Identification of phosphate transporter NaPi2b as MX35 cancer antigen by modified SEREX approach
In this study we describe the identification of sodium-dependent phosphate transporter NaPi2b as MX35 cancer-associated antigen. To achieve this goal we have screened extensively a cDNA expressing library from ovarian cancer cell line OVCAR3 with monoclonal antibody MX35. To further confirm the auth...
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| Опубліковано в: : | Біополімери і клітина |
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| Дата: | 2008 |
| Автори: | , , , , , , , , , |
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| Мова: | Англійська |
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Інститут молекулярної біології і генетики НАН України
2008
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| Назва журналу: | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| Цитувати: | Identification of phosphate transporter NaPi2b as MX35 cancer antigen by modified SEREX approach / R.G. Kiyamova, V.S. Gryshkova, V.S. Usenko, Y.S. Khozaenko, V.A. Gurtovyy, B. Yin, G. Ritter, L. Old, I.T. Gout, V.V. Filonenko // Біополімери і клітина. — 2008. — Т. 24, № 3. — С. 218-224. — Бібліогр.: 28 назв. — англ. |
Репозитарії
Digital Library of Periodicals of National Academy of Sciences of Ukraine| _version_ | 1859464725990998016 |
|---|---|
| author | Kiyamova, R.G. Gryshkova, V.S. Usenko, V.S. Khozaenko, Y.S. Gurtovyy, V.A. Yin, B. Ritter, G. Old, L. Gout, I.T. Filonenko, V.V. |
| author_facet | Kiyamova, R.G. Gryshkova, V.S. Usenko, V.S. Khozaenko, Y.S. Gurtovyy, V.A. Yin, B. Ritter, G. Old, L. Gout, I.T. Filonenko, V.V. |
| citation_txt | Identification of phosphate transporter NaPi2b as MX35 cancer antigen by modified SEREX approach / R.G. Kiyamova, V.S. Gryshkova, V.S. Usenko, Y.S. Khozaenko, V.A. Gurtovyy, B. Yin, G. Ritter, L. Old, I.T. Gout, V.V. Filonenko // Біополімери і клітина. — 2008. — Т. 24, № 3. — С. 218-224. — Бібліогр.: 28 назв. — англ. |
| collection | DSpace DC |
| container_title | Біополімери і клітина |
| description | In this study we describe the identification of sodium-dependent phosphate transporter NaPi2b as MX35 cancer-associated antigen. To achieve this goal we have screened extensively a cDNA expressing library from ovarian cancer cell line OVCAR3 with monoclonal antibody MX35. To further confirm the authenticity of this finding, we showed that bacterially and baculovirally expressed NaPi2b is specifically recognized by MX35 antibody. Moreover, the validity of these results was verified in a parallel study involving affinity purification and mass spectrometry. The epitope for MX35 monoclonal antibody was mapped to the largest extracellular loop of NaPi2b. Taken together, this study uncovers the identity of MX35 antigen and provides molecular tools for studying its function in normal and cancer tissues.
Натрій-залежний транспортер NaPi2b ідентифіковано як MX35 пухлинний антиген. Для цього здійснено скринування кДНК-експресуючої біблеотеки, отриманої з клітин OVCAR3, моноклональними антитілами MX35. Щоб підтвердити ці дослідження, встановлено, що NaPi2b, експресований в бактеріях та клітинах комах, специфічно розпізнається MX35 антитілами. Крім того, виявлено ділянку, яка містить епітоп для моноклональних антитіл на великому позаклітинному домені NaPi2b. Таким чином, нами визначено антиген MX35, що забезпечує молекулярну основу для подальшого вивчення його функції в нормальних тканинах та при раку.
Натрий-зависимый транспортер NaPi2b идентифицирован как MX35 опухлевый антиген. Для достижения этого осуществлено скринирование кДНК-экспрессирующей библиотеки, полученной из OVCAR3 клеток, моноклональными антителами. Чтобы подтвердить эти результаты, установлено, что NaPi2b, экспрессированный в бактериях и в клетках насекомых, специфично узнается антителами MX35. Определен участок на внеклеточном домене NaPi2b, содержащий эпитоп для антител. Таким образом, нами идентифицирован антиген MX35, что создает молекулярную основу для дальнейшего изучения его функции в нормальных и раковых тканях.
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| first_indexed | 2025-11-24T04:44:52Z |
| format | Article |
| fulltext |
Identification of phosphate transporter NaPi2b as MX35
cancer antigen by modified SEREX approach
R. G. Kiyamova, V. S. Gryshkova, V. S. Usenko, Y. S. Khozaenko1, V. A. Gurtovyy1,
B. Yin2, G. Ritter2, L. Old2, I. T. Gout3, V. V. Filonenko
Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine
150 Zabolotnogo Str., Kyiv 03680, Ukraine
1BIONTEC
52A/88 Komsomol’ska Str., Dnipropetrovs’k, 49000, Ukraine
2Ludwig Institute for Cancer Research, New-York branch at Memorial Sloan-Kettering Cancer Center
1275 York Avenue, BOX 32 New York, NY 10021-6007, USA
3Department of Biochemistry and Molecular Biology, University College London
Gower Street, London WC1E6BT, UK
r.g.kiyamova@imbg.org.ua
In this study we describe the identification of sodium-dependent phosphate transporter NaPi2b as MX35
cancer-associated antigen. To achieve this goal we have screened extensively a cDNA expressing library
from ovarian cancer cell line OVCAR3 with monoclonal antibody MX35. To further confirm the authenticity
of this finding, we showed that bacterially and baculovirally expressed NaPi2b is specifically recognized by
MX35 antibody. Moreover, the validity of these results was verified in a parallel study involving affinity
purification and mass spectrometry. The epitope for MX35 monoclonal antibody was mapped to the largest
extracellular loop of NaPi2b. Taken together, this study uncovers the identity of MX35 antigen and provides
molecular tools for studying its function in normal and cancer tissues.
Keywords: MX35 monoclonal antibodies, ovarian cancer, sodium-dependent phosphate cotransporter
NaPi2b.
Introduction. The identification and molecular
characterization of tumor-associated markers and
antigens is central for the development new diagnostic
and therapeutic interventions in cancer. Monoclonal
antibody technology was exploited extensively in
1970s for the identification of antigens that distinguish
normal cells from cancer cells and could be used as
diagnostic markers or as targets for immunotherapy.
Mouse monoclonal antibody MX35 was developed
more than 20 years ago by immunizing mice with
ovarian carcinoma cells and designated screening for
tumor-associated antigens. Further analysis indicated
that MX35 antigen is overexpressed in 80 % cases of
human epithelial ovarian cancer, but shows restricted
expression in normal tissues. Biochemical and
immunohistochemical studies revealed that MX35
mAb recognizes cell surface glycoprotein of about
95 kDa which is overexpressed in 80 % ovarian cancer
specimens [1]. Moreover, MX35 antigen was found to
be stabilized by disulfide bond formation [2].
218
ISSN 0233-7657. Á³îïîë³ìåðè ³ êë³òèíà. 2008. Ò. 24. ¹ 3
Ó R. G. KIYAMOVA, V. S. GRYSHKOVA, V. S. USENKO,
Y. S. KHOZAENKO, V. A. GURTOVYY, B. YIN, G. RITTER,
L. OLD, I. T. GOUT, V. V. FILONENKO, 2008
Clinical studies with Fab fragments of radiolabeled
MX35 antibody suggest therapeutic potential in
patients with ovarian cancer [3]. Ovarian cancer is the
most common gynecologic malignancy that usually
becomes far advanced before it is diagnosed.
Therefore, it is associated with poor prognosis and
survival rate. So far, only few tumor-associated
markers and antigens specific for ovarian cancer have
been identified. Undoubtedly, MX35 antigen is one of
them. However, the progress on studying the function
of MX35 antigen in normal and cancer cell lines or
tissues has been held back by unknown molecular
identity of this antigen.
Here we describe the identification of MX35
antigen as sodium-dependent phosphate transporter
NaPi2b. For this purpose, we performed extensive
screening of OVCAR-3 expression library with MX35
mAb. Molecular identity of MX35 antigen was further
confirmed by immunoblotting of bacterially and
baculovirally expressed NaPi2b with MX35 antibody.
The identification of MX35 antigen as NaPi2b opens
broad perspectives for future studies, especially on
establishing the link between phosphate homeostasis,
cellular metabolism, and cancer growth.
Materials and Methods. Cell lines and antibodies.
All cell lines used in this study were obtained from the
cell bank of the Ludwig Institute for Cancer Research,
New York Branch at Memorial Sloan-Kettering Cancer
Center. Murine mAb MX35 (IgG1) was purified from
hybridoma supernatant by protein G chromatography.
Immunoscreening of OVCAR-3 expression library.
cDNA expression library from OVCAR-3 cell line was
constructed using ZAP Express® cDNA Synthesis Kit
and ZAP Express® cDNA Gigapack III Gold Cloning
Kit («Stratagene», USA). A total of 1×106 recombinant
phages were screened with MX35 monoclonal
antibody. For the primary screening, XL-1 Blue MRF
cells were infected with 6×103 phage particles, mixed
with top agarose and plated onto 150 mm Petri dishes.
The expression of recombinant proteins was induced by
the addition of 1 mM IPTG. The transfer of the proteins
to the nitrocellulose membrane Hybond-C was carried
out overnight at 37 °C. The membranes were then
blocked with 5 % non-fat dried milk (NFDM) for 1 hr at
room temperature and incubated with MX35 mAb
(5 mg/ml, in 0.2 % NFDM/TBS) for 15 hrs at room
temperature. After washing in TBS, the membranes
were incubated with alkaline phosphatase conjugated
Fc-fragment specific goat anti-human IgG («Sigma»,
USA) and processed for 4-nitro blue tetrazolium
chloride/5-bromo-4-chloro-3-indolil-phosphate color
development. Positive clones were extracted from agar
and stored in 0.5 ml of SM buffer (0.1 M NaCl, 10 mM
MgSO4, 50 mM Tris-HCl, 0.01 % gelatine) containing
20 ml chloroform. MX35 positive clones were subjected
to secondary screening as described above. Isolated
positive phages were converted into pBK-CMV
phagemid using in vivo excision protocol
(«Stratagene», USA). Rescued plasmid DNAs were
purified according to manufacturer’s instruction
(«Qiagen», USA) and subjected to restriction analysis
and DNA sequencing.
Cloning, expression, and purification of
GST/NaPi2b fusion proteins in bacteria. A cDNA
fragment, corresponding to the largest extracellular
loop (ECL) of human NaPi2b cotransporter (188–361
aa) was PCR amplified and cloned into pGEX4T1
vector in frame with GST. Briefly, EcoRI and BamHI
restriction sites were introduced at the 5' and 3' of PCR
amplified NaPi2b DNA fragments respectively. The
expression of GST/NaPi2b-L fusion protein in BL21
DE3 cells was induced by 1 mM IPTG for 3 hrs at
37 °C. Cells were harvested by centrifugation and
disrupted by sonication in lysis buffer (25 mM Tris
HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5 % Triton
and protease inhibitor cocktail («Boehringer
Mannheim», Germany). Purification of GST-NaPi2b-L
from the insoluble fraction was performed using
electro-elution from the gel. The purity of eluted
GST/NaPi2b-L was analyzed by SDS-PAGE
electrophoresis.
Expression and purification of His/NaPi2b-L
protein in baculoviral system. A cDNA fragment,
corresponding to the ECL of human NaPi2b
cotransporter (188–361 aa) was PCR amplified and
subcloned into pFastBac-His vector using EcoRI and
BamHI sites. The recombinant baculovirus was
generated using Bac-to-Bac expression system
(«Invitrogen», USA). Sf9 insect cells growing in IPL41
medium (10 % FBS, 50 units/ml penicillin and 50 mg/ml
streptomycin) were infected with recombinant
His/NaPi2b-L baculovirus for 3 days. Collected cells
219
IDENTIFICATION OF PHOSPHATE TRANSPORTER NAPI2B AS MX35 CANCER ANTIGEN
were lysed in buffer containing 50 mM Tris HCl, pH
8.5, 100 mM KCl, 5 mM b-mercaptoethanol, 1% NP40,
1 mM PMSF, 10 mM benzamidine, 1 mg/ml pepstain.
The lysates were then centrifuged at 10000 rpm for
15 min and clarified supernatant probed with Ni-NTA
sepharose for 1 hour at 4 oC. Affinity purification of
His/NaPi2b-L protein on Ni-NTA sepharose was
performed according to manufacturer’s recommendati-
ons («Qiagen»). The quality of purified His/NaPi2b-L
was analyzed by SDS-PAGE electrophoresis.
Western-blot analysis. For immunoblotting,
purified recombinant proteins or cell lysates were
separated by SDS-PAGE and transferred onto PVDF
membrane. The membrane was blocked for 1 hr in
PBST (PBS with 0.05 % Tween 20) and incubated
overnight at 4 °C with MX35 antibodies (1 mg/ml).
After washing three times in PBST the membrane was
incubated with anti-mouse IgG antibody («Promega»,
USA) at 1:2000 dilutions for 2 hrs. Finally, the
membranes were washed with PBST and probed with
chemiluminescence reagent (ECL, «Amersham
Biosciences», UK) for 2 min, and then exposed to the
X-ray film.
Antibody depletion procedure. 50 mg of MX35 Mab
was diluted in 2.5 ml PBST and incubated with 100 ml
of Glutathione Sepharose coupled with 0.5 mg
GST/NaPi2b-L or GST alone overnight at 4 îC.
Unbound fractions of mAbs were used for
immunohistochemical analysis of ovarian cancer
samples according to standard protocol.
Immunohistochemistry. Representative sections of
ovarian tumors were prepared from paraffin blocks.
Endogenous peroxidase was quenched with H2O2 (3 %)
in methanol. After blocking of non-specific staining
with avidin-biotin blocking solution (Vector
Laboratories, Burlinghame, CA, USA), tissue sections
were incubated overnight at 4 °C with MX35 mAb (10
mg/ml) and MX35 mAb depleted against
GST/NaPi2B-L or GST alone. Then, sections were
incubated with biotinylated secondary antibodies for 2
hrs at room temperature (1:400, goat anti-mouse
biotinylated IgG, «Sigma»), followed by the addition of
avidin-biotin-peroxidase compex (Vector Labora-
tories, Burlinghame, CA, USA; 30 min, room
temperature) and the development with diamino-
benzidine solution. Haematoxylin was used for
counterstaining. The sections were examined using
Zeiss Universal microscope («Zeiss», Germany).
Bioinformatics. CLUSTALW (1.82) software
(www.ebi.ac.uk/clustalw/) was used for multiple
sequence alignment of DNA and protein sequences
from MX35 positive clones. B-cell epitope prediction
software (www.imtech.res.in/raghara/bcepred) was
used for predicting MX35 epitope.
Results and Discussion. The search for MX35
antigen was carried out in the frame of a collaborative
consortium involving research laboratories from the
Ludwig Institute for Cancer Research (New York and
Uppsala Branches) and the Institute of Molecular
Biology and Genetics. To identify MX35 antigen, we
employed a modified version of SEREX methodology
(Serological identification of recombinantly expressed
clones). We have used this methodology extensively to
search for novel tumor-associated antigens in various
types of human cancer [4–7]. Taking into account that
MX35 antigen is expressed at high level in ovarian
cancer and ovarian cancer cell line OVCAR-3 in
particular, the expression library from OVCAR-3 cells
was generated and probed with MX35 monoclonal
antibody as described in Material and Methods.
Screening of 1×106 plagues with affinity purified MX35
IgGs allowed us to isolate a panel of weakly positive
clones. Second- and third-round screenings confirmed
specificity towards MX35 mAb only for three clones,
which we termed RK1, RK4, and RK6. The
immunoreactivity of RK1 positive clone in primary and
secondary screenings is shown in Fig. 1, A and B (see
plating). Following excision from the phagemid, the
cDNA inserts from rescued plasmids were analysed
using restriction digestion and DNA sequencing.
Bioinformatic analysis of obtained sequences revealed
that RK1 clone encodes sodium-dependent phosphate
transporter NaPi2b (AF146796), while clones RK4 and
RK6 possess overlapping sequences of zinc-finger
protein 638 (BC083513) (Fig. 1, C, see plating, Fig. 2,
A, see plating). Literature-based analysis of both
proteins indicated that sodium-dependent phosphate
transporter NaPi2b (also known as NaPi3b, Npt2,
SLC34A2) exhibits significant similarities to MX35
antigen. First of all, NaPi2b is a cell-surface
glycoprotein (690 aa) with approximately 8
transmembrane domains (SMART, Uniprot T-KB
220
KIYAMOVA R. G. ET AL
database). Secondly, similar to MX35 antigen fully
glycosylated form of NaPi2b runs on SDS-PAGE as a
95 kDa protein [8]. Moreover, NaPi2b has a predicted
basic charge 13.0 (Bioinformatic harvester database)
while MX35 antigen does not bind to DEAE sephadex
column suggesting its basic charge as well [2]. Finally,
NaPi2b and MX35 antigen exhibit similar patterns of
expression at protein and mRNA level in normal and
cancer tissues [1, 9]. In contrast to NaPi2b, zinc-finger
protein 638 shows nucleocytoplasmic localization and
exhibits different biochemical properties and
expression profiles when compared to MX35 antigen
[10]. The function of this protein is proposed to be
involved in packaging, transferring, and processing of
mRNA transcripts. Interestingly, zinc-finger protein
638 is also known as a CTCL tumor antigen se33-1 or
cutaneous T-cell lymphoma associated antigen se33-1.
Taking this into account, we decided to further
investigate the specificity of NaPi2b recognition by
MX35 antibody using various bioinformatic and
experimental approaches. Sequence analysis and
alignment of type II family of sodium-dependent
phosphate transporters suggest that NaPi2b might
possess 8 transmembrane domains mostly located
within two Na+/Pi cotransporter domains, while both
the N- and C-terminal regions face intracellular
compartment. The topology of potential
transmembrane domain implies the existence of a large
extracellular loop located between transmembrane
domains 3 and 4 [11]. Since MX35 mAb recognizes the
antigen on the surface of immunoreactive cells, one
may predict the location of the MX35 epitope within
one of the extracellular loops of NaPi2b. To check this
assumption, we have expressed the largest extracellular
loop of NaPi2b (188–361 aa) in bacterial and
baculoviral system with GST and His-tag respectively.
The expression of recombinant GST/NaPi2b-ECL in
bacteria and His/NaPi2b-ECL in insect cells was
confirmed by immunoblotting with anti-GST or
anti-His-tag antibodies (unpublished data). When
expressed in bacteria, GST/NaPi2b-ECL is found
mainly in the insoluble fraction, while baculovirally
produced His/NaPi2b-ECL shows very good solubility.
Initially, we probed the lysates of bacterial cells
expressing GST alone or GST/NaPi2b-ECL with
MX35 mAb. The data presented in Fig. 3, A, a indicate
clearly that immunoreactive band of approximately 45
kDa is specifically recognized with MX35 mAb in cells
expressing GST/NaPi2b-ECL, but not GST alone.
Notably, the molecular weight of GST/NaPi2b-ECL is
around 45 kDa. Then, we demonstrated that
baculovirally expressed His/NaPi2b-ECL is also
recognized by MX35 antibody (Fig. 3, B). Notably, the
molecular weight of GST/NaPi2b-ECL is around 45
221
IDENTIFICATION OF PHOSPHATE TRANSPORTER NAPI2B AS MX35 CANCER ANTIGEN
Fig. 3. A – expression of GST/NaPi2b-ECL in bacteria was induced by 1 mM IPTG. Total lysates and soluble fractions were resolved by
SDS-PAGE and probed with MX35 mAb; B – Sf9 cells were infected with recombinant baculovirus which drives the expression of
His/NaPi2b-ECL or wild type virus. Total lysates and soluble fractions of infected cells were resolved by SDS-PAGE and immunoblotted
with MX35 mAb
kDa while the molecular weight His/NaPi2b-ECL is
around 21 kDa. Taken together, these results provide
strong evidence that NaPi2b is a strong candidate for
MX35 antigen and that the epitope is located within the
largest extracellular loop. Moreover, the identification
of NaPi2b as a potential MX35 antigen in the
expression screening was further confirmed by affinity
puri fication followed by mass spectrometry analysis
[12].
The results of 2nd and 3rd round screening
undoubtedly demonstrated that MX35 mAb
recognition of RK4 and RK6 clones was specific, but
much weaker when compared to RK clone. These
results suggest that all three clones possess sequences
which correspond or are highly homologous to MX35
epitope. To test this assumption, we aligned amino acid
sequences from the largest extracellular loop of NaPi2b
and zinc-finger protein 638 using ClustalW software.
This analysis allowed us to identify a region which
exhibited very high level of homology between two
proteins (Fig. 2, B, see plating). Further studies are
required to map the epitope for MX35 antibody more
precisely.
As mentioned above, MX35 is overexpressed in
ovarian cancer. Therefore, we employed immuno-
histochemical approach to examine staining of ovarian
carcinoma samples with MX35 mAb under various
experimental conditions, including depletion of MX35
mAb immunoreactivity with recombinant NaPi2b. As
shown in Fig. 4, A (see plating), the apical surface of
ovarian carcinoma is strongly stained with MX35 mAb.
However, pretreatment of MX35 mAb with the access
of GST/NaPi2b-ECL, as described in Materials and
Methods, abolished the immunoreactivity of the
depleted sample in ovarian carcinoma (Fig. 4, B, see
plating). When GST alone was used for depletion of
MX35 mAb, no significant changes in the strength of
immunoreactive signal in ovarian carcinoma samples
was observed (unpublished data). These findings
provided further evidence that NaPi2b might function
as a tumor-associated antigen in ovarian cancer and that
MX35 epitope is localized in the extracellular loop of
NaPi2b. We have recently developed a panel of
monoclonal antibodies towards recombinant ECL of
NaPi2b (188–361 aa), which recognize endogenous
antigen in various immunological assays [13] and that
may be further developed as novel immunotherapeutic
agents to treat cancer.
As a member of type II family of sodium-dependent
phosphate transporters, NaPi2b has been implicated in
maintaining cellular homeostasis of inorganic
phosphate in various organs, including lungs, testis,
liver, salivary glands, secreting mammary gland,
osteoblasts, etc. [9, 14–18]. The transporting function
of NaPi2B is tightly controlled by mitogens,
glucocorticoids, vitamins, and metabolic factors [8,
19–25]. Deregulation of NaPi2b activity has been
recently associated with pulmonary and testicular
microlithiasis [26]. So far, there are only few reports
linking NaPi2b to malignant growth. The studies
involving DNA microarrays and SAGE analysis found
NaPi2b to be overexpressed in ovarian and papillary
thyroid cancer [27, 28]. The identification of NaPi2b as
a potential MX35 cancer-associated antigen should
give further incentives for the investigation on the role
of this transporter in the regulation of cell growth,
proliferation, and survival in health and cancer.
Acknowledgments. This study was supported in
part by grants from the National Academy of Sciences
of Ukraine and the Kerr Program, the Ludwig Institute
for Cancer Research. R. Kiyamova was supported by
short term fellowship from FEBS for conducting this
study.
Ð. Ã. ʳÿìî âà, Â. Ñ. Ãðèø êî âà, Â. Ñ. Óñåí êî, Þ. Ñ. Õîæàºíêî,
Â. À. Ãóð òî âèé, Á. Âèí, Ã. Ðèò òåð, Ë. Îëä, ². Ò. Ãóò,
Â. Â. Ô³ëî íåí êî
²äåí òèô³êàö³ÿ ôîñ ôàò íî ãî òðàíñ ïîð òå ðà NaPi2b ÿê ïóõ ëèí íî ãî
àí òè ãå íó MX35 ç âè êî ðèñ òàí íÿì ìî äèô³êî âà íî ãî ìå òî äó
SEREX
Ðå çþ ìå
Íàòð³é-çà ëåæ íèé òðàíñ ïîð òåð NaPi2b ³äåí òèô³êî âà íî ÿê
MX35 ïóõ ëèí íèé àí òè ãåí. Äëÿ öüî ãî çä³éñíå íî ñêðè íó âàí íÿ
êÄÍÊ-åêñïðå ñó þ ÷î¿ á³áëå î òå êè, îò ðè ìà íî¿ ç êë³òèí OVCAR3,
ìî íîê ëî íàëü íè ìè àí òèò³ëàìè MX35. Ùîá ï³äòâåð äè òè ö³
äîñë³äæåí íÿ, âñòà íîâ ëå íî, ùî NaPi2b, åêñïðå ñî âà íèé â áàê -
òåð³ÿõ òà êë³òè íàõ êî ìàõ, ñïå öèô³÷íî ðîçï³çíàºòüñÿ MX35 àí -
òèò³ëàìè. Êð³ì òîãî, âè ÿâ ëå íî ä³ëÿí êó, ÿêà ì³ñòèòü åï³òîï
äëÿ ìî íîê ëî íàëü íèõ àí òèò³ë íà âå ëè êî ìó ïî çàêë³òèí íî ìó äî -
ìåí³ NaPi2b. Òà êèì ÷è íîì, íàìè âèç íà ÷å íî àí òè ãåí MX35, ùî
çà áåç ïå ÷óº ìî ëå êó ëÿð íó îñíî âó äëÿ ïîä àëü øî ãî âèâ ÷åí íÿ éîãî
ôóíêö³¿ â íîð ìàëü íèõ òêà íè íàõ òà ïðè ðàêó.
Êëþ ÷îâ³ ñëî âà: ìî íîê ëî íàëüí³ àí òèò³ëà MX35, ðàê
ÿº÷íèê³â, ôîñ ôàò íèé íàòð³é-çà ëåæ íèé êîò ðàí ñïîð òåð
NaPi2b.
222
KIYAMOVA R. G. ET AL
Ð. Ã. Êè ÿ ìî âà, Â. C. Ãðèø êî âà, Â. C. Óñåí êî, Þ. Ñ. Õî æà åí êî,
Â. À. Ãóð òî âîé, Á. Âèí, Ã. Ðèò òåð, Ë. Îëä, È. T. Ãóò,
Â. B. Ôè ëî íåí êî
Èäåí òè ôè êà öèÿ ôîñ ôàò íî ãî òðàíñ ïîð òå ðà NaPi2b êàê
îïóõ ëå âî ãî àí òè ãå íà MX35 ñ èñ ïîëü çî âà íè åì
ìî äè ôè öè ðî âàí íî ãî ìå òî äà SEREX
Ðå çþ ìå
Íàò ðèé-çà âè ñè ìûé òðàíñ ïîð òåð NaPi2b èäåí òè ôè öè ðî âàí
êàê MX35 îïóõ ëå âûé àí òè ãåí. Äëÿ äîñ òè æå íèÿ ýòî ãî îñó ùå -
ñòâëå íî ñêðè íè ðî âà íèå êÄÍÊ-ýêñ ïðåñ ñè ðó þ ùåé áèá ëè î òå êè,
ïî ëó ÷åí íîé èç OVCAR3 êëå òîê, ìî íîê ëî íàëü íû ìè àí òè òå ëà -
ìè. ×òî áû ïîä òâåð äèòü ýòè ðå çóëü òà òû, óñòà íîâ ëå íî, ÷òî
NaPi2b, ýêñ ïðåñ ñè ðî âàí íûé â áàê òå ðè ÿõ è â êëåò êàõ íà ñå êî -
ìûõ, ñïå öè ôè÷ íî óçíà åò ñÿ àí òè òå ëà ìè MX35. Îïðå äå ëåí ó÷àñ -
òîê íà âíåê ëå òî÷ íîì äî ìå íå NaPi2b, ñî äåð æà ùèé ýïè òîï äëÿ
àí òè òåë. Òà êèì îá ðà çîì, íàìè èäåí òè ôè öè ðî âàí àí òè ãåí
MX35, ÷òî ñî çäà åò ìî ëå êó ëÿð íóþ îñíî âó äëÿ äàëü íåé øå ãî èç -
ó÷å íèÿ åãî ôóíê öèè â íîð ìàëü íûõ è ðà êî âûõ òêà íÿõ.
Êëþ ÷å âûå ñëî âà: ìî íîê ëî íàëü íûå àí òè òå ëà MX35, ðàê ÿè÷ -
íè êîâ, ôîñ ôàò íûé íà òðèé-çà âè ñè ìûé êîò ðàí ñïîð òåð NaPi2b.
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ÓÄÊ 577.2, 577.27
Íàä³éøëà äî ðå äàêö³¿ 10.10.07
224
KIYAMOVA R. G. ET AL
|
| id | nasplib_isofts_kiev_ua-123456789-157682 |
| institution | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| issn | 0233-7657 |
| language | English |
| last_indexed | 2025-11-24T04:44:52Z |
| publishDate | 2008 |
| publisher | Інститут молекулярної біології і генетики НАН України |
| record_format | dspace |
| spelling | Kiyamova, R.G. Gryshkova, V.S. Usenko, V.S. Khozaenko, Y.S. Gurtovyy, V.A. Yin, B. Ritter, G. Old, L. Gout, I.T. Filonenko, V.V. 2019-06-20T17:52:30Z 2019-06-20T17:52:30Z 2008 Identification of phosphate transporter NaPi2b as MX35 cancer antigen by modified SEREX approach / R.G. Kiyamova, V.S. Gryshkova, V.S. Usenko, Y.S. Khozaenko, V.A. Gurtovyy, B. Yin, G. Ritter, L. Old, I.T. Gout, V.V. Filonenko // Біополімери і клітина. — 2008. — Т. 24, № 3. — С. 218-224. — Бібліогр.: 28 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.0007A3 https://nasplib.isofts.kiev.ua/handle/123456789/157682 577.2, 577.27 In this study we describe the identification of sodium-dependent phosphate transporter NaPi2b as MX35 cancer-associated antigen. To achieve this goal we have screened extensively a cDNA expressing library from ovarian cancer cell line OVCAR3 with monoclonal antibody MX35. To further confirm the authenticity of this finding, we showed that bacterially and baculovirally expressed NaPi2b is specifically recognized by MX35 antibody. Moreover, the validity of these results was verified in a parallel study involving affinity purification and mass spectrometry. The epitope for MX35 monoclonal antibody was mapped to the largest extracellular loop of NaPi2b. Taken together, this study uncovers the identity of MX35 antigen and provides molecular tools for studying its function in normal and cancer tissues. Натрій-залежний транспортер NaPi2b ідентифіковано як MX35 пухлинний антиген. Для цього здійснено скринування кДНК-експресуючої біблеотеки, отриманої з клітин OVCAR3, моноклональними антитілами MX35. Щоб підтвердити ці дослідження, встановлено, що NaPi2b, експресований в бактеріях та клітинах комах, специфічно розпізнається MX35 антитілами. Крім того, виявлено ділянку, яка містить епітоп для моноклональних антитіл на великому позаклітинному домені NaPi2b. Таким чином, нами визначено антиген MX35, що забезпечує молекулярну основу для подальшого вивчення його функції в нормальних тканинах та при раку. Натрий-зависимый транспортер NaPi2b идентифицирован как MX35 опухлевый антиген. Для достижения этого осуществлено скринирование кДНК-экспрессирующей библиотеки, полученной из OVCAR3 клеток, моноклональными антителами. Чтобы подтвердить эти результаты, установлено, что NaPi2b, экспрессированный в бактериях и в клетках насекомых, специфично узнается антителами MX35. Определен участок на внеклеточном домене NaPi2b, содержащий эпитоп для антител. Таким образом, нами идентифицирован антиген MX35, что создает молекулярную основу для дальнейшего изучения его функции в нормальных и раковых тканях. en Інститут молекулярної біології і генетики НАН України Біополімери і клітина Структура та функції біополімерів Identification of phosphate transporter NaPi2b as MX35 cancer antigen by modified SEREX approach Идентификация фосфатного транспортера NaPi2b как опухлевого антигена MX35 с использованием модифицированного метода SEREX Ідентифікація фосфатного транспортера NaPi2b як пухлинного антигену MX35 з використанням модифікованого методу SEREX Article published earlier |
| spellingShingle | Identification of phosphate transporter NaPi2b as MX35 cancer antigen by modified SEREX approach Kiyamova, R.G. Gryshkova, V.S. Usenko, V.S. Khozaenko, Y.S. Gurtovyy, V.A. Yin, B. Ritter, G. Old, L. Gout, I.T. Filonenko, V.V. Структура та функції біополімерів |
| title | Identification of phosphate transporter NaPi2b as MX35 cancer antigen by modified SEREX approach |
| title_alt | Идентификация фосфатного транспортера NaPi2b как опухлевого антигена MX35 с использованием модифицированного метода SEREX Ідентифікація фосфатного транспортера NaPi2b як пухлинного антигену MX35 з використанням модифікованого методу SEREX |
| title_full | Identification of phosphate transporter NaPi2b as MX35 cancer antigen by modified SEREX approach |
| title_fullStr | Identification of phosphate transporter NaPi2b as MX35 cancer antigen by modified SEREX approach |
| title_full_unstemmed | Identification of phosphate transporter NaPi2b as MX35 cancer antigen by modified SEREX approach |
| title_short | Identification of phosphate transporter NaPi2b as MX35 cancer antigen by modified SEREX approach |
| title_sort | identification of phosphate transporter napi2b as mx35 cancer antigen by modified serex approach |
| topic | Структура та функції біополімерів |
| topic_facet | Структура та функції біополімерів |
| url | https://nasplib.isofts.kiev.ua/handle/123456789/157682 |
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