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Identification of roles for peptide: N-glycanase and endo-beta-N-acetylglucosaminidase (Engase1p) during protein N-glycosylation in human HepG2 cells.

Chantret I, Fasseu M, Zaoui K, Le Bizec C, Sadou Yayé H, Dupré T, Moore SE - PLoS ONE (2010)

Bottom Line: During mammalian protein N-glycosylation, 20% of all dolichol-linked oligosaccharides (LLO) appear as free oligosaccharides (fOS) bearing the di-N-acetylchitobiose (fOSGN2), or a single N-acetylglucosamine (fOSGN), moiety at their reducing termini.Saccharomyces cerevisiae does not possess ENGase activity and expression of human Engase1p in the png1Delta deletion mutant, in which fOS are reduced by over 98%, partially restored fOS generation.The fully mannosylated structures that occur in the Ngly1p-dependent fOSGN2 pool indicate an ERAD process that does not require N-glycan trimming.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U773, Centre de Recherche Bichat Beaujon, Paris, France; Université Paris 7 Denis Diderot, site Bichat, Paris, France. isabelle.chantret@inserm.fr

ABSTRACT

Background: During mammalian protein N-glycosylation, 20% of all dolichol-linked oligosaccharides (LLO) appear as free oligosaccharides (fOS) bearing the di-N-acetylchitobiose (fOSGN2), or a single N-acetylglucosamine (fOSGN), moiety at their reducing termini. After sequential trimming by cytosolic endo beta-N-acetylglucosaminidase (ENGase) and Man2c1 mannosidase, cytosolic fOS are transported into lysosomes. Why mammalian cells generate such large quantities of fOS remains unexplored, but fOSGN2 could be liberated from LLO by oligosaccharyltransferase, or from glycoproteins by NGLY1-encoded Peptide-N-Glycanase (PNGase). Also, in addition to converting fOSGN2 to fOSGN, the ENGASE-encoded cytosolic ENGase of poorly defined function could potentially deglycosylate glycoproteins. Here, the roles of Ngly1p and Engase1p during fOS metabolism were investigated in HepG2 cells.

Methods/principal findings: During metabolic radiolabeling and chase incubations, RNAi-mediated Engase1p down regulation delays fOSGN2-to-fOSGN conversion, and it is shown that Engase1p and Man2c1p are necessary for efficient clearance of cytosolic fOS into lysosomes. Saccharomyces cerevisiae does not possess ENGase activity and expression of human Engase1p in the png1Delta deletion mutant, in which fOS are reduced by over 98%, partially restored fOS generation. In metabolically radiolabeled HepG2 cells evidence was obtained for a small but significant Engase1p-mediated generation of fOS in 1 h chase but not 30 min pulse incubations. Ngly1p down regulation revealed an Ngly1p-independent fOSGN2 pool comprising mainly Man(8)GlcNAc(2), corresponding to approximately 70% of total fOS, and an Ngly1p-dependent fOSGN2 pool enriched in Glc(1)Man(9)GlcNAc(2) and Man(9)GlcNAc(2) that corresponds to approximately 30% of total fOS.

Conclusions/significance: As the generation of the bulk of fOS is unaffected by co-down regulation of Ngly1p and Engase1p, alternative quantitatively important mechanisms must underlie the liberation of these fOS from either LLO or glycoproteins during protein N-glycosylation. The fully mannosylated structures that occur in the Ngly1p-dependent fOSGN2 pool indicate an ERAD process that does not require N-glycan trimming.

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Related in: MedlinePlus

Examination of fOS, and N-glycans after NGLY1-3 or Z-vad mediated reduction of Ngly1p activity in control and castanospermine-treated cells.A. HepG2 cells were transfected with 25 pmoles of control siRNA (Ctrl), or 25 pmoles of NGLY1-3. Three days after transfection, cells were pulse-radiolabeled with [2-3H]mannose for 30 min and cellular EndoH-released [2-3H]N-glycans (N-glycans) and [2-3H]fOS (fOS) were prepared and analysed by HPLC. Whereas the whole fOS fractions were analysed, only 25% of the N-glycan fractions were examined by HPLC. The elution positions of standard oligosaccharides are indicated above the HPLC profiles and the abbreviations used are defined in the legend for Fig 2. B. HepG2 cells were transfected with 50 pmoles of control siRNA (Ctrl), or 25 pmoles each of NGLY1-3 and 25 pmoles of control siRNA (NGLY), or 25 pmoles each of ENG-3 and 25 pmoles of control siRNA (ENG), or 25 pmoles each of NGLY1-3 and ENG-3 (ENG + NGLY). Three days after transfection, cells were preincubated for 30 min with 2 mM castanospermine (CST), and where indicated, with 40 µM Z-vad-fmk (Z-vad). Cells were then pulse-radiolabeled with [2-3H]mannose for 30 min and cellular EndoH-released [2-3H]N-glycans (GP) and [2-3H]fOS (fOS) were prepared and examined by TLC. The scanned TLC lanes are from the same fluorograph, but due to uneven migration, the scans were aligned manually to facilitate interpretation of data. Closed and open arrowheads indicate the migration positions of components bearing one or two residues, respectively, of GlcNAc at their reducing end. The abbreviations are: G3M9; Glc3Man9GlcNAc1-2, G3M8; Glc3Man8GlcNAc1-2. C. These experiments were performed once.
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pone-0011734-g005: Examination of fOS, and N-glycans after NGLY1-3 or Z-vad mediated reduction of Ngly1p activity in control and castanospermine-treated cells.A. HepG2 cells were transfected with 25 pmoles of control siRNA (Ctrl), or 25 pmoles of NGLY1-3. Three days after transfection, cells were pulse-radiolabeled with [2-3H]mannose for 30 min and cellular EndoH-released [2-3H]N-glycans (N-glycans) and [2-3H]fOS (fOS) were prepared and analysed by HPLC. Whereas the whole fOS fractions were analysed, only 25% of the N-glycan fractions were examined by HPLC. The elution positions of standard oligosaccharides are indicated above the HPLC profiles and the abbreviations used are defined in the legend for Fig 2. B. HepG2 cells were transfected with 50 pmoles of control siRNA (Ctrl), or 25 pmoles each of NGLY1-3 and 25 pmoles of control siRNA (NGLY), or 25 pmoles each of ENG-3 and 25 pmoles of control siRNA (ENG), or 25 pmoles each of NGLY1-3 and ENG-3 (ENG + NGLY). Three days after transfection, cells were preincubated for 30 min with 2 mM castanospermine (CST), and where indicated, with 40 µM Z-vad-fmk (Z-vad). Cells were then pulse-radiolabeled with [2-3H]mannose for 30 min and cellular EndoH-released [2-3H]N-glycans (GP) and [2-3H]fOS (fOS) were prepared and examined by TLC. The scanned TLC lanes are from the same fluorograph, but due to uneven migration, the scans were aligned manually to facilitate interpretation of data. Closed and open arrowheads indicate the migration positions of components bearing one or two residues, respectively, of GlcNAc at their reducing end. The abbreviations are: G3M9; Glc3Man9GlcNAc1-2, G3M8; Glc3Man8GlcNAc1-2. C. These experiments were performed once.

Mentions: To gain more insight into fOS generation during Ngly1p down regulation [2-3H]glycoproteins and [2-3H]fOS were examined under different experimental conditions indicated in Fig 5A and B. In this experiment, although Ngly1p down-regulation produced the expected effect on the distribution of fOS (Fig 5A), the N-glycan profile differed little from that derived from control cell incubations (Fig 5B). It was noted that EndoH-released N-glycans comprised 22.0%, 30.9% and 17.9% Glc1Man9GlcNAc, Man9GlcNAc and Man8GlcNAc, respectively, for the control RNAi-transfected cells, and 22.8%, 32.2% and 15.6%, respectively, for NGLY1-3 transfected cells. As the Ngly1p-dependent fOSGN2 pool is enriched in Glc1Man9GlcNAc2 whose N-linked counterpart can interact with the lectins calnexin and calreticulin [2], we asked whether or not formation of the Ngly1p-dependent fOSGN2 pool required such N-glycan/lectin interactions. It is known that in order for glycoproteins to interact with calnexin and calreticulin, ER glucosidase-dependent trimming of triglucosylated N-glycans must occur [4]. Accordingly, the effect of ER glucosidase inhibition by castanospermine (CST) on Ngly1p-mediated fOS generation was investigated. Fig 5C demonstrates the presence of an Ngly1p-dependent pool of Glc3Man9GlcNAc2 and an Ngly1p-independent pool of Glc3Man8GlcNAc2 during ER glucosidase inhibition. In the same incubations it can be seen that the ratio of N-linked Glc3Man9GlcNAc2 to Glc3Man8GlcNAc2 remains similar under all the experimental conditions. Thus, an Ngly1p-dependent fOS pool was detected in the absence of monoglucosylated N-glycans. Furthermore, irrespective of glucosidase inhibition, the Ngly1p-dependent fOS pools are predominantly fully mannosylated whereas the Ngly1p-independent pools comprise oligosaccharides containing mainly 8 residues of mannose.


Identification of roles for peptide: N-glycanase and endo-beta-N-acetylglucosaminidase (Engase1p) during protein N-glycosylation in human HepG2 cells.

Chantret I, Fasseu M, Zaoui K, Le Bizec C, Sadou Yayé H, Dupré T, Moore SE - PLoS ONE (2010)

Examination of fOS, and N-glycans after NGLY1-3 or Z-vad mediated reduction of Ngly1p activity in control and castanospermine-treated cells.A. HepG2 cells were transfected with 25 pmoles of control siRNA (Ctrl), or 25 pmoles of NGLY1-3. Three days after transfection, cells were pulse-radiolabeled with [2-3H]mannose for 30 min and cellular EndoH-released [2-3H]N-glycans (N-glycans) and [2-3H]fOS (fOS) were prepared and analysed by HPLC. Whereas the whole fOS fractions were analysed, only 25% of the N-glycan fractions were examined by HPLC. The elution positions of standard oligosaccharides are indicated above the HPLC profiles and the abbreviations used are defined in the legend for Fig 2. B. HepG2 cells were transfected with 50 pmoles of control siRNA (Ctrl), or 25 pmoles each of NGLY1-3 and 25 pmoles of control siRNA (NGLY), or 25 pmoles each of ENG-3 and 25 pmoles of control siRNA (ENG), or 25 pmoles each of NGLY1-3 and ENG-3 (ENG + NGLY). Three days after transfection, cells were preincubated for 30 min with 2 mM castanospermine (CST), and where indicated, with 40 µM Z-vad-fmk (Z-vad). Cells were then pulse-radiolabeled with [2-3H]mannose for 30 min and cellular EndoH-released [2-3H]N-glycans (GP) and [2-3H]fOS (fOS) were prepared and examined by TLC. The scanned TLC lanes are from the same fluorograph, but due to uneven migration, the scans were aligned manually to facilitate interpretation of data. Closed and open arrowheads indicate the migration positions of components bearing one or two residues, respectively, of GlcNAc at their reducing end. The abbreviations are: G3M9; Glc3Man9GlcNAc1-2, G3M8; Glc3Man8GlcNAc1-2. C. These experiments were performed once.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2909182&req=5

pone-0011734-g005: Examination of fOS, and N-glycans after NGLY1-3 or Z-vad mediated reduction of Ngly1p activity in control and castanospermine-treated cells.A. HepG2 cells were transfected with 25 pmoles of control siRNA (Ctrl), or 25 pmoles of NGLY1-3. Three days after transfection, cells were pulse-radiolabeled with [2-3H]mannose for 30 min and cellular EndoH-released [2-3H]N-glycans (N-glycans) and [2-3H]fOS (fOS) were prepared and analysed by HPLC. Whereas the whole fOS fractions were analysed, only 25% of the N-glycan fractions were examined by HPLC. The elution positions of standard oligosaccharides are indicated above the HPLC profiles and the abbreviations used are defined in the legend for Fig 2. B. HepG2 cells were transfected with 50 pmoles of control siRNA (Ctrl), or 25 pmoles each of NGLY1-3 and 25 pmoles of control siRNA (NGLY), or 25 pmoles each of ENG-3 and 25 pmoles of control siRNA (ENG), or 25 pmoles each of NGLY1-3 and ENG-3 (ENG + NGLY). Three days after transfection, cells were preincubated for 30 min with 2 mM castanospermine (CST), and where indicated, with 40 µM Z-vad-fmk (Z-vad). Cells were then pulse-radiolabeled with [2-3H]mannose for 30 min and cellular EndoH-released [2-3H]N-glycans (GP) and [2-3H]fOS (fOS) were prepared and examined by TLC. The scanned TLC lanes are from the same fluorograph, but due to uneven migration, the scans were aligned manually to facilitate interpretation of data. Closed and open arrowheads indicate the migration positions of components bearing one or two residues, respectively, of GlcNAc at their reducing end. The abbreviations are: G3M9; Glc3Man9GlcNAc1-2, G3M8; Glc3Man8GlcNAc1-2. C. These experiments were performed once.
Mentions: To gain more insight into fOS generation during Ngly1p down regulation [2-3H]glycoproteins and [2-3H]fOS were examined under different experimental conditions indicated in Fig 5A and B. In this experiment, although Ngly1p down-regulation produced the expected effect on the distribution of fOS (Fig 5A), the N-glycan profile differed little from that derived from control cell incubations (Fig 5B). It was noted that EndoH-released N-glycans comprised 22.0%, 30.9% and 17.9% Glc1Man9GlcNAc, Man9GlcNAc and Man8GlcNAc, respectively, for the control RNAi-transfected cells, and 22.8%, 32.2% and 15.6%, respectively, for NGLY1-3 transfected cells. As the Ngly1p-dependent fOSGN2 pool is enriched in Glc1Man9GlcNAc2 whose N-linked counterpart can interact with the lectins calnexin and calreticulin [2], we asked whether or not formation of the Ngly1p-dependent fOSGN2 pool required such N-glycan/lectin interactions. It is known that in order for glycoproteins to interact with calnexin and calreticulin, ER glucosidase-dependent trimming of triglucosylated N-glycans must occur [4]. Accordingly, the effect of ER glucosidase inhibition by castanospermine (CST) on Ngly1p-mediated fOS generation was investigated. Fig 5C demonstrates the presence of an Ngly1p-dependent pool of Glc3Man9GlcNAc2 and an Ngly1p-independent pool of Glc3Man8GlcNAc2 during ER glucosidase inhibition. In the same incubations it can be seen that the ratio of N-linked Glc3Man9GlcNAc2 to Glc3Man8GlcNAc2 remains similar under all the experimental conditions. Thus, an Ngly1p-dependent fOS pool was detected in the absence of monoglucosylated N-glycans. Furthermore, irrespective of glucosidase inhibition, the Ngly1p-dependent fOS pools are predominantly fully mannosylated whereas the Ngly1p-independent pools comprise oligosaccharides containing mainly 8 residues of mannose.

Bottom Line: During mammalian protein N-glycosylation, 20% of all dolichol-linked oligosaccharides (LLO) appear as free oligosaccharides (fOS) bearing the di-N-acetylchitobiose (fOSGN2), or a single N-acetylglucosamine (fOSGN), moiety at their reducing termini.Saccharomyces cerevisiae does not possess ENGase activity and expression of human Engase1p in the png1Delta deletion mutant, in which fOS are reduced by over 98%, partially restored fOS generation.The fully mannosylated structures that occur in the Ngly1p-dependent fOSGN2 pool indicate an ERAD process that does not require N-glycan trimming.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U773, Centre de Recherche Bichat Beaujon, Paris, France; Université Paris 7 Denis Diderot, site Bichat, Paris, France. isabelle.chantret@inserm.fr

ABSTRACT

Background: During mammalian protein N-glycosylation, 20% of all dolichol-linked oligosaccharides (LLO) appear as free oligosaccharides (fOS) bearing the di-N-acetylchitobiose (fOSGN2), or a single N-acetylglucosamine (fOSGN), moiety at their reducing termini. After sequential trimming by cytosolic endo beta-N-acetylglucosaminidase (ENGase) and Man2c1 mannosidase, cytosolic fOS are transported into lysosomes. Why mammalian cells generate such large quantities of fOS remains unexplored, but fOSGN2 could be liberated from LLO by oligosaccharyltransferase, or from glycoproteins by NGLY1-encoded Peptide-N-Glycanase (PNGase). Also, in addition to converting fOSGN2 to fOSGN, the ENGASE-encoded cytosolic ENGase of poorly defined function could potentially deglycosylate glycoproteins. Here, the roles of Ngly1p and Engase1p during fOS metabolism were investigated in HepG2 cells.

Methods/principal findings: During metabolic radiolabeling and chase incubations, RNAi-mediated Engase1p down regulation delays fOSGN2-to-fOSGN conversion, and it is shown that Engase1p and Man2c1p are necessary for efficient clearance of cytosolic fOS into lysosomes. Saccharomyces cerevisiae does not possess ENGase activity and expression of human Engase1p in the png1Delta deletion mutant, in which fOS are reduced by over 98%, partially restored fOS generation. In metabolically radiolabeled HepG2 cells evidence was obtained for a small but significant Engase1p-mediated generation of fOS in 1 h chase but not 30 min pulse incubations. Ngly1p down regulation revealed an Ngly1p-independent fOSGN2 pool comprising mainly Man(8)GlcNAc(2), corresponding to approximately 70% of total fOS, and an Ngly1p-dependent fOSGN2 pool enriched in Glc(1)Man(9)GlcNAc(2) and Man(9)GlcNAc(2) that corresponds to approximately 30% of total fOS.

Conclusions/significance: As the generation of the bulk of fOS is unaffected by co-down regulation of Ngly1p and Engase1p, alternative quantitatively important mechanisms must underlie the liberation of these fOS from either LLO or glycoproteins during protein N-glycosylation. The fully mannosylated structures that occur in the Ngly1p-dependent fOSGN2 pool indicate an ERAD process that does not require N-glycan trimming.

Show MeSH
Related in: MedlinePlus