Anti-KS: identification of autoantibodies to asparaginyl-transfer RNA synthetase associated with interstitial lung disease.

Autoantibodies to five of the aminoacyl-transfer RNA (tRNA) synthetases have been described, and each is associated with a syndrome of inflammatory myopathy with interstitial lung disease (ILD) and arthritis. Serum KS, from a patient with ILD and inflammatory arthritis without evidence of myositis, immunoprecipitated a tRNA that was distinct from that precipitated by any described anti-synthetase or other reported tRNA-related Abs, along with a protein of 65 kDa. KS serum and IgG fraction each showed significant (88%) inhibition of asparaginyl-tRNA synthetase (AsnRS) activity, but not of any of the other 19 aminoacyl-tRNA synthetase activities. Among 884 patients with connective tissue diseases tested, only two other sera were found to immunoprecipitate tRNAs and proteins of identical gel mobility. These two and KS showed identical immunodiffusion lines using HeLa cell extract. The new sera significantly inhibited AsnRS without significant effects on other synthetases tested. Both patients had ILD but neither had evidence of myositis. These data strongly suggest that these three sera have autoantibodies to AsnRS, representing a sixth anti-synthetase. Anti-KS was more closely associated with ILD than with myositis. Further study of this Abs might prove useful in dissecting the stimuli responsible for the genesis of anti-synthetase autoantibodies.

Analysis of the Mi-2 autoantigen of dermatomyositis.

OBJECTIVE: To determine the biochemical structure and antigenic components of Mi-2 autoantigen, the target of autoantibodies in 15-20% of dermatomyositis patients. METHODS: Immunoprecipitation from 35S-labeled HeLa cell extract, immunoblotting, and purification from bovine thymus by immunoaffinity chromatography. RESULTS: All 46 sera that had anti-Mi-2 autoantibodies demonstrated by immunodiffusion immunoprecipitated a major protein of approximately 240 kd. Additional proteins of 200, 150, 72, 65, 63, 50, and 34 kd appeared to be part of the antigen. Fractions of purified bovine Mi-2 with antigenic activity showed high molecular weight bands comparable with immunoprecipitated HeLa Mi-2. Twenty-four of 47 anti-Mi-2 positive sera reacted with the 240-kd protein by immunoblot against anti-Mi-2 immunoprecipitates. CONCLUSION: Mi-2 antigen consists of multiple proteins, of which the 240-kd protein appears to be the major reactive component.

Primary structure and functional expression of human Glycyl-tRNA synthetase, an autoantigen in myositis.

Two identical cDNAs encoding human glycyl-tRNA synthetase were isolated from K562 and HEL cell lambda gt11 libraries by screening with "anti-EJ" autoantibodies from a patient with dermatomyositis, previously shown to specifically inhibit this enzyme. The sequenced cDNA had 2,385 base pairs, with a predicted protein of 685 amino acids and M(r) 77,500. The antigen reactive with anti-EJ was immunoaffinity-purified from HeLa cells, and two proteolytic peptides matched areas of the predicted amino acid sequence. The sequence was 61.6% identical with that of silk moth glycyl-tRNA synthetase. Several very highly conserved regions were identified, including the single class II synthetase motif region and an N-terminal region similar to one found in at least three other synthetases. Near identity in other regions suggested that they also had important functions. Expression of the cDNA in Escherichia coli yielded a protein of expected size that reacted with the autoantibodies and was active as glycyl-tRNA synthetase.

Analysis of the specificity of anti-PM-Scl autoantibodies.

OBJECTIVE: To compare the specificity of anti-PM-Scl autoantibodies in serum samples from 43 patients with myositis, scleroderma, or both. METHODS: Anti-PM-Scl immunoprecipitates from HeLa cell extract were used as antigen for immunoblot analyses to determine the antigenic components. A series of complementary DNA fragments was expressed in Escherichia coli for immunoblot examination of the reaction with the 100-kd protein. RESULTS: The immunoblot against immunoprecipitates was sensitive and specific for detecting reactions with components of the PM-Scl antigen: 42 of 43 sera (97.7%) reacted with the 100-kd, 27 of 43 (62.8%) with the 70-kd, and 5 of 43 (11.6%) with the 37-kd protein (not previously recognized as antigenic). Forty-one sera reacted with N-terminal protein S1 (amino acids 11-437), 39 with central protein S2 (amino acids 439-749), and 24 with C-terminal protein S3 (amino acids 750-882). Of 42 sera tested, 28 (66.7%) reacted most strongly with S1, and 6 (14.3%) reacted most strongly with S2. Absorption studies implied additional, conformational epitopes not present on the bacterially expressed antigen. CONCLUSION: There was an overall similarity in reactivity to the PM-Scl antigen, but there were differences in the reactivity to the 70-kd and 37-kd proteins, as well as in the relative strength of the reactivity to the S2 protein.

Reaction of anti-OJ autoantibodies with components of the multi-enzyme complex of aminoacyl-tRNA synthetases in addition to isoleucyl-tRNA synthetase.

Autoantibodies to five aminoacyl-tRNA synthetases have been reported, and all have been associated with a syndrome of myositis and interstitial lung disease. Four of these synthetases exist free in the cytoplasm, but the fifth, isoleucyl-tRNA synthetase (recognized by anti-OJ autoantibodies), is a component of the multi-enzyme complex containing at least seven synthetases. In an effort to better understand the origins of these antibodies, we examined sera from 11 patients with anti-OJ autoantibodies for evidence of reaction with other components of the complex. All sera showed a characteristic pattern of 10 proteins bands by immunoprecipitation from HeLa cell extract. 10 of 11 sera significantly inhibited isoleucyl-tRNA synthetase enzyme activity. Serum and IgG from four patients also inhibited leucyl-tRNA synthetase activity, and serum and IgG from two inhibited lysyl-tRNA synthetase. Immunoblotting experiments supported reaction of the two sera with lysyl-tRNA synthetase, and revealed additional reactivity of three sera with a 160-kD component believed to be glutaminyl-tRNA synthetase. Despite reaction of some sera with additional synthetases, the immunoprecipitated tRNA appeared the same with all sera, and functioned as tRNA(ile). While reaction with more than one synthetase was seen with some anti-OJ sera, all synthetases targeted by anti-OJ sera were components of the complex, rather than unassociated synthetases. These findings suggest that an initial autoantibody response against isoleucyl-tRNA synthetase was followed by extension to involve other components of the synthetase complex. These observations may have implications for understanding the generation of antisynthetase autoantibodies.

Antibodies to glycyl-transfer RNA synthetase in patients with myositis and interstitial lung disease.

OBJECTIVE: We have previously described anti-EJ antibodies, and provided evidence that these antibodies react with glycyl-transfer RNA (gly-tRNA) synthetase. The aim of the present study was to identify patients with anti-EJ antibodies and describe the clinical associations of the antibody, in particular, whether it is associated with the syndrome of myositis and interstitial lung disease (ILD) that has been previously associated with autoantibodies to the aminoacyl-tRNA synthetases for histidine, threonine, and alanine. METHODS: Sera from patients with suspected or proven polymyositis or dermatomyositis (DM), sera with anticytoplasmic patterns, and control sera were tested for anti-EJ antibodies by immunoprecipitation (IPP). Positive sera and controls were tested for the ability to inhibit gly-tRNA synthetase by preincubation of the enzyme source with the serum. RESULTS: Anti-EJ antibodies were demonstrated in the sera of 5 patients, by IPP of characteristic tRNAs and protein. Original serum EJ and each of the new sera significantly inhibited the enzymatic activity of gly-tRNA synthetase but not histidyl-tRNA synthetase. All 5 of the new patients had inflammatory myopathy, a typical DM rash, and ILD. One, who had an overlap syndrome with systemic lupus erythematosus, had anti-EJ at least 4 months before the development of clinical myositis. Arthritis and Raynaud's phenomenon, other features associated with antisynthetases, were also seen. CONCLUSION: Anti-EJ is associated with the syndrome of myositis and lung disease that is seen in association with other antisynthetases. The finding of specific inhibition of gly-tRNA synthetase by all anti-EJ-positive sera strongly supports the identification of EJ antigen as gly-tRNA synthetase.