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Identification of GPI-anchored protein releasing factor using GPI-anchored GFP

使用 GPI 锚定 GFP 鉴定 GPI 锚定蛋白释放因子

基本信息

批准号：
13670118
负责人：
KONDOH Gen
金额：
$ 2.3万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

项目摘要

To clarify the fate of glyeosylphosphstidylinositol (GPI) in mammals, we developed GPI-anchored green fluorescent protein (GFP-GPI) and transgenic mice carrying this fusion constract. When it was introduced to culture cells, the GFP-GPI protein was correctly sorted to plasma membranes and mierosomes depending on GPI biosynthesis. Transgenic mice carrying GFP-GPI were found to show a broad transgene expression. Historogically, a prominent polarized localization of GFP-GPI protein was observed in various epithelia, nervous system and liver, as well as non-polarized presence in non-epthelial tissues.Surprisingly, the GFP-GPI protein showed high level secretion in exocrine glands and testis. What is the biological significance of this phenotype? How are GPI-anchored proteins released from membranes? To answer to these questions, we attempted to isolate which converts GPI-anchored proteins from the membrane attached form to the soluble form.Processing and turnover of transmembrane polypeptides and extra-cellular matrix proteins are performed by particular metalloproteases, contributing to multiple biological processes at the cell surface. However the regulation of -anchored proteins, a class of proteins anchoring to the outer-leaflet of membrane lipid bilayer via glycophospholipid moiety, was not further clarified.Here we found a metalloprotease, the angiotensin converting enzyme (ACE), release GPI-anchored proteins from the cell surface. ACE known to be a key regulator of blood pressure homeostasis cleaves various soluble small peptides, notably angiotensin I and bradykinin, thereby changing their biological activities and leading up-regulation of the blood pressure. The novel activity we found here suggests that ACE also contributes to membrane protein turnover and acts on more broad biological processes.

为了阐明糖基磷脂酰肌醇（GPI）在哺乳动物中的命运，我们开发了GPI锚定的绿色荧光蛋白（GFP-GPI）和携带该融合蛋白的转基因小鼠。当将其引入培养细胞时，GFP-GPI蛋白根据GPI生物合成被正确地分选到质膜和微粒体。发现携带GFP-GPI的转基因小鼠显示广泛的转基因表达。GFP-GPI蛋白在上皮细胞、神经系统和肝脏中有明显的极化定位，在非上皮组织中无极化定位，在外分泌腺和睾丸中有高水平的分泌。这种表型的生物学意义是什么？GPI锚定蛋白如何从膜中释放出来？为了回答这些问题，我们试图分离出将GPI锚定蛋白从膜附着形式转化为可溶性形式的蛋白。跨膜多肽和细胞外基质蛋白的加工和周转由特定的金属蛋白酶进行，有助于细胞表面的多种生物过程。然而，GPI-锚定蛋白（通过糖磷脂部分锚定在膜脂双层外叶的一类蛋白）的调控还没有进一步阐明，在这里我们发现了一种金属蛋白酶，血管紧张素转换酶（ACE），从细胞表面释放GPI-锚定蛋白。已知ACE是血压稳态的关键调节剂，其切割各种可溶性小肽，特别是血管紧张素I和缓激肽，从而改变它们的生物活性并导致血压的上调。我们在这里发现的新活性表明，ACE也有助于膜蛋白的周转，并作用于更广泛的生物过程。