title: The relative contribution of mannose salvage pathways to glycosylation in PMI-deficient mouse embryonic fibroblast cells.
abstract: Mannose for mammalian glycan biosynthesis can be imported directly from the medium, derived from glucose or salvaged from endogenous or external glycans. All pathways must generate mannose 6-phosphate, the activated form of mannose. Imported or salvaged mannose is directly phosphorylated by hexokinase, whereas fructose 6-phosphate from glucose is converted to mannose 6-phosphate by phosphomannose isomerase (PMI). Normally, PMI provides the majority of mannose for glycan synthesis. To assess the contribution of PMI-independent pathways, we used PMI-null fibroblasts to study N-glycosylation of DNase I, a highly sensitive indicator protein. In PMI-null cells, imported mannose and salvaged mannose make a significant contribution to N-glycosylation. When these cells were grown in mannose-free medium along with the mannosidase inhibitor, swainsonine, to block the salvage pathways, N-glycosylation of DNase I was almost completely eliminated. Adding approximately 13 microm mannose to the medium completely restored normal glycosylation. Treatment with bafilomycin A(1), an inhibitor of lysosomal acidification, also markedly reduced N-glycosylation of DNase I, but in this case only 8 microm mannose was required to restore full glycosylation, indicating that a nonlysosomal source of mannose made a significant contribution. Glycosylation levels were greatly also reduced in glycoconjugate-free medium, when endosomal membrane trafficking was blocked by expression of a mutant SKD1. From these data, we conclude that PMI-null cells can salvage mannose from both endogenous and external glycoconjugates via lysosomal and nonlysosomal degradation pathways.
authors.affiliation: Department of Applied Biological Science, Tokyo University of Agriculture and Technology, Japan.
authors.forename: Naonobu
authors.initials: N
authors.lastname: Fujita
authors.forename: Ayako
authors.initials: A
authors.lastname: Tamura
authors.forename: Aya
authors.initials: A
authors.lastname: Higashidani
authors.forename: Takashi
authors.initials: T
authors.lastname: Tonozuka
authors.forename: Hudson H
authors.initials: HH
authors.lastname: Freeze
authors.forename: Atsushi
authors.initials: A
authors.lastname: Nishikawa
mesh_terms: D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004622:Embryo, Mammalian / Q000166:cytology; D056827:Endosomal Sorting Complexes Required for Transport; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D047468:Immunoprecipitation; D018942:Macrolides / Q000494:pharmacology; D008358:Mannose / Q000378:metabolism*; D008359:Mannose-6-Phosphate Isomerase / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D017026:Swainsonine / Q000494:pharmacology
chemical_list: D056827:Endosomal Sorting Complexes Required for Transport; D006023:Glycoproteins; D018942:Macrolides; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D049452:Green Fluorescent Proteins; C040929:bafilomycin A1; D006026:Glycoside Hydrolases; D000251:Adenosine Triphosphatases; D000074183:ATPases Associated with Diverse Cellular Activities; C089217:Vps4b protein, mouse; D008359:Mannose-6-Phosphate Isomerase; D008358:Mannose; D017026:Swainsonine