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.
The classification of antinuclear antibodies (ANAs) is important for diagnosis and prognosis and for understanding the molecular pathology of autoimmune disease. Many of the proteins that associate with RNA in the ribonucleoprotein (RNP) complexes of the spliceosome have been found to react with some types of ANA , including proteins of the heterogeneous nuclear RNP (hnRNP) complex that associate with newly transcribed pre-mRNA. Autoantibodies to the A2, B1, and B2 proteins of hnRNP found in some patients may be markers of several overlap syndromes . However, ANAs with specificity for these proteins as well as for the D protein also appear to occur in many distinct connective-tissue diseases, although epitope specificities may differ . ANAs with specificity for the C component of hnRNP (consisting of the C1 and C2 proteins) have to our knowledge so far been described in only one case . We here describe the approach taken to unambiguously identify the C1/C2 proteins as ANA targets in the sera of some patients.
To determine the fine specificity of sera containing an unusual speckled ANA-staining pattern using a combination of 2D gel electrophoresis and MS.
Patient sera were screened for ANAs by indirect immunofluorescence microscopy on HEp-2 cells (cultured carcinoma cells). Sera with an unusual, very regular, speckled ANA pattern were tested for reactivity with components of nuclear extracts of HeLa cells that were separated by one-dimensional (1D) or 2D gel electrophoresis or by reversed-phase high-performance liquid chromatography (HPLC). IgG reactivity was assessed by immunoblotting. Reactive protein spots from 2D separations were excised from the gels and subjected to in-gel digestion with trypsin for subsequent peptide mapping, partial peptide sequencing, and protein identification by MS and tandem MS on a hybrid electrospray ionization/quadrupole/time-of-flight (ESI-Q-TOF) mass spectrometer [5,6,7].
We observed a strong nuclear staining pattern (titer >1280) with the characteristic even-sized coarse speckles and no staining of nucleoli in sera from three patients. On immunoblots of nuclear extracts from HeLa cells, these sera stained two distinct bands, at Mr 42 000 and 41 000. There activity strongly resembled that of the patient originally described by Stanek et al . The antigens were enriched by fractionating the extract using reversed-phase HPLC on a C4 column, and the two reactive spots on 2D separations were excised for identification. The two components appeared to be of approximately the same isoelectric points, although their molecular masses differed by approximately 2000. Peptide-mass mapping was performed by matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) MS on the tryptic peptide mixture generated by digestion of the two excised proteins. The database search suggested that the two proteins were C1/C2 hnRNPs (Swissprot accession number P07910). The identity of the proteins was further confirmed by tandem MS using an ESI-Q-TOF instrument. One peptide carrying two positive charges (m/z 580.32 Da), corresponding to a peptide mass of 1158.7 Da, was selected as a precursor ion and partially sequenced by collisional fragmentation. The fragmented peptide was found to represent the tryptic fragment VDSLLENLEK, ie amino acids 207-216 (C2 protein numbering). Four other peptides were partially sequenced and all of them matched the human C1/C2 hnRNP sequence. The theoretical masses of C1 and C2 are 32.0 and 33.3 kDa, respectively. The difference between the two sequences is a 13-amino-acid insert in C2 between positions 107 and 108 of C1. The presence of a specific tryptic fragment in the MALDI-TOF peptide-mass map from the higher-molecular-mass spot containing a 13-amino-acid insert that was not present in the lower-molecular-mass spot, further demonstrated that the two components represented the two isoforms of the C class of hnRNPs.
The patient whose case prompted us to investigate the specificities of these antibodies was a 72-year-old man who had arthralgias and oligoarthritis but did not fulfill the criteria for rheumatoid arthritis and did not have dermatological complaints. The reactivity of various patient groups to the C1/C2 hnRNP autoantigens was subsequently tested by immunoblotting of HeLa-cell nuclear extracts. Of 59 patients with rheumatoid arthritis, 19 with polymyositis, 33 with scleroderma, and 10 with psoriatic arthritis, none had IgG antibodies reacting with the two bands. Of sera from 139 consecutive patients who had moderately to strongly positive speckled ANA patterns shown by indirect immunofluorescence on HEp-2 cells, only two reacted with the C1/C2 hnRNP bands in immunoblotting. One of these was from a young woman (22 years old) whose complaints of muscle tenderness were not explained by objective findings or abnormal laboratory test results. The third patient that we identified through ANA screening followed by immunoblotting was a 54-year-old male who was being treated with methotrexate for long-standing polymyositis in addition to psoriasis and possible osteoporosis.
The results confirm the existence of anti-C1/C2 antibodies in some patients with speckled ANAs. The antigens were identified through the use of biochemical methods using high-resolution separation techniques combined with mass-spectrometry peptide mapping and database searches. As a general approach, this is a powerful way to identify new antigens using small amounts of material without the need for conventional protein sequencing. The approach does require, however, that the proteins can be found in databases, that they are not extensively post-translationally modified, that they can be digested enzymatically, and that they can be isolated in appropriately pure form by the separation technique used.
It is not known at present if the C1/C2 antibodies may have pathogenic relevance and/or relate to specific diagnoses or subsets within the group of connective-tissue diseases. It does appear that the reactivity is quite rare among ANA-positive patients, and therefore many patients will have to be examined to determine these issues. The fact that the antibodies to the C1/C2 hnRNPs are revealed by indirect immunofluorescence would indicate that the epitopes are accessible in intact, fixed HEp-2 cells and thus probably reside outside the nucleic-acid-binding domains that would be expected to be covered by RNA.