Ho L, Fukuchi K i, Younkin S G
Department of Neurosciences, Case Western Reserve University, Cleveland, Ohio 44106, USA.
J Biol Chem. 1996 Nov 29;271(48):30929-34. doi: 10.1074/jbc.271.48.30929.
The insoluble amyloid deposited extracellularly in the brains of patients with Alzheimer's disease (AD) is composed of amyloid beta protein, a approximately 4-kDa secreted protein that is derived from a set of large proteins collectively referred to as the amyloid beta protein precursor (betaAPP). During normal processing the betaAPP is cleaved by beta secretase, producing a large NH2-terminal secreted derivative (sAPPbeta) and a COOH-terminal fragment beginning at Abeta1, which is subsequently cleaved by gamma secretase releasing secreted Abeta. Most secreted Abeta is Abeta1-40, but approximately 10% of secreted Abeta is Abeta1-42. Alternative betaAPP cleavage by alpha secretase produces a slightly longer NH2-terminal secreted derivative (sAPPalpha) and a COOH-terminal fragment beginning at Abeta17, which is subsequently cleaved by gamma secretase releasing a approximately 3-kDa secreted form of Abeta (P3). Several of the betaAPP isoforms that are produced by alternative splicing contain a 56-amino acid Kunitz protease inhibitor (KPI) domain known to inhibit proteases such as trypsin and chymotrypsin. To determine whether the KPI domain influences the proteolytic cleavages that generate Abeta, we compared Abeta production in transfected cells expressing human KPI-containing betaAPP751 or KPI-free betaAPP695. We focused on Abetas ending at Abeta42 because these forms appear to be most relevant to AD. Using specific sandwich enzyme-linked immunosorbent assays, we analyzed full-length Abeta1-42 and total Abeta ending at Abeta42 (Abeta1-42 + P3(42)). In addition, we analyzed the large secreted derivatives produced by alpha secretase (sAPPalpha) and beta secretase (sAPPbeta). In mouse teratocarcinoma (P19) cells expressing betaAPP695 or betaAPP751, expression of the KPI-containing betaAPP751 resulted in the secretion of a lower percentage of P3(42) and sAPPalpha and a correspondingly higher percentage of Abeta1-42 and sAPPbeta. Similar results were obtained in human embryonic kidney (293) cells. These results indicate that expression of the KPI domain reduces alpha secretase cleavage so that less P3 and relatively more full-length Abeta are produced. Thus, in human brain and in animal models of AD, the amount of KPI-containing betaAPP produced may be an important factor influencing Abeta deposition.
阿尔茨海默病（AD）患者大脑中细胞外沉积的不溶性淀粉样蛋白由淀粉样β蛋白组成，它是一种约4 kDa的分泌蛋白，源自一组统称为淀粉样β蛋白前体（βAPP）的大蛋白。在正常加工过程中，βAPP被β分泌酶切割，产生一个大的NH2末端分泌衍生物（sAPPβ）和一个从Aβ1开始的COOH末端片段，该片段随后被γ分泌酶切割，释放出分泌型Aβ。大多数分泌的Aβ是Aβ1-40，但约10%的分泌型Aβ是Aβ1-42。α分泌酶对βAPP的另一种切割产生一个稍长的NH2末端分泌衍生物（sAPPα）和一个从Aβ17开始的COOH末端片段，该片段随后被γ分泌酶切割，释放出一种约3 kDa的分泌型Aβ（P3）。通过可变剪接产生的几种βAPP异构体含有一个56个氨基酸的Kunitz蛋白酶抑制剂（KPI）结构域，已知该结构域可抑制诸如胰蛋白酶和胰凝乳蛋白酶等蛋白酶。为了确定KPI结构域是否影响产生Aβ的蛋白水解切割，我们比较了在表达含人KPI的βAPP751或不含KPI的βAPP695的转染细胞中Aβ的产生情况。我们关注以Aβ42结尾的Aβ，因为这些形式似乎与AD最相关。使用特异性夹心酶联免疫吸附测定法，我们分析了全长Aβ1-42和以Aβ42结尾的总Aβ（Aβ1-42 + P3(42)）。此外，我们分析了由α分泌酶（sAPPα）和β分泌酶（sAPPβ）产生的大分泌衍生物。在表达βAPP695或βAPP751的小鼠畸胎瘤（P19）细胞中，含KPI的βAPP751的表达导致P3(42)和sAPPα的分泌百分比降低，相应地Aβ1-42和sAPPβ的分泌百分比升高。在人胚肾（293）细胞中也获得了类似的结果。这些结果表明，KPI结构域的表达减少了α分泌酶的切割，从而产生较少的P3和相对较多的全长Aβ。因此，在人类大脑和AD动物模型中，含KPI的βAPP的产生量可能是影响Aβ沉积的一个重要因素。
