tailieunhanh - Báo cáo Y học: Secretion of a peripheral membrane protein, MFG-E8, as a complex with membrane vesicles A possible role in membrane secretion

MFG-E8 (milk fat globule-EGF factor 8) is a peripheral membrane glycoprotein, which is expressed abundantly in lactating mammary glands and is secreted in association with fat globules. This protein consists of two-repeated EGF-like domains, a mucin-like domain and two-repeated discoidinlike domains (C-domains), and contains an integrin-binding motif (RGD sequence) in the EGF-like domain. To clarify the role of each domain on the peripheral association with the cell membrane, several domain-deletion mutants of MFG-E8 were expressed in COS-7 cells. . | Eur. J. Biochem. 269 1209-1218 2002 FEBS 2002 Secretion of a peripheral membrane protein MFG-E8 as a complex with membrane vesicles A possible role in membrane secretion Kenji Oshima1 Naohito Aoki1 Takeo Kato2 Ken Kitajima1 and Tsukasa Matsuda1 1Graduate School of Bioagricultural Sciences Nagoya University Nagoya Japan 2Food Research Institute Aichi Prefectural Government Nagoya Japan MFG-E8 milk fat globule-EGF factor 8 is a peripheral membrane glycoprotein which is expressed abundantly in lactating mammary glands and is secreted in association with fat globules. This protein consists of two-repeated EGF-like domains a mucin-like domain and two-repeated discoidin-like domains C-domains and contains an integrin-binding motif RGD sequence in the EGF-like domain. To clarify the role of each domain on the peripheral association with the cell membrane several domain-deletion mutants of MFG-E8 were expressed in COS-7 cells. The immunofluo-rescent staining of intracellular and cell-surface proteins and biochemical analyses of cell-surface-biotinylated and secreted proteins demonstrated that both of the two C-domains were required for the membrane association. During the course of these studies for domain functions MFG-E8 but not C-domain deletion mutants was shown to be secreted as membrane vesicle complexes. By size-exclusion chromatography and ultracentrifugation analyses the complexes were characterized to have a high-molecular mass low density and higher sedimentation velocity and to be detergentsensitive. Not only such a exogenously expressed MFG-E8 but also that endogenously expressed in a mammary epithelial cell line COMMA-1D was secreted as the membrane vesicle-like complex. Scanning electron microscopic analyses revealed that MFG-E8 was secreted into the culture medium in association with small membrane vesicles with a size from 100 to 200 nm in diameter. Furthermore the expression of MFG-E8 increased the number of these membrane vesicle secreted into the .

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