Role of glycosylation in the biosynthesis and activity of rabbit testicular angiotensin-converting enzyme.
Role of glycosylation in the biosynthesis and activity of rabbit testicular angiotensin-converting enzyme.
复制标题
糖基化在兔睾丸血管紧张素转换酶的生物合成和活性中的作用。
作者：
Kasturi,S;Jabbar,MA;Sen,GC;Sen,I
血管紧张素转换酶（ACE）是一种I型糖蛋白，通过其羧基端附近的疏水结构域锚定在质膜上。ACE的具有酶活性的胞外结构域通过其膜锚定羧基末端区域的裂解去除而从细胞中缓慢释放。在本研究中，我们研究了N-和O-糖基化在兔睾丸ACE的细胞内转运和细胞外切割分泌中的作用。对于ACE表达，我们使用了体外翻译系统、永久转染的小鼠细胞系以及用牛痘病毒-T7 RNA聚合酶驱动的表达载体瞬时转染的人和中国仓鼠细胞。通过测试其对特定糖苷酶的敏感性来分析ACE的糖修饰。用化学抑制剂和蛋白糖基化缺陷突变细胞系抑制细胞蛋白糖基化。我们的实验表明，新合成的ACE获得N-和O-连接的糖之前，其切割分泌和糖基化的完全阻断导致糖基化不足的ACE的快速细胞内周转。然而，在没有N-连接的复合糖和O-连接的糖的情况下合成的ACE可以进行正常的转运和切割-分泌，并且糖基化不足的蛋白质具有酶活性。15.1），一种肽基二肽酶，通过从其无活性前体血管紧张素I产生血管加压素血管紧张素II和通过使血管抑制肽缓激肽失活，在维持心血管稳态中起重要作用（Skeggs等，1981; Erdos & Yang，1967; Cushman & Ondetti，1980）。血管紧张素转换酶（ACE）1具有两种同工酶形式，肺（P）和睾丸（T），其以组织特异性方式表达（Soffer，1981; El-Dorry等人，1982 a，B）。这两种同工酶在结构上相关并由同一基因编码（Kumar等人，1989，1991; Thekirkara等人，1992; Soubrier等人，1988;伯恩斯坦等人，1989;霍华德等人，1990; Hubert等人，1991年）。兔ACEt具有737个氨基酸残基，ACEp具有1309个残基（Kumar et
Angiotensin-converting enzyme (ACE) is a type I glycoprotein anchored in the plasma membrane by a hydrophobic domain near its carboxyl terminus. The enzymatically active extracellular domain of ACE is slowly released from the cell by cleavage-removal of its membrane-anchoring carboxyl-terminal region. In the present study, we investigated the role of N-and O-glycosylation in intracellular transport and extracellular cleavage-secretion of rabbit testicular ACE. For ACE expression, we used an ir. vitro translation system, a permanently transfected mouse cell line, and human and Chinese hamster cells transiently transfected with vaccinia virus-T7 RNA polymerase-driven expression vectors. Sugar modifications of ACE were analyzed by testing its sensitivity to specific glycosidases. Cellular protein glycosylation was inhibited byusing chemical inhibitors and a mutant cell line defective in protein glycosylation. Our experiments demonstrated that newly synthesized ACE acquires both N-and O-linked sugars before its cleavage-secretion and complete blockage of glycosylation results in rapid intracellular turnover of underglycosylated ACE. However, ACE synthesized without N-linked complex sugars and O-linked sugars can undergo normal transport and cleavage-secretion, and the underglycosylated protein is enzymatically active.Angiotensin-converting enzyme (EC 3.4. 15.1), a peptidyl dipeptidase, plays an important role in maintaining cardio-vascular homeostasis by generating the vasopressor angiotensin II from its inactive precursor angiotensin I and by inactivating the vasodepressor peptide bradykinin (Skeggs et al., 1981; Erdos & Yang, 1967; Cushman & Ondetti, 1980). Angiotensin-converting enzyme (ACE) 1 has two isozymic forms, pulmonary (P) and testicular (T), which are expressed in a tissue-specific fashion (Soffer, 1981; El-Dorry et al., 1982a, b). The two isozymes are structurally related and encoded by the same gene (Kumar et al., 1989, 1991; Thekkumkara et al., 1992; Soubrier et al., 1988; Bernstein et al., 1989; Howard et al., 1990; Hubert et al., 1991). Rabbit ACEt has 737 amino acid residues, and ACEp has 1309 residues (Kumar et