Department of Rheumatology, Tokyo Medical University Hospital, Tokyo, Japan.
PLoS One. 2022 Mar 23;17(3):e0265687. doi: 10.1371/journal.pone.0265687. eCollection 2022.
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.
黏膜中针对瓜氨酸化肽的免疫反应被认为在类风湿关节炎（RA）前发病期向临床明显 RA 的转变中发挥重要作用。虽然有报道表明唾液中存在抗瓜氨酸化肽抗体，但很少有研究报告在人唾液中检测到瓜氨酸化肽。本研究旨在鉴定人唾液中的瓜氨酸化肽，并探讨其临床意义。收集了 11 例 RA 患者和 20 例健康个体的唾液样本。使用抗修饰瓜氨酸（AMC）抗体检测瓜氨酸化肽。对健康个体的唾液进行二维蛋白质电泳以分离瓜氨酸化肽，然后通过基质辅助激光解吸/电离飞行时间质谱和肽质量指纹图谱分析质谱进行分析。通过免疫沉淀（IP）-western 印迹验证结果。定量分析与抗角蛋白 13（CK13）和 AMC 抗体沉淀的条带的信号强度。用 AMC 抗体产生的条带的信号强度比除以用抗 CK13 抗体产生的条带的信号强度，计算瓜氨酸化 CK13（Cit-CK13）比值。在健康个体的唾液中检测到一个对应分子量约为 50 kDa 的瓜氨酸化肽带，并通过质谱和 IP-western 印迹鉴定为 CK13。RA 患者和健康参与者的唾液 Cit-CK13 比值无显著差异（p = 0.605）。这是第一项表明 Cit-CK13 存在于人唾液中的研究，并且 RA 患者和健康个体的 Cit-CK13 比值无显著差异，表明唾液 Cit-CK13 含量与 RA 发展之间可能没有关联。Cit-CK13 在口腔中的生理和病理作用及其对黏膜免疫的反应性仍不清楚，将成为进一步研究的课题。
