Cleavage of the ADAMTS13 propeptide is not required for protease activity
Cleavage of the ADAMTS13 propeptide is not required for protease activity
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DOI：
10.1074/jbc.m309872200
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发表时间：
2003-11-21
影响因子：
4.8
通讯作者：
Sadler, JE
中科院分区：
文献类型：
--
作者：
Majerus, EM;Zheng, XL;Sadler, JE
ADAMTS13属于“具有血栓反应蛋白重复序列的崩解素和金属蛋白酶”家族，并将血管性血液病因子多聚体切割成更小的形式。对于一些相关的蛋白酶，正常的折叠和酶促潜伏期依赖于在生物合成过程中被蛋白水解过程去除的NH2末端前肽。然而，ADAMTS13前肽异常短且保守性差，表明它可能不具有这些功能。ADAMTS13在转染的HeLa细胞中半时间为7 h分泌，限速步骤从内质网输出。该前肽的缺失不影响活性ADAMTS13的分泌，表明该前肽在折叠过程中是不可缺少的。Furin以两种方式被证明对ADAMTS13前肽加工是足够的。首先，furin共识识别位点的突变阻止了HeLa细胞中前肽的切割，导致前ADAMTS13的分泌。其次，缺乏furin的LoVo细胞分泌的ADAMTS13前肽完整，furin共转染恢复了前肽的切割。在两种细胞系中，分泌的原ADAMTS13对血管性血友病因子具有正常的蛋白水解活性。在共表达ADAMTS13和von Willebrand因子的细胞中，pro ADAMTS13在细胞内切割pro von Willebrand因子。因此，ADAMTS13前肽不需要折叠或分泌，也不执行维持酶潜伏期的常见功能。
ADAMTS13 belongs to the " a disintegrin and metalloprotease with thrombospondin repeats" family, and cleaves von Willebrand factor multimers into smaller forms. For several related proteases, normal folding and enzymatic latency depend on an NH2- terminal propeptide that is removed by proteolytic processing during biosynthesis. However, the ADAMTS13 propeptide is unusually short and poorly conserved, suggesting it may not perform these functions. ADAMTS13 was secreted from transfected HeLa cells with a half- time of 7 h and the rate- limiting step was exported from the endoplasmic reticulum. Deletion of the propeptide did not impair the secretion of active ADAMTS13, indicating that the propeptide is dispensable for folding. Furin was shown to be sufficient for ADAMTS13 propeptide processing in two ways. First, mutation of the furin consensus recognition site prevented propeptide cleavage in HeLa cells and resulted in secretion of pro- ADAMTS13. Second, furin- deficient LoVo cells secreted ADAMTS13 with the propeptide intact, and cotransfection with furin restored propeptide cleavage. In both cell lines, secreted pro- ADAMTS13 had normal proteolytic activity toward von Willebrand factor. In cells coexpressing both ADAMTS13 and von Willebrand factor, pro- ADAMTS13 cleaved pro- von Willebrand factor intracellularly. Therefore, the ADAMTS13 propeptide is not required for folding or secretion, and does not perform the common function of maintaining enzyme latency.