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

为了阐明glyeosylphosphstidylinositol （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）和携带该融合结构的转基因小鼠。当GFP-GPI蛋白被引入培养细胞时，GFP-GPI蛋白根据GPI的生物合成被正确地分选到质膜和粒质体上。结果发现，携带GFP-GPI的转基因小鼠具有广泛的转基因表达。从历史上看，GFP-GPI蛋白在各种上皮、神经系统和肝脏中都有明显的极化定位，在非上皮组织中也有非极化存在。令人惊讶的是，GFP-GPI蛋白在外分泌腺和睾丸中显示出高水平的分泌。这种表型的生物学意义是什么？gpi锚定蛋白是如何从膜上释放的？为了回答这些问题，我们试图分离gpi锚定蛋白从膜附着形式转化为可溶性形式。跨膜多肽和细胞外基质蛋白的加工和转换是由特定的金属蛋白酶完成的，有助于细胞表面的多种生物过程。然而，锚定蛋白（一类通过糖磷脂片段锚定在膜脂双分子层外小叶上的蛋白）的调控尚不清楚。在这里，我们发现金属蛋白酶，血管紧张素转换酶（ACE），从细胞表面释放gpi锚定蛋白。ACE是血压稳态的关键调节因子，可切割多种可溶性小肽，特别是血管紧张素I和缓激肽，从而改变其生物活性，导致血压上调。我们在这里发现的新活性表明，ACE也有助于膜蛋白的周转，并在更广泛的生物过程中起作用。

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.