Inhibition of sodium-dependent phosphate transporter NaPi2b function with MX35 antibody
Sodium-dependent phosphate transporter NaPi2b is involved in transport of inorganic phosphate and the maintenance of phosphate homeostasisin human body. NaPi2b wasrecently identified as a marker of ovarian cancer, termed MX35. Monoclonal antibody (mAb) against transporter NaPi2b, called MX35, demon...
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| Cite this: | Inhibition of sodium-dependent phosphate transporter NaPi2b function with MX35 antibody / V.S. Gryshkova, V.V. Filonenko, R.G. Kiyamova // Вiopolymers and Cell. — 2011. — Т. 27, № 3. — С. 193-198. — Бібліогр.: 24 назв. — англ. |
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Gryshkova, V.S. Filonenko, V.V. Kiyamova, R.G. 2019-06-18T12:04:51Z 2019-06-18T12:04:51Z 2011 Inhibition of sodium-dependent phosphate transporter NaPi2b function with MX35 antibody / V.S. Gryshkova, V.V. Filonenko, R.G. Kiyamova // Вiopolymers and Cell. — 2011. — Т. 27, № 3. — С. 193-198. — Бібліогр.: 24 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.0000B9 https://nasplib.isofts.kiev.ua/handle/123456789/156363 577.2, 577.27 Sodium-dependent phosphate transporter NaPi2b is involved in transport of inorganic phosphate and the maintenance of phosphate homeostasisin human body. NaPi2b wasrecently identified as a marker of ovarian cancer, termed MX35. Monoclonal antibody (mAb) against transporter NaPi2b, called MX35, demonstrated therapeutic efficacy in radioimmunotherapy of patients with ovarian cancer. Aim. The present experiments explored whether MX35 antibody can affect function of NaPi2b to transport inorganic phosphate ions in cellular models. Methods. HEK293 cells stably expressing wild type NaPi2b and mutant NaPi2b_T330V, which could not be recognized by MX35 antibody, were incubated with MX35 antibody and analyzed by phosphate uptake assay. Results. Cells expressing wild type NaPi2b showed reduced phosphate uptake after incubation with MX35 antibody at concentration 50 µg/ml. No significant changes in phosphate transport were detected for cells expressing NaPi2b_T330V at the same experimental conditions. Conclusions. Our results demonstrate 1,8-fold decrease of NaPi2b-mediated phosphate transport in HEK293 cells stably expressing wild type NaPi2b after MX35 antibody application, which can be considered as a specific inhibitor of NaPi2b function. Keywords: NaPi2b, MX35 antibody. Натрій-залежний транспортер фосфату NaPi2b бере участь у перенесенні неорганічного фосфату та підтриманні фосфатного гомеостазу в організмі людини. Нещодавно NaPi2b описано як маркер раку яєчника МХ35. Моноклональні антитіла (МКАТ) проти транспортера NaPi2b, які отримали назву МХ35, виявляють терапевтичну ефективність при лікуванні хворих на рак яєчника. Мета даної роботи полягала в тому, щоб з’ясувати, чи впливає зв’язування МКАТ МХ35 з NaPi2b на його функцію транспортувати іони неорганічного фосфату в клітинних моделях. Методи. Клітини лінії НЕК293, що стабільно експресують дикий тип NaPi2b та мутантну форму NaPi2b_T330V, яка не розпізнається антитілами МХ35, інкубували з антитілами МХ35, після чого аналізували поглинання клітинами радіоактивно міченого фосфату. Результати. Після інкубації з антитілами МХ35 у концентрації 50 мкг/мл відмічено значне зменшення поглинання фосфату клітинами, які експресують дикий тип NaPi2b. Для клітин, що експресують мутантну форму NaPi2b_T330V, за аналогічних експериментальних умов значних змін у поглинанні фосфату не виявлено. Висновки. Наші дані свідчать про 1,8-разове зниження NaPi2b-залежного транспорту фосфату в результаті інкубації з МКАТ МХ35, які можуть слугувати специфічним інгібітором функції NaPi2b. Ключові слова: NaPi2b, моноклональні антитіла МХ35. Натрий-зависимый транспортер фосфата NaPi2b участвует в переносе неорганического фосфата и поддержании фосфатного гомеостаза в организме человека. Недавно NaPi2b описан как маркер рака яичника МХ35. Моноклональные антитела (МКАТ) против транспортера NaPi2b, названные МХ35, проявляют терапевтическую эффективность при лечении больных раком яичника. Цель данной работы состояла в выяснении того, влияет ли связывание МКАТ МХ35 с NaPi2b на его функцию транспортировать ионы неорганического фосфата в клеточных моделях. Методы. Клетки линии НЕК293, стабильно экспрессирующие дикий тип NaPi2b и мутантную форму NaPi2b_T330V, не распознаваемую антителами МХ35, инкубировали с антителами МХ35, после чего анализировали поглощение клетками радиоактивно меченного фосфата. Результаты. После инкубации с антителами МХ35 в концентрации 50 мкг/мл отмечено значительное уменьшение поглощения фосфата клетками, экспрессирующими дикий тип NaPi2b. Для клеток, экспрессирующих мутантную форму транспортера NaPi2b_T330V, не выявлено существенных изменений в поглощении фосфата при аналогичных экспериментальных условиях. Выводы. Наши результаты свидетельствуют о 1,8-кратном снижении NaPi2b-зависимого транспорта фосфата в результате инкубации с МКАТ МХ35, которые можно рассматривать как специфический ингибитор функции NaPi2b. Ключевые слова: NaPi2b, моноклональные антитела МХ35. en Інститут молекулярної біології і генетики НАН України Вiopolymers and Cell Structure and Function of Biopolymers Inhibition of sodium-dependent phosphate transporter NaPi2b function with MX35 antibody Інгібування функції натрій-залежного транспортера фосфатів NaPi2b специфічними антитілами MX35 Ингибирование функции натрий-зависимого транспортера фосфатов NaPi2b специфическими антителами MX35 Article published earlier |
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| title |
Inhibition of sodium-dependent phosphate transporter NaPi2b function with MX35 antibody |
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Inhibition of sodium-dependent phosphate transporter NaPi2b function with MX35 antibody Gryshkova, V.S. Filonenko, V.V. Kiyamova, R.G. Structure and Function of Biopolymers |
| title_short |
Inhibition of sodium-dependent phosphate transporter NaPi2b function with MX35 antibody |
| title_full |
Inhibition of sodium-dependent phosphate transporter NaPi2b function with MX35 antibody |
| title_fullStr |
Inhibition of sodium-dependent phosphate transporter NaPi2b function with MX35 antibody |
| title_full_unstemmed |
Inhibition of sodium-dependent phosphate transporter NaPi2b function with MX35 antibody |
| title_sort |
inhibition of sodium-dependent phosphate transporter napi2b function with mx35 antibody |
| author |
Gryshkova, V.S. Filonenko, V.V. Kiyamova, R.G. |
| author_facet |
Gryshkova, V.S. Filonenko, V.V. Kiyamova, R.G. |
| topic |
Structure and Function of Biopolymers |
| topic_facet |
Structure and Function of Biopolymers |
| publishDate |
2011 |
| language |
English |
| container_title |
Вiopolymers and Cell |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| format |
Article |
| title_alt |
Інгібування функції натрій-залежного транспортера фосфатів NaPi2b специфічними антитілами MX35 Ингибирование функции натрий-зависимого транспортера фосфатов NaPi2b специфическими антителами MX35 |
| description |
Sodium-dependent phosphate transporter NaPi2b is involved in transport of inorganic phosphate and the maintenance of phosphate homeostasisin human body. NaPi2b wasrecently identified as a marker of ovarian cancer,
termed MX35. Monoclonal antibody (mAb) against transporter NaPi2b, called MX35, demonstrated therapeutic
efficacy in radioimmunotherapy of patients with ovarian cancer. Aim. The present experiments explored whether MX35 antibody can affect function of NaPi2b to transport inorganic phosphate ions in cellular models.
Methods. HEK293 cells stably expressing wild type NaPi2b and mutant NaPi2b_T330V, which could not be recognized by MX35 antibody, were incubated with MX35 antibody and analyzed by phosphate uptake assay.
Results. Cells expressing wild type NaPi2b showed reduced phosphate uptake after incubation with MX35 antibody at concentration 50 µg/ml. No significant changes in phosphate transport were detected for cells expressing NaPi2b_T330V at the same experimental conditions. Conclusions. Our results demonstrate 1,8-fold decrease of NaPi2b-mediated phosphate transport in HEK293 cells stably expressing wild type NaPi2b after
MX35 antibody application, which can be considered as a specific inhibitor of NaPi2b function.
Keywords: NaPi2b, MX35 antibody.
Натрій-залежний транспортер фосфату NaPi2b бере участь у
перенесенні неорганічного фосфату та підтриманні фосфатного гомеостазу в організмі людини. Нещодавно NaPi2b описано як
маркер раку яєчника МХ35. Моноклональні антитіла (МКАТ)
проти транспортера NaPi2b, які отримали назву МХ35, виявляють терапевтичну ефективність при лікуванні хворих на рак
яєчника. Мета даної роботи полягала в тому, щоб з’ясувати, чи
впливає зв’язування МКАТ МХ35 з NaPi2b на його функцію транспортувати іони неорганічного фосфату в клітинних моделях.
Методи. Клітини лінії НЕК293, що стабільно експресують дикий
тип NaPi2b та мутантну форму NaPi2b_T330V, яка не розпізнається антитілами МХ35, інкубували з антитілами МХ35,
після чого аналізували поглинання клітинами радіоактивно міченого фосфату. Результати. Після інкубації з антитілами МХ35
у концентрації 50 мкг/мл відмічено значне зменшення поглинання
фосфату клітинами, які експресують дикий тип NaPi2b. Для
клітин, що експресують мутантну форму NaPi2b_T330V, за аналогічних експериментальних умов значних змін у поглинанні фосфату не виявлено. Висновки. Наші дані свідчать про 1,8-разове зниження NaPi2b-залежного транспорту фосфату в результаті
інкубації з МКАТ МХ35, які можуть слугувати специфічним інгібітором функції NaPi2b.
Ключові слова: NaPi2b, моноклональні антитіла МХ35.
Натрий-зависимый транспортер фосфата NaPi2b участвует в
переносе неорганического фосфата и поддержании фосфатного
гомеостаза в организме человека. Недавно NaPi2b описан как
маркер рака яичника МХ35. Моноклональные антитела (МКАТ)
против транспортера NaPi2b, названные МХ35, проявляют терапевтическую эффективность при лечении больных раком яичника. Цель данной работы состояла в выяснении того, влияет ли
связывание МКАТ МХ35 с NaPi2b на его функцию транспортировать ионы неорганического фосфата в клеточных моделях. Методы. Клетки линии НЕК293, стабильно экспрессирующие дикий
тип NaPi2b и мутантную форму NaPi2b_T330V, не распознаваемую антителами МХ35, инкубировали с антителами МХ35, после
чего анализировали поглощение клетками радиоактивно меченного фосфата. Результаты. После инкубации с антителами
МХ35 в концентрации 50 мкг/мл отмечено значительное уменьшение поглощения фосфата клетками, экспрессирующими дикий
тип NaPi2b. Для клеток, экспрессирующих мутантную форму
транспортера NaPi2b_T330V, не выявлено существенных изменений в поглощении фосфата при аналогичных экспериментальных условиях. Выводы. Наши результаты свидетельствуют о
1,8-кратном снижении NaPi2b-зависимого транспорта фосфата в результате инкубации с МКАТ МХ35, которые можно рассматривать как специфический ингибитор функции NaPi2b.
Ключевые слова: NaPi2b, моноклональные антитела МХ35.
|
| issn |
0233-7657 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/156363 |
| citation_txt |
Inhibition of sodium-dependent phosphate transporter NaPi2b function with MX35 antibody / V.S. Gryshkova, V.V. Filonenko, R.G. Kiyamova // Вiopolymers and Cell. — 2011. — Т. 27, № 3. — С. 193-198. — Бібліогр.: 24 назв. — англ. |
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2025-11-25T20:31:29Z |
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| fulltext |
STRUCTURE AND FUNCTION OF BIOPOLYMERS
Inhibition of sodium-dependent phosphate transporter
NaPi2b function with MX35 antibody
V. S. Gryshkova, V.V. Filonenko, R. G. Kiyamova
Institute of Molecular Biology and Genetics NAS of Ukraine
150, Akademika Zabolotnoho St., Kyiv, Ukraine, 03680
filonenko@imbg.org.ua
Sodium-dependent phosphate transporter NaPi2b is involved in transport of inorganic phosphate and the main-
tenance of phosphate homeostasis in human body. NaPi2b was recently identified as a marker of ovarian cancer,
termed MX35. Monoclonal antibody (mAb) against transporter NaPi2b, called MX35, demonstrated therapeutic
efficacy in radioimmunotherapy of patients with ovarian cancer. Aim. The present experiments explored whe-
ther MX35 antibody can affect function of NaPi2b to transport inorganic phosphate ions in cellular models.
Methods. HEK293 cells stably expressing wild type NaPi2b and mutant NaPi2b_T330V, which could not be re-
cognized by MX35 antibody, were incubated with MX35 antibody and analyzed by phosphate uptake assay.
Results. Cells expressing wild type NaPi2b showed reduced phosphate uptake after incubation with MX35 an-
tibody at concentration 50 µg/ml. No significant changes in phosphate transport were detected for cells expres-
sing NaPi2b_T330V at the same experimental conditions. Conclusions. Our results demonstrate 1,8-fold de-
crease of NaPi2b-mediated phosphate transport in HEK293 cells stably expressing wild type NaPi2b after
MX35 antibody application, which can be considered as a specific inhibitor of NaPi2b function.
Keywords: NaPi2b, MX35 antibody.
Introduction. Sodium-dependent phosphate transpor-
ter NaPi2b (SLC34A2, NaPiIIb, Npt2) is normally ex-
pressed in mammalian small intestine at the apical side
of enterocyte’s brush-border membranes. NaPi2b par-
ticipates in the transcellular inorganic phosphate (Pi)
absorption, contributing to the maintenance of phos-
phate homeostasis in the organism [1–3]. The studies
on conditional knockout mice demonstrated that
NaPi2b contributes to > 90 % of total active phosphate
absorption [4].
The attempts to generate NaPi2b homozygous de-
ficient mice resulted in death of embryos in uterus soon
after implantation, indicating that NaPi2b is the major
Pi transporter during mouse embryonic development
[5].
The expression of NaPi2b mRNA was revealed by
Northern blot analysis in many human normal tissues
including lung, kidney, liver, prostate, pancreas, testis,
ovary, thymus [1, 2]. The expression of NaPi2b on pro-
tein level has been detected in normal human salivary
glands by Western blotting with polyclonal antibodies
against NaPi2b [6].
It was reported that alterations in NaPi2b expres-
sion are linked to several human abnormalities. In
lungs, NaPi2b is the only phosphate transporter that is
highly expressed, and it is involved in uptake of phos-
phate for synthesis of surfactant proteins. Deregulation
of the NaPi2b transporter function as a result of muta-
tions in SLC34A2 gene may lead to the pulmonary
alveolar microlithiasis, autosomal recessively inherited
disease, characterized by deposition of calcium-phos-
phate precipitates in lungs [7, 8].
193
ISSN 0233–7657. Biopolymers and Cell. 2011. Vol. 27. N 3. P. 193–198
Institute of Molecular Biology and Genetics, NAS of Ukraine, 2011
The increased expression of SLC34A2 gene in can-
cer was detected in ovarian and papillary thyroid tu-
mors [9, 10]. Recently, it has been demonstrated that
the expression level of SLC34A2 gene is increased in
human breast tumors [11]. Low dietary Pi uptake, well-
known regulator of NaPi2b expression and function,
was shown to cause lung tumorigenesis in K-ras (LA1)
model mice on the background of increased NaPi2b
expression [12].
Previously, we identified NaPi2b as an ovarian
cancer antigen MX35 by screening cDNA library from
OVCAR3 cells with the MX35 antibody [13]. This
antibody was generated from mice immunized with a
cocktail of human ovarian carcinoma cells and showed
reactivity with approximately 90 % samples of human
epithelial ovarian cancers [14]. Heterogeneous expres-
sion profile of MX35/NaPi2b protein was further con-
firmed for epithelial ovarian tumors of different histo-
morphological types [15]. The radiolabelled MX35
antibody was used in clinical trials and was shown to
possess the therapeutic effect in patients with ovarian
cancer [16, 17]. However, the unlabelled MX35 anti-
body did not have any therapeutic effect on tumor gro-
wth in model mice [18].
Playing an essential role in phosphate homeostasis
in a human body NaPi2b is considered as a target for in-
hibition of hyperphosphatemia in patients with chronic
renal failure [19]. There are some Pi-containing com-
pounds that have been reported as inhibitors of sodium-
dependent phosphate transport including phospho-
noformic acid, 2V-phosphophloretin, and JTP-59557
[20–22]. But all mentioned inhibitors are not strictly
specific for sodium-dependent phosphate transport me-
diated by NaPi2b.
The identification of MX35 ovarian cancer antigen
as sodium-dependent transporter NaPi2b gave an op-
portunity to investigate the impact of specific antibody
MX35 on the transport function of NaPi2b. This study
aimed to analyze the effect of MX35 antibody on the
functioning of sodium-dependent phosphate transpor-
ter NaPi2b using cellular models and to determine whe-
ther this antibody can function as a specific inhibitor of
NaPi2b-mediated phosphate transport.
Materials and methods. Constructs, stable cell
line generation. Generation of HEK293 cells stably ex-
pressing wild type and mutant forms of NaPi2b was
previously described [23]. Briefly, human NaPi2b
cDNA (NaPi2b_WT) was cloned into pcDNA3.1(+)
(«Invitrogen», USA) in the frame with EE-tag. A mu-
tant form of NaPi2b (pcDNA3.1(+)/NaPi2b_T330V)
was obtained by site-directed mutagenesis. HEK293
cells were transfected with recombinant plasmids
(pcDNA3.1(+)/NaPi2b_WT, pcDNA3.1(+)/NaPi2b_
T330V or empty vector) using FuGene («Roshe»,
Switzerland). Cells were cultured in the presence of
G418 antibiotic («Gibco», UK) at the concentration of
1 mg/ml for 7–10 days to eliminate nontransfected cells
before splitting.
Phosphate uptake analysis. HEK293 cells were
placed on 96-well plate at 3⋅104 cells/well and incubated
with the MX35 antibody for different time (6, 12, 24,
48, 96 h). The MX35 antibody was kindly provided by
Dr. Gerd Ritter (Ludwig Institute for Cancer research,
USA). After medium was removed cells were washed 3
times with 200 µl of wash solution (137 mM NaCl,
5.4 mM KCl, 2.8 mM CaCl2, 1.2 mM MgSO4, 14 mM
Tris HCl (pH 7.4)). 50 µl of assay solution (0.1 mM
KH2PO4, 137 mM NaCl, 5.4 mM KCl, 2.8 mM CaCl2,
1.2 mM MgSO4, 14 mM Tris HCl (pH 7.4)) with [33P]
orthophosphate (1 µCi/ml) was added to each well and
incubated at room temperature. Then, assay solution
was removed and cells were rinsed 3 times with 150 µl
of ice-cold stop solution (137 mM NaCl, 14 mM Tris
HCl (pH 7.4)). Cells were lysed in 50 µl of lysis buffer
(0.01 M Tris HCl, pH 7.5, 0.15 M NaCl, 0.01 M MgCl2,
0.5 % NP-40) and 25 µl of cell lysate was used to de-
termine radioactivity in a liquid scintillation counter
(«Perkin Elmer», USA). The rest of cell lysate was
used to determine protein concentration using BCA
protein assay kit («Pierce Biotechnology», USA). To
inhibit sodium-dependent phosphate transport phos-
phonoformic acid («Sigma», USA) was added to the
assay solution at 5 mM concentration.
Western blot analysis. HEK293 cells were lysed in
buffer containing 10 mM Tris-HCI, pH 7.5, 150 mM
NaCl, 10 mM MgCl2, 0.5 % NP-40 and a mixture of
Halt Protease Inhibitor Cocktail («Pierce», USA). Pro-
tein concentration was estimated by Comassie Assay
(«Pierce»). Protein samples were separated by 10 %
SDS/PAGE and electrotransferred on polyvinylidene
difluoride (PVDF) membrane («Millipore», USA).
The membranes were blocked with 3 % BSA in 1 ×
194
GRYSHKOVA V. S., FILONENKO V. V., KIYAMOVA R. G.
× PBST for 1 h at RT and incubated with the MX35
antibody at 4 °C overnight. After incubation with
HRP-conjugated goat anti-mouse IgG («Promega»,
USA) the membranes were developed using the ECL
system («Amersham», Sweden) and then exposed to
Agfa X-ray film.
Statistical analysis. Data are provided as means ±
± SEM. All experiments were repeated 3 times; in all
repetitions qualitatively similar data were obtained.
Results and discussion. Sodium-dependent phos-
phate transporter NaPi2b, which plays an essential role
in the maintenance of phosphate homeostasis in the
human body, was revealed as ovarian cancer marker
MX35 [13]. It has been shown that the radiolabelled
MX35 antibody recognizing NaPi2b has therapeutic
effect in patients with ovarian cancer and F(ab)2 frag-
ments of the MX35 antibody specifically localize in
micrometastatic loci within peritoneal cavity [16, 17].
Knowledge about the function of MX35 antigen pro-
vided the basis for the investigation of the MX35 an-
tibody impact on the phosphate transport function of
MX35/NaPi2b protein.
To investigate the function of NaPi2b as a trans-
porter of inorganic phosphate we applied a phosphate
uptake assay using HEK293 cells expressing the wild
type and mutant form of NaPi2b as well as HEK293
cells transfected with empty pcDNA3.1(+) plasmid as a
control. In the predicted topology of NaPi2b, both N-
and C-terminal tails face cytoplasm, exposing loops of
various length to the extracellular and intracellular
compartments including a large extracellular loop
(188–361 aa). Previously, we have shown that the epi-
tope recognized by MX35 antibody is located within
the predicted extracellular domain (311–340 aa) [13,
24]. Further studies have shown that the mutation
T330V in the large extracellular loop of NaPi2b trans-
porter is crucial for the recognition of NaPi2b by the
MX35 antibody [23]. Western blot analysis of lysates
of cell lines stably expressing the wild type and mutant
form (NaPi2b_T330V) of transporter clearly indicated
that mutant NaPi2b_T330V was not recognized by the
MX35 antibody whereas both wild type and mutant
form were recognized well by the EE-tag specific an-
tibody (Fig. 1).
We applied a phosphate uptake assay to investigate
the NaPi2b transport function in the cells expressing
wild type NaPi2b and in the control cell line. In time-
course study, the human NaPi2b-mediated Pi uptake in-
creased linearly up to 30 min, thus 15 min uptake time
was used to evaluate phosphate uptake in further ex-
periments (data not shown).
At this uptake time we observed 2.5-fold increase
in Pi uptake in the cells expressing wild type NaPi2b
(10.6 ± 0.6 nmol/mg protein) in contrast to the cells
transfected with empty pcDNA3.1(+) plasmid (4.2 ±
± 0.4 nmol/mg protein) (Fig. 2). The cells expressing
the mutant form NaPi2b_T330V transported inorganic
phosphate with the same efficiency as the cells expres-
sing the wild type of NaPi2b (11.5 ± 0.7 nmol/mg
protein) (Fig. 2). The use of well-known inhibitor of
sodium-dependent phosphate transport phosphonofor-
195
INHIBITION OF SODIUM-DEPENDENT PHOSPHATE TRANSPORTER NAPI2B FUNCTION WITH MX35 ANTIBODY
90 kDa
$-actin
NaPi2b
1 2 3 1 2 3
a b
Fig. 1. Western blot analysis of HEK293 cells expressing wild type
NaPi2b (1), mutant form NaPi2b_T330V (2) and cells transfected with
empty pcDNA3.1(+) vector (3) by anti-EE-tag (a) and MX35 (b)
antibodies; β-actin was used as a loading control
0
2
4
6
8
10
12
1 2 3 1 2 3
a b
Fig. 2. The results of phosphate uptake assay on HEK293 stable cell
lines expressing wild type NaPi2b (1), mutant form NaPi2b_T330V (2)
and cells transfected with empty pcDNA3.1.(+) vector (3) (a); phos-
phate uptake assay of the same cell lines after treatment with phos-
phonoformic acid at 5 mM concentration (b). Cells were incubated in
assay solution for 15 min at RT
mic acid at 5 mM concentration in assay solution led to
the total abolishment of Pi uptake in all the investigated
cell lines (Fig. 2).
To determine the effect of MX35 antibody on
NaPi2b-mediated transport we performed the phospha-
te uptake experiments for all mentioned cell lines after
incubation with the MX35 antibody at different con-
centrations (5, 10, 20, and 50 µg/ml) and different time
(6, 12, 24, 48, 96 h) (data not shown). We detected a
considerable inhibition of phosphate uptake in the cells
expressing wild type of NaPi2b incubated for 24h with
the MX35 antibody at concentration of 50 µg/ml. In
this case 1,8-fold decrease in sodium-dependent phos-
phate uptake was observed for the wild type NaPi2b
(incubation with antibody – 10.52 ± 0.65 nmol/mg;
incubation without antibody – 5.6 ± 0.45 nmol/mg). At
the same time phosphate transport in the cell line trans-
fected with empty pcDNA3.1(+) plasmid remained
unchanged (incubation with antibody – 4.5 ± 0.3 nmol/
mg protein; incubation without antibody – 4.2 ±
± 0.4 nmol/mg) (Fig. 3).
To confirm the specificity of this inhibition we
evaluated the effect of MX35 antibody on the cells ex-
pressing the mutant form NaPi2b_T330V, which is not
recognized by the MX35 antibody. No significant
changes were detected in the phosphate uptake mea-
surements in the cells expressing NaPi2b_T330V after
incubation with the MX35 antibody (11.5 ± 0.7 nmol/
mg protein; 10 ± 0.5 nmol/mg protein) (Fig. 3). Our
data clearly demonstrate that the MX35 antibody spe-
cific to NaPi2b transporter significantly decreased so-
dium-dependent phosphate uptake in the cellular mo-
dels used in this study.
The mechanism of remarkable inhibition of so-
dium-dependent phosphate transport by the antibody
specific to extracellular domain of NaPi2b still remains
unknown. It should be further investigated whether the
incubation of the cells expressing NaPi2b with the
MX35 antibody may lead to down regulation of
NaPi2b protein decreasing the number of transporter
units on cell surface or the binding of MX35 antibody
can cause conformational changes in transporter struc-
ture affecting its function and vice versa it can prevent
conformational changes necessary for transporter func-
tioning.
Conclusions. Using cellular models expressing the
wild type and mutant NaPi2b_T330V transporter we
showed that the MX35 antibody directed against
NaPi2b significantly decreased phosphate uptake me-
diated by NaPi2b and, thus, can be considered as a spe-
cific inhibitor of NaPi2b transport function.
Acknowledgments. V. Gryshkova was supported
by Boehringer Inhelheim Fonds (BIF) travel grant to
perform the part of this work at the University College
London under I. Gout supervision.
В. С. Гриш ко ва, В. В. Філо нен ко, Р. Г. Кіямо ва
Інгібу ван ня функції натрій-за леж но го транс пор те ра фос фатів
NaPi2b спе цифічни ми ан титілами MX35
Ре зю ме
Натрій-за леж ний транс пор тер фос фа ту NaPi2b бере участь у
пе рене сенні не органічно го фос фа ту та підтри манні фос фат но -
го го ме ос та зу в організмі лю ди ни. Не що дав но NaPi2b опи са но як
мар кер раку яєчни ка МХ35. Мо нок ло нальні ан титіла (МКАТ)
про ти транс пор те ра NaPi2b, які от ри ма ли на зву МХ35, ви яв ля -
ють те ра пев тич ну ефек тивність при ліку ванні хво рих на рак
яєчни ка. Мета да ної ро бо ти по ля га ла в тому, щоб з’я су ва ти, чи
впливає зв’я зу ван ня МКАТ МХ35 з NaPi2b на його функцію транс -
пор ту ва ти іони не органічно го фос фа ту в клітин них мо де лях.
Ме то ди. Клітини лінії НЕК293, що стабільно експре су ють ди кий
тип NaPi2b та му тан тну фор му NaPi2b_T330V, яка не роз-
пізнається ан титілами МХ35, інку бу ва ли з ан титілами МХ35,
після чого аналізу ва ли по гли нан ня кліти на ми радіоак тив но міче -
но го фос фа ту. Ре зуль та ти. Після інку бації з ан титілами МХ35
у кон цен трації 50 мкг/мл відмічено знач не змен шен ня по гли нан ня
фос фа ту кліти на ми, які експре су ють ди кий тип NaPi2b. Для
клітин, що експре су ють му тан тну фор му NaPi2b_T330V, за ана -
логічних ек спе ри мен таль них умов знач них змін у по гли нанні фос -
фа ту не ви яв ле но. Вис нов ки. Наші дані свідчать про 1,8-раз ове
196
GRYSHKOVA V. S., FILONENKO V. V., KIYAMOVA R. G.
0
2
4
6
8
10
12
a b c
1
2
Fig. 3. The results of phosphate uptake assay on stable cell lines ex-
pressing wild type NaPi2b (a), mutant form NaPi2b_T330V (b) and
control cell line (c) without (1) and with (2) MX35 antibody at concen-
tration 50 µg/ml during 24 h
знижен ня NaPi2b-за леж но го транс пор ту фос фа ту в ре зуль таті
інку бації з МКАТ МХ35, які мо жуть слу гу ва ти спе цифічним інгі-
біто ром функції NaPi2b.
Клю чові сло ва: NaPi2b, мо нок ло нальні ан титіла МХ35.
В. С. Гриш ко ва, В. В. Фи ло нен ко, Р. Г. Ки я мо ва
Инги би ро ва ние функ ции на трий-за ви си мо го транс пор те ра
фос фа тов NaPi2b спе ци фи чес ки ми ан ти те ла ми MX35
Ре зю ме
Нат рий-за ви си мый транс пор тер фос фа та NaPi2b учас тву ет в
пе ре но се не орга ни чес ко го фос фа та и под дер жа нии фос фат но го
го ме ос та за в орга низ ме че ло ве ка. Не дав но NaPi2b опи сан как
мар кер рака яич ни ка МХ35. Мо нок ло наль ные ан ти те ла (МКАТ)
про тив транс пор те ра NaPi2b, на зван ные МХ35, про яв ля ют те -
ра пев ти чес кую эф фек тив ность при ле че нии боль ных ра ком яич -
ни ка. Цель дан ной ра бо ты со сто я ла в вы яс не нии того, вли я ет ли
свя зы ва ние МКАТ МХ35 с NaPi2b на его функ цию транс пор ти ро -
вать ионы не орга ни чес ко го фос фа та в кле точ ных мо де лях. Ме -
то ды. Клет ки ли нии НЕК293, ста биль но экс прес си ру ю щие ди кий
тип NaPi2b и му тан тную фор му NaPi2b_T330V, не рас поз на ва е -
мую ан ти те ла ми МХ35, ин ку би ро ва ли с ан ти те ла ми МХ35, по сле
чего ана ли зи ро ва ли по гло ще ние клет ка ми ра ди о ак тив но ме чен -
но го фос фа та. Ре зуль та ты. Пос ле ин ку ба ции с ан ти те ла ми
МХ35 в кон цен тра ции 50 мкг/мл от ме че но зна чи тель ное умень -
ше ние по гло ще ния фос фа та клет ка ми, экс прес си ру ю щи ми ди кий
тип NaPi2b. Для кле ток, экс прес си ру ю щих му тан тную фор му
транс пор те ра NaPi2b_T330V, не вы яв ле но су щес твен ных из ме -
не ний в по гло ще нии фос фа та при ана ло гич ных экс пе ри мен таль -
ных усло ви ях. Вы во ды. Наши ре зуль та ты сви де т ельству ют о
1,8-крат ном сни же нии NaPi2b-за ви си мо го транс пор та фос фа -
та в ре зуль та те ин ку ба ции с МКАТ МХ35, ко то рые мож но рас -
смат ри вать как спе ци фи чес кий ин ги би тор функ ции NaPi2b.
Клю че вые сло ва: NaPi2b, мо нок ло наль ные ан ти те ла МХ35.
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