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

(1)建立了利用抗体特异性和配基特异性亲和层析技术高效制备68 kDa丝氨酸蛋白酶及其相关酶的方法。(2)建立了模拟人脑细胞外基质的体外系统，用于分析这些酶的功能。(3)天然β-底物的聚集和降解分析。当C端底物与4型胶原共孵育时，高分子量聚集产物的生成效率最高。另一方面，含有APP胞外结构域的N端底物是该酶的良好底物，蛋白降解的效率似乎与β-淀粉样蛋白中的糖共轭结合有关。(4)糖修饰酶分析表明，人脑68 kDa丝氨酸蛋白酶含有硫酸乙酰肝素糖偶联物。这一发现表明底物和蛋白酶都被糖偶联物修饰，这表明它们在体内蛋白降解的特异性和拓扑限制方面具有重要意义。(5)编码该酶的cDNA的结构分析。(6)未来展望：通过抗体特异性和配基特异性亲和层析从人脑中制备出几种具有相似生化性质的蛋白酶。鉴于一系列丝氨酸蛋白酶参与凝血，对这些酶的进一步研究似乎很重要。分离和鉴定特定的抑制物对于了解这些酶的功能也很有意义。

(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.