Purification and characterization of PLUNC from human - Tracheobronchial secretions
Purification and characterization of PLUNC from human - Tracheobronchial secretions
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DOI：
10.1165/rcmb.2003-0142oc
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发表时间：
2004-02-01
影响因子：
6.4
通讯作者：
Whitney, PL
中科院分区：
文献类型：
--
作者：
Campos, MA;Abreu, AR;Whitney, PL
为了研究分泌到气道中的蛋白质，我们使用了来自原代人气道上皮细胞的分泌物，在气液界面处重新分化，以及来自手术期间插管的患者。培养的细胞分泌物的主要蛋白质是乙醇可溶性的。这种蛋白质进行纯化，分析Edman降解，基质辅助激光解吸电离的飞行时间质谱的胰蛋白酶消化酶，和蛋白质印迹的二维电泳凝胶使用抗血清对纯化的制剂。经鉴定，该蛋白为腭、肺、鼻上皮克隆蛋白（plate-lung-nasal epithelium clone protein，PLE 1）。该蛋白质具有多个截短分子，这一模式也见于气管抽吸物。磷脂酰肌醇在水（50 μ g/ml）或50 mM NaCl中溶解性差，但在75%乙醇（> 380 μ g/ml）中更易溶解。在空气-液体界面培养的第二周期间，血小板分泌显著增加，并且随着时间的推移继续增加。免疫组化结果显示，人气道上皮和粘膜下腺体表达PLR。虽然PLP 4属于细菌宿主防御蛋白的脂多糖（LPS）结合蛋白（LBP）和杀菌/通透性增加蛋白家族，但纯化的PLP 4不能与LBP竞争结合LPS，而多粘菌素B（一种已知的LPS-LBP结合抑制剂）确实干扰结合。本研究表明plunc基因产物在体内和体外均能表达，并详细介绍了其纯化方法，同时提供了其分泌物生化特性的基本信息。
To study proteins secreted into the airway, we used secretions from primary human airway epithelial cells, re-differentiated at the air-liquid interface, and from patients intubated during surgery. A major protein of the cultured cell secretions was ethanol soluble. This protein was purified, analyzed by Edman degradation, matrix-assisted laser-desorption ionization time-of-flight mass spectroscopy of tryptic digests, and Western blots of two-dimensional electrophoresis gels using antisera against the purified preparation. The protein was identified as palate, lung, nasal epithelium clone protein (PLUNC). The protein had multiple truncated molecules, a pattern also seen in tracheal aspirates. PLUNC was poorly soluble in water (50 mug/ml) or in 50 mM NaCl but was more soluble in 75% ethanol (> 380 mug/ml). PLUNC secretion dramatically increased during the second week in air-liquid interface culture and continued to increase over time. Immunohistochemistry showed that PLUNC was expressed in human airway epithelium and submucosal glands. Although PLUNC is in the lipopolysaccharide (LPS) binding protein (LBP) and bactericidal/permeability-increasing protein family of antibacterial host defense proteins, purified PLUNC failed to compete with LBP for the binding of LPS, whereas polymyxin B, a known inhibitor of LPS-LBP binding, did interfere with binding. This study showed that plunc gene product is expressed both in vivo and in vitro, detailed a method for its purification and provided basic information on its biochemical properties in secretions.