An Attempt to Identify Amyloid Precursor Protein Processing Pathway Involving Lysosomal System by Vesicle Specific Protein

溶酶体途径被认为是淀粉样蛋白前体蛋白（APP）片段和β淀粉样蛋白（Abeta）的形成位点。然而，溶酶体是细胞内交通流汇合的地方。我们在此描述了一种通过囊泡特异性蛋白1识别溶酶体系统参与APP加工途径的尝试。针对运输囊泡特异性蛋白产生抗体：有网格蛋白包被的囊泡和非网格蛋白包被的囊泡，称为运输囊泡的包被蛋白包被的囊泡。我们成功地培养了针对网格蛋白氨基酸序列同源的合成肽和β - cop2的抗体。康那霉素A （Con A）对APP蛋白水解过程的影响：康那霉素A通过网格蛋白包被的囊泡抑制从细胞表面到胞内的选择性内吞作用。1)培养肌细胞的组织病理学研究：在Con A作用12小时后，培养的肌细胞的质膜上出现了更多的空泡，直到24小时时，空泡数量增加。2) APP和Abeta定位的免疫病理研究：Con A诱导的空泡用抗APP和抗Abeta抗体标记。用网格蛋白抗体也获得了许多这样的液泡。有趣的是，β - cop抗体在未经Con A处理的细胞质中标记出独特的点状结构。用Con A处理培养的肌细胞，空泡用β - cop抗体标记。但内质网特异性抗ergic 53和高尔基体特异性抗garactosyltransferase蛋白均无免疫反应。APP蛋白水解过程的生化研究：对有/无Con a处理的培养肌细胞进行代谢标记，用针对APP和Abeta的特异性抗体免疫沉淀细胞裂解液中的APP和Abeta。随后，洗脱液进行电泳分离，然后进行自电泳。未经Con A处理的细胞裂解液中检测到4kda Abeta片段，Con A处理后该4kda Abeta片段的强度增加。我们的数据表明，非网格蛋白包被的囊泡到酸性区室，溶酶体，可能在淀粉样蛋白片段和β的产生中起重要作用，除了网格蛋白包被的囊泡。少

The lysosomal pathway has been suggested as the site of formation for amyloidogenic amyloid precursor protein (APP) fragment and amyloid beta protein (Abeta). Lysosomes are, however, meeting places in which several streams of intracellular traffic converge. We describe here an attempt to identify APP processing pathway involving lysosomal system by vesicle specific protein1. Antibodies raised against transport vesicle specific proteins : There are clathrin-coated vesicle, and non-clathrin coated vesicle, designated as coatmer protein-coated vesicle of transport vesicles. We were successful in raising antibodies against synthetic peptides homologous to amino acid sequence of clathrin, and beta-COP.2. Proteolytic processing of APP in cultured human myocyte treated with/without concanamycin A (Con A) : Con A inhibits selectively endocytosis from cell surface to intracsllular compartments via clathrin-coated vesicles.1) Histopathological studies of cultured myocytes : Vacuoles appeared ben … More eath the plasma membrane in the cultured myocytes 12 hours after Con A treatment, and increase in number until up to 24 hours.2) Immunopathological studies of APP and Abeta localization : Con A induced vacuoles were labeled with anti-APP,and anti-Abeta antibodies. A number of these vacuoles were also atained with clathrin antibody. Interestingly, beta-COP antibody labeled unique dot-like structures in the cytoplasm without Con A treatment. Cultured myocytes treated with Con A,vacuoles were labeled with beta-COP antibody. But no immunoreactivities were observed with anti-ERGIC 53, specific for endoplasmic reticulum, and with anti-garactosyltransferase, specific protein to Golgi apparatus.3. Biochemical studies on proteolytic processing of APP : Metabolic labeling of cultured myocytes were performed of cultured myocytes treated with/without Con A.APP and Abeta in the cell lysates were immunoprecipitated with specific antibodies against APP,and Abeta. Subsequently, eluates were separated on electrophoresis, and then subjected to autoragiography. 4 kDa Abeta fragment was detected in cell lysate without Con A treatment, and this 4 kDa Abeta fragment increased their intensity after Con A treatment.Our data indicate that non-clathrin coated vesicles to acidic compartments, lysosomes, may play an important role in the generation of amyloidogenic fragments and Abeta, in addition to clathrin-coated vesicles. Less