Abstract
BackgroundAnti-citrullinated protein antibodies (ACPAs) are highly specific for rheumatoid arthritis (RA). In vivo, ACPAs target peptidyl-citrulline epitopes (cit-) in a variety of proteins (cit-prot-ACPAs) and derived peptides (cit-pept-ACPAs) generated via the peptidylarginine deiminase (PAD) isoenzymes. We aimed to identify a cell line with self-citrullination capacity, to describe its autoantigenic citrullinome, and to test it as a source of autocitrullinated proteins and peptides.MethodsHuman cell lines were screened for cit-proteins by Western blot. PAD isoenzymes were identified by RT-PCR. Autocitrullination of ECV304 was optimized, and the ECV304 autocitrullinomes immunoprecipitated by sera from three RA patients were characterized by mass spectrometry. Cit-pept-ACPAs were detected using anti-CCP2 ELISA and cit-prot-ACPAs, by an auto-cit-prot-ECV304 ELISA. Sera from 177 RA patients, 59 non-RA rheumatic disease patients and 25 non-disease controls were tested.ResultsOf the seven cell lines studied, only ECV304 simultaneously overexpressed PAD2 and PAD3 and its extracts reproducibly autocitrullinated self and non-self-proteins. Proteomic analysis of the cit-ECV304 products immunoprecipitated by RA sera, identified novel cit-targets: calreticulin, profilin 1, vinculin, new 14–3-3 protein family members, chaperones, and mitochondrial enzymes. The auto-cit-prot-ECV304 ELISA had a sensitivity of 50% and a specificity of 95% for RA diagnosis.ConclusionsECV304 cells overexpress two of the PAD isoenzymes capable of citrullinating self-proteins. These autocitrullinated cells constitute a basic and clinical research tool that enable the detection of cit-prot-ACPAs with high diagnostic specificity and allow the identification of the specific cit-proteins targeted by individual RA sera.
Highlights
Anti-citrullinated protein antibodies (ACPAs) are highly specific for rheumatoid arthritis (RA)
ECV304: a cell line capable of self‐citrullination First, we investigated the presence of peptidylarginine deiminase (PAD) activity capable of producing cit-proteins in the lysates of seven human cell lines and assessed cit-prot-epitopes by Western blots (WB) using a single RA serum with high anti-cyclic-citrullinated-peptide 2 (CCP2) titer
PAD2 and PAD3 in ECV304 promote the citrullination of proteins recognized by antibodies in RA sera
Summary
Anti-citrullinated protein antibodies (ACPAs) are highly specific for rheumatoid arthritis (RA). ACPAs target peptidyl-citrulline epitopes (cit-) in a variety of proteins (cit-prot-ACPAs) and derived peptides (cit-peptACPAs) generated via the peptidylarginine deiminase (PAD) isoenzymes. ACPAs represent a heterogeneous group of antibodies recognizing a variety of cit-epitopes either on proteins (cit-prot-ACPAs) or peptides (cit-pept-ACPAs) [4]. Studies of human ACPA specificities show that anti-CCP2 ACPAs are not cit-protein specific and target widely cross-reactive epitopes on cit-peptides/ cit-proteins candidates [6]. Cit-prot-epitopes designed as “private” are defined by the amino acids neighbouring citrulline residues on the targeted autoantigenic cit-protein. As pathogenic autoantibodies in RA should target “private” cit-prot-epitopes in the joints, those epitopes would be ideally detected using whole cit-proteins [8, 9]
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