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The U1 small nuclear ribonucleoproteins (nRNPs) are common targets of autoantibodies in lupus and other autoimmune diseases. However, the etiology and progression of autoimmune responses directed against these antigens are not well understood. Using a unique collection of serial human samples from before and after nRNP antibody development, we investigated early humoral events in the development of anti-nRNP autoimmunity.
Lupus patients with sera available from both before and after nRNP antibody precipitin development were identified from the Oklahoma Clinical Immunology Serum Repository. Antibodies in the serial samples were analyzed by ELISA, Western blotting, solid-phase epitope mapping and competition assays.
The first detected nRNP antibodies targeted 6 common initial epitopes in nRNP A, 2 in nRNP C and 9 in nRNP 70K. The initial epitopes of nRNP A and nRNP C were significantly enriched for proline (p=0.0004, p=0.048) and shared up to 95% sequence homology. The initial nRNP 70K humoral epitopes differed from nRNP A and C. The initial antibodies to nRNP A and nRNP C were cross-reactive with the Sm B′-derived peptide PPPGMRPP. Antibody binding against all three nRNP subunits diversified significantly over time.
nRNP A and nRNP C autoantibodies initially targeted restricted, proline-rich motifs. Antibody binding subsequently spread to other epitopes. The similarity and cross-reactivity between the initial targets of nRNP and Sm autoantibodies identifies a likely commonality in etiology and a focal point for intermolecular epitope spreading.
Systemic lupus erythematosus (SLE) is a systemic autoimmune disease of complex and incompletely understood etiology (reviewed in (1, 2)). Antibodies against the nRNP complex are found in sera from 21-47% of SLE patients, and antibodies against the Smith antigen (Sm) are found in sera from approximately 5-30% of SLE patients (3-5). The components of the nRNP complex, nRNP 70K, nRNP A, and nRNP C, may each be targeted by antibodies. In contrast to Sm autoantibodies, which are almost exclusively found in SLE patient sera, nRNP autoantibodies are often detected in patients with other autoimmune disorders, including mixed connective tissue disease (MCTD), Raynaud's phenomenon and scleroderma. nRNP 70K antibodies are normally associated with MCTD, while antibodies against the other subunits are more common in SLE (6, 7).
Despite considerable effort, the development of anti-nRNP antibodies in human SLE is inadequately understood. Studies in murine models implicate both nRNP A and nRNP 70K in the initiation of nRNP antibodies (8, 9). A study investigating the order of nRNP antibody development in human rheumatic disease shows that nRNP 70 K antibodies appear first (10). However, this study includes both SLE patients and other individuals with nRNP antibodies, possibly obscuring the primary pathway in SLE. Epitopes of nRNP proteins targeted in an established autoimmune response are mapped (11-22), but the key initial epitopes, which herald the onset of the loss of tolerance, have not been investigated or identified in detail. The understanding of the human SLE-specific pattern of nRNP antibody development is therefore still far from complete.
More is known about the initial humoral immune response to Sm B′ in SLE patients. Sm B′ antibodies initially target the amino acid sequence PPPGMRPP (23-25), and then diversify first to a repeated, proline-rich region and eventually targeting a variety of autoantigens in a process termed epitope spreading (26-28). The close physical proximity and regions of similar amino acid sequences of Sm and nRNP proteins, as well as the temporal linkage of antibody appearance to these proteins, suggest that autoantibodies to nRNP and Sm B′ may have common originating events in select subsets of SLE patients.
Serial serum samples from SLE patients who had at least one sample that was negative for nRNP autoimmunity and a later sample in which nRNP antibodies were present provided a unique opportunity to examine the initiation and development of nRNP antibodies. The experiments evaluate the hypotheses that nRNP humoral autoimmunity in SLE begins with a limited number of epitopes, that this response diversifies over time, and that the development of nRNP and Sm humoral autoimmunity are intertwined in a subset of individuals.
This project was carried out in accord with the Helsinki Declaration and approved by the institutional review boards of the Oklahoma Medical Research Foundation (OMRF) and the Oklahoma University Health Sciences Center (OUHSC). Uniquely coded, serial serum samples from 20 SLE patients that met the revised SLE classification criteria (29) and had samples available from both before and after the development of nRNP autoantibodies were obtained from the Oklahoma Clinical Immunology Serum Repository (Oklahoma City, OK). Sera from over 27,000 individuals with rheumatic diseases, of which 1,794 were positive for nRNP antibodies at some time point, have been collected in this repository. The twenty studied patients had a mean of 6.3 samples available (range 2 to 19) spanning an average 8.0 years (range 2-20 years). All patients were classified with systemic lupus erythematosus per American College of Rheumatology criteria (29, 30). Patients with mixed connective tissue disease who did not meet at least 4 of the ACR classification criteria for SLE were excluded from evaluation. Sera from 11 unaffected volunteers were used as controls.
All samples were tested for autoantibodies against nRNP by Ochterlony immunodiffusion assay by the OMRF Clinical Immunology Laboratory (Oklahoma City, OK) according to published methods (31), and by standard ELISA as described previously (14, 32). Briefly, 1 μg of nRNP antigen purified from calf thymus (Immunovision, Springdale, AR) was coated onto each well of 96-well polystyrene plates. Serum samples were diluted 1:100 and 1:1000 and incubated in the antigen-coated wells at room temperature (RT) for 2 h. Alkaline phosphatase goat anti-human IgG conjugate (Jackson Immunoresearch Laboratories, West Grove, PA) was allowed to bind for 16 h at 4°C, followed by the addition of para-nitrophenyl phosphate disodium (PNPP) (Sigma-Aldrich, St. Louis, MO) as the colorimetric substrate. Absorbance was measured at 410 nm by using a Dynex MRX II ELISA reader when the OD410 of the positive control was 1.0, which enabled standardization of results among multiple plates.
Full-length nRNP A (gene ID 6626) cloned into the pRSET-B vector (Invitrogen, Carlsbad, CA) was received from C. Lutz (University of Medicine and Dentistry of New Jersey) (33) and transformed into BL21 gold E. coli (Stratagene, Carlsbad, CA). The coding sequence for nRNP 70K (gene ID 6625) was cloned from HeLa cDNA by using PCR with the primer set forward 5′ ACG CGT CGA CAT GAC CCA GTT CCT GCC GC 3′, reverse 5′ ATA AGA ATG CGG CCG CTC ACT CCG GCG CAG CCT C 3′, and ligated into the expression vector pET28b (Novagen, Carlsbad, CA). The protein was expressed in BL21Star competent cells (Invitrogen). The plasmid containing the nRNP C (gene ID 6631) insert was obtained from ATCC as I.M.A.G.E clone # 6391327 and was ligated into the expression vector pET28c at the Sal1 and Not1 sites and expressed in Bl21Gold (DE3) pLysS competent cells (Stratagene). All proteins were expressed with 6-His tags. Expression of recombinant antigens was induced with 0.5 mM isopropyl-β-D-thiogalactoside at 37°C for 4 h. After sonication, the expressed recombinant proteins were purified from the cells by using Ni-NTA affinity columns (Qiagen, Valencia, CA) (33).
The linear peptides PPPGMMPV (nRNP C109-118), KIPPTPFS (nRNP C61-68), GPAPGMRPP (nRNP C117-125), MPPPGMIPP (nRNP A164-172), PPPGLAPG (nRNP A171-178), PRDPIPYLPP (nRNP 70K15-24), EFEDRPDAPPTRA (nRNP 70K47-59) and ERPGPSPLPH (nRNP 70K221-230) were used for inhibition analysis of the nRNP autoantibodies. The Sm-derived linear peptide PPPGMRPP (Sm B′191-198) was used for testing of cross-reactivity and PPPPPPPP (poly-proline) was used as a negative control. Peptides were synthesized by the OUHSC Molecular Biology and Proteomics Facility (Oklahoma City, OK).
HeLa cell extracts or recombinant nRNP A, C or 70K proteins were subjected to electrophoresis in 12.5% polyacrylamide gels and transferred to a nitrocellulose membrane. Consistent protein loading and transfer were confirmed by staining with fast green (Fisher Scientific, Pittsburg, PA). Blots were cut into strips and blocked with 3% (w/v) dry milk in TBS. Sera were incubated at a 1:200 dilution with individual membrane strips for 2 h at RT. After washing, alkaline phosphatase goat anti-human IgG conjugate was added for 3 h. Serum antibody binding to specific proteins was visualized by the addition of the nitrotetrazolium blue/5-bromo-4-chloro-3-indolyl phosphate substrate (Fisher Scientific, Waltham, MA).
Western blot analysis was used to determine peptide inhibition of antibody binding. Recombinant nRNP A, nRNP C or nRNP 70K were electrophoresed and transferred to nitrocellulose membranes as described previously. Serum samples were diluted 1:50-1:20,000 and then incubated with 40 μg/ml of peptide for 2 h at room temperature. A concentration of 40 μg/ml was experimentally determined to be the penultimate dilution in a dilution series of 10, 20, and 40 μg/ml peptide (data not shown). In additional experiments, the nRNP 70K-positive samples were also incubated with poly-lysine (Fisher Scientific) at concentrations of 100, 500, and 1000 μg /ml. The peptide pre-absorbed sera were incubated with the nitrocellulose-bound antigens, and antibody binding was detected as described above. Blots were scanned and band intensity was quantified by using Scion Image software for Windows (Scion Corporation, Fredrick, MA). Western blot experiments were repeated in triplicate, with average values used for comparisons. Inhibition is likely to be under-represented due to the compressed dynamic range of the Western blot readout. Indeed, incubation with full-length recombinant nRNP at 40 μg/ml only showed a mean inhibition of 41%.
Decapeptides overlapping by eight amino acids representing the protein sequences of nRNP 70K (locus NP_003080) (34) and nRNP C (locus PO9234) (35), as well as maximally overlapping octapeptides encompassing the sequence of nRNP A (locus PO 9012) (36) were constructed on the ends of polyethylene pins by using Fmoc solid-phase peptide chemistry as previously described (14, 23). Serum antibody binding to each peptide in the solid-phase assay was detected by using a modified ELISA technique. Briefly, individual solid-phase peptides were incubated with patient sera for 2 h at RT. Peptides were washed and then incubated with anti-human IgG alkaline phosphatase conjugate (Jackson Immunoresearch Laboratories) overnight at 4°C. After washing, peptides were incubated at 37°C with PNPP substrate (Sigma-Aldrich, St. Louis, MO) until positive control wells had OD405 of 1.0. A well-characterized positive control serum was used to normalize the results among multiple plates.
Antibody binding to peptides in the solid-phase assays was considered significant if the mean absorbance of the positive samples was at least 3 standard deviations above the mean absorbance of the controls. Epitopes were defined as one or more positively bound, overlapping peptide. Chi square analysis was used to analyze significant differences in amino acid prevalence and the number of peptides recognized by initial and late samples. The Mann-Whitney U test was used to compare inhibition of antibody binding. Comparisons that resulted in a p value of <0.05 were considered significant.
Twenty SLE patients were found for which samples were available from both before and after appearance of nRNP antibodies by precipitin. These samples included the following patients: 11 African American, 6 European American, 2 American Indian, and 1 Asian, 18 females and 2 males. The average age (SD) was 43.5 (12.6) years. The patients met an average of 5.7 (range 4-10) ACR SLE classification criteria (29, 30).
The presence of anti-nRNP antibodies was confirmed by nRNP antigen-specific ELISA and Western blot analysis. For every case, at least one sample was observed to be negative for nRNP antibodies by precipitin, ELISA and Western blot, and nRNP antibodies were detected in at least one subsequent sample by using precipitin and ELISA. The average time (SD) between the last negative and the first positive samples was 3.5 (2.7) years (range 2 months to 9.8 years). Patients were observed from 1 to 14 years after the development of anti-nRNP. No significant differences in the anti-nRNP appearance times were noted when the methods of nRNP antibody detection were compared.
Western blot analysis with HeLa cell extracts as the antigen source was used to detect antibodies against the nRNP subunits nRNP A, nRNP C or nRNP 70K. Antibody appearance against either a single nRNP protein (8/15) or multiple proteins at once (7/15) was common in the initial nRNP-positive sera. Antibodies against single nRNP proteins were most commonly targeted to nRNP A (4/8), with two patients each developing initial antibodies against only nRNP C or nRNP 70K. Five samples that were positive for nRNP in precipitin and ELISA did not have detectable antibodies against nRNP subunits in denaturing Western blots. Antibody binding to nRNP subunits diversified in subsequent samples, with 11/15 individual patients positive for antibodies to multiple subunits in the last available samples. Of the patients with antibodies against a single subunit, one had antibodies against nRNP C and three had antibodies directed towards nRNP A.
Sufficient quantities of serum were available to map the initial binding of the first nRNP A-positive sera from 6 SLE patients by using solid-phase peptide analysis. The autoantibodies detected in these patients along with the order of autoantibody appearance are provided in Table 1. Antibody binding was analyzed for each unique octapeptide in the nRNP A sequence (Figure 1). The mean antibody binding pattern of SLE patients was compared to the mean pattern obtained from 4 normal volunteers and 3 anti-nRNP-negative individuals who later developed anti-nRNP.
Twelve peptides comprising 6 epitopes were identified from the first nRNP A-positive samples (Figure 2A). The initial nRNP A epitopes were significantly enriched for proline (χ2 = 12.5, p=0.0004) compared to the peptides that were not targeted by autoantibodies. No other amino acids were significantly enriched in the early epitopes. The most commonly bound epitope GQPPYMPPPGMIPPPGLPG (aa 159-178) was recognized by 5/6 (88.3%) of the nRNP-positive samples. The only sample that did not bind to this peptide did not significantly bind to any sequential peptide sequence tested. This sequence, especially PPPGMIPP, bears a striking resemblance to the prominent initial epitope PPPGMRP(G)P found in Sm B'.
To determine the proportion of the anti-nRNP A initial antibody response accounted for by binding to the epitope at aa 159-178, the peptides nRNPA164-172 and nRNPA171-178 were tested for the ability to inhibit binding to recombinant nRNP A. Binding was significantly inhibited by both nRNPA164-172 and by a combination of both peptides; mean inhibition was 17.0% for nRNPA164-172 (p=0.025) and 33.2% for the combination of both peptides (p=0.0091). Inhibition of binding in individual samples of up to 75% was observed (Figure 2C).
Evolution of the humoral fine specificity for several lupus autoantigens is described (25, 26, 37-39). The extent of epitope spreading in antibodies to nRNP A was examined by comparing the initial nRNP antibody-positive sample to the antibody binding profile in sera collected a mean (SD) of 2.3 (1.4) years after this sample (Figure 1). The later response was significantly more diverse than the initial response; 40 out of the 276 octapeptides bound in the later response and 12 octapeptides were recognized by the first nRNP A autoreactive sera. (Figure 2D)(χ2=6.068, p=0.014). The pattern of antibody binding, with prominent binding to the N-terminus, that was found in the late sera was substantially different from that of the initial sera.
Antibodies to nRNP C were most commonly found in tandem with nRNP A antibodies. The sequence of nRNP C resembles that of nRNP A in the prevalence of large proline-rich regions. Initial autoantibody binding to nRNP C was examined by using solid-phase mapping of sera with the first detectable nRNP C antibodies, collected a mean (SD) of 3.6 (3.4) years after the last negative sample. The mean antibody binding pattern in the initial nRNP C-positive sera from 6 SLE patients was compared to the mean antibody pattern of 5 normal controls and 6 SLE patients before onset of nRNP C antibodies (Figure 1). The autoantibody development pattern found in these patients is provided in Table 1.
Epitope mapping revealed significant antibody binding to only 2 initial epitopes of nRNP C (Figure 3). These epitopes are located at aa 57-66, (FQQGKIPPTP) and aa 109-128, (PPPGMMPVGPAPGMRPPMGG). Both epitopes were recognized by antibodies from 3/6 of the samples. One sample bound only to three contiguous decapeptides whose mean was not significantly increased overall (aa 63-78, PPTPFSAPPPAGAM). One sample did not bind to any of the sequential epitopes. The initial epitopes targeted by nRNP C autoantibodies were significantly enriched for proline compared to the epitopes in the unbound regions (χ2=3.90, p<0.48). No other amino acids were significantly enriched in the initial epitopes.
Antibody binding to recombinant nRNP C was significantly inhibited by incubation with peptides corresponding to the initial epitopes. Incubation with a mixture of the peptides nRNP C 61-68, nRNP C 109-118 and nRNP C 117-125, encompassing both of the commonly bound initial epitopes, resulted in a mean 40.3% inhibition and up to 74.5% inhibition in individual samples (Figure 3C, p=0.034). Inhibition with the peptide nRNP C61-68 only inhibited antibody binding in two serum samples, but binding in those two samples was inhibited by 56% and 90%. Interestingly, the time interval between the last negative and first positive sample for nRNP C was only 1.2 and 1.75 years, respectively, for these two individuals, while the mean interval for the remaining samples was 5.2 years. This short interval in the samples that were highly inhibited by incubation with the peptides supports the idea that these epitopes are more important early in the autoantibody response, which diversifies over time. Incubation with nRNP C109-118 inhibited binding of 3/6 samples by more than 20%. nRNPC117-125 exhibited the strongest individual inhibitory effect of the three peptides; 5/6 samples were inhibited by more than 25% (p=0.034) (Figure 3C).
Autoantibody binding to nRNP C diversified considerably with time. Serum samples from 5 patients drawn an average (SD) of 4.1 (5.2) years after the initially reactive sample significantly bound to 23 individual decapeptides, while only 4 decapeptides were bound by the first positive samples (χ2= 14.6, p=0.0001) (Figure 3D). Individual late nRNP C-positive samples recognized between 18 and 25 decapeptides with an average (SD) of 20.2 (2.8).
The fine antibody specificities of the negative and first reactive sera of three patients were mapped by using decapeptides overlapping by 8 amino acids and encompassing the entire nRNP 70K sequence (Figure 1). The antibody development pattern of these three individuals shows diverse first epitopes, including some enriched for proline and some enriched for basic amino acids (Table 1). An average (SD) of 1.67 (1.15) years existed between the negative samples and the first nRNP 70K-binding samples. The initial sera bound to 21 common decapeptides, comprising 9 epitopes (Figure 4B). Individual samples bound to 5, 41, and 66 decapeptides. The epitope at aa 63-84 (REERMERKRREKIERRQQEVET) was bound by all 3 samples. The highest average absorbance was found in epitope 4 (aa 217-234 SRYDERPGPSPLPHRDRD), which was bound by 2/3 samples.
Unlike the initial epitopes in nRNP A and nRNP C humoral immunity, in which proline-rich sequences were predominant, the initial epitopes in nRNP 70K were diverse. However, 5/9 initial epitopes were highly basic, having theoretical isoelectric points of 10.67 (epitope 2), 12.48 (epitope 3), 11.83 (epitope 5), 11.42 (epitope 6), and 10.67(epitope 8). The theoretical pI of the nRNP 70K protein is 9.94. The initial epitopes as a whole were not significantly enriched for any amino acid.
To determine whether the antibody binding to the basic epitopes was a result of charge-charge interactions rather than sequence specificity, the three first 70K antibody-positive sera were incubated with poly-lysine and inhibition of binding was tested by Western blot. No decrease in antibody binding was observed after incubation with poly-lysine at concentrations up to 1 mg/ml (Data not shown).
Serial serum samples from 2 years after the development of nRNP autoimmunity were available for all three patients that developed anti-nRNP 70K. Epitope mapping was performed on these samples revealing epitope spreading (Figure 1). The total number of decapeptides that were significantly bound by comparing the mean of the late samples to the mean of the negative samples increased from to 66 from 21 decapeptides identified by comparing the mean of the first positive sample to the negative samples (χ2= 27.9, p<0.0001) (Figure 4C).
The proline enrichment that was observed in the initial epitopes of nRNP A and nRNP C was especially interesting since a proline-rich epitope, PPPGMRP(G)P, has been described as a crucial, early humoral epitope in the development of autoimmunity to Sm B′ (23, 24). Portions of the most commonly bound initial epitope of nRNP A, GQPPYMPPPGMIPPPGLPG (nRNP A159-178), are nearly identical to the repeated motif PPPGMRPP from Sm B′, as are the 2 initial epitopes from nRNP C FQQGKIPPTP (nRNP C57-66) and PPPGMMPVGPAPGMRPPMGG (nRNP C109-128). Given the tight temporal association of antibodies to nRNP and Sm, antibodies against these complexes may develop at the same time and under the same initiating conditions. If this is the case, the earliest antibodies would likely cross-react between the two protein subsets.
We examined the extent of cross-reactivity between the antibodies that bound to the initial epitopes in nRNP A or nRNP C with the Sm B′-derived peptide. The initial anti-nRNP A- or nRNP C-positive patient sera were incubated with the Sm B′-derived peptide PPPGMRPP or with poly-proline as a negative control. Incubation with PPPGMRPP significantly inhibited binding to nRNP A (p=0.0068) (Figure 5A). Incubation with PPPGMRPP significantly eliminated the total binding to nRNP C (p=0.0087) (Figure 5B). No inhibition of binding to recombinant nRNP 70 K was observed after incubation of the first positive sera with PPPGMRPP (data not shown).
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, 25). 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, 40). Antibodies against proline-rich regions from nRNP A, nRNP C, Sm B′, and nRNP 70K can cross-react (16, 17, 41), 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 (11-13) 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 (14-17). 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, 44), 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, 18). By using fine resolution peptide mapping, this study identified the specific amino acids bound in larger, previously described nRNP 70K epitopes (19, 20, 45). An epitope previously found in the mature nRNP 70K humoral immune responses is aa 135-194 (21, 22). 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, 47), 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, 49), 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 (11-22, 45). Antibodies against epitopes identified through this method make up a large proportion of the total antibody population (24, 25). 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, 7). 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.
The authors thank Terri McHugh and Jody Gross for their technical assistance. We are grateful for the kind gift of the expression plasmid for nRNP A (U1A) from Carol Lutz, PhD (University of Medicine and Dentistry of New Jersey). We thank Kristina Wasson-Blader, PhD, for review and revision of the manuscript. We also thank the Oklahoma University Health Sciences Center Molecular Biology-Proteomics facility for peptide synthesis.
This work was supported by grants from the National Institutes of Health (AR48940, AR045084, RR15577, AR053483, T32AI007633, RR020143), and the Lou Kerr Chair in Biomedical Research at OMRF.