Identification of myeloperoxidase, α-defensin and calgranulin in calcium oxalate renal stones
Identification of myeloperoxidase, α-defensin and calgranulin in calcium oxalate renal stones
复制标题
DOI：
10.1016/j.cca.2007.05.015
复制
发表时间：
2007-09-01
影响因子：
5
通讯作者：
Shafqat, Jawed
中科院分区：
文献类型：
--
作者：
Mushtaq, Shamim;Siddiqui, Anwar Ali;Shafqat, Jawed
背景：为了了解结石形成的机制，确定构成尿路结石的大分子的特征是必不可少的。对草酸钙(CaOx)肾结石的内、外基质蛋白质进行了鉴定。方法：分离CaOx肾结石的内、外基质，用含有十二烷基硫酸钠和β-巯基乙醇的缓冲液提取蛋白质。分别用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法和反相高效液相色谱法对蛋白质进行分析和纯化。用MALDI TOF质谱仪对凝胶和蛋白质组分的蛋白质条带进行了测序。采用ELISA法、免疫印迹法和狭缝印迹法对结石患者的结石和尿液中的蛋白质进行鉴定。通过聚集仪观察其对CaOx结晶的影响，评价了所鉴定的蛋白质作为有效的促进剂或抑制剂的潜力。结果：内核提取物主要以相对分子质量在12-14 kDa范围内的蛋白质种类为主。然而，在结石形成者和非结石形成者的内核和外基质以及尿液中也检测到一条66 kDa的条带，被鉴定为骨桥蛋白。采用反相高效液相色谱法对低相对分子质量蛋白质进行纯化。串联质谱仪鉴定为髓过氧化物酶A(MPO-A)、α-防御素和钙颗粒蛋白。EL ISA、Western印迹和狭缝印迹免疫分析进一步证实它们仅存在于核内，而不存在于外基质中。浊度分析显示低分子量肾结石蛋白促进CaOx晶体聚集。结论：持续性高草酸尿导致肾小管上皮细胞损伤，导致这些抗炎蛋白的释放。这些蛋白质可能首先被吸附在CaOx晶体上，从而成为成核过程的一部分，从而导致内部基质的形成。(C)2007 Elsevier B.V.保留所有权利。
Background: In order to understand the mechanism of stone genesis, it is essential to determine the characteristics of macromolecules constituting the urinary stones. We characterized proteins from the inner core and outer matrix of calcium oxalate (CaOx) renal stones.Methods: Inner core and outer matrix of CaOx renal stones were separated and proteins were extracted with a buffer containing SDS and beta-mercaptoethanol. Proteins were analyzed and purified by SDS-PAGE and RP-HPLC respectively. The protein bands from gel and protein fractions were sequenced by MALDI TOF mass spectrometry. ELISA, western and slot blot immunoassays were performed to confirm the identity of the proteins in stones and urine of the stone formers. The potential of the identified protein as an effective promoter or inhibitor was assessed by observing their effects on CaOx crystallization using aggregometer.Results: The inner core extract predominantly exhibited protein species in the molecular weight range of 12-14 kDa. However, a 66 kDa band, identified as osteopontin was also detected in the inner core along with outer matrix and in the urine of stone formers and non stone formers. Purification of low molecular weight proteins was carried out by reversed phase HPLC. Tandem mass spectrometry analysis identified them as myeloperoxidase chain A (MPO-A), alpha-defensin, and calgranulin. ELISA, western blot and slot-blot immuno-assays further confirmed their presence restricted to the inner core and not in the outer matrix. Turbidity assays showed that low molecular weight renal stone proteins promoted the aggregation of CaOx crystals.Conclusions: Persistent hyperoxaluria leads to tubular epithelial injury, resulting in the release of these anti-inflammatory proteins. These proteins could have been first adsorbed on CaOx crystals thereby become a part of nucleation process leading to inner matrix formation. (c) 2007 Elsevier B.V. All rights reserved.