Tsuzuki K, Fukatsu R, Takamaru Y, Fujii N, Takahata N
Department of Microbiology, Sapporo Medical University School of Medicine, Japan.
Brain Res. 1994 Oct 3;659(1-2):213-20. doi: 10.1016/0006-8993(94)90881-8.
The principal neuropathological feature of Alzheimer's disease is extracellular deposition of approximately 4-kDa proteinous fragment, designated as beta-amyloid peptides (beta/A4 peptides) derived by proteolytic cleavage from amyloid precursor protein (APP), a large cell-surface receptor-like protein. There has been evidence that APP is proteolytically degraded in the secretory and endosomal/lysosomal pathways. The pathway in which APP is cleaved to generate beta/A4 peptides is still not identified. To clarify the intracellular processing of APP into the generation of beta/A4 peptides, we detected and characterized potentially amyloidogenic or non-amyloidogenic fragments using newly established monoclonal and polyclonal antibodies in the cultured cells with or without leupeptin, potent lysosomal protease inhibitor of lysosome. APP fragments of 50 and 20 kDa containing full-length beta/A4 peptides were identified in the cultured cells. Immunoblot analysis, biochemical study for specific marker enzyme activity of the fractions obtained from subcellular fractionation, sucrose density gradient centrifugation indicated that the 50-kDa APP fragment was produced in the compartment closely related to endosomal/lysosomal system. Our data suggest that the endosomal/lysosomal pathway is involved in the processing and generation of beta/A4 peptides.
阿尔茨海默病的主要神经病理学特征是细胞外沉积一种约4 kDa的蛋白质片段，称为β-淀粉样肽（β/A4肽），它是由淀粉样前体蛋白（APP）经蛋白水解切割产生的，APP是一种大型细胞表面受体样蛋白。有证据表明APP在分泌途径和内体/溶酶体途径中被蛋白水解降解。APP被切割产生β/A4肽的途径仍未明确。为了阐明APP在细胞内加工生成β/A4肽的过程，我们使用新建立的单克隆抗体和多克隆抗体，在有或没有亮抑酶肽（一种有效的溶酶体蛋白酶抑制剂）的培养细胞中，检测并鉴定了潜在的淀粉样生成或非淀粉样生成片段。在培养细胞中鉴定出了含有全长β/A4肽的50 kDa和20 kDa的APP片段。免疫印迹分析、对亚细胞分级分离得到的各组分特定标记酶活性的生化研究、蔗糖密度梯度离心表明，50 kDa的APP片段是在内体/溶酶体系统密切相关的区室中产生的。我们的数据表明，内体/溶酶体途径参与了β/A4肽的加工和生成。
