Characterization and use of the ECV304 autoantigenic citrullinome to understand anti-citrullinated protein/peptide autoantibodies in rheumatoid arthritis

Abstract Background Anti-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...

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Main Authors: Natalia Regine de França, Henri André Ménard, Maximilien Lora, Zhijie Zhou, Joyce Rauch, Carol Hitchon, Luís Eduardo Coelho Andrade, Inés Colmegna
Format: Article
Language:English
Published: BMC 2022-01-01
Series:Arthritis Research & Therapy
Subjects:
Online Access:https://doi.org/10.1186/s13075-021-02698-2
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author Natalia Regine de França
Henri André Ménard
Maximilien Lora
Zhijie Zhou
Joyce Rauch
Carol Hitchon
Luís Eduardo Coelho Andrade
Inés Colmegna
author_facet Natalia Regine de França
Henri André Ménard
Maximilien Lora
Zhijie Zhou
Joyce Rauch
Carol Hitchon
Luís Eduardo Coelho Andrade
Inés Colmegna
author_sort Natalia Regine de França
collection DOAJ
description Abstract Background Anti-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. Methods Human 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. Results Of 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. Conclusions ECV304 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.
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spelling doaj.art-fa4f26124b0349d6892f18d4dfcd9a1b2022-12-21T21:19:57ZengBMCArthritis Research & Therapy1478-63622022-01-0124111110.1186/s13075-021-02698-2Characterization and use of the ECV304 autoantigenic citrullinome to understand anti-citrullinated protein/peptide autoantibodies in rheumatoid arthritisNatalia Regine de França0Henri André Ménard1Maximilien Lora2Zhijie Zhou3Joyce Rauch4Carol Hitchon5Luís Eduardo Coelho Andrade6Inés Colmegna7Division of Rheumatology, Department of Medicine, McGill University, The Research Institute of the McGill University Health CentreDivision of Rheumatology, Department of Medicine, McGill University, The Research Institute of the McGill University Health CentreDivision of Rheumatology, Department of Medicine, McGill University, The Research Institute of the McGill University Health CentreDivision of Rheumatology, Department of Medicine, McGill University, The Research Institute of the McGill University Health CentreDivision of Rheumatology, Department of Medicine, McGill University, The Research Institute of the McGill University Health CentreSection of Rheumatology, Department of Medicine, University of ManitobaDivision of Rheumatology, Paulista School of Medicine, Federal University of Sao PauloDivision of Rheumatology, Department of Medicine, McGill University, The Research Institute of the McGill University Health CentreAbstract Background Anti-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. Methods Human 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. Results Of 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. Conclusions ECV304 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.https://doi.org/10.1186/s13075-021-02698-2ECV304AutocitrullinomePeptidylarginine deiminasesACPAsRheumatoid arthritis
spellingShingle Natalia Regine de França
Henri André Ménard
Maximilien Lora
Zhijie Zhou
Joyce Rauch
Carol Hitchon
Luís Eduardo Coelho Andrade
Inés Colmegna
Characterization and use of the ECV304 autoantigenic citrullinome to understand anti-citrullinated protein/peptide autoantibodies in rheumatoid arthritis
Arthritis Research & Therapy
ECV304
Autocitrullinome
Peptidylarginine deiminases
ACPAs
Rheumatoid arthritis
title Characterization and use of the ECV304 autoantigenic citrullinome to understand anti-citrullinated protein/peptide autoantibodies in rheumatoid arthritis
title_full Characterization and use of the ECV304 autoantigenic citrullinome to understand anti-citrullinated protein/peptide autoantibodies in rheumatoid arthritis
title_fullStr Characterization and use of the ECV304 autoantigenic citrullinome to understand anti-citrullinated protein/peptide autoantibodies in rheumatoid arthritis
title_full_unstemmed Characterization and use of the ECV304 autoantigenic citrullinome to understand anti-citrullinated protein/peptide autoantibodies in rheumatoid arthritis
title_short Characterization and use of the ECV304 autoantigenic citrullinome to understand anti-citrullinated protein/peptide autoantibodies in rheumatoid arthritis
title_sort characterization and use of the ecv304 autoantigenic citrullinome to understand anti citrullinated protein peptide autoantibodies in rheumatoid arthritis
topic ECV304
Autocitrullinome
Peptidylarginine deiminases
ACPAs
Rheumatoid arthritis
url https://doi.org/10.1186/s13075-021-02698-2
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