Glycosylphosphatidylinositol-anchored proteins are not required for crosslinking-mediated endocytosis or transfection of avidin bioconjugates into biotinylated cells.
Even though glycosylphosphatidylinositol (GPI)-anchored proteins lack direct structural contact with the intracellular space, these ubiquitously expressed surface receptors activate signaling cascades and endocytosis when crosslinked by extracellular ligands. Such properties may be due to their association with membrane microdomains composed of glycosphingolipids, cholesterol and some signaling proteins. In this study, we hypothesize that GPI proteins may be required for crosslinking-mediated endocytosis of extracellular bioconjugates. To test this hypothesis, we first biotinylated the surface membranes of native K562 erythroleukemia cells versus K562 cells incapable of surface GPI protein expression. We then compared the entry of fluorescently labeled avidin or DNA condensed on polyethylenimine-avidin bioconjugates into the two biotinylated cell populations. Using fluorescence microscopy, nearly 100% efficiency of fluorescent avidin endocytosis was demonstrated in both cell types over a 24 h period. Surprisingly, plasmid DNA transfer was slightly more efficient among the biotinylated GPI-negative cells as measured by the expression of green fluorescence protein. Our findings that GPI proteins are not required for the endocytosis of avidin bioconjugates into biotinylated cells suggest that endocytosis associated with general membrane crosslinking may be due to overall reorganization of the membrane domains rather than GPI protein-specific interactions.
Laboratory of Chemical Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
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Wojda
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Miller
D001360:Avidin / Q000737:chemistry / Q000235:genetics; D001710:Biotin / Q000737:chemistry; D003432:Cross-Linking Reagents; D004705:Endocytosis; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D017261:Glycosylphosphatidylinositols / Q000096:biosynthesis / Q000737:chemistry*; D004915:Leukemia, Erythroblastic, Acute; D008565:Membrane Proteins / Q000737:chemistry; D008856:Microscopy, Fluorescence; D011094:Polyethyleneimine / Q000737:chemistry; D014162:Transfection; D014407:Tumor Cells, Cultured
D003432:Cross-Linking Reagents; D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; D001360:Avidin; D001710:Biotin; D011094:Polyethyleneimine; D016650:Fluorescein-5-isothiocyanate