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

L.F. Minigulova ^a*, V.S. Skripova ^a**, A.K. Nurgalieva ^a***, D.V. Savenkova ^a****, A.S. Kozlova ^a*****, N.I. Akberova ^a******, D.D. Reshetnikova ^a*******, L.A. Savinska ^b********, O.M. Garifulin ^b*********, M.V. Bogdanov ^{a,c***********}, R.G. Kiyamova ^a***********

^aKazan Federal University, Kazan, 420008 Russia

^bInstitute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, Kiev, 03680 Ukraine

^cUniversity of Texas Health Science Center at Houston, Houston, TX 77030 USA

E-mail: ^*minigulovalf@gmail.com, ^**vsk190@gmail.com, ^***alsina97@mail.ru, ^****darina.sava1@gmail.com, ^*****hellgatedoctor@yandex.com, ^******nakberova@mail.ru, ^*******reshetnikovaddm@gmail.com, ^********savinska@gmail.com, ^*********oleg.garifulin@gmail.com, ^**********mikhail.v.bogdanov@uth.tmc.edu, ^***********kiyamova@mail.ru

Received August 26, 2020

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DOI: 10.26907/2542-064X.2020.4.529-540

For citation: Minigulova L.F., Skripova V.S., Nurgalieva A.K., Savenkova D.V., Kozlova A.S., Akberova N.I., Reshetnikova D.D., Savinska L.A., Garifulin O.M., Bogdanov M.V., Kiyamova R.G. Recognition of the sodium-dependent phosphate transporter NaPi2b by monoclonal antibodies N-NaPi2b in ovarian cancer cells. Uchenye Zapiski Kazanskogo Universiteta. Seriya Estestvennye Nauki, 2020, vol. 162, no. 4, pp. 529–540. doi: 10.26907/2542-064X.2020.4.529-540. (In Russian)

Abstract

The sodium-dependent phosphate cotransporter NaPi2b encoded by the SLC34A2 gene is a transmembrane protein that belongs to the family of phosphate transporters SLC34 and is involved in maintaining phosphate homeostasis in the human body. The phosphate transporter NaPi2b is expressed in several normal tissues and body organs, as well as in malignant neoplasms of the ovary, lung, colorectal, and other types of cancer. The membrane protein NaPi2b is a target for therapeutic humanized antibodies Rebmab200, XMT-1535, and XMT-1536 in cancer cells. These antibodies were created based on MX35 antibodies and are directed against the large extracellular domain of the transporter NaPi2b. Since the location of the N-terminal domain of NaPi2b in the cell has not been experimentally confirmed, this work aimed to analyze the recognition of the phosphate transporter NaPi2b in ovarian cancer cells OVCAR-4 and OVCAR-8 by antibodies against the N-terminal domain of the transporter using Western blot analysis and confocal microscopy.

Keywords: SLC34A2, NaPi2b, N-terminal domain of NaPi2b, monoclonal antibodies, ovarian cancer

Acknowledgments. This study was supported by the Russian Foundation for Basic Research (project no. 19-34-90173) and in part by the Russian Government Program of Competitive Growth of Kazan Federal University. Confocal laser microscope images were taken with the support of the Russian Science Foundation (project no. 20-14-00166).

Figure Captions

Fig. 1. Analysis of the NaPi2b expression in ovarian cancer cells OVCAR-4 and OVCAR-8. GAPDH was used as loading control.

Fig. 2. Visualization of the N-terminal domain of NaPi2b using confocal laser microscopy in ovarian cancer cells OVCAR-4 with and without cell permeabilization: a) fixed cells treated with N-NaPi2b antibodies (15/1); b) fixed cells stained with DAPI; c) superimposed image for a and b; d) fixed and permeabilized with Triton X-100 cells treated with N-NaPi2b antibodies (15/1); e) fixed and permeabilized with Triton X-100 cells stained with DAPI; f) superimposed image for d and e.

Fig. 3. Visualization of the N-terminal domain of NaPi2b using confocal laser microscopy in ovarian cancer cells OVCAR-8 with and without cell permeabilization: fixed cells treated with N-NaPi2b antibodies (15/1); b) fixed cells stained with DAPI; c) superimposed image for a and b; d) fixed and permeabilized with Triton X-100 cells treated with N-NaPi2b antibodies (15/1); e) fixed and permeabilized with Triton X-100 cells stained with DAPI; f) superimposed image for d and e.

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