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

本文对乳脂球膜（MFGM）糖蛋白的结构分析、与刷边膜的相互作用、合成及翻译后修饰进行了研究，以阐明其生理功能。所得结果总结如下：(1)与刷边膜相互作用：牛MFGM糖蛋白在还原和非还原条件下分离，转移到PVDF膜上。将印迹的糖蛋白与小鼠和大鼠刷状缘膜制剂孵育，用碱性磷酸酶活性染色检测印迹的MFGM糖蛋白与刷状缘膜的结合情况。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边制剂进行进一步分析。(2) MFGM糖蛋白的糖链及其修饰酶：为了阐明MFGM糖蛋白糖链在乳汁和初乳中糖转移酶，尤其是半乳糖转移酶（GalTase）与刷状边界膜相互作用中的作用。建立了一种高灵敏度的半乳糖酶活性测定方法。结果表明，半乳糖基化在初乳中是自发发生的，半乳糖基化的靶底物是MFGM糖蛋白。(3) MFGM糖蛋白的生物合成：分析小鼠乳腺中MFGM糖蛋白及其基因的表达，提示高脂喂养导致乳脂生物合成减少，但不影响MFGM糖蛋白的合成。我们还发现，仅在哺乳期MFG-E8的EGF-like结构域和C结构域之间，含有o链糖链的Pro/Thr-rich结构域还被掺入。进一步分析了信号分子在乳腺上皮细胞中的表达和作用方式，以及蛋白磷酸酶对乳蛋白（包括MFGM糖蛋白）基因表达的负调控作用。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