Biochemistry and Biophysics Reports 15 (2018) 76–80

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Biochemistry and Biophysics Reports
journal homepage: www.elsevier.com/locate/bbrep

Epitope mapping of an anti-alpha thalassemia/mental retardation syndrome
X-linked monoclonal antibody AMab-6

T

Mika K. Kanekoa, Shinji Yamadaa, Shunsuke Itaia,b, Yoshikazu Furusawaa,c,d, Takuro Nakamuraa,
Miyuki Yanakaa, Saori Handaa, Kayo Hisamatsua, Yoshimi Nakamuraa, Masato Fukuid,⁎
Hiroyuki Haradab, Yukinari Katoa,c,aDepartment of Antibody Drug Development, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan
Department of Oral and Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku,
Tokyo 113-8510, Japanc
New Industry Creation Hatchery Center, Tohoku University, 2-1, Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, JapandZENOAQ RESOURCE CO., LTD., 1-1 Tairanoue, Sasagawa, Asaka-machi, Koriyama, Fukushima 963-0196, Japanb

A R T I C LE I N FO

A B S T R A C T

Keywords:
ATRX
AMab-6
Epitope mapping

The alpha-thalassemia/mental-retardation-syndrome-X-linked (ATRX) gene is located on the q arm of the X
chromosome. ATRX gene mutations were ﬁrst discovered in pancreatic neuroendocrine tumors, and subsequently in other cancer subtypes, including gliomas. Molecular subgrouping of gliomas has been more important
than conventional histological classiﬁcations. Mutations in the isocitrate dehydrogenase (IDH), telomerase reverse transcriptase (TERT) promoter, and ATRX and the codeletion of chromosomes 1p/19q are used as biomarkers for diagnosing the subtypes of diﬀuse gliomas. We recently developed a sensitive monoclonal antibody(mAb) AMab-6 against ATRX by immunizing mice with recombinant human ATRX. AMab-6 can help to detect
ATRX mutations via Western blotting and immunohistochemical analyses. In this study, we characterized thebinding epitope of AMab-6 using enzyme-linked immunosorbent assay (ELISA), Western blotting, and immunohistochemical analysis, and found that Gln2368 of ATRX is critical for AMab-6 binding to ATRX. Ourﬁndings could be applied to the production of more functional anti-ATRX mAbs.

1. Introduction
The alpha-thalassemia/mental-retardation-syndrome-X-linked (ATRX)
gene is located on the q arm of the X chromosome. ATRX gene mutations
were ﬁrst discovered in pancreatic neuroendocrine tumors [1], and subsequently in other cancer subtypes, including gliomas. Gliomas are themost frequently occurring brain tumors and have a heterogeneous molecular background [2]. Molecular subgrouping of gliomas using mutationsin isocitrate dehydrogenase (IDH) 1/2, TERT promoter, and ATRX andcodeletion of 1p/19q as biomarkers stratiﬁes patients into distinct groups
that are more prognostically assessed compared with conventional histological classiﬁcations [1,3–5]. These molecular subtypes are clinically important because treatment strategies can be planned in accordance withmolecular subtype along with the World Health Organization (WHO)tumor grading system. The 2016 WHO Classiﬁcation of Tumors of theCentral Nervous System (2016 WHO CNS) is both conceptually andpractically more advanced than the 2007 WHO CNS [6,7]. The 2016 WHO
CNS uses molecular parameters, in addition to histological assessments todeﬁne many tumor entities, presents major restructuring of diﬀuse
gliomas, medulloblastomas, and other embryonal tumors; and incorporates
new entities that are deﬁned using both histology results and molecularfeatures.
The loss of ATRX mRNA and protein in gliomas is caused by an ATRX
mutation. Loss of the ATRX protein can be diagnosed by immunohistochemistry using anti-ATRX antibodies [8,9]; however, nearly all
studies on ATRX protein have used polyclonal antibodies [10] because
highly sensitive monoclonal antibodies (mAbs) against human ATRX
protein had not been established. Recently, we established a novel antiATRX mAb, AMab-6, which is very useful in enzyme-linked immunosorbent assay (ELISA), Western blot, and immunohistochemical
analyses [11]. In this study, we characterized the binding epitope of AMab6 using ELISA, Western blot analysis, and immunohistochemical analyses.

Abbreviations: ATRX, alpha-thalassemia/mental-retardation-syndrome-X-linked; mAb, monoclonal antibody; ELISA, enzyme-linked immunosorbent assay; PBS,
phosphate-buﬀered saline; DAB, 3,3-diaminobenzidine tetrahydrochloride⁎
Corresponding author at: New Industry Creation Hatchery Center, Tohoku University, 2-1, Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan.
E-mail address: yukinarikato@med.tohoku.ac.jp (Y. Kato).
https://doi.org/10.1016/j.bbrep.2018.07.003
Received 15 June 2018; Received in revised form 1 July 2018; Accepted 2 July 2018
2405-5808/ © 2018 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license(http://creativecommons.org/licenses/BY/4.0/).

Biochemistry and Biophysics Reports 15 (2018) 76–80

M.K. Kaneko et al.

Fig. 1. Production of ATRX deletion mutants. Three ATRX C-terminal deletion mutants and three ATRX N-terminal deletion mutants were produced. Four ATRX
peptides were also synthesized. Black bars, the deletion mutants or synthesized peptides, which were detected by AMab-6; white bar, the deletion mutants orsynthesized peptides, which were not detected by AMab-6.

2. Materials and methods

2.3. Western blot analyses

2.1. Plasmid preparation

Competent Escherichia coli TOP-10 cells (Thermo Fisher ScientiﬁcInc.) were transformed and cultured overnight at 37 °C in LB medium(Thermo Fisher Scientiﬁc Inc.) containing 100 μg/mL ampicillin (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). Cell pellets
were resuspended in phosphate buﬀered solution with 1% Triton X-100
and 50 μg/mL aprotinin (Sigma-Aldrich Corp.). Lysates (10 μg) wereboiled in sodium dodecyl sulfate sample buﬀer (Nacalai Tesque, Inc.,
Kyoto, Japan). The proteins were electrophoresed on 5–20% polyacrylamide gels (FUJIFILM Wako Pure Chemical Corporation) andtransferred onto a polyvinylidene diﬂuoride (PVDF) membrane (Merck
KGaA, Darmstadt, Germany). After blocking with 4% skim milk
(Nacalai Tesque, Inc.), the membrane was ﬁrst incubated with AMab-6[11] or NZ-1 (anti-PA tag) [13] and then with peroxidase-conjugatedanti-mouse or anti-rat antibody (1:1000 diluted; Agilent Technologies
Inc.) and developed using the Pierce Western Blotting Substrate Plus
(Thermo Fisher Scientiﬁc Inc.) or the ImmunoStar LD Chemiluminescence Reagent (FUJIFILM Wako Pure Chemical Corporation) using a
Sayaca-Imager (DRC Co. Ltd., Tokyo, Japan).

Human ATRX cDNA (Accession No. AB102641) encoding aminoacids 2273–2413, 2273–2378, 2273–2343, 2273–2308, 2309–2413,
2344–2413, and 2379–2413 were obtained using polymerase chain
reaction (PCR) with cDNA derived from human lung cDNA as a template. The primer sets for ATRX are summarized in Supplementary
Table 1. ATRXs were subcloned into the expression vector pMAL-c2(New England Biolabs Inc., Beverly, MA, USA) with MAP (GDGMVPPGIEDK) [12] and PA (GVAMPGAEDDVV) [13] tags using the In-Fusion
PCR Cloning Kit (Takara Bio., Inc., Shiga, Japan). Substitution of ATRX
amino acids 2273–2413 with either alanine or glycine was performed
using a QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent
Technologies Inc., Santa Clara, CA, USA). This construct was veriﬁed bydirect DNA sequencing.

2.2. Enzyme-linked immunosorbent assay
Synthesized ATRX peptides using PEPScreen (Sigma-Aldrich Corp.,
St. Louis, MO, USA) were immobilized on Nunc Maxisorp 96-well immunoplates (Thermo Fisher Scientiﬁc Inc., Waltham, MA, USA) at
10 μg/mL for 30 min at 37 °C. After blocking with SuperBlock T20 (PBS)
Blocking Buﬀer (Thermo Fisher Scientiﬁc Inc.), the plates were incubated with 10 μg/mL puriﬁed AMab-6 followed by a 1:2000 dilutionof peroxidase-conjugated anti-mouse IgG (Agilent Technologies Inc.).
The enzymatic reaction was conducted using 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, USA). These reactions were performed at 37 °C using a
total sample volume of 50–100 μL.

2.4. Immunohistochemical analyses
This study examined one patient with oral cancer who underwent
surgery at Tokyo Medical and Dental University. The Tokyo Medical
and Dental University Institutional Review Board reviewed and approved the use of the human cancer tissues, and written informed
consent was obtained from the patient. Histological sections (4-μm
thick) were directly autoclaved for 20 min in citrate buﬀer (pH 6.0;
Nichirei Biosciences, Inc., Tokyo, Japan). After blocking with
SuperBlock T20 (PBS) Blocking Buﬀer (Thermo Fisher Scientiﬁc Inc.),
the sections were incubated with 5 μg/mL AMab-6 or 5 μg/mL AMab-6
plus 5 μg/mL peptides for 1 h at room temperature and treated using an
EnVision+ Kit (Agilent Technologies Inc.) for 30 min. Color was77Biochemistry and Biophysics Reports 15 (2018) 76–80

M.K. Kaneko et al.

developed using 3,3′-diaminobenzidine tetrahydrochloride (DAB;
Agilent Technologies Inc.) for 2 min, and counterstained with hematoxylin (FUJIFILM Wako Pure Chemical Corporation).

Table 1
Determination of AMab-6 Epitope by ELISA.

3. Results and discussion
Immunohistochemistry is a robust and widely available method
used to assess genetic changes at the molecular level using deﬁned
protocols and materials [14,15]. The important molecules for subtype
diagnosis of diﬀuse gliomas are mutations of IDH1/2, TERT promoter,and ATRX and the codeletion of 1p/19q among the many molecularparameters [16]. Mutations of IDH1/2 [14] and ATRX [8,10] can be
accurately detected using this method. We recently developed bothanti-mutated IDH mAbs [17] and an anti-ATRX mAb, AMab-6 [11].
Several mAbs against IDH mutants include HMab-1/HMab-2 against
IDH1-R132H and multi-speciﬁc mAbs MsMab-1/MsMab-2 against
IDH1/2 mutations [18–21].
As shown in Fig. 1, we produced three C-terminal deletion mutants(dC2378, dC2343, and dC2308) and three N-terminal deletion mutants(dN2309, dN2344, and dN2379). Western blot analysis demonstrated
that AMab-6 detected dC2378, dN2309, and dN2344 but not dC2343,
dC2308, and dN2379 (Fig. 2A), indicating that the N-terminus of theAMab-6-epitope exits between amino acids 2344 and 2379, and the Cterminus of the AMab-6-epitope exits between amino acids 2343 and2378. Next, we produced the following four peptides: pp2344–2363(ATRX amino acids 2344–2363), pp2349–2368 (ATRX amino acids
2349–2368), pp2354–2373 (ATRX amino acids 2354–2373), andpp2359–2378 (ATRX amino acids 2359–2378) as depicted in Fig. 1.
ELISA demonstrated that AMab-6 detected pp2354–2373 andpp2359–2378, and did not react with pp2344–2363 or pp2349–2368(Table 1).

PeptideSequence

AMab-62344–2363
2349–2368
2354–2373
2359–2378
K2359A
E2360A
N2361A
M2362A
N2363A
L2364AS2365A
E2366A
A2367G
Q2368A
V2369A
Q2370A
A2371G
L2372A
A2373GAVRIQPLEDIISAVWKENMNPLEDIISAVWKENMNLSEAQISAVWKENMNLSEAQVQALA
KENMNLSEAQVQALALSRQA
AENMNLSEAQVQALALSRQA
KANMNLSEAQVQALALSRQA
KEAMNLSEAQVQALALSRQA
KENANLSEAQVQALALSRQA
KENMALSEAQVQALALSRQA
KENMNASEAQVQALALSRQA
KENMNLAEAQVQALALSRQA
KENMNLSAAQVQALALSRQA
KENMNLSEGQVQALALSRQA
KENMNLSEAAVQALALSRQA
KENMNLSEAQAQALALSRQA
KENMNLSEAQVAALALSRQA
KENMNLSEAQVQGLALSRQA
KENMNLSEAQVQAAALSRQA
KENMNLSEAQVQALGLSRQA

−
−
＋＋＋
＋＋＋
＋＋＋
＋＋＋
＋＋＋
＋＋＋
＋＋＋
＋＋＋
＋＋＋
＋＋＋
＋＋＋
＋
＋＋＋
＋＋＋
＋＋＋
＋＋＋
＋＋＋

＋＋＋, OD655 ≧ 1.0; ＋＋, 0.6 ≦ OD655＜1.0.
＋, 0.1 ≦ OD655＜0.6; -, OD655＜0.1.

We further synthesized the following 15 peptides of ATRX point
mutations: K2359A, E2360A, N2361A, M2362A, N2363A, L2364A,
S2365A, E2366A, A2367G, Q2368A, V2369A, Q2370A, A2371G,
L2372A, and A2373G (Table 1). ELISA demonstrated that AMab-6 reacted strongly with nearly all point mutants; however, it reacted very
weakly with Q2368A, indicating that Gln2368 is important for AMab-6
binding to ATRX protein.
We produced the following 11 ATRX point mutants: N2363A,

Fig. 2. Epitope mapping of AMab-6 using deletion mutants and point mutants of ATRX. Cell lysates (10 μg) of deletion mutants (A) and point mutants (B) were
electrophoresed and proteins were transferred onto PVDF membranes. After blocking, membranes were incubated with 1 μg/mL of AMab-6 or 1 μg/mL of anti-PA tag(clone: NZ-1) followed by peroxidase-conjugated anti-mouse or rat IgG.
78

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M.K. Kaneko et al.

Fig. 3. Immunohistochemistry using oral cancer tissue. Oral cancer tissues were autoclaved for 20 min in a citrate buﬀer. Sections were then incubated with 5 μg/
mL AMab-6 or 5 μg/mL AMab-6 plus 5 μg/mL peptides and stained using an EnVision+ kit. Color development was performed using 3,3′-diaminobenzidine tetrahydrochloride. Sections were then counterstained with hematoxylin. Notes: scale bar = 100 µm; HE, hematoxylin and eosin.

References

L2364A, S2365A, E2366A, A2367G, Q2368A, V2369A, Q2370A,
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AMab-6 did not detect mutant Q2368A (Fig. 2B), conﬁrming that
Gln2368 is important for AMab-6 binding to ATRX protein.
ATRX mutation has been reported in gliomas in which the ATRX
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usually detected in nearly all cancers, including oral cancers, via immunohistochemistry using anti-ATRX antibodies because ATRX mutations have not been reported in those cancers [22]. In this study, we
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In conclusion, Gln2368 of ATRX is critical for AMab-6 binding. Ourﬁndings can be applied for the production of more functional anti-ATRX
mAbs.

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Funding
This research was supported in part by AMED under Grant
numbers: JP18am0101078 (Y.K.), JP18am0301010 (Y.K.), andJP18ae0101028 (Y.K.), and by JSPS KAKENHI Grant Number
17K07299 (M.K.K.) and Grant Number 16K10748 (Y.K.).
Conﬂict of interest
The authors declare no conﬂicts of interest involving this article.
Appendix A. Transparency document
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.bbrep.2018.07.003.
Appendix A. Supplementary material
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.bbrep.2018.07.003.
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