Adsorptive pinocytosis of phosphorylated oligosaccharides by human fibroblasts.
Adsorptive pinocytosis of lysosomal enzymes by human fibroblasts depends on phosphomannosyl recognition markers on the enzymes and on high affinity receptors on the cell surface. To define the role of phosphorylated oligosaccharides in enzyme recognition, we studied the pinocytosis of [2-3H]mannose-labeled oligosaccharides purified from glycoproteins secreted by fibroblasts. Uptake of the oligosaccharides was inhibited 97% by 2 mM mannose-6-phosphate, 33% by 2 mM glucose 6-phosphate, and 5% or less by 2 mM alpha-methylmannoside, mannose, galactose, or L-fucose. The oligosaccharides were separated into neutral and five anionic species by chromatography on quaternary aminoethyl-Sephadex, characterized, and compared for susceptibility to pinocytosis. Treatment of the phosphorylated oligosaccharides with alkaline phosphatase before or after mild acid hydrolysis demonstrated that they contained one or two phosphates in phosphodiester linkage (covered) or phosphomonoester linkage (uncovered), or two phosphates, one in monoester linkage and one in diester linkage. Neutral oligosaccharides and those with one covered phosphate were not taken up by fibroblasts. Species with one uncovered phosphate or two covered phosphates showed low but detectable uptake. Oligosaccharides isolated as species with two uncovered phosphates, or those converted to this form by mild acid hydrolysis, were taken up 30-fold greater than the lower uptake forms during a 12-h incubation. Thus, oligosaccharides with two uncovered phosphates were far better ligands for the phosphomannosyl receptor than other oligosaccharides on acid hydrolases secreted by fibroblasts and initial rates of uptake of these oligosaccharides were comparable to those reported for several "high uptake" lysosomal enzymes.
D002478:Cells, Cultured; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D006020:Glycopeptides / Q000032:analysis; D006600:Hexosephosphates / Q000494:pharmacology; D006601:Hexoses / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D009844:Oligosaccharides / Q000378:metabolism*; D010766:Phosphorylation; D010873:Pinocytosis / Q000187:drug effects; D013661:Tay-Sachs Disease / Q000378:metabolism*
D006020:Glycopeptides; D006600:Hexosephosphates; D006601:Hexoses; D009844:Oligosaccharides