Diverse antibody recognition patterns of the multiple Sm-D antigen polypeptides.

Anti-Sm antibodies are intrinsically associated with systemic lupus erythematosus. The major targets are the so-called B and D polypeptides. The number of Sm targets increased upon the report that SDS-PAGE conditions could be manipulated to display not one, but three Sm-D polypeptides. To characterize the relative reactivities of Sm-D1, Sm-D2, and Sm-D3, both human and murine autoantibodies were screened. These sera displayed two distinct patterns of reactivity. The Sm-D1/D3 pattern was at least four times more common than Sm-D1/D2/D3 recognition. The predominant immunoreactive protein was Sm-D1. We screened over 40 monoclonal antibodies that were derived from MRL mice and were selected as anti-Sm positive. Of these, 27 reacted with at least one Sm-D polypeptide by protein blot, but in contrast to the MRL sera, none of these antibodies reacted with Sm-D2. Rather, there were two recognition patterns of approximately equal abundance, against Sm-D1/D3 or Sm-D1 alone. Last, we explored the immune response to isolated Sm-D (containing all three D antigens) from rabbit thymus. The autoantibody produced reacted only with Sm-D2. There is accumulating evidence that the anti-Sm response is at least partially antigen-driven. The details of the intragroup relationships within the Sm-D family of proteins provide further insight into the Sm autoimmune response.

Isolation and comparison of natural and recombinant human CENP-A autoantigen.

Anticentromere antibodies (ACA) are associated with systemic sclerosis (scleroderma) patients exhibiting the more benign or so called limited manifestation of the disease (lSSc). ACA reactivity is directed against multiple polypeptide targets, the smallest of which is designated CENP-A. CENP-A is not an abundant cellular constituent; therefore to maximize recovery, we developed a protocol with a minimum of steps to isolate CENP-A from a human cell line. The trace cellular amount of this protein clearly dictated the production of its recombinant counterpart to facilitate determination of the role of the CENP-A antigen in scleroderma pathogenesis. Here we describe the eukaryotic expression of CENP-A cDNA using baculovirus-mediated infection of insect cells. The non-fusion recombinant protein spans the natural residues of the human CENP-A protein and rCENP-A followed the same chromotographic sequence for purification as did the natural source. The availability of the bona fide antigen provided a critical standard upon which to document authenticity of the recombinant polypeptide. The two forms of this antigen have been compared and shown to exhibit similar physical and antigenic properties.

Analysis of human T cell and B cell responses against U small nuclear ribonucleoprotein 70-kd, B, and D polypeptides among patients with systemic lupus erythematosus and mixed connective tissue disease.

OBJECTIVE: To analyze T and B cell reactivity with U small nuclear RNP (snRNP) 70-kd, B, and D polypeptides among patients with connective tissue disease (CTD) and to examine the functional characteristics of snRNP-reactive T cell clones. METHODS: We used an snRNP enzyme-linked immunosorbent assay and immunoblotting to characterize antibodies in patients' sera. We used human recombinant fusion proteins 70 kd, B, and D to stimulate and clone snRNP-reactive T cells from CTD patients. We analyzed the cell surface phenotype, antigenic specificity, and cytokine profiles of T cell clones. RESULTS: Patients showed T cell responsiveness to snRNP polypeptides that paralleled their autoantibody reactivities. A total of 256 clones were generated, and clones were identified which were specific for the 70-kd, B, or D polypeptides. Clones expressed a T helper cell phenotype, and were found to produce substantial quantities of both interleukin-4 (IL-4) and interferon-gamma, and lesser quantities of IL-2 and IL-6. CONCLUSION: These results show that CTD patients have clonable circulating snRNP-reactive T cells that parallel the specificity of snRNP-reactive antibodies in their sera. The snRNP-reactive T cells exhibit a helper cell phenotype and produce cytokines which are important in B cell help and differentiation.

Screening of SLE sera using purified recombinant Sm-D1 protein from a baculovirus expression system.

The Sm-D1 polypeptide is a major target of autoantibodies diagnostic for systemic lupus erythematosus (SLE). The cDNA encoding the human antigen was expressed as a full-length, nonfusion protein using a eukaryotic baculovirus expression system. This recombinant version of Sm-D1 (rSm-D1) was purified to apparent homogeneity by a combination of differential extraction steps and FPLC chromatography. A direct antibody-binding ELISA was developed using the purified antigen. There was 96% correlation between the rSm-D1 and bona fide Sm-D1 from either HeLa cells or rabbit thymus when tested against Sm-positive patient sera by ELISA. The baculovirus-expressed Sm-D1 is reactive not only with patient anti-Sm sera, but also with anti-Sm monoclonal antibodies. Our results suggest that this rSm-D1 mimics the bona fide sources, providing a valuable addition to the roster of antigens available for SLE screening, epitope mapping and overall structure study.

Analysis of genes for human snRNP Sm-D1 protein and identification of the promoter sequence which shows segmental homology to the promoters of Sm-E and U1 snRNA genes.

The Sm core proteins of U1, U2, U4/U6 and U5 snRNPs include B(B1), B'(B2), N(B3), D1, D2, D3, E, F and G polypeptides. We have isolated genomic clones encoding the Sm-D1 protein using the Sm-D1 cDNA as probe. Southern blotting and DNA sequencing analysis of these clones revealed the presence of an Sm-D1 multigene family in the human genome. Three gene members have been identified. Two of the genes are without introns and contain mutations compared to the cDNA sequence. They appear to be processed pseudogenes. The third gene, termed SNRPD1, shares 100% identity to the cDNA sequence including both 5'- and 3'-untranslated regions (UTR); it contains three introns. Analysis of the 5'-flanking region of the SNRPD1 gene revealed promoter activity, suggesting this is the functional gene that encodes the Sm-D1 protein. The promoter activity was localized in a 0.38 kb PstI fragment using CAT reporter gene fusion assays. Addition of an SV40 enhancer element did not enhance the transcription directed by that fragment. Sequence comparison of the 0.38 kb promoter sequence with the promoters of the Sm-E gene and U1 snRNA genes revealed several homologous motifs, suggesting that genes encoding the snRNP components may be coordinately regulated.

Detection of anticentromere antibodies using recombinant human CENP-A protein.

OBJECTIVE: To evaluate CENP-A reactivity with anticentromere antibodies (ACA) using recombinant protein (rCENP-A). METHODS: Human CENP-A antigen was overexpressed in insect cells using the baculovirus system. We tested for ACA activity against the full-length recombinant polypeptide by immunoblot and by enzyme-linked immunosorbent assay (ELISA). RESULTS: Of the ACA+ sera studied (n = 38), 95% were positive when tested against the rCENP-A in the ELISA system. Of the ACA- sera (n = 100), only 2% gave false-positive results in the assay. There was good correlation between the recombinant and bona fide antigens in assaying for ACA reactivity. CONCLUSION: CENP-A is a significant ACA target. The availability of the rCENP-A assay is a valuable adjunct to the previously described rCENP-B assay in analyses of the clinical significance of ACA.

Antibody reactivity to the HRES-1 endogenous retroviral element identifies a subset of patients with systemic lupus erythematosus and overlap syndromes. Correlation with antinuclear antibodies and HLA class II alleles.

OBJECTIVE: To evaluate the correlation between the presence of antibodies to an endogenous retroviral element-encoded nuclear protein autoantigen, HRES-1, and the presence of other antinuclear antibodies and HLA class II alleles in patients with systemic lupus erythematosus (SLE) and overlap syndromes. METHODS: Antibody reactivities to native and recombinant proteins and synthetic peptides were assessed by counterimmunoelectrophoresis, enzyme-linked immunosorbent assay, and Western blotting. HLA class II alleles were determined by oligonucleotide typing. RESULTS: Forty-eight percent of the 153 patients with autoimmune disease, and 52% of the subgroup with SLE, had HRES-1 antibodies. In contrast, 3.6% of 111 normal donors, and none of 42 patients with the acquired immunodeficiency syndrome or 50 asymptomatic human immunodeficiency virus 1-infected patients, had HRES-1 antibodies. Chi-square analyses revealed a significant association between anti-HRES-1 and anti-RNP and an inverse correlation between HRES-1 and Ro/La autoantibodies in patients with SLE or overlap syndromes. Antigenic epitopes of HRES-1 and the retroviral gag-related region of the 70-kd protein component of U1 small nuclear RNP, which share 3 consecutive highly charged amino acids (Arg-Arg-Glu), an additional Arg, and functionally similar Arg/Lys residues, represent cross-reactive epitopes between the two proteins. Selective removal of HRES-1 antibodies from sera of HRES-1-seropositive/RNP-seropositive patients by absorption on recombinant HRES-1/glutathione-S-transferase-conjugated agarose beads had no effect on anti-RNP reactivities. A comparative multivariate analysis of HLA class II genes revealed a differential segregation of DQB1 alleles in HRES-1-seropositive versus HRES-1-seronegative patients (P = 0.04). While a relative increase of DQB1*0402 among HRES-1-seropositive patients was noted across ethnic groups (P = 0.02), a decrease of DQB1*0201 and DQB1*0301 was found in white HRES-1-seropositive patients (P = 0.04). CONCLUSION: Autoantibodies to HRES-1 are detectable in a distinct subset of patients with autoimmune disease, primarily in those who do not have antibodies to Ro and La. Anti-HRES-1 and anti-RNP reactivities are mediated by cross-reactive but separate antibody molecules. HLA-DQB genes, rather than HLA-DRB or DQA genes, may have a more significant influence on generation of these antinuclear autoantibodies.

Autoimmunity and filariasis. Autoantibodies against cytoplasmic cellular proteins in sera of patients with onchocerciasis.

Onchocerciasis or "river blindness" is a vector-borne tropical disease resulting from infection with the filarial nematode Onchocerca volvulus. Disease manifestations include dermatitis, rheumatic complaints, and blindness. Recent findings have suggested an autoimmune etiology for the occurrence of chorioretinopathy, a disease of the eye which together with sclerosing keratoconjunctivitis is responsible for approximately 400,000 onchocerciasis-related cases of blindness. The identification of onchocerciasis as an important cause of tropical rheumatism prompted us to evaluate serologically the presence of systemic autoimmune disease in onchocerciasis patients and local controls from a hyperendemic area in Sierra Leone. In both groups there was a marked autoimmune response against cytoplasmic non-RNA-associated proteins consisting of autoantibodies against five major Ag with respective m.w. of 35, 51, 64, 83, and 110 kDa. These five proteins are novel autoantigens that could be distinguished from calreticulin, the human homologue of the onchocercal Ag RAL-1, and known autoantigens such as the 50-kDa La/SS-B or 52- and 60-kDa Ro/SS-A proteins by immunoblotting and ELISA assays. Furthermore, autoantibody reactivity against calreticulin was significantly higher in O. volvulus-infected individuals than in endemic controls. Autoantibody reactivity against the five major autoantigens, anti-calreticulin reactivity, and antibody reactivity against the 65-kDa arthritis-associated mycobacterial heat shock protein were intercorrelated as parts of an onchocerciasis-associated autoimmune response. The implication of autoimmunity in the disease pathogenesis of onchocerciasis could have important consequences for future research on therapeutical regimens, pathogenetic mechanisms, and serological diagnosis of onchocerciasis.

Protein blot assays specific for the discrimination of the centromere autoantigen, CENP-A, from human cells.

The Western or protein blot has proven to be a valuable resource in detecting discrete, immunoreactive antigen targets associated with a variety of autoimmune diseases. As the roster of autoantigens has expanded, it has become increasingly common to tailor specific gel or blot conditions to a particular polypeptide antigen. Two such assays are reported here as applied to the fractionation and visualization of human centromere protein (CENP-A), a centromere autoantigen associated with the rheumatic disease, systemic sclerosis. The centromere antigens are effectively solubilized in the presence of 1 M MgCl2 to allow for further purification. CENP-A copurifies with the histone proteins, primarily H3 and H4. The two CENP-A-specific protein blot assays separate CENP-A from the histone proteins and enhance CENP-A immunoreactivity. The first assay is based on the use of acid-urea gels with a Triton X-100 concentration chosen to maximize separation of CENP-A from all the histones. The second assay is based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis to differentiate two very basic proteins of similar molecular weight, namely CENP-A and histone H3. For each gel system, a selective choice of associated immunoblot parameters allows for the reproducible discrimination of the CENP-A antigen.

Human snRNP polypeptide D1 promotes pre-mRNA splicing in yeast and defines nonessential yeast Smd1p sequences.

Parallel investigations of yeast and metazoan pre-mRNA splicing have documented enormous complexity in the nucleic acid and protein components of the cellular splicing apparatus, the spliceosome. The degree to which yeast and metazoan spliceosomal proteins differ in composition and structure is currently unknown. In this report we demonstrate that the human small nuclear ribonucleoprotein (snRNP) polypeptide D1 complements the cell lethality, splicing deficiency, and snRNA instability phenotypes associated with a yeast smd1 null allele. Mutational analysis of yeast SMD1, guided by a comparison of the predicted yeast and human proteins, reveals that a large, nonconserved portion of Smd1p is dispensable for biological activity. These observations firmly establish D1 as an essential component of the cellular splicing apparatus and suggest that yeast and metazoa are remarkably similar in the polypeptides guiding early snRNP assembly.

Mapping of the immunoreactive domains of a small nuclear ribonucleoprotein-associated Sm-D autoantigen.

The Sm-D(D1) small nuclear ribonucleoprotein (snRNP) polypeptide is a major target of autoantibodies diagnostic for systemic lupus erythematosus. The cDNA encoding the protein from Raji cells was expressed in Escherichia coli as a fusion protein with anthranilate synthase (TrpE-Sm-D). When tested by protein blot, the recombinant polypeptide was strongly immunoreactive under defined blotting conditions, which appear to facilitate the refolding of the polypeptide into a native conformation. Multiple translational fusions between the trpE gene and fragments encompassing the length of the Sm-D coding sequence were constructed for epitope mapping. The results describe two general patterns of anti-Sm reactivity: (i) antibodies that recognize only the full-length antigen and are presumably directed against discontinuous epitopes, and (ii) antibodies that recognize the carboxy terminus of the antigen which embodies an extended/charged structure.

Overproduction of a human snRNP-associated Sm-D autoantigen in Escherichia coli and Saccharomyces cerevisiae.

To conduct functional and autoimmunity studies, we overproduced human Sm-D1 (hSm-D1), a small nuclear ribonucleoprotein 'core' protein and autoantigen, in Escherichia coli and Saccharomyces cerevisiae. Optimal expression in these organisms was achieved by designing vectors that synthesized abundant hSm-D1 mRNA under the control of the strong, regulatable promoters: T7 phi 10 (E. coli) and GAL1 (yeast). In addition, efficient translation initiation of the hSm-D1 coding sequence was effected in E. coli by utilizing a two-cistron approach; for expression in yeast, we created a 5' untranslated leader whose sequence was based on the consensus of highly expressed genes in S. cerevisiae. The hSm-D1 protein accumulated at high levels in both bacteria and yeast, representing, respectively, approx. 10% and 7% of the total protein. However, in comparison with the authentic protein, the recombinant hSm-D1 displayed different immunoreactive determinants as assessed by Western blot. We thus conclude that certain hSm-D1 immunologic properties are most likely dependent on posttranslational modifications that take place in the cells of higher eukaryotes.

Cross-reactivity of the B/B' subunit of the Sm ribonucleoprotein autoantigen with proline-rich polypeptides.

Using recombinant fusion proteins representing different regions of the human Sm B/B' polypeptide, the 4B4 monoclonal anti-Sm antibody was found to bind a C-terminus epitope that is proline-rich. 4B4 cross-reacted with the p24 gag protein of HIV-1 and with other polypeptides rich in proline residues, including collagen. BALB/c mice immunized with human collagen not only produced antibodies to the immunizing antigen but also antibodies to Sm. This immune mouse serum also recognized C-terminus B/B' fusion proteins. These data suggest that the Sm B/B' antigen contains a poly-Pro epitope that is shared by several autoantigens and retroviral proteins. These sites may be important in the induction of autoantibodies through molecular mimicry.

High-level bacterial expression, purification and characterization of human calreticulin.

To investigate its cellular function and role in autoimmune disease pathogenesis, we have bacterially expressed human calreticulin, a major calcium-binding protein in the endoplasmic reticulum and a human autoantigen. This is the first report describing the heterologous expression of calreticulin from any source. The recombinant calreticulin constituted approximately 32% of the soluble Escherichia coli proteins, and was purified to apparent homogeneity by ion exchange and hydrophobic liquid chromatography. As does the bona fide protein, the recombinant calreticulin binds calcium and undergoes changes in its conformation upon Zn2+ binding. We take this as a strong indication that the folding of the E.coli-expressed calreticulin is very similar, if not identical, to that of the authentic protein. Moreover, the bacterially expressed calreticulin readily reacted with anti-human and anti-rabbit antibodies, and the anti-recombinant calreticulin antibodies immunoreacted with HeLa calreticulin. The availability of this expression system will allow us to carry out site-specific and deletion mutagenesis analysis in structure--function studies of calreticulin.

Characterization of the autoantigen calreticulin.

Anti-Ro/SS-A antibodies are commonly found in the sera of patients with Sjögren's syndrome and SLE. These antibodies also occur in the mothers of children with neonatal lupus and congenital heart block. Ro/SS-A is a ribonucleoprotein complex whose cellular function remains unknown. To study its cellular function and to characterize its immunoreactivity, we have used an oligonucleotide designed after the published amino terminal sequence of a putative 60-kDa Ro/SS-A autoantigen to isolate its cDNA. This cDNA encodes a polypeptide that is the human homologue of calreticulin, a calcium binding protein of the endoplasmatic reticulum. The encoded polypeptide also shows a 64.4% identity with RAL-1, an Ag of the river blindness pathogen Onchocerca volvulus. Contrary to the data published by other authors, our results indicate that calreticulin is not a Ro/SS-A autoantigen. Moreover, we show that anticalreticulin autoantibodies occur in the sera of patients with SLE and patients with onchocerciasis.

Antigenic domains on the U1 small nuclear ribonucleoprotein-associated 70K polypeptide: a comparison of regions selectively recognized by human and mouse autoantibodies and by monoclonal antibodies.

Antigenic regions on the U1 small nuclear ribonucleoprotein (snRNP)-associated 70K polypeptide recognized by human and mouse autoantibodies or by monoclonal antibodies were identified and compared. Using a set of 70K fusion proteins as antigen in enzyme-linked immunosorbent assay and immunoblotting revealed that serum autoantibodies of human and of MRL/Mp mouse origin recognized a common region of the 70K polypeptide. Monoclonal anti-70K antibodies derived from a patient with mixed connective tissue disease, from an autoimmune MRL/Mp mouse, and from a BALB/c mouse immunized with purified U1 snRNP were all shown to bind to a part of the 70K polypeptide rich in charged residues and different from the region recognized by most human and MRL/Mp mouse serum autoantibodies.

Heterologous expression and epitope mapping of a human small nuclear ribonucleoprotein-associated Sm-B'/B autoantigen.

The U snRNP associated B'/B polypeptides are primary targets of Sm autoantibodies in patients with systemic lupus erythematosus. We have bacterially expressed a Sm-B'/B autoantigen from Raji cells as a fusion with the anthranilate synthase protein from Escherichia coli. The recombinant Sm-B'/B fusion displays comparable immunologic reactivity to the native protein when tested with both monoclonal and polyclonal antibodies. To map Sm-B'/B epitopes, we constructed a series of 12 anthranilate synthase fusions spanning different regions of Sm-B'/B and tested such fusions on immunoblots against a panel of characterized sera. In this manner, we have identified six epitopes, five of which overlap the proline-rich carboxyl-terminus of the protein. Some of these epitopes appear to be conformational. The human sera tested can be divided, according to the epitopes they recognize, into six groups. Finally, we have shown that anti-Sm recognition of the (U1)RNP-specific A protein is attributable to cross-reactivity between the Sm-B'/B and A autoantigens.