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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

Effects of Engase1p and Man2c1p down regulation on steady-state levels of cytosolic and MBC fOS in HepG2 cells.fOS recovered from both the cytosol and the MBC fractions of SLO-permeabilized, control siRNA (Control), ENG-3 or M2C1-1 -transfected cells, were derivatized with 2-AP, and analyzed by HPLC before (− EndoH) and after digestion with EndoH from S. plicatus (+ EndoH). A. HPLC profiles of the cytosolic PA-derivatized fOS obtained before (Blue traces) and after EndoH (Red traces) digestion from control siRNA (Left panel) or ENG-3 (Right panel) transfected HepG2 cells. The open arrowheads indicate the migration positions of fOSGN2-AP derivatives whose appearance is abrogated after EndoH digestion. Only the region of the chromatograms that display significant differences before and after EndoH digestion is shown. B. The EndoH treated fOS from the cytosol (Upper panel) and membrane bound compartments MBC (Lower panel) were resolved by HPLC and the migration positions of standard radioactive oligosaccharide 2-AP derivatives are indicated, and where known, the isomeric configuration of the standard structures are indicated (Green circles; mannose, blue squares; N-acetylglucosamine). The asteriks indicate peaks corresponding to oligosaccharides whose abundance does not change under the different conditions. In both A and B the background noise associated with the ENG-3 profiles is higher than in either the control or M2C1-3 traces because the fluorescence scale was amplified to take into account the smaller amount of cells recovered from ENG-3 transfected cultures. This experiment was performed once, but a preliminary experiment, in which cells were not permeabilised with SLO, was performed and found to give qualitatively similar results.
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pone-0011734-g003: Effects of Engase1p and Man2c1p down regulation on steady-state levels of cytosolic and MBC fOS in HepG2 cells.fOS recovered from both the cytosol and the MBC fractions of SLO-permeabilized, control siRNA (Control), ENG-3 or M2C1-1 -transfected cells, were derivatized with 2-AP, and analyzed by HPLC before (− EndoH) and after digestion with EndoH from S. plicatus (+ EndoH). A. HPLC profiles of the cytosolic PA-derivatized fOS obtained before (Blue traces) and after EndoH (Red traces) digestion from control siRNA (Left panel) or ENG-3 (Right panel) transfected HepG2 cells. The open arrowheads indicate the migration positions of fOSGN2-AP derivatives whose appearance is abrogated after EndoH digestion. Only the region of the chromatograms that display significant differences before and after EndoH digestion is shown. B. The EndoH treated fOS from the cytosol (Upper panel) and membrane bound compartments MBC (Lower panel) were resolved by HPLC and the migration positions of standard radioactive oligosaccharide 2-AP derivatives are indicated, and where known, the isomeric configuration of the standard structures are indicated (Green circles; mannose, blue squares; N-acetylglucosamine). The asteriks indicate peaks corresponding to oligosaccharides whose abundance does not change under the different conditions. In both A and B the background noise associated with the ENG-3 profiles is higher than in either the control or M2C1-3 traces because the fluorescence scale was amplified to take into account the smaller amount of cells recovered from ENG-3 transfected cultures. This experiment was performed once, but a preliminary experiment, in which cells were not permeabilised with SLO, was performed and found to give qualitatively similar results.

Mentions: A detailed analysis of the steady state levels of cytosolic fOS in HepG2 cells has been reported, and although fOSGN2 were not detected, a complex mixture of glucosylated and nonglucosylated fOSGN was described [38]. In another report down-regulation of Man2c1p using an siRNA strategy in HEK-293 cells caused an accumulation of the substrates of this enzyme (Glc1-0Man9-7GlcNAc) but little reduction in its limit digest product (Man5GlcNAc) steady state level, indicating an alternative, quantitatively important, mechanism for the generation of this latter component in these cells [17]. In order to further examine the consequences of Engase1p down-regulation on fOS metabolism, fOS steady state levels were examined in the cytosol and MBC of cells transfected with ENG-3 and compared to fOS levels observed in cells transfected with control and M2C1-1 siRNA duplexes. Total fOS were extracted from the different compartments and derivatised with 2-AP. The resulting fOS-AP derivatives were then resolved by HPLC before and after digestion with EndoH: only fOSGN2-AP are cleaved and loose their fluorescent tag. It was observed that the EndoH treatment only modified fOS elution profiles in a region of the chromatographs corresponding to the elution times of Glc1Man9GlcNAc2-AP and Man9-7GlcNAc2-AP standards, and this region of the elution profiles is shown in Fig 3A. Differences in the superimposed HPLC profiles (− EndoH/+ EndoH) as shown in Fig 3A for the cytosol fractions of control-transfected (left panel) and ENG-3-transfected (right panel) cells reveal the presence of only small quantities of fOSGN2 in control cells and 6–11 fold increases in these components in ENG-3-transfected cells (Table 1). The major species that accumulate in the ENG-3-transfected cells appear to elute similarly to standard Glc1Man9GlcNAc2-AP, Man9GlcNAc2-AP and Man8GlcNAc2-AP. By contrast to the metabolic radiolabeling experiments shown in Fig 1, small quantities of an oligosaccharide behaving as Man7GlcNAc2-AP were also noted in the ENG-3-transfected cells (Fig 3A, right panel). In addition, the HPLC profiles indicate that the peak corresponding to Man8GlcNAc2-AP is not symmetrical and comprises a shoulder on its leading edge indicating the presence of a second component (Fig 3A, right panel). At present it is not clear whether this second minor Man8GlcNAc2-AP component and the Man7GlcNAc2-AP structure represent slow processing of Glc1-0Man9-8GlcNAc2 oligosaccharides by cytosolic mannosidase or whether they arise directly from either LLO or glycoprotein. In cells transfected with M2C1-1, large quantities of interfering fOSGN impeded unambiguous detection of fOSGN2 (results not shown). Finally, with the amount of starting material used, fOSGN2 could not be clearly identified in MBC fractions. Fig 3B shows the chromatographs obtained when the EndoH-treated samples containing only fOSGN-AP were analysed. A detailed analysis of the changes in fOSGN-AP caused by the different siRNA duplexes is beyond the scope of the present report, but two important observations can be underlined. First, as well as their capacity to increase large cytosolic fOS levels (Glc1-0Man9-7GlcNAc2 for ENG-3: Fig 3A and Glc1-0Man9-7GlcNAc for M2C1-1: Fig 3B, green trace) ENG-3 and M2C1-1 siRNA duplexes reduce the steady state concentration of cytosolic Man5GlcNAc, and do so with a relative potency that mirrors their ability to provoke the accumulation of fOSGN2 and untrimmed fOSGN, respectively. Second, data presented in Table 1 indicate that for large fOS, ∼5–15% of the total cellular amount (Cyt + MBC) of each component is associated with the MBC, however this value increases to 50–90% for oligosaccharides that co migrate with standard Man4-3GlcNAc. This observation is consistent with previous results indicating a predominantly lysosomal localization of fOSGN possessing less than 5 residues of mannose [11]. Results presented in both Fig 3B and Table 1 demonstrate that in addition to reducing levels of cytosolic Man5GlcNAc, both the ENG-3 and M2C1-1 siRNA duplexes reduce the levels of Man4-3GlcNAc that occur in the MBC. To summarize, the effects of ENG-3 and M2C1-1 siRNA duplexes on steady state levels of fOS in HepG2 cells are consistent with the hypothesis that Engase1p and Man2c1p have a concerted action in regulating the subcellular trafficking of fOS in HepG2 cells.


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)

Effects of Engase1p and Man2c1p down regulation on steady-state levels of cytosolic and MBC fOS in HepG2 cells.fOS recovered from both the cytosol and the MBC fractions of SLO-permeabilized, control siRNA (Control), ENG-3 or M2C1-1 -transfected cells, were derivatized with 2-AP, and analyzed by HPLC before (− EndoH) and after digestion with EndoH from S. plicatus (+ EndoH). A. HPLC profiles of the cytosolic PA-derivatized fOS obtained before (Blue traces) and after EndoH (Red traces) digestion from control siRNA (Left panel) or ENG-3 (Right panel) transfected HepG2 cells. The open arrowheads indicate the migration positions of fOSGN2-AP derivatives whose appearance is abrogated after EndoH digestion. Only the region of the chromatograms that display significant differences before and after EndoH digestion is shown. B. The EndoH treated fOS from the cytosol (Upper panel) and membrane bound compartments MBC (Lower panel) were resolved by HPLC and the migration positions of standard radioactive oligosaccharide 2-AP derivatives are indicated, and where known, the isomeric configuration of the standard structures are indicated (Green circles; mannose, blue squares; N-acetylglucosamine). The asteriks indicate peaks corresponding to oligosaccharides whose abundance does not change under the different conditions. In both A and B the background noise associated with the ENG-3 profiles is higher than in either the control or M2C1-3 traces because the fluorescence scale was amplified to take into account the smaller amount of cells recovered from ENG-3 transfected cultures. This experiment was performed once, but a preliminary experiment, in which cells were not permeabilised with SLO, was performed and found to give qualitatively similar results.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0011734-g003: Effects of Engase1p and Man2c1p down regulation on steady-state levels of cytosolic and MBC fOS in HepG2 cells.fOS recovered from both the cytosol and the MBC fractions of SLO-permeabilized, control siRNA (Control), ENG-3 or M2C1-1 -transfected cells, were derivatized with 2-AP, and analyzed by HPLC before (− EndoH) and after digestion with EndoH from S. plicatus (+ EndoH). A. HPLC profiles of the cytosolic PA-derivatized fOS obtained before (Blue traces) and after EndoH (Red traces) digestion from control siRNA (Left panel) or ENG-3 (Right panel) transfected HepG2 cells. The open arrowheads indicate the migration positions of fOSGN2-AP derivatives whose appearance is abrogated after EndoH digestion. Only the region of the chromatograms that display significant differences before and after EndoH digestion is shown. B. The EndoH treated fOS from the cytosol (Upper panel) and membrane bound compartments MBC (Lower panel) were resolved by HPLC and the migration positions of standard radioactive oligosaccharide 2-AP derivatives are indicated, and where known, the isomeric configuration of the standard structures are indicated (Green circles; mannose, blue squares; N-acetylglucosamine). The asteriks indicate peaks corresponding to oligosaccharides whose abundance does not change under the different conditions. In both A and B the background noise associated with the ENG-3 profiles is higher than in either the control or M2C1-3 traces because the fluorescence scale was amplified to take into account the smaller amount of cells recovered from ENG-3 transfected cultures. This experiment was performed once, but a preliminary experiment, in which cells were not permeabilised with SLO, was performed and found to give qualitatively similar results.
Mentions: A detailed analysis of the steady state levels of cytosolic fOS in HepG2 cells has been reported, and although fOSGN2 were not detected, a complex mixture of glucosylated and nonglucosylated fOSGN was described [38]. In another report down-regulation of Man2c1p using an siRNA strategy in HEK-293 cells caused an accumulation of the substrates of this enzyme (Glc1-0Man9-7GlcNAc) but little reduction in its limit digest product (Man5GlcNAc) steady state level, indicating an alternative, quantitatively important, mechanism for the generation of this latter component in these cells [17]. In order to further examine the consequences of Engase1p down-regulation on fOS metabolism, fOS steady state levels were examined in the cytosol and MBC of cells transfected with ENG-3 and compared to fOS levels observed in cells transfected with control and M2C1-1 siRNA duplexes. Total fOS were extracted from the different compartments and derivatised with 2-AP. The resulting fOS-AP derivatives were then resolved by HPLC before and after digestion with EndoH: only fOSGN2-AP are cleaved and loose their fluorescent tag. It was observed that the EndoH treatment only modified fOS elution profiles in a region of the chromatographs corresponding to the elution times of Glc1Man9GlcNAc2-AP and Man9-7GlcNAc2-AP standards, and this region of the elution profiles is shown in Fig 3A. Differences in the superimposed HPLC profiles (− EndoH/+ EndoH) as shown in Fig 3A for the cytosol fractions of control-transfected (left panel) and ENG-3-transfected (right panel) cells reveal the presence of only small quantities of fOSGN2 in control cells and 6–11 fold increases in these components in ENG-3-transfected cells (Table 1). The major species that accumulate in the ENG-3-transfected cells appear to elute similarly to standard Glc1Man9GlcNAc2-AP, Man9GlcNAc2-AP and Man8GlcNAc2-AP. By contrast to the metabolic radiolabeling experiments shown in Fig 1, small quantities of an oligosaccharide behaving as Man7GlcNAc2-AP were also noted in the ENG-3-transfected cells (Fig 3A, right panel). In addition, the HPLC profiles indicate that the peak corresponding to Man8GlcNAc2-AP is not symmetrical and comprises a shoulder on its leading edge indicating the presence of a second component (Fig 3A, right panel). At present it is not clear whether this second minor Man8GlcNAc2-AP component and the Man7GlcNAc2-AP structure represent slow processing of Glc1-0Man9-8GlcNAc2 oligosaccharides by cytosolic mannosidase or whether they arise directly from either LLO or glycoprotein. In cells transfected with M2C1-1, large quantities of interfering fOSGN impeded unambiguous detection of fOSGN2 (results not shown). Finally, with the amount of starting material used, fOSGN2 could not be clearly identified in MBC fractions. Fig 3B shows the chromatographs obtained when the EndoH-treated samples containing only fOSGN-AP were analysed. A detailed analysis of the changes in fOSGN-AP caused by the different siRNA duplexes is beyond the scope of the present report, but two important observations can be underlined. First, as well as their capacity to increase large cytosolic fOS levels (Glc1-0Man9-7GlcNAc2 for ENG-3: Fig 3A and Glc1-0Man9-7GlcNAc for M2C1-1: Fig 3B, green trace) ENG-3 and M2C1-1 siRNA duplexes reduce the steady state concentration of cytosolic Man5GlcNAc, and do so with a relative potency that mirrors their ability to provoke the accumulation of fOSGN2 and untrimmed fOSGN, respectively. Second, data presented in Table 1 indicate that for large fOS, ∼5–15% of the total cellular amount (Cyt + MBC) of each component is associated with the MBC, however this value increases to 50–90% for oligosaccharides that co migrate with standard Man4-3GlcNAc. This observation is consistent with previous results indicating a predominantly lysosomal localization of fOSGN possessing less than 5 residues of mannose [11]. Results presented in both Fig 3B and Table 1 demonstrate that in addition to reducing levels of cytosolic Man5GlcNAc, both the ENG-3 and M2C1-1 siRNA duplexes reduce the levels of Man4-3GlcNAc that occur in the MBC. To summarize, the effects of ENG-3 and M2C1-1 siRNA duplexes on steady state levels of fOS in HepG2 cells are consistent with the hypothesis that Engase1p and Man2c1p have a concerted action in regulating the subcellular trafficking of fOS in HepG2 cells.

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