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

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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