title: Glycosylation inhibition reduces cholesterol accumulation in NPC1 protein-deficient cells.
abstract: Lysosomes are lined with a glycocalyx that protects the limiting membrane from the action of degradative enzymes. We tested the hypothesis that Niemann-Pick type C 1 (NPC1) protein aids the transfer of low density lipoprotein-derived cholesterol across this glycocalyx. A prediction of this model is that cells will be less dependent upon NPC1 if their glycocalyx is decreased in density. Lysosome cholesterol content was significantly lower after treatment of NPC1-deficient human fibroblasts with benzyl-2-acetamido-2-deoxy-α-D-galactopyranoside, an inhibitor of O-linked glycosylation. Direct biochemical measurement of cholesterol showed that lysosomes purified from NPC1-deficient fibroblasts contained at least 30% less cholesterol when O-linked glycosylation was blocked. As an independent means to modify protein glycosylation, we used Chinese hamster ovary ldl-D cells defective in UDP-Gal/UDP-GalNAc 4-epimerase in which N- and O-linked glycosylation can be controlled. CRISPR generated, NPC1-deficient ldl-D cells supplemented with galactose accumulated more cholesterol than those in which sugar addition was blocked. In the absence of galactose supplementation, NPC1-deficient ldl-D cells also transported more cholesterol from lysosomes to the endoplasmic reticulum, as monitored by an increase in cholesteryl [(14)C]-oleate levels. These experiments support a model in which NPC1 protein functions to transfer cholesterol past a lysosomal glycocalyx.
authors.affiliation: Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305-5307.
authors.forename: Jian
authors.initials: J
authors.lastname: Li
authors.affiliation: Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305-5307.
authors.forename: Maika S
authors.initials: MS
authors.lastname: Deffieu
authors.affiliation: Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305-5307.
authors.forename: Peter L
authors.initials: PL
authors.lastname: Lee
authors.affiliation: Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305-5307.
authors.forename: Piyali
authors.initials: P
authors.lastname: Saha
authors.affiliation: Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305-5307 pfeffer@stanford.edu.
authors.forename: Suzanne R
authors.initials: SR
authors.lastname: Pfeffer
mesh_terms: D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects / Q000235:genetics; D016466:CHO Cells; D002238:Carbohydrate Epimerases / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins; D002784:Cholesterol / Q000235:genetics / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D005690:Galactose / Q000031:analogs & derivatives / Q000494:pharmacology; D019276:Glycocalyx / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008247:Lysosomes / Q000235:genetics / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000172:deficiency*; D000077296:Niemann-Pick C1 Protein
chemical_list: C411946:1-benzyl-2-acetamido-2-galactopyranoside; D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; C106881:NPC1 protein, human; D000077296:Niemann-Pick C1 Protein; D002784:Cholesterol; D002238:Carbohydrate Epimerases; D005690:Galactose
keywords: cholesterol; glycocalyx; lysosomal glycoprotein; lysosomal storage disease