The alternatively spliced Kunitz protease inhibitor domain alters amyloid beta protein precursor processing and amyloid beta protein production in cultured cells
The alternatively spliced Kunitz protease inhibitor domain alters amyloid beta protein precursor processing and amyloid beta protein production in cultured cells
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DOI：
10.1074/jbc.271.48.30929
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发表时间：
1996-11-29
影响因子：
4.8
通讯作者：
Younkin, SG
中科院分区：
文献类型：
--
作者：
Ho, LB;Fukuchi, K;Younkin, SG
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，这是一种类似于4 kDa分泌蛋白的蛋白质，由一系列统称为淀粉样β蛋白前体(βAPP)的大蛋白衍生而来。在正常的加工过程中，βAPP被β分泌酶切割，产生一个大的NH2末端分泌的衍生物(SAPβ)和一个从Aβ1开始的COOH末端片段，随后被释放的分泌的Aβ的伽马分泌酶切割。分泌最多的Aβ是Aβ1-40，但与10%分泌的Aβ相似的是Aβ1-42。选择性βAPP被α分泌酶切割产生一个略长的NH2末端分泌的衍生物(SAPPα)和一个从Aβ17开始的COOH末端片段，随后被伽马分泌酶切割，释放类似3 kDa的分泌形式的Aβ(P3)，通过选择性剪接产生的几个βAPP亚型包含一个56氨基酸的kunitz蛋白酶抑制(KPI)结构域，已知能抑制胰蛋白酶和胰凝乳酶等蛋白酶，为了确定KPI结构域是否影响产生Aβ的蛋白水解性切割，我们比较了表达含KPI的βAPP751和不含KPI的βAPP695的细胞中Aβ的产生。我们关注A测试版S在A测试版42结束，因为这些表格似乎与AD最相关。利用特异性夹心酶联免疫吸附试验，我们分析了全长Aβ1-42和以Aβ42结尾的总Aβ(Aβ1-42+P3(42))。此外，我们还分析了由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的小鼠畸胎癌细胞(P19)中，含有KPI的βAPP751的表达导致P3(42)和Sappα的分泌百分比降低，而Aβ1-42和Sappβ的分泌百分比相应增加。在人类胚胎肾脏(293)细胞中也得到了类似的结果。这些结果表明，KPI结构域的表达减少了α分泌酶的切割，从而产生了较少的P3和相对较多的全长Aβ。因此，在人脑和AD动物模型中，含有KPI的βAPP的生成量可能是影响Aβ沉积的重要因素。
The insoluble amyloid deposited extracellularly in the brains of patients with Alzheimer's disease (AD) is composed of amyloid beta protein, a similar to 4-kDa secreted protein that is derived from a set of large proteins collectively referred to as the amyloid beta protein precursor (beta APP). During normal processing the beta APP is cleaved by beta secretase, producing a large NH2-terminal secreted derivative (sAPP beta) and a COOH-terminal fragment beginning at A beta 1, which is subsequently cleaved by gamma secretase releasing secreted A beta. Most secreted A beta is A beta 1-40, but similar to 10% of secreted A beta is A beta 1-42. Alternative beta APP cleavage by alpha secretase produces a slightly longer NH2-terminal secreted derivative (sAPP alpha) and a COOH-terminal fragment beginning at A beta 17, which is subsequently cleaved by gamma secretase releasing a similar to 3-kDa secreted form of A beta (P3), Several of the beta APP 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 A beta, we compared A beta production in transfected cells expressing human KPI-containing beta APP751 or KPI-free beta APP695. We focused on A beta s ending at A beta 42 because these forms appear to be most relevant to AD. Using specific sandwich enzyme-linked immunosorbent assays, we analysed full-length A beta 1-42 and total A beta ending at A beta 42 (A beta 1-42 + P3(42)). In addition, we analysed the large secreted derivatives produced by alpha secretase (sAPP alpha) and beta secretase (sAPP beta). In mouse teratocarcinoma (P19) cells expressing beta APP695 or beta APP751, expression of the KPI-containing beta APP751 resulted in the secretion of a lower percentage of P3(42) and sAPP alpha and a correspondingly higher percentage of A beta 1-42 and sAPP beta. 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 A beta are produced. Thus, in human brain and in animal models of AD, the amount of KPI-containing beta APP produced may be an important factor influencing A beta deposition.