat
IgG, respectively. (B) Schematic illustration of DaMab-8 epitope. Black bars, deletion mutants detected by DaMab-8; whitebars, deletion mutants not detected by DaMab-8.
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4Fisher Scientific, Inc.,), incubated with DaMab-8 (1 mg/mL)
for 1 hour at the room temperature, and then treated using theEnvision Kit (Agilent Technologies, Inc.) for 30 minutes.
The tissue sections were stained using 3,3¢-diaminobenzidine
tetrahydrochloride (DAB; Agilent Technologies, Inc.) for 2
minutes, and counterstaining was performed using hematoxylin (FUJIFILM Wako Pure Chemical Corporation).
Results and Discussion

Several anti-DGKa mAbs are commercially available andare reportedly useful in Western blotting and for immunohistochemical analyses.(11) Furthermore, we have developed
DaMab-2 (mouse IgG1, kappa), a specific mAb against DGKa,
which is extremely useful in immunocytochemical analysis.(7)The binding epitopes of DaMab-2 were determined to beCys246, Lys249, Pro252, and Cys253 of DGKa.(8) Unfortunately, DaMab-2 was not applicable for immunohisto-SANO ET AL.

chemical analysis. We have previously immunized mice withrecombinant DGKa and developed several anti-DGKclones.(7) One of these clones, DaMab-8, recognized only
DGKa in ELISA and showed no reaction with other isozymes, such as DGKg, DGKz, DGKZ, and DGKd (data notshown). DGKa was reported to be expressed in T lymphocytes
abundantly, in which it facilitates the nonresponsive state
known as clonal anergy.(5) Immunohistochemical screening
revealed that DaMab-8 was extremely useful in immunohistochemical analysis for T cells in oropharyngeal squamous cell
carcinomas (Fig. 1).
We next moved on to the determination of the binding
epitope of DaMab-8. As shown in Figure 2, we produced 10 Nterminal deletion mutants of DGKa (i.e., dN541, dN561,dN581, dN601, dN621, dN641, dN661, dN681, dN701, anddN721). Western blotting demonstrated that DaMab-8 detected dN541, dN561, dN581, and dN601 but not dN621,dN641, dN661, dN681, dN701, and dN721, although allFIG. 4. Epitope mapping of DaMab-8 using point mutants of DGKa. (A) Immunoprecipitates of point mutants were
electrophoresed and transferred onto PVDF membranes. After blocking, the membranes were incubated with 1 mg/mL ofDaMab-8 and anti-PA tag (NZ-1), followed by incubation with peroxidase-conjugated anti-mouse IgG and biotin-conjugatedanti-rat IgG, respectively. (B) Schematic illustration of DaMab-8 epitope. (C) Schematic illustration of DaMab-2 and DaMab-8epitopes. Red amino acids, strong reaction with DaMab-8; brown amino acids, weak reaction with DaMab-8. PVDF, polyvinylidene difluoride.

EPITOPE OF DAMAB-8

deletion mutants were detected by an anti-PA tag mAb, NZ1 (Fig. 2A), indicating that the N-terminus of the DaMab-8epitope exists between amino acids 601 and 621 (Fig. 2B).
Western blotting of the additional deletion mutants ofDGKa—that is, dN606, dN611, and dN616—demonstrated
that DaMab-8 detected dN606 and dN611, but not dN616,
although all deletion mutants were detected by an anti-PA
tag mAb, NZ-1 (Fig. 3A), thereby indicating that the Nterminus of the DaMab-8-epitope exists between aminoacids 611 and 616 (Fig. 3B).
Accordingly, we produced the following 26 DGKa point
mutants: M605A, H606A, G607A, G608A, S609A, N610A,L611A, W612A, G613A, D614A, T615A, R616A, R617A,P618A, H619A, G620A, D621A, I622A, Y623A, G624A,I625A, N626A, Q627A, A628G, L629A, and G630A. Western blotting demonstrated that the anti-PA tag mAb, NZ-1,detected all point mutants (Fig. 4A). In contrast, DaMab-8
strongly detected M605A, H606A, G607A, G608A, S609A,T615A, R616A, R617A, P618A, G620A, D621A, I622A,I625A, N626A, Q627A, A628G, L629A, and G630A; weaklydetected N610A, W612A, and D614A; and did not detect mutants L611A, G613A, H619A, Y623A, and G624A (Fig. 4A).
DaMab-8 epitopes are summarized in Figure 4B.
In conclusion, Asn610, Leu611, Trp612, Gly613, Asp614,His619, Tyr623, and Gly624 are important for facilitating
DaMab-8 binding to the DGKa protein. This epitope exists inthe catalytic domain of DGKa, whereas DaMab-2 binds toZn-finger (Fig. 4C). DaMab-8 could be advantageous forimmunohistochemical analyses toward clarifying the distribution of DGKa-expressing T cells in every pathophysiological tissue. Furthermore, these findings could be applied
for the production of more functional anti-DGKa mAbs.
Acknowledgments

We thank Shunsuke Itai, Miyuki Yanaka, Kayo Hisamatsu,
Saori Handa, Yoshimi Nakamura, and Maki Takahashi for
their excellent technical assistance, and Shinji Yamada forhis helpful discussion. This research was supported, in part,by AMED under Grant Numbers: JP18am0101te078 (Y.K.),
JP18am0301010 (Y.K.), and JP18ae0101028 (Y.K.), and by
JSPS KAKENHI Grant Numbers 17K07299 (M.K.K.) and16K10748 (Y.K.).
Author Disclosure Statement

Y.K. received research funding from Ono PharmaceuticalCo., Ltd. The other authors have no conflict of interest.
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Address correspondence to:
Yukinari Kato
New Industry Creation Hatchery CenterTohoku University
2-1, Seiryo-machi, Aoba-ku
Sendai 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: January 30, 2019
Accepted: March 22, 2019