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Heterogeneous Nuclear Ribonucleoproteins C1/C2 Identified

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Heterogeneous Nuclear Ribonucleoproteins C1/C2 Identified http://arthritis-research.com/content/2/5/407 Primary research Heterogeneous nuclear ribonucleoproteins C1/C2 identified as autoantigens by biochemical and mass spectrometric methods Niels HH Heegaard, Martin R Larsen*, Terri Muncrief, Allan Wiik and Peter Roepstorff* Department of Autoimmunology, Statens Serum Institut, Copenhagen, and *Institute of Molecular Biology, University of Southern Denmark, Odense University, Denmark Received: 5 April 2000 Arthritis Res 2000, 2:407–414 Revisions requested: 27 April 2000 The electronic version of this article can be found online at Revisions received: 18 May 2000 http://arthritis-research.com/content/2/5/407 Accepted: 6 June 2000 Published: 11 July 2000 © Current Science Ltd (Print ISSN 1465-9905; Online ISSN 1465-9913) Statement of findings The antigenic specificity of an unusual antinuclear antibody pattern in three patient sera was identified after separating HeLa-cell nuclear extracts by two-dimensional (2D) gel electrophoresis and localizing the antigens by immunoblotting with patient serum. Protein spots were excised from the 2D gel and their contents were analyzed by matrix-assisted laser desorption-ionization (MALDI) or nanoelectrospray ionization time-of-flight (TOF) tandem mass spectrometry (MS) after in-gel digestion with trypsin. A database search identified the proteins as the C1 and C2 heterogeneous nuclear ribonucleoproteins. The clinical spectrum of patients with these autoantibodies includes arthritis, psoriasis, myositis, and scleroderma. None of 59 patients with rheumatoid arthritis, 19 with polymyositis, 33 with scleroderma, and 10 with psoriatic arthritis had similar antibodies. High-resolution protein-separation methods and mass-spectrometric peptide mapping in combination with database searches are powerful tools in the identification of novel autoantigen specificities. Keywords: antinuclear antibodies, autoantibodies, heterogeneous nuclear ribonucleoproteins C1/C2, mass spectrometry Synopsis Introduction: The classification of antinuclear antibodies proteins as well as for the D protein also appear to occur in (ANAs) is important for diagnosis and prognosis and for many distinct connective-tissue diseases, although epitope understanding the molecular pathology of autoimmune specificities may differ [3]. ANAs with specificity for the C disease. Many of the proteins that associate with RNA in the component of hnRNP (consisting of the C1 and C2 proteins) ribonucleoprotein (RNP) complexes of the spliceosome have have to our knowledge so far been described in only one been found to react with some types of ANA [1], including case [4]. We here describe the approach taken to proteins of the heterogeneous nuclear RNP (hnRNP) unambiguously identify the C1/C2 proteins as ANA targets complex that associate with newly transcribed pre-mRNA. in the sera of some patients. Autoantibodies to the A2, B1, and B2 proteins of hnRNP Aims: To determine the fine specificity of sera containing an found in some patients may be markers of several overlap unusual speckled ANA-staining pattern using a combination of syndromes [2]. However, ANAs with specificity for these 2D gel electrophoresis and MS. 1D = one-dimensional; 2D = two-dimensional; ANA = antinuclear antibody; CHAPS = 3-([3-cholamidopropyl]-dimethylammonio)-1-propane sulfonate; ELISA = enzyme-linked immunoassay; ESI-Q-TOF = electrospray ionization/quadrupole/time-of-flight; hnRNP = heterogeneous nuclear RNP; HPLC = high-performance liquid chromatography; MALDI-MS = matrix-assisted laser desorption-ionization/mass spectrometry; MALDI-TOF = matrix-assisted laser desorption-ionization time-of-flight; MS = mass spectrometry; NRDP = non-redundant protein sequence database; RNP = ribonucleoprotein; SDS = sodium dodecyl sulfate. Arthritis Research Vol 2 No 5 Heegaard et al Methods: Patient sera were screened for ANAs by indirect amino-acid insert that was not present in the lower-molecular- immunofluorescence microscopy on HEp-2 cells (cultured mass spot, further demonstrated that the two components carcinoma cells). Sera with an unusual, very regular, speckled represented the two isoforms of the C class of hnRNPs. ANA pattern were tested for reactivity with components of The patient whose case prompted us to investigate the nuclear extracts of HeLa cells that were separated by one- specificities of these antibodies was a 72-year-old man who dimensional (1D) or 2D gel electrophoresis or by reversed-phase had arthralgias and oligoarthritis but did not fulfill the criteria for high-performance liquid chromatography (HPLC). IgG reactivity rheumatoid arthritis and did not have dermatological was assessed by immunoblotting. Reactive protein spots from complaints. The reactivity of various patient groups to the 2D separations were excised from the gels and subjected to in- C1/C2 hnRNP autoantigens was subsequently tested by gel digestion with trypsin for subsequent peptide mapping, immunoblotting of HeLa-cell nuclear extracts. Of 59 patients partial peptide sequencing, and protein identification by MS and with rheumatoid arthritis, 19 with polymyositis, 33 with tandem MS on a hybrid electrospray ionization/quadrupole/time- scleroderma, and 10 with psoriatic arthritis, none had IgG of-flight (ESI-Q-TOF) mass spectrometer [5–7]. antibodies reacting with the two bands. Of sera from 139 Results: We observed a strong nuclear staining pattern (titer consecutive patients who had moderately to strongly positive >1280) with the characteristic even-sized coarse speckles speckled ANA patterns shown by indirect immunofluorescence and no staining of nucleoli in sera from three patients. On on HEp-2 cells, only two reacted with the C1/C2 hnRNP immunoblots of nuclear extracts from HeLa cells, these sera bands in immunoblotting. One of these was from a young stained two distinct bands, at Mr 42 000 and 41 000. The woman (22 years old) whose complaints of muscle tenderness reactivity strongly resembled that of the patient originally were not explained by objective findings or abnormal laboratory described by Stanek et al [4]. The antigens were enriched by test results. The third patient that we identified through ANA fractionating the extract using reversed-phase HPLC on a C4 screening followed by immunoblotting was a 54-year-old male column, and the two reactive spots on 2D separations were who was being treated with methotrexate for long-standing excised for identification. The two components appeared to polymyositis in addition to psoriasis and possible osteoporosis. be of approximately the same isoelectric points, although Discussion: The results confirm the existence of anti-C1/C2 their molecular masses differed by approximately 2000. antibodies in some patients with speckled ANAs. The antigens Peptide-mass mapping was performed by matrix-assisted were identified through the use of biochemical methods using laser desorption-ionization time-of-flight (MALDI-TOF) MS on high-resolution separation techniques combined with mass- the tryptic peptide mixture generated by digestion of the two spectrometry peptide mapping and database searches. As a excised proteins. The database search suggested that the general approach, this is a powerful way to identify new two proteins were C1/C2 hnRNPs (Swissprot accession antigens using small amounts of material without the need for number P07910). The identity of the proteins was further conventional protein sequencing. The approach does require, confirmed by tandem MS using an ESI-Q-TOF instrument. however, that the proteins can be found in databases, that they One peptide carrying two positive charges (m/z 580.32 Da), are not extensively post-translationally modified, that they can corresponding to a peptide mass of 1158.7 Da, was selected be digested enzymatically, and that they can be isolated in as a precursor ion and partially sequenced by collisional appropriately pure form by the separation technique used. fragmentation. The fragmented peptide was found to It is not known at present if the C1/C2 antibodies may have represent the tryptic fragment VDSLLENLEK, ie amino acids pathogenic relevance and/or relate to specific diagnoses or 207–216 (C2 protein numbering). Four other peptides were subsets within the group of connective-tissue diseases. It does partially sequenced and all of them matched the human appear that the reactivity is quite rare among ANA-positive C1/C2 hnRNP sequence. The theoretical masses of C1 and patients, and therefore many patients will have to be examined C2 are 32.0 and 33.3 kDa, respectively. The difference to determine these issues. The fact that the antibodies to the between the two sequences is a 13-amino-acid insert in C2 C1/C2 hnRNPs are revealed by indirect immunofluorescence between positions 107 and 108 of C1. The presence of a would indicate that the epitopes are accessible in intact, fixed specific tryptic fragment in the MALDI-TOF peptide-mass HEp-2 cells and thus probably reside outside the nucleic-acid- map from the higher-molecular-mass spot containing a 13- binding domains that would be expected to be covered by RNA. Full article Introduction various protein antigens associated with ANAs characteris- The determination of the molecular and submolecular tically represent molecules that bind nucleic acids. Thus, specificity of ANAs is important for diagnosis and progno- many of the proteins that associate with RNA in the RNP sis in clinical medicine and for understanding the molecular complexes of the spliceosome have been found to react pathology of autoimmune disease.
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