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

本研究从结构分析、与刷状缘膜的相互作用、乳脂球膜糖蛋白的合成和翻译后修饰等方面对乳脂球膜糖蛋白进行了研究，旨在阐明乳脂球膜糖蛋白的生理功能。所得结果总结如下。(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，并转移至PVDF膜。将印迹的MFGM糖蛋白与小鼠和大鼠刷状缘膜孵育，并进行碱性磷酸酶活性染色以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和亲丁酸蛋白，染色较弱。需要使用几种不同的刷状缘制备物进行进一步分析。(2)MFGM糖蛋白的糖链和修饰酶：阐明MFGM糖蛋白糖链的作用 关于我们 本文研究了乳和初乳中MFGM与刷状缘膜、糖转移酶特别是半乳糖基转移酶（GalTase）相互作用的机制。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，初乳中半乳糖基化反应是自发发生的，其靶底物是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，并且表明高脂喂养诱导的乳脂生物合成的减少不影响MFGM糖蛋白的合成。此外，我们还分析了乳腺上皮细胞中信号分子的表达和作用方式，以及蛋白磷酸酶在MFGM糖蛋白等乳蛋白基因表达负调控中的作用。少

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