Research paperAnti-SAE antibodies in autoimmune myositis: Identification by unlabelled protein immunoprecipitation in an Italian patient cohort☆
Highlights
► A non radiolabelled immunoprecipitation for anti-SAE antibody detection was set up. ► The method's specificity was confirmed by immunoblotting on immunoprecipitates. ► Sera from five patients, affected with dermatomyositis, resulted anti-SAE positive. ► Clinical features in anti-SAE positive or negative adult DM patients were compared. ► Anti-SAE positive patients had mainly skin and muscle manifestations.
Introduction
Polymyositis (PM) and dermatomyositis (DM) are autoimmune idiopathic inflammatory myopathies (IIM) occurring with heterogeneous clinical features. The clinical criteria of Bohan and Peter, 1975a, Bohan and Peter, 1975b are still used for classification of the disease; however, the identification of autoantibodies in patient sera can help to define some disease subsets with distinctive clinical manifestations. Autoantibodies found in patients with autoimmune myositis can be schematically distinguished into myositis specific antibodies (MSAs) and myositis associated antibodies (MAAs). MSAs are highly selective and usually mutually exclusive and are directed against cytoplasmic or nuclear components involved in key regulatory intracellular processes, including protein synthesis, gene transcription and protein translocation. The three best defined antibody specificities target mainly the cytoplasmic enzymes aminoacyl-tRNA synthetases (ARS) (Zampieri et al., 2005), the nuclear helicase protein Mi-2 (Ghirardello et al., 2005a, Hengstman et al., 2006b), and the cytoplasmic complex signal recognition particle (SRP) (Brouwer et al., 2001, Hengstman et al., 2006a).
Anti-Mi-2 is the autoantibody most clearly associated with DM; however, during the last few years novel autoantibodies have been described, above all in DM, thus contributing to a better characterization of MSA profile (Ghirardello et al., 2010, Mammen, 2010). The newly identified DM specific autoantibodies are anti-p155/140, anti-CADM-140, anti-MJ and anti-SAE (Small ubiquitin-like modifier activating enzyme) (Ghirardello et al., 2011, Gunawardena et al., 2008a). Anti-p155/140 autoantibodies were observed both in adult and juvenile DM (Chinoy et al., 2007, Kaji et al., 2007, Targoff et al., 2006) where they were associated with severe skin involvement; in adult, anti-p155/140 autoantibodies were associated with cancer (Gunawardena et al., 2008b). The target antigen is the nuclear protein TIF1-γ, participating in transcription regulation, cell growth control and cell differentiation (Yan et al., 2004). Anti-MJ reactivity was found exclusively in the juvenile form of DM and was associated with subcutaneous calcinosis (Espada et al., 2009, Gunawardena et al., 2009b, Guseinova et al., 2011, Marco Puche et al., 2010, Mathiesen et al., 2010). Its target antigen, NXP-2, is involved in RNA metabolism and in nuclear maintenance architecture (Kimura et al., 2002). Anti-CADM-140 antibodies were found in clinically amyopathic DM and were associated with acute, rapidly progressive interstitial lung disease (ILD) (Sato et al., 2005, Sato et al., 2009). They are directed against the RIG-I-like receptor IFIH1/MDA5 (Melanoma Differentiation-Associated Gene 5), which plays an important role in innate immune responses against RNA viruses (Nakashima et al., 2010, Yoneyama et al., 2010, Zampieri et al., 2006).
Anti-SAE antibodies were first identified in a preliminary study by Betteridge et al., 2007, and the same group then confirmed and characterized these autoantibodies in a larger cohort of IIM patients from United Kingdom (Betteridge et al., 2009). Screening of sera from IIM patients by radiolabelled 35S immunoprecipitation revealed anti-SAE autoantibodies exclusively in adult DM, with a prevalence of 8%. The target antigen of anti-SAE antibodies is the Small Ubiquitin-like MOdifier (SUMO) Activating Enzyme, composed of two subunits SAE1 and SAE2 of apparent molecular weight of 40 and 90 kDa, respectively. SUMO is a small protein, structurally similar to ubiquitin, involved in post-translational modification of numerous target proteins by a process termed “sumoylation.” By binding to the target proteins, SUMO forms stable conjugates and mediates various cell processes, including gene transcription, nucleocytoplasmic transport, subcellular compartmentalization, genome stability (Desterro et al., 1999, Wilkinson and Henley, 2010).
The gold standard technique for MSA detection in patient sera is protein immunoprecipitation (IP) from radiolabelled cell extracts. The advantages of IP include a higher sensitivity and specificity than other laboratory tests. Moreover the procedure detects antigens in their native conformation and also allows the precipitation of molecular components complexed with the target antigen (Tan and Peebles, 1993). However, radioisotopes are used only in equipped laboratories and under rigorous control. The aim of this study was to set up an IP technique using non radiolabelled cell extracts for autoantibody identification in the sera of myositis patients, in order to determine any MSA undetectable by currently used methods. In particular, non radiolabelled IP has been applied to the detection of anti-SAE antibodies in our myositis cohort.
Section snippets
Materials and methods
Sera from 130 patients with autoimmune myositis (75 adult DM and 12 juvenile DM, 43 PM), 30 healthy subjects and 53 disease controls with other connective tissue diseases (22 systemic lupus erythematosus, 10 rheumatoid arthritis, 9 psoriatic arthritis, 7 Sjogren's syndrome, 3 systemic sclerosis, 1 undifferentiated connective tissue disease, 1 mixed connective tissue disease) were screened by an in-house unlabelled IP. In the same sera MSAs and MAAs were routinely determined by immunoblotting and
Detection of autoantibodies in the sera by unlabelled protein IP
Reference positive sera with known antibody reactivity were tested by unlabelled protein IP to evaluate the method's analytical specificity. Sera positive for anti-histidyl, anti-alanyl, and anti-isoleucyl-tRNA synthetase (Jo-1, PL12 and OJ, respectively), anti-Ro/SSA, anti-La/SSB and anti-Ku immunoprecipitated the respective autoantigens, whereas sera from healthy controls did not immunoprecipitate any specific antigen (Fig. 1). The method was not optimal or inadequate for the identification
Discussion
This report describes the identification of anti-SAE antibodies in an Italian myositis patient cohort. We set up a non radiolabelled protein IP technique for identification of anti-SAE antibodies in the sera of myositis patients. This immunological technique can be considered a valuable alternative to radiolabelled IP and a promising analytical approach for the detection of MSAs or MAAs in patient sera. Using our protocol we were unable to identify some antibody reactivities, probably due to
Competing interests
The authors have declared no conflict of interest.
Acknowledgments
This study was supported by a grant from “Ministero dell'istruzione, dell'Università e della Ricerca” [Grant number 60A07-1030/09], Italy.
We thank Mr. Fabrizio Milani for his technical assistance.
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2022, Handbook of Systemic Autoimmune DiseasesCitation Excerpt :The prevalence of ILD in patients with anti-ARS is around 80%, whereas up to 50% of patients with anti-MDA5 from Western countries and almost 75% from Asian countries develop this complication [27–29]. Anti-SAE has also been related to a high ILD rate in Asian patients (50%–70%) [30,31], while this association seems weaker in Western countries (0%–18%) [32,33]. Conversely, ILD is extremely rare in patients with other MSAs, such as anti-TIF1ɣ, anti-HMGCR, anti-Mi2, and anti-SRP [26].
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Funding: This work was supported by “Ministero dell'Istruzione e della Ricerca,” grant no. 60A07-2184/10, Italy. The founding source had no involvement in the conduction of the research and/or preparation of the article.