Secretion of a peripheral membrane protein, MFG-E8, as a complex with membrane vesicles.
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 immunofluorescent 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 detergent-sensitive. 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 culture medium. These results suggest a possible role of MFG-E8 in the membrane vesicle secretion, such as budding or shedding of plasma membrane (microvesicles) and exocytosis of endocytic multivesicular bodies (exosomes).
Oshima
Kenji
K
Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan.
Aoki
Naohito
N
Kato
Takeo
T
Kitajima
Ken
K
Matsuda
Tsukasa
T
D000818:Animals; D000954:Antigens, Surface; D002460:Cell Line; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D006017:Glycolipids / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism*; D066292:Lipid Droplets; D008321:Mammary Glands, Animal / Q000166:cytology / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008566:Membranes / Q000378:metabolism; D051379:Mice; D008894:Milk Proteins; D009154:Mutation; D010718:Phosphatidylserines / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion
D000954:Antigens, Surface; D006017:Glycolipids; D006023:Glycoproteins; D008562:Membrane Glycoproteins; C490919:Mfge8 protein, mouse; D008894:Milk Proteins; D010718:Phosphatidylserines; D011994:Recombinant Proteins; C110367:milk fat globule