Localization of presenilin-nicastrin complexes and gamma-secretase activity to the trans-Golgi network.
Localization of presenilin-nicastrin complexes and gamma-secretase activity to the trans-Golgi network.
复制标题
早老素-尼克斯特林复合物和γ-分泌酶活性在跨高尔基体网络中的定位。
DOI：
10.1046/j.1471-4159.2003.01616.x
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发表时间：
2003
影响因子：
4.7
通讯作者：
Velji,Jamel
中科院分区：
文献类型：
--
作者：
Siman,Robert;Velji,Jamel
大量的生物化学和遗传学证据表明，早老素是γ-分泌酶的催化组分，γ-分泌酶是负责产生阿尔茨海默氏症淀粉样β蛋白的蛋白酶。然而，早老素在早期分泌区室和γ-分泌酶底物在晚期分泌区室和质膜的差异定位（“空间悖论”）反驳了这一观点。我们通过研究nicastrin（另一种假定的γ-分泌酶组分）的定位及其与早老素-1结合成蛋白水解活性复合物来研究这个问题。糖苷酶分析表明，nicastrin以多种糖型存在，并且末端被唾液酸化，这是一种通常与trans-Golgi网络相关的修饰。通过密度梯度分离和免疫荧光显微镜证实了nicastrin向trans-Golgi网络的运输。然而，在早老素缺乏的细胞中，nicastrin的运输和成熟是异常的，因为蛋白质被限制在早期分泌区室并且未能被唾液酸化。成熟的唾液酸化nicastrin intrans-Golgi网络组分与早老素-1的N-和C-末端片段定量复合，而存在于早期分泌室中的未成熟nicastrin则不是。此外，反式高尔基体网络组分含有γ-分泌酶底物β-淀粉样前体蛋白C83，并富含早老素依赖性γ-分泌酶蛋白水解活性。结果通过证明早老素-nicastrin复合物和早老素依赖性γ分泌酶活性共定位于晚期分泌室来解决明显的空间矛盾。这些发现进一步证明了含早老素的复合物是γ-分泌酶，并表明早老素也调节γ-分泌酶的组装。
Abundant biochemical and genetic evidence suggests that presenilins are catalytic components of γ‐secretase, the protease responsible for generating the Alzheimer amyloid β‐protein. However, the differential localization of presenilins to early secretory compartments and γ‐secretase substrates to late secretory compartments and the plasma membrane (the ‘spatial paradox’) argues against this view. We investigated this issue by studying the localization of nicastrin, another putative γ‐secretase component, and its association with presenilin‐1 into proteolytically active complexes. Glycosidase digests revealed that nicastrin exists in multiple glycoforms and is terminally sialylated, a modification often associated with thetrans‐Golgi network. Trafficking of nicastrin to thetrans‐Golgi network was confirmed by density gradient fractionation and immunofluorescence microscopy. In presenilin‐deficient cells, however, nicastrin trafficking and maturation were abnormal, as the protein was restricted to early secretory compartments and failed to be sialylated. Mature sialylated nicastrin intrans‐Golgi network fractions was complexed quantitatively with N‐ and C‐terminal fragments of presenilin‐1, whereas immature nicastrin present in early secretory compartments was not. Additionally,trans‐Golgi network fractions contained the γ‐secretase substrate β‐amyloid precursor protein C83 and were enriched in presenilin‐dependent γ‐secretase proteolytic activity. The results resolve the apparent spatial paradox by demonstrating that presenilin–nicastrin complexes and presenilin‐dependent γ‐secretase activity are co‐localized to a late secretory compartment. The findings provide further evidence that presenilin‐containing complexes are the γ‐secretase, and indicate that presenilins also regulate γ‐secretase assembly.