Analysis of the mechanisms of beta-amyloid accumulation by 68kDa serine protease with beta-secretase activity

具有β-分泌酶活性的68kDa丝氨酸蛋白酶积累β-淀粉样蛋白的机制分析

• 批准号: 07670173
• 负责人: MATSUMOTO Akira
• 金额: $ 1.15万
• 依托单位: KOBE UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07670173/
• 关键词: beta-amyloid; serine protease; aggregation; Alzheimer's disease; proteolysis; cDNA; heparan sulfate; extracellular matrix; 脳老化; 分子生物学; βセクレターゼ; 細胞外マトリックス; 陰性荷電; ヘパラン硫酸; 複合糖質

(1) Establishment of the efficient preparation method for the 68kDa serine protease and its related enzymes utilizing antibody-specific and ligand-specific affinity chromatography.(2) Establishment of an in vitro system mimicking human brain extracellular matrix to analyze functions of these proteases.(3) Analysis of aggregation and degradation of natural Abeta-harboring substrates. Highmolecular weight aggregated product was most efficiently generated when C-terminal substrates were co-incubated with collagen type 4. On the other hand, N-terminal substrates with extracellular domains of APP were excellent substrates for this protease, and it seems that the efficiency of proteolysis is related to the glycoconjugate binding do main within beta-amyloid.(4) Analysis using sugar-modifying enzymes revealed that human brain 68kDa serine protease contains heparan sulfate giycoconjugates. This finding implies that both substrate and protease are modified by glycoconjugates, suggesting their significance in specificity and topological restriction of proteolysis in vivo.(5) Structural analysis of cDNA encoding the protease.(6) Future prospects : Several proteases with similar biochemical properties were prepared from human brain by antibody-specific and ligand-specific affinity chromatography. Further study of these enzymes seems to be important in view of a series of serine proteases participating in blood coagulation. Isolation and identification of specific inhibitors are also of interest to understand the functions of these proteases.

(1)建立了利用抗体特异性和配体特异性亲和层析高效制备68 kDa丝氨酸蛋白酶及其相关酶的方法。(2)体外模拟人脑细胞外基质系统的建立以分析这些蛋白酶的功能。(3)天然Abeta-携带基质的聚集和降解的分析。高分子量聚集产物是最有效地产生时，C-末端底物共孵育与胶原蛋白4型。另一方面，N-末端底物与APP的胞外结构域是这种蛋白酶的优良底物，并且似乎蛋白水解的效率与β-淀粉样蛋白内的糖缀合物结合有关。(4)用糖修饰酶分析显示人脑68 kDa丝氨酸蛋白酶含有硫酸乙酰肝素糖缀合物。这一发现意味着底物和蛋白酶都被糖缀合物修饰，表明它们在体内蛋白水解的特异性和拓扑限制中的意义。(5)编码蛋白酶的cDNA的结构分析。(6)未来前景：利用抗体特异性和配体特异性亲和层析技术，从人脑中制备了几种生化性质相似的蛋白酶。鉴于一系列丝氨酸蛋白酶参与血液凝固，对这些酶的进一步研究似乎是重要的。特异性抑制剂的分离和鉴定对于理解这些蛋白酶的功能也是有意义的。

项目成果

Akira Matsumoto: "The 68 kDa β-secretase with heparan sulfate is expressed in serum and lymphocyte cytosol of normal aged and Alzheimer's disease patient" Alzheimer's Research. 2. 115-120 (1996)

Akira Matsumoto：“68 kDa β-分泌酶与硫酸乙酰肝素在正常老年人和阿尔茨海默病患者的血清和淋巴细胞胞浆中表达”阿尔茨海默病研究，2. 115-120 (1996)。

Akira Matsumoto: "A serine protease in Alzheimer's disease cells cleaves a 16K-peptide with fianking residues upstream to beta-amyloid-N-terminus as natural substrate" Neuroscience Letters. 195. 171-174 (1995)

Akira Matsumoto：“阿尔茨海默病细胞中的丝氨酸蛋白酶会裂解 16K 肽，其上游残基位于 β-淀粉样蛋白 N 末端作为天然底物”《神经科学快报》。

Matsumoto, A.: "Molecular cloning of human cDNA with a sequence highly similar to that of the dihydrofolate reductase gene in brain libraries derived from Alzheimer's disease patients" Eur. J. Biochem.230. 337-343 (1995)

Matsumoto, A.：“人类 cDNA 的分子克隆，其序列与阿尔茨海默氏病患者脑文库中的二氢叶酸还原酶基因高度相似”。

松本 明: "アルツハイマー病におけるカルシウム依存性プロテアーゼ" Clinical calcium. 15. 1287-1290 (1995)

Akira Matsumoto：“阿尔茨海默病中的钙依赖性蛋白酶”临床钙。15。1287-1290（1995）

Akira Matsumoto: "The 68 kDa beta-secretase with haparan sulfate is expressed in serum and lymphocyte cytosol of normal aged and Alzheimer's disease patient" Alzheimer's Research. 2. 115-120 (1996)

Akira Matsumoto：“68 kDa β-分泌酶与硫酸乙酰肝素在正常老年人和阿尔茨海默病患者的血清和淋巴细胞胞浆中表达”阿尔茨海默病研究。