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.
References
- Levi M., Gratton E., Forster I.C., Hernando N., Wagner C.A., Biber J., Sorribas V., Murer H. Mechanisms of phosphate transport. Nat. Rev. Nephrol., 2019, vol. 15, no. 8, pp. 482–500. doi: 10.1038/s41581-019-0159-y.
- 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. Identification of phosphate transporter NaPi2b as MX35 cancer antigen by modified SEREX approach. Biopolym. Cell, 2008, vol. 4, no. 3, pp. 218–224. doi: 10.7124/bc.0007A3.
- Yin B.W.T., Kiyamova R., Chua R., Caballero O.L., Gout I., Gryshkova V., Bhaskaran N., Souchelnytskyi S., Hellman U., Filonenko V., Jungbluth A.A., Odunsi K., Lloyd K.O., Old L.J., Ritter G. Monoclonal antibody MX35 detects the membrane transporter NaPi2b (SLC34A2) in human carcinomas. Cancer Immun., 2008, vol. 8, art. 3, pp. 1–9.
- Gryshkova V., Goncharuk I., Gurtovyy V., Khozhayenko Y., Nespryadko S., Vorobjova L., Usenko V., Gout I., Filonenko V., Kiyamova R. The study of phosphate transporter NAPI2B expression in different histological types of epithelial ovarian cancer. Exp. Oncol., 2009, vol. 31, no. 1, pp. 37–42.
- Shyian M., Gryshkova V., Kostianets O., Gorshkov V., Gogolev Y., Goncharuk I., Nespryadko S., Vorobjova L., Filonenko V., Kiyamova R. Quantitative analysis of SLC34A2 expression in different types of ovarian tumors. Exp. Oncol., 2011, vol. 33, no. 2, pp. 94–98.
- Kiyamova R., Shyian M., Lyzogubov V.V., Usenko V.S., Gout T., Filonenko V. Immunohistochemical analysis of NaPi2b protein (MX35 antigen) expression and subcellular localization in human normal and cancer tissues. Exp. Oncol., 2011, vol. 33, no. 3, pp. 157–161.
- Zhang Z., Ye S., Zhang M., Wu J., Yan H., Li X., He J. High expression of SLC34A2 is a favorable prognostic marker in lung adenocarcinoma patients. Tumour Biol., 2017, vol. 39, no. 7, art. 1010428317720212, pp. 1–7. doi: 10.1177/1010428317720212.
- Liu L., Yang Y., Zhou X., Yan X., Wu Z. Solute carrier family 34 member 2 overexpression contributes to tumor growth and poor patient survival in colorectal cancer. Biomed. Pharmacother., 2018, vol. 99, pp. 645–654. doi: 10.1016/j.biopha.2018.01.124.
- Santos M.L., Yeda F.P., Tsuruta L.R., Horta B.B., Pimenta A.A., Degaki T.L., Soares I.C., Tuma M.C., Okamoto O.K., Alves V.A.F., Old L.J., Ritter G., Moro A.M. Rebmab200, a humanized monoclonal antibody targeting the sodium phosphate transporter NaPi2b displays strong immune mediated cytotoxicity against cancer: A novel reagent for targeted antibody therapy of cancer. PLoS ONE, 2013, vol. 8, no. 7, art. e70332, pp. 1–10. doi: 10.1371/journal.pone.0070332.
- Mosher R., Poling L.L., Bergstrom D.A. Compositions and methods for predicting response to NaPi2b-targeted therapy. Patent no. WO 2019/060542 A2. 2019. 97 p.
- Bodyak N., Yurkovetskiy A., Yin M., Gumerov D., Bollu R., Conlon P., Gurijala V.R., McGillicuddy D., Stevenson C., Ter-Ovanesyan E., Park P.U., Poling L., Lee W., DeVit M., Xiao D., Qin L., Lowinger T.B., Bergstrom D.A. Discovery and preclinical development of a highly potent NaPi2b-targeted antibody-drug conjugate (ADC) with significant activity in patient-derived non-small cell lung cancer (NSCLC) xenograft models. Cancer Res., 2016, vol. 76, no. 14, suppl.: Proc. 107th Annu. Meet. Am. Assoc. Cancer Res.; Apr. 16–20, 2016; New Orleans, LA. Philadelphia (PA). abstr. no. 1194. doi: 10.1158/1538-7445.AM2016-1194.
- Lituiev D.S., Kiyamova R.G. Mutations in the gene of human type IIb sodium-phosphate cotransporter SLC34A2. Biopolym. Cell, 2010, vol. 26, no. 1, pp. 13–22. doi: 10.7124/bc.00013F.
- Kiyamova R., Gryshkova V., Ovcharenko G., Lituyev D., Malyuchik S., Usenko V., Khozhayenko Y., Gurtovyy V., Yin B., Ritter G., Old L., Filonenko V., Gout I. Development of monoclonal antibodies specific for the human sodium-dependent phosphate co-transporter NaPi2b. Hybridoma (Larchmt), 2008, vol. 27, no. 4, pp. 277–284. doi: 10.1089/hyb.2008.0015.
- Charneski C.A., Hurst L.D. Positive charge loading at protein termini is due to membrane protein topology, not a translational ramp. Mol. Biol. Evol., 2014, vol. 31, no. 1, pp. 70–84. doi: 10.1093/molbev/mst169.
- Hartmann E., Rapoport T.A., Lodish H.F. Predicting the orientation of eukaryotic membrane-spanning proteins. Proc. Natl. Acad. Sci. U. S. A., 1989, vol. 86, no. 15, pp. 5786–5790. doi: 10.1073/pnas.86.15.5786.
- Bogdanov M., Dowhan W., Vitrac H. Lipids and topological rules governing membrane protein assembly. Biochim. Biophys. Acta, 2014, vol. 1843, no. 8, pp. 1475–1488. doi: 10.1016/j.bbamcr.2013.12.007.
- Gryshkova V., Lituiev D., Savinska L., Ovcharenko G., Gout I., Filonenko V., Kiyamova R. Generation of monoclonal antibodies against tumor-associated antigen MX35/sodium-dependent phosphate transporter NaPi2b. Hybridoma (Larchmt), 2011, vol. 30, no. 1, pp. 37–42. doi: 10.1089/hyb.2010.0064.
- Fenollar-Ferrer C., Patti M., Knöpfel T., Werner A., Forster I.C., Forrest L.R. Structural fold and binding sites of the human Na+-phosphate cotransporter NaPi-II. Biophys. J., 2014, vol. 106, no. 6, pp. 1268–1279. doi: 10.1016/j.bpj.2014.01.043.
- Lanaspa M.A., Caldas Y.A., Breusegem S.Y., Andrés-Hernando A., Cicerchi C., Levi M., Sorribas V. Inorganic phosphate modulates the expression of the NaPi-2a transporter in the trans-Golgi network and the interaction with PIST in the proximal tubule. Biomed. Res. Int., 2013, vol. 2013, art. 513932, pp. 1–9. doi: 10.1155/2013/513932.
- Giral H., Cranston D., Lanzano L., Caldas Y., Sutherland E., Rachelson J., Dobrinskikh E., Weinman E.J., Doctor R.B., Gratton E., Levi M. NHE3 regulatory factor 1 (NHERF1) modulates intestinal sodium-dependent phosphate transporter (NaPi-2b) expression in apical microvilli. J. Biol. Chem., 2012, vol. 287, no. 42, pp. 35047–35056. doi: 10.1074/jbc.M112.392415.
- Dobson L., Reményi I., Tusnády G.E. CCTOP: A Consensus Constrained TOPology prediction web server. Nucleic Acids Res., 2015, vol. 43, no. W1, pp. W408–W412. doi: 10.1093/nar/gkv451.
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