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.

The Post-Translational Modifications of Human Salivary Peptides and Proteins Evidenced by Top-Down Platforms

“…A recent paper compared the levels of citrullinated proteins levels in the saliva of RA patients with respect to healthy controls, also studying their correlation with periodontal status and a temporomandibular joint-associated disorder [ 178 ]. Confirming previous data [ 175 ], no differences in citrullinated protein levels were found between RA patients and healthy controls, apart from the differences in periodontal status observed between groups. However, in RA, the salivary anti-cyclic citrullinated peptide levels were in correlation with periodontal disease staging, considering that the bacteria involved in its pathogenesis produce non-physiological citrullination by a family of PAD enzymes very similar to human PADs [ 179 ].…”

“…PAD 2 is the isoenzyme acting in salivary and secretory glands, as well as in other cell types and tissues, targeting myelin basic protein, C-X-C motif chemokine 10 and 11, vimentin, actin, glial fibrillary acidic protein, S100-A3, and histones H3 and H4 [ 173 ]. The importance of the detection of anti-citrullinated protein antibodies (ACPAs) in serum for the diagnosis of RA [ 173 , 175 ] has increased the interest of their application to saliva analysis for the characterization of citrullinated proteins.…”

“…Yasuda et al identified citrullinated cytokeratin 13 in saliva samples of RA patients and healthy subjects by two-dimensional electrophoresis, silver staining, and immunoprecipitation Western blotting [ 175 ]. In the same paper, the origin of the pre- or post-secretion of citrullinated cytokeratin 13 in saliva is also discussed based on calcium concentration data inside salivary gland cells and secreted saliva, since PAD enzyme catalysis is calcium-dependent.…”

Proteomics analysis of lung tissue reveals protein makers for the lung injury of adjuvant arthritis rats

“…Proteomics, which is a technique that directly quantifies and identifies all proteins in cells and tissues, is currently one of the more comprehensive and effective tools for the identification of drug therapeutic targets and diagnostic markers by the monitoring of changes in protein expression ( 17 ). It has been widely used to study the pathogenesis and diagnostic markers of certain rheumatic diseases, including RA ( 18 ), ankylosing spondylitis ( 19 ) and systemic lupus erythematosus ( 20 ). Therefore, proteomics ( 17 ) is helpful to investigate the underlying pathogenesis and the key biomarkers in RA lung injury.…”