The role of proline P325 residue in the recognition of the MX35 epitope of the sodium-dependent phosphate transporter NaPi2b by monoclonal antibodies in ovarian carcinoma cells

Introduction. One of the key components of cell membranes are membrane proteins, which provide a wide range of functions – from transport and signal transmission to coordination of intercellular interactions. Among them, of particular interest is the sodium-dependent phosphate transporter NaPi2b, which plays an important role in maintaining phosphate homeostasis and is characterized by an increased content in a number of tumor cells. The large extracellular domain (ECD) of NaPi2b contains the MX35 epitope, which is of significant interest in the context of the development of monoclonal antibodies for targeted therapy of ovarian and lung carcinoma. Recognition of the MX35 epitope depends on the conformation of the large ECD, which is influenced by disulfide bonds and glycosylation. Between cysteine residues C322 and C328, there is a proline residue P325, which we hypothesize may contribute to the conformation of the NaPi2b large ECD by forming a disulfide bond between C322 and C328, potentially affecting the interaction of monoclonal antibodies with the MX35 epitope.

Aim. To study the impact of the proline residue at position 325 in the large ECD of the transporter NaPi2b on the interaction of monoclonal antibodies L3(28/1) with the MX35 epitope.

Materials and methods. The human ovarian epithelial carcinoma cell lines OVCAR-8 and OVCAR-4 were used in the study. By site-directed mutagenesis, the proline residue P325 of NaPi2b was replaced with an alanine residue, resulting in OVCAR-8 cells stably expressing the mutant variant of NaPi2bp.P325A. The effect of the p.P325A substitution in NaPi2b on the interaction of monoclonal antibodies L3(28/1) with the MX35 epitope was analyzed using western blotting and laser confocal microscopy. To assess the impact of the p.P325A mutation on the formation of disulfide bonds in the NaPi2b large ECD, cysteine residues thiol groups were modified using maleimide-containing compounds.

Results. It was found that the substitution of p.P325A in NaPi2b did not significantly affect the recognition of the MX35 epitope by L3(28/1) antibodies, as shown by both western blot analysis and confocal microscopy. The number of disulfide bonds in the large ECD of the mutant NaPi2bp.P325A form was unchanged compared to wild-type NaPi2b.

Conclusion. Substitution p.P325A in the NaPi2b transporter does not have a significant effect on the recognition of the MX35 epitope by antibodies or the formation of disulfide bonds in the NaPi2b large ECD. Future studies could focus on a more detailed investigation of the roles of other substitutions in the NaPi2b large ECD and their influence on the epitope’s accessibility to antibodies.

1. Guo L., Wang S., Li M., Cao Z. Accurate classification of membrane protein types based on sequence and evolutionary information using deep learning. BMC Bioinformatics 2019;20:1–17. DOI: 10.1186/s12859-019-3275-6

2. Krogh A., Larsson B., Von Heijne G., Sonnhammer E.L. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 2001;305(3):567–80. DOI: 10.1006/jmbi.2000.4315

3. Overington J.P., Al-Lazikani B., Hopkins A.L. How many drug targets are there? Nat Rev Drug Discov 2006;5(12):993–6. DOI: 10.1038/nrd2199

4. Forster I.C. The molecular mechanism of SLC34 proteins: insights from two decades of transport assays and structure-function studies. Pflügers Arch 2019;471(Pt 2):15–42. DOI: 10.1007/s00424-018-2207-z

5. Bulatova L., Savenkova D., Nurgalieva A. et al. Toward a topology based therapeutic design of membrane proteins: validation of NaPi2b topology in live ovarian cancer cells. Front Mol Biosci 2022;9:895911. DOI: 10.3389/fmolb.2022.895911

6. Vlasenkova R., Nurgalieva A., Akberova N. et al. Characterization of SLC34A2 as a potential prognostic marker of oncological diseases. Biomolecules 2021;11(12):1878. DOI: 10.3390/biom11121878

7. Soares I.C., Simões K., de Souza J.E.S. et al. In silico analysis and immunohistochemical characterization of NaPi2b protein expression in ovarian carcinoma with monoclonal antibody MX35. Appl Immunohistochem Mol Morphol 2012;20(2):165–72. DOI: 10.1097/pai.0b013e318228e232

8. Kiyamova R., Shyian M., Lyzogubov V.V. et al. Immunohistochemical analysis of NaPi2b protein (MX35 antigen) expression and subcellular localization in human normal and cancer tissues. Exp Oncol 2011;33(3):157–61.

9. Gryshkova V., Goncharuk I., Gurtovyy V. et al. The study of phosphate transporter NaPi2b expression in different histological types of epithelial ovarian cancer. Exp Oncol 2009;31(1):37–42.

10. Rangel L.B.A., Sherman-Baust C.A., Wernyj R.P. et al. Characterization of novel human ovarian cancer-specific transcripts (HOSTs) identified by serial analysis of gene expression. Oncogene 2003;22(46):7225–32. DOI: 10.1038/sj.onc.1207008

11. Yin B.W.T., Kiyamova R., Chua R. et al. Monoclonal antibody MX35 detects the membrane transporter NaPi2b (SLC34A2) in human carcinomas. Cancer Immun 2008;8:3.

12. Horsley E., Jabeen A., Veillard N. et al. Preclinical development of NaPi2b-PL2202, a novel camptothecin-based antibody-drug conjugate targeting solid tumors expressing NaPi2b. Cancer Res 2024;84(6_Suppl):5085. DOI: 10.1158/1538-7445.AM2024-5085

13. Fessler S., Dirksen A., Collins S.D. et al. XMT-1592, a site-specific dolasynthen-based NaPi2b-targeted antibody-drug conjugate for the treatment of ovarian cancer and lung adenocarcinoma. Cancer Res 2020;80(16_Suppl):2894. DOI: 10.1158/1538-7445.am2020-2894

14. Kiyamova R., Minigulova L.F., Skripova V. et al. N-glycosylation status of membrane phosphate transporter NaPi2b is crucial for its epitope recognition by monoclonal antibody in tumour cells. Ann Oncol 2020;31(5):S1227–8. DOI: 10.1016/j.annonc.2020.08.2193

15. Korotaeva A.V., Bulatova L.F., Vlasenkova R.A., Kiyamova R.G. Recognition of the Na-dependent phosphate transporter NaPi2b by monoclonal antibodies in bacterial and eukaryotic cells. Biotekhnologiya = Biotechnology 2022;38(5):66–72. (In Russ.). DOI: 10.56304/S023427582205009X

16. Minigulova L.F., Skripova V.S., Nurgalieva A.K. et al. Recognition of the sodium-dependent phosphate transporter NaPi2b by monoclonal antibodies N-NaPi2b in ovarian cancer cells. Uchenye zapiski Kazanskogo universiteta = Academic notes of Kazan University 2020;162(4):529–40. (In Russ.). DOI: 10.26907/2542-064X.2020.4.529-540

17. Gryshkova V., Lituiev D., Savinska L. et al. Generation of monoclonal antibodies against tumor-associated antigen MX35 sodium-dependent phosphate transporter NaPi2b. Hybridoma (Larchmt) 2011;30(1):37–42. DOI: 10.1089/hyb.2010.0064

18. Bulatova L.F., Skripova V.S., Nurgalieva A.K. et al. Structurally constrained tumor-specific epitope within the largest extracellular domain of sodium-dependent phosphate transporter NaPi2b. Ann Oncol 2021;32(5):S368–9. DOI: 10.1016/j.annonc.2021.08.304

19. Bulatova L.F., Skripova V.S., Korotaeva A.V. et al. Recognition of mutant forms of the sodium-dependent phosphate transporter NaPi2b by monoclonal antibodies in ovarian cancer cells. Kazanskiy meditsinskiy zhurnal = Kazan medical journal 2022;103(4):608–16. (In Russ.). DOI: 10.17816/KMJ2022-608

20. Nadeau V.G., Deber C.M. Structural impact of proline mutations in the loop region of an ancestral membrane protein. Biopolymers 2016;106(1):37–42. DOI: 10.1002/bip.22765

21. Kiyamova R., Gryshkova V., Ovcharenko G. et al. Development of monoclonal antibodies specific for the human sodium-dependent phosphate co-transporter NaPi2b. Hybridoma 2008;27(4):277–84. DOI: 10.1089/hyb.2008.0015

22. Boichuk S., Dunaev P., Skripova V. et al. Unraveling the mechanisms of sensitivity to anti-FGF therapies in imatinib resistant gastrointestinal stromal tumors (GIST) lacking secondary KIT mutations. Cancers (Basel) 2023;15(22):5354. DOI: 10.3390/cancers15225354.

23. McKinnon L.J., Fukushima J., Endow J.K. et al. Membrane chaperoning of a thylakoid protease whose structural stability is modified by the protonmotive force. Plant Cell 2020;32(5):1589–609. DOI: 10.1105/tpc.19.00797

24. Krieger F., Möglich A., Kiefhaber T. Effect of proline and glycine residues on dynamics and barriers of loop formation in polypeptide chains. J Am Chem Soc 2005;127(10):3346–52. DOI: 10.1021/ja042798i

25. Schmitt S., Mai I., Machui P. et al. TUB-040, a novel Napi2b targeting ADC built with ethynylphosphonamidate conjugation chemistry, demonstrates high and long-lasting anti-tumor efficacy via Topoisomerase-I inhibition and excellent tolerability predictive of a wide therapeutic window in humans. Cancer Res 2024; 84(6_Suppl):2622. DOI: 10.1158/1538-7445.AM2024-2622

26. Gryshkova V.S., Filonenko V.V., Kiyamova R.G. Inhibition of sodium-dependent phosphate transporter NaPi2b function with MX35 antibody. Biopolym Cell 2011;27:193–8. DOI: 10.7124/bc.0000B9