Identification of human urinary trefoil factor 1 as a novel calcium oxalate crystal growth inhibitor
Identification of human urinary trefoil factor 1 as a novel calcium oxalate crystal growth inhibitor
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DOI：
10.1172/jci25342
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发表时间：
2005-12-01
影响因子：
15.9
通讯作者：
Thongboonkerd, V
中科院分区：
文献类型：
--
作者：
Chutipongtanate, S;Nakagawa, Y;Thongboonkerd, V
以前对抑制肾结石形成的蛋白质的研究已经确定了相对少量的特征良好的抑制剂。鉴别其他结石抑制剂将增加对肾结石发病机制和病理生理学的了解。我们结合了传统的。生化的方法与质谱（MS）的最新进展，以确定一种新的草酸钙（CaOx）晶体生长抑制剂在正常人尿液。阴离子蛋白通过DEAE吸附分离，并通过HiLoad 16/60 Superdex 75凝胶过滤分离。通过阴离子交换色谱分离纯化了一个对CaOx晶体生长具有有效抑制活性的组分。保留抑制活性的2个亚组分中的蛋白质通过基质辅助激光解吸/电离飞行时间MS和电喷雾电离四极杆飞行时间串联MS鉴定为人三叶因子1（TFF 1）。Western blot分析证实了质谱蛋白鉴定。对尿TFF 1的功能研究表明，其抑制效力与肾钙蛋白相似。尿TFF 1的抑制活性是剂量依赖性的，并被TFF 1抗血清抑制。抗C-末端抗体特别有效，与我们提出的模型一致，其中TFF 1的4个C-末端谷氨酸残基与钙离子相互作用以防止CaOx晶体生长。特发性CaOx肾结石患者尿液中TFF 1的浓度和相对量显著低于对照组（浓度为2.5倍，相对量为5- 22倍）。这些数据表明，TFF 1是一种新的有效的CaOx晶体生长抑制剂，在肾结石的潜在病理生理作用。
Previous research on proteins that inhibit kidney stone formation has identified a relatively small number of well-characterized inhibitors. Identification of additional stone inhibitors would increase understanding of the pathogenesis and patbophysiology of nephrolithiasis. We have combined conventional. biochemical. methods with recent advances in mass spectrometry (MS) to identify a novel calcium oxalate (CaOx) crystal growth inhibitor in normal human urine. Anionic proteins were isolated by DEAE adsorption and separated by HiLoad 16/60 Superdex 75 gel filtration. A fraction with potent inhibitory activity against CaOx crystal growth was isolated and purified by anion exchange chromatography. The protein in 2 subfractions that retained inhibitory activity was identified by matrix-assisted laser desorption/ionization-time-of-flight MS and electrospray ionizadon-quadrupole-time-of-flight tandem MS as human trefoil factor 1 (TFF1). Western blot analysis confirmed the mass spectrometric protein identification. Functional studies of urinary TFF1 demonstrated that its inhibitory potency was similar to that of nephrocalcin. The inhibitory activity of urinary TFF1 was dose dependent and was inhibited by TFF1 antisera. Anti-C-terminal antibody was particularly effective, consistent with our proposed model in which the 4 C-terminal glutamic residues of TFF1 interact with calcium ions to prevent CaOx crystal growth. Concentrations and relative amounts of TFF1 in the urine of patients with idiopathic CaOx kidney stone were significantly less (2.5-fold for the concentrations and 5- to 22-fold for the relative amounts) than those found in controls. These data indicate that TFF1 is a novel potent CaOx crystal growth inhibitor with a potential pathophysiological role in nephrolithiasis.