Glycobiology Advance Access originally published online on January 24, 2008
Glycobiology 2008 18(3):210-224; doi:10.1093/glycob/cwn003
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Review |
Free oligosaccharide regulation during mammalian protein N-glycosylation
1 INSERM, U773, Centre de Recherche Biomédicale Bichat-Beaujon, CRB3, BP 416, F-75018, Paris, France
2 Université Paris 7 Denis Diderot, site Bichat, BP 416, F-75018, Paris, France
3 To whom correspondence should be addressed: Tel: +33-1-44-85-63-54; Fax: +33-1-46-77-02-33; e-mail: moore{at}bichat.inserm.fr
Received on November 5, 2007; revised on January 21, 2008; accepted on January 21, 2008
 |
Abstract |
|---|
Top
Abstract
IntroductionDuring protein N-glycosylation in mammalian cells, free oligosaccharides (fOS) are generated from lipid-linked oligosaccharides by a pyrophosphatase activity and oligosaccharyltransferase and from misfolded glycoproteins by peptide:N-glycanase in both the ER and cytoplasm. Trafficking machinery comprising oligosaccharide-specific ER and lysosomal transporters, an endo-β-N-acetyl-glucosaminidase, and the cytosolic M2C1 mannosidase drives a flux of fOS from the ER to cytoplasm and from the cytoplasm into lysosomes where fOS are degraded. Transport of fOS out of the ER is normally efficient and if inhibited causes fOS to be secreted via the Golgi apparatus. By contrast, fOS clearance from the cytosol into lysosomes is less efficient resulting in low micromolar concentrations of fOS in the cytoplasm. Structural analysis of cytosolic fOS reveals oligosaccharide families whose relative abundance highlights the importance of different ER-associated degradation (ERAD) pathways for misfolded glycoproteins and suggests that in liver cells substantial amounts of glycoproteins destined for ERAD may transit early compartments of the Golgi apparatus. Glycoprotein quality control and ERAD are controlled by N-glycan/lectin interactions and the fOS trafficking pathway would seem to ensure that fOS do not interfere with these processes which occur in both the ER and cytoplasm. Although Saccharomyces cerevisiae strains harbouring mutations in genes of the yeast fOS metabolic pathway do not display obvious phenotypes, mammalian fOS are quantitatively more important and the processes leading to their regulation are more complex, raising the possibility that distinct phenotypes will be seen in mammalian cells or animals in which fOS metabolism is modified.
Key words: endoplasmic reticulum-associated protein degradation / ERAD / free oligosaccharides / protein N-glycosylation / subcellular transport
|
Introduction |
|---|
Protein N-glycosylation can be divided into three phases. The first concerns the synthesis of an oligosaccharide on the lipid carrier dolichol to generate the lipid-linked oligosaccharide (LLO) sugar donor Glc3Man9GlcNAc2-PP-dolichol (Burda and Aebi 1999
). The second includes the transfer of the oligosaccharide moiety of this LLO onto nascent glycoproteins (Kelleher and Gilmore 2006) in the lumen of the ER. Chaperone-assisted glycoprotein folding and quality control processes then allow either correctly folded glycoproteins to be targetted to their final subcellular destinations (Helenius and Aebi 2004), or the elimination of terminally misfolded glycoproteins from the endomembrane system (Ruddock and Molinari 2006). The third phase includes recycling of the lipid carrier (Rush et al. 2007) and elimination of oligosaccharide waste generated during protein N-glycosylation (Moore 1999). In fact, during protein N-glycosylation, oligomannose-type oligosaccharides (see orange lines in Figure 1), which are called free oligosaccharides (fOS) in order to distinguish them from their N- and lipid-linked counterparts, are generated in both the ER and cytoplasm (Verbert and Cacan 1999; Spiro 2004). Under normal circumstances fOS are eventually targetted to lysosomes via a nonvesicular trafficking pathway that is distinct from the vesicular pathways required for glycoconjugate delivery to lysosomes (see orange and brown arrows in Figure 1). The quantitative importance of this catabolic route is highlighted by the observation that up to 20% of the storage oligosaccharides identified in the tissues and urine of lysosomal 
-mannosidosis patients (Norden et al. 1974; Yamashita et al. 1980; Egge et al. 1982) have structures characteristic of those generated by the fOS trafficking pathway (Moore 1999; Winchester 2005). Presently, it is not known whether or not fOS have physiological roles to play in either the ER or cytoplasm.
|
Here, the origins, subcellular trafficking and ultimate fates of fOS under normal and abnormal conditions are reviewed. In addition, the ways in which the study of fOS regulation can complement the more mechanistic approaches that are used to examine glycosylation-related processes of the early secretory pathway are highlighted.
|
Protein N-glycosylation in yeast and mammalian cells |
|---|
ER events are similar in yeast and mammalian cells
The biosynthesis of glycoproteins bearing N-linked carbohydrate units is a common function of both yeast (Burda and Aebi 1999
) and mammalian ER (Helenius and Aebi 2004) and the initial steps of this process are shown in Figure 2. The transfer of oligosaccharide from LLO onto nascent glycoproteins is carried out by oligosaccharyltransferase (OST), a multi-protein complex situated in the lumen of the ER (Kelleher and Gilmore 2006). The dolichol cycle is completed by a series of reactions in which the OST-generated dolichol-PP is converted to dolichol-P (Rush et al. 2007) before being flipped accross the ER membrane in order that its phosphate group is re-exposed to the cytoplasm (Schenk et al. 2001). Several studies indicate that dolichol-P is a rate-limiting intermediate during protein glycosylation (Rosenwald et al. 1990). After protein glycosylation, N-glycans play important roles during glycoprotein folding (Spiro 2004), quality control (Ruddock and Molinari 2006), and the removal of terminally misfolded glycoproteins from the ER (Romisch 2005).
|
ER-associated protein degradation
Early studies (Ward et al. 1995
; Werner et al. 1996) demonstrated a lysosome-independent process for the elimination of misfolded glycoproteins from the ER that involved proteolysis by the cytoplasmically located proteasome (Goldberg 1995). A mechanism for this process was suggested by the demonstration that a human cytomegalovirus viral protein promotes the association of an MHC class I glycoprotein with the ER-protein-conducting channel (sec61 complex) causing the MHC class I molecule to be dislocated into the cytosol (Wiertz et al. 1996). Inhibition of proteasomal degradation of the class I molecule revealed it to be deglycosylated by a peptide:N-glycanase (PNGase) because isoelectric focussing of the deglycosylated class I molecule revealed it to possess an extra negative charge when compared to the nonglycosylated protein (Wiertz et al. 1996)—an observation consistent with an PNGase action which, upon hydrolyzing the amide bond of the linkage Asn residue, converts the Asn into Asp while liberating a molecule of NH3. Numerous yeast and mammalian cell-misfolded glycoproteins are now known to be degraded by similar proteasome-dependent processes (de Virgilio et al. 1998; Dusseljee et al. 1998; Mancini et al. 2000; Gong et al. 2005; Bailey et al. 2007) which are collectively called ER-associated protein degradation (ERAD) processes (Kawaguchi and Ng 2007). Finally, proteasome-independent ERAD processes have now been described (Cabral et al. 2000; Donoso et al. 2005) in which proteolytic events occur in the lumen of the ER. Another proteasome-independent ERAD process for the elimination of ER-situated protein aggregrates involves the lysosomal/autophagic pathway (Fujita et al. 2007).
Glycoprotein maturation in the Golgi apparatus is different in yeast and mammalian cells
In mammalian cells, correctly folded glycoproteins are transported to the Golgi apparatus where their N-glycans are modified by mannosidases and glycosyltransferases to yield complex, sialic acid, and fucose- and galactose-containing structures (Kornfeld R and Kornfeld S 1985). The modification of yeast glycoproteins in the Golgi apparatus is quite different (Dean 1999). First of all, Saccharomyces cerevisiae does not appear to contain Golgi mannosidases (Herscovics and Orlean 1993), and second, glycosyltransferase activity in this organelle, which is restricted to mannose addition, leads to the generation of glycoproteins bearing either core-type, or polymannose N-linked glycans (Dean 1999).
|
Phosphorylated fOS are generated from LLO |
|---|
During early studies on the involvement of LLO intermediates in protein glycosylation it was noted, after radiolabeling mouse myeloma microsomes with GDP-[14C]Man, that whereas radioactive LLO and glycoproteins could be recovered from an insoluble particulate material, radioactive Man5GlcNAc2-P could be isolated from the water-soluble fraction (Hsu et al. 1974
). In fact, in the same experiments, this oligosaccharide structure was observed linked to both dolichol and glycoproteins, and it was proposed that whereas Man5GlcNAc2-PP-dolichol was the oligosaccharide donor for protein glycosylation, the Man5GlcNAc2-P was a degradation product of LLO (Hsu et al. 1974). Similar findings were reported by other groups (Cacan et al. 1980; Oliver et al. 1975a,b; Oliver and Hemming 1975). Indeed, it later became apparent that Man5GlcNAc2-PP-dolichol is a truncated LLO and its oligosaccharide moiety is transferred onto a protein inefficiently and only under conditions of cellular stress. Later, experiments were carried out on CHO-derived B3F7 cells that have a mutation in the DPM1 gene (see Figure 2) causing an accumulation of the LLO Man5GlcNAc2-PP-dolichol. These cells generate increased amounts of phosphorylated fOS (fOS-P) compared to control cells and, in addition to the expected Man5GlcNAc2-P, elaborate Man2GlcNAc2-P (Cacan et al. 1992). The fact that these were the only two fOS-P detected, despite the presence of small amounts of more fully mannosylated and glucosylated LLO intermediates, led this group (Cacan et al. 1992) to examine the hypothesis that fOS-P are liberated on the cytosolic face of the ER where Man1-5GlcNAc2-PP-dolichol intermediates are known to be synthesized (see Figure 2). Indeed, specific permeabilization of the plasma membranes of the mutant cells revealed that fOS-P could only be recovered from the cytosolic compartment (Cacan and Verbert 1997). The ensemble of these results led to the notion that excess Man1-5GlcNAc2-PP-dolichol species are hydrolyzed at the cytosolic face of the ER by a pyrophosphatase activity in order to stop immature LLO from being translocated into the lumen of the ER and utilized by OST (Cacan and Verbert 1997; 1999). This hypothesis is also attractive because dolichol-P is a rate-limiting intermediate in protein glycosylation (Rosenwald et al. 1990); accordingly any dolichol-P ‘tied up’ in surplus immature LLO intermediates could potentially severely restrict the amount of dolichol-P available for LLO biosynthesis and other dolichol-P-requiring pathways. However, the situation appears to be more complex because several previous studies by the same authors demonstrated the presence of Man9GlcNAc2-P in normal mouse splenocytes or rat lymphocytes (Cacan et al. 1980, 1987, 1989; Hoflack et al. 1981). Additionally, the generation of Man8GlcNAc2-P was noted during radiolabeling of yeast microsomal LLO with GDP-[14C]Man (Belard et al. 1988). As Man6-9GlcNAc2-PP-dolichol intermediates are thought to be synthesized in the lumen of the ER, these observations suggest that either there is also a pyrophosphatase in the lumen of the ER or, under certain conditions, these intermediates can be flipped back from the lumenal to the cytosolic face of the ER (see Figure 2). To further complicate matters, skin biopsy fibroblasts from patients with type I congenital disorders of glycosylation (CDG I) generate the same low levels of fOS-P as those found in skin biopsy fibroblasts from normal children (Dancourt, J. and Moore,S.E.H., unpublished observations) despite displaying abnormal accumulations of Man5GlcNAc2-PP-dolichol (CDG Ie; mutation in DPM1, Dancourt et al. 2006), Man7GlcNAc2-PP-dolichol (CDG Ig; mutation in ALG12, Chantret et al. 2002), Man9GlcNAc2-PP-dolichol (CDG Ic; mutation in Alg6), and (Glc1)Man9GlcNAc2-PP-dolichol (CDG Ih; mutation in Alg8, Chantret et al. 2003). To summarize, fOS-P (Man2-9GlcNAc2-P) have been detected in many cell lines and found to occur at abnormally high levels in certain cell-lines-harbouring deficits in LLO biosynthesis. Clearly, identification of the putative pyrophosphatase will be crucial to our understanding of the physiological relevance of fOS-P liberation. In fact, in a preliminary characterization, a bivalent cation-requiring, pyrophosphate-, NAD+-, and bacitracin-sensitive activity capable of this reaction was reported to occur in yeast microsomes (Belard et al. 1988). |
Liberation of neutral fOS from LLO |
|---|
When intact hen oviduct microsomes were radiolabeled with GDP-[14C]Man and UDP-[14C]Glc, substantial amounts of neutral fOS were generated during the course of the protein glycosylation reactions (Hanover et al. 1982
; Hanover and Lennarz 1981). These fOS contained the di-N-acetylchitobiose moiety at their reducing termini (fOS-GN2) and possessed the same sugar structure as that found in the mature triglucosylated LLO (see Figure 2). Neutral fOS-GN2 was released into the lumen of intact microsomes, and the quantity of released fOS was higher in experiments where the microsomes had been frozen prior to use (Hanover et al. 1982; Hanover and Lennarz 1981). Later, employing bovine thyroid microsomes it was shown that Glc3Man9GlcNAc2 was released from Glc3Man9GlcNAc2-PP-dolichol by an enzymic reaction (Anumula and Spiro 1983). In this same study it was demonstrated that dephosphorylation of fOS-P could not account for the appearance of these neutral fOS (Anumula and Spiro 1983). Furthermore, Glc3Man9GlcNAc2 generation closely followed protein glycosylation, and could, like the latter process, be inhibited by the addition of EDTA to the incubation mixtures (Anumula and Spiro 1983). Finally, it was found, as has been shown for protein glycosylation, that when a nonglucosylated LLO was used as radioactive substrate, fOS-GN2 release was 10-fold less efficient when compared to the release observed when glucosylated LLO were used. The similarities between oligosaccharide transfer from LLO onto a protein and the hydrolysis of LLO to liberate fOS-GN2 suggested that both events were carried out by OST (Anumula and Spiro 1983). These results were consolidated and extended by demonstrating that the addition of a small peptide containing the N-glycosylation consensus sequence to microsome incubation mixtures abolished both the generation of fOS and the glycosylation of protein (Spiro MJ and Spiro RG 1991). Interestingly, fOS generation was found to be more susceptible to inhibition by Triton X-100 than protein glycosylation. The ensemble of these results led to the hypothesis that when peptide glycosylation acceptor sites are limiting, the oligosaccharide-primed catalytic subunit of OST preferentially transfers its oligosaccharide onto a water molecule resulting in the release of a neutral fOS into the lumen of the ER (Figure 2). This process was suggested to be a mechanism whereby the quantity of LLO is controlled (Anumula and Spiro 1983; Spiro MJ and Spiro RG 1991). More recently, it has been reported that when SLO-permeabilized cells (which are unable to generate endogenous OST polypeptide acceptors) are incubated with castanospermine (CST, an inhibitor of ER glucosidases, see legend to Figure 2, Spiro MJ and Spiro RG 1991) and mannose-6-phosphate (M6P), Glc3Man9GlcNAc2-PP-dolichol becomes unstable with the liberation of Glc3Man9GlcNAc2 (Gao et al. 2005). This effect was abolished upon the addition of exogenous peptide acceptor indicating that M6P-evoked fOS liberation and peptide glycosylation deplete the same LLO pool. M6P-evoked fOS liberation seems to be highly specific for both M6P and triglucosylated LLO (Gao et al. 2005). Under normal physiological conditions cellular M6P levels appear to be too low to destabilize LLO and thus affect protein N-glycosylation. By contrast, cellular concentrations of M6P are elevated in cells from patients with CDG Ia in which conversion of M6P to mannose-1-P (M1P) is hindered by mutations in the gene encoding phosphomannomutase 2 (PMM2, see Figure 2). Accordingly, it has been proposed that as well as reduced quantities of GDP-Man in CDG Ia cells, increased M6P levels may also contribute to glycoprotein hypoglycosylation seen in these patients (Gao et al. 2005). In a mouse model for CDG Ib in which the interconversion of fructose-6-phosphate and M6P is abolished by disruption of the PMI gene (DeRossi et al. 2006), M6P was found to accumulate in embryonic fibroblasts. In these cells, although LLO stability and fOS generation were not investigated, protein N-glycosylation appeared to be normal. To conclude, evidence suggests that under certain circumstances OST can promote the cleavage of Glc3Man9GlcNAc2-PP-dolichol to yield Glc3Man9GlcNAc2 presumably inside the lumen of the ER. However, the physiological relevance of this process and how it can be regulated by factors such as increased M6P and decreased polypeptide acceptor availability remain to be explored. |
The generation of fOS from misfolded glycoproteins |
|---|
Many metabolic radiolabeling studies suggest that the majority of neutral fOS are not generated from LLO as described above but arise from the deglycosylation of glycoproteins rapidly after their synthesis. During the metabolic radiolabeling of either intact cells (Cacan et al. 1992
; Moore and Spiro 1994; Villers et al. 1994; Cacan and Verbert 1999) or tissue slices (Anumula and Spiro 1983), and to a lesser extent broken cell preparations (Anumula and Spiro 1983; Villers et al. 1994), fOS bearing a single residue of GlcNAc at their reducing termini (fOS-GN) were observed. During equilibrium metabolic radiolabeling of tissues or cells, or after-chase incubations, these fOS-GN were always found to be in large excess of fOS-P and fOS-GN2. In fact, protein synthesis inhibitors reduce the incorporation of [2-3H]Man into LLO, fOS-GN2, and fOS-P, but the effect of these agents was found to be more pronounced on the generation of glycoproteins and fOS-GN (Spiro MJ and Spiro RJ 1991; Duvet et al. 1998). In S. cerevisiae, despite provoking a 4-fold increase in [2-3H]Man incorporation into LLO, cycloheximide (CHX) inhibited radiolabel incorporation into glycoprotein and fOS by greater than 95% demonstrating that in yeast the bulk of fOS originate from glycoprotein deglycosylation (Chantret et al. 2003).
Free OS-GN can be derived from misfolded glycoproteins
CHO cells harboring a deficiency in the first glucosyltransferase (see Figure 2, Alg6) of the LLO biosynthetic pathway (Quellhorst et al. 1999) are unable to synthesize glucosylated LLO but the N-glycans of misfolded glycoproteins are recognized and specifically monoglucosylated by UDP-glucose:glycoprotein glucosyltransferase (UGGT) as a part of ER-associated glycoprotein quality control (Spiro 2004). In these cells, monoglucosylated fOS can be used as markers for misfolded glycoprotein deglycosylation (Cacan et al. 2001), and analysis of the metabolically radiolabeled fOS generated during pulse-chase experiments revealed the presence of cytosolic monoglucosylated fOS-GN (Cacan et al. 2001). As already mentioned, the disposal of glycoproteins via ERAD is thought to involve glycoprotein deglycosylation by a PNGase activity that would yield fOS-GN2 rather than the above-mentioned fOS-GN. In fact, mammalian cell cytosol is also known to contain an endo-β-N-acetylglucosaminidase (ENGase) activity that could liberate fOS-GN from glycoproteins. However, this same enzyme could also convert PNGase-generated fOS-GN2 into fOS-GN.
The PNGase, Png1p, deglycosylates misfolded glycoproteins in yeast and mammalian cells and generates the bulk of fOS in S. cerevisiae
Both biochemical evidence and data bank searches indicate that S. cerevisiae does not possess an ENGase. With the characterization of a soluble S. cerevisiae PNGase activity (Suzuki et al. 1998) and subsequent identification of the encoding gene (PNG1, yeast ORF YPL096w, Suzuki et al. 2000), in vivo studies have shown that Png1p is responsible for cytosolic de-N-glycosylation of misfolded glycoproteins (Hirsch et al. 2003). In vitro studies with purified yeast Png1p show that the enzyme preferentially deglycosylates misfolded glycoproteins (Hirsch et al. 2004; Joshi et al. 2005), and in vivo experiments reveal that the disposal of a carboxypeptidase Y mutant (Suzuki et al. 2000) and ricin A chain (Kim et al. 2006) in the png1
strain is slower than that observed in wild-type S. cerevisiae. The mechanism behind the stabilization of ERAD substrates in the yeast png1 strain is suggested from in vitro experiments which show that deglycosylation of artificial substrates by yeast PNGase facilitates subsequent proteasome degradation (Hagihara et al. 2007). In agreement with the above observations, when the S. cerevisiae png1 strain is metabolically radiolabeled with [2-3H]Man, a 
75% decrease in fOS (mainly Man8GlcNAc2, see Figure 3), with respect to the isogenic wt strain, is noted (Chantret et al. 2003). At present the origins of the two Png1p-independent fOS pools that are described in Figure 3 are not understood, but because CHX inhibits fOS generation by greater than 95% it would appear that these Png1p-independent species are also derived from glycoprotein perhaps by an as yet unidentified PNGase activity. Mammalian homologues (Suzuki et al. 2003) of yeast PNG1 have been identified and the role of this enzyme in deglycosylating some ERAD substrates has been confirmed in vivo using shRNA (Blom et al. 2004) and the Png1p inhibitor Z-VAD-fmk (Misaghi et al. 2004). However, in contrast to the situation with yeast, the impact of Png1p on general fOS generation in mammalian cells has not been reported.
|
Are glycoproteins deglycosylated by the cytosolic ENGase in mammalian cells?
A rat liver cytosolic endo-N-acetyl-β-D-glucosaminidase (ENGase) capable of cleaving the di-N-acetylchitobiose moiety of glycopeptides containing polymannose-, or nonsialylated N-acetyllactosamine-type glycans has been described (Pierce et al. 1979
, 1980; Lisman et al. 1985). This ENGase, which has a neutral pH optimum, was later identified in different human tissues (Overdijk et al. 1981), hen oviduct (Kato et al. 1997), and Caenorhabditis elegans (Kato et al. 2002). More refined specificity studies demonstrated that the ENGase is capable of hydrolyzing the di-N-acetylchitobiose moiety of branched Man5GlcNAc2 (comprising mannose residues 3, 4, 5, 8, and 10, see Figure 2) when occurring as either a fOS or a glycopeptide (Kato et al. 1997). The same structure when occurring in a glycoprotein was less susceptible to cleavage. The enzyme preferred oligomannose-type structures to hybrid- or complex-type oligosaccharides (Kato et al. 1997, 2002). Finally, oligosaccharides containing 6–9 mannose residues were preferred to those containing 3–5 mannose residues (Kato et al. 2002). ENGase was cloned in C. elegans using an EST showing homology to Mucor hiemalis endo-β-N-acetylglucosaminidase (Endo-M, Kato et al. 2002). Soon after, two human ENGase ESTs were identified in silico using sequence data derived from the purified hen oviduct enzyme (Suzuki et al. 2002). These two transcripts could arise by alternative splicing, but transient expression of the larger transcript (Gene Bank accession number: AJ397822
[GenBank]
coding for a 743-amino-acid protein) in COS-7 cells generated a soluble activity capable of hydrolyzing glycopeptides (Suzuki et al. 2002). Although the specificity of the ENGase suggests that glycoproteins are not the best substrates, is it possible that this enzyme generates fOS-GN from misfolded glycoproteins in the cytosol? Several considerations suggest that this might not be the case. First, the proteasome is more active toward deglycosylated substrates and in vitro studies suggest that even the residual GlcNAc residue that remains linked to the polypeptide after an ENGase action is capable of impeding proteasome activity (Hagihara et al. 2007). Second, the fate of the resulting glycopeptides bearing single N-linked GlcNAc residues would present a problem as cytosolic PNGase is not able to deglycosylate such structures (Hagihara et al. 2007). Third, MDBK cells do not possess a cytosolic ENGase activity but rather manifest a cytosolic chitobiase activity which, although it is active toward fOS-GN2, is incapable of acting upon glycopeptides or glycoproteins (Cacan et al. 1996). Therefore these cells do not require a cytosolic ENGase activity and this circumstantial evidence is not in favor of ENGase playing an important role in cytosolic glycoprotein deglycosylation in mammalian tissues. To conclude, the nature of the cytosolic ENGase suggests that it may not have a major deglycosylating role but rather acts as a chitobiase in the cytosol and that glycoprotein deglycosylation is accomplished by PNGase.
Where does Png1p-mediated glycoprotein deglycosylation occur?
Mammalian and yeast PNG1 genes encode soluble enzymes that do not possess N-terminal signal peptides (Suzuki et al. 2000). However, both yeast and mammalian Png1p are, as well-being localized in the cytosol, associated with the ER. Fluorescence microscopy studies in yeast demonstrate that Png1p localization overlaps with that of the proteasome (Suzuki et al. 2000) which is often localized within the nucleus and found to be associated with the ER (Russell et al. 1999). It is now known that Png1p binds to Rad23p (Suzuki et al. 2001) which contains an ubiquitin-like domain at its N-terminus enabling it to bind to the 26S proteasome. Mammalian Png1p has been reported to form complexes with many proteins including the mammalian homolog of Rad23p, and fluorescence microscopy demonstrates that GFP-coupled Png1p also localizes to both the cytoplasm and ER in COS-1 cells (Park et al. 2001). In a more detailed study using subcellular fractionation and immunofluoresence in Hela cells, human Png1p labeling was detected in the cytoplasm but also found to be associated with the ER (Katiyar et al. 2004). These data demonstrate that Png1p is a cytosolic enzyme that forms complexes with proteins attached to the cytoplasmic face of the ER. Indeed, inhibition of the proteasome has often been observed to lead to the appearance of deglycosylated proteins in the cytosol (Bebok et al. 1998; de Virgilio et al. 1998; Wiertz et al. 1996; Gong et al. 2005). However, such experiments cannot exclude the possibility that the deglycosylation event occurred in the lumen of the ER. On the other hand, although some data demonstrate deglycosylated proteins to be associated with microsomal membranes, to the best of our knowledge no study has conclusively demonstrated the presence of a deglycosylated protein within the lumen of the ER (Yu et al. 1997). Surprisingly then, as will be discussed later, there is some evidence that fOS-GN2 are released from glycoproteins in the ER.
|
The transport and subcellular trafficking of free OS in mammalian cells |
|---|
In the preceeding sections evidence was presented indicating that fOS are generated from glycoproteins and LLO in both the cytosol and the early endomembrane system. Initial studies on the fate of fOS demonstrated that these structures are not secreted from mammalian cells under normal conditions. This observation was perplexing in the light of experiments which showed that small glycopeptides artificially generated in the lumen of the ER are rapidly secreted into the extracellular medium via the Golgi apparatus (Wieland et al. 1987
; van Leyen et al. 1994). Why do small glycopeptides bearing N-linked oligomannose units behave so differently to fOS that are also generated in the ER?
ER-to-cytosol transport of fOS
Using 2[3H]Man-radiolabeled and SLO-permeabilized HepG2 cells evidence was obtained for the transport of neutral fOS-GN2 from the lumen of the ER into the cytosol (Moore et al. 1995). The cytosol-depleted cells were found to contain a single fOS species, Man9GlcNAc2 (mostly derived from LLO). Reincubation of the permeabilized cells in either the absence or presence of ATP demonstrated an ATP-dependent processing of Man9GlcNAc2 to Man8GlcNAc2 (probably by ER mannosidase I) and an ATP-dependent transport of both of these components into the cytosolic compartment (Moore et al. 1995). The transport process is inhibited by mannose and various mannosides such as benzyl mannose but not by GlcNAc or di-N-acetylchitobiose (Moore 1998). Under conditions where efficient ER-to-cytosol fOS-GN2 transport was observed, there was no transport of glycotripeptide (Moore et al. 1995). Usually ER-generated fOS-GN2 are rapidly deglucosylated by ER-glucosidases I and II (see Figure 2). When cells were radiolabeled in the presence of CST prior to permeabilization, it was noted that triglucosylated fOS-GN2 were not transported (Moore et al. 1995). The ER-to-cytosol transport of fOS was found to be dependent upon the presence of low amounts of Ca2+ in the incubation media and ATP-dependent transport was completely abolished by thapsigargin, a potent inhibitor of the ER-situated Ca2+/Mg2+ ATPase (Moore 1998). As the calcium ionophores A23187
[GenBank]
and ionomycin also inhibited transport, it has been proposed that fOS export from the ER into the cytosol is dependent upon a thapsigargin-sensitive Ca2+ store in the lumen of the ER (Moore 1998). The ensemble of these results demonstrates that the ER membrane possesses transport machinery capable of exporting fOS from the lumen of the ER into the cytosol.
Trimming of fOS in the cytosol
The above considerations, taken along with an action of the cytosolic PNGase, suggest that fOS-GN2 would rapidly accumulate in the cytoplasm. However, in HepG2 cells, pulse-chase studies followed by permeabilization of the plasma membrane with SLO reveal that cytosolic fOS-GN2 are rapidly converted into fOS-GN by the previously discussed ENGase activity without being demannosylated (Moore and Spiro 1994). By contrast, cytosolic fOS-GN are partially demannosylated to yield an oligosaccharide identical to the limit digest structure generated from Man9GlcNAc by purified cytosolic mannosidase in vitro (Moore and Spiro 1994).
The Cytosolic M2C1 Mannosidase.
A soluble, neutral mannosidase, distinct from lysosomal enzymes was detected in rat liver many years ago (Marsh and Gourlay 1971; Shoup and Touster 1976; Tulsiani and Touster 1987). More recently the properties of the human (al Daher et al. 1992), bovine and feline (De Gasperi et al. 1992), rat (Haeuw et al. 1991; Grard et al. 1994), and quail (Oku and Hase 1991; Oku et al. 1991) enzymes have been described. They are several fold more active toward fOS-GN than to fOS-GN2 (Kumano et al. 1996; Oku and Hase 1991), indicating that they act preferentially on the products of cytosolic ENGase. Furthermore, the purified enzyme was capable of only partially demannosylating Man9GlcNAc to yield linear Man5GlcNAc (containing mannose residues 3–7, see Figure 2) whose mannose configuration is different to that of N-linked Man5GlcNAc (containing mannose residues 3, 4, 5, 8 and 10, see Figure 2) generated by Golgi mannosidase I.
The Cloning of M2C1.
A rat liver microsomal enzyme whose biochemical properties are similar to those described for the cytosolic mannosidase was purified (Bischoff and Kornfeld 1983) and, later, the gene corresponding to this activity was cloned (Bischoff et al. 1990). However, the gene was found not to encode a signal sequence and after expression in COS cells gave rise to a 107-kD polypeptide that was largely confined to the cytosol and not the ER (Bischoff et al. 1990). Two lines of evidence now show that the M2C1 cDNA first described by Bischoff et al. encodes an enzyme involved in processing cytosolic fOS. First, when tagged human M2C1 is expressed in P. pastoris it can be purified and shown to have identical characteristics to those ascribed to cytosolic mannosidase (Kuokkanen et al. 2007). Mouse M2C1 has been overexpressed in COS cells and a cytosolic extract from these cells displayed an increased capacity to generate Man5GlcNAc from Man9GlcNAc (Costanzi et al. 2006). Second, specific down regulation of the mRNA encoding this protein using RNAi inhibits cytosolic fOS processing (Suzuki et al. 2006). In one immunofluorescence microscopy study a cytosolic localization for M2C1 was noted (Suzuki et al. 2006), whereas in a later study a punctiform staining pattern, showing no overlap with ER and Golgi and lysosomal markers, was observed (Kuokkanen et al. 2007). Finally, the relationship of cytosolic M2C1 to the microsomal protein (now known as ER mannosidase II, Weng and Spiro 1996) with similar biochemical (Bischoff and Kornfeld 1983; Weng and Spiro 1996) and immunological (Weng and Spiro 1996) characteristics remains to be clarified. It has been proposed that the cytosolic form of the enzyme is translocated into the ER after proteolytic cleavage (Weng and Spiro 1996).
The S. cerevisiae. Vacuolar Mannosidase Ams1p is Homologous to Mammalian M2C1 and is Required for the Catabolism of Png1p-Generated fOS in Yeast.
Sequence data revealed that mammalian M2C1 is related to Ams1p, the S. cerevisiae vacuolar mannosidase (Bischoff et al. 1990). The yeast vacuolar mannosidase is also synthesized in the cytosol and, after oligomerization, is transfered to the vacuole (Yoshihisa and Anraku 1990) by the autophagic and Cytosol-to-Vacuole Targetting (CVT) pathways (Hutchins and Klionsky 2001) where it undergoes proteolytic cleavage. In fact, it has now been shown that the catabolism of the Png1p-dependent population of fOS in S. cerevisiae requires the growth-related increase in Ams1p expression, and in ams1p cells the catabolism of this pool of fOS is abolished (Chantret et al. 2003). Despite the implication of Ams1p in fOS catabolism, this process is little affected in the vma1 strain which is deficient in vacuolar acidification (see Figure 3). Accordingly, as Ams1p is known to be active in the cytoplasm (Hutchins and Klionsky 2001) it is possible that Ams1p-dependent fOS degradation occurs in this compartment. It should also be noted that no structural studies have been performed on the products that can be generated from Man8GlcNAc2 by Ams1p, and the ultimate fate of such products remain obscure. Finally, what happens to the two Png1p-independent fOS pools that are seen in S. cerevisiae? The pool comprising small fOS (marked with asterisks in Figure 3) appears to be disposed of by unknown enzymes in the vacuole because its rate of decay is retarded in the vma1 strain deficient in vacuolar acidification. The pool containing mainly Man8GlcNAc2 has a short half-life and disappears without the appearance of intermediates suggesting that it may be generated and disposed of along the secretory pathway.
The lysosomal free OS importer
After several hours of chase, pulse-radiolabeled fOS are cleared from the cytosol without apparent trimming beyond Man5GlcNAc (Moore and Spiro 1994). However, at these longer chase times smaller fOS transiently appear in a membrane-bound subcellular compartment suggesting that the cytosolically trimmed fOS are transferred to the lysosome in order to be completely degraded (Moore and Spiro 1994). Later, it was shown that when either lysosomal acidification is blocked by the vacuolar H+ ATPase inhibitor concanamycin A, or lysosomal -mannosidase is inhibited by 0.1 µM swainsonine (SW), the fOS-GN that are lost from the cytosol during chase incubations are recovered in a subcellular compartment which cosediments with lysosomes during Percoll density centrifugation (Saint-Pol et al. 1997). The transfer of fOS-GN from cytosol to lysosome is not blocked by inhibitors of macroautophagy and is energy dependent (Saint-Pol et al. 1997). An in vitro transport assay, employing Man5GlcNAc as substrate and Percoll-purified lysosomes, reveals a saturable ATP-dependent (Kuptake, 22.3 µM, Vmax, 7.1 fmol/min/unit β-hexosaminidase) transport process. Transport is specific for partially demannosylated fOS (Man3GlcNAc > Man4GlcNAc > Man5GlcNAc >> Man9GlcNAc) and is unable to transport fOS-P (Saint-Pol et al. 1999). Studies on the fate of metabolically radiolabeled cytosolic triglucosylated fOS in HepG2 cells (Moore and Spiro 1994) and steady-state levels of glucosylated fOS in intact mouse lymphoma HL60 cells (Mellor et al. 2004) suggest that these components are also poor substrates for the lysosomal fOS transporter. Interestingly, in vitro assays demonstrated that the transport of fOS-GN is not inhibited by mannose or various mannosides but is blocked by GlcNAc (Saint-Pol et al. 1999). In fact the most potent inhibitors of lysosomal fOS-GN import are small chitooligosaccharides (Saint-Pol et al. 1999). Furthermore it was shown that the transport apparatus recognizes predominantly the free-reducing GlcNAc residue of fOS-GN. Accordingly, reduced fOS-GN are poor transport substrates and reduced chitobiose does not efficiently inhibit the transport of fOS-GN into lysosomes (Saint-Pol et al. 1999). The specificity of the lysosomal fOS import apparatus therefore indicates that it must work in conjunction with the cytosolic glycosidases (see Normal cell in Figure 4) in order to effectively clear the cytosol of the larger ER-type fOS-GN2. The consequences of impaired transport of fOS from the cytosol into the lysosome are not clear but evidence suggests that triglucosylated fOS-GN do not accumulate indefinitely in ER-glucosidase I-deficient CHO, Lec23, cells but are cleared from the cell by an as yet undefined mechanism (Durrant and Moore 2002). In mice treated with N-butyl deoxynojirimycin, an inhibitor of both ER and lysosomal glucosidase activities (Saul et al. 1983; Andersson et al. 2004), triglucosylated fOS-GN can be recovered from the tissues, serum, and urine (Mellor et al. 2004). The origin of the urine and serum oligosaccharides is not clear. Because triglucosylated fOS-GN could arise during an ERAD process that involves elements of the lysosomal/autophagic pathway (Fujita et al. 2007), it is possible that they have a similar fate to lysosomal storage oligosaccharides which are also found in the serum and urine of affected patients (see Figure 1).
|
Why such a complex catabolic pathway?
As shown in Figure 4 (Normal cell), trafficking machinery comprising oligosaccharide-specific ER and lysosomal transporters, an ENGase and the cytosolic M2C1 mannosidase, drives a flux of fOS from the ER to cytoplasm and from the cytoplasm into lysosomes where fOS are degraded. It has been proposed that in mammalian cells this sophisticated, energy-requiring pathway ensures the rapid segregation of fOS and glycoproteins so that the former structures do not interfere with the N-glycan-dependent aspects of glycoprotein maturation along the secretory pathway (Moore 1999
). |
Complex-type fOS-GN2 are secreted when ER-to-cytosol fOS transport is blocked |
|---|
When CST is used to provoke an ER accumulation of glucosylated fOS-GN2 in HepG2 cells, fOS-GN2 can be recovered from the extracellular medium (Durrant and Moore 2002
). Most of these fOS-GN2 are secreted by the classical secretory pathway because (1) they do not come into contact with cytosolic ENGase; (2) the extracellular structures were deglucosylated by Golgi endomannosidase which mediates glucosidase-independent deglucosylation of glycoproteins in HepG2 cells (Moore and Spiro 1990) and further processed by Golgi enzymes to yield complex, sialic acid-containing fOS-GN2; and (3) their secretion was blocked by agents (monensin and brefeldin A) known to perturb transport through the Golgi apparatus (Durrant and Moore 2002). Similar observations have been made in glucosidase I-deficient Lec23 CHO cells, except that these cells do not possess endomannosidase and so secretory fOS-GN2 are not converted into complex-type structures (Durrant and Moore 2002). In mice treated with the glucosidase inhibitor butyl-deoxynojirimycin (BuDNJ), the fOS Glc1Man7GlcNAc2 was found in heart, kidney, lung, liver, brain, and spleen as well as serum and urine (Alonzi et al. 2007). Whether or not this species is exported from cells via the classical secretory pathway or a Golgi-independent pathway is not clear. In the same study complex, sialic acid-containing fOS-GN2 were not examined (Alonzi et al. 2007). |
Have the unexpected effects of ER-to-cytosol fOS transport inhibition unmasked a novel ERAD pathway? |
|---|
Although triglucosylated fOS-GN2 cannot undergo ER-to-cytosol transport, cells with impaired glucosidase I activity do generate triglucosylated fOS-GN in the cytosol (Moore and Spiro 1994
; Durrant and Moore 2002; Mellor et al. 2004; Alonzi et al. 2007) and this has been attributed to the translocation of misfolded glycoproteins out of the ER into the cytosol where triglucosylated fOS-GN2 are liberated by Png1p (see Figure 4, CST-treated cell). However, this sequence of events may not be the same for all glycoproteins. When a temperature sensitive mutant of the VSV G protein is expressed in HepG2 cells, the glycoprotein is rapidly degraded upon incubation of the cells at the nonpermissive temperature. Because the VSV G glycoprotein is expressed at very high levels, the fOS that are generated during its degradation account for greater than 90% of total cellular fOS (Spiro MJ and Spiro RG 2001). Furthermore, the majority of these glycoprotein-derived fOS are fOS-GN, compatible with their having been processed by the cytosolic ENGase. But were these fOS generated in the cytosol or in the lumen of the ER? To answer this question the same experiments were performed in the presence of CST in order to provoke the release of triglucosylated fOS from the VSV glycoprotein (Spiro MJ and Spiro RG 2001). If these triglucosylated fOS are generated in the ER, their egress from this compartment would be blocked. It was found that the ensuing triglucosylated fOS were fOS-GN2 and therefore no longer possessed structures compatible with cytosolic processing, suggesting that deglycosylation by a PNGase had occurred in the lumen of the ER (Spiro MJ and Spiro RG 2001). More recently, using benzylmannose to inhibit ER-to-cytosol fOS transport in CHO cells, a pool of glycoprotein-derived fOS-GN2 was detected (Duvet et al. 2004). Using the same arguments as employed above, the existence of an ER lumenal PNGase-dependent deglycosylation process was proposed. Interestingly, the lumenal degradation pathway was favoured in wild-type CHO cells whereas a cytosolic deglycosylation process became evident in mutant or glucose-starved CHO cells that generate truncated LLO (Duvet et al. 2004). Therefore the consequences of pharmacological manipulation of the ER-to-cytosol transport of fOS has led two groups to suggest that a PNGase activity is operational in the early endomembrane system. These are interesting findings because there is increasing evidence for a proteasome-independent ERAD pathway occuring within these compartments (Cabral et al. 2000; Spiro 2004; Donoso et al. 2005). But how can this result be reconciled with the cytoplasmic localization of Png1p? First, before the identification of PNG1 several distinct PNGase activities were identified in mammalian cells. Png1p was probably first identified in stationary mouse L-929 fibroblasts (Suzuki et al. 1994a,b). Later, two other PNGase activities were identified in logarithmically growing L-929 fibroblasts, and one of the soluble activities appeared to be glycosylated (Chang et al. 1997). More recently, a rat microsome PNGase activity that has its catalytic site oriented toward the lumen of microsomal vesicles has been proposed to have a role in the de-N-glycosylation of misfolded glycoproteins (Weng and Spiro 1997). Accordingly, there is preliminary evidence for the presence of at least two distinct PNGase activities and some data indicating the presence of an ER-luminal PNGase activity; however, the relationship of these poorly characterized activities to the well-estabished Png1p is presently not understood. Second, if Png1p accounts for all PNGase activity, it may generate lumenal fOS-GN2 subsequent to being translocated into the ER (Spiro 2004). Third, if it is postulated that at the point where glycoprotein dislocation occurs, the ER is not delimited by membrane but by protein components of the dislocation complex, Png1p could be associated with the cytosolic face of the complex while at the same time having access to the lumen of the ER. A gating structure comprising cytosolic components of the dislocation complex would be required in order to permit stretches of deglycosylated polypeptide to be extruded from the ER while retaining fOS-GN2 in the lumen. |
In HepG2 cells up to 17% of cytosolic fOS appear to originate from misfolded glycoproteins that have transited early Golgi compartments |
|---|
Insights into fOS generation, trafficking, and metabolism have been obtained by determining the steady-state structures and concentrations of cytosolic fOS in hen oviduct (Iwai et al. 1999
), perfused mouse liver (Ohashi et al. 1999), and cultured HepG2 cells (Yanagida et al. 2006). In all cases fOS-GN were more abundant than fOS-GN2, and the linear Man5GlcNAc (containing mannose residues 3–7, see Figure 2) was found to be the predominant structure isolated from all the cytosolic fractions examined. In order to gain further insights into cytosolic fOS processing and the N-glycan structures of glycoproteins destined for ERAD, studies on cytosolic fOS derived from control and SW-treated HepG2 cells were performed (Yanagida et al. 2006). Two major fOS families could be identified (Figure 5). The first corresponds to fOS whose structures are consistent with their generation from (Glc1)Man9-8GlcNAc by the cytosolic mannosidase only (Figure 5, structures highlighted in yellow) because, in the absence of SW, their structures are similar to those known to be generated when Man9-8GlcNAc is digested with cytosolic mannosidase in vitro (al Daher et al. 1992). The second family, revealed in SW-treated cells, comprises fOS whose structures indicate a Golgi -mannosidase and/or Golgi endo -mannosidase (see Figure 4, CST-treated cell) action in addition to cytosolic mannosidase processing (Figure 5, structures highlighted in magenta). The existence of this second fOS family can be explained by the fact that some misfolded glycoproteins pass through the Golgi complex before returning to the ER for translocation into the cytosol for deglycosylation and proteolysis (Frenkel et al. 2003; Hosokawa et al. 2007). Assuming that the bulk of cytosolic fOS are derived from misfolded glycoproteins and that SW itself does not perturb ERAD processes, then it can be calculated that up to 17% of HepG2 cell cytosolic fOS are perhaps generated from misfolded glycoproteins that have recycled back from early Golgi compartments prior to ERAD (Yanagida et al. 2006). Similarly, analysis of Png1p-released fOS-GN2 structures (Chantret et al. 2003) recovered from the metabolically radiolabeled yeast ams1 strain (Figure 5) indicates the presence of fOS whose structure has been modified by the Och1p mannosyltransferase that is known to reside in the early Golgi apparatus of yeast.
|
|
Potential functions of fOS: Where and how should we be looking? |
|---|
fOS-GN2 oligosaccharides have a short half-life in mammalian cells and it is thought that their clearance from the ER is efficient. By contrast, mouse liver cytosol contains 3.4 nmol and 0.5 nmol per gram wet tissues of fOS-GN and fOS-GN2 (Ohashi et al. 1999
), respectively, and if it is assumed that the cytosol occupies 10% of the tissue volume then the concentrations of fOS-GN and fOS-GN2 in the cytosol are 40 µM and 5 µM, respectively. These values are similar to the Kd values for cytosolic PNGase (10 µM for Man3GlcNAc2, Suzuki et al. 1995), ENGase (Kato et al. 1997), and the Kuptake of the lysosomal fOS importer (22 µM for Man5GlcNAc, Saint-Pol et al. 1999). It would seem from steady-state concentrations of fOS-GN and fOS-GN2 in the cytosol that ENGase activity assures low cytosolic fOS-GN2 concentrations (in cytosol preparations derived from HepG2 cells, fOS-GN2 could not be detected), and because (Glc1)Man5GlcNAc are always predominant cytosolic fOS, the lysosomal fOS transport is rate limiting for their degradation. Mammalian Png1p possess C-terminal carbohydrate binding domains of unknown function which are not present in the yeast enzyme (Suzuki et al. 1994a,b). Mammalian cells also contain cytosolic F-box proteins (Fbs1 and Fbs2) possessing lectin-like domains (Hagihara et al. 2007) that play roles in preventing misfolded glycoprotein aggregation in the cytosol and presenting glycoprotein ERAD substrates to the PNGase/proteasome complex (Yoshida et al. 2002, 2003 , 2005; 2007). The Fbs1 lectin binds to glycoprotein substrates via the chitobiose-containing core of N-glycans and the physiological relevance of fOS interactions with this protein is unclear but merits discussion. Fbs1 does not appear to interact with fOS-GN in vitro (Hagihara et al. 2007), and furthermore fully mannosylated fOS-GN2 bind less well than the core oligosaccharide, Man3GlcNAc2 (Hagihara et al. 2007). Accordingly, under normal conditions the cytosolic ENGase probably assures that fOS-GN2 do not interfere with Fbs1 binding to its glycoprotein substrates. Presently therefore, it is not known whether or not the cytoplasm of mammmalian cells contains lectins that could transduce information encoded by changing fluxes of fOS-GN.
What are the phenotypes of cells and organisms deficient in fOS regulation?
A C. elegans strain in which the ENGase gene is disrupted possessed a slightly shorter lifespan than that of control organisms (Kato et al. 2007). A distinct hyphal morphology was observed in a Neurospora crassa strain deficient in Png1p (Seiler and Plamann 2003). Yeast strains deficient in proteins involved in fOS production (Suzuki et al. 2000) and degradation (Kuranda and Robbins 1987; Cueva et al. 1990) do not manifest obvious phenotypes. These observations should be treated with caution as nothing is known about fOS metabolism in the former organisms, and although some aspects of yeast fOS regulation resemble those of mammalian cells, there are striking differences and it appears as though mammalian fOS are quantitatively more important, and that the processes involved in mammalian fOS regulation are more complex. In Jurkat (Qu et al. 2006) and human nasopharyngeal carcinoma (Yue et al. 2004) cells in which M2C1 is down regulated using an antisense strategy, striking changes in cell morphology and adhesion have been reported. In these studies fOS metabolism was not investigated and the effects of M2C1 down regulation could be ascribed to the capacity of this enzyme to intervene in glycoprotein processing rather than to its role in clearing fOS from the cytosol (Weng and Spiro 1996; Cabral et al. 2000). A mutation in the gene-encoding ER-glucosidase I was demonstrated to be the underlying cause of the only known case of CDG IIb (De Praeter et al. 2000). This fatal disease progressed rapidly and presented a clinical picture in which liver and brain were severely affected. ER-glucosidase inhibition leads to profound changes in fOS regulation (see Figure 4, CST-treated cell) and it could be postulated that abnormal intracellular accumulations of triglucosylated fOS in this patient contributed to the severe pathology (Durrant and Moore 2002). However, this proposition is difficult to evaluate as glucosidase inhibition is known to have profound effects on many aspects of glycoprotein maturation along the secretory pathway all of which could contribute to the disease process seen in this patient (Spiro 2004).
|
Concluding remarks |
|---|
The available published data on fOS regulation in mammalian and yeast cells have been reviewed, and working hypotheses for the generation and subcellular trafficking of these species in mammalian cells are presented. Although S. cerevisiae is a useful model to study the generation of fOS from LLO and glycoproteins, further work will be required in order to know whether or not this organism possesses ER and vacuolar fOS transport activities. The mammalian cell models must now be built upon by identifying at the molecular level the pyrophosphatase activity that liberates fOS-P, and the ER and lysosomal fOS transporters. Other interesting problems need to be addressed. For example, although several mechanisms have been proposed for the generation of fOS, it is not yet clear whether or not they can account for all fOS generation observed in mammalian tissue culture cells. In particular, what is the nature of the process that gives rise to glycoprotein-derived fOS-GN2 in the lumen of the ER?
Finally, research into fOS regulation will continue to be a rich source of information for cell biologists studying early events of the secretory pathway, and the development of cellular and animal models presenting with specific perturbations in fOS regulation may, on the one hand, lead to the identification of roles for these molecules in cellular homeostasis and, on the other, help in the discovery of potential human diseases associated with faulty fOS control.
|
Funding |
|---|
The authors' laboratory is supported by: Institut National de la Santé et de la Recherche Médicale (INSERM); The Mizutani Foundation; GIS – Institut des maladies rares/INSERM funded French CDG Research Network; EUROGLYCANET (LSHM-CT-2005-51231); La Fondation pour la Recherche Médicale (FRM). S.E.H.M. has been awarded a Hospital/INSERM Contrat d’Interface.
|
Conflict of interest statement |
|---|
None declared.
|
Acknowledgements |
|---|
We thank Bryan Winchester for valuable dicussions. Jean-Pierre Laigneau (CRB3) performed the art work.
|
Abbreviations |
|---|
BuDNJ, butyl-deoxynojirimycin; CDG, Congenital disorder of glycosylation; CHO, Chinese hamster ovary; CHX, cycloheximide; CPY, carboxypeptidase Y; CST, castanospermine; ENGase, endo-β-N-acetylglucosaminidase; ER, endoplasmic reticulum; ERAD, endoplasmic reticulum-associated degradation; EST, expressed sequence tag; fOS, free oligosaccharide; fOS-GN1, fOS bearing a single GlcNAc at their reducing termini; fOS-GN2, fOS bearing the di-N-acetylchitobiose moiety at their reducing termini; fOS-P, phosphorylated free oligosaccharide; Glc, glucose; GlcNAc, N-acetylglucosamine; LLO, lipid-linked oligosaccharide; Man, mannose; ManNAc, N-acetylmannosamine; MHC, major histocompatibility complex; M1P, mannose-1-phosphate; M2C1, cytosolic mannosidase; M6P, mannose-6-phosphate; OS, oligosaccharide; OST, oligosaccharyltransferase; PNGase, peptide:N-glycanase; PNP-mannose, p-nitrophenyl-
-D-mannopyranoside; RNAi, interfering RNA; shRNA, small hairpin RNA; SDS–PAGE, SDS polyacrylamide gel electrophoresis; SLO, Streptolysin O; SW, swainsonine; TCR, T-cell antigen receptor; UGGT, UDP-glucose: glycoprotein glucosyltransferase; VSV, vesicular stomatitis virus. |
References |
|---|
al Daher S, De Gasperi R, Daniel P, Hirani S, Warren C, Winchester B. Substrate specificity of human liver neutral alpha-mannosidase. Biochem J (1992) 286(Part 1):47–53.[Web of Science][Medline]
Alonzi DS, Neville DC, Lachmann RH, Dwek RA, Butters TD. Glucosylated free oligosaccharides are biomarkers of endoplasmic reticulum alpha-glucosidase inhibition. Biochem J (2008) 409:571–580.[CrossRef][Web of Science][Medline]
Andersson U, Smith D, Jeyakumar M, Butters TD, Borja MC, Dwek RA, Platt FM. Improved outcome of N-butyldeoxygalactonojirimycin-mediated substrate reduction therapy in a mouse model of Sandhoff disease. Neurobiol Dis (2004) 16:506–515.[CrossRef][Web of Science][Medline]
Anumula KR, Spiro RG. Release of glucose-containing polymannose oligosaccharides during glycoprotein biosynthesis. Studies with thyroid microsomal enzymes and slices. J Biol Chem (1983) 258:15274–15282.
Bailey D, Barreca C, O'Hare P. Trafficking of the bZIP transmembrane transcription factor CREB-H into alternate pathways of ERAD and stress-regulated intramembrane proteolysis. Traffic (2007) 8:1796–1814.[CrossRef][Web of Science][Medline]
Bebok Z, Mazzochi C, King SA, Hong JS, Sorscher EJ. The mechanism underlying cystic fibrosis transmembrane conductance regulator transport from the endoplasmic reticulum to the proteasome includes Sec61beta and a cytosolic, deglycosylated intermediary. J Biol Chem (1998) 273:29873–29878.
Belard M, Cacan R, Verbert A. Characterization of an oligosaccharide-pyrophosphodolichol pyrophosphatase activity in yeast. Biochem J (1988) 255:235–242.[Web of Science][Medline]
Bischoff J, Kornfeld R. Evidence for an alpha-mannosidase in endoplasmic reticulum of rat liver. J Biol Chem (1983) 258:7907–7910.
Bischoff J, Moremen K, Lodish HF. Isolation, characterization, and expression of cDNA encoding a rat liver endoplasmic reticulum alpha-mannosidase. J Biol Chem (1990) 265:17110–17117.
Blom D, Hirsch C, Stern P, Tortorella D, Ploegh HL. A glycosylated type I membrane protein becomes cytosolic when peptide:N-glycanase is compromised. Embo J (2004) 23:650–658.[CrossRef][Web of Science][Medline]
Burda P, Aebi M. The dolichol pathway of N-linked glycosylation. Biochim Biophys Acta (1999) 1426:239–257.[Medline]
Cabral CM, Choudhury P, Liu Y, Sifers RN. Processing by endoplasmic reticulum mannosidases partitions a secretion-impaired glycoprotein into distinct disposal pathways. J Biol Chem (2000) 275:25015–25022.
Cacan R, Cecchelli R, Verbert A. Catabolic pathway of oligosaccharide-diphospho-dolichol. Study of the fate of the oligosaccharidic moiety in mouse splenocytes. Eur J Biochem (1987) 166:469–474.[Web of Science][Medline]
Cacan R, Dengremont C, Labiau O, Kmiecik D, Mir AM, Verbert A. Occurrence of a cytosolic neutral chitobiase activity involved in oligomannoside degradation: A study with Madin-Darby bovine kidney (MDBK) cells. Biochem J (1996) 313(Part 2):597–602.[Web of Science][Medline]
Cacan R, Duvet S, Labiau O, Verbert A, Krag SS. Monoglucosylated oligomannosides are released during the degradation process of newly synthesized glycoproteins. J Biol Chem (2001) 276:22307–22312.
Cacan R, Hoflack B, Verbert A. Fate of oligosaccharide-lipid intermediates synthesized by resting rat-spleen lymphocytes. Eur J Biochem (1980) 106:473–479.[Web of Science][Medline]
Cacan R, Lepers A, Belard M, Verbert A. Catabolic pathway of oligosaccharide-diphospho-dolichol. Subcellular sites of the degradation of the oligomannoside moiety. Eur J Biochem (1989) 185:173–179.[Web of Science][Medline]
Cacan R, Verbert A. Free oligomannosides produced during the N-glycosylation process: Origin, intracellular trafficking and putative rôles. Trends Glycosci Glycotechnol (1997) 9:365–377.
Cacan R, Verbert A. Free and N-linked oligomannosides as markers of the quality control of newly synthesized glycoproteins. Biochem Biophys Res Commun (1999) 258:1–5.[CrossRef][Web of Science][Medline]
Cacan R, Villers C, Belard M, Kaiden A, Krag SS, Verbert A. Different fates of the oligosaccharide moieties of lipid intermediates. Glycobiology (1992) 2:127–136.
Chang T, Suzuki T, Inoue S, Inoue Y. Change of expression of different peptide N-glycanases at different stages of cell growth. FASEB J (1997) 11:A1247.
Chantret I, Dancourt J, Dupre T, Delenda C, Bucher S, Vuillaumier-Barrot S, Ogier de Baulny H, Peletan C, Danos O, Seta N, Durand G, Oriol R, Codogno P, Moore SEH. A deficiency in dolichyl-P-glucose: Glc1Man9GlcNAc2-PP-dolichyl alpha3-glucosyltransferase defines a new subtype of congenital disorders of glycosylation. J Biol Chem (2003) 278:9962–9971.
Chantret I, Dupre T, Delenda C, Bucher S, Dancourt J, Barnier A, Charollais A, Heron D, Bader-Meunier B, Danos O, Seta N, Durand G, Oriol R, Codogno P, Moore SEH. Congenital disorders of glycosylation type Ig is defined by a deficiency in dolichyl-P-mannose: Man7GlcNAc2-PP-dolichyl mannosyltransferase. J Biol Chem (2002) 277:25815–25822.
Chantret I, Frenoy JP, Moore SEH. Free-oligosaccharide control in the yeast Saccharomyces cerevisiae: Roles for peptide:N-glycanase (Png1p) and vacuolar mannosidase (Ams1p). Biochem J (2003) 373:901–908.[CrossRef][Web of Science][Medline]
Costanzi E, Balducci C, Cacan R, Duvet S, Orlacchio A, Beccari T. Cloning and expression of mouse cytosolic alpha-mannosidase (Man2c1). Biochim Biophys Acta (2006) 1760:1580–1586.[Medline]
Cueva R, Bordallo C, Suarez Rendueles P. Biochemical and genetic analysis of an alpha-mannosidase mutant from Saccharomyces cerevisiae. FEMS Microbiol Lett (1990) 57:153–157.[Medline]
Dancourt J, Vuillaumier-Barrot S, de Baulny HO, Sfaello I, Barnier A, le Bizec C, Dupre T, Durand G, Seta N, Moore SEH. A new intronic mutation in the DPM1 gene is associated with a milder form of CDG Ie in two French siblings. Pediatr Res (2006) 59:835–839.[CrossRef][Web of Science][Medline]
Daniel PF, Evans JE, De Gasperi R, Winchester B, Warren CD. A human lysosomal alpha(1–6)-mannosidase active on the branched trimannosyl core of complex glycans. Glycobiology (1992) 2:327–336.
De Gasperi R, al Daher S, Winchester BG, Warren CD. Substrate specificity of the bovine and feline neutral alpha-mannosidases. Biochem J (1992) 286(Part 1):55–63.[Web of Science][Medline]
De Praeter CM, Gerwig GJ, Bause E, Nuytinck LK, Vliegenthart JF, Breuer W, Kamerling JP, Espeel MF, Martin JJ, De Paepe AM, Chan NW, Dacremont GA, Van Coster RN. A novel disorder caused by defective biosynthesis of N-linked oligosaccharides due to glucosidase I deficiency. Am J Hum Genet (2000) 66:1744–1756.[CrossRef][Web of Science][Medline]
de Virgilio M, Weninger H, Ivessa NE. Ubiquitination is required for the retro-translocation of a short-lived luminal endoplasmic reticulum glycoprotein to the cytosol for degradation by the proteasome. J Biol Chem (1998) 273:9734–9743.
Dean N. Asparagine-linked glycosylation in the yeast Golgi. Biochim Biophys Acta (1999) 1426:309–322.[Medline]
Dean RT. Accelerated fluid endocytosis and re-exocytosis by lysosomal storage disease fibroblasts. Exp Cell Res (1984) 151:563–566.[CrossRef][Web of Science][Medline]
DeRossi C, Bode L, Eklund EA, Zhang F, Davis JA, Westphal V, Wang L, Borowsky AD, Freeze HH. Ablation of mouse phosphomannose isomerase (Mpi) causes mannose 6-phosphate accumulation, toxicity, and embryonic lethality. J Biol Chem (2006) 281:5916–5927.
Donoso G, Herzog V, Schmitz A. Misfolded BiP is degraded by a proteasome-independent endoplasmic-reticulum-associated degradation pathway. Biochem J (2005) 387:897–903.[CrossRef][Web of Science][Medline]
Durrant C, Moore SEH. Perturbation of free oligosaccharide trafficking in endoplasmic reticulum glucosidase I-deficient and castanospermine-treated cells. Biochem J (2002) 365:239–247.[CrossRef][Web of Science][Medline]
Dusseljee S, Wubbolts R, Verwoerd D, Tulp A, Janssen H, Calafat J, Neefjes J. Removal and degradation of the free MHC class II beta chain in the endoplasmic reticulum requires proteasomes and is accelerated by BFA. J Cell Sci (1998) 111(Part 15):2217–2226.[Abstract]
Duvet S, Foulquier F, Mir AM, Chirat F, Cacan R. Discrimination between lumenal and cytosolic sites of deglycosylation in endoplasmic reticulum-associated degradation of glycoproteins by using benzyl mannose in CHO cell lines. Glycobiology (2004) 14:841–849.
Duvet S, Labiau O, Mir AM, Kmiecik D, Krag SS, Verbert A, Cacan R. Cytosolic deglycosylation process of newly synthesized glycoproteins generates oligomannosides possessing one GlcNAc residue at the reducing end. Biochem J (1998) 335(Part 2):389–396.[Web of Science][Medline]
Egge H, Michalski JC, Strecker G. Heterogeneity of urinary oligosaccharides from mannosidosis: Mass spectrometric analysis of permethylated Man9, Man8, and Man7 derivatives. Arch Biochem Biophys (1982) 213:318–326.[CrossRef][Web of Science][Medline]
Frenkel Z, Gregory W. Endoplasmic reticulum-associated degradation of mammalian glycoproteins involves sugar chain trimming to Man6–5GlcNAc2. J Biol Chem (2003) 278:34119–34124.
Fujita E, Kouroku Y, Isoai A, Kumagai H, Misutani A, Matsuda C, Hayashi YK, Momoi T. Two endoplasmic reticulum-associated degradation (ERAD) systems for the novel variant of the mutant dysferlin: Ubiquitin/proteasome ERAD(I) and autophagy/lysosome ERAD(II). Hum Mol Genet (2007) 16:618–629.
Gao N, Shang J, Lehrman MA. Analysis of glycosylation in CDG-Ia fibroblasts by fluorophore-assisted carbohydrate electrophoresis: Implications for extracellular glucose and intracellular mannose 6-phosphate. J Biol Chem (2005) 280:17901–17909.
Goldberg AL. Functions of the proteasome: The lysis at the end of the tunnel. Science (1995) 268:522–523.
Gong Q, Keeney DR, Molinari M, Zhou Z. Degradation of trafficking-defective long QT syndrome type II mutant channels by the ubiquitin-proteasome pathway. J Biol Chem (2005) 280:19419–19425.
Grard T, Saint-Pol A, Haeuw JF, Alonso C, Wieruszeski JM, Strecker G, Michalski JC. Soluble forms of alpha-D-mannosidases from rat liver. Separation and characterization of two enzymic forms with different substrate specificities. Eur J Biochem (1994) 223:99–106.[Web of Science][Medline]
Haeuw JF, Strecker G, Wieruszeski JM, Montreuil J, Michalski JC. Substrate specificity of rat liver cytosolic alpha-D-mannosidase. Novel degradative pathway for oligomannosidic type glycans. Eur J Biochem (1991) 202:1257–1268.[Web of Science][Medline]
Hagihara S, Goda K, Matsuo I, Ito Y. Analysis of ER-associated glycoprotein degradation using synthetic glycopeptide probes. Biochem Biophys Res Commun (2007) 360:357–362.[CrossRef][Web of Science][Medline]
Hagihara S, Miyazaki A, Matsuo I, Tatami A, Suzuki T, Ito Y. Fluorescently labeled inhibitor for profiling cytoplasmic peptide:N-glycanase. Glycobiology (2007) 17:1070–1076.
Hanover JA, Elting J, Mintz GR, Lennarz WJ. Temporal aspects of the N- and O-glycosylation of human chorionic gonadotropin. J Biol Chem (1982) 257:10172–10177.
Hanover JA, Lennarz WJ. Transmembrane assembly of membrane and secretory glycoproteins. Arch Biochem Biophys (1981) 211:1–19.[CrossRef][Web of Science][Medline]
Helenius A, Aebi M. Roles of N-linked glycans in the endoplasmic reticulum. Annu Rev Biochem (2004) 73:1019–1049.[CrossRef][Web of Science][Medline]
Herscovics A, Orlean P. Glycoprotein biosynthesis in yeast. Faseb J (1993) 7:540–550.[Abstract]
Hirsch C, Blom D, Ploegh HL. A role for N-glycanase in the cytosolic turnover of glycoproteins. Embo J (2003) 22:1036–1046.[CrossRef][Web of Science][Medline]
Hirsch C, Misaghi S, Blom D, Pacold ME, Ploegh HL. Yeast N-glycanase distinguishes between native and non-native glycoproteins. EMBO Rep (2004) 5:201–206.[CrossRef][Web of Science][Medline]
Hoflack B, Cacan R, Verbert A. Dolichol pathway in lymphocytes from rat spleen. Influence of the glucosylation on the cleavage of dolichyl diphosphate oligosaccharides into phosphooligosaccharides. Eur J Biochem (1981) 117:285–290.[Web of Science][Medline]
Hosokawa N, You Z, Tremblay LO, Nagata K, Herscovics A. Stimulation of ERAD of misfolded null Hong Kong alpha1-antitrypsin by Golgi alpha1,2-mannosidases. Biochem Biophys Res Commun (2007) 362:626–632.[CrossRef][Web of Science][Medline]
Hsu AF, Baynes JW, Heath EC. The role of a dolichol-oligosaccharide as an intermediate in glycoprotein biosynthesis. Proc Natl Acad Sci USA (1974) 71:2391–2395.
Hutchins MU, Klionsky DJ. Vacuolar localization of oligomeric alpha-mannosidase requires the cytoplasm to vacuole targeting and autophagy pathway components in Saccharomyces cerevisiae. J Biol Chem (2001) 276:20491–20498.
Iwai K, Mega T, Hase S. Detection of Man5GlcNAc and related free oligomannosides in the cytosol fraction of hen oviduct. J Biochem (Tokyo) (1999) 125:70–74.
Joshi S, Katiyar S, Lennarz WJ. Misfolding of glycoproteins is a prerequisite for peptide: N-glycanase mediated deglycosylation. FEBS Lett (2005) 579:823–826.[CrossRef][Web of Science][Medline]
Katiyar S, Li G, Lennarz WJ. A complex between peptide:N-glycanase and two proteasome-linked proteins suggests a mechanism for the degradation of misfolded glycoproteins. Proc Natl Acad Sci USA (2004) 101:13774–13779.
Kato T, Fujita K, Takeuchi M, Kobayashi K, Natsuka S, Ikura K, Kumagai H, Yamamoto K. Identification of an endo-beta-N-acetylglucosaminidase gene in Caenorhabditis elegans and its expression in Escherichia coli. Glycobiology (2002) 12:581–587.
Kato T, Hatanaka K, Mega T, Hase S. Purification and characterization of endo-beta-N-acetylglucosaminidase from hen oviduct. J Biochem (Tokyo) (1997) 122:1167–1173.
Kato T, Kitamura K, Maeda M, Kimura Y, Katayama T, Ashida H, Yamamoto K. Free oligosaccharides in the cytosol of Caenorhabditis elegans are generated through endoplasmic reticulum-golgi trafficking. J Biol Chem (2007) 282:22080–22088.
Kawaguchi S, Ng DT. SnapShot: ER-associated protein degradation pathways. Cell (2007) 129:1230.[Medline]
Kelleher DJ, Gilmore R. An evolving view of the eukaryotic oligosaccharyltransferase. Glycobiology (2006) 16:47R–62R.
Kim I, Ahn J, Liu C, Tanabe K, Apodaca J, Suzuki T, Rao H. The Png1-Rad23 complex regulates glycoprotein turnover. J Cell Biol (2006) 172:211–219.
Kornfeld R, Kornfeld S. Assembly of asparagine-linked oligosaccharides. Annu Rev Biochem (1985) 54:631–664.[CrossRef][Web of Science][Medline]
Kumano M, Omichi K, Omichi K, Hase S. Substrate specificity of bovine liver cytosolic neutral alpha-mannosidase activated by Co2+. J Biochem (Tokyo) (1996) 119:991–997.
Kuokkanen E, Smith W, Makinen M, Tuominen H, Puhka M, Jokitalo E, Duvet S, Berg T, Heikinheimo P. Characterization and subcellular localization of human neutral class II{alpha}-mannosidase cytosolic enzymes/free oligosaccharides/glycosidehydrolase family 38/M2C1/N-glycosylation. Glycobiology (2007) 17:1084–1093.
Kuranda MJ, Robbins PW. Cloning and heterologous expression of glycosidase genes from Saccharomyces cerevisiae. Proc Natl Acad Sci USA (1987) 84:2585–2589.
Lisman JJ, Van Der Wal CJ, Overdijk B. Endo-N-acetyl-beta-D-glucosaminidase activity in rat liver. Studies on substrate specificity, enzyme inhibition, subcellular localization and partial purification. Biochem J (1985) 229:379–385.[Web of Science][Medline]
Mancini R, Fagioli C, Fra AM, Maggioni C, Sitia R. Degradation of unassembled soluble Ig subunits by cytosolic proteasomes: Evidence that retrotranslocation and degradation are coupled events. Faseb J (2000) 14:769–778.
Marsh CA, Gourlay GC. Evidence for a non-lysosomal alpha-mannosidase in rat liver homogenates. Biochim Biophys Acta (1971) 235:142–148.[Medline]
Mellor HR, Neville DC, Harvey DJ, Platt FM, Dwek RA, Butters TD. Cellular effects of deoxynojirimycin analogues: Inhibition of N-linked oligosaccharide processing and generation of free glucosylated oligosaccharides. Biochem J (2004) 381:867–875.[CrossRef][Web of Science][Medline]
Misaghi S, Pacold ME, Blom D, Ploegh HL, Korbel GA. Using a small molecule inhibitor of peptide:N-glycanase to probe its role in glycoprotein turnover. Chem Biol (2004) 11:1677–1687.[CrossRef][Web of Science][Medline]
Moore SEH. Transport of free polymannose-type oligosaccharides from the endoplasmic reticulum into the cytosol is inhibited by mannosides and requires a thapsigargin-sensitive calcium store. Glycobiology (1998) 8:373–381.
Moore SEH. Oligosaccharide transport: Pumping waste from the ER into lysosomes. Trends Cell Biol (1999) 9:441–446.[CrossRef][Web of Science][Medline]
Moore SEH, Bauvy C, Codogno P. Endoplasmic reticulum-to-cytosol transport of free polymannose oligosaccharides in permeabilized HepG2 cells. Embo J (1995) 14:6034–6042.[Web of Science][Medline]
Moore SEH, Spiro RG. Demonstration that Golgi endo-alpha-D-mannosidase provides a glucosidase-independent pathway for the formation of complex N-linked oligosaccharides of glycoproteins. J Biol Chem (1990) 265:13104–13112.
Moore SEH, Spiro RG. Intracellular compartmentalization and degradation of free polymannose oligosaccharides released during glycoprotein biosynthesis. J Biol Chem (1994) 269:12715–12721.
Norden NE, Lundblad A, Svensson S, Autio S. Characterization of two mannose-containing oligosaccharides isolated from the urine of patients with mannosidosis. Biochemistry (1974) 13:871–874.[CrossRef][Web of Science][Medline]
Ohashi S, Iwai K, Mega T, Hase S. Quantitation and isomeric structure analysis of free oligosaccharides present in the cytosol fraction of mouse liver: Detection of a free disialobiantennary oligosaccharide and glucosylated oligomannosides. J Biochem (Tokyo) (1999) 126:852–858.
Oku H, Hase S. Studies on the substrate specificity of neutral alpha-mannosidase purified from Japanese quail oviduct by using sugar chains from glycoproteins. J Biochem (Tokyo) (1991) 110:982–989.
Oku H, Hase S, Ikenaka T. Purification and characterization of neutral alpha-mannosidase that is activated by Co2+ from Japanese quail oviduct. J Biochem (Tokyo) (1991) 110:29–34.
Oliver GJ, Harrison J, Hemming FW. The mannosylation of dolichol-diphosphate oligosaccharides in relation to the formation of oligosaccharides and glycoproteins in pig-liver endoplasmic reticulum. Eur J Biochem (1975) 58:223–229.[Web of Science][Medline]
Oliver GJ, Harrison J, Hemming FW. The mannosylation of dolichol diphosphate oligosaccharides and the formation of oligosaccharides and glycoproteins in pig liver microsomal preparations. Biochem Soc Trans (1975) 3:666–668.[Web of Science][Medline]
Oliver GJ, Hemming FW. The transfer of mannose to dolichol diphosphate oligosaccharides in pig liver endoplasmic reticulum. Biochem J (1975) 152:191–199.[Web of Science][Medline]
Overdijk B, Van Der Kroef WM, Lisman JJ, Pierce RJ, Montreuil J, Spik G. Demonstration and partial characterization of endo-N-acetyl-beta-D-glucosaminidase in human tissues. FEBS Lett (1981) 128:364–366.[CrossRef][Web of Science][Medline]
Park H, Suzuki T, Lennarz WJ. Identification of proteins that interact with mammalian peptide:N-glycanase and implicate this hydrolase in the proteasome-dependent pathway for protein degradation. Proc Natl Acad Sci USA (2001) 98:11163–11168.
Pierce RJ, Spik G, Montreuil J. Cytosolic location of an endo-N-acetyl-beta-D-glucosaminidase activity in rat liver and kidney. Biochem J (1979) 180:673–676.[Web of Science][Medline]
Pierce RJ, Spik G, Montreuil J. Demonstration and cytosolic location of an endo-N-acetyl-beta-D-glucosaminidase activity towards an asialo-N-acetyl-lactosaminic-type substrate in rat liver. Biochem J (1980) 185:261–264.[Web of Science][Medline]
Qu L, Ju JY, Chen SL, Shi Y, Xiang ZG, Zhou YQ, Tian Y, Liu Y, Zhu LP. Inhibition of the alpha-mannosidase Man2c1 gene expression enhances adhesion of Jurkat cells. Cell Res (2006) 16:622–631.[CrossRef][Web of Science][Medline]
Quellhorst GJ Jr, O’Rear JL, Cacan R, Verbert A, Krag SS. Nonglucosylated oligosaccharides are transferred to protein in MI8-5 Chinese hamster ovary cells. Glycobiology (1999) 9:65–72.
Reddy A, Caler EV, Andrews NW. Plasma membrane repair is mediated by Ca(2+)-regulated exocytosis of lysosomes. Cell (2001) 106:157–169.[CrossRef][Web of Science][Medline]
Romisch K. Endoplasmic reticulum-associated degradation. Annu Rev Cell Dev Biol (2005) 21:435–456.[CrossRef][Web of Science][Medline]
Rosenwald AG, Stoll J, Krag SS. Regulation of glycosylation. Three enzymes compete for a common pool of dolichyl phosphate in vivo. J Biol Chem (1990) 265:14544–14553.
Ruddock LW, Molinari M. N-Glycan processing in ER quality control. J Cell Sci (2006) 119:4373–4380.
Rush JS, Gao N, Lehrman MA, Waechter CJ. Recycling of dolichyl monophosphate to the cytoplasmic leaflet of the ER after the cleavage of dolichyl pyrophosphate on the lumenal monolayer. J Biol Chem (2008) 283:4087–4093.
Russell SJ, Steger KA, Johnston SA. Subcellular localization, stoichiometry, and protein levels of 26 S proteasome subunits in yeast. J Biol Chem (1999) 274:21943–21952.
Saint-Pol A, Bauvy C, Codogno P, Moore SEH. Transfer of free polymannose-type oligosaccharides from the cytosol to lysosomes in cultured human hepatocellular carcinoma HepG2 cells. J Cell Biol (1997) 136:45–59.
Saint-Pol A, Codogno P, Moore SEH. Cytosol-to-lysosome transport of free polymannose-type oligosaccharides. Kinetic and specificity studies using rat liver lysosomes. J Biol Chem (1999) 274:13547–13555.
Saul R, Chambers JP, Molyneux RJ, Elbein AD. Castanospermine, a tetrahydroxylated alkaloid that inhibits beta-glucosidase and beta-glucocerebrosidase. Arch Biochem Biophys (1983) 221:593–597.[CrossRef][Web of Science][Medline]
Schenk B, Fernandez F, Waechter CJ. The in(side) and out(side) of dolichyl phosphate biosynthesis and recycling in the endoplasmic reticulum. Glycobiology (2001) 11:61R–70R.
Seiler S, Plamann M. The genetic basis of cellular morphogenesis in the filamentous fungus Neurospora crassa. Mol Biol Cell (2003) 14:4352–4364.
Shoup VA, Touster O. Purification and characterization of the alpha-D-mannosidase of rat liver cytosol. J Biol Chem (1976) 251:3845–3852.
Spiro MJ, Spiro RG. Potential regulation of N-glycosylation precursor through oligosaccharide-lipid hydrolase action and glucosyltransferase-glucosidase shuttle. J Biol Chem (1991) 266:5311–5317.
Spiro MJ, Spiro RG. Release of polymannose oligosaccharides from vesicular stomatitis virus G protein during endoplasmic reticulum-associated degradation. Glycobiology (2001) 11:803–811.
Spiro RG. Role of N-linked polymannose oligosaccharides in targeting glycoproteins for endoplasmic reticulum-associated degradation. Cell Mol Life Sci (2004) 61:1025–1041.[CrossRef][Web of Science][Medline]
Suzuki T, Hara I, Nakano M, Shigeta M, Nakagawa T, Kondo A, Funakoshi Y, Taniguchi N. Man2C1, an alpha-mannosidase, is involved in the trimming of free oligosaccharides in the cytosol. Biochem J (2006) 400:33–41.[CrossRef][Web of Science][Medline]
Suzuki T, Kitajima K, Inoue S, Inoue Y. Does an animal peptide: N-glycanase have the dual role as an enzyme and a carbohydrate-binding protein? Glycoconj J (1994) 11:469–476.[CrossRef][Web of Science][Medline]
Suzuki T, Kitajima K, Inoue S, Inoue Y. Occurrence and biological roles of ‘proximal glycanases’ in animal cells. Glycobiology (1994) 4:777–789.
Suzuki T, Kitajima K, Inoue Y, Inoue S. Carbohydrate-binding property of peptide: N-glycanase from mouse fibroblast L-929 cells as evaluated by inhibition and binding experiments using various oligosaccharides. J Biol Chem (1995) 270:15181–15186.
Suzuki T, Kwofie MA, Lennarz WJ. Ngly1, a mouse gene encoding a deglycosylating enzyme implicated in proteasomal degradation: Expression, genomic organization, and chromosomal mapping. Biochem Biophys Res Commun (2003) 304:326–332.[CrossRef][Web of Science][Medline]
Suzuki T, Park H, Hollingsworth NM, Sternglanz R, Lennarz WJ. PNG1, a yeast gene encoding a highly conserved peptide: N-glycanase. J Cell Biol (2000) 149:1039–1052.
Suzuki T, Park H, Kitajima K, Lennarz WJ. Peptides glycosylated in the endoplasmic reticulum of yeast are subsequently deglycosylated by a soluble peptide: N-glycanase activity. J Biol Chem (1998) 273:21526–21530.
Suzuki T, Park H, Kwofie MA, Lennarz WJ. Rad23 provides a link between the Png1 deglycosylating enzyme and the 26 S proteasome in yeast. J Biol Chem (2001) 276:21601–21607.
Suzuki T, Yano K, Sugimoto S, Kitajima K, Lennarz WJ, Inoue S, Inoue Y, Emori Y. Endo-beta-N-acetylglucosaminidase, an enzyme involved in processing of free oligosaccharides in the cytosol. Proc Natl Acad Sci USA (2002) 99:9691–9696.
Tulsiani DR, Touster O. Substrate specificities of rat kidney lysosomal and cytosolic alpha-D-mannosidases and effects of swainsonine suggest a role of the cytosolic enzyme in glycoprotein catabolism. J Biol Chem (1987) 262:6506–6514.
van Leyen K, Widemann M, Wieland F. Glycotripeptides are released by yeast but not by mammalian microsomes. FEBS Lett (1994) 355:147–150.[CrossRef][Web of Science][Medline]
Verbert A, Cacan R. Trafficking of oligomannosides released during N-glycosylation: A clearing mechanism of the rough endoplasmic reticulum. Biochim Biophys Acta (1999) 1473:137–146.[Medline]
Villers C, Cacan R, Mir AM, Labiau O, Verbert A. Release of oligomannoside-type glycans as a marker of the degradation of newly synthesized glycoproteins. Biochem J (1994) 298(Part 1):135–142.[Web of Science][Medline]
Ward CL, Omura S, Kopito RR. Degradation of CFTR by the ubiquitin-proteasome pathway. Cell (1995) 83:121–127.[CrossRef][Web of Science][Medline]
Weng S, Spiro RG. Endoplasmic reticulum kifunensine-resistant alpha-mannosidase is enzymatically and immunologically related to the cytosolic alpha-mannosidase. Arch Biochem Biophys (1996) 325:113–123.[CrossRef][Web of Science][Medline]
Weng S, Spiro RG. Demonstration of a peptide: N-glycosidase in the endoplasmic reticulum of rat liver. Biochem J (1997) 322(Part 2):655–661.[Web of Science][Medline]
Werner ED, Brodsky JL, McCracken AA. Proteasome-dependent endoplasmic reticulum-associated protein degradation: An unconventional route to a familiar fate. Proc Natl Acad Sci USA (1996) 93:13797–13801.
Wieland FT, Gleason ML, Serafini TA, Rothman JE. The rate of bulk flow from the endoplasmic reticulum to the cell surface. Cell (1987) 50:289–300.[CrossRef][Web of Science][Medline]
Wiertz EJ, Jones TR, Sun L, Bogyo M, Geuze HJ, Ploegh HL. The human cytomegalovirus US11 gene product dislocates MHC class I heavy chains from the endoplasmic reticulum to the cytosol. Cell (1996) 84:769–779.[CrossRef][Web of Science][Medline]
Winchester B. Lysosomal metabolism of glycoproteins. Glycobiology (2005) 15:1R–15R.
Yamashita K, Tachibana Y, Mihara K, Okada S, Yabuuchi H, Kobata A. Urinary oligosaccharides of mannosidosis. J Biol Chem (1980) 255:5126–5133.
Yanagida K, Natsuka S, Hase S. Structural diversity of cytosolic free oligosaccharides in the human hepatoma cell line, HepG2. Glycobiology (2006) 16:294–304.
Yoshida Y, Adachi E, Fukiya K, Iwai K, Tanaka K. Glycoprotein-specific ubiquitin ligases recognize N-glycans in unfolded substrates. EMBO Rep (2005) 6:239–244.[CrossRef][Web of Science][Medline]
Yoshida Y, Chiba T, Tokunaga F, Kawasaki H, Iwai K, Suzuki T, Ito Y, Matsuoka K, Yoshida M, Tanaka K, Tai T. E3 ubiquitin ligase that recognizes sugar chains. Nature (2002) 418:438–442.[CrossRef][Medline]
Yoshida Y, Murakami A, Iwai K, Tanaka K. A neural-specific F-box protein Fbs1 functions as a chaperone suppressing glycoprotein aggregation. J Biol Chem (2007) 282:7137–7144.
Yoshida Y, Tokunaga F, Chiba T, Iwai K, Tanaka K, Tai T. Fbs2 is a new member of the E3 ubiquitin ligase family that recognizes sugar chains. J Biol Chem (2003) 278:43877–43884.
Yoshihisa T, Anraku Y. A novel pathway of import of alpha-mannosidase, a marker enzyme of vacuolar membrane, in Saccharomyces cerevisiae. J Biol Chem (1990) 265:22418–22425.
Yu H, Kaung G, Kobayashi S, Kopito RR. Cytosolic degradation of T-cell receptor alpha chains by the proteasome. J Biol Chem (1997) 272:20800–20804.
Yue W, Jin YL, Shi GX, Liu Y, Gao Y, Zhao FT, Zhu LP. Suppression of 6A8 alpha-mannosidase gene expression reduced the potentiality of growth and metastasis of human nasopharyngeal carcinoma. Int J Cancer (2004) 108:189–195.[CrossRef][Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  Y. Li, W. Fang, L. Zhang, H. Ouyang, H. Zhou, Y. Luo, and C. Jin Class IIC {alpha}-mannosidase AfAms1 is required for morphogenesis and cellular function in Aspergillus fumigatus Glycobiology, June 1, 2009; 19(6): 624 - 632. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||