Ro60 Peptides Induce Antibodies to Similar Epitopes Shared Among Lupus-Related Autoantigens

Abstract

The coexistence of autoantibodies to ribonucleoproteins (RNP) in sera of patients with systemic lupus erythematosus has been attributed to intermolecular determinant spreading among physically associated proteins. Recently, we showed that murine Ab responses to rRo60 or Ro60 peptides were diversified unexpectedly to small nuclear RNP. In this investigation, the mechanisms for this autoantibody diversification were examined. Intramolecular determinant spreading was demonstrated in mice immunized with human or mouse Ro60316–335. Immune sera depleted of anti-peptide Ab immunoprecipitated Ro60-associated mY1 and mY3 RNA and remained reactive to a determinant on Ro60128–285. Absorption with the immunogen depleted the immune sera completely of anti-Golgi complex Ab (inducible only with human Ro60316–335) and anti-La Ab, and reduced substantially Ab to SmD and 70-kDa U1RNP. Mouse rRo60 completely inhibited the immune sera reactivity to La, SmD, and 70-kDa U1RNP. However, La, SmD, and 70-kDa U1RNP preferentially inhibited the antiserum reactivities to these Ags, respectively. Affinity-purified anti-La Ab were reactive with Ro60, La, SmD, and 70-kDa U1RNP. These results provide evidence that a population of the induced autoantibodies recognized determinants shared by these autoantigens. Lack of sequence homology between Ro60316–335 and La, SmD, or 70-kDa U1RNP suggests that these determinants are conformational. Interestingly, similar cross-reactive autoantibodies were found in NZB/NZW F1 sera. Thus, a single molecular mimic may generate Ab to multiple RNP Ags. Furthermore, cross-reactive determinants shared between antigenic systems that are not associated physically (Ro/La RNP and small nuclear RNP) may be important in the generation of autoantibody diversity in systemic lupus erythematosus.

Evidence for Multiple Shared Antigenic Determinants within Ro60 and Other Lupus-Related Ribonucleoprotein Autoantigens in Human Autoimmune Responses

“…Immunization of inbred strains of mice with human and mouse Ro60, as well as with selected peptides derived from these autoantigens, elicited cross-reactive IgG Ab responses to La, SmD, and the U1 RNP 70-kDa protein (12). Critically, similar cross-reactive Abs were observed in spontaneously autoimmune NZB/NZW F 1 mice, ruling out the possibility that the observations were an artifact of immunization (13). Subsequent studies revealed that cross-reactive IgG Ab responses to polypeptides within the small nuclear RNP complex can be readily induced in mice immunized with SmD, SmB, and A-RNP (14).…”

“…Our lab has made some observations in this regard, which also indicate that additional mechanisms may contribute toward Ab diversification (12,13). Immunization of inbred strains of mice with human and mouse Ro60, as well as with selected peptides derived from these autoantigens, elicited cross-reactive IgG Ab responses to La, SmD, and the U1 RNP 70-kDa protein (12).…”

“…However, several unexpected patterns of apparent diversification have been reported, indicating the need to revisit the issue of autoantibody diversification (10,11). Previous work in our lab using murine models of lupus made it evident that, while epitope spreading was an important and widespread phenomenon in the diversification of the immune response, there also existed an extensive and unappreciated level of cross-reactivity between several seemingly nonhomologous RNP autoantigens (12)(13)(14). Cross-reactivity between Ro60 and SmD was particularly striking because it linked immune responses to proteins within two different antigenic systems.…”

A SmD Peptide Induces Better Antibody Responses to Other Proteins within the Small Nuclear Ribonucleoprotein Complex than to SmD Protein via Intermolecular Epitope Spreading

“…Reactivity to whole snRNP particle was determined by immunoprecipitation using cell extracts from metabolically labeled WEHI 7.1 cells with [ 35 S]methionine (14). To determine cross-reactivity between A-RNP and immunizing peptides, absorption and elution studies were performed as previously described (16,18). Briefly, 5 l of pooled sera were absorbed with 50 l of A-RNP coupled-Sepharose beads.…”

Key Residues of a Major Cytochrome P4502D6 Epitope Are Located on the Surface of the Molecule

“…B cell epitopes, unlike T cell epitopes, are usually conformational, i.e., highly dependent on the three-dimensional structure of the protein (21)(22)(23)(24)(25)(26). The presence of conformational epitopes on CYP2D6 was first suggested by Duclos-Vallee et al (10) who were unable to reverse inhibition of the CYP2D6 enzymatic activity by preincubation of LKM1-positive sera with synthetic peptides spanning known linear epitopes.…”