Development of recombinant peptides to inhibit PAD4 enzyme involved in the pathogenesis of rheumatoid arthritis

Purpose: To develop a recombinant inhibitor based on VH (variable heavy chain) model to reduce the activity of the PAD4 enzyme responsible for the pathogenesis of rheumatoid arthritis. Materials and Methods: PAD4 gene was synthesized after optimisation of the sequence for E. coli . The synthesized PAD4 gene was cloned into the pET-30a vector and transferred into E. coli BL21 (DE3), recombinantly produced PAD4 protein was purified by IMAC. CD-1 mice were immunized with purified PAD4 protein. Immunized mouse spleens were removed,total RNA was isolated. cDNA synthesis and VH amplification were done using primers for VH amptification. VH amplicons were cloned into the pComb3-hy vector constructed in the study and transferred into E. coli XL1-Blue to create a VH library. The bacteria containing the VH library in pComb3-hy vector were infected with M13K07 phage and the VH-displaying phage library was obtained. VH-expressing phages with affinity for PAD4 from the VH library were selected by panning. Sequence analysis was done for one of the VH showing affinity for PAD4 named as VH4. A three-dimensional structure was obtained using the amino acid sequence of VH 4 and modeling was performed to investigate its in silico interaction with PAD4. Inhibition of the citrullinating activity of PAD4 by VH4 was tested in-vitro by fluorescence-based monitoring. The morphological effect of VH4 inhibition of the citrullinating activity of PAD4 on the HeLa mammalian cell line was evaluated. The cytotoxicity assay of VH4 was performed with HeLa cells using WST-1. Results: Cloning of PAD4 was confirmed by colony PCR and sequence analysis of the insert. Production and purification of recombinant PAD4 was confirmed by obtaining a band around 75 kDa in SDS-PAGE analysis. The phage with affinity for PAD4 was obtained from the VH display phage library created against PAD4. The molecular weight of this VH named as VH4 is 13 kDa and in silico analysis showed that it forms 6 hydrogen bonds with PAD4. It was shown to reduce citrullinating activity of PAD4 by 30% in in-vitro experiments, and in experiments with a mammalian cell line, it prevented PAD4-induced morphological disorder and did not cause cytotoxic effects. Conclusion: In this study a recombinant inhibitor of PAD4 was obtained, which is important in the pathogenesis of rheumatoid arthritis with its citrullination activity. Using amino acid sequence of the recombinant inhibitor of PAD4, three-dimensional structure and in silico interaction of this inhibitor with PAD4 were shown. Further studies including animal model studies should be done to investigate its potential to be used for treatment of rheumatoid arthritis. Keywords: Rheumatoid arthritis, peptidyl arginine deiminase (PAD), phage display, recombinant peptide