Clinical Evaluation of Autoantibodies to a Novel PM/Scl Peptide
Clinical Evaluation of Autoantibodies to a Novel PM/Scl Peptide
Anti-PM/Scl antibodies represent a specific serological marker for a subset of patients with scleroderma (Scl) and polymyositis (PM), and especially with the PM/Scl overlap syndrome (PM/Scl). Anti-PM/Scl reactivity is found in 24% of PM/Scl patients and is found in 3–10% of Scl and PM patients. The PM/Scl autoantigen complex comprises 11–16 different polypeptides. Many of those proteins can serve as targets of the anti-PM/Scl B-cell response, but most frequently the PM/Scl-100 and PM/Scl-75 polypeptides are targeted. In the present study we investigated the clinical relevance of a major alpha helical PM/Scl-100 epitope (PM1-α) using a newly developed peptide-based immunoassay and compared the immunological properties of this peptide with native and recombinant PM/Scl antigens. In a technical comparison, we showed that an ELISA based on the PM1-α peptide is more sensitive than common techniques to detect anti-PM/Scl antibodies such as immunoblot, indirect immunofluorescence on HEp-2 cells and ELISA with recombinant PM/Scl polypeptides. We found no statistical evidence of a positive association between anti-PM1-α and other antibodies, with the exception of known PM/Scl components. In our cohort a negative correlation could be found with anti-Scl-70 (topoisomerase I), anti-Jo-1 (histidyl tRNA synthetase) and anti-centromere proteins. In a multicenter evaluation we demonstrated that the PM1-α peptide represents a sensitive and reliable substrate for the detection of a subclass of anti-PM/Scl antibodies. In total, 22/40 (55%) PM/Scl patients, 27/205 (13.2%) Scl patients and 3/40 (7.5%) PM patients, but only 5/288 (1.7%) unrelated controls, tested positive for the anti-PM1-α peptide antibodies. These data indicate that anti-PM1-α antibodies appear to be exclusively present in sera from PM/Scl patients, from Scl patients and, to a lesser extent, from PM patients. The anti-PM1-α ELISA thus offers a new serological marker to diagnose and discriminate different systemic autoimmune disorders.
Systemic autoimmune diseases such as scleroderma (Scl), polymyositis (PM), rheumatoid arthritis, systemic lupus erythematosus (SLE) and mixed connective tissue disease are characterized by the occurrence of circulating antibodies to defined intracellular targets. Some of these autoantibodies represent useful diagnostic markers for a variety of systemic autoimmune diseases.
Antibodies targeting the PM/Scl complex serve as a marker for the PM/Scl overlap syndrome, where they are found in 24% of sera, but they are also seen in 8% of PM patients and in 3% of Scl patients. The PM/Scl complex was identified as the human counterpart of the yeast exosome and consists of 11–16 polypeptides with molecular masses ranging from 20 to 110 kDa. PM/Scl-100, the human equivalent of the yeast Rrp6p, has been cloned by two independent groups and its key function during the 5.8 S rRNA end formation has been described.
In previous studies, the human immune response targeting the PM/Scl complex has been reported to be predominantly directed against two polypeptides with apparent molecular masses of 100 kDa and 75 kDa. In the past it has been shown that nearly all PM/Scl-positive sera contain autoantibodies to the 100 kDa protein and that only about 50–60% react with the 75 kDa protein. A more recent study has shown that the PM/Scl-75 protein contains a previously unidentified N-terminal region that is important for the antigenicity of the protein. The reactivity of sera with this new isoform of PM/Scl-75c is similar to the conventional PM/Scl-100 protein. Several other components of the human exosome, including hRrp4p, hRrp40p, hRrp41, hRrp42p, hRrp46p and hCsl4p, are also recognized by anti-PM/Scl antibodies, but to a lesser extent.
In several studies during the past decade, we and others have attempted to identify the epitopes on PM/Scl-100 that are recognized by the cognate autoantibodies. The prime reactivity of anti-PM/Scl-100 sera was localized to a domain of the protein represented by amino acids 231–245 using membrane-bound peptide arrays. The amino acids contributing to the antibody binding were identified by mutational analysis. Based on these observations and on secondary structure predictions, a local alpha-helical structure has been proposed for this major PM/Scl-100 epitope.
The aim of this study was to develop an ELISA with a 15-mer peptide comprising the PM/Scl-100 major epitope as a substrate, and to evaluate its sensitivity and specificity for the detection of anti-PM/Scl antibodies.
Anti-PM/Scl antibodies represent a specific serological marker for a subset of patients with scleroderma (Scl) and polymyositis (PM), and especially with the PM/Scl overlap syndrome (PM/Scl). Anti-PM/Scl reactivity is found in 24% of PM/Scl patients and is found in 3–10% of Scl and PM patients. The PM/Scl autoantigen complex comprises 11–16 different polypeptides. Many of those proteins can serve as targets of the anti-PM/Scl B-cell response, but most frequently the PM/Scl-100 and PM/Scl-75 polypeptides are targeted. In the present study we investigated the clinical relevance of a major alpha helical PM/Scl-100 epitope (PM1-α) using a newly developed peptide-based immunoassay and compared the immunological properties of this peptide with native and recombinant PM/Scl antigens. In a technical comparison, we showed that an ELISA based on the PM1-α peptide is more sensitive than common techniques to detect anti-PM/Scl antibodies such as immunoblot, indirect immunofluorescence on HEp-2 cells and ELISA with recombinant PM/Scl polypeptides. We found no statistical evidence of a positive association between anti-PM1-α and other antibodies, with the exception of known PM/Scl components. In our cohort a negative correlation could be found with anti-Scl-70 (topoisomerase I), anti-Jo-1 (histidyl tRNA synthetase) and anti-centromere proteins. In a multicenter evaluation we demonstrated that the PM1-α peptide represents a sensitive and reliable substrate for the detection of a subclass of anti-PM/Scl antibodies. In total, 22/40 (55%) PM/Scl patients, 27/205 (13.2%) Scl patients and 3/40 (7.5%) PM patients, but only 5/288 (1.7%) unrelated controls, tested positive for the anti-PM1-α peptide antibodies. These data indicate that anti-PM1-α antibodies appear to be exclusively present in sera from PM/Scl patients, from Scl patients and, to a lesser extent, from PM patients. The anti-PM1-α ELISA thus offers a new serological marker to diagnose and discriminate different systemic autoimmune disorders.
Systemic autoimmune diseases such as scleroderma (Scl), polymyositis (PM), rheumatoid arthritis, systemic lupus erythematosus (SLE) and mixed connective tissue disease are characterized by the occurrence of circulating antibodies to defined intracellular targets. Some of these autoantibodies represent useful diagnostic markers for a variety of systemic autoimmune diseases.
Antibodies targeting the PM/Scl complex serve as a marker for the PM/Scl overlap syndrome, where they are found in 24% of sera, but they are also seen in 8% of PM patients and in 3% of Scl patients. The PM/Scl complex was identified as the human counterpart of the yeast exosome and consists of 11–16 polypeptides with molecular masses ranging from 20 to 110 kDa. PM/Scl-100, the human equivalent of the yeast Rrp6p, has been cloned by two independent groups and its key function during the 5.8 S rRNA end formation has been described.
In previous studies, the human immune response targeting the PM/Scl complex has been reported to be predominantly directed against two polypeptides with apparent molecular masses of 100 kDa and 75 kDa. In the past it has been shown that nearly all PM/Scl-positive sera contain autoantibodies to the 100 kDa protein and that only about 50–60% react with the 75 kDa protein. A more recent study has shown that the PM/Scl-75 protein contains a previously unidentified N-terminal region that is important for the antigenicity of the protein. The reactivity of sera with this new isoform of PM/Scl-75c is similar to the conventional PM/Scl-100 protein. Several other components of the human exosome, including hRrp4p, hRrp40p, hRrp41, hRrp42p, hRrp46p and hCsl4p, are also recognized by anti-PM/Scl antibodies, but to a lesser extent.
In several studies during the past decade, we and others have attempted to identify the epitopes on PM/Scl-100 that are recognized by the cognate autoantibodies. The prime reactivity of anti-PM/Scl-100 sera was localized to a domain of the protein represented by amino acids 231–245 using membrane-bound peptide arrays. The amino acids contributing to the antibody binding were identified by mutational analysis. Based on these observations and on secondary structure predictions, a local alpha-helical structure has been proposed for this major PM/Scl-100 epitope.
The aim of this study was to develop an ELISA with a 15-mer peptide comprising the PM/Scl-100 major epitope as a substrate, and to evaluate its sensitivity and specificity for the detection of anti-PM/Scl antibodies.
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