Parvathy S, Hussain I, Karran E H, Turner A J, Hooper N M
School of Biochemistry and Molecular Biology, University of Leeds, U.K.
Biochemistry. 1999 Jul 27;38(30):9728-34. doi: 10.1021/bi9906827.
The amyloid precursor protein (APP) is proteolytically processed predominantly by alpha-secretase to release the ectodomain (sAPPalpha). In this study, we have addressed the cellular location of the constitutive alpha-secretase cleavage of endogenous APP in a neuronal cell line. Incubation of the neuroblastoma cell line IMR32 at 20 degrees C prevented the secretion into the medium of soluble wild-type APP cleaved by alpha-secretase as revealed by both immunoelectrophoretic blot analysis with a site-specific antibody and immunoprecipitation following metabolic labeling of the cells. No sAPPalpha was detected in the cell lysates following incubation of the cells at 20 degrees C, indicating that alpha-secretase does not cleave APP in the secretory pathway prior to or within the trans-Golgi network. Parallel studies using an antibody that recognizes specifically the neoepitope revealed on soluble APP cleaved by beta-secretase indicated that this enzyme was acting intracellularly. alpha-Secretase is a zinc metalloproteinase susceptible to inhibition by hydroxamate-based compounds such as batimastat [Parvathy, S., et al. (1998) Biochemistry 37, 1680-1685]. Incubation of the cells with a cell-impermeant, biotinylated hydroxamate inhibitor inhibited the release of sAPPalpha by >92%, indicating that alpha-secretase is cleaving APP almost exclusively at the cell surface. The observation that alpha-secretase cleaves APP at the cell surface, while beta-secretase can act earlier in the secretory pathway within the neuronal cell line indicates that there must be strict control mechanisms in place to ensure that APP is normally cleaved primarily by alpha-secretase in the nonamyloidogenic pathway to produce the neuroprotective sAPPalpha.
淀粉样前体蛋白（APP）主要通过α-分泌酶进行蛋白水解加工，以释放胞外结构域（sAPPα）。在本研究中，我们探讨了内源性APP在神经元细胞系中组成型α-分泌酶切割的细胞定位。神经母细胞瘤细胞系IMR32在20℃孵育时，可防止经α-分泌酶切割的可溶性野生型APP分泌到培养基中，这通过位点特异性抗体的免疫电泳印迹分析以及细胞代谢标记后的免疫沉淀得以证实。细胞在20℃孵育后，细胞裂解物中未检测到sAPPα，这表明α-分泌酶在分泌途径中，在反式高尔基体网络之前或之内不会切割APP。使用特异性识别由β-分泌酶切割的可溶性APP上出现的新表位的抗体进行的平行研究表明，该酶在细胞内起作用。α-分泌酶是一种锌金属蛋白酶，易受基于异羟肟酸酯的化合物（如batimastat）抑制[Parvathy, S., 等人（1998年）《生物化学》37卷，1680 - 1685页]。用细胞不可渗透的生物素化异羟肟酸酯抑制剂孵育细胞，可使sAPPα的释放抑制>92%，这表明α-分泌酶几乎仅在细胞表面切割APP。α-分泌酶在细胞表面切割APP，而β-分泌酶可在神经元细胞系的分泌途径中更早发挥作用，这一观察结果表明，必须存在严格的控制机制，以确保APP在非淀粉样生成途径中通常主要由α-分泌酶切割，从而产生神经保护作用的sAPPα。
