75P - Possible significance of the proline at position 325 in the recognition of the MX35 epitope of the NaPi2b transporter by monoclonal antibodies

Background

The sodium-dependent phosphate transporter NaPi2b is a promising target for the treatment of malignant tumors due to its increased expression in several oncological diseases. NaPi2b is a target for therapeutic monoclonal antibodies directed against the MX35 epitope within the large extracellular domain (ECD) of the NaPi2b. We assume that this epitope has tumor-specific conformation that is formed due to the creation of disulfide bonds between four cysteine residues at positions 303, 322, 328 and 350. The proline residue at position 325 lies between cysteines 322 and 328 in the region of the MX35 epitope of NaPi2b. We assume that proline at this position due to its formation of a bend in the structure of the peptide participates in the formation of a disulfide bond between cysteines at positions 322 and 328 that is critical in the recognition of the MX35 epitope by antibodies. To verify this, the mutant form with the proline replacement should be evaluated using antibodies against the MX35 epitope in Western blot and microscopy analysis. The aim of this work was to create a genetic construct encoding the mutant form of the NaPi2b transporter with the replacement of proline with alanine at position 325.

Methods

To carry out site-directed mutagenesis, we selected primers with the replacement of triplets encoding proline at position 325 with a triplet encoding alanine in the SLC34A2 (encoding transporter NaPi2b) gene. The resulting PCR product was transformed into E.coli cells of the XL1-Blue strain and plasmid DNA was isolated. To confirm the mutation, samples were sequenced using the Sanger method.

Results

As a result of our work, we created a genetic construct with the replacement of proline with alanine at position 325 of the NaPi2b transporter, which will be further test in Western blot and microscopy analysis by monoclonal antibodies.

Conclusions

The obtained results supported by the Kazan Federal University Strategic Academic Leadership Program (PRIORITY-2030) are important for further study of the mechanism of recognition of the MX35 NaPi2b epitope by monoclonal antibodies.

Editorial acknowledgement

Clinical trial identification

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 the Kazan Federal University Strategic Academic Leadership Program (PRIORITY-2030).

Disclosure

All authors have declared no conflicts of interest.