Isolation and characterization of a novel trypsin-like protease found in rat bronchiolar epithelial Clara cells. A possible activator of the viral fusion glycoprotein.
Isolation and characterization of a novel trypsin-like protease found in rat bronchiolar epithelial Clara cells. A possible activator of the viral fusion glycoprotein.
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DOI：
10.1016/s0021-9258(18)42250-8
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发表时间：
1992-07
期刊：
影响因子：
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通讯作者：
H. Kido;Y. Yokogoshi;K. Sakai;M. Tashiro;Y. Kishino;A. Fukutomi;N. Katunuma
中科院分区：
文献类型：
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作者：
H. Kido;Y. Yokogoshi;K. Sakai;M. Tashiro;Y. Kishino;A. Fukutomi;N. Katunuma
用一系列标准层析方法从大鼠肺组织中纯化得到一种与大鼠细支气管上皮Clara细胞相关的新型胰蛋白酶样蛋白酶，命名为Tryptase Clara。该酶在凝胶过滤上的表观分子量为180 +/- 16 kDa，在还原条件下十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上的表观分子量为30 +/- 1.5 kDa。其等电点为pH 4.75。对模型肽底物的研究表明，该酶优先识别单个精氨酸切割位点，最有效地切割Boc-Gln-Ala-Arg-4-甲基香豆酰-7-酰胺，并且该底物的最适pH为7.5。该酶被抑肽酶、二异丙基氟磷酸盐、抗痛剂、亮抑酶肽和Kunitz型大豆胰蛋白酶抑制剂强烈抑制，但仅被Bowman-Birk大豆胰蛋白酶抑制剂、苯甲脒和α 1-抗胰蛋白酶轻微抑制。免疫组织化学研究表明，该酶是专门位于细支气管上皮克拉拉细胞和共定位与表面活性剂。免疫反应性蛋白质的分子量为28.5 kDa的气道分泌物中也检测到蛋白质印迹分析，这表明30 kDa的蛋白酶在克拉拉细胞的处理之前或之后，其分泌。流感病毒血凝素的蛋白水解裂解是病毒变得具有传染性的先决条件。类胰蛋白酶Clara显示出以剂量依赖性方式切割血凝素并激活甲型流感病毒的感染性。这些结果表明，该酶是一种可能的激活剂的非活性病毒融合糖蛋白在呼吸道，从而负责肺致病性的病毒。
A novel trypsin-like protease associated with rat bronchiolar epithelial Clara cells, named Tryptase Clara, was purified to homogeneity from rat lung by a series of standard chromatographic procedures. The enzyme has apparent molecular masses of 180 +/- 16 kDa on gel filtration and 30 +/- 1.5 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Its isoelectric point is pH 4.75. Studies with model peptide substrates showed that the enzyme preferentially recognizes a single arginine cleavage site, cleaving Boc-Gln-Ala-Arg-4-methylcoumaryl-7-amide most efficiently and having a pH optimum of 7.5 with this substrate. The enzyme is strongly inhibited by aprotinin, diisopropylfluorophosphate, antipain, leupeptin, and Kunitz-type soybean trypsin inhibitor, but inhibited only slightly by Bowman-Birk soybean trypsin inhibitor, benzamidine, and alpha 1-antitrypsin. Immunohistochemical studies indicated that the enzyme is located exclusively in the bronchiolar epithelial Clara cells and colocalized with surfactant. An immunoreactive protein with a molecular mass of 28.5 kDa was also detected in airway secretions by Western blotting analyses, suggesting that the 30-kDa protease in Clara cells is processed before or after its secretion. Proteolytic cleavage of the hemagglutinin of influenza virus is a prerequisite for the virus to become infectious. Tryptase Clara was shown to cleave the hemagglutinin and activate infectivity of influenza A virus in a dose-dependent way. These results suggest that the enzyme is a possible activator of inactive viral fusion glycoprotein in the respiratory tract and thus responsible for pneumopathogenicity of the virus.