Surface membrane biotinylation efficiently mediates the endocytosis of avidin bioconjugates into nucleated cells.
Here we demonstrate that biotin covalently attached to cell surface obligates existing receptors to endocytose avidin bioconjugates into nucleated cells. Incubation of fluorescein-labeled avidin with biotinylated cell lines resulted in uniform and rapid surface attachment and endocytosis compared with no detectable association of the avidin-conjugated dye with unbiotinylated cells. Uptake was detected within minutes with efficiencies approaching 100% in cell lines and freshly obtained peripheral blood mononuclear cells. After 24 h, avidin was barely detectable on the surface of the nucleated cells. In marked contrast, fluorescent avidin remained exclusively on the external membrane of erythrocytes after 24 h. To investigate biotin-mediated endocytosis for the delivery of DNA, we prepared polyethylenimine-avidin (PEI-avidin) conjugates. Surface biotinylation significantly increased the transfection efficiencies of PEI-avidin condensed plasmid DNA coding green fluorescent protein (GFP) to the level of transferrin-receptor targeted gene delivery (15-20% GFP positive cells in culture after 48 h). The increase in transfection efficiency was blocked by the addition of free avidin or biotin to the culture medium. Biotin covalently bound to cell surface membrane proteins efficiently mediates the entry of avidin bioconjugates into nucleated cells.
Wojda
U
U
Laboratory of Chemical Biology, National Institute of Diabetes and Digestive and Kidney Diseases and Metabolic Disease Branch, National Institutes of Health, Bethesda, Maryland 20892, USA.
Goldsmith
P
P
Miller
J L
JL
D001360:Avidin / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D001710:Biotin / Q000494:pharmacology; D019921:Biotinylation; D001773:Blood Cells / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004705:Endocytosis; D019793:Fluorescein; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D006801:Humans; D019169:Jurkat Cells; D004915:Leukemia, Erythroblastic, Acute; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive; D008164:Luminescent Proteins / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured
D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D001360:Avidin; D049452:Green Fluorescent Proteins; D001710:Biotin; D004247:DNA; D019793:Fluorescein