By using serial sera in our clinical immunology repository collected from more than 27,000 individuals, we identified SLE patients who progressed from nRNP antibody negative to nRNP autoantibody positive. This resource allowed the investigation of the initial targets of the humoral nRNP autoimmune response. We identified six common, early epitopes of nRNP A in these sera. One of these epitopes, QPPYMPPPGMIPPPGLPG (aa 159-178), was recognized by every initial sera that had detectable binding to sequential epitopes, and was bound more strongly than the other initial epitopes. We found that the early response to nRNP C commonly bound only two epitopes, both of which were proline-rich. nRNP 70K early antibody binding showed predominance of basic amino acids and was mainly dissimilar to nRNP C and nRNP A binding.
Proline-rich sequences were commonly bound by the first autoantibodies against all three nRNP proteins. The proline-rich sequences that were bound by initial antibodies from nRNP A and C were 44-74% similar to each other and to the sequence PPPGMRPP, which is the initial humoral epitope targeted in Sm B′ humoral autoimmunity and the major target of anti-Sm B′ antibodies (24
). Enrichment for proline-rich motifs in the initial antibody binding may be due to a number of factors, including potentially high surface expression, protein conformation or cross-reactivity with similar sequences in other antigens.
The inhibition of binding to nRNP proteins after incubation with PPPGMRPP indicates that cross-reactive antibodies make up a major portion of the initial autoantibody response to nRNP A and C in this patient collection. Cross-reactivity of antibodies is suggested as a mechanism for the development or spreading of autoimmunity through molecular mimicry (39
). Antibodies against proline-rich regions from nRNP A, nRNP C, Sm B′, and nRNP 70K can cross-react (16
), and immunization of mice with the SmB′-derived sequence leads to lupus-like autoimmunity (42
). It is plausible that antibodies against these similar proline-rich regions could facilitate epitope spreading between nRNP and Sm proteins.
Comparison of the present study with studies of the mature nRNP A response (11
) reveals that the pattern of antibody binding changes from the initial to mature response, in which antibody binding is focused on the N-terminal sequences. This shift of pattern as the antibody response matures suggests that separate selective forces may drive the antibody specificity as time passes. For example, the initial response may be due to cross-reactivity with environmental triggers, while the later antibody specificity may be determined by other factors such as access to the binding sites on the antigen, selective antigen processing/presentation or preferential V-D-J recombination. These findings indicate that care should be taken in attempting to gain insight into the etiology of autoimmune processes by studying samples collected late in the disease process.
Antibody binding to nRNP C was initially directed against just two regions. These initial epitopes were also found in other studies that mapped the mature nRNP C epitopes (14
). Unlike epitope spreading in nRNP A, the epitope spreading found in nRNP C does not diminish the role of the initial epitopes in the mature humoral response. On the contrary, the epitopes from amino acids 109-128 remain predominant in the mature antibody binding pattern.
The initial response to nRNP 70K was not similar to the responses to nRNP A and nRNP C. Interestingly, nRNP 70K immunity is more strongly associated with mixed connective tissue disease than with lupus (43
), and the difference in initial antibody binding patterns may reflect different pathogenic processes. The initial epitopes that we discovered in nRNP 70K humoral autoimmunity were consistent with other studies of nRNP 70K humoral epitopes. (12
). By using fine resolution peptide mapping, this study identified the specific amino acids bound in larger, previously described nRNP 70K epitopes (19
). An epitope previously found in the mature nRNP 70K humoral immune responses is aa 135-194 (21
). Although the initial nRNP-positive sera did not bind this region, the later samples did, indicating the importance of epitope spreading in the development of autoimmunity.
The targeting of proline-rich, similar epitopes in early humoral autoimmunity may be partially due to environmental factors. Epstein-Barr virus (EBV) infection is associated with SLE with a high degree of significance (46
), and the EBNA-1 protein has a sequence, PPPGRRP, that is very similar to the initial epitopes of nRNP A and C identified in the current study (48
). Immunization with the EBNA-1-derived peptide on a branching poly-lysine backbone causes lupus-like autoimmunity (48
), as does DNA vaccination of mice with EBNA-1 (50
). The similarity of epitopes from four major autoantigens targeted in lupus with the EBNA-1 sequence suggests that the role for EBNA-1 in lupus may be more extensive than was previously thought, and could help to explain the association of EBV infection with the development of lupus, even in patients that do not develop antibodies against Sm.
These studies used a powerful resource, the OMRF clinical immunology serum collection. While a prospective longitudinal study may yield more data than this retrospective study, obtaining sufficient samples and participation for such a study is impractical. Using the serum collection allowed for a cost-effective means to approach these questions. An additional limitation of the study is that solid-phase peptide assays may miss conformational epitopes while they do provide high resolution of the antibody binding pattern. Although this is a concern, the level of agreement between the current and previous studies performed by using the peptide assay with studies of protein fragments and larger peptides is high (11
). Antibodies against epitopes identified through this method make up a large proportion of the total antibody population (24
). Indeed, in this study incubation with the most predominant peptides detected by solid-phase epitope mapping led to a mean decrease in antibody binding of over 30% for both nRNP A and nRNP C.
This study identifies a consistent proline-rich motif in early humoral autoimmunity to nRNP proteins; a motif shared with early autoimmunity to Sm B′. The data support a role for this motif in epitope spreading in the initiation and development of the autoantibody response to nRNP proteins. The motif is absent in early nRNP 70K antibody targets, suggesting that the development of nRNP 70K autoantibodies follows a different path than antibodies against the other nRNP components. These findings are consistent with the previously observed close temporal association of nRNP and Sm autoantibodies (51
), and with the association of nRNP 70K and nRNP A antibodies with different clinical outcomes (6
). These results identify a key pattern in the development of a number of lupus-associated autoimmune specificities in human disease, and support the possibility that molecular mimicry is involved in the initiation of autoimmunity against these components in naturally occurring human SLE.