Processing of pulmonary surfactant protein B by napsin and cathepsin H
Processing of pulmonary surfactant protein B by napsin and cathepsin H
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DOI：
10.1074/jbc.m312029200
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发表时间：
2004-04-16
影响因子：
4.8
通讯作者：
Weaver, TE
中科院分区：
文献类型：
--
作者：
Ueno, T;Linder, S;Weaver, TE
表面活性蛋白B（SP-B）是肺表面活性物质的重要组成部分。SP-B在肺泡II型细胞中作为前原蛋白合成，并通过NH 2-和COOH-末端肽的裂解加工成成熟肽。已经提出了一种N-乙酰基蛋白酶来切割NH 2-末端前肽，产生25-kDa的中间体。Napsin是一种在肺泡II型细胞中表达的乙酰化蛋白酶，早在妊娠第16天，SP-B表达和加工开始前1天，就在胎肺匀浆中检测到Napsin。Napsin被定位于多泡体，SP-B前蛋白在II型细胞加工的网站。孵育SP-B前蛋白从II型细胞与粗膜提取物从napsin转染的细胞导致在增强的水平的25 kDa的中间体。纯化的napsin在成熟SP-B的NH 2端上游22个氨基酸处的Leu（178）和Pro（179）之间的NH 2端前肽内切割重组SP-B/EGFP融合蛋白。组织蛋白酶H是一种半胱氨酸蛋白酶，也参与前SP-B加工，切割成熟SP-B NH 2末端上游13个氨基酸的SP-B/EGFP融合蛋白。Napsin不裂解COOH-末端肽，而组织蛋白酶H裂解成熟SP-B和COOH-末端肽之间的边界，并在其他几个网站内的COOH-末端肽。通过小干扰RNA敲低napsin导致II型细胞中成熟SP-B和成熟SP-C的水平降低。这些结果表明，napsin，组织蛋白酶H，和至少一种其他酶参与成熟的生物活性SP-B肽。
Surfactant protein B (SP-B) is an essential constituent of pulmonary surfactant. SP-B is synthesized in alveolar type II cells as a preproprotein and processed to the mature peptide by the cleavage of NH2- and COOH-terminal peptides. An aspartyl protease has been suggested to cleave the NH2- terminal propeptide resulting in a 25-kDa intermediate. Napsin, an aspartyl protease expressed in alveolar type II cells, was detected in fetal lung homogenates as early as day 16 of gestation, 1 day before the onset of SP-B expression and processing. Napsin was localized to multivesicular bodies, the site of SP-B proprotein processing in type II cells. Incubation of SP-B proprotein from type II cells with a crude membrane extract from napsin-transfected cells resulted in enhanced levels of a 25-kDa intermediate. Purified napsin cleaved a recombinant SP-B/EGFP fusion protein within the NH2- terminal propeptide between Leu(178) and Pro(179), 22 amino acids upstream of the NH2 terminus of mature SP-B. Cathepsin H, a cysteine protease also implicated in pro-SP-B processing, cleaved SP-B/EGFP fusion protein 13 amino acids upstream of the NH2 terminus of mature SP-B. Napsin did not cleave the COOH-terminal peptide, whereas cathepsin H cleaved the boundary between mature SP-B and the COOH-terminal peptide and at several other sites within the COOH-terminal peptide. Knockdown of napsin by small interfering RNA resulted in decreased levels of mature SP-B and mature SP-C in type II cells. These results suggest that napsin, cathepsin H, and at least one other enzyme are involved in maturation of the biologically active SP-B peptide.