Cathepsin L1, the major protease involved in liver fluke (Fasciola hepatica) virulence -: Propeptide cleavage sites and autoactivation of the zymogen secreted from gastrodermal cells
Cathepsin L1, the major protease involved in liver fluke (Fasciola hepatica) virulence -: Propeptide cleavage sites and autoactivation of the zymogen secreted from gastrodermal cells
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DOI：
10.1074/jbc.m308831200
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发表时间：
2004-04-23
影响因子：
4.8
通讯作者：
Donnelly, S
中科院分区：
文献类型：
--
作者：
Collins, PR;Stack, CM;Donnelly, S
对参与肝片吸虫致病的主要组织蛋白酶L1半胱氨酸蛋白酶的分泌和激活进行了研究。在培养肝片吸虫成虫的培养液中，只有经过充分加工的成熟酶才能被检测到。然而，免疫细胞化学研究表明，不活跃的原蛋白L1被包裹在寄生虫肠道内的上皮细胞的分泌小泡中。这些观察表明，原蛋白L1的加工和激活发生在这些细胞分泌到酸性肠腔之后。37 kDa原蛋白L1在巴斯德毕赤酵母中的表达表明，前肽中保守的GXNXFXD基序中的分子间加工事件产生了活性30 kDa的中间体。进一步的激活是通过降低pH到5.0来启动的，包括将37 kDa和30 kDa的形式逐步加工成其他中间体，最后加工成完全成熟的24.5 kDa的组织蛋白L，并增加1到2个氨基酸。用甘氨酸(26)取代半胱氨酸(26)构建的L活性位点突变体Proathepsin不能自动加工。然而，[Gly(26)]原组织蛋白酶L被外源野生型组织蛋白酶L加工成一个成熟的酶加上N端连接的10个氨基酸。这种外源加工发生在没有形成30 kDa中间形式的情况下。结果表明，通过去除前肽激活原蛋白L1可以通过不同的途径发生，这发生在寄生虫的肠道内，在那里，蛋白酶在食物消化中发挥作用，并从那里释放出来，作为一种活性酶，用于额外的体外作用。
The secretion and activation of the major cathepsin L1 cysteine protease involved in the virulence of the helminth pathogen Fasciola hepatica was investigated. Only the fully processed and active mature enzyme can be detected in medium in which adult F. hepatica are cultured. However, immunocytochemical studies revealed that the inactive procathepsin L1 is packaged in secretory vesicles of epithelial cells that line the parasite gut. These observations suggest that processing and activation of procathepsin L1 occurs following secretion from these cells into the acidic gut lumen. Expression of the 37-kDa procathepsin L1 in Pichia pastoris showed that an intermolecular processing event within a conserved GXNXFXD motif in the propeptide generates an active 30-kDa intermediate form. Further activation of the enzyme was initiated by decreasing the pH to 5.0 and involved the progressive processing of the 37 and 30-kDa forms to other intermediates and finally to a fully mature 24.5 kDa cathepsin L with an additional 1 or 2 amino acids. An active site mutant procathepsin L, constructed by replacing the Cys(26) with Gly(26), failed to autoprocess. However, [Gly(26)] procathepsin L was processed by exogenous wild-type cathepsin L to a mature enzyme plus 10 amino acids attached to the N terminus. This exogenous processing occurred without the formation of a 30-kDa intermediate form. The results indicate that activation of procathepsin L1 by removal of the propeptide can occur by different pathways, and that this takes place within the parasite gut where the protease functions in food digestion and from where it is liberated as an active enzyme for additional extracorporeal roles.