Structural analysis of the asparagine-linked oligosaccharides from three lysosomal enzymes of Dictyostelium discoideum. Evidence for an unusual acid-stable phosphodiester.
Lysosomal enzymes of the slime mold Dictyostelium discoideum contain mannose 6-phosphate and bind with high affinity to the phosphomannosyl receptor of human fibroblasts. In this study, we have partially characterized the Asn-linked oligosaccharide units present on these enzymes. [3H]Mannose-labeled alpha-D-mannosidase, beta-D-glucosidase, and beta-D-N-acetylglucosaminidase were purified from the spent growth medium of strain AX3 and glycopeptides were prepared by pronase digestion. Approximately 75% of the glycopeptides contained sulfate residues. These could be removed by solvolysis without degrading the underlying oligosaccharide. Following solvolysis (but not before), the oligosaccharides could be released by endo-beta-N-acetylglucosaminidase H, indicating the presence of high mannose-type units. Greater than 85% of the oligosaccharides contained one or two mannose 6-phosphate residues in the form of an unusual acid-stable phosphodiester. About 3% of the oligosaccharides contained phosphomonoesters and only 6% were neutral species. The major neutral oligosaccharide eluted in the position of Man9GlcNAc when analyzed by high performance liquid chromatography whereas the minor species appeared to be 1-2 residues larger. Acetolysis of the major phosphorylated fractions revealed that molecules with a single mannose 6-phosphate contained the phosphomannosyl residue on the branch linked alpha 1,6 to the beta-linked mannose whereas molecules with two phosphomannosyl residues had the residues on this branch as well as the branch linked alpha 1,3 to the beta-linked mannose. The mechanism of mannose phosphorylation in the slime mold must differ from that of mammalian cells since the phosphomannosyl residues are present as acid-resistant phosphodiesters rather than acid-labile phosphodiesters.
D001216:Asparagine; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D002854:Chromatography, Paper; D004023:Dictyostelium / Q000201:enzymology*; D006596:Hexosaminidases / Q000032:analysis; D008247:Lysosomes / Q000201:enzymology*; D008361:Mannosidases / Q000032:analysis; D009844:Oligosaccharides / Q000032:analysis*; D043323:alpha-Mannosidase; D001617:beta-Glucosidase / Q000032:analysis; D001619:beta-N-Acetylhexosaminidases
D009844:Oligosaccharides; D001216:Asparagine; D006596:Hexosaminidases; D008361:Mannosidases; D001617:beta-Glucosidase; D043323:alpha-Mannosidase; D001619:beta-N-Acetylhexosaminidases