title: Abnormal accumulation and recycling of glycoproteins visualized in Niemann-Pick type C cells using the chemical reporter strategy.
abstract: Niemann-Pick type C (NPC) disease is characterized by impaired cholesterol efflux from late endosomes and lysosomes and secondary accumulation of lipids. Although impaired trafficking of individual glycoproteins and glycolipids has been noted in NPC cells and other storage disorders, there is currently no effective way to monitor their localization and movement en masse. Using a chemical reporter strategy in combination with pharmacologic treatments, we demonstrate a disease-specific and previously unrecognized accumulation of a diverse set of glycoconjugates in NPC1-null and NPC2-deficient fibroblasts within endocytic compartments. These labeled vesicles do not colocalize with the cholesterol-laden compartments of NPC cells. Experiments using the endocytic uptake marker dextran show that the endosomal accumulation of sialylated molecules can be largely attributed to impaired recycling as opposed to altered fusion of vesicles. Treatment of either NPC1-null or NPC2-deficient cells with cyclodextrin was effective in reducing cholesterol storage as well as the endocytic accumulation of sialoglycoproteins, demonstrating a direct link between cholesterol storage and abnormal recycling. Our data further demonstrate that this accumulation is largely glycoproteins, given that inhibitors of O-glycan initiation or N-glycan processing led to a significant reduction in staining intensity. Taken together, our results provide a unique perspective on the trafficking defects in NPC cells, and highlight the utility of this methodology in analyzing cells with altered recycling and turnover of glycoproteins.
authors.affiliation: Department of Chemistry, University of Georgia, Athens, GA 30602, USA.
authors.forename: Ngalle Eric
authors.initials: NE
authors.lastname: Mbua
authors.forename: Heather
authors.initials: H
authors.lastname: Flanagan-Steet
authors.forename: Steven
authors.initials: S
authors.lastname: Johnson
authors.forename: Margreet A
authors.initials: MA
authors.lastname: Wolfert
authors.forename: Geert-Jan
authors.initials: GJ
authors.lastname: Boons
authors.forename: Richard
authors.initials: R
authors.lastname: Steet
mesh_terms: D001386:Azides / Q000493:pharmacokinetics; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism; D003911:Dextrans / Q000493:pharmacokinetics; D011992:Endosomes / Q000378:metabolism; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D005688:Galactosamine / Q000493:pharmacokinetics; D006001:Glycoconjugates / Q000235:genetics / Q000378:metabolism; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008247:Lysosomes / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D000077296:Niemann-Pick C1 Protein; D052556:Niemann-Pick Disease, Type C / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D021381:Protein Transport / Q000502:physiology; D012795:Sialoglycoproteins / Q000378:metabolism; D033921:Vesicular Transport Proteins; D047392:beta-Cyclodextrins / Q000494:pharmacology
chemical_list: D001386:Azides; D002352:Carrier Proteins; D003911:Dextrans; D006001:Glycoconjugates; D006023:Glycoproteins; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; C106881:NPC1 protein, human; C104585:NPC2 protein, human; D000077296:Niemann-Pick C1 Protein; D012795:Sialoglycoproteins; D033921:Vesicular Transport Proteins; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D005688:Galactosamine; D002784:Cholesterol