Tsukuba T, Sakai H, Yamada M, Maeda H, Hori H, Azuma T, Akamine A, Yamamoto K
Department of Pharmacology, Kyushu University Faculty of Dentistry, Higashi-ku, Fukuoka.
J Biochem. 1996 Jan;119(1):126-34. doi: 10.1093/oxfordjournals.jbchem.a021197.
Cathepsin E (CE) is the only known aspartic proteinase that exists as a homodimer consisting of two fully catalytically active monomers, which are covalently bound by a disulfide bond between two cysteine residues at the NH2-terminal region (Cys43 in human pro-CE). To understand the physiological significance of the dimer formation, the monomeric mutant of human CE was constructed by site-directed mutagenesis (Cys43 ->Ser43) and expressed in Chinese hamster ovary (CHO) cells. Immunolocalization of the mutant protein at both the light and electron microscopic levels revealed the monomeric CE to be associated predominantly with the endoplasmic reticulum and the non-lysosomal endocytic organelles. The cellular localization of the monomeric protein was compatible with that of the wild-type (dimeric form) of recombinant human CE expressed in the same cells. The monomeric protein was generated primarily as the 46-kDa pro-CE with a high-mannose-type oligosaccharide chain in the cells. In addition to the maximal activation at around pH 3.5, a substantial proportion of the monomeric pro-CE was converted to the mature form by incubation at pH 7 and 37 degrees C for 5 min. In contrast, the dimeric pro-CE was scarcely activated by treatment at pH7. Although catalytic properties of the in vitro-activated monomeric CE appeared to be indistinguishable from those of the dimeric forms of natural and recombinant CE, the monomeric form was more unstable to pH and temperature changes than these dimeric forms. These results indicate that the dimerization of CE is not necessarily required for proper folding to express activity, correct intracellular localization and carbohydrate modification, but that it may be essential to structurally stabilize the molecule in vivo.
组织蛋白酶E（CE）是唯一已知的天冬氨酸蛋白酶，它以同二聚体形式存在，由两个完全具有催化活性的单体组成，这两个单体通过NH2末端区域两个半胱氨酸残基（人pro-CE中的Cys43）之间的二硫键共价结合。为了了解二聚体形成的生理意义，通过定点诱变构建了人CE的单体突变体（Cys43→Ser43），并在中国仓鼠卵巢（CHO）细胞中表达。在光学和电子显微镜水平对突变蛋白进行免疫定位显示，单体CE主要与内质网和非溶酶体胞吞细胞器相关。单体蛋白的细胞定位与在相同细胞中表达的重组人CE野生型（二聚体形式）的定位一致。单体蛋白在细胞中主要以带有高甘露糖型寡糖链的46 kDa pro-CE形式产生。除了在pH 3.�左右的最大激活外，相当一部分单体pro-CE在pH 7和37℃孵育5分钟后转化为成熟形式。相比之下，二聚体pro-CE在pH 7处理时几乎不被激活。尽管体外激活的单体CE的催化特性似乎与天然和重组CE的二聚体形式没有区别，但单体形式比这些二聚体形式对pH和温度变化更不稳定。这些结果表明，CE的二聚化对于正确折叠以表达活性、正确的细胞内定位和糖基化修饰不一定是必需的，但它可能对于在体内结构稳定分子至关重要。
