Identification of Cellular Compartments Involved in Processing of Cathepsin E in Primary Cultures of Rat Microglia
Identification of Cellular Compartments Involved in Processing of Cathepsin E in Primary Cultures of Rat Microglia
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大鼠小胶质细胞原代培养物中参与组织蛋白酶 E 加工的细胞区室的鉴定
DOI：
10.1046/j.1471-4159.1998.70052045.x
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发表时间：
1998
影响因子：
4.7
通讯作者：
Kenji Yamamoto
中科院分区：
文献类型：
--
作者：
D. F. Sastradipura;H. Nakanishi;T. Tsukuba;K. Nishishita;H. Sakai;Y. Kato;T. Gotow;Y. Uchiyama;Kenji Yamamoto
摘要：组织蛋白酶 E 是一种主要的非溶酶体细胞内天冬氨酸蛋白酶，定位于各种细胞区室，如质膜、内体样细胞器和内质网 (ER)。为了了解组织蛋白酶 E 进入其适当细胞目的地的分离机制，本研究开始确定大鼠脑小胶质细胞原代培养物中该酶的生物合成、加工和细胞内定位以及蛋白水解成熟的位点。免疫组织化学和免疫印迹分析表明，组织蛋白酶 E 在各种脑细胞类型中的小胶质细胞中含量最丰富，其中该酶主要以成熟酶形式存在。免疫电子显微镜研究表明，该酶主要存在于内体样液泡中，部分存在于跨高尔基体区域和内质网管腔的囊泡中。在用[35S]蛋氨酸标记的原代培养的小胶质细胞中，30分钟脉冲后，>95%的标记组织蛋白酶E由46-kDa多肽（还原形式）代表。大部分在追踪后 4 小时内通过 44-kDa 中间体蛋白水解加工成 42-kDa 成熟形式。该过程被巴弗洛霉素 A1（一种液泡型 H+-ATP 酶的特异性抑制剂）完全抑制。 Brefeldin A 是一种分泌蛋白从内质网运输到高尔基复合体的阻断剂，也能抑制组织蛋白酶 E 的加工并增强其降解。脉冲标记后，组织蛋白酶 E 对内切糖苷酶 H 表现出完全敏感性，而成熟的酶几乎获得了对内切糖苷酶 H 和 F 的抗性。本研究提供了第一个证据，表明小胶质细胞中的组织蛋白酶 E 首先是作为带有高甘露糖寡糖的无活性前体合成的，然后加工成具有活性的成熟酶 通过中间形式形成复合型寡糖，最终的蛋白水解成熟步骤发生在类内体的酸性区室中。
Abstract: Cathepsin E is a major nonlysosomal, intracellular aspartic proteinase that localizes in various cellular compartments such as the plasma membrane, endosome‐like organelles, and the endoplasmic reticulum (ER). To learn the segregation mechanisms of cathepsin E into its appropriate cellular destinations, the present studies were initiated to define the biosynthesis, processing, and intracellular localization as well as the site of proteolytic maturation of the enzyme in primary cultures of rat brain microglia. Immunohistochemical and immunoblot analyses revealed that cathepsin E was the most abundant in microglia among various brain cell types, where the enzyme existed predominantly as the mature enzyme. Immunoelectron microscopy studies showed the presence of the enzyme predominantly in the endosome‐like vacuoles and partly in the vesicles located in the trans‐Golgi area and the lumen of ER. In the primary cultured microglial cells labeled with [35S]methionine, >95% of labeled cathepsin E were represented by a 46‐kDa polypeptide (reduced form) after a 30‐min pulse. Most of it was proteolytically processed via a 44‐kDa intermediate to a 42‐kDa mature form within 4 h of chase. This processing was completely inhibited by bafilomycin A1, a specific inhibitor of vacuolar‐type H+‐ATPase. Brefeldin A, a blocker for the traffic of secretory proteins from the ER to the Golgi complex, also inhibited the processing of procathepsin E and enhanced its degradation. Procathepsin E, after pulse‐labeling, showed complete susceptibility to endoglycosidase H, whereas the mature enzyme almost acquired resistance to endoglycosidases H as well as F. The present studies provide the first evidence that cathepsin E in microglia is first synthesized as the inactive precursor bearing high‐mannose oligosaccharides and processed to the active mature enzyme with complex‐type oligosaccharides via the intermediate form and that the final proteolytic maturation step occurs in endosome‐like acidic compartments.
DOI：
--
发表时间：
1994
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Xu,H;Shields,D
通讯作者：
Shields,D