Identification of prodomain determinants involved in ADAMTS-1 biosynthesis
Identification of prodomain determinants involved in ADAMTS-1 biosynthesis
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DOI：
10.1074/jbc.m313151200
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发表时间：
2004-08-06
影响因子：
4.8
通讯作者：
Leduc, R
中科院分区：
文献类型：
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作者：
Longpré, JM;Leduc, R
金属蛋白酶 ADAMTS-1（具有 I 型血小板反应蛋白基序的解整合素和金属蛋白酶）与 ADAMTS 家族的其他成员类似，最初被合成为酶原 proADAMTS-1，在前结构域/催化结构域连接处由前蛋白转化酶类弗林蛋白酶家族的丝氨酸蛋白酶进行蛋白水解加工。本研究的目的是鉴定在 ADAMTS-1 加工中起重要作用的前结构域残基，并确定酶原加工所需的转化酶的身份。为了深入了解特定前结构域残基在 ADAMTS-1 生物合成中的推定作用，我们在表达 proADAMTS-1 野生型和前结构域突变体的瞬时转染人胚胎肾 293 细胞中进行了生物合成标记实验。表达野生型 ADAMTS-1 的细胞最初产生 110 kDa 的酶原形式，随后转化为 87 kDa 的形式，这也在培养基中检测到。尽管 PACE4 和 PC6B 等转化酶加工 proADAMTS-1，但我们发现弗林蛋白酶是产生成熟 ADAMTS-1 87-kDa 部分的最有效的酶。对 ADAMTS-1 前结构域（RRNR173 和 RKKR235）内发现的两个推定的弗林蛋白酶识别序列进行定点诱变表明，Arg(235) 是唯一的加工位点。使用高尔基干扰剂布雷菲德菌素 A 和莫能菌素表明，proADAMTS-1 的裂解发生在高尔基体分泌之前。其他 ADAMTS 成员的前结构域内的保守残基暗示它们可能充当成熟决定簇。用丙氨酸替换所选残基 Cys(106)、Tyr(108)、Gly(110)、Cys(125) 和 Cys(181) 以及包含 137-144 序列的残基显着影响酶的生物合成谱。我们的结果表明，ADAMTS-1 前结构域中除弗林蛋白酶切割位点以外的保守残基参与其生物合成。
The metalloprotease ADAMTS-1 (a disintegrin and metalloprotease with thrombospondin type I motif), similarly to other members of the ADAMTS family, is initially synthesized as a zymogen, proADAMTS-1, that undergoes proteolytic processing at the prodomain/catalytic domain junction by serine proteinases of the furin-like family of proprotein convertases. The goals of this study were to identify residues of the prodomain that play an essential role in ADAMTS-1 processing and to determine the identity of the convertase required for zymogen processing. To gain insight into the putative roles of specific prodomain residues in ADAMTS-1 biosynthesis, we performed biosynthetic labeling experiments in transiently transfected human embryonic kidney 293 cells expressing wild-type and prodomain mutants of proADAMTS-1. Cells expressing wild-type ADAMTS-1 initially produced a 110-kDa zymogen form that was later converted to an 87-kDa form, which was also detected in the media. Although convertases such as PACE4 and PC6B processed proADAMTS-1, we found that furin was the most efficient enzyme at producing the mature ADAMTS-1 87-kDa moiety. Site-directed mutagenesis of the two putative furin recognition sequences found within the ADAMTS-1 prodomain (RRNR173 and RKKR235) revealed that Arg(235) was the sole processing site. Use of the Golgi disturbing agent, Brefeldin A, and monensin suggests that the cleavage of proADAMTS-1 takes place in the Golgi apparatus prior to its secretion. Conserved residues within the prodomain of other ADAMTS members hinted that they might act as maturation determinants. Replacement with alanine of selected residues Cys(106), Tyr(108), Gly(110), Cys(125), and Cys(181) and residues encompassing the 137-144 sequence significantly affected the biosynthetic profile of the enzyme. Our results suggest that conserved residues other than the furin cleavage site in the prodomain of ADAMTS-1 are involved in its biosynthesis.