Processing of proendothelin-1 at the C-terminus of big endothelin-1 is essential for proteolysis by endothelin-converting enzyme-1 in vivo
Processing of proendothelin-1 at the C-terminus of big endothelin-1 is essential for proteolysis by endothelin-converting enzyme-1 in vivo
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DOI：
10.1111/j.1432-1033.1997.00520.x
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发表时间：
1997-03-01
期刊：
影响因子：
--
通讯作者：
Masaki, T
中科院分区：
文献类型：
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作者：
Kido, T;Sawamura, T;Masaki, T
内皮素 1 的生产被认为是一个三步过程，包括将内皮素原前体初步蛋白水解裂解为大内皮素 1-Lys-Arg，通过羧肽酶进行 C 末端修剪，然后通过内皮素转化酶 (ECE) 将大内皮素 1 肽进一步加工为内皮素 1。为了进一步阐明内皮素-1生物合成的加工机制，我们在内皮素-1 cDNA中引入点突变，将人前内皮素-1原中弗林蛋白酶样转化酶识别基序（Arg49或Arg89）的-4位置的精氨酸替换为甘氨酸。当突变的 cDNA 在中国仓鼠卵巢 (CHO)-K1 细胞中表达时，它们无法在突变的处理信号处被处理，这表明前内皮素原-1 的 Arg-Ser-Lys-Arg 基序被 CHO-K1 弗林蛋白酶样转化酶识别。与 ECE-1 cDNA 共转染表明，Arg52 处的切割对于 ECE-1 的切割而言并不是必需的，但 Arg92 处的切割至关重要。虽然内皮素 1 的高分子质量形式是通过 ECE-1 加工产生的，且在 Arg52 处没有裂解，但它不会在内皮素 (A) 受体表达细胞中引起 Ca2+ 瞬态。总之，弗林蛋白酶样转化酶在 Arg92 处的预先裂解对于 ECE-1 在 Trp73 处裂解以产生成熟的内皮素-1 是绝对必要的。
Production of endothelin-1 is thought to be a three-step process consisting of an initial proteolytic cleavage of the proendothelin-1 precursor to big endothelin-1-Lys-Arg, C-terminal trimming by a carboxypeptidase and further processing of the big endothelin-1 peptide to endothelin-1 by endothelin-converting enzyme (ECE). To further clarify the mechanism of processing in the biosynthesis of endothelin-1, we introduced a point mutation into endothelin-1 cDNA to replace the Arg in the -4 position of the recognition motifs of furin-like convertase in human preproendothelin-1 (Arg49 or Arg89) by Gly. When mutant cDNAs were expressed in Chinese hamster ovary (CHO)-K1 cells, they failed to be processed at the mutated processing signal, suggesting that the Arg-Ser-Lys-Arg motifs of preproendothelin-1 are recognized by CHO-K1 furin-like convertase. Co-transfection with ECE-1 cDNA revealed that cleavage at Arg52 is not essential for cleavage by ECE-1, but that cleavage at Arg92 is critical. Although a high-molecular-mass form of endothelin-1 is produced by processing by ECE-1 without cleavage at Arg52, it did not evoke Ca2+ transient in endothelin(A)-receptor-expressing cells. In conclusion, prior cleavage at Arg92 by furin-like convertase is absolutely necessary for cleavage by ECE-1 at Trp73 to produce mature endothelin-1.