Anti-SAE antibodies in autoimmune myositis: Identification by unlabelled protein immunoprecipitation in an Italian patient cohort

Abstract

Introduction

Myositis specific autoantibodies (MSAs) are useful in the diagnosis of idiopathic inflammatory myopathies and in the definition of disease subsets. The aim of this study was to set up an unlabelled protein immunoprecipitation technique for MSA identification in the sera of myositis patients, in order to identify and investigate new antibody reactivity, undetectable by currently used methods.

Methods

Sera of 183 patients with connective tissue diseases (75 adult dermatomyositis, 12 juvenile dermatomyositis, 43 polymyositis, 53 other connective tissue diseases) and 30 healthy controls were screened by an in-house procedure of unlabelled protein immunoprecipitation. In the same sera MSAs and myositis associated antibodies were determined by immunoblotting and immunoprecipitation for RNA.

Results

The analytical specificity of unlabelled protein immunoprecipitation was demonstrated by testing reference sera with known antibody reactivity.

Sera from five patients, affected with dermatomyositis (5/75 = 7%), immunoprecipitated two proteins of 40 and 90 kDa apparent molecular weights respectively, consistent with the subunits of the small ubiquitin like modifier activating enzyme heterodimer (SAE1/SAE2). The identity of putative SAE immunoprecipitated proteins was confirmed by immunoblotting on immunoprecipitates using commercial monospecific antibodies to SAE1 and SAE2. Major clinical features were compared between anti-SAE positive and negative patients. Interestingly, anti-SAE positive patients had mainly skin and muscle manifestations while dysphagia, interstitial lung disease, arthritis and constitutional symptoms were absent.

Conclusions

Unlabelled protein immunoprecipitation is a specific analytical approach, appropriate for the identification of the recently described anti-SAE autoantibody. We confirmed the role of anti-SAE antibody as marker of dermatomyositis.

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.