Nagai Naoko, Habuchi Hiroko, Kitazume Shinobu, Toyoda Hidenao, Hashimoto Yasuhiro, Kimata Koji
Institute for Molecular Science of Medicine, Aichi Medical University, Yazako, Nagakute, Aichi 480-1195, Japan.
J Biol Chem. 2007 May 18;282(20):14942-51. doi: 10.1074/jbc.M610691200. Epub 2007 Mar 15.
The enzymes involved in glycosaminoglycan chain biosynthesis are mostly Golgi resident proteins, but some are secreted extracellularly. For example, the activities of heparan sulfate 6-O-sulfotransferase (HS6ST) and heparan sulfate 3-O-sulfotransferase are detected in the serum as well in the medium of cell lines. However, the biological significance of this is largely unknown. Here we have investigated by means of monitoring green fluorescent protein (GFP) fluorescence how C-terminally GFP-tagged HS6STs that are stably expressed in CHO-K1 cell lines are secreted/shed. Brefeldin A and monensin treatments revealed that the N-terminal hydrophobic domain of HS6ST3 is processed in the endoplasmic reticulum or cis/medial Golgi. Treatment of HS6ST3-GFP-expressing cells with various protease inhibitors revealed that the cell-permeable beta-secretase inhibitor N-benzyloxycarbonyl-Val-Leu-leucinal (Z-VLL-CHO) specifically inhibits HS6ST secretion, although this effect was specific for HS6ST3 but not for HS6ST1 and HS6ST2. However, Z-VLL-CHO treatment did not increase the molecular size of the HS6ST3-GFP that accumulated in the cell. Z-VLL-CHO treatment also induced the intracellular accumulation of SP-HS6ST3(-TMD)-GFP, a modified secretory form of HS6ST3 that has the preprotrypsin leader sequence as its N-terminal hydrophobic domain. Diminishment of beta-secretase activity by coexpressing the amyloid precursor protein of a Swedish mutant, a potent beta-secretase substrate, also induced intracellular HS6ST3-GFP accumulation. Moreover, Z-VLL-CHO treatment increased the 6-O-sulfate (6S) levels of HS, especially in the disaccharide unit of hexuronic acid-GlcNS(6S). Thus, the HS6ST3 enzyme in the Golgi apparatus and therefore the 6-O sulfation of heparan sulfates in the cell are at least partly regulated by beta-secretase via an indirect mechanism.
参与糖胺聚糖链生物合成的酶大多是驻留在高尔基体的蛋白质，但有些酶会分泌到细胞外。例如，在血清以及细胞系培养基中均可检测到硫酸乙酰肝素6-O-磺基转移酶（HS6ST）和硫酸乙酰肝素3-O-磺基转移酶的活性。然而，其生物学意义在很大程度上尚不清楚。在此，我们通过监测绿色荧光蛋白（GFP）荧光，研究了在CHO-K1细胞系中稳定表达的C末端带有GFP标签的HS6STs是如何被分泌/脱落的。布雷菲德菌素A和莫能菌素处理表明，HS6ST3的N末端疏水结构域在内质网或顺式/中间高尔基体中被加工。用各种蛋白酶抑制剂处理表达HS6ST3-GFP的细胞表明，细胞可渗透的β-分泌酶抑制剂N-苄氧羰基-Val-Leu-亮氨醛（Z-VLL-CHO）特异性抑制HS6ST的分泌，尽管这种作用对HS6ST3具有特异性，而对HS6ST1和HS6ST2则没有。然而，Z-VLL-CHO处理并未增加细胞内积累的HS6ST3-GFP的分子大小。Z-VLL-CHO处理还诱导了SP-HS6ST3(-TMD)-GFP在细胞内的积累，SP-HS6ST3(-TMD)-GFP是HS6ST3的一种修饰分泌形式，其N末端疏水结构域为前胰蛋白酶原前导序列。通过共表达瑞典突变体的淀粉样前体蛋白（一种有效的β-分泌酶底物）来降低β-分泌酶活性，也会诱导细胞内HS6ST3-GFP的积累。此外，Z-VLL-CHO处理增加了硫酸乙酰肝素的6-O-硫酸盐（6S）水平，尤其是在己糖醛酸-GlcNS(6S)的二糖单元中。因此，高尔基体中的HS6ST3酶以及细胞中硫酸乙酰肝素的6-O硫酸化至少部分是由β-分泌酶通过间接机制调控的。
