Cell Epitopes of RoSSA

A major goal of immunochemistry is to define the molecular basis of receptor recognition, which requires the analysis of structure and function of (1) the receptor, (2) the ligand, and (3) the interface between the two. The portion of the antigen that is recognized by an antibody during the binding reaction is called "antigenic determinant" or "epitope". Structurally defined antibody-protein complexes share common features: 15-22 amino acid residues contact each other on both antibody and antigen molecules (Laver et al. 1990, Smith-Gill 1994). Epitopes are usually classified as either continuous or discontinuous, consisting of linear peptides or amino acid residues brought together by folding of the polypeptide chain, respectively.

Patients with autoimmune connective tissue diseases such as SLE characteristically produce autoantibodies that recognize continuous or discontinuous B-cell epitopes.A considerable heterogeneity of epitopes has been targeted by patient sera (Chou et al. 1992, van Venrooij et al. 1994). Properties of autoantigenic epitopes seem to be similar to those of foreign protein antigens in that they reside in hydrophilic and highly conserved domains (Elkon et al. 1988). However, ANAs are often directed against functional regions of the conserved antigenic molecules (Chan and Tan 1989), and autoantigens of systemic rheumatic diseases are generally either basic or contain extended, multivalent, charge-rich sequence motifs such as coiled-coils (Dohlman et al. 1993).

Numerous studies described the definition of important epitopes on the RoRNP complex. Autoepitopes of Ro/SSA60 have been reported by several authors using different biochemical methods. The most commonly used methods are to test autoimmune sera (a) with successively truncated recombinant fragments of the autoantigen by immunoblot or immunoprecipitation or (b) with overlapping synthetic peptides in ELISA. Although detection techniques differed between the studies, positions of major epitopes within Ro/SSA60 and Ro/SSA52 were found to be consistent. The major antigenic region of Ro/SSA60 resides in the middle of the protein, between amino acid residues 150 and 320 (Saitta et al. 1994, Scofield and Harley 1991, Wahren et al. 1996). Accordingly, sera from most patients with NLE, SCLE, and Sjogren's syndrome recognized an epitope within residues 139 and 326 (McCauliffe et al. 1994). The major epitope of Ro/SSA52 has been mapped to the center of the protein within residues 136-292 (Buyon et al. 1994, Frank et al. 1994, Kato et al. 1995, Dorner et al. 1996). It turned out that the major antigenic determinant of the Ro/SSA60 antigen is the most hydrophilic and presumably the most exposed part of the protein. The most frequently identified antigenic domains of Ro/SSA52 contain a putative coiled-coil domain (Kastner et al. 1992) and a leucine zipper motif (Chan et al. 1991, Itoh et al. 1991). Taken together, the data suggest that the similarities in the autoimmune response against Ro/SSA as observed with different (a) patient sera and (b) detection methods results from an immune response that is stimulated by endogenous autoantigens. Such immune responses are also referred to as antigen driven.

The idea that Ro/SSA autoantibodies generate during antigen-driven immune responses is corroborated by results from animal experiments. In contrast to the epitope mapping with patient sera, monoclonal antibodies raised in mice recognized various antigenic regions on Ro/SSA60 and Ro/SSA52 that were not confined to the middle part but that are distributed all over the proteins (Schmitz et al. 1997, Veld-hoven et al. 1995). Immunization of mice (Topfer et al. 1995) and rabbits (Scofield et al. 1996) with recombinant Ro/SSA antigens or synthetic Ro/SSA peptides leads to intramolecular and intermolecular spreading of the immune reactivity, referred to as epitope spreading. It is thought that once an autoimmune response to one antigenic determinant has developed it might spread to associated proteins. However, to date, it is far from clear whether such an epitope spreading (a) occurs in patients during the onset of systemic autoimmune responses or (b) merely represents an artifact resulting from immunization of animals with exogenous proteins.

To address the issue of discontinuous epitopes on the RoRNP particle, Saitta et al. (Saitta et al. 1994) generated 10 overlapping recombinant polypeptides of the human 60-kDa Ro/SSA protein and compared their reactivities to immunoprecipitation of in vitro translated, native Ro60. Patient sera that immunoprecipitated full-length Ro/SSA60 had low or undetectable anti-Ro/SSA60 titers by ELISA and immunoblot-ting using recombinant antigens. These results suggest that discontinuous epitopes are the predominant targets of anti-Ro/SSA60 autoantibodies. It is important to note that during immunoprecipitation and immunofluorescence, the autoantigens retain their native structure, whereas in ELISA and immunoblotting, the substrate proteins are more or less denatured, which favors the exclusive detection of linear epitopes. Since ANAs in general represent very useful tools for immunoprecipitation, it was concluded that they preferentially recognize discontinuous epitopes on RNP complexes. Using ribonuclease protection assays,Wolin and Steitz (Wolin and Steitz 1984) discovered the major epitope of anti-Ro/SSA antibodies on the RoRNP particle. For each of the three human Ro RNAs whose sequence was known at that time, the most highly protected portion was found in immunoprecipitates corresponding to the lower section of the stem formed by base pairing the 5' and 3' ends of the RNA (see Fig. 24.2). These results confirmed that the complex of Ro/SSA60 and the lower stem of Ro RNA forms a major, discontinuous epitope for anti-Ro/SSA autoantibodies.

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