Department of Mental Health Promotion, Osaka University Graduate School of Medicine, Toyonaka, Japan.
Department of Pharmacotherapeutics II, Faculty of Pharmacy, Osaka Medical and Pharmaceutical University, Takatsuki, Japan.
Psychogeriatrics. 2023 Mar;23(2):311-318. doi: 10.1111/psyg.12933. Epub 2023 Jan 23.
Amyloid-β peptide is well-known as a pathogen of Alzheimer's disease, but its precursor, amyloid-beta precursor protein (APP), remains unexplained 30 years after its discovery. APP has two homologues called amyloid precursor-like protein 1 (APLP1) and amyloid precursor-like protein 2 (APLP2), and shares a similar structural organisation with them and has partially overlapping functions. APP family proteins are essential for survival, shown by the crossbreeding analysis of knockout mice of APP family molecules, including APLP1 and APLP2. APLP2 is known to play the most important role among them, but the molecular metabolism of APLP2 is only partially understood. Here, we analysed ectodomain shedding and γ-secretase cleavage of APLP2 by molecular biological and biochemical techniques.
We analysed the culture supernatant of HEK293 cells overexpressing APLP2 and human cerebrospinal fluid. For the analysis of secreted APLP2 fragments, we raised the OA603 antibody that reacts with the juxtamembrane domain of APLP2. Substrate cleavage sites were identified by matrix assisted laser desorption/ionisation mass spectrometry.
By overexpressing in HEK293 cells, APLP2 undergoes ectodomain shedding at three sites in the extracellular region by α- and β-secretase-like activity and then is intramembranously cleaved at three sites by γ-secretase. In particular, in shedding, α-secretase-like activity was dominant in HEK cells. Surprisingly, in human cerebrospinal fluid, APLP2-derived metabolic fragments were mainly cleaved by β-secretase-like activity, not by α-secretase-like activity. Because APP is also mainly cleaved by beta-site amyloid precursor protein cleaving enzyme 1 in neurons and APLP1 is expressed exclusively in neurons, these findings suggest that APP family proteins may play a common role via β-secretase-like cleavage in the central nerve system.
Thus, these findings may contribute to a better understanding of the role of APP family proteins in Alzheimer's disease.
淀粉样β肽是阿尔茨海默病的已知病原体，但在发现其 30 年后，其前体淀粉样β前体蛋白（APP）仍然没有得到解释。APP 有两个同源物，称为淀粉样前体样蛋白 1（APLP1）和淀粉样前体样蛋白 2（APLP2），它们与 APP 具有相似的结构组织，具有部分重叠的功能。APP 家族蛋白对生存至关重要，这可以从 APP 家族分子（包括 APLP1 和 APLP2）的基因敲除小鼠的杂交分析中看出。已知 APLP2 发挥着最重要的作用，但 APLP2 的分子代谢仅部分被理解。在这里，我们通过分子生物学和生化技术分析了 APLP2 的外显子脱落和 γ-分泌酶切割。
我们分析了过表达 APLP2 和人脑脊液的 HEK293 细胞培养上清液。为了分析分泌的 APLP2 片段，我们制备了与 APLP2 跨膜结构域反应的 OA603 抗体。基质辅助激光解吸/电离质谱用于鉴定底物切割位点。
通过在 HEK293 细胞中过表达，APLP2 在细胞外区域的三个位点通过 α-和 β-分泌酶样活性进行外显子脱落，然后通过 γ-分泌酶在三个位点进行跨膜切割。特别是在脱落中，HEK 细胞中α-分泌酶样活性占主导地位。令人惊讶的是，在人脑脊液中，APLP2 衍生的代谢片段主要通过β-分泌酶样活性而不是 α-分泌酶样活性被切割。因为 APP 也主要在神经元中被β-位点淀粉样前体蛋白裂解酶 1 切割，而 APLP1 仅在神经元中表达，这些发现表明 APP 家族蛋白可能通过中枢神经系统中的β-分泌酶样切割发挥共同作用。
因此，这些发现可能有助于更好地理解 APP 家族蛋白在阿尔茨海默病中的作用。
Zotero 插件