MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 38, Number 4, 2019ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2019.0020

Epitope Mapping of Anti-Diacylglycerol Kinase Zeta
Monoclonal Antibody DzMab-1
for Immunohistochemical Analyses

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Masato Sano,1 Mika K. Kaneko,1 and Yukinari Kato1,2

The diacylglycerol kinases (DGKs) catalyze the phosphorylation of the cell membrane lipid diacylglycerol(DG), which is important in lipid biochemistry and signal transduction into phosphatidic acid. DG-mediatedsignal transduction downstream of the T cell receptor has been reported to be terminated by DGKz, 1 of 10
DGK isoforms in most cases. We previously established an anti-DGKz monoclonal antibody (mAb) DzMab-1(rat IgG1, kappa), which reacts with both mouse DGKz and human DGKz (hDGKz). In this study, we characterized the binding epitope of DzMab-1 using Western blotting, and found that Met1 and Pro3 residues ofhDGKz are important for facilitating DzMab-1 binding to hDGKz. Furthermore, DzMab-1 was shown to be
useful for immunohistochemical analyses for formalin-fixed paraffin-embedded HeLa cells. These findings
could be applied for the production of more functional anti-hDGKz mAbs.
Keywords: diacylglycerol kinase, DGKz, monoclonal antibody, epitope mapping

Introduction

Diacylglycerol kinases (DGKs) phosphorylate thecell membrane lipid diacylglycerol (DG) into phosphatidic acid.(1–3) DG functions as an important second
messenger in T cells.(4) DG-mediated signal transduction
downstream of T cell receptors (TCR) was shown to be terminated by 2 of the 10 DGK isoforms, DGKa and DGKz.(5)Especially, DGKz is known to be the dominant isoform.(6)T cells deficient in either DGKa or DGKz are hyperresponsive, leading to enhanced proliferation and secretion ofcytokines in response to TCR activation.(7–9) CD8+ T cells
deficient in DGKs exhibit enhanced activity against xenografts after adoptive transfer of T cells when expressing
TCRs or chimeric antigen receptors specific for tumor antigens.(10) Furthermore, targeting DGKz may increase the efficacy of adoptive T cell and immune checkpoint therapies inthe treatment of leukemia.(11) In this study, we characterized
the binding epitope of an anti-DGKz monoclonal antibody(mAb), DzMab-1 using Western blotting.

wise, lysates were immunoprecipitated using amylose resin(New England Biolabs, Inc., Beverly, MA) and boiled in SDS
sample buffer (Nacalai Tesque, Inc.). The samples wereelectrophoresed on 5%–20% polyacrylamide gels (NacalaiTesque, Inc. or FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) 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
(anti-PA tag)(12) for 1 hour, followed by biotin-conjugatedanti-rat IgG (1:1000 dilution; Agilent Technologies, Inc.,
Santa Clara, CA) for 30 minutes, and further incubated withVECTASTAIN Elite ABC HRP Kit (Vector Laboratories,Inc., Burlingame, CA) for 30 minutes. The membrane wasfinally 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 were performed atroom temperature.
Immunohistochemical analyses

Materials and Methods
Western blotting

Lysates were boiled in sodium dodecyl sulfate (SDS)
sample buffer (Nacalai Tesque, Inc., Kyoto, Japan). Other1
2

Cell blocks of HeLa cells (American Type Culture Collection, Manassas, VA) were produced using iPGell (Genostaff Co., Ltd., Tokyo, Japan). Histological sections of 4-mm
thickness were deparaffinized in xylene, then rehydrated,
and were autoclaved in EnVision FLEX Target Retrieval

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.
New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan.

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SANO ET AL.

FIG. 1. Epitope mapping of DzMab-1 using C-terminal deletion mutants of hDGKz. (A) Cell lysates of C-terminal
deletion mutants of hDGKz were electrophoresed and transferred onto membranes. After blocking, the membranes wereincubated with 1 mg/mL of DzMab-1 or anti-PA tag (NZ-1). (B) Schematic illustration of DzMab-1 epitope. DGK, diacylglycerol kinase; hDGKz, human DGKz.
Solution High pH (Agilent Technologies, Inc.) for 20 minutes
and then blocked using SuperBlock T20 (PBS) Blocking
Buffer (Thermo Fisher Scientific, Inc., Waltham, MA). Samples were incubated with DzMab-1 (5 mg/mL) for 1 hour atroom temperature, followed by biotin-conjugated anti-rat
IgG (1:1000 dilution; Agilent Technologies, Inc.) for 1 hour,and further incubated with VECTASTAIN Elite ABC HRP
Kit (Vector Laboratories, Inc.) for 1 hour. The tissue sections
were stained using 3,3¢-diaminobenzidine tetrahydrochloride(DAB; Agilent Technologies Inc.) for 5 minutes and counterstained using hematoxylin (FUJIFILM Wako Pure Chemical Corporation).
Results and Discussion

Previously, DzMab-1 (rat IgG1, kappa), which can reactwith both mouse and human DGKz, was established.(13)Western blot analysis demonstrated that DzMab-1 did notreact with DGKa or DGKg, indicating that DzMab-1 specifically reacts with human DGKz (hDGKz).
We herein characterized the binding epitope of DzMab-1using Western blotting. As shown in Figure 1, we produced
seven C-terminal deletion mutants of hDGKz (i.e., dC700,dC600, dC500, dC400, dC300, dC200, and dC100). Western
blotting demonstrated that DzMab-1 detected all deletionmutants, which were also detected by an anti-PA tag mAb,
NZ-1 (Fig. 1A), indicating that the N-terminus of the DzMab1-epitope exists between 1st amino acid (aa) and 100th aa(Fig. 1B).
We next produced nine N-terminal deletion mutants ofhDGKz-dC200 (i.e., dN10, dN20, dN30, dN40, dN50, dN60,

FIG. 2. Epitope mapping of DzMab-1 using N-terminal
deletion mutants of hDGKz. Immunoprecipitates (A) or celllysates (B) of N-terminal deletion mutants of hDGKz-dC200
were electrophoresed and transferred onto membranes. After
blocking, the membranes were incubated with 1 mg/mL ofDzMab-1 or anti-PA tag (NZ-1). (C) Schematic illustrationof DzMab-1 epitope.

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EPITOPE OF DZMAB-1

FIG. 3. Epitope mapping of DzMab-1 using point mutantsof hDGKz. (A) Immunoprecipitates of point mutants ofhDGKz-dC200 were electrophoresed and transferred ontomembranes. After blocking, the membranes were incubatedwith 1 mg/mL of DzMab-1 or anti-PA tag (NZ-1). (B) Schematic illustration of DzMab-1 epitope. Met1 and Pro3 areimportant for DzMab-1 binding to hDGKz.
dN70, dN80, and dN90). Western blotting demonstrated that
DzMab-1 did not react with those deletion mutants; in contrast, anti-PA tag mAb, NZ-1, detected them (Fig. 2A, B),
indicating that the N-terminus of the DzMab-1-epitope exists
between 1st aa and 10th aa (Fig. 2C).
We produced the following 10 point mutants usinghDGKz-dC200: M1A, E2A, P3A, R4A, D5A, G6A, S7A,P8A, E9A, and A10G. Western blotting demonstrated thatthe anti-PA tag mAb, NZ-1, detected all point mutants

FIG. 4. Immunohistochemical analyses of DzMab-1 for
FFPE HeLa cells. Sections were incubated with 5 mg/mL ofDzMab-1 (A) or blocking buffer (B). Scale bar = 100 mm.

177(Fig. 3A). In contrast, DzMab-1 did not detect M1A andP3A. These results are summarized in Figure 3B.
In our previous reports, DzMab-1 was shown to be usefulfor immunocytochemical analyses.(13) In this study, we investigated whether DzMab-1 is also applicable for immunohistochemical analyses. As depicted in Figure 4, DzMab-1
reacted with HeLa cells in immunohistochemical analyses
using formalin-fixed paraffin-embedded (FFPE) cells, indicating that DzMab-1 could also be useful for FFPE samplesof patient tissues.
In conclusion, Met1 and Pro3 residues of hDGKz are important for facilitating DzMab-1 binding to hDGKz. Thesefindings could be applied for the production of more functional anti-hDGKz mAbs.
Acknowledgments

This research was supported in part by AMED under grant
numbers JP18am0101078 (Y.K.), JP18am0301010 (Y.K.),
and JP18ae0101028 (Y.K.), and by JSPS KAKENHI grant
numbers 17K07299 (M.K.K.), 16K10748 (Y.K.), and19K07705 (Y.K.).
Author Disclosure Statement

Y.K. received research funding from Ono PharmaceuticalCo., Ltd. The other authors have no conflicts of interest.
References

1. Topham MK, and Epand RM: Mammalian diacylglycerol
kinases: Molecular interactions and biological functions ofselected isoforms. Biochim Biophys Acta 2009;1790:416–424.
2. Goto K, Hozumi Y, Nakano T, Saino SS, and Kondo H:
Cell biology and pathophysiology of the diacylglycerol
kinase family: Morphological aspects in tissues and organs.
Int Rev Cytol 2007;264:25–63.
3. Eichmann TO, and Lass A: DAG tales: The multiple facesof diacylglycerol—Stereochemistry, metabolism, and signaling. Cell Mol Life Sci 2015;72:3931–3952.
4. Joshi RP, and Koretzky GA: Diacylglycerol kinases:
Regulated controllers of T cell activation, function, anddevelopment. Int J Mol Sci 2013;14:6649–6673.
5. Krishna S, and Zhong X: Role of diacylglycerol kinases in
T cell development and function. Crit Rev Immunol 2013;
33:97–118.
6. Joshi RP, Schmidt AM, Das J, Pytel D, Riese MJ, Lester M,Diehl JA, Behrens EM, Kambayashi T, and Koretzky GA:
The zeta isoform of diacylglycerol kinase plays a predominant role in regulatory T cell development and TCRmediated ras signaling. Sci Signal 2013;6:ra102.
7. Zhong XP, Hainey EA, Olenchock BA, Jordan MS, Maltzman JS, Nichols KE, Shen H, and Koretzky GA: Enhanced T cell responses due to diacylglycerol kinase zetadeficiency. Nat Immunol 2003;4:882–890.
8. Zha Y, Marks R, Ho AW, Peterson AC, Janardhan S,Brown I, Praveen K, Stang S, Stone JC, and Gajewski TF:
T cell anergy is reversed by active Ras and is regulated bydiacylglycerol kinase-alpha. Nat Immunol 2006;7:1166–1173.
9. Olenchock BA, Guo R, Carpenter JH, Jordan M, TophamMK, Koretzky GA, and Zhong XP: Disruption of diacylglycerol metabolism impairs the induction of T cellanergy. Nat Immunol 2006;7:1174–1181.

178

Downloaded by Yukinari Kato from www.liebertpub.com at 08/13/19. For personal use only.

10. Riese MJ, Wang LC, Moon EK, Joshi RP, Ranganathan
A, June CH, Koretzky GA, and Albelda SM: Enhanced
effector responses in activated CD8+ T cells deficientin diacylglycerol kinases. Cancer Res 2013;73:3566–3577.
11. Jing W, Gershan JA, Holzhauer S, Weber J, Palen K,McOlash L, Pulakanti K, Wesley E, Rao S, Johnson BD,and Riese MJ: T cells deficient in diacylglycerol kinase zeta
are resistant to PD-1 inhibition and help create persistenthost immunity to leukemia. Cancer Res 2017;77:5676–5686.
12. Kato Y, Kaneko MK, Kuno A, Uchiyama N, Amano
K, Chiba Y, Hasegawa Y, Hirabayashi J, Narimatsu H,
Mishima K, and Osawa M: Inhibition of tumor cell-induced
platelet aggregation using a novel anti-podoplanin antibody reacting with its platelet-aggregation-stimulating
domain. Biochem Biophys Res Commun 2006;349:1301–1307.
13. Nakano T, Ogasawara S, Tanaka T, Hozumi Y, Sano M,
Sayama Y, Yamada S, Kaneko MK, Kato Y, and Goto K:

SANO ET AL.

DzMab-1: Anti-human DGKzeta monoclonal antibody for
immunocytochemistry. Monoclon Antib Immunodiagn
Immunother 2019;38:179–182.

Address correspondence to:
Yukinari Kato
New Industry Creation Hatchery Center
Tohoku University2-1, Seiryo-machi, Aoba-ku
Sendai
Miyagi 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: April 23, 2019
Accepted: July 26, 2019