Fine specificity of autoantibodies to calreticulin: epitope mapping and characterization

Abstract

SUMMARYExtracellular calreticulin (CRT) as well as anti-CRT antibodies have been reported in patients with various autoimmune disorders and CRT has been implicated in`epitope spreading' to other autoantigens such as the Ro/SS-A complex. In addition, antibodies against parasite forms of the endoplasmic reticulum chaperone, CRT, have been found in patients suffering from onchocerciasis and schistosomiasis. In this study, we screened sera for anti-CRT antibodies from patients with active and inactive systemic lupus ertythematosus (SLE) and primary or secondary Sjo Ègren's syndrome. Approximately 40% of all SLE patients were positive for anti-CRT antibodies. The antigenic regions of CRT were determined using full length CRT and fragments of CRT prepared in yeast and Escherichia coli, respectively. Synthetic 15mer peptides corresponding to the major autoantigenic region of CRT (amino acids 1±289), each one overlapping by 12 amino acids, were used to map the B cell epitopes on the CRT protein recognized by autoimmune sera. Major antigenic epitopes were found to be associated with the N-terminal half of the protein in 69% of the SLE sera from active disease patients, while the Cdomain was not antigenic. Major epitopes were found to be reactive with antibodies in sera from SLE patients with both active and inactive disease, spanning different regions of the N and P-domains. Sera from both healthy and disease controls and primary Sjo Ègren's syndrome patients were non-reactive to these sequences. Limited proteolysis of CRT with two major leucocyte serine proteases, elastase and cathepsin G, demonstrated that an N-terminal region of CRT is resistant to digestion. Interestingly, some of the epitopes with the highest reactivity belong to the fragments of the protein which bind to C1q and inhibit complement activation. Whether C1q association with CRT is a pathological or protective interaction between these two proteins is currently under investigation.

The pathophysiology of photosensitivity in lupus erythematosus

“…Interestingly, the surface blebs of apoptotic keratinocytes bind C1q, an early component of the complement cascade (132). The only C1q binding protein that has been identified in apoptotic blebs to date is calreticulin (98) and autoantibodies to calreticulin can interfere with this binding (133). The binding of C1q to apoptotic cells has been postulated to facilitate the clearance of these cells by macrophages, which express a C1q cell surface receptor (134,135).…”

“…The identification of a dominant grp78 binding motif on Ro52 (178) and the coexistence of grp78, Ro52 and Ro60 in the surface membrane blebs of apoptotic cells (98) support this view. Interestingly, autoantibodies directed towards grp94/gp96 and calreticulin have also been detected in the sera of patients with SLE (133,179,180) and could perhaps be generated via a similar mechanism.…”

Vaccination with antioxidant enzymes confers protective immunity against challenge infection with Schistosoma mansoni

“…Analyses of the reactivity of specific antibody isotypes against these peptides were aimed to identify relevant B cell epitopes that could be used in a subunit vaccine (Joshi et al 2000, Tallima et al 2003. Synthetic peptides have been widely used for the evaluation of both B and T cell epitopes responsible for (or that could lead to) protection against other pathogens, including Schistosoma (Reynolds et al 1992, 1994, Eggleton et al 2000, Joshi et al 2000. We demonstrated that BALB/c mice vaccinated with SmCT-SOD developed high levels of antibodies directed mainly to the SmCT-SOD derived peptide 3 (Pep 3), which included amino acid residues 64-83 .…”

The angiogenesis inhibitor vasostatin is regulated by neutrophil elastase–dependent cleavage of calreticulin in AML patients

“…8,9 The various mechanisms contributing to intracellular CRT delocalization and the effects of CRT presentation on the cell surface for induction of immunogenic cell death and apoptosis recently have been studied intensely. [8][9][10] In contrast, the extracellular expression and regulation of CRT are poorly understood. In this report, we investigated the regulation and function of CRT in the serum of patients with AML.…”