Asparagine-linked Oligosaccharides Protect Lamp-1 and Lamp-2 from Intracellular Proteolysis

Kundra, Robin; Kornfeld, Stuart

doi:10.1074/jbc.274.43.31039

Cited by 179 publications

(125 citation statements)

References 40 publications

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“…Kundra and Kornfeld (28) removed Asn-linked oligosaccharides from lysosomal membrane glycoproteins and found that Asn-linked glycans protect LAMP proteins from the action of proteases; they also found that LAMP1 and LAMP2 are not required for maintaining the acidic pH, density, membrane stability, or degradative capacity of lysosomes (28). Similar findings were obtained upon analysis of LAMP1/LAMP2 double-deficient fibroblasts (29).…”

supporting

confidence: 69%

Glycosylation inhibition reduces cholesterol accumulation in NPC1 protein-deficient cells

Deffieu

Lee

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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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 ...

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supporting

confidence: 69%

Glycosylation inhibition reduces cholesterol accumulation in NPC1 protein-deficient cells

Deffieu

Lee

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…An interaction of N-glycan with the polypeptide chain can induce a ␤-turn structure (7). Also, glycoproteins are more stable and more resistant to proteolysis than their corresponding unglycosylated counterparts (42,46). For example, the normally fully glycosylated wild-type ␤-subunit of the apical transport enzyme, the gastric H-K-ATPase, is more resistant to proteolysis by trypsin (13) or proteinase K (5) than its glycosylation-deficient mutants.…”

Section: Role Of N-glycans In Protein Folding Stability and Qualitymentioning

confidence: 99%

Role of N-glycosylation in trafficking of apical membrane proteins in epithelia

Vagin

Kraut²,

Sachs³

2009

American Journal of Physiology-Renal Physiology

179

157

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ized distribution of plasma membrane transporters and receptors in epithelia is essential for vectorial functions of epithelia. This polarity is maintained by sorting of membrane proteins into apical or basolateral transport containers in the transGolgi network and/or endosomes followed by their delivery to the appropriate plasma membrane domains. Sorting depends on the recognition of sorting signals in proteins by specific sorting machinery. In the present review, we summarize experimental evidence for and against the hypothesis that N-glycans attached to the membrane proteins can act as apical sorting signals. Furthermore, we discuss the roles of N-glycans in the apical sorting event per se and their contribution to folding and quality control of glycoproteins in the endoplasmic reticulum or retention of glycoproteins in the plasma membrane. Finally, we review existing hypotheses on the mechanism of apical sorting and discuss the potential roles of the lectins, VIP36 and galectin-3, as putative apical sorting receptors. apical sorting; apical membrane retention; H-K-ATPase ␤-subunit; lectin EPITHELIAL CELLS CARRY OUT vectorial transport that requires polarized distribution of transporters and receptors to apical or basolateral membrane domains. These domains are separated by tight junctions that connect neighboring cells in the epithelial monolayer and act as diffusion barriers to prevent mixing of apical and basolateral membrane components (22). Asymmetric distribution of plasma membrane proteins is accomplished by their sorting into apical and basolateral containers in the trans-Golgi network (TGN) and/or endosomes followed by vectorial transport of these containers and insertion and retention of the proteins in the appropriate plasma membrane domains. Sorting depends on recognition of apical and basolateral sorting signals within the proteins by cellular sorting machinery (20,58,59,75,76).Numerous studies have indicated that both O-and N-glycans attached to the extracellular domains of some membrane proteins are important for apical location of these proteins. This review will focus on the role of N-glycans in polarized distribution of plasma membrane proteins in epithelia. The role of O-glycans in apical sorting has been described in several recent excellent reviews (18, 71) and will not be discussed further here.A putative role of N-glycans as apical sorting signals was postulated more than 10 years ago (24, 31). However, this hypothesis remains controversial primarily because N-glycans are important for the processes that precede or follow the actual sorting event, such as protein folding, quality control, endoplasmic reticulum (ER)-associated degradation, ER-to-Golgi trafficking, and retention of glycoproteins in the apical membrane. Therefore, merely examining the effect of altering the number or the nature of N-linked glycans on the relative abundance of the glycoprotein in the apical membrane, as has been done in many of the studies reported, does not allow one to distinguish between effects on apica...

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“…Cells were then placed in serum-free DMEM with lysosome inhibitors pepstatin A and leupeptin (both at 1 or 5 M; Calbiochem) alone or in combination as described previously (24). Cells were incubated for 4 h and then harvested for Western blot analysis.…”

Section: Methodsmentioning

confidence: 99%

The Tetraspan Protein EMP2 Regulates Expression of Caveolin-1

Forbes¹,

Wadehra

Mareninov³

et al. 2007

Journal of Biological Chemistry

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Caveolin-1 is the primary component of caveolae and functions in a variety of intracellular activities, including membrane trafficking and signal transduction. EMP2 (epithelial membrane protein 2) is a tetraspan protein recently identified as a novel regulator of caveolin-1 expression. In this study, we analyzed the mechanism of EMP2-mediated caveolin-1 regulation. In NIH 3T3 cells and in the human retinal pigment epithelium cell line (ARPE-19), EMP2 regulates caveolin-1 transcription and more substantially its protein levels. EMP2-mediated down-regulation of caveolin-1 does not affect caveolin-1 translational efficiency, phosphorylation, or proteasome-mediated degradation. Analysis of caveolin-1 protein half-life indicates the EMP2-mediated loss of caveolin-1 occurs rapidly. Protease inhibition and laser confocal microscopy associates this fate with specific intracellular compartmentalization, including early lysosomal delivery. These findings elucidate a new mechanism of caveolin-1 regulation and define an additional role for EMP2 as a key regulator of cell membrane composition.

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Asparagine-linked Oligosaccharides Protect Lamp-1 and Lamp-2 from Intracellular Proteolysis

Cited by 179 publications

References 40 publications

Glycosylation inhibition reduces cholesterol accumulation in NPC1 protein-deficient cells

Glycosylation inhibition reduces cholesterol accumulation in NPC1 protein-deficient cells

Role of N-glycosylation in trafficking of apical membrane proteins in epithelia

The Tetraspan Protein EMP2 Regulates Expression of Caveolin-1

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