Regulation of ganglioside biosynthesis by enzyme complex formation of glycosyltransferases
Regulation of ganglioside biosynthesis by enzyme complex formation of glycosyltransferases
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DOI：
10.1021/bi0259958
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发表时间：
2002-09-24
期刊：
影响因子：
2.9
通讯作者：
Yu, RK
中科院分区：
文献类型：
--
作者：
Bieberich, E;MacKinnon, S;Yu, RK
神经节苷脂生物合成中的三种关键调节酶，唾液酸转移酶I（ST 1）、唾液酸转移酶II（ST 2）和N-乙酰氨基半乳糖转移酶I（GaINAcT），已在F-11 A细胞中表达为与绿色、黄色或红色荧光蛋白（GFP、YFP或RFP）的融合蛋白。F-11 A细胞是鼠神经母细胞瘤F-11细胞的亚株，其仅含有低内源性ST 2和GaINAcT活性。融合蛋白的亚细胞定位已被确定通过荧光显微镜，和神经节苷脂组成的这些细胞进行了分析，通过高效薄层色谱法（HPTLC）。ST 2-GFP（85 kDa）显示出明显的高尔基体定位，而ST 1-YFP（85 kDa）和GalNAcT-RFP（115 kDa）广泛分布于ER和高尔基体中。未转染的F-11 A细胞主要含有GM 3，而ST 2或GaINAcT的稳定转染导致b系列复合神经节苷脂（BCGs）的主要表达。该结果表明，ST 2的表达增强内源性GaINAcT的活性，反之亦然。该反应的特异性已经通过用洗涤剂溶解的酶进行的体外活性测定来验证，表明在ST 2和GaINAcT之间形成酶复合物，但不与ST 1形成酶复合物。复合物的形成也已经通过在用GalNAcT-HA-RFP瞬时转染时ST 2-GFP的免疫共沉淀和通过局限于高尔基体的GFP至RFP FRET信号来验证。FRET分析还表明，ST 2-GFP紧密结合芘标记的GM 3，但不ST 1。我们假设ST 2-GM 3复合物与GaINAcT相关，导致高尔基体中GM 3向GD 3和BCGs的转化增强。两者合计，我们的研究结果支持的概念，神经节苷脂的生物合成受到严格调控的糖基转移酶复合物在ER和/或高尔基体的形成。
Three key regulatory enzymes in ganglioside biosynthesis, sialyltransferase I (ST1), sialyltransferase II (ST2), and N-acetylgalactosaminyltransferase I (GaINAcT), have been expressed as fusion proteins with green, yellow, or red fluorescent protein (GFP, YFP, or RFP) in F-11A cells. F-11A cells are a substrain of murine neuroblastoma F-11 cells that contain only low endogenous ST2 and GaINAcT activity. The subcellular localization of the fusion proteins has been determined by fluorescence microscopy, and the ganglioside composition of these cells was analyzed by high-performance thin-layer chromatography (HPTLC). ST2-GFP (85 kDa) shows a distinct Golgi localization, whereas ST1-YFP (85 kDa) and GalNAcT-RFP (115 kDa) are broadly distributed in ER and Golgi. Untransfected F-11A cells contain mainly GM3, whereas stable transfection with ST2 or GaINAcT results in the predominant expression of b-series complex gangliosides (BCGs). This result indicates that the expression of ST2 enhances the activity of endogenous GaINAcT and vice versa. The specificity of this reaction has been verified by in vitro activity assays with detergent-solubilized enzymes, suggesting the formation of an enzyme complex between ST2 and GaINAcT but not with ST1. Complex formation has also been verified by co-immunoprecipitation of ST2-GFP upon transient transfection with GalNAcT-HA-RFP and by GFP-to-RFP FRET signals that are confined to the Golgi. FRET analysis also suggests that ST2-GFP binds tightly to pyrene-labeled GM3 but not to ST1. We hypothesize that an ST2-GM3 complex is associated with GaINAcT, resulting in the enhanced conversion of GM3 to GD3 and BCGs in the Golgi. Taken together, our results support the concept that ganglioside biosynthesis is tightly regulated by the formation of glycosyltransferase complexes in the ER and/or Golgi.