Molecular Biological Studies on Trafficking and Processing of Amyloid Precursor Protein

研究了淀粉样前体蛋白（APP）在细胞内的运输和加工以产生淀粉样β蛋白（Abeta）。1.针对细胞内区室特异性蛋白的抗体：我们成功地制备了针对细胞内区室特异性蛋白、ERGIC 53、半乳糖基转移酶、组织蛋白酶D的特异性抗体。2. APP在培养细胞中的运输：APP和Abeta免疫反应阳性细胞位于细胞表面，胞浆内可见泡状结构。蛋白质印迹分析显示，微粒体中有50、20 kDa的淀粉样蛋白片段和4 kDa的A β。3.布雷菲德菌素A和莫能菌素对APP加工的影响：布雷菲德菌素A和莫能菌素能够抑制蛋白质从内质网（ER）向高尔基体的运输，以及从高尔基体向另一个细胞区室的运输。在布雷菲德菌素A处理下，培养细胞的细胞质中出现许多空泡，并且用抗ERGIC 53免疫标记空泡。用莫能菌素处理，空泡出现在核周细胞质，并与抗APP和抗高尔基体抗体染色。Western blot分析显示Abeta 4 kDa条带的强度降低。4.野生型APP 770和Swedish突变体cDNA转染的HeLa细胞中APP的运输：在培养的HeLa细胞中，野生型APP 770和Swedish突变体APP过表达。APP免疫反应性似乎增加ER，细胞表面，和这些转染细胞的细胞质中的囊泡。用北方酶联免疫吸附试验（ELISA）检测APP mRNA和Swedish型mRNA，结果显示APP mRNA和Swedish型mRNA比质粒对照增加了约3 ~ 6倍，我们的数据提供了新的证据，表明APP可能在内质网或溶酶体中加工产生Abeta，或者两者都参与。

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.