Recognition of mutant forms of the sodium-dependent phosphate transporter NaPi2b by ­monoclonal antibodies in ovarian cancer cells

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Abstract

Background. The sodium-dependent phosphate transporter NaPi2b is expressed in a number of tumors and serves as a target for monoclonal antibody therapy. The NaPi2b transporter has a large extracellular domain containing 4 cysteines and an MX35 epitope recognized by the corresponding monoclonal antibodies.

Aim. To study the recognition of the MX35 epitope in cysteine mutants C303 and C350 of the sodium-dependent phosphate transporter NaPi2b by monoclonal antibodies in ovarian cancer cells.

Material and methods. Ovarian cancer cells of the OVCAR-8 line were transfected with plasmids encoding mutant forms of the NaPi2b transporter. The recognition of these forms by monoclonal antibodies was studied by Western blot and flow cytometry. Statistical analysis of flow cytometry data was performed using Pearson’s Chi-squared test with Yates’ correction.

Results. Western blot analysis of lysates of OVCAR-8 cells expressing the wild-type NaPi2b transporter demonstrated the presence of a specific signal only in samples without the addition of dithiothreitol, while in all samples with NaPi2b cysteine mutants, even without the addition of dithiothreitol, no specific signal from monoclonal antibodies was detected. The flow cytometry results showed that proportion of OVCAR-8 cells with positive staining of the MX35 epitope with antibodies is less when expressing the NaPi2b mutants compared to the wild type.

Conclusion. Cysteines C303 and C350 are involved in disulfide bonds formation, which is of key importance for the formation of the conformation of the extracellular domain of the sodium-dependent phosphate transporter NaPi2b, which ensures the MX35 epitope recognition by monoclonal antibodies.

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About the authors

Leisan F. Bulatova

Kazan (Volga region) Federal University

Email: bulatovalef@gmail.com
ORCID iD: 0000-0001-6696-8477

Junior Researcher, research laboratory “Biomarker”, Institute of Fundamental Medicine and Biology

Russian Federation, Kazan, Russia

Vera S. Skripova

Kazan (Volga region) Federal University

Email: vsskripova@gmail.com
ORCID iD: 0000-0002-6342-0390

Junior Researcher, research laboratory “Biomarker”, Institute of Fundamental Medicine and Biology

Russian Federation, Kazan, Russia

Arina V. Korotaeva

Kazan (Volga region) Federal University

Email: ArVKorotaeva@stud.kpfu.ru
ORCID iD: 0000-0002-2253-7488

Student, Institute of Fundamental Medicine and Biology

Russian Federation, Kazan, Russia

Mikhail V. Bogdanov

Kazan (Volga region) Federal University; The University of Texas Health Science Center

Email: mikhail.v.bogdanov@uth.tmc.edu
ORCID iD: 0000-0002-6093-0926

Cand. Sci. (Biol.), Senior Research Fellow, research laboratory “Biomarker”, Institute of Fundamental Medicine and Biology; Prof., Depart. of Biochemistry and Molecular Biology

United States, Kazan, Russia; Houston, USA

Ramziya G. Kiyamova

Kazan (Volga region) Federal University

Author for correspondence.
Email: kiyamova@mail.ru
ORCID iD: 0000-0002-2547-2843

D. Sci. (Biol.), Prof., Depart. of biochemistry, biotechnology and pharmacology, chief researcher, research laboratory “Biomarker”, Institute of Fundamental Medicine and Biology

Russian Federation, Kazan, Russia

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Supplementary files

Supplementary Files
Action
1. Рис. 1. Вестерн-блот анализ мутантных форм натрий-зависимого фосфатного транспортёра NaPi2b c заменами цистеинов на аланин в положениях 303 и 350 в клетках рака яичника OVCAR-8. А. Анализ с моноклональными антителами N-NaPi2b (15/1); Б. Анализ с моноклональными антителами L2(20/3); ДТТ — дитиотреитол; GAPDH — глицеральдегид-3-фосфатдегидрогеназа

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2. Рис. 3. Анализ мутантных форм натрий-зависимого фосфатного транспортёра NaPi2b с помощью проточной цитометрии. А–Г. Точечные диаграммы результатов проточной цитометрии клеток рака яичника OVCAR-8, эктопически экспрессирующих NaPi2b дикого типа (А, В) и его мутантную форму (Б, Г) с заменой остатка цистеина С303 на остаток аланина (NaPi2b_C303A). А и Б. Анализ с моноклональными антителами L2(20/3). В и Г. Анализ с моноклональными антителами N-NaPi2b (15/1). Д. Мозаичный плот сравнения распознавания эпитопа MX35 NaPi2b дикого типа и его мутантной формы NaPi2b_C303A антителами L2(20/3). Статистический анализ выполнен с помощью критерия χ2 Пирсона с поправкой Йейтса; ***p <0,001

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3. Рис. 4. Анализ мутантных форм натрий-зависимого фосфатного транспортёра NaPi2b с помощью проточной цитометрии. А–Г. Точечные диаграммы результатов проточной цитометрии клеток рака яичника OVCAR-8, эктопически экспрессирующих NaPi2b дикого типа (А, В) и его мутантную форму (Б, Г) с заменой остатка цистеина С350 на остаток аланина (NaPi2b_C350A). А и Б. Анализ с моноклональными антителами L2(20/3). В и Г. Анализ с моноклональными антителами N-NaPi2b (15/1). Д. Мозаичный плот сравнения распознавания эпитопа MX35 NaPi2b дикого типа и его мутантной формы NaPi2b_C350A антителами L2(20/3). Статистический анализ выполнен с помощью критерия χ2 Пирсона с поправкой Йейтса; ***p <0,001

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