Zhou W, Scott S A, Shelton S B, Crutcher K A
Department of Neurosurgery, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0515, USA.
Neuroscience. 2006 Dec;143(3):689-701. doi: 10.1016/j.neuroscience.2006.08.019. Epub 2006 Sep 25.
Proteolysis of apolipoprotein E (apoE) may be involved in the pathogenesis of Alzheimer's disease (AD). We previously identified aspartic protease(s) as possibly contributing to the proteolysis of apoE in human brain homogenates. The current study used biochemical and immunohistochemical methods to examine whether cathepsin D (catD) and cathepsin E (catE), candidate aspartic proteases, may be involved in apoE proteolysis. CatD was found to proteolyze both lipid-free recombinant full-length human apoE and lipidated human plasma full-length apoE (apoE4/dipalmitoylphosphatidylcholine-reconstituted discs). CatE was found to proteolyze lipid-free recombinant human apoE to a much greater extent than lipidated apoE. This proteolysis, as well as proteolysis of human apoE added to brain homogenates from apoE-deficient mice, was inhibited by pepstatin A (an aspartic protease inhibitor), but not by phenylmethanesulfonyl fluoride (a serine protease inhibitor). The major apoE fragment obtained with catD included the receptor-binding domain and had an apparent molecular weight similar to that found in human brain homogenates. There was little immunoreactivity for catE in AD brain tissue sections. In contrast, qualitative and quantitative analyses of immunostained sections of the frontal cortex revealed that catD and apoE are colocalized in a subset of predominantly dense-core neuritic plaques and in some neurofibrillary tangles. A positive correlation was observed between estimated duration of illness and the percentage of apoE-positive plaques that were also catD-positive. These results suggest that aspartic proteases, catD in particular, may be involved in proteolysis of apoE and perhaps contribute to the generation of apoE fragments previously implicated in AD pathology.
载脂蛋白E（apoE）的蛋白水解作用可能参与阿尔茨海默病（AD）的发病机制。我们之前已确定天冬氨酸蛋白酶可能与人脑匀浆中apoE的蛋白水解有关。本研究采用生化和免疫组化方法，以检测天冬氨酸蛋白酶的候选者组织蛋白酶D（catD）和组织蛋白酶E（catE）是否参与apoE的蛋白水解。研究发现，catD能使无脂质的重组人全长apoE和脂质化的人血浆全长apoE（apoE4/二棕榈酰磷脂酰胆碱重构盘）发生蛋白水解。研究发现，catE对无脂质的重组人apoE的蛋白水解作用比对脂质化apoE的蛋白水解作用要强得多。这种蛋白水解作用以及添加到apoE缺陷小鼠脑匀浆中的人apoE的蛋白水解作用，均受到胃蛋白酶抑制剂A（一种天冬氨酸蛋白酶抑制剂）的抑制，但不受苯甲基磺酰氟（一种丝氨酸蛋白酶抑制剂）的抑制。用catD获得的主要apoE片段包括受体结合结构域，其表观分子量与人脑匀浆中发现的相似。AD脑组织切片中catE的免疫反应性较弱。相比之下，额叶皮质免疫染色切片的定性和定量分析显示，catD和apoE共定位于主要为致密核心的神经炎性斑块的一个亚群以及一些神经原纤维缠结中。在估计的病程与也是catD阳性的apoE阳性斑块的百分比之间观察到正相关。这些结果表明，天冬氨酸蛋白酶，尤其是catD，可能参与apoE的蛋白水解，并且可能有助于生成先前与AD病理学相关的apoE片段。
