Detection of salivary citrullinated cytokeratin 13 in healthy individuals and patients with rheumatoid arthritis by proteomics analysis

Abstract

The immune response to citrullinated peptides in the mucosa has been suggested to play an important role in the transition from pre-onset rheumatoid arthritis (RA) to clinically evident RA. Although there are reports indicating the presence of anti-citrullinated peptide antibodies in the saliva, few studies have reported citrullinated peptide detection in human saliva. This study aimed to identify citrullinated peptides in human saliva and discuss their clinical significance. Saliva samples were collected from 11 patients with RA and from 20 healthy individuals. Citrullinated peptides were detected using an anti-modified citrulline (AMC) antibody. Saliva from the healthy individuals was subjected to two-dimensional protein electrophoresis to isolate citrullinated peptides, which were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and mass spectrometry by peptide mass fingerprinting. The results were corroborated by immunoprecipitation (IP)-western blotting. The signal intensities of the bands precipitated with anti-cytokeratin 13 (CK13) and AMC antibodies were quantified. The signal intensity ratio of the band produced by the AMC antibody was divided by that of the band produced by the anti-CK13 antibody to calculate the citrullinated CK13 (Cit-CK13) ratio. A citrullinated peptide band corresponding to a molecular weight of approximately 50 kDa was detected in the saliva of healthy individuals, and identified as CK13 via mass spectrometry and IP-western blotting. No significant difference was observed between the salivary Cit-CK13 ratios of patients with RA and healthy participants (p = 0.605). This is the first study to show that Cit-CK13 is present in human saliva, and that there is no significant difference between the Cit-CK13 ratios of patients with RA and healthy individuals, suggesting that salivary Cit-CK13 content and RA development may not be associated. The physiological and pathological roles of Cit-CK13 in the oral cavity, and its responsiveness to mucosal immunity, remain unknown and will be the subject of further investigation.