Gross Cystic Disease Fluid Protein-15(GCDFP-15)/Prolactin-Inducible Protein (PIP) as Functional Salivary Biomarker for Primary Sjögren's Syndrome.
Gross Cystic Disease Fluid Protein-15(GCDFP-15)/Prolactin-Inducible Protein (PIP) as Functional Salivary Biomarker for Primary Sjögren's Syndrome.
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DOI：
10.4172/2157-7412.1000140
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发表时间：
2013-06-15
期刊：
影响因子：
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通讯作者：
Baldini C
中科院分区：
文献类型：
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作者：
Gallo A;Martini D;Sernissi F;Giacomelli C;Pepe P;Rossi C;Riveros P;Mosca M;Alevizos I;Baldini C
Gross cystic disease fluid protein-15（GCDFP-15）/prolactin-inducible protein（PIP）是一种分泌性腺泡糖蛋白，分子量为14 KDa，我们最近通过蛋白质组学分析发现，与健康志愿者相比，原发性干燥综合征（pSS）患者唾液样本中的GCDFP-15/PIP显著降低。(1)为了验证我们先前关于大量pSS受试者中GCDFP-15/PIP蛋白减少的数据（2），将蛋白质组学结果与互补免疫测定结合，以便更好地阐明GCDFP-15/PIP在pSS外分泌体病中的病理生理学相关性（3），评估GCDFP-15/PIP的腺体表达和腺体GCDFP-15/PIP的水平，为了验证所观察到的唾液中GCDFP-15/PIP的减少是否可能与蛋白质产生的减少有关，在患者的小唾液腺（MSG）活检中检测PIP mRNA。采用SELDI-TOF-MS、2DE、MALDI-TOF-MS等多种蛋白质组学技术对123例pSS、no-SS干燥综合征患者和性别年龄匹配的健康志愿者的唾液样本进行分析。GCDFP-15/PIP的表达随后通过蛋白质印迹分析进行验证。然后对小唾液腺（MSG）活检组织中的GCDFP-15/PIP进行真实的PCR和免疫组化。通过使用互补蛋白质组学分析，我们发现16547 m/z的推定峰是能够区分患者和健康对照的pSS的最佳独立生物标志物之一，灵敏度为96%，特异性为70%，全局交叉验证误差为29%。我们鉴定了峰值为GCDFP-15/PIP蛋白，并证实了与无SS干燥症受试者和健康对照相比，pSS患者中GCDFP-15/PIP的强度显著较低（p<0.0001）。GCDFP-15/PIP表达也与唾液流速（r=0.312，p=0.023）和MSG活检病灶评分（r=-0.377，p=0.04）相关。最后，免疫组织化学证实，GCDFP-15/PIP染色在粘液腺泡中微弱，并且真实的时间PCR显示，当与无SS干燥症受试者和健康对照相比时，pSS患者中的GCDFP-15/PIP mRNA显著较低（p=0.023），从而支持以下假设：所观察到的pSS唾液中GCDFP-15/PIP的减少可能与蛋白质产生的减少有关。在这项研究中，通过不同的互补组学技术，我们证实了GCDFP-15/PIP作为pSS的新生物标志物的潜在作用。这一发现也可能在功能上很重要，因为GCDFP-15/PIP先前已被证明与水通道蛋白5（AQP 5）结合，水通道蛋白5是一种唾液腺水通道，对唾液形成至关重要，已知其在pSS中下调。探索GCDFP-15/PIP/AQP 5轴可能有助于更好地了解pSS中唾液腺功能障碍的机制。
Gross cystic disease fluid protein-15(GCDFP-15)/prolactin-inducible protein (PIP) is a secretory acinar glycoprotein of 14 KDa which we have recently described as significantly lower in salivary samples of patients with primary Sjögren’s syndrome (pSS) in comparison to healthy volunteers by proteomic analysis. (1) to validate our previous data on the decrease of GCDFP-15/PIP protein in a larger number of subjects with pSS (2) to integrate the proteomic results with complementary immunoassays in order better clarify the pathophysiological relevance of GCDFP-15/PIP in pSS exocrinopathy (3) to assess both the glandular expression of the GCDFP-15/PIP and the levels of glandular GCDFP-15/PIP mRNA in the patients’ minor salivary gland (MSG) biopsies in order to verify whether the observed reduction of GCDFP-15/PIP in saliva may be related to a decrease in the protein production. A total of 123 salivary samples from patients affected by pSS, no-SS sicca syndrome and sex- age-matched healthy volunteers were analyzed by different proteomic techniques (SELDI-TOF-MS, 2DE, MALDI-TOF-MS). The expression of GCDFP-15/PIP was then validated by western blot analysis. Real Time PCR and immunohistochemistry for GCDFP-15/PIP in the minor salivary glands (MSG) biopsies were then carried out. By using complementary proteomic analysis we found that a putative peak of 16547 m/z was among the best independent biomarkers for pSS able to discriminate between patients and healthy controls with a sensitivity of 96 % and a specificity of 70%, with a global cross validated error of 29%. We identified the peak as the GCDFP-15/PIP protein and verified that the intensity of GCDFP-15/PIP was significantly lower in pSS patients when compared to both no-SS sicca subjects and healthy controls (p<0.0001). GCDFP-15/PIP expression also correlated with both the salivary flow rate (r=0.312, p=0.023) and MSG biopsies focus score (r=−0.377, p=0.04). Finally, immunohistochemistry confirmed that GCDFP-15/PIP staining was faint in mucus acini and Real Time PCR showed that GCDFP-15/PIP mRNA was significantly lower in pSS patients when compared to both no-SS sicca subjects and healthy controls (p=0.023) thus supporting the hypothesis that the observed reduction of GCDFP-15/PIP in pSS saliva may be related to a decrease in the protein production. In this study by different complementary-omic techniques we confirmed the potential role of GCDFP-15/PIP as a novel biomarker for pSS. This finding might also be functionally important as GCDFP-15/PIP has previously been shown to bind to Aquaporin 5 (AQP5), a salivary gland water channel, critical to saliva formation that is known to be downregulated in pSS. It is likely that exploring the GCDFP-15/PIP/AQP5 axis will help better understand the mechanism of salivary gland dysfunction in pSS.