Analvsis of localization of a glycosyltransferase and transfort of glycolipids by gene manipulation

通过基因操作分析糖基转移酶的定位和糖脂的转移

• 批准号: 06670146
• 负责人: FURUKAWA Keiko
• 金额: $ 1.28万
• 依托单位: Nagasaki University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06670146/
• 关键词: glycosyltransferase; N-glycosylation; localization; gene modification; ganglioside; Golgi局在; 基質特異性; Golgi膜

The amino acid sequence deduced from the cloned human cDNA of beta-1,4-N-acetyl-galactosaminyltransferase (GalNAc-T ; EC2.4.1.92) gene predicted three potential sites for N-linked glycosylation. Although many glycosyltransferases isolated contain from 2 to 6 N-glycosylation sites, their significance has not been adequately demonstrated. To clarify the roles of N-glycosylation in GalNAc-T function, we generated a series of mutant cDNAs, in which some or all of the glycosylation recognition sites were eliminated by polymerase chain reaction( PCR)-mediated site directed mutagenesis. Using transcription/translation in vitro, we confirmed the all potential N-glycosylation sites could be used. Although cell lines transfected with mutant cDNAs showed equivalent levels of GalNAcbeta1*4 (NeuAcalpha2*3) Galbeta1*4GlcCer (G_<M2>) to that of the wild-type, the extracts from mutant cDNA transfectants demonstrated lower enzyme activity than in the wild-type. The decrease in enzyme activity was more evident as the number of deglycosylated sites increased, with about 90% decrease in a totally deglycosylated mutant. The enzyme kinetics analysis revealed no significant change of K_m among wild-type and mutant cDNA products. The intracellular localization of GalNAc-T expressed in transfectants with wild-type or mutant cDNAs also showed a similar perinuclear pattern (Golgi pattern). These results suggest that N-linked carbohydrates on GalNAc-T are required for regulating the stability of the enzyme structure.

从β-1，4-N-乙酰氨基半乳糖转移酶（GalNAc-T ; EC2.4.1.92）基因的克隆人cDNA推导的氨基酸序列预测了N-连接糖基化的三个潜在位点。虽然许多分离的糖基转移酶含有2至6个N-糖基化位点，但它们的重要性尚未得到充分证明。为了阐明N-糖基化在GalNAc-T功能中的作用，我们产生了一系列突变的cDNA，其中部分或全部糖基化识别位点通过聚合酶链反应（PCR）介导的定点突变被消除。使用体外转录/翻译，我们证实了所有潜在的N-糖基化位点都可以使用。虽然用突变cDNA转染的细胞系显示出与野生型相同水平的GalNAc β 1 *4（NeuAcalpha 2 *3）Galbeta 1 *4GlcCer（G_<M2>），但突变cDNA转染子的提取物显示出比野生型低的酶活性。随着去糖基化位点数量的增加，酶活性的降低更加明显，在完全去糖基化的突变体中降低约90%。酶动力学分析表明，野生型和突变型cDNA产物的Km值无明显变化。在具有野生型或突变cDNA的转染子中表达的GalNAc-T的细胞内定位也显示出类似的核周模式（高尔基模式）。这些结果表明，N-连接的碳水化合物的GalNAc-T是需要调节的酶结构的稳定性。

项目成果

Kogo Takamiya: "T cell receptor-mediated stimulatoin of mouse thymocytes induces up-regulation of the GM2/GD2 synthase gene" FEBS Letters. 358. 79-83 (1995)

Kogo Takamiya：“T 细胞受体介导的小鼠胸腺细胞刺激诱导 GM2/GD2 合酶基因上调”FEBS Letters。

Hamagichi,M.,et al.: "The effects the site-directed removal of N-glycosylation sites from GM2/DM2 synthase on its function." Biochemical J.312. 273-280 (1995)

Hamagichi,M.,et al.：“从 GM2/DM2 合酶中定点去除 N-糖基化位点对其功能的影响。”

Takamiya,K.: "T cell receptor-mediated stimulation of mouse thymocytes induces up-regulation of the GM2/GD2 synthase gene." FEBS Lett.358. 79-83 (1995)

Takamiya,K.：“T 细胞受体介导的小鼠胸腺细胞刺激可诱导 GM2/GD2 合酶基因上调。”

Lutz, M.S., et al.: "Cloned beta1,4 N-acetylgalactosaminyltransferase synthesizes GA2 as well as gangliosides GM2 and GD2. GM3 synthesis has priority over GA2 synthesis for utilization of lactosylceramide substrate in vivo." The Journal of Biological Chem

Lutz, M.S. 等人：“克隆的 β1,4 N-乙酰半乳糖氨基转移酶合成 GA2 以及神经节苷脂 GM2 和 GD2。对于体内乳糖神经酰胺底物的利用，GM3 合成优先于 GA2 合成。”

Yamashiro, S., et al.: "Substrate specificity of beta1,4-N-acetylgalactosaminyltransferase in vitro and in cDNA-transfected cells. GM2/GD2 synthase efficiently generates asialo-GM2 in certain cells." The Journal of Biological Chemistry. 270. 6149-6155 (19

Yamashiro, S. 等人：“β1,4-N-乙酰半乳糖胺基转移酶在体外和 cDNA 转染细胞中的底物特异性。GM2/GD2 合酶在某些细胞中有效生成去唾液酸-GM2。”