Abstract 81P
Background
The search for anticancer therapy targets is the main task of oncology. One hopeful target is a membrane glycoprotein the sodium-dependent phosphate transporter NaPi2b overexpressed in ovarian carcinomas. NaPi2b is an antigen for the MX35 antibody which recognizes an epitope in the large extracellular domain (ECD) of the NaPi2b mostly in tumor but not in normal cells. The ECD contains 4 cysteines at positions 303, 322, 328, 350 and potential N-glycosylation sites at positions 295, 308, 313, 321, 335, 340. We formulated a hypothesis that the MX35 epitope of NaPi2b has a tumor-specific nature which availability for antibodies depends on disulfide bonds and N-glycosylation of the ECD. The aim of this work is to study the role of disulfide bonds and N-glycosylation in the recognition of the MX35 epitope of NaPi2b in intact ovarian cancer cells using confocal fluorescence microscopy.
Methods
We simulated the disulfide bonds reduction or partial deglycosylation of the ECD of NaPi2b by replacing cysteine at positions 303, 322, 328, 350 and asparagine at positions 295, 308, 313, 321, 335, 340 with alanine. Mutant forms were studied by confocal fluorescence microscopy of intact OVCAR8 ovarian cancer cells transfected by corresponding mutant constructs with subsequent assessment of the fluorescence intensity recognition of the mutant forms relative to the wild-type NaPi2b.
Results
The substitution of each cysteine at positions 303, 322, 328, 350 of NaPi2b results in a reduction of the MX35 epitope recognition by monoclonal antibodies. Substitution of the asparagine at position 313 increases the MX35 epitope recognition by monoclonal antibodies.
Conclusions
We showed that the recognition of the MX35 epitope of NaPi2b depends on the ECD conformation due to the presence of disulfide bonds between cysteines at positions 303, 322, 328, 350 and glycosylation of asparagine at position 313. Since malignant transformation of cells is accompanied by changes in the tumor microenvironment, including oxidative stress, we suggest this may lead to a rearrangement of disulfide bonds between cysteines and a change in the ECD conformation when the MX35 epitope becomes more accessible to antibodies in tumor cells, making it a potential tumor-specific epitope.
Legal entity responsible for the study
Research Laboratory \"Biomarker\", Institute of Fundamental Medicine and Biology, Kazan Federal University.
Funding
This work has been supported by Russian Science Foundation, Russia (project no. 20-14-00166) and the Kazan Federal University Strategic Academic Leadership Program (PRIORITY-2030).
Disclosure
All authors have declared no conflicts of interest.