Ladror U S, Snyder S W, Wang G T, Holzman T F, Krafft G A
Abbott Laboratories, Abbott Park, Illinois 60064.
J Biol Chem. 1994 Jul 15;269(28):18422-8.
Amyloid beta (A beta) is a 39-43-residue protein that originates from proteolysis of the beta-protein precursor (beta PP) and accumulates in senile plaques in brains of Alzheimer's disease (AD) patients. Mutant beta PP, which incorporates an AD-causing double mutation at positions 687-688, has been shown to enhance A beta production in transfected cells. In this work we investigate the susceptibility of the mutant beta PP sequence to proteolytic cleavage by proteinases from human brain. Internally quenched fluorogenic substrates were used that encompass the NH2-terminal sequence of A beta from wild-type beta PP, the double mutant, and the two single substitutions. Proteinase activity in brain extract cleaved the mutant substrate 100-fold faster than the wild-type substrate and the partial mutants 25-fold faster. The major cleavage site in all substrates was at the amyloidogenic Asp1 site. The brain activity appeared to be cathepsin D (CD), as indicated by similarities to purified CD in 1) the rate and site of substrates cleavage, 2) the pH optima, and 3) the sensitivity to pepstatin A. The increased activity against the mutant substrate was not shared by cathepsins B and C, pepsin, HIV proteinase, and Candida albicans Asp-proteinase. Furthermore, CD cleaved a substrate that incorporates the COOH terminus of A beta at positions equivalent to Thr43 and Ala42, at ratios of 68% and 32%, respectively. CD degraded A beta 1-40 into six fragments but A beta 1-42 was completely resistant to digestion, probably because of its aggregation characteristics. These results indicate that CD is capable of producing the cleavages resulting in A beta production and that it may prove to be a suitable therapeutic target.
淀粉样β蛋白（Aβ）是一种由β-蛋白前体（βPP）经蛋白水解产生的39 - 43个氨基酸残基的蛋白质，它会在阿尔茨海默病（AD）患者大脑的老年斑中积聚。已证明，在687 - 688位含有导致AD的双突变的突变型βPP，能增强转染细胞中Aβ的产生。在本研究中，我们探究了突变型βPP序列对人脑海蛋白酶解切割的敏感性。使用了内部淬灭的荧光底物，其包含野生型βPP、双突变体以及两个单取代突变体Aβ的NH2末端序列。脑提取物中的蛋白酶活性切割突变底物的速度比野生型底物快100倍，比部分突变体底物快25倍。所有底物的主要切割位点都在淀粉样变的Asp1位点。脑内活性似乎是组织蛋白酶D（CD），这体现在以下几个方面与纯化的CD相似：1）底物切割的速度和位点；2）最适pH值；3）对胃蛋白酶抑制剂A的敏感性。组织蛋白酶B和C、胃蛋白酶、HIV蛋白酶以及白色念珠菌天冬氨酸蛋白酶对突变底物没有显示出这种增强的活性。此外，CD切割了一个包含Aβ羧基末端、相当于Thr43和Ala42位置的底物，切割比例分别为68%和32%。CD将Aβ1 - 40降解为六个片段，但Aβ1 - 42完全抵抗消化，这可能是由于其聚集特性。这些结果表明，CD能够产生导致Aβ产生的切割作用，并且可能被证明是一个合适的治疗靶点。
