Association of autoimmunity to peptidyl arginine deiminase type 4 with genotype and disease severity in rheumatoid arthritis

Abstract

Objective: Protein citrullination is an important posttranslational modification recognized by rheumatoid arthritis (RA)-specific autoantibodies. One of the citrullinating enzymes, peptidyl arginine deiminase type 4 (PAD-4), is genetically associated with development of RA in some populations, although the mechanism(s) mediating this effect are not yet clear. There have been descriptions of anti-PAD-4 autoantibodies in different rheumatic diseases. This study was undertaken to investigate whether anti-PAD-4 antibodies are specific to RA, are associated with disease phenotype or severity, and whether PAD-4 polymorphisms influence the anti-PAD-4 autoantibody response.

Methods: Sera from patients with established RA, patients with other rheumatic diseases, and healthy adults were assayed for anti-PAD-4 autoantibodies by immunoprecipitation of in vitro-translated PAD-4. The epitope(s) recognized by PAD-4 autoantibodies were mapped using various PAD-4 truncations. PAD-4 genotyping was performed on RA patients with the TaqMan assay. Joint erosions were scored from hand and foot radiographs using the Sharp/van der Heijde method.

Results: PAD-4 autoantibodies were found in 36-42% of RA patients, and were very infrequent in controls. Recognition by anti-PAD-4 autoantibodies required the 119 N-terminal amino acids, which encompass the 3 nonsynonymous polymorphisms associated with disease susceptibility. Strikingly, the anti-PAD-4 immune response was associated with the RA susceptibility haplotype of PADI4. Anti-PAD-4 antibodies were associated with more severe joint destruction in RA.

Conclusion: Our findings indicate that anti-PAD-4 antibodies are specific markers of RA, independently associated with more severe disease, suggesting that an anti-PAD-4 immune response may be involved in pathways of joint damage in this disease. Polymorphisms in the PADI4 gene influence the immune response to the PAD-4 protein, potentially contributing to disease propagation.

Figure 1

Peptidyl arginine deiminase type 4 (PAD-4) is a frequent, specific target in rheumatoid arthritis (RA). A, ³⁵S-methionine–labeled PAD-4 generated by in vitro transcription and translation (IVTT) was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and visualized by fluorography. B, For each immunoprecipitation, 1 μl of PAD-4 generated by IVTT was mixed with 1 μl of patient serum. Results obtained using sera from 17 patients in the RA pilot group (lanes 1–17) are shown. C, Immunoprecipitation with PAD-4 generated by IVTT was performed as described above, using sera from 16 normal controls (lanes 3–18). The RA sera used in lanes 1 and 2 in B were included as reference sera in the immunoprecipitations shown in lanes 1 and 2 of C.

Figure 2

Both subtypes of PAD-4 autoantibodies require the N-terminal domain encompassing the polymorphic residues for recognition. A, Schematic representation of the PAD-4 constructs used for the immunoprecipitation studies shown in B. Red vertical lines denote S55, A82, and A112 in wild-type (WT) PAD-4. Blue vertical lines denote G55, V82, and G112 in polymorphic PAD-4. The ability of type I and type II PAD-4 antibodies to immunoprecipitate the different constructs is summarized on the right. B, Equally labeled amounts (assessed by densitometry) of different ³⁵S-methionine–labeled PAD-4 products were immunoprecipitated with representative type I or type II PAD-4 autoantibody–positive RA sera. All constructs in the lower panel were fusion proteins containing glutathione S-transferase (GST). None of the sera recognized GST alone (results not shown). For each of the different IVTT constructs, a gel sample consisting of one-fifth of the volume used for immunoprecipitation (lanes labeled with an asterisk) was electrophoresed adjacent to the paired immunoprecipitations. Type I sera recognized only full-length (FL) PAD-4 constructs, and removal of either the N- or C-terminal domains prevented recognition. Type II sera immunoprecipitated all constructs containing the 119 N-terminal amino acids of PAD-4. SNP = single-nucleotide polymorphism (see Figure 1 for other definitions).