Molecular Biological Studies on Trafficking and Processing of Amyloid Precursor Protein

对淀粉样前体蛋白(APP)在细胞内的转运和加工以产生淀粉样β蛋白(Abeta)进行了研究。1.针对细胞内特定蛋白的抗体：我们成功地制备了针对细胞内特异性蛋白ERGIC53、半乳糖基转移酶、组织蛋白酶D的特异性抗体。2.培养细胞的APP转运：APP和Abeta免疫反应位于细胞表面，胞浆中有囊泡结构。蛋白质印迹分析显示，微粒体组分中有50、20 kDa的淀粉样变性片段，以及4 kDa的Abeta片段。3.灯盏花素A和莫能菌素对APP加工的影响：灯盏花素A和莫能菌素能抑制蛋白质从内质网到高尔基体、从高尔基体到另一个细胞隔室的运输。在灯盏花素A处理下，培养细胞胞浆内出现大量空泡，并用抗ERGIC53进行免疫标记。莫能菌素处理后，核周细胞质内出现空泡，并被抗APP和抗高尔基体抗体染色。4.野生型APP770和瑞典突变型APP在培养的HeLa细胞中高表达APP。APP免疫反应阳性细胞在内质网、细胞表面和胞浆内的小泡中均有增强。Northern酶联免疫吸附试验检测到APP基因和瑞典型基因的表达水平较质粒对照增加约3~6倍，为APP可能在内质网或溶酶体中产生Aβ提供了新的证据，也表明APP可能参与内质网和溶酶体的合成。

Intracellular trafficking and processing of amyloid precursor protein (APP) to generate amyloid beta protein (Abeta) was studied.1.Antibodies against specific protein in intracellular compartments : We were successful in raising specific antibodies against intracellular compartment specific protein, ERGIC53, galactosyltransferase, cathepsin D.2.APP trafficking in cultured cells : APP and Abeta immunoreactivities were observed in cell surface, and vesicular structures in cytoplasm. Western blot analyzes showed 50,20 kDa amyloidogenic fragments, and 4 kDa, apparently Abeta, in microsomal fraction.3.Effect of brefeldin A and monensin on APP processing : Brefeldin A and monensin are able to inhibit protein trafficking from endoplasmic reticulum (ER) to Golgi apparatus, and from Golgi apparatus to another cellular compartments. Under brefeldin A treatment, numerous vacuoles appeared in the cytoplasm of the cultured cells, and the vacuoles were immunolabeled with anti-ERGIC53. With monensin treatment, vacuoles appeared in perinuclear cytoplasm, and were stained with anti-APP and anti-Golgi antibodies. Western blot analysis showed reduced intensity of Abeta 4 kDa band.4.APP trafficking in Hela cells transfected with wild type APP770, and Swedish mutant cDNA : Wild type APP770 and Swedish mutant APP were overexpressed in cultured HeLa cells. APP immunoreactivities appeared to increase in ER,cell surface, and vesicles in the cytoplasm of these transfected cells. APP mRNA and Swedish type mRNA were determined with Northern ELISA,and turned out to increase about 3 to 6 fold compared to plasmid controls.Our data provide new evidence indicating that APP may process to generate Abeta either in ER,or lysosomes, or both compartments may be involved.