Uptake of neoglycoproteins via membrane lectin(s) of L1210 cells evidenced by quantitative flow cytofluorometry and drug targeting.
The presence and the sugar specificity of membrane lectins on the cell surface of mouse L1210 leukemia cells were demonstrated by using various neoglycoproteins (glycosylated serum albumin) substituted with fluorescein or methotrexate. Neoglycoproteins were prepared by reaction of glycosidophenylisothiocyanates with bovine serum albumin. The binding of neoglycoproteins to L1210 cells depends on the nature of the carried sugar and on the number of bound sugar residues per neoglycoprotein molecule. The best results were obtained with fucosylated serum albumin containing 25 +/- 5 residues of fucose. The amount of cell-associated fluorescein-labeled neoglycoproteins was several fold higher at 37 degrees C than at 4 degrees C suggesting a specific endocytotic process. The membrane lectin-mediated endocytosis was further demonstrated by showing that the cell-associated fluorescence upon cell incubation in the presence of fluorescein-labeled neoglycoproteins at 37 degrees C increased several fold after addition of monensin, a proton/sodium ionophore known to raise the pH of endosomes and lysosomes. The analysis of fluorescein-labeled neoglycoproteins association to the L1210 cells were achieved by quantitative flow cytofluorometry after standardization with calibrated polystyrene sulfonate beads carrying various amounts of 1-(fluoresceinylthioureido)-4,8-diazalicosane. In addition, the cytotoxicity of neoglycoprotein-bound methotrexate was shown to be related to the presence and to the nature of the carried sugar: fucosylated serum albumin was shown to be the most efficient neoglycoprotein carrier, and to have a cytotoxicity close to that of anti L1210 cell IgM monoclonal antibody carrying methotrexate.
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D017127:Glycation End Products, Advanced; D006863:Hydrogen-Ion Concentration; D037102:Lectins; D007939:Leukemia L1210 / Q000378:metabolism* / Q000473:pathology; D008727:Methotrexate / Q000633:toxicity; D051379:Mice; D012709:Serum Albumin / Q000378:metabolism*; D013861:Thiocyanates; D000093423:Glycated Serum Albumin
D005452:Fluoresceins; D005456:Fluorescent Dyes; D017127:Glycation End Products, Advanced; D037102:Lectins; D012709:Serum Albumin; D013861:Thiocyanates; D016650:Fluorescein-5-isothiocyanate; D008727:Methotrexate; D000093423:Glycated Serum Albumin