Development of an in vitro model to study the role of disulfide bonds in the largest extracellular domain of the sodium-dependent phosphate transporter NaPi2b in OVCAR-8 ovarian carcinoma cells

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Introduction. The sodium-dependent phosphate transporter NaPi2b is a promising target for targeted antitumor therapy. There is the largest extracellular domain (ECD) containing a cryptic MX35 epitope, against which therapeutic antibodies have been developed and are undergoing preclinical and clinical trials. The accessibility of the MX35 epitope to antibodies is higher in tumor cells and depends on the conformation of the ECD, determined by disulfide bonds between cysteine residues C303, C322, C328 and C350. The number of these disulfide bonds and cysteine residues that participate in the NaPi2b ECD conformation maintaining, regulation of its transport activity and stability is unknown. Isolation and purification of transmembrane proteins, including NaPi2b, for structural and functional studies is difficult, therefore it is necessary to develop an in vitro model to study the formation of disulfide bonds in the ECD region of the NaPi2b transporter and their role in ensuring the availability of the cryptic MX35 epitope and transporter activity in living cells.Aim. To create a panel of clonal sublines of human ovarian carcinoma OVCAR-8 containing recombinant variants of the wild-type NaPi2b transporter, as well as with single and double substitutions of cysteine residues in the ECD region with alanine residues.Materials and methods. OVCAR-8 ovarian carcinoma cells that do not express the NaPi2b transporter gene were transduced with lentiviral particles carrying nucleotide sequences encoding the wild-type NaPi2b transporter or its mutant variants with single and double substitutions of cysteine residues C303, C322, C328 and C350 with alanine residues to simulate reduction of potential disulfide bonds between them. After selecting transduced cells, clonal sublines were obtained, in the lysates of which the content of recombinant variants of the NaPi2b transporter was assessed using Western blot analysis and dot blot analysis.Results. A panel of 9 clonal sublines of OVCAR-8 ovarian carcinoma containing the wild-type recombinant NaPi2b transporter and its mutant variants was obtained. The effect of the introduced amino acid substitutions on the content and electrophoretic mobility of the NaPi2b transporter was noted.Conclusion. The resulting panel of clonal sublines can be used as an in vitro model to study the conformation of the ECD transporter NaPi2b, determined by disulfide bonds, which will reveal the mechanism of formation of the cryptic MX35 epitope and shed light on the role of ECD in the regulation of NaPi2b transport activity. Understanding the mechanism of formation of the cryptic MX35 epitope will make it possible to find new cryptic epitopes in the extracellular domains of transmembrane proteins, which can be used as targets for antitumor therapy.