Immune recognition of lysyl-tRNA synthetase and isoleucyl-tRNA synthetase by anti-OJ antibody-positive sera
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.
References (49)
- et al.
Idiopathic inflammatory myopathies and the anti-synthetase syndrome: a comprehensive review
Autoimmun. Rev.
(2014) - et al.
Autoantibodies in idiopathic inflammatory myopathies: clinical associations and laboratory evaluation by mono- and multispecific immunoassays
Autoimmun. Rev.
(2019) - et al.
Anti-OJ autoantibodies: rare or underdetected?
Autoimmun. Rev.
(2019) - et al.
Autoantibodies to Su/Argonaute 2 in Japanese patients with inflammatory myopathy
Clin. Chim. Acta
(2017) Would a new name hasten the acceptance of amyopathic dermatomyositis (dermatomyositis siné myositis) as a distinctive subset within the idiopathic inflammatory dermatomyopathies spectrum of clinical illness?
J. Am. Acad. Dermatol.
(2002)- et al.
Interstitial lung disease with autoantibodies against aminoacyl-tRNA synthetases in the absence of clinically apparent myositis
Semin. Arthritis Rheum.
(1996) - et al.
UKMyonet contributors, Frequency, mutual exclusivity and clinical associations of myositis autoantibodies in a combined European cohort of idiopathic inflammatory myopathy patients
J. Autoimmun.
(2019) - et al.
Clinical characteristics of patients with anti-aminoacyl-tRNA synthetase antibody positive idiopathic interstitial pneumonia
Respir. Med.
(2017) - et al.
Testing for myositis specific autoantibodies: comparison between line blot and immunoprecipitation assays in 57 myositis sera
J. Immunol. Methods
(2016) Selecting an appropriate method for expressing a recombinant protein
Methods Enzymol.
(2009)