Peptidylarginine deiminase (PAD, EC 3.5.3.15) enzymes catalyze the conversion of protein-bound arginine to citrulline. This post-translational modification may have a big impact on the structure and function of the target protein. In this review, we will discuss the effects of citrullination and its involvement in several human diseases, including rheumatoid arthritis and multiple sclerosis. So far, four isotypes of PAD have been described in mammals. We describe the existence of PAD in non-mammalian vertebrates and the existence of a fifth mammalian PAD. In addition, tissue-specific expression, genomic organization and evolutionary conservation of the different PAD isotypes will be discussed in detail. This article contains supplementary material which may be viewed at the BioEssays website at http://www.interscience.wiley.com/jpages/0265-9247/suppmat/2003/25/v25.1106.html.

Background: Antibodies directed to proteins containing the non-standard amino acid citrulline, are extremely specific for rheumatoid arthritis (RA). Peptidylcitrulline can be generated by post-translational conversion of arginine residues. This process, citrullination, is catalysed by a group of calcium dependent peptidylarginine deiminase (PAD) enzymes. Objective: To investigate the expression and activity of four isotypes of PAD in peripheral blood and synovial fluid cells of patients with RA. Results: The data presented here show that citrullination of proteins by PAD enzymes is a process regulated at three levels: transcription-in peripheral blood PAD2 and PAD4 mRNAs are expressed predominantly in monocytes; PAD4 mRNA is not detectable in macrophages, translation-translation of PAD2 mRNA is subject to differentiation stage-specific regulation by its 39 UTR, and activation-the PAD proteins are only activated when sufficient Ca 2+ is available. Such high Ca 2+ concentrations are normally not present in living cells. In macrophages, which are abundant in the inflamed RA synovium, vimentin is specifically citrullinated after Ca 2+ influx. Conclusion: PAD2 and PAD4 are the most likely candidate PAD isotypes for the citrullination of synovial proteins in RA. Our results indicate that citrullinated vimentin is a candidate autoantigen in RA.

This study provides evidence that smoking enhances PAD2 expression in the bronchial mucosal and alveolar compartment, with consequent generation of citrullinated proteins in the latter. Smoking is an environmental factor that may lead to citrulline autoimmunity in genetically susceptible subjects.

Objective. To examine synovial fluid as a site for generating citrullinated antigens, including the candidate autoantigen citrullinated ␣-enolase, in rheumatoid arthritis (RA).Methods. Synovial fluid was obtained from 20 patients with RA, 20 patients with spondylarthritides (SpA), and 20 patients with osteoarthritis (OA). Samples were resolved using sodium dodecyl sulfatepolyacrylamide gel electrophoresis, followed by staining with Coomassie blue and immunoblotting for citrullinated proteins, ␣-enolase, and the deiminating enzymes peptidylarginine deiminase type 2 (PAD-2) and PAD-4. Proteins from an RA synovial fluid sample were separated by 2-dimensional electrophoresis, and each protein was identified by immunoblotting and mass spectrometry. Antibodies to citrullinated ␣-enolase peptide 1 (CEP-1) and cyclic citrullinated peptide 2 were measured by enzyme-linked immunosorbent assay.Results. Citrullinated polypeptides were detected in the synovial fluid from patients with RA and patients with SpA, but not in OA samples. Alpha-enolase was detected in all of the samples, with mean levels of 6.4 ng/ l in RA samples, 4.3 ng/ l in SpA samples, and <0.9 ng/ l in OA samples. Two-dimensional electrophoresis provided evidence that the ␣-enolase was citrullinated in RA synovial fluid. The citrullinating enzyme PAD-4 was detected in samples from all 3 disease groups. PAD-2 was detected in 18 of the RA samples, in 16 of the SpA samples, and in none of the OA samples. Antibodies to CEP-1 were found in 12 of the RA samples (60%), in none of the SpA samples, and in 1 OA sample.Conclusion. These results highlight the importance of synovial fluid for the expression of citrullinated autoantigens in inflammatory arthritis. Whereas the expression of citrullinated proteins is a product of inflammation, the antibody response remains specific for RA.

Cancer/testis-associated genes (CTAs) are a subgroup of tumor antigens with a restricted expression in testis and malignancies. During the last decade, many of these immunotherapy candidate genes have been discovered using various approaches. Most of these genes are localized on the X-chromosome, often as multigene families. Methylation status seems to be the main, but not the only regulator of their specific expression pattern. In testis, CTAs are exclusively present in cells of the germ cell lineage, though there is a lot of variation in the moment of expression during different stages of sperm development. Likewise, there is also a lot of heterogeneity in the expression of CTAs in melanoma samples. Clues regarding functionality of CTAs for many of these proteins point to a role in cell cycle regulation or transcriptional control. Better insights in the function of these genes may shed light on the link between spermatogenesis and tumor growth and could be of use in anti-tumor therapies. This review outlines the CTA family and focuses on their expression and putative function during male germ cell development and melanocytic tumor progression.