RESUMEN
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.
Asunto(s)
Autoantígenos/aislamiento & purificación , Proteínas Cromosómicas no Histona/aislamiento & purificación , Immunoblotting/métodos , Fraccionamiento Celular , Núcleo Celular/química , Proteína A Centromérica , Electroforesis en Gel de Poliacrilamida , Células HeLa , Histonas/aislamiento & purificación , Humanos , Cloruro de Magnesio/farmacología , Octoxinol , SolubilidadRESUMEN
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.
Asunto(s)
Autoantígenos/inmunología , Ribonucleoproteínas Nucleares Pequeñas/inmunología , Secuencia de Aminoácidos , Western Blotting , Clonación Molecular , ADN/genética , Epítopos , Humanos , Datos de Secuencia Molecular , Pruebas de Precipitina , Proteínas Recombinantes de Fusión/inmunología , Mapeo Restrictivo , Ribonucleoproteínas Nucleares Pequeñas/genética , Proteínas Nucleares snRNPRESUMEN
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.
Asunto(s)
Autoantígenos/genética , Escherichia coli/genética , Plásmidos/genética , Ribonucleoproteínas/genética , Saccharomyces cerevisiae/genética , Autoantígenos/biosíntesis , Autoantígenos/inmunología , Secuencia de Bases , Western Blotting , Clonación Molecular , Humanos , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Ribonucleoproteínas/inmunología , Ribonucleoproteínas Nucleares Pequeñas , Proteínas Nucleares snRNPRESUMEN
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.
Asunto(s)
Proteínas de Unión al Calcio/genética , Proteínas Recombinantes/genética , Secuencia de Aminoácidos , Secuencia de Bases , Calcio/metabolismo , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/inmunología , Proteínas de Unión al Calcio/aislamiento & purificación , Calreticulina , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Plásmidos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Zinc/metabolismoRESUMEN
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.
Asunto(s)
Autoantígenos/genética , Proteínas de Unión al Calcio/inmunología , Lupus Eritematoso Sistémico/inmunología , Oncocercosis/inmunología , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Southern Blotting , Western Blotting , Proteínas de Unión al Calcio/genética , Calreticulina , Clonación Molecular , ADN/genética , Genes , Humanos , Datos de Secuencia Molecular , Peso Molecular , Oligonucleótidos/química , Biosíntesis de Proteínas , Procesamiento Proteico-Postraduccional , ARN Mensajero/genética , Ribonucleoproteínas/genética , Ribonucleoproteínas/inmunologíaRESUMEN
Anti-Sm is an antibody specificity often associated with the autoimmune disease systemic lupus erythematosus. The polypeptides Sm-B'/B (estimated molecular mass 27 and 26 kDa, respectively) are primary targets of Sm antibodies. Sm-B'/B are part of the core polypeptides of small ribonucleoprotein particles (snRNP) involved in pre-mRNA splicing. Sm-B'/B share the same amino-terminal sequence as we determined by microsequence analyses of the purified polypeptides. Oligonucleotide probes based on that sequence were used to isolate seven clones from a human lymphoblastoid cDNA library in lambda gt10. The clones contained a single coding region for a protein of approximately 25 kDa. The predicted amino-terminal sequence was identical to that of the isolated Sm-B'/B polypeptides. In vitro translation experiments produced a protein immunoreactive with human polyclonal anti-Sm antibodies. The isolation of only one unique cDNA sequence suggests that Sm-B'/B may be post-translational variants encoded by a single message. The specific structural features which distinguish Sm-B' from Sm-B have yet to be determined. Northern blot analysis confirmed the diverse tissue and species distribution expected for these immunologically conserved polypeptides. The Sm-B'/B primary sequence is rich in proline (20%) and glycine (15%) residues. The prolines are concentrated in the carboxyl-terminal half of the protein and display a repetitive unit that is shared with other snRNP and nucleic acid binding proteins. Analysis of these arrays suggests an eight residue proline-rich consensus sequence with potential as either an RNA binding domain, or as a site of protein/protein interaction.
Asunto(s)
Autoantígenos/aislamiento & purificación , ADN/aislamiento & purificación , Péptidos/aislamiento & purificación , Ribonucleoproteínas/aislamiento & purificación , Secuencia de Aminoácidos , Autoantígenos/biosíntesis , Autoantígenos/genética , Secuencia de Bases , Northern Blotting , Clonación Molecular , Células HeLa , Humanos , Datos de Secuencia Molecular , Biosíntesis de Péptidos , Péptidos/genética , Pruebas de Precipitina , Biosíntesis de Proteínas , Conformación Proteica , Ribonucleoproteínas/biosíntesis , Ribonucleoproteínas/genética , Ribonucleoproteínas Nucleares Pequeñas , Proteínas Nucleares snRNPRESUMEN
Antibodies to the Sm-D polypeptide antigen are closely associated with the rheumatic disease systemic lupus erythematosus. Sm-D exists in the cell as one of the core proteins of the small nuclear ribonucleoprotein complexes implicated in RNA processing. We have isolated a cDNA clone, D45-2, coding for the Sm-D human nuclear antigen by screening a human B-lymphocyte cDNA library with synthetic oligonucleotide probes. The 1633-base-pair clone contains an open reading frame (ORF) 357 nucleotides long, capable of encoding a 13,282-dalton polypeptide. The Sm-D coding region is initiated at an AUG codon downstream from a sequence with excellent match to the consensus for the eukaryotic ribosome-binding site. The Sm-D ORF is preceded by a 150-nucleotide-long untranslated leader and followed by a 1126-nucleotide-long untranslated region containing four putative poly(A) signals. The predicted amino acid sequence reveals a (Gly-Arg)9 repeated motif at the C terminus, which may constitute one of the Sm-D immunoreactive determinants. Moreover, this C terminus shows interesting features: (i) a good homology to protamines as expected for a nucleic acid binding protein and (ii) a striking similarity to a region in the Epstein-Barr nuclear antigen.
Asunto(s)
Autoantígenos/genética , Ribonucleoproteínas Nucleares Pequeñas , Secuencia de Aminoácidos , Antígenos Virales/genética , Linfocitos B/análisis , Secuencia de Bases , ADN/genética , Antígenos Nucleares del Virus de Epstein-Barr , Humanos , Datos de Secuencia Molecular , Protaminas/genética , Homología de Secuencia de Ácido Nucleico , Proteínas Nucleares snRNPRESUMEN
One of the major proteins in human serum capable of binding DNA has been shown to be factor B of the alternative pathway of complement activation. This protein, designated DNA-binding protein-2 (DBP-2), is recognized by antisera directed against both factor B and its activated form, fragment Bb. Its m.w., charge microheterogeneity, and amino acid composition correspond closely with reported values for those properties of factor B. A radioimmunoassay was used to estimate the serum concentration of DBP-2 at 266 +/- 83 microgram/ml, which also corresponds with the level of factor B normally present in the serum. DBP-2 functions as factor B in the activation of factor B-depleted serum. Limited proteolytic treatment of DBP-2 produced a fragment pattern resembling that of factor B both in the m.w. of the fragments and their electrophoretic mobilities. By means of DNA affinity chromatography of the fragments produced by trypsinization, the DNA-binding domain of DBP-2 was localized.