Binding of phosphorylated oligosaccharides to immobilized phosphomannosyl receptors.
We have purified phosphomannosyl-enzyme receptors from bovine liver on an affinity column composed of glycoproteins isolated from Dictyostelium discoideum secretions. Binding of human fibroblast beta-hexosaminidase B to receptors reconstituted into phosphatidylcholine liposomes was 1) specifically inhibited by mannose 6-phosphate, but not mannose 1-phosphate or glucose 6-phosphate, and 2) had properties similar to the previously reported binding of enzyme to receptors on cell surfaces and isolated membranes. In order to determine the structural features of the phosphomannosyl recognition marker required for receptor recognition, we covalently coupled purified receptor to an agarose gel bead support for affinity chromatography of phosphorylated, high mannose-type oligosaccharides isolated from fibroblast secretions radiolabeled with [2-3H]mannose. Neutral oligosaccharides and oligosaccharides containing one or two phosphates in phosphodiester linkage were not retained by the receptor column. By contrast, oligosaccharides bearing one phosphomonoester moiety were retarded on the column; those bearing two phosphomonoesters were bound to the column and were eluted with 10 mM mannose 6-phosphate. The binding of the oligosaccharides to the immobilized receptor correlates with their ability to be pinocytosed by fibroblasts and shows that the preferred recognition marker for the phosphomannosyl-enzyme receptor is a high mannose-type oligosaccharide chain bearing two uncovered phosphomannosyl groups.
D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D004023:Dictyostelium; D006023:Glycoproteins; D008099:Liver / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism*; D010766:Phosphorylation; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear; D013329:Structure-Activity Relationship
D006023:Glycoproteins; D009844:Oligosaccharides; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; C034304:cation-dependent mannose-6-phosphate receptor