Lysosomal enzyme phosphorylation in mouse lymphoma cell lines with altered asparagine-linked oligosaccharides.

The targeting of acid hydrolases to lysosomes involves phosphorylation of mannose residues on these enzymes.To determine whether alterations in the structure of the oligosaccharide acceptor affect formation of the mannose 6-phosphate recognition marker, two mouse lymphoma cell lines altered in the assembly of asparagine-linked oligosaccharides were studied.Cells were labeled with [2-3 H]mannose and the phosphorylated oligosaccharides were isolated and characterized.PhaR2.7 cells, which are deficient in glucosidase II activity, formed less oligosaccharide-bound mannose 6-phosphate than parent cells.The major phosphorylated oligosaccharide formed by Pha R 2 ' 7 contained a single phosphodiester located on the branch linked al,6 to the fl-linked mannose; the majority of these molecules were glucosylated.Relatively few oligosaccharides containing two phosphate esters were isolated and those formed were not glucosylated.This suggests that glucose residues present on high mannose oligosaccharides prevent phosphorylation of mannose residues on the branch linked al,3 to the Bf-linked mannose.In contrast, Thy-1-cells synthesized a normal level of oligosaccharide-bound mannose 6-phosphate despite forming truncated, high mannose-type units.These oligosaccharides contained only four mannose residues and a single phosphate, present as either a phosphodiester or phosphomonoester.,f-Galactosidase isolated from Thy-1-cells bound to human fibroblast membranes and was endocytosed by intact fibroblasts to a similar extent as parent enzyme.These findings demonstrate that altered high mannose-type oligosaccharides on lysosomal enzymes are phosphorylated.However, the extent of phosphorylation and the distribution of the esters is dependent upon the structure of the acceptor oligosaccharide.In addition, the presence of monophosphorylated oligosaccharides on acid hydrolases is sufficient for their recognition and translocation by the mannose 6-phosphate receptor.In many cell types the targeting of newly synthesized acid hydrolases to lysosomes is mediated by phosphomannosyl residues present on high mannose-type oligosaccharide units