Lin Bei, Makiko Saito, Masao Iwamori
Department of Obstetrics and Gynecology, the Second Affiliated Hospital of China Medical University, Shenyang 110004, China.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 2005 Dec;27(6):761-6.
To compare the substrate specificity of three murine GDP fucose: beta-galactoside alpha1,2-fucosyltransferases (alpha1,2-FT).
Three members of MFUT- I, -II and -III, coding for a alpha1,2-FT, a GDP-fucose, were cloned from a cDNA of murine small intestine by reverse transcription-polymerase chain reaction. The coding regions were ligated into mammalian expression vector pcDNA 3.1 (pcDNA3.1-MFUT-I, pcDNA3.1-MFUT- II , and pcDNA3.1-MFUT- III) and were transiently transfected into COS-7 cells using a cellphect transfection kit. Then the cells were analyzed for expression and function of alpha1,2-FT and the substrate specificity of three alpha1,2-FT was compared.
MFUT- I, -II, and -III exhibited sequence homology with human H (77%), Se (79%), and Sec1 (75%) genes, respectively. COS-7 cells transfected with pcDNA3.1-MFUT- I and pcDNA3.1-MFUT- II showed alpha1,2-FT activity, but no activity was detected in COS-7 cells transfected with pcDNA3.1- MFUT-III. MFUT- II showed alpha1,2-FT activity with both asialo-monosialoteterahexosyl ganglioside (GA1) and monosialoteterahexosyl ganglioside (GM1) as substrates to produce fucosyl GA1(FGA1) and fucosyl GM1(FGM1), respectively, but MFUT- I only showed alpha1,2-FT activity with GA1. The relative activity of MFUT- II with GA1 was 80-90-folds higher compared with MFUT- I, and the relative activity of MFUT- II with GA1 was 10-20-folds higher than that of GM1. The fucosyltransferase encoded by the MFUT- II gene showed the enzyme activity not only responsible for the synthesis of type 4-H antigens FGA1 and FGM1, but also responsible for the synthesis of type 1-H and 2-H antigens with lactotetraosylceramide and neolactotetraosylceramide as substrates.
MFUT- II is the main alpha1,2-FT in mouse and MFUT- II can product type 4-H antigen FGA1 and FGM1, but MFUT- I only synthesizes FGA1. MFUT-III has no alpha1,2-FT activity.
比较三种小鼠GDP岩藻糖：β-半乳糖苷α1,2-岩藻糖基转移酶（α1,2-FT）的底物特异性。
通过逆转录-聚合酶链反应从鼠小肠cDNA中克隆出编码α1,2-FT（一种GDP-岩藻糖）的MFUT-I、-II和-III的三个成员。将编码区连接到哺乳动物表达载体pcDNA 3.1（pcDNA3.1-MFUT-I、pcDNA3.1-MFUT-II和pcDNA3.1-MFUT-III）中，并使用细胞fect转染试剂盒将其瞬时转染到COS-7细胞中。然后分析细胞中α1,2-FT的表达和功能，并比较三种α1,2-FT的底物特异性。
MFUT-I、-II和-III分别与人H（77%）、Se（79%）和Sec1（75%）基因具有序列同源性。用pcDNA3.1-MFUT-I和pcDNA3.1-MFUT-II转染的COS-7细胞显示出α1,2-FT活性，但用pcDNA3.1-MFUT-III转染的COS-7细胞未检测到活性。MFUT-II以脱唾液酸单唾液酸四己糖神经节苷脂（GA1）和单唾液酸四己糖神经节苷脂（GM1）为底物均显示出α1,2-FT活性，分别产生岩藻糖基GA1（FGA1）和岩藻糖基GM1（FGM1），但MFUT-I仅以GA1显示出α1,2-FT活性。MFUT-II对GA1的相对活性比MFUT-I高80-90倍，MFUT-II对GA1的相对活性比GM1高10-20倍。由MFUT-II基因编码的岩藻糖基转移酶不仅显示出负责合成4-H型抗原FGA1和FGM1的酶活性，还负责以乳糖四糖神经酰胺和新乳糖四糖神经酰胺为底物合成1-H型和2-H型抗原。
MFUT-II是小鼠中的主要α1,2-FT，MFUT-II可产生4-H型抗原FGA1和FGM1，但MFUT-I仅合成FGA1。MFUT-III没有α1,2-FT活性。
