Purification and characterization of defensins from cystic fibrosis sputum
Purification and characterization of defensins from cystic fibrosis sputum
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DOI：
10.1007/s000110050114
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发表时间：
1997-03-01
影响因子：
6.7
通讯作者：
Caughey, GH
中科院分区：
文献类型：
--
作者：
Soong, LB;Ganz, T;Caughey, GH
目的和设计：囊性纤维化（CF）痰液中含有大量的中性粒细胞，其最丰富的颗粒蛋白是防御素。在吞噬溶酶体内，防御素杀死微生物;然而，细胞外防御素对人类细胞是有毒的。为了开始探索防御素损害CF气道的可能性，本研究探讨了CF sputum.Materials中防御素的浓度和性质：作为生物材料的来源，其中检测防御素的水平，脓痰收集从人CF住院支气管炎恶化。方法：采用免疫分析法测定CF痰液中无细胞成分中防御素的含量。从痰液中酸提防御素，经Sephacryl S-200凝胶过滤层析和反相高效液相色谱（HPLC）纯化。CF防御素的毒性通过将纯化的防御素与在无血清medium.Results中培养的CF气管细胞系孵育来测试：在5名CF患者中，痰防御素水平范围从300到> 1600 μ g/ml，高于先前报道的任何人分泌物或流体中的水平，并且大大超过体外对哺乳动物细胞有毒的浓度。通过酸性尿素-PAGE和N-末端测序判断，HPCL-纯化的CF痰防御素是纯的，其显示防御素-1、-2和-3以类似于4：2：1的比例的混合物。在> 10 μ g/ml的纯化的混合物是有毒的CF气管细胞培养在无血清medium.Conclusions细胞数量的减少和增加的渗透性来判断：这项研究表明，CF痰中的防御素是完整的，足够丰富，他们可能会损害气道上皮细胞。
Objective and Design: Cystic fibrosis (CF) sputum contains large numbers of neutrophils whose most abundant granule proteins are defensins. Within phagolysosomes, defensins kill microbes; however, extracellular defensins can be toxic to human cells. To begin to explore the possibility that defensins damage CF airways, this study examines the concentration and properties of defensins in CF sputum.Materials: As a source of biological material in which to assay levels of defensins, purulent sputum was collected from persons with CF hospitalized with exacerbations of bronchitis. Purulent CF sputum was also a source of material for purification of defensins.Methods: Defensin concentration in the cell-free component of CF sputum was measured by immunoassay. Defensins acid-extracted from sputum were purified by Sephacryl S-200 gel filtration chromatography and reverse phase-high pressure liquid chromatography (HPLC). Toxicity of CF defensins was tested by incubating the purified defensins with a line of CF tracheal cells cultured in serum-free medium.Results: In 5 patients with CF, sputum defensin levels ranged from 300 to > 1600 mu g/ml, higher than levels previously reported in any human secretion or fluid and greatly exceeding concentrations toxic to mammalian cells in vitro. HPCL-purified CF sputum defensins were pure as judged by acid urea-PAGE and N-terminal sequencing, which revealed a mixture of defensins-1, -2 and -3 at ratios of similar to 4:2:1. At > 10 mu g/ml the purified mixture was toxic for a line of CF tracheal cells cultured in serum-free medium, as judged by reductions in cell numbers and increased permeability to trypan blue.Conclusions: This study suggests that defensins in CF sputum are intact and sufficiently abundant that they may damage airway epithelium.