Recognition of mutant forms of the sodium-dependent phosphate transporter NaPi2b by monoclonal antibodies in ovarian cancer cells
- Authors: Bulatova L.F.1, Skripova V.S.1, Korotaeva A.V.1, Bogdanov M.V.1,2, Kiyamova R.G.1
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Affiliations:
- Kazan (Volga region) Federal University
- The University of Texas Health Science Center
- Issue: Vol 103, No 4 (2022)
- Pages: 608-616
- Section: Experimental medicine
- URL: https://kazanmedjournal.ru/kazanmedj/article/view/105242
- DOI: https://doi.org/10.17816/KMJ2022-608
- ID: 105242
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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, RussiaVera 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, RussiaArina 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, RussiaMikhail 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, USARamziya 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, RussiaReferences
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