Higaki J, Catalano R, Guzzetta A W, Quon D, Navé J F, Tarnus C, D'Orchymont H, Cordell B
Scios, Inc., Mountain View, California 94043, USA.
J Biol Chem. 1996 Dec 13;271(50):31885-93. doi: 10.1074/jbc.271.50.31885.
The events leading to the formation of beta-amyloid (betaA4) from its precursor (betaAPP) involve proteolytic cleavages that produce the amino and carboxyl termini of betaA4. The enzyme activities responsible for these cleavages have been termed beta- and gamma-secretase, respectively, although these protease(s) have not been identified. Since betaA4 is known to possess heterogeneity at both the amino and carboxyl termini, beta- and gamma-secretases may actually be a collection of proteolytic activities or perhaps a single proteolytic enzyme with broad amino acid specificity. We investigated the role of cathepsin D in the processing of betaAPP since this enzyme has been widely proposed as a gamma-secretase candidate. Treatment of a synthetic peptide that spans the gamma-secretase site of betaAPP with human cathepsin D resulted in the cleavage of this substrate at Ala42-Thr43. A sensitive liquid chromatography/mass spectrometry technique was also developed to further investigate the ability of cathepsin D to process longer recombinant betaAPP substrates (156 and 100 amino acids of betaAPP carboxyl terminus) in vitro. The precise cathepsin D cleavage sites within these recombinant betaAPP substrates were identified using this technique. Both recombinant substrates were cleaved at the following sites: Leu49-Val50, Asp68-Ala69, Phe93-Phe94. No cleavages were observed at putative gamma-secretase sites: Val40-Ile41 or Ala42-Thr43, suggesting that cathepsin D is not gamma-secretase as defined by these betaA4 termini. Under conditions where the betaAPP156 substrate was first denatured prior to cathepsin D digestion, two additional cleavage sites near the amino terminus of betaA4, Glu-3-Val-2 and Glu3-Phe4, were observed, indicating that cathepsin D cleavage of betaAPP is influenced by the structural integrity of the substrate. Taken together, these results indicate that in vitro, cathepsin D is unlikely to function as gamma-secretase; however, the ability of this enzyme to efficiently cleave betaAPP substrates at nonamyloidogenic sites within the molecule may reflect a role in betaAPP catabolism.
由其前体（β淀粉样前体蛋白，betaAPP）形成β淀粉样蛋白（betaA4）的过程涉及蛋白水解切割，产生betaA4的氨基和羧基末端。尽管尚未鉴定出负责这些切割的蛋白酶，但负责这些切割的酶活性分别被称为β-分泌酶和γ-分泌酶。由于已知betaA4在氨基和羧基末端均具有异质性，β-和γ-分泌酶实际上可能是一系列蛋白水解活性，或者可能是一种具有广泛氨基酸特异性的单一蛋白水解酶。我们研究了组织蛋白酶D在betaAPP加工过程中的作用，因为该酶已被广泛认为是γ-分泌酶的候选者。用人组织蛋白酶D处理跨越betaAPP的γ-分泌酶位点的合成肽，导致该底物在Ala42-Thr43处被切割。还开发了一种灵敏的液相色谱/质谱技术，以进一步研究组织蛋白酶D在体外加工更长的重组betaAPP底物（betaAPP羧基末端的156和100个氨基酸）的能力。使用该技术鉴定了这些重组betaAPP底物内组织蛋白酶D的确切切割位点。两种重组底物均在以下位点被切割：Leu49-Val50、Asp68-Ala69、Phe93-Phe94。在假定的γ-分泌酶位点Val40-Ile41或Ala42-Thr43未观察到切割，这表明根据这些betaA4末端定义，组织蛋白酶D不是γ-分泌酶。在组织蛋白酶D消化之前先将betaAPP156底物变性的条件下，在betaA4氨基末端附近观察到另外两个切割位点Glu-3-Val-2和Glu3-Phe4，这表明组织蛋白酶D对betaAPP的切割受底物结构完整性的影响。综上所述，这些结果表明，在体外，组织蛋白酶D不太可能发挥γ-分泌酶的功能；然而，该酶在分子内非淀粉样生成位点有效切割betaAPP底物的能力可能反映了其在betaAPP分解代谢中的作用。
