Structural studies of the phosphorylated high mannose-type oligosaccharides on human beta-glucuronidase

Phosphomannosyl residues present on numerous acid hydrolases serve a critical role in mediating the endocytosis and intracellular transport of these glycoproteins. Previous work established that the mannose 6-phosphate on lysosomal enzymes is present on endoglycosidase H releasable oligosaccharides and that much of the phosphate is in diester linkage. In order to determine the number and location of the phosphates as well as the precise arrangement of the neutral sugar residues, we examined the structures of the phosphorylated oligosaccharides from a single acid hydrolase, human beta-glucuronidase isolated from spleen. The beta-glucuronidase-derived phosphorylated oligosaccharides are all high mannose-type oligosaccharides whose linkages correspond to previously described prototypical high mannose structures. They contain 1 or 2 moieties of mannose 6-phosphate/oligosaccharide. The major species contains 1 phosphate in diester linkage and represents approximately 63% of the phosphorylated oligosaccharides. Only 15% of the phosphorylated oligosaccharides have their phosphate exclusively in monoester linkage. The phosphate(s) present on these molecules is heterogeneous in location, but all of the phosphate present on the branch linked to the 3-carbon of the beta-linked mannose is found on its innermost alpha-1,2-linked mannose. Analysis of the phosphate-covering moiety showed it to be alpha-linked N-acetylglucosamine in most, if not all, cases.