Immune recognition of lysyl-tRNA synthetase and isoleucyl-tRNA synthetase by anti-OJ antibody-positive sera

https://doi.org/10.1016/j.jaut.2021.102680Get rights and content

Highlights

  • In vitro translated recombinant isoleucyl/lysyl tRNA synthetases were used in ELISA.

  • The ELISA results correlated well with anti-OJ antibody immunoprecipitation results.

  • In vitro translated recombinants were useful for detecting anti-OJ antibodies.

Abstract

Objective

Anti-aminoacyl-tRNA synthetase (anti-ARS) antibodies are useful for identifying a clinical subset of patients with idiopathic inflammatory myopathies (IIMs). Anti-OJ antibodies, which recognize multi-enzyme synthetase complexes including isoleucyl-tRNA synthetase (IARS) and lysyl-tRNA synthetase (KARS), are among the anti-ARS antibodies. Although testing antibodies to other ARSs have been used clinically, no validated immunoassays for detecting anti-OJ antibodies are available. We aimed to establish an anti-OJ ELISA.

Methods

Serum samples were collected from 279 patients with IIMs and 22 patients with idiopathic interstitial pneumonia. Sixty-four of the samples that had been confirmed to be negative for anti-OJ by standard immunoprecipitation were used as the negative control, and 12 anti-OJ-positive reference sera were used as the positive control. Antibodies to IARS and KARS were assayed by ELISA using biotinylated recombinant proteins generated by in vitro transcription/translation.

Results

The anti-OJ-positive sera strongly reacted with the KARS and IARS recombinant proteins in ELISA. Although all 12 reference sera were positive in the anti-KARS ELISA, 4 of the 64 anti-OJ-negative sera were also weakly positive. The sensitivity and the specificity were 100% and 93.8%, respectively. Since our anti-KARS ELISA performed well, showing a high agreement with the results for immunoprecipitation (Cohen's κ > 0.8), the remaining 237 samples were also tested. Thirteen anti-KARS-positive sera were newly found by ELISA, all of which were anti-OJ positive by immunoprecipitation.

Conclusion

Immunoassays for detecting anti-OJ antibodies using KARS and IARS recombinant proteins were developed. Our ELISAs performed well, with very high agreement of the results by immunoprecipitation and can be applied to the first reliable, easy-to-use measurement assays for anti-OJ antibodies.

Introduction

Anti-aminoacyl-tRNA synthetase (anti-ARS) antibodies are myositis-specific autoantibodies (MSAs) that help to diagnose, subset, and predict complications and prognoses in idiopathic inflammatory myopathies (IIMs). Since the myositis of anti-ARS-positive patients is characterized by a unique set of non-myopathic manifestations, including interstitial lung disease (ILD), fever, Raynaud's phenomenon, mechanic's hands, and arthralgia, the patients are classified as having anti-synthetase syndrome (ASS) [1]. To date, autoantibodies have been identified to 8 kinds of ARSs, including Jo-1 (histidyl), PL-7 (threonyl), PL-12 (alanyl), EJ (glycyl), OJ (isoleucyl), KS (asparaginyl), Zo (phenylalanyl), and Ha (tyrosyl) [1].

Although immunoprecipitation assays are recognized as the gold standard for detecting each anti-ARS, they are time- and labor-intensive and require specialists to interpret the results. Recently, a number of diverse commercial immunoassays have been used for identifying MSAs, including anti-ARS antibodies [2]. However, the validation of these new assays (line-blot and dot-blot) is insufficient and their actual performances are not always favorable for certain autoantibody specificities [3,4]. An anti-ARS antibody ELISA using a mixture of five recombinant ARSs (Jo-1, PL-7, PL-12, EJ, and KS) (MESACUP™ anti-ARS test; MBL, Nagoya, Japan) was developed, was validated with an immunoprecipitation assay, and is available worldwide. Although attempts were made to include the OJ antigen (isoleucyl-tRNA synthetase, IARS) in the anti-ARS assay, the recombinant IARS protein could not be included because it was not well recognized by the anti-OJ-positive sera [5]. Unfortunately, commercially available line/dot-blots show very low specificity and sensitivity for anti-OJ [3,6].

Anti-OJ antibodies recognize a multi-enzyme synthetase complex (MSC) that contains 9 synthetases and 3 non-catalytic components [7]. Since anti-OJ-positive sera were found to inhibit the enzymatic activity of IARS most strongly in an aminoacylation assay [8], IARS has been considered as the major target of anti-OJ antibodies. The epitope(s) of anti-OJ antibodies has been thought to be dependent on the conformation of the MSC, based on the poor reactivity in immunoblotting using HeLa cell extract [8] and immunoassays using recombinant protein [5]. If this interpretation is correct, it may be possible to detect anti-OJ antibodies if proteins are used in their more native forms. The present study aimed to develop an immunoassay for detecting anti-OJ antibodies using recombinant lysyl-tRNA synthetase (KARS) protein and IARS protein expressed using an in vitro transcription and translation system.

Section snippets

Patients and sera

Serum samples were collected from 279 Japanese patients with IIM (218 with adult dermatomyositis (DM), 18 with juvenile DM (JDM), 29 with polymyositis (PM), and 14 with myositis overlap syndrome (OL)), and from 22 Japanese patients with idiopathic interstitial pneumonia (IIP). The 218 patients consisted of 110 patients with classical DM, 81 with clinically amyopathic DM (CADM), and 27 with cancer-associated DM. The male:female ratio was 92:210. One hundred seventy-one of the 301 sera were from

Components of OJ multi-enzyme complex by immunoprecipitation assays using reference sera

The anti-OJ-positive sera characteristically immunoprecipitated 3 synthetases at high molecular weights corresponding to glutamyl-proryl-tRNA synthetase (EPRS), IARS, and leucyl-tRNA synthetase (LARS), and other components of MSC (Fig. 1). All anti-OJ-positive sera immunoprecipitated the three proteins in addition to less intense bands of other components of the complex.

Development of ELISAs using recombinant IARS and KARS proteins to test for anti-OJ antibody

We previously reported an in-house ELISA to detect various MSAs [16,17]. We tested the anti-OJ positive sera using TnT

Discussion

IIMs are a group of systemic autoimmune diseases that include PM, DM, and inclusion body myopathies [22]. Several MSAs are associated with certain clinical subsets of IIM, and they are useful tools for predicting the prognosis of IIMs [22]. Anti-ARS antibodies have been found to be specific for PM and DM and to associate strongly with the complication of ILD [1]. Not only can IIM patients be positive for anti-ARS antibodies, but so can apparent IIP patients, because ILD often precedes the

Conclusion

In conclusion, we confirmed the reactivity of our recombinant proteins of KARS as well as IARS by using anti-OJ antibodies. Epitopes of anti-OJ antibodies have been considered to be largely conformation-dependent for a long time, which would make it difficult to construct conventional assays for detecting them. Although the chemical structure of our recombinants is not fully clarified, the present study clearly shows that anti-OJ antibodies can be detected using KARS or IARS proteins produced

Author statement

Y.M.: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Resources, Data curation, Writing - Original Draft, Writing - Review & Editing. Y.Y., K.Y., Y.O., K.N., T.M., S.K., A.M., M.O., T.T., Y. Todoroki: Resources, Data Curation. Y.K., M.K., Y. Tanaka: Supervision. M.S.: Methodology, Validation, Formal analysis, Investigation, Resources, Data curation, Writing - Review & Editing. M.A.: Conceptualization, Resources, Writing - Review & Editing.

Funding

This research received no specific grants from funding agencies in the public, commercial, or not-for-profit sectors.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The authors thank Dr. Koji Tamakoshi (Nagoya University Graduate School of Medicine) for his critical reading of the manuscript, and Ms. Eri Yamamoto (Nagoya University Graduate School of Medicine), Ms. Tomoko Hasegawa, and Dr. Shin Tanaka (University of Occupational and Environmental Health) for their technical assistance.

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