Abstract 26P
Background
The search for tumor-specific targets among membrane proteins is important for the development for new effective anticancer drugs because membrane proteins represent 60% of drug targets. Transmembrane proteins in cancer cells exhibit an altered pattern of post-translational modifications and generally contain a large extracellular domains with multiple glycosylation and sulfhydryl sites with unknown function. We analyze the features of membrane protein which are required for recognition by monoclonal antibodies using the sodium-dependent phosphate transporter NaPi2b, containing a large extracellular domain 4 (EMD4, 250-360 a.a.) with 4 cysteines and 6 potential asparagine-linked glycosylation sites which can potentially contribute to foldability of conformationally exposed epitope corresponding to this domain.
Methods
Each of the 4 cysteines and 6 asparagines have been substituted EMD4 by alanine by site-directed mutagenesis. The epitope recognition and ability to take up an inorganic phosphate by wild-type and mutant forms of the NaPi2b ectopically expressed in ovary cancer OVCAR-8 cells has been analyzed with L2 (20/3) antibodies using western blotting, confocal microscopy, flow cytometry and transport assay with 32P-orthophosphate.
Results
We demonstrated an importance of disulfide bonds and N-glycosylation in maintenance of unique supersecondary or tertiary structure of the EMD4 which can contribute to accessibility of corresponding epitope to monoclonal antibodies. Intriguing involvement of conformation sensitive EMD4 in the kinetics of NaPi2b transport via re-arrangement of disulfide bonds may provide a unique regulatory conformational switch mechanism.
Conclusions
The unique structural features required for of NaPi2b EMD4 recognition by analytical and therapeutic monoclonal antibodies, specifically accumulating in cancer cells in vivo, allowed us to postulate an existence of a sulfhydryl bond constrained and glycosylated tumor-specific conformationally exposed epitope within NaPi2b EMD4 representing an excellent targets for specific therapeutic antibodies.
Clinical trial identification
Editorial acknowledgement
Legal entity responsible for the study
The authors.
Funding
This work is part of Kazan Federal University Strategic Academic Leadership Program and Russian Science Foundation project No 20-14-00166.
Disclosure
All authors have declared no conflicts of interest.