title: Alterations to N-linked oligosaccharides which affect intracellular transport rates and regulated secretion but not sorting of lysosomal acid phosphatase in Dictyostelium discoideum.
abstract: The importance of N-linked oligosaccharides and their associated modifications in the transport, sorting, and secretion of lysosomal acid phosphatase was investigated using three mutant Dictyostelium cell lines. These mutants synthesize altered N-linked oligosaccharides with the following properties: (i) in strain HL244 carbohydrate side chains lack mannose 6-sulfate residues, (ii) in strain M31 the side chains retain the two alpha-1,3-linked glucose residues resulting in less sulfate and methylphosphate modifications, and (iii) in strain HL243 the nonglucosylated branches are missing three of the outer mannose sugars and the oligosaccharides contain fewer sulfate and phosphate modifications. Lysosomal enzymes in both HL243 and HL244 are also missing a shared epitope termed common antigen-1 (CA-1), which consists in part of mannose 6-sulfate moieties. No increases were observed in the secretion of radiolabeled acid phosphatase or acid phosphatase activity during growth in any of the mutant cell lines, suggesting that the enzyme was correctly sorted to lysosomes. In support of this, Percoll gradient fractionations and indirect immunofluorescence microscopy indicated that acid phosphatase was transported to lysosomes in all cell lines. However, radiolabel pulse chase protocols indicated that newly synthesized acid phosphatase was transported out of the endoplasmic reticulum (ER) and into lysosomes at a two- to threefold slower rate in HL243 and at a sixfold slower rate in M31. The rate of transport of acid phosphatase from the ER to the Golgi was reduced only twofold in M31 as determined by digestion of newly synthesized enzyme with endoglycosidose H. This suggests that certain alterations in carbohydrate structure may only slightly affect transport of the enzyme from the ER to the Golgi but these alterations may greatly delay transport from the Golgi or post-Golgi compartments to lysosomes. Finally all three mutants secreted acid phosphatase at significantly lower rates than the wild-type strain when growing cells were placed in a buffered salt solution (conditions which stimulate the secretion of mature lysosomally localized enzymes). In contrast, alpha-mannosidase was secreted with similar kinetics from the mutant and wild-type strains. Together, these results suggest that the mechanism(s) operating to sort acid phosphatase in Dictyostelium can tolerate a wide range of changes in N-linked oligosaccharides including a reduction in phosphate and the absence of CA-1 and sulfate, while in contrast, these same alterations can profoundly influence the rate of transport of acid phosphatase from the ER and post-ER compartments to lysosomes as well as the secr
authors.affiliation: Department of Microbiology and Immunology, LSU Medical Center, Shreveport 71130.
authors.forename: J M
authors.initials: JM
authors.lastname: Bush
authors.forename: D L
authors.initials: DL
authors.lastname: Ebert
authors.forename: J A
authors.initials: JA
authors.lastname: Cardelli
mesh_terms: D000118:Acetylglucosaminidase; D000135:Acid Phosphatase / Q000096:biosynthesis / Q000235:genetics*; D004023:Dictyostelium / Q000201:enzymology / Q000235:genetics*; D005455:Fluorescent Antibody Technique; D006031:Glycosylation; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology*; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D009154:Mutation; D009844:Oligosaccharides / Q000096:biosynthesis*; D011499:Protein Processing, Post-Translational; D013347:Subcellular Fractions / Q000201:enzymology
chemical_list: D009844:Oligosaccharides; D000135:Acid Phosphatase; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase