PROCESSING OF SURFACTANT PROTEIN-B PROPROTEIN BY A CATHEPSIN-D-LIKE PROTEASE
PROCESSING OF SURFACTANT PROTEIN-B PROPROTEIN BY A CATHEPSIN-D-LIKE PROTEASE
复制标题
DOI：
10.1152/ajplung.1992.263.1.l95
复制
发表时间：
1992-07-01
影响因子：
--
通讯作者：
DEY, C
中科院分区：
文献类型：
--
作者：
WEAVER, TE;LIN, S;DEY, C
表面活性蛋白B(SP-B)是一种相对分子质量(M(R))=8000的疏水性多肽，与肺表面活性物质磷脂有关。SP-B是由肺泡II型上皮细胞合成的一种M(R)=42,000的前蛋白，它至少需要两次蛋白水解酶才能产生79个残基的成熟SP-B肽。我们以前已经报道过，NH2末端前肽的裂解产生M(R)=25,000的加工中间体，发生在接近分泌的时间。在本研究中，我们证明了从稳定表达的中国仓鼠卵巢细胞中分离出的SP-B蛋白可以被粗制的II型细胞膜部分处理到M(R)=25000，而不是被完整的II型细胞或II型细胞条件培养液处理到M(R)=25000。用II型细胞膜制剂在体外处理该前蛋白，释放出M(R)=16,000-17,000的单一多肽，并用针对该前体的前肽的抗原表位的抗血清进行检测。用Na2CO3裂解制备的II型细胞膜提取SP-B的加工活性，最适pH为5.0-6.0，并被10(-7)M胃抑素A抑制，表明前体的NH2端肽被天冬氨酸蛋白酶切割。与这一假设一致，粗制的II型细胞膜制剂对SP-B的加工可被针对天冬氨酸蛋白酶组织蛋白酶D的抗血清阻断；此外，纯化的组织蛋白酶D有效地处理M(R)=25,000的SP-B前体。总之，这些结果表明，SP-B前体的NH2末端前肽的裂解是由组织蛋白酶D或定位于II型上皮细胞分泌途径中的组织蛋白酶D样酶介导的。
Surfactant protein B (SP-B) is a hydrophobic peptide of relative molecular weight (M(r)) = 8,000 that is associated with pulmonary surfactant phospholipids. SP-B is synthesized by the alveolar type II epithelial cell as a proprotein of M(r) = 42,000 which requires at least two proteolytic cleavages to generate the 79 residue mature SP-B peptide. We have previously reported that cleavage of the NH2-terminal propeptide, to generate a processing intermediate of M(r) = 25,000, occurs in close temporal approximation to secretion. In the present study we demonstrate that SP-B proprotein, isolated from stably transfected Chinese hamster ovary cells, is processed to M(r) = 25,000 by a crude type II cell membrane fraction but not by intact type II cells or type II cell conditioned media. In vitro processing of the proprotein by the type II cell membrane preparation resulted in release of a single peptide of M(r) = 16,000-17,000, which was detected by antiserum directed against antigenic epitopes in propeptide of the precursor. SP-B processing activity was extracted by Na2CO3 lysis of the type II cell membrane preparation, had a pH optimum of 5.0-6.0, and was inhibited by 10(-7) M pepstatin A, suggesting that the NH2-terminal peptide of the precursor is cleaved by an aspartyl protease. Consistent with this hypothesis, processing of SP-B by a crude type II cell membrane preparation was blocked by antiserum directed against the aspartyl protease cathepsin D; further, purified cathepsin D efficiently processed the SP-B precursor to M(r) = 25,000. Collectively these results suggest that cleavage of the NH2-terminal propeptide of the SP-B precursor is mediated by cathepsin D or a cathepsin D-like protease localized within the secretory pathway of the type II epithelial cell.