The I Protein of the Heterogeneous Nuclear Ribonucleoprotein Complex is a Novel Dog Nuclear Autoantigen (original) (raw)
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cDNA cloning of a human autoimmune nuclear ribonucleoprotein antigen
Proceedings of the National Academy of Sciences, 1985
Sera from patients with systemic lupus erythematosus and other autoimmune disorders contain antibodies against nuclear proteins. One such autoantibody system, known as Sm, reacts with antigens associated with small nuclear RNA molecules. In this paper we report the use of Sm autoantibodies to isolate a cDNA clone for the mRNA of one of these nuclear antigens. A HeLa cell cDNA library was screened by message selection followed by autoantibody reaction of cell-free translation products. This led to the identification of a cDNA clone, p281, containing sequences complementary to mRNA for an Sm autoantibody-reactive, 11,000 Mr protein. This cloned Sm antigen comigrated with the small nuclear RNA-associated protein known as "E" and reacted with four out of four Sm autoantibodies that precipitate E protein from total mRNA translation products. RNA gel blot hybridization with clone p281 DNA revealed a poly(A)+ mRNA of -600 nucleotides in human and marmoset (New World primate) cells. Southern blot hybridization of HeLa cell and human
Autoantibodies to histone, DNA and nucleosome antigens in canine systemic lupus erythematosus
Clinical & Experimental Immunology, 2008
SUMMARY Dogs can develop systemic lupus erythematosus syndromes that are clinically similar to those seen in humans. In contrast, previous observations suggest differences in their autoantibody reactivity patterns against histones and DNA which are components of the nucleosome in chromatin. The objective of this study was to assess comprehensively the levels of autoantibodies against histone, DNA and nucleosome antigens in a population of lupus dogs. The specificities of antibodies in lupus and control dog sera were determined using IgM- and IgG-specific reagents in an ELISA against a variety of chromatin antigens. When compared with control sera, IgG antibodies to individual histones H1, H2A, H3 and H4 were significantly higher in the lupus group. In contrast, we did not detect IgG antibodies specific for H2B, H2A-H2B, DNA, H2A-H2B-DNA or nucleosomes in lupus dogs. There was no significant increase in any of the IgM specificities tested. Therefore, the reactivity pattern to nucleos...
Nucleolar proteins identified in human cells as antigens by sera from dogs with autoimmune disorders
Experimental Cell Research, 1989
In the course of a systematic screening of sera from dogs suffering from autoimmune disorders, three sera were shown by indirect immunofluorescence to characteristically label the nucleoli and nucleoplasm of human cell lines (Hep-2 and HeLa). This pattern of staining persisted throughout the cell cycle, except for mitosis when the fluorescence was localized in extrachromosomal areas. By immunoblotting nuclear and subnuclear fractions, three polypeptides of 110,000, 95,000, and 45,000 Da apparent molecular weight were identified, which reacted with all three sera. By means of aftinity purification, it was shown that an antibody specific for any one of the three proteins also reacts with the two others. This antigenic cross-reactivity suggested regions of structural homology shared by the three proteins. Indeed, treatment of nucleoli with high concentrations of DNase I containing residual proteolytic activity resulted in the disappearance of the llO-and 95-kDa proteins and the concomitant appearance of a doublet of 45-kDa proteins. Subnuclear localization studies indicated that all three polypeptides were located in both nucleoli and nucleoplasm. Significantly, the llO-kDa protein differs from the major nucleolar protein, nucleolin, by its electrophoretic mobility in two-dimensional gels, its location in nucleoli and in nucleoplasm, its absence in nucleolar organizer regions of chromosomes, and its differential solubihty of DNase I. Therefore, the three antigenically related species reported in this study constitute a new class of nucleolar proteins. @ 1989 Academic press, IN.
Journal of Clinical Immunology, 1987
We have previously reported the purification of Sm and RNP antigens from goat liver and identified two polypeptides of molecular weights 70 and 80-90 kd as RNP specific and of 14 and 30 kd as Sm specific. In this communication the effect of ribonuclease and trypsin on Sm and RNP antigens was studied at the polypeptide level. We found that the RNP antigenic determinant polypeptides of 70 and 80-90 kd are lost as a result of such treatment, whereas there is no effect on the Sm-specific 14-and 30-kd polypeptides. The role of RNA in the antigenicity of Sm and RNP was studied by dissociation and reconstitution studies. The antigens were fractionated into protein and RNA and the individual fractions were tested for Sm and RNP activity by counterimmunoelectrophoresis (CIE) and enzyme-linked immunosorbent assay (ELISA). The RNA fraction did not react alone with anti-Sm and anti-RNP sera with either of the assays. Conversely when the protein fraction was tested by CIE, only Sm antigenicity was detectable. In the ELISA both Sm and RNP activities were demonstrated in the protein fraction. These results show that the presence of RNA is important in the immunoprecipitation reactions involving only RNP antigen, whereas Sm activity is independent of RNA. In addition, when the reaction is carried out by an assay involving primary antigen-antibody reaction (e.g., ELISA), RNP antibodies react with protein fractions alone, without the presence of RNA. We also report the gtycoprotein nature of Sm-specific polypeptides. The antigen was found to react specifically with concanavatin A (Con A), indicating the presence of glycosyl and/or mannosyl residues. The observed glycoprotein nature of the Sm-specific polypeptides possibly explains their remarkable stability, unlike RNP-specific polypeptides, which are susceptible to proteolytic attack.
Journal of Clinical Investigation, 1992
RA33 is a nuclear autoantigen with an apparent molecular mass of33 kD. Autoantibodies against RA33 are found in about 30% of sera from RA patients, but only occasionally in sera from patients with other connective tissue diseases. To characterize RA33, the antigen was purified from HeLa cell nuclear extracts to more than 90% homogeneity by affinity chromatography on heparin-Sepharose and by chromatofocusing. Sequence analysis of five tryptic peptides revealed that their sequences matched corresponding sequences of the A2 protein of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex. Furthermore, RA33 was shown to be present in the 40S hnRNP complex and to behave indistinguishably from A2 in binding to single stranded DNA. In summary, these data strongly indicate that RA33 and A2 are the same protein, and thus identify on a molecular level a new autoantigen. (J. Clin.
Heterogeneous Nuclear Ribonucleoprotein H1, a Novel Nuclear Autoantigen
Clinical Chemistry, 2009
BACKGROUND: Serum samples from patients with autoimmune connective tissue diseases that show a finely speckled antinuclear antibody (ANA) on indirect immune-fluorescence often have antibodies against unknown nuclear target antigens. To search for such autoantigens we applied a proteomic approach using sera from patients with a high ANA titer (Ն640) and finely speckled fluorescence but in whom no antibodies to extractable nuclear antigens (ENA) could be identified.
Journal of Autoimmunity, 2003
Objective: To gain insights on initial stages of the autoimmune response in lupus prone mice taking advantage of new sensitive and quantitative techniques for the detection of autoantibodies specific for RNA-(ribonucleoproteins) and DNA-protein (chromatin) complexes. Methods: DNA and nucleosome antibodies were detected by ELISA, antibodies to SmB, U1A-RNP, Ro52, Ro60 and La by a new radioligand assay, using de novo synthesized radio-labeled antigens. Results: Analysis of anti-chromatin (including anti-nucleosome, anti-dsDNA and anti-histone antibodies) and of anti-snRNP antibodies (including anti-U1A-RNP, anti-SmB, anti-Ro52, anti-Ro60, anti-La antibodies) was performed in sequential sera from B/W, MRL+/+, MRL Yaa and MRL lpr/lpr mice. In a cohort of 105 MRL+/+ mice of different ages, 59, 51, and 57 mice were positive for anti-nucleosome, anti-SmB and anti-U1A-RNP, respectively. None of them was positive for anti-dsDNA. Importantly, antibody positivities were not randomly distributed but were significantly clustered in individual mice. Appearance of DNA-and RNA-protein complex antibodies started at w18-20 weeks of age, preceding that of the anti-dsDNA (or anti-histone) antibodies that only started at 30-32 weeks. Anti-nucleosome, anti-SmB and anti-U1A-RNP antibody responses did not display any cross-reactivity as demonstrated by inhibition and adsorption experiments. Conclusion: These data indicate that anti-nucleosome and anti-snRNP antibodies appear early and concomitantly in lupus prone mice even though they do not share any cross-reactivity. These results fit with the assumption that their production is triggered by tightly physically associated nucleosomes and snRNP autoantigens contained in the same apoptotic bodies.
Antibodies against the proliferating cell nuclear antigen (PCNA) was first discovered in the sera of systemic lupus erythematosus (SLE) patients. However, the reactivity and specificity of anti-PCNA autoantibodies are still unclear. To investigate the property of anti-PCNA autoantibodies, we conducted an ELISA screening of the anti-PCNA autoantibodies in sera of SLE patients. Eighteen out of 191 SLE sera were found to be positive for anti-PCNA antibodies giving a frequency of nearly 10%. Among the positive sera, a sample with the highest titer of anti-PCNA autoantibody preferentially recognizes the wild-type PCNA as compared to the Y114A mutation which contains a single amino acid substitution at 114 and fails to form the toroidal structure. Moreover, the autoantibody purified from this serum identifies only the free PCNA in crude mammalian cell extracts but not other associated cellular components. This finding raises a possibility that immunostaining with the human anti-PCNA autoantibodies in previous studies might have only partially PCNAs in tissues.