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Studies on functions of milk-fat globule membrane glycoproteins with the guidance of interaction of brush border membrane

刷状缘膜相互作用指导乳脂球膜糖蛋白功能研究

基本信息

批准号：
10660121
负责人：
MATSUDA Tsukasa
金额：
$ 2.56万
依托单位：
NAGOYA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 2000
项目状态：
已结题

项目摘要

I this research the structural analyses, the interaction to brush border membrane, the synthesis and post-translational modification of the milk-fat globule membrane (MFGM) glycoproteins were investigated in order to elucidate physiological roles of the MFGM glycoproteins. The results obtained are summarized as follows. (1) Interaction to brush border membrane : Bovine MFGM glycoproteins were separated under reduced and nonreduced conditions, and transferred to PVDF membrane. The blotted glycoproteins were incubated with mouse and rat brush border membrane preparations, and the alkaline-phosphatase activity-staining was done to detect binding of brush border membrane to the blotted MFGM glycoproteins. Several MFGM glycoproteins, including MFG-8 and butyrophilin, were stained weakly. Further analysis would be required by using several different preparations of brush border. (2) Sugar chains of MFGM glycoproteins and modifying enzymes : To elucidate roles of sugar chains of MFGM glycopro … More teins in the interaction between MFGM and brush border membrane, sugar transferases, especially galactosyl transferase (GalTase), in milk and colostrum were investigated. A highly sensitive assay method for GalTase activity assay was established. By using this method it was suggested that galactosylation happened spontaneously in colostrum and the some target substrates of the galactosylation were MFGM glycoproteins. (3) Biosynthesis of MFGM glycoproteins : Expression of MFGM glycoprotein and their genes in mouse mammary gland was analyzed, and the decrease in milk fat biosynthesis induced by high fat feeding was suggested not to affect the MFGM glycoprotein synthesis. It was also found that the Pro/Thr-rich domain with O-linked sugar chains was additionally incorporated into between the EGF-like and C domains of MFG-E8 only at the lactation stage.Furthermore, expression and mode of action of signaling molecules in mammary epithelial cells were analyzed, and the contribution of protein phosphatases on the negative regulation of gene expression of milk proteins including MFGM glycoproteins. Less

本研究对乳脂球膜糖蛋白的结构分析、与刷状缘膜的相互作用、糖蛋白的合成及翻译后修饰等方面进行了研究，旨在阐明乳脂球膜糖蛋白的生理功能。所获得的结果总结如下。(1)与刷状缘膜的相互作用：在还原和非还原条件下分离牛MFGM糖蛋白，并转移到PVDF膜上。将印迹的糖蛋白与小鼠和大鼠的刷状缘膜共孵育，碱性磷酸酶染色检测刷状缘膜与印迹的MFGM糖蛋白的结合。几种MFGM糖蛋白，包括MFG-8和ButyroPhilin，染色较弱。需要使用几种不同的刷子边缘制备方法进行进一步分析。(2)MFGM糖蛋白糖链及其修饰酶：阐明MFGM糖蛋白糖链…的作用研究了乳汁和初乳中糖转移酶，特别是半乳糖基转移酶(GalTase)与MFGM相互作用的更多因素。建立了一种高灵敏度的半乳糖苷酶活性测定方法。结果表明，初乳中的半乳糖基化是自发发生的，其靶标底物为MFGM糖蛋白。(3)MFGM糖蛋白的生物合成：分析了MFGM糖蛋白及其基因在小鼠乳腺中的表达，认为高脂喂养导致乳脂生物合成减少不影响MFGM糖蛋白的合成。此外，研究还发现，只有在哺乳期，MFG-E8的富含Pro/Thr结构域的O-连接糖链才被附加到EGF样区和C区之间。此外，还分析了信号分子在乳腺上皮细胞中的表达和作用方式，以及蛋白磷酸酶在包括MFGM糖蛋白在内的乳蛋白基因表达负调控中的作用。较少