MOLECULAR BIOLOGICAL STUDIES OF APP PROCESSING

阿尔茨海默病的主要神经病理特征是细胞外沉积约4 kDa的蛋白质片段，命名为淀粉样β多肽(Abeta)，由淀粉样前体蛋白(APP)--大细胞表面受体样蛋白--分解而来。已有证据表明，APP在分泌途径和内体/溶酶体途径中被蛋白质降解。其中APP被切割以产生Abeta的途径仍未确定。为了阐明APP在细胞内产生Abeta的过程，我们使用新建立的单抗和多克隆抗体来检测和表征潜在的淀粉样变或非淀粉样变片段，这些抗体是针对APP770序列推导出的八种不同的合成肽，以及从含有或不含有亮肽蛋白的培养细胞中获得的亚细胞组分，亮肽素是溶酶体的有效溶酶体蛋白酶抑制剂。在培养的细胞中发现了含有全长Abeta的50 kDa和20 kDa的APP片段。免疫印迹分析、亚细胞分级、蔗糖密度梯度离心法和免疫组织化学观察表明，50 kDa的APP片段产生于与内体/溶酶体系统密切相关的区段。我们的数据表明，内体/溶酶体途径参与了Abeta的处理和生成。

The principal neuropathological feature of Alzheimer's disease is extracellular deposition of about 4 kDa proteinous fragment, designated as amyloid beta peptides (Abeta) derived by proteolytic cleavage from amyloid precursor protein (APP), large cell surface receptor like protein. There have been evidences that APP is proteolytically degraded in the secretory pathway, and endosomal/lysosomal pathway. The pathway is not still identified, in which APP is cleaved to generate Abeta. To clarify the intracellular processing of APP into the generation of Abeta, we detected and characterized potentially amyloidogenic or non-amyloidogenic fragments using newly established monoclonal and polyclonal antibodies, which were raised against eight different synthetic peptides deduced from the sequence of APP770 cDNA,with subcellular fractions obtained from the cultured cells with or without leupeptin, potent lysosomal protease inhibitor of lysosome. APP fragments of 50 and 20 kDa molecular mass containing full-length Abeta 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, together with immunohistochemical observations 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 Abeta.