The effects of altered N-linked oligosaccharide structures on maturation and targeting of lysosomal enzymes in Dictyostelium discoideum.
We have examined the relationship of N-linked oligosaccharide structures to the proper targeting and proteolytic processing of two lysosomal enzymes, alpha-mannosidase and beta-glucosidase, in the slime mold Dictyostelium discoideum. Two different mutant strains, HL241 and HL243, each synthesize the same nonglucosylated, truncated, lipid-linked oligosaccharide precursor, Man6GlcNAc2. [3H]Mannose-labeled N-linked oligosaccharides were studied following their release from immunoprecipitated alpha-mannosidase and beta-glucosidase by digestion with peptide:N-glycosidase F. The oligosaccharides from both mutants resembled each other, but they were smaller and contained fewer anionic groups than those from the wild-type. The oligosaccharides from the mutants strains were reduced in sulfate and Man-6-P content, and all Man-6-P was in the form of acid-stable phosphodiesters. Pulse-chase radiolabeling experiments using [35S] methionine indicated that the precursor forms of both enzymes were smaller than wild-type, and that this difference was due solely to differences in N-linked oligosaccharides. The precursor forms of the enzymes were not over-secreted, but appeared to be proteolytically processed into mature forms at approximately 50% the rate of wild-type. This is mainly due to their prolonged retention in the rough endoplasmic reticulum, but, ultimately, both enzymes were properly targeted to lysosomes. These studies indicate that a reduction in the amount of sulfation, phosphorylation or size of the N-linked oligosaccharides in these mutants is not critical for the proteolytic processing and targeting of the lysosomal enzymes, but that these changes may influence their rate of exit from the rough endoplasmic reticulum.
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La Jolla Cancer Research Foundation, Cancer Research Center, California 92037.
Koza-Taylor
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Saunders
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Cardelli
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D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D002852:Chromatography, Ion Exchange; D004023:Dictyostelium / Q000201:enzymology* / Q000235:genetics; D005959:Glucosidases / Q000096:biosynthesis*; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology*; D008361:Mannosidases / Q000096:biosynthesis* / Q000235:genetics; D009154:Mutation; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011499:Protein Processing, Post-Translational; D043323:alpha-Mannosidase; D001617:beta-Glucosidase / Q000096:biosynthesis* / Q000235:genetics
D009844:Oligosaccharides; D005959:Glucosidases; D008361:Mannosidases; D001617:beta-Glucosidase; D043323:alpha-Mannosidase