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

Abstract

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.

Keywords

• napi2b
• mx35 epitope
• proline residue p325
• monoclonal antibody
• ovarian carcinoma