Boland K, Behrens M, Choi D, Manias K, Perlmutter D H
Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
J Biol Chem. 1996 Jul 26;271(30):18032-44. doi: 10.1074/jbc.271.30.18032.
There is now extensive evidence that amyloid-beta peptide is toxic to neurons and that its cytotoxic effects can be attributed to a domain corresponding to amyloid-beta 25-35, GSNKGAIIGLM. We have shown recently that the serine proteinase inhibitor (serpin)-enzyme complex receptor (SEC-R), a receptor initially identified for binding of alpha1-antitrypsin (alpha1-AT) and other serine protease inhibitors, also recognizes the amyloid-beta 25-35 domain. In fact, by recognizing the amyloid-beta 25-35 domain, SEC-R mediates cell surface binding, internalization, and degradation of soluble amyloid-beta peptide. In this study, we examined the possibility that SEC-R mediates the neurotoxic effect of amyloid-beta peptide. A series of peptides based on the sequences of amyloid-beta peptide and alpha1-AT was prepared soluble in dimethyl sulfoxide or insoluble in water and examined in assays for SEC-R binding, for cytotoxicity in neuronal PC12 cells and murine cortical neurons in primary culture, and for aggregation in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The results show that amyloid-beta peptide 25-35 and amyloid-beta peptide 1-40 prepared soluble in dimethyl sulfoxide compete for binding to SEC-R, are nontoxic, and migrate as monomers in SDS-PAGE analysis. In contrast, the same peptides aged in water did not compete for binding to SEC-R but were toxic and migrated as aggregates in SDS-PAGE. An all-D-amyloid-beta 25-35 peptide was not recognized at all by SEC-R but retained full toxic/aggregating properties. Using a series of deleted, substituted, and chimeric ambeta/alpha1-AT peptides, toxicity correlated well with aggregation but poorly with SEC-R recognition. In a subclone of PC12 cells which developed resistance to the toxic effect of aggregated amyloid-beta 25-35 there was a 2.5-3-fold increase in the number of SEC-R molecules/cell compared with the parent PC12 cell line. These data show that SEC-R does not mediate the cytotoxic effect of aggregated amyloid-beta peptide. Rather, SEC-R could play a protective role by mediating clearance and catabolism of soluble, monomeric amyloid-beta peptide, if soluble amyloid-beta peptide proves to be an in vivo precursor of the insoluble, toxic peptide.
目前有大量证据表明β淀粉样肽对神经元有毒性，其细胞毒性作用可归因于与β淀粉样肽25 - 35（GSNKGAIIGLM）相对应的结构域。我们最近发现，丝氨酸蛋白酶抑制剂（serpin）-酶复合物受体（SEC-R）最初被确定为可结合α1-抗胰蛋白酶（α1-AT）和其他丝氨酸蛋白酶抑制剂，它也能识别β淀粉样肽25 - 35结构域。事实上，通过识别β淀粉样肽25 - 35结构域，SEC-R介导可溶性β淀粉样肽在细胞表面的结合、内化及降解。在本研究中，我们探究了SEC-R介导β淀粉样肽神经毒性作用的可能性。制备了一系列基于β淀粉样肽和α1-AT序列的肽，这些肽可溶于二甲基亚砜或不溶于水，并对其进行SEC-R结合检测、对原代培养的神经元PC12细胞和小鼠皮质神经元的细胞毒性检测以及十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析中的聚集检测。结果表明，溶于二甲基亚砜的β淀粉样肽25 - 35和β淀粉样肽1 - 40竞争结合SEC-R，无毒，且在SDS-PAGE分析中以单体形式迁移。相比之下，在水中老化的相同肽不竞争结合SEC-R，但有毒，且在SDS-PAGE中以聚集体形式迁移。全D型β淀粉样肽25 - 35根本不被SEC-R识别，但保留了全部毒性/聚集特性。使用一系列缺失、取代和嵌合的β淀粉样肽/α1-AT肽，毒性与聚集密切相关，但与SEC-R识别关系不大。在对聚集的β淀粉样肽25 - 35的毒性作用产生抗性的PC12细胞亚克隆中，与亲代PC12细胞系相比，每个细胞中SEC-R分子数量增加了2.5 - 3倍。这些数据表明，SEC-R不介导聚集的β淀粉样肽的细胞毒性作用。相反，如果可溶性β淀粉样肽被证明是不溶性有毒肽的体内前体，SEC-R可能通过介导可溶性单体β淀粉样肽的清除和分解代谢发挥保护作用。
