title: Calreticulin binds preferentially with B cell linear epitopes of Ro60 kD autoantigen, enhancing recognition by anti-Ro60 kD autoantibodies.
abstract: Calreticulin is a molecular chaperone to newly synthesized polypeptides. Previous studies suggested that calreticulin is probably a protein member of the Ro/La RNP complex. The aims of this study were (a) to investigate whether linear B cell epitopes of the Ro/La RNP complex are bound to calreticulin and (b) if the complex peptide-calreticulin is recognized specifically by anti-Ro autoantibodies. Calreticulin was isolated from either human or pig spleen using a multi-step purification method and found to interact preferentially with biotinylated peptides derived from the sequence of the Ro60 kD 175-184aa(10p) and 216-232aa(17p). The interaction of the peptide-calreticulin complex was favoured by the combination of heat treatment, divalent cations and ATP. La/SSB epitopes did not react with calreticulin. Peptides corresponding to La/SSB epitopes as well as the common epitope of Sm did not interact with calreticulin. Thirty-eight anti-Ro60 KD positive and 23 anti-Ro60 kD negative sera of patients with systemic lupus erythematosus (SLE) and primary Sjögren's syndrome (pSS) were tested. All anti-Ro60 kD positive sera bound the complex calreticulin-17p, while 95% of the same sera had activity against the complex calreticulin - 10p. Tested individually, calreticulin, pep10p and pep17p presented very low reactivity (8%, 11% and 29%, respectively) against anti-Ro60 kD positive sera. Anti-Ro60 KD negative sera did not exhibit significant reactivity either with calreticulin, 10rho and 17rho or with the complexes calreticulin - 10p and calreticulin-17p (<5%). These results suggest that calreticulin can induce conformation-dependent recognition of the Ro60 kD epitopes, leading eventually to their recognition by autoantibodies. This is the first time that such a relationship is shown between a chaperone protein and fragments of an intracellular autoantigen. This work also provides insights into the understanding of mechanisms for autoantibody production. Furthermore, this association can be proved useful for the development of new sensitive assays for autoantibody detection.
authors.affiliation: Department of Pathophysiology, School of Medicine, National University of Athens, 11527 Athens, Greece.
authors.forename: E V
authors.initials: EV
authors.lastname: Staikou
authors.forename: J G
authors.initials: JG
authors.lastname: Routsias
authors.forename: A A
authors.initials: AA
authors.lastname: Makri
authors.forename: A
authors.initials: A
authors.lastname: Terzoglou
authors.forename: M
authors.initials: M
authors.lastname: Sakarellos-Daitsiotis
authors.forename: C
authors.initials: C
authors.lastname: Sakarellos
authors.forename: G
authors.initials: G
authors.lastname: Panayotou
authors.forename: H M
authors.initials: HM
authors.lastname: Moutsopoulos
authors.forename: A G
authors.initials: AG
authors.lastname: Tzioufas
mesh_terms: D000818:Animals; D001323:Autoantibodies / Q000032:analysis / Q000276:immunology*; D001324:Autoantigens; D001327:Autoimmune Diseases / Q000276:immunology* / Q000378:metabolism; D001402:B-Lymphocytes / Q000276:immunology*; D037282:Calreticulin / Q000302:isolation & purification / Q000378:metabolism*; D016022:Case-Control Studies; D000939:Epitopes / Q000378:metabolism*; D006801:Humans; D008180:Lupus Erythematosus, Systemic / Q000276:immunology; D011485:Protein Binding; D020733:RNA, Small Cytoplasmic; D012261:Ribonucleoproteins / Q000276:immunology*; D017411:Ribonucleoproteins, Small Nuclear / Q000276:immunology; D012859:Sjogren's Syndrome / Q000276:immunology; D013552:Swine; D013696:Temperature; D055517:snRNP Core Proteins; D000097585:SS-B Antigen; D000099207:SS-A Antigen
chemical_list: D001323:Autoantibodies; D001324:Autoantigens; D037282:Calreticulin; D000939:Epitopes; D020733:RNA, Small Cytoplasmic; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear; D055517:snRNP Core Proteins; D000097585:SS-B Antigen; D000099207:SS-A Antigen; C116006:RO60 protein, human