Neurobiology of Disease Laboratory, Genetics and Aging Research Unit, MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, USA.
Mol Neurodegener. 2010 Dec 23;5:61. doi: 10.1186/1750-1326-5-61.
The voltage-gated sodium channel β2 subunit (Navβ2) is a physiological substrate of BACE1 (β-site APP cleaving enzyme) and γ-secretase, two proteolytic enzymes central to Alzheimer's disease pathogenesis. Previously, we have found that the processing of Navβ2 by BACE1 and γ-secretase regulates sodium channel metabolism in neuronal cells. In the current study we identified the BACE1 cleavage sites in human Navβ2.
We found a major (147-148 L↓M, where ↓ indicates the cleavage site) and a minor (144145 L↓Q) BACE1 cleavage site in the extracellular domain of human Navβ2 using a cell-free BACE1 cleavage assay followed by mass spectrometry. Next, we introduced two different double mutations into the identified major BACE1 cleavage site in human Navβ2: 147LM/VI and 147LM/AA. Both mutations dramatically decreased the cleavage of human Navβ2 by endogenous BACE1 in cell-free BACE1 cleavage assays. Neither of the two mutations affected subcellular localization of Navβ2 as confirmed by confocal fluorescence microscopy and subcellular fractionation of cholesterol-rich domains. Finally, wildtype and mutated Navβ2 were expressed along BACE1 in B104 rat neuroblastoma cells. In spite of α-secretase still actively cleaving the mutant proteins, Navβ2 cleavage products decreased by ~50% in cells expressing Navβ2 (147LM/VI) and ~75% in cells expressing Navβ2 (147LM/AA) as compared to cells expressing wildtype Navβ2.
We identified a major (147-148 L↓M) and a minor (144-145 L↓Q) BACE1 cleavage site in human Navβ2. Our in vitro and cell-based results clearly show that the 147-148 L↓M is the major BACE1 cleavage site in human Navβ2. These findings expand our understanding of the role of BACE1 in voltage-gated sodium channel metabolism.
电压门控钠离子通道 β2 亚基（Navβ2）是 BACE1（β 位淀粉样前体蛋白裂解酶）和 γ 分泌酶的生理底物，这两种蛋白水解酶是阿尔茨海默病发病机制的核心。此前，我们发现 BACE1 和 γ 分泌酶对 Navβ2 的加工调节神经元细胞中钠离子通道的代谢。在本研究中，我们鉴定了人 Navβ2 中的 BACE1 切割位点。
我们使用无细胞 BACE1 切割测定法结合质谱法，在人 Navβ2 的细胞外结构域中发现了一个主要（147-148 L↓M，其中↓表示切割位点）和一个次要（144-145 L↓Q）BACE1 切割位点。接下来，我们在人 Navβ2 中鉴定的主要 BACE1 切割位点引入了两个不同的双突变：147LM/VI 和 147LM/AA。这两种突变都显著降低了无细胞 BACE1 切割测定中人 Navβ2 的切割。两种突变均不影响 Navβ2 的亚细胞定位，这一点通过共聚焦荧光显微镜和富含胆固醇的区域的亚细胞分级分离得到证实。最后，野生型和突变型 Navβ2 与 B104 大鼠神经母细胞瘤细胞中的 BACE1 一起表达。尽管 α-分泌酶仍能有效地切割突变蛋白，但与表达野生型 Navβ2 的细胞相比，表达 Navβ2（147LM/VI）的细胞中的 Navβ2 切割产物减少了约 50%，而表达 Navβ2（147LM/AA）的细胞中的 Navβ2 切割产物减少了约 75%。
我们鉴定了人 Navβ2 中的一个主要（147-148 L↓M）和一个次要（144-145 L↓Q）BACE1 切割位点。我们的体外和基于细胞的结果清楚地表明，147-148 L↓M 是人 Navβ2 中的主要 BACE1 切割位点。这些发现扩展了我们对 BACE1 在电压门控钠离子通道代谢中的作用的理解。
