Occurrence of a cytosolic neutral chitobiase activity involved in oligomannoside degradation: a study with Madin-Darby bovine kidney (MDBK) cells

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The Chinese hamster ovary mutant MI8 -5 is known to synthesize Man 9 GlcNAc 2 -P-P-dolichol rather than the fully glucosylated lipid intermediate Glc 3 Man 9 GlcNAc 2 -P-P-dolichol. This nonglucosylated oligosaccharide lipid precursor is used as donor for N-glycosylation. In this paper we demonstrate that a significant part of the glycans bound to the newly synthesized glycoproteins in MI8 -5 cells are monoglucosylated. The presence of monoglucosylated glycans on glycoproteins determines their binding to calnexin as part of the quality control machinery. Furthermore, we point out the presence of Glc 1 Man 5 GlcNAc 1 in the cytosol of MI8 -5 cells. This indicates that part of the monoglucosylated glycoproteins can be directed toward a deglycosylation process that occurs in the cytosol. Besides studies on glycoprotein degradation based on the disappearance of protein moieties, MI8 -5 cells can be used as a tool to elucidate the various step leading to glycoprotein degradation by studying the fate of the glycan moieties.A key reaction of N-glycosylation is the transfer en bloc of a Glc 3 Man 9 GlcNAc 2 oligosaccharide from a lipid intermediate to an Asn residue in the Asn-Xaa-Ser(Thr) consensus sequence of a nascent protein. This N-glycosylation process is immediately followed by sequential deglucosylation leading to Man 9 GlcNAc 2 protein. It is now clearly established that oligosaccharide trimming in the ER 1 is intimately linked to the quality control process, leading to the degradation of misfolded glycoproteins or unassembled multimeric proteins. Indeed the presence of monoglucosylated oligomannosides on glycoproteins retained in the ER is essential for their binding to the molecular chaperones calnexin and calreticulin (1, 2). The presence of a glucosyl residue is controlled by reglucosylation-deglucosylation cycles involving UDP-glucose : glycoprotein glucosyltransferase acting as a folding sensor (3) and glucosidase II, respectively. It has been demonstrated that glucose trimming and reglucosylation cycles determine the association of a glycoprotein with calnexin, as well as its folding (4,5).During this quality control, part of newly synthesized glycoproteins are degraded after translocation of the glycosylated polypeptide chains from the ER to the cytosol. As first demonstrated by Wiertz et al. (6), using labeled protein with an amino acid precursor, this retrotranslocation is accompanied by the removal of the glycans by a cytosolic peptide N-glycanase (PNGase), such as the one isolated by Suzuki et al. (7). The relationship between the location of the enzyme and the degradation process has been discussed by Karaivanova and Spiro (8). However, it is worth mentioning that few labs follow the fate of newly synthesized proteins by labeling with [2-3 H]mannose. In this case, the N-glycosylation process is accompanied by the release of glycans from either lipid intermediates or from newly synthesized glycoproteins (9 -11). The trafficking of the released oligomannosides from both origins leads to the formatio...

Section: Discussionmentioning

confidence: 85%

Monoglucosylated Oligomannosides Are Released during the Degradation Process of Newly Synthesized Glycoproteins

Cacan

Duvet

Labiau

et al. 2001

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“…8), the interaction of free OS with the lysosomal transporter bears similarities to the interactions between free OS and cytosolic glycosidases. Accordingly, the cytosolic chitobiase (15) by definition interacts with the reducing di-N-acetylchitobiose moiety of free OS, but more surprisingly, the cytosolic mannosidase has been found to act only on free OS bearing a single GlcNAc residue at their reducing termini (20). Therefore, these two enzymes and the cytosol-to-lysosome free OS transporter interact with free OS in a manner that involves surveillance of the reducing terminus of the oligosaccharide.…”

Section: The Lysosomal Free Os Transporter Displays a Unique Specificmentioning

confidence: 99%

“…In fact, neutral free OS are subject to sequential processing (15)(16)(17) by a previously characterized cytosolic endo-H-like activity (18) or chitobiase (15) and cytosolic mannosidase (19 -21) to yield a free OS containing five residues of mannose and a single reducing N-acetylglucosamine residue. It has now been demonstrated that this free OS is transferred to lysosomes in order to be degraded (17).…”

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confidence: 99%

Cytosol-to-lysosome Transport of Free Polymannose-type Oligosaccharides

Saint‐Pol

Codogno

Moore

1999

In hepatocellular carcinoma HepG2 cells, free polymannose-type oligosaccharides appearing in the cytosol during the biosynthesis and quality control of glycoproteins are rapidly translocated into lysosomes by an as yet poorly defined process (Saint-Pol, A., Bauvy, C., Codogno, P., and Moore, S. E. H. (1997) J. Cell Biol. 136, 45-59). Here, we demonstrate an ATP-dependent association of [2-3 H]mannose-labeled Man 5 GlcNAc with isolated rat liver lysosomes. This association was only observed in the presence of swainsonine, a mannosidase inhibitor, which was required for the protection of sedimentable, but not nonsedimentable, Man 5 GlcNAc from degradation, indicating that oligosaccharides were transported into lysosomes. Saturable high affinity transport (K uptake , 22.3 M, V max , 7.1 fmol/min/unit of ␤-hexosaminidase) was dependent upon the hydrolysis of ATP but independent of vacuolar H ؉ /ATPase activity. Transport was inhibited strongly by NEM and weakly by vanadate but not by sodium azide, and, in addition, the sugar transport inhibitors phloretin, phloridzin, and cytochalasin B were without effect on transport. Oligosaccharide import did not show absolute specificity but was selective toward partially demannosylated and dephosphorylated oligosaccharides, and, furthermore, inhibition studies revealed that the free reducing GlcNAc residue of the oligosaccharide was of critical importance for its interaction with the transporter. These results demonstrate the presence of a novel lysosomal free oligosaccharide transporter that must work in concert with cytosolic hydrolases in order to clear the cytosol of endoplasmic reticulum-generated free oligosaccharides.Free polymannose-type oligosaccharides (free OS) 1 are a byproduct of the biosynthesis of dolichol oligosaccharides (1-3) and the glycosylation of glycoproteins (3, 4). Whereas phosphorylated free OS (Man 5 GlcNAc 2 -P) are released from dolichol oligosaccharides on the cytoplasmic face of the ER (5), neutral free OS (Glc 3-1 Man 9 -8 GlcNAc 2 ) are liberated from oligosaccharide-lipid in the lumen of the ER (1, 6). After deglucosylation, these neutral free OS do not follow the secretory pathway but are routed to the cytosol (7) by an ATP-and calcium-requiring translocation process that can be inhibited by mannose but not by N-acetylglucosamine (8). Glycopepetides (9, 10) and misfolded glycoproteins (11,12) are also translocated out of the ER into the cytosol, where they can be deglycosylated by an Nglycanase (13, 14), giving rise to further cytosolic free OS.The rapid appearance of substantial quantities of free OS in the cytosol of cells during the biosynthesis and quality control of glycoproteins suggested that the cytosol must possess elements capable of the disposal of these osmotically active components. In fact, neutral free OS are subject to sequential processing (15-17) by a previously characterized cytosolic endo-H-like activity (18) or chitobiase (15) and cytosolic mannosidase (19 -21) to yield a free OS containing five residues of mannose and a ...

“…The remaining free oligosaccharides (fOS) bear two GlcNAc residues at their reducing ends (called fOSGN2). They are subsequently trimmed by the ␤-endo-N-acetylglucosaminidase (ENGase), resulting in the formation of oligosaccharides possessing a single GlcNAc residue at their reducing ends (called fOSGN) (5). fOSGN may be also released directly from glycoproteins and glycopeptides by the direct action of ENGase (6).…”

mentioning

confidence: 99%

“…Misfolded glycoproteins originating in the lumen of the ER move to the cytosol to be degraded (4). An N-glycanase (PNGase) cleaves the N-linked glycans (5), which are reduced to Man 9 GlcNAc by an ENGase (6). Man 9 GlcNAc is then trimmed down to Man 5 GlcNAc by Man2c1 (7) prior to transfer into the lysosome (8).…”

mentioning

confidence: 99%

Accumulation of Free Oligosaccharides and Tissue Damage in Cytosolic α-Mannosidase (Man2c1)-deficient Mice

Paciotti

Persichetti

Klein

et al. 2014

Background: Cytosolic ␣-mannosidase (Man2c1) is involved in the catabolism of free oligosaccharides (fOS). Results: Man2c1-deficient mice accumulate fOS in tissues and show biochemical and histological alterations in the CNS, liver, and intestine. Conclusion: Man2c1 plays an essential role in the catabolism of fOS. Significance: Man2c1-deficient mice could be a useful tool to examine the biological significance of Man2c1.