Brasch Frank, Ten Brinke Anja, Johnen Georg, Ochs Matthias, Kapp Nadine, Müller Klaus M, Beers Michael F, Fehrenbach Heinz, Richter Joachim, Batenburg Joseph J, Bühling Frank
Division of Electron Microscopy, Department of Anatomy, University of Göttingen, Germany.
Am J Respir Cell Mol Biol. 2002 Jun;26(6):659-70. doi: 10.1165/ajrcmb.26.6.4744.
Surfactant protein C (SP-C) is synthesized by type II pneumocytes as a 21-kD propeptide (proSP-C) which is proteolytically processed to a 4.2-kD dipalmitoylated protein. To characterize the processing of proSP-C and the role of the cysteine protease cathepsin H, we studied the localization of proSP-C and cathepsin H in human as well as proSP-C in rat lungs, the enzymatic cathepsin H activity in isolated rat lamellar bodies, and the cleavage of human proSP-C by purified cathepsin H. Using antisera directed against the N-terminal E(11)-R(23) (NPROSP-C(11-23)), the C-terminal G(162)-G(174) domain (CPROSP-C(162-174)) of proSP-C, and against cathepsin H, immunogold labeling identified all three in electron-dense multivesicular bodies, but only NPROSP-C(11-23) and cathepsin H in composite as well as lamellar bodies of type II pneumocytes. Immuno double-labeling further distinguished electron-dense vesicles containing cathepsin H or electron light vesicles/multivesicular bodies containing proSP-C. Isolated lamellar bodies contained enzymatically active cathepsin H, a 6-kD proSP-C processing intermediate detected only by NPROSP-C(11-23), and mature SP-C. Using enzyme activities comparable to those in isolated lamellar bodies, purified cathepsin H generated a partially N-terminal processed proSP-C intermediate in vitro. In conclusion, our results indicate that after the fusion of electron-dense vesicles containing cathepsin H and electron-light vesicles or multivesicular bodies containing proSP-C, cathepsin H is involved in the first N-terminal processing step of proSP-C in electron-dense multivesicular bodies of type II pneumocytes.
表面活性蛋白C（SP-C）由II型肺细胞合成为一种21-kD的前体肽（proSP-C），该前体肽经蛋白水解加工成一种4.2-kD的二棕榈酰化蛋白。为了表征proSP-C的加工过程以及半胱氨酸蛋白酶组织蛋白酶H的作用，我们研究了proSP-C和组织蛋白酶H在人肺中的定位以及proSP-C在大鼠肺中的定位、分离的大鼠板层小体中的组织蛋白酶H酶活性，以及纯化的组织蛋白酶H对人proSP-C的切割。使用针对proSP-C的N端E(11)-R(23)（NPROSP-C(11-23)）、C端G(162)-G(174)结构域（CPROSP-C(162-174)）以及针对组织蛋白酶H的抗血清，免疫金标记在电子致密多囊泡体中鉴定出了这三者，但仅在II型肺细胞的复合小体和板层小体中鉴定出了NPROSP-C(11-23)和组织蛋白酶H。免疫双标记进一步区分了含有组织蛋白酶H的电子致密囊泡或含有proSP-C的电子淡囊泡/多囊泡体。分离的板层小体含有酶活性的组织蛋白酶H、仅由NPROSP-C(11-23)检测到的6-kD proSP-C加工中间体以及成熟的SP-C。使用与分离的板层小体中相当的酶活性，纯化的组织蛋白酶H在体外产生了一种部分N端加工的proSP-C中间体。总之，我们的结果表明，在含有组织蛋白酶H的电子致密囊泡与含有proSP-C的电子淡囊泡或多囊泡体融合后，组织蛋白酶H参与了II型肺细胞电子致密多囊泡体中proSP-C的首个N端加工步骤。
