MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY
Volume 38, Number 1, 2019ª Mary Ann Liebert, Inc.
DOI: 10.1089/mab.2018.0047

Original Articles

Epitope Mapping of Antidiacylglycerol Kinasea Monoclonal Antibody DaMab-2

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Masato Sano, Mika K. Kaneko, and Yukinari Kato

Diacylglycerol kinase (DGK) is responsible for the enzymatic conversion of diacylglycerol (DG) to phosphatidic acid (PA). Both DG and PA serve as signaling molecules; therefore, DGK functions as a key enzyme
between DG- and PA-mediated signaling. DGKa, one of the 10 DGK isozymes, is involved in T cell function
and has been shown to localize in the cytoplasm and nucleus. Furthermore, DGKa translocates to the plasma
membrane in response to T cell receptor stimulation. Recently, we developed a specific monoclonal antibody(mAb), DaMab-2 (mouse IgG1, kappa), against DGKa. DaMab-2 is very useful in immunocytochemical
analysis using HeLa cells. In this study, we characterized the binding epitope of DaMab-2 using Western blot
and revealed that Cys246, Lys249, Pro252, and Cys253 of DGKa are important for DaMab-2 binding to theDGKa protein. Our findings can be applied for the production of more functional anti-DGKa mAbs.
Keywords: diacylglycerol kinase, DGKa, monoclonal antibody, epitope mapping

mice show an altered activity of TCR signaling and hyperproliferation.(13) DGKa localizes in the cytoplasm andtranslocates to the plasma membrane in response to externalstimuli.(14)
Because no specific anti-DGKa monoclonal antibody(mAb) was available to detect human DGKa by immunostaining, the localization of the native protein was not clarified. Recently, we reported a novel antihuman DGKa mAb,
DaMab-2 (mouse IgG1, kappa), which is very useful in immunocytochemical analysis using HeLa cells. In this study,
we characterized the binding epitope of DaMab-2 using
Western blot.

Introduction

Diacylglycerol 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 serves as a second messenger that activates the conventional and novel types of the protein kinase C(PKC) family, RasGRP, Unc-13, and canonical transient receptor potential channels.(2,3) PA acts as a messenger molecule that activates hypoxia-inducible factor-1a, atypical
PKCz, and mammalian target of rapamycin. Therefore, DGKserves as a key enzyme between DG- and PA-mediatedpathways.
In mammalian species, DGK constitutes an enzyme family
comprising 10 isozymes.(1,2) Each isozyme possesses a distinct molecular structure and subcellular localization pattern.
DGKa is the first identified enzyme of 80 kDa that contains
an EF-hand motif (Ca2+-binding site), a Zn finger (C1 domain, DG-binding site), and a catalytic domain. Various cell
types and tissues express DGKa in normal cells, including
glial cells, vascular cells, hepatocytes, adrenal medullarycells, fibroblasts, and lymphocytes, or in several cancer cells,such as melanomas and sarcomas.(3–11) DGKa regulates cellproliferation in response to IL-2 stimulation in T cells(3) andis involved in T cell receptor (TCR) signaling by modulating
RasGRP activity.(12) T cells isolated from DGKa-deficient

Materials and Methods
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)using the In-Fusion HD Cloning kit (Takara Bio, Inc., Shiga,Japan), named pMAL-c2-DGKa-PA. The deletion mutantsof DGKa were produced by polymerase chain reaction(PCR) and subcloned into pMAL-c2 with PA tag using the InFusion HD Cloning kit. The substitution of DGKa aminoacids 240–260 with either alanine or glycine in dC320 of
DGKa was performed using the QuikChange Lightning SiteDirected Mutagenesis Kit (Agilent Technologies, Inc., Santa

Department of Antibody Drug Development, Tohoku University Graduate School of Medicine, Sendai, Japan.

8EPITOPE OF DAMAB-2

9Clara, CA). These constructs were verified by direct DNA
sequencing.

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Western blot analyses

Competent Escherichia coli TOP-10 cells (Thermo FisherScientific, Inc., Waltham, MA) were transformed and cultured overnight at 37C in Luria-Bertani (LB) medium(Thermo Fisher Scientific) containing 100 mg/mL ampicillin(FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). Cell pellets were resuspended in phosphate-bufferedsaline with 1% Triton X-100 and 50 mg/mL aprotinin (SigmaAldrich Corp.). Lysates were immunoprecipitated using
amylose resin (New England Biolabs, Inc.) and boiled insodium dodecyl sulfate sample buffer (Nacalai Tesque, Inc.,Kyoto, Japan). The samples were electrophoresed on 5%–
20% polyacrylamide gels (FUJIFILM Wako Pure ChemicalCorporation) and transferred onto a polyvinylidene difluoride
membrane (Merck KGaA, Darmstadt, Germany). After
blocking with 4% skim milk (Nacalai Tesque, Inc.) for 1 h atroom temperature, the membrane was incubated withDaMab-2(17) overnight at 4C, NZ-1 (anti-PA tag) for 1 h atroom temperature, or LpMab-13 (anti-PA tag) for 1 h at roomtemperature(18) and then with peroxidase-conjugated antimouse IgG (1:2000 dilution; Agilent Technologies, Inc.) orantirat IgG (1:10,000 dilution; Sigma-Aldrich Corp.) for 1 hat room temperature. The membrane was finally developed
using ImmunoStar LD Chemiluminescence Reagent (FUJIFILM Wako Pure Chemical Corporation) using SayacaImager (DRC Co., Ltd., Tokyo, Japan).
Results and Discussion

Several anti-DGKa mAbs are commercially available andhave been reported to be useful in Western blot and immunocytochemical analyses. However, recombinant or overexpressed DGKa has been usually used in these studies.

FIG. 1. Production of DGKa deletion mutants.
(A) Immunoprecipitates of deletion mutants were electrophoresed and transferred onto PVDF membranes. Afterblocking, membranes were incubated with 20 mg/mL DaMab-2
or 1 mg/mL anti-PA tag (NZ-1) followed by peroxidaseconjugated antimouse or antirat IgG. (B) Schematic illustrationof DaMab-2 epitope. Black bars, deletion mutants detected byDaMab-2; white bars, deletion mutants not detected by DaMab2. hDGKa, human diacylglycerol kinase a; MBP, maltosebinding protein; PA, phosphatidic acid; PVDF, polyvinylidene
difluoride.

Table 1. Epitope Mapping of DaMab-2 Using Point Mutants
Point mutants
Y240A
T241A
V242A
H243A
D244A
Q245A
C246A
A247G
M248A
K249A
A250G
L251A
P252A
C253A
E254A
V255A
S256A
T257A
Y258A
A259G
K260A

Sequence

DaMab-2

LpMab-13

ATVHDQCAMKALPCEVSTYAKYAVHDQCAMKALPCEVSTYAKYTAHDQCAMKALPCEVSTYAKYTVADQCAMKALPCEVSTYAKYTVHAQCAMKALPCEVSTYAKYTVHDACAMKALPCEVSTYAKYTVHDQAAMKALPCEVSTYAKYTVHDQCGMKALPCEVSTYAKYTVHDQCAAKALPCEVSTYAKYTVHDQCAMAALPCEVSTYAKYTVHDQCAMKGLPCEVSTYAKYTVHDQCAMKAAPCEVSTYAKYTVHDQCAMKALACEVSTYAKYTVHDQCAMKALPAEVSTYAKYTVHDQCAMKALPCAVSTYAKYTVHDQCAMKALPCEASTYAKYTVHDQCAMKALPCEVATYAKYTVHDQCAMKALPCEVSAYAKYTVHDQCAMKALPCEVSTAAKYTVHDQCAMKALPCEVSTYGKYTVHDQCAMKALPCEVSTYAA++++++++++++++++++++++++++++++++++++++

Italic letters show the substitution of amino acid.

10

SANO ET AL.
Acknowledgments

This research was supported, in part, by AMED undergrant numbers JP18am0101078 (Y.K.), JP18am0301010(Y.K.), and JP18ae0101028 (Y.K.), and by JSPS KAKENHI
grant number 17K07299 (M.K.K.) and grant number16K10748 (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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References

FIG. 2. Epitope mapping of DaMab-2 using deletion andpoint mutants of DGKa. (A) Immunoprecipitates of point
mutants were electrophoresed and transferred onto PVDF
membranes. After blocking, membranes were incubatedwith 20 mg/mL DaMab-2 or 1 mg/mL anti-PA tag (LpMab13) followed by peroxidase-conjugated antimouse IgG.
(B) Schematic illustration of DaMab-2 epitope. Red underlined letters, the critical amino acids for DaMab-2recognition.
Recently, we immunized mice with recombinant DGKa anddeveloped a clone (DaMab-2).(17) DaMab-2 recognized only
DGKa in enzyme-linked immunosorbent assay and did notreact with other isozymes, such as DGKg, DGKz, DGKZ,and DGKd. Furthermore, DaMab-2 is very useful in immunocytochemical analysis using HeLa cells.
As shown in Figure 1, we produced 10 C-terminal deletionmutants of DGKa (dC320, dC300, dC280, dC260, dC240,dC220, dC200, dC180, dC160, and dC140). Western blot
demonstrated that DaMab-2 detected dC320, dC300, dC280,
and dC260 but not dC240, dC220, dC200, dC180, dC160,
and dC140, although all deletion mutants were detected by ananti-PA tag mAb, NZ-1 (Fig. 1A), indicating that the Cterminus of the DaMab-2-epitope exists between amino acids
240 and 260. These results are summarized in Figure 1B.
We next produced the following 21 DGKa point mutants:
Y240A, T241A, V242A, H243A, D244A, Q245A, C246A,A247G, M248A, K249A, A250G, L251A, P252A, C253A,
E254A, V255A, S256A, T257A, Y258A, A259G, andK260A (Table 1). Western blot demonstrated that the anti-PA
tag mAb, LpMab-13, detected Q245A, C246A, A247G,M248A, K249A, A250G, L251A, P252A, C253A, E254A,V255A, and S256A (Fig. 2A). In contrast, DaMab-2 strongly
detected Q245A, A247G, M248A, A250G, L251A, E254A,
V255A, and S256A, but did not detect mutants C246A andK249A and weakly detected P252A and C253A (Fig. 2A).
Both DaMab-2 and LpMab-13 recognized the other point
mutants, such as Y240A, T241A, V242A, H243A, D244A
T257A, Y258A, A259G, and K260A (Table 1). DaMab-2
epitope is summarized in Figure 2B.
In conclusion, Cys246, Lys249, Pro252, and Cys253 areimportant for DaMab-2 binding to the DGKa protein. Ourfindings can be applied for the production of more functionalanti-DGKa mAbs.

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Address correspondence to:
Yukinari Kato
Department of Antibody Drug Development
Tohoku University Graduate School of Medicine
New Industry Creation Hatchery Center
Tohoku University
2-1, Seiryo-machi, Aoba-ku
Sendai 980-8575
JapanE-mail: yukinarikato@med.tohoku.ac.jp
Received: November 14, 2018
Accepted: December 10, 2018