Background/Purpose:

One unique feature of rheumatoid arthritis (RA) is the presence of anti-citrullinated protein antibody (ACPA). Protein citrullination, a process mediated by peptidyl arginine deiminases, such as PAD4, not only yields antigens recognized by ACPA but also contributes to RA through several other mechanisms including promotion of neutrophil extracellular traps (NET) formation. A C-to-T single nucleotide polymorphism (SNP) located at position 1858 of human PTPN22, a protein tyrosine phosphatase, carries an odds ratio of 2-3 and, together with HLA alleles containing shared epitopes, synergistically increases the risk of RA 20- to 30-fold. The effect of this SNP is limited to ACPA+ RA. An explanation for this unique association is still lacking. We postulate that PTPN22 and the C1858T SNP, which converts an arginine (R620) to a tryptophan, influence protein citrullination.

Methods:

The level of citrullinated proteins in wild type and PTPN22-deficient cells, as well as PBMC from healthy donors carrying or not carrying the risk T allele was examined with Western blotting using F95 antibody and anti-citrullinated histone H3.  Co-immunoprecipitation was used to examine the physical interaction between PTPN22 and PAD4. The formation of neutrophil extracellular traps was analyzed with Sy-tox uptake and immunocytochemistry.

Results:

Impaired expression of PTPN22 resulted in hypercitrullination in mouse and human immune cells. This effect was partly mediated by PAD4.  PTPN22 did not regulate the expression or tyrosine phosphorylation of PAD4. Instead, PTPN22 physically interacted with and suppressed the activity of PAD4 independently of its phosphatase activity. Conversion of the R620 to a tryptophan disrupted the interaction between PTPN22 and PAD4, and completely ablated the ability of PTPN22 to suppress protein citrullination. Accordingly, the risk T allele is associated with hypercitrullination in PBMC and heightened propensity to form NET in healthy donors.

Conclusion: PTPN22 is a natural inhibitor of PAD, and the R620 is critical for this non-phosphatase function of PTPN22. The C1858T-mediated conversion of R620 to tryptophan increases the risk of ACPA+ RA through hypercitrullination and heightened propensity to form NET. Our data also identify a novel pathway regulating PAD activity and establish a molecular connection between the two RA risk genes PTPN22 and PAD4.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/inhibition-of-pad4-activity-and-the-formation-of-neutrophil-extracellular-traps-via-ptpn22-but-not-its-rheumatoid-arthritis-prone-w620-variant/