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.L ow-density lipoprotein-derived cholesterol is delivered to cells by receptor-mediated endocytosis and transport to lysosomes. Within lysosomes, cholesterol esters are hydrolyzed and cholesterol is exported to the cytoplasm for cellular use (1). Cholesterol export requires two lysosomal glycoproteins: NPC1 and NPC2 (2, 3). Patients carrying homozygous mutations in either of these proteins present with Niemann-Pick type C (NPC) disease, a neurological disorder that is associated with massive accumulation of unesterified cholesterol and glycosphingolipids in lysosomes (2-4).NPC2 is a small, soluble, cholesterol binding protein that is thought to pick up cholesterol both from lipoprotein lipase and from the abundant, intralumenal membranes present within lysosomes (5, 6). NPC1 is a much larger glycoprotein that contains 13 transmembrane domains and three, relatively large, lumenally oriented domains (2). NPC1's first (N-terminal), luminal domain binds cholesterol directly (7,8); the second domain can bind NPC2 in a cholesterol-dependent manner and has been proposed to facilitate transfer of cholesterol from NPC2 onto NPC1's N-terminal domain (9).The requirement for NPC1 and NPC2 proteins for cholesterol export from lysosomes is somewhat enigmatic because cholesterol can generally partition freely into membrane bilayers (10). The limiting membrane of lysosomes is lined predominantly by densely packed, highly glycosylated, lysosomal membrane proteins; their oligosaccharides are thought to protect the phospholipid bilayer from hydrolytic destruction (11,12). This glycoprotein coat ...

Clues to the mechanism of cholesterol transfer from the structure of NPC1 middle lumenal domain bound to NPC2

“…To verify the physiological importance of NPC1 MLD residues predicted to mediate NPC2 binding from the complex structure we introduced MLD mutations into the closely related, full-length mNPC1 membrane protein and tested whether the mutant proteins could rescue the cholesterol accumulation phenotype seen in NPC1 −/− cells (20). In these experiments, cholesterol accumulation was monitored using Alexa 647-conjugated Perfringolysin O* that binds cholesterol directly (20).…”

“…In these experiments, cholesterol accumulation was monitored using Alexa 647-conjugated Perfringolysin O* that binds cholesterol directly (20).…”

“…Altogether, the NPC2-NPC1 transfer system can facilitate cholesterol transfer across the lysosomal glycocalyx (∼8 nm; ref. 20) and initiate a "pocket brigade" for cholesterol transport (17).…”

Structure of human Niemann–Pick C1 protein

“…The NTD is linked to TM 1 and the rest of NPC1 by a loop consisting of residues 247-266 that includes eight conserved prolines (17). The height of the protein may help NPC1 reach through the glycocalyx that lines the inner surface of the lysosome membrane (15,41). This would permit the NTD to reorient in such a way as to allow cholesterol transfer to the transmembrane domain (state 3).…”

N-glycome of the Lysosomal Glycocalyx is Altered in Niemann-Pick Type C Disease (NPC) Model Cells

“…Moreover, it has been recently shown that modulation of glycosylation of APP, a key protein in the pathogenesis of AD, may represent a potential target for AD therapy (18). It has been previously shown that glycoproteins accumulate in NPC model (19), and that blocking the O-linked glycosylation lowers cholesterol levels and increases the number of lysosomes (20), thus rescuing the NPC cellular defects. In the present work, we studied N-glycosylation profile of the lysosomal membrane proteins in NPC1-null cells vs. wt-cells.…”