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

溶酶体途径已被认为是淀粉样蛋白生成淀粉样前体蛋白（APP）片段和淀粉样β蛋白（Abeta）的形成位点。然而，溶酶体是几种细胞内运输流汇合的地方。我们在这里描述了一个尝试，以确定APP加工途径涉及溶酶体系统的囊泡特异性蛋白1。针对转运囊泡特异性蛋白产生的抗体：有网格蛋白包被的囊泡和非网格蛋白包被的囊泡，称为转运囊泡的包被蛋白包被囊泡。结果：1.成功地制备了针对网格蛋白氨基酸序列同源的合成肽和β-COP的抗体。用/不用ConA处理的培养人心肌细胞中APP的蛋白水解过程：ConA通过网格蛋白包被的囊泡选择性地抑制从细胞表面到细胞内的内吞作用。 ...更多信息 2）APP和Abeta定位的免疫病理学研究：用抗APP和抗Abeta抗体标记ConA诱导的空泡。用网格蛋白抗体也能观察到许多空泡。有趣的是，β-COP抗体在没有Con A处理的情况下标记细胞质中独特的点样结构。用ConA处理培养的心肌细胞，用β-COP抗体标记空泡。内质网特异性抗ERGIC 53和高尔基体特异性抗半乳糖基转移酶均未观察到免疫反应性. APP蛋白水解过程的生物化学研究：对用/不用Con A处理的培养的肌细胞进行培养的肌细胞的代谢标记。用针对APP和Abeta的特异性抗体对细胞裂解物中的APP和Abeta进行免疫沉淀。随后，在电泳上分离洗脱液，然后进行自显影。未用Con A处理的细胞裂解液中检测到4kDa Abeta片段，并且该4kDa Abeta片段在Con A处理后增强其强度。我们的数据表明，除了网格蛋白包被的囊泡之外，非网格蛋白包被的囊泡到酸性隔室，溶酶体，可能在淀粉样蛋白生成片段和Abeta的产生中起重要作用。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