Manser E, Fernandez D, Lim L
Molecular Neurobiology Laboratory, National University of Singapore.
Biochem J. 1991 Dec 15;280 ( Pt 3)(Pt 3):695-701. doi: 10.1042/bj2800695.
Carboxypeptidase E (CPE) catalyses the removal of C-terminal basic amino acids and is implicated in the processing of peptides and hormones in secretory vesicles. The enzyme has been isolated in multiple forms from both soluble and membrane-bound compartments, and has been demonstrated to be co-secreted with peptides from pancreatic and adrenal cells. To address the question regarding the origin of the multiple forms of the enzyme, we have transfected a construct containing the cDNA for human CPE under the control of the murine-sarcoma-virus enhancer and metallothionein promoter into the C6 rat glioma cell line, which itself has extremely low levels of CPE expression. Within transfectants, multiple forms of the enzyme have been detected by Western blotting, and their sizes are comparable with that in pituitary. Fractionation of the intracellular forms of CPE with Triton X-114 at various pH values indicates that the membrane-bound, but not the soluble, form is amphipathic; this difference probably arises from post-translational modification of the enzyme. Interestingly, the secreted enzyme is found to have the amphipathic characteristics of the membrane-bound form. By modification of the cDNA sequence, enzyme lacking N-terminal and C-terminal domains have been expressed: deletion of the 'pro' region of CPE, leading to loss of the signal cleavage site, gives a more hydrophobic but active enzyme which is not efficiently secreted from the cell; deletion of a C-terminal region gives a secreted form of the enzyme which no longer exhibits amphipathic behaviour. Our data indicate that a single mRNA species can give rise to the multiple forms of CPE enzyme that have been identified and that the C6 cells are able to carry out the intracellular processing events to generate these forms.
羧肽酶E（CPE）催化去除C末端碱性氨基酸，并参与分泌小泡中肽和激素的加工过程。该酶已从可溶性和膜结合区室中以多种形式分离出来，并已证明与胰腺和肾上腺细胞分泌的肽共同分泌。为了解决关于该酶多种形式起源的问题，我们将一个构建体转染到C6大鼠胶质瘤细胞系中，该构建体包含在鼠肉瘤病毒增强子和金属硫蛋白启动子控制下的人CPE cDNA，而C6细胞系本身的CPE表达水平极低。在转染细胞内，通过蛋白质免疫印迹法检测到该酶的多种形式，其大小与垂体中的相当。在不同pH值下用Triton X-114对细胞内形式的CPE进行分级分离表明，膜结合形式而非可溶性形式具有两亲性；这种差异可能源于该酶的翻译后修饰。有趣的是，发现分泌的酶具有膜结合形式的两亲性特征。通过对cDNA序列进行修饰，表达了缺失N末端和C末端结构域的酶：缺失CPE的“前体”区域，导致信号切割位点丢失，产生一种更疏水但有活性的酶，该酶不能有效地从细胞中分泌出来；缺失C末端区域产生一种分泌形式的酶，该酶不再表现出两亲性行为。我们的数据表明，单一的mRNA种类可以产生已鉴定出的多种形式的CPE酶，并且C6细胞能够进行细胞内加工过程以产生这些形式。
