GLYCOSYLATION OF AMYLOID PRECURSOR PROTEINS

阿尔茨海默病是一种表现为人类大脑退行性疾病 临床上由痴呆症引起。病理上，一种不寻常的蛋白质沉积， β-淀粉样蛋白(BetaA4)存在于脑血管系统和老年人 斑块。BetaA4是一种由40个氨基酸组成的多肽，由蛋白质降解而成。 一种名为β-淀粉样前体蛋白的较大前体的切割 (BetaAPP)。β-APP是一种具有1-2个N连接的跨膜蛋白 低聚糖和15-20个O-连接的低聚糖。 糖基化在细胞内生物合成中的作用 贩运，表面表达的BetaAPP尚不清楚。此外, 目前尚不清楚BetaAPP糖基化的改变是否会影响 BetaAPP的蛋白质分解处理导致BetaA4的形成增加。 这个项目将通过使用两个细胞培养模型来研究这些问题 系统和从人脑和脑脊液中提纯的BetaAPP。 人畸胎癌细胞系NT2/D1可被诱导 维甲酸孵育诱导神经元分化。这些细胞 合成大量的BetaAPP及其异构体和电泳谱 分化后，BetaAPP的迁移率发生改变。因此， NT2/D1细胞将使我们能够对人的糖基化进行生化分析 并检验这些细胞分化的假说 结果该蛋白质上的寡糖发生了修饰。在……里面 此外，它们将允许我们检验这样一个假设，即改变 分化的NT2/D1细胞中BetaAPP的糖基化改变 这种蛋白质的合成、分泌和蛋白质分解过程。 转人BetaAPP基因中国仓鼠卵巢(CHO)野生型细胞 CDNAs也表达大量的BetaAPP。因此，通过使用 糖基化抑制剂，并通过将BetaAPP CDNA导入凝集素- 具有明确蛋白糖基化缺陷的CHO耐药细胞系，WE 会改变BetaAPP的糖基化。这种方法将使我们能够 检验N-和O-连接的寡糖共价结合的假设 在调节合成、分泌和蛋白分解过程中起重要作用 BetaAPP的加工。 最后，将找到的结果与单元格相关联将非常重要 用人脑发现的培养模型系统。BetaAPP将是 从人脑和脑脊液中提纯并结合 分析了低聚糖。这将使我们能够检验假设 人脑BetaAPP糖基化在衰老过程中发生变化，并在 阿尔茨海默病患者。 这些研究很可能阐明糖基化蛋白的作用。 BetaAPP在阿尔茨海默病的病理生物学中发挥作用。

Alzheimer's disease is a human cerebral degenerative disease manifested clinically by dementia. Pathologically, deposits of an unusual protein, Beta amyloid (BetaA4), are found in the cerebral vasculature and in senile plaques. BetaA4 is a 40 amino acid peptide which is derived by proteolytic cleavage from a larger precursor termed Beta-amyloid precursor protein (BetaAPP). Beta APP is a transmembrane protein with 1-2 N-linked oligosaccharides and 15-20 O-linked oligosaccharides. The role that glycosylation plays in the biosynthesis, intracellular trafficking, and surface expression of BetaAPP is not clear. In addition, it is not known whether alterations in BetaAPP glycosylation can influence proteolytic processing of BetaAPP leading to increased BetaA4 formation. This project will examine these issues by using two cell culture model systems and BetaAPP purified from human brain and cerebrospinal fluid. The NT2/D1 human teratocarcinoma cell line can be induced to undergo neuronal differentiation by incubation with retinoic acid. These cells synthesize abundant amounts of BetaAPP and the isoforms and electrophoretic mobility of BetaAPP are altered following differentiation. Therefore, NT2/D1 cells will allow us to biochemically analyze glycosylation of human BetaAPP and to examine the hypothesis that differentiation of these cells results in modification of the oligosaccharides on this protein. In addition, they will allow us to test the hypothesis that altering the glycosylation of BetaAPP in differentiated NT2/D1 cells changes the synthesis, secretion, and proteolytic processing of this protein. Wild-type Chinese hamster ovary (CHO) cells transfected with human BetaAPP CDNA also express abundant amounts of BetaAPP. Thus, by using glycosylation inhibitors and by transfecting BetaAPP CDNA into lectin- resistant CHO cell lines with defined defects in protein glycosylation, we will alter the glycosylation of BetaAPP. This approach will allow us to test the hypothesis that covalently bound N- and O-linked oligosaccharides are important in modulating the synthesis, secretion, and proteolytic processing of BetaAPP. Finally, it will be important to correlate the results found with the cell culture model systems to those found with human brain. BetaAPP will be purified from human brain and cerebrospinal fluid and the bound oligosaccharides analyzed. This will allow us to test the hypothesis that glycosylation of human brain BetaAPP changes during aging and is altered in patients with Alzheimer's disease. These studies are likely to clarify the role that the glycosylation of BetaAPP plays in the pathobiology of Alzheimer's disease.