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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

Quantitation of Ngly1p-dependent and -independent fOS pools in HepG2 cells.Cells were transfected with 50 pmoles of control siRNA (Ctrl and Z-vad), 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, where indicated, cells were preincubated for 30 min with 40 µM Z-vad-fmk (Z-vad). Cells were then pulse-radiolabeled in either the absence or presence of Z-vad with [2-3H]mannose for 30 min (Pulse, left panels) or pulse-radiolabeled for 30 min prior to conducting a 1 h chase incubation (Chase, right panels). [2-3H]lipid-linked oligosaccharides, [2-3H]N-glycans released by EndoH from both cellular and medium glycoproteins and [2-3H]fOS were prepared and quantitated by scintillation counting. After summing the radioactivity associated with the above described fractions to generate total radioactivity incorporation into cells. [2-3H]fOS were examined by TLC. In order to take into account the differences in total incorporation of radiolabel into cells cultivated under the different conditions, the fraction of total fOS that was loaded onto the TLC was adjusted to take into account the ratio of the total cellular radioactivity for a given incubation to that recovered from the incubation incorporating least radioactivity. The migration positions of standard oligosaccharides are shown to the left of the chromatograms and the abbreviations used are as described in Fig 2. After elution of the oligosaccharide components from TLC plates and quantitation by scintillation counting, inhibition of total fOS appearance with respect to the control was calculated and is shown under the appropriate TLC lanes. The percentage inhibitions of individual oligosaccharides (Glc1Man9GlcNAc2: G1M9, Man9GlcNAc2: M9, Man8GlcNAc2: M8) observed in the ENG + NGLY and ENG + Z-vad conditions with respect to the ENG condition are shown to the right of the chromatograms. The scanned TLC lanes are from the same fluorograph, but due to uneven migration, the scans were aligned manually to facilitate interpretation of data. This experiment was repeated 4 times and the error bars represent the standard deviation.
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pone-0011734-g006: Quantitation of Ngly1p-dependent and -independent fOS pools in HepG2 cells.Cells were transfected with 50 pmoles of control siRNA (Ctrl and Z-vad), 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, where indicated, cells were preincubated for 30 min with 40 µM Z-vad-fmk (Z-vad). Cells were then pulse-radiolabeled in either the absence or presence of Z-vad with [2-3H]mannose for 30 min (Pulse, left panels) or pulse-radiolabeled for 30 min prior to conducting a 1 h chase incubation (Chase, right panels). [2-3H]lipid-linked oligosaccharides, [2-3H]N-glycans released by EndoH from both cellular and medium glycoproteins and [2-3H]fOS were prepared and quantitated by scintillation counting. After summing the radioactivity associated with the above described fractions to generate total radioactivity incorporation into cells. [2-3H]fOS were examined by TLC. In order to take into account the differences in total incorporation of radiolabel into cells cultivated under the different conditions, the fraction of total fOS that was loaded onto the TLC was adjusted to take into account the ratio of the total cellular radioactivity for a given incubation to that recovered from the incubation incorporating least radioactivity. The migration positions of standard oligosaccharides are shown to the left of the chromatograms and the abbreviations used are as described in Fig 2. After elution of the oligosaccharide components from TLC plates and quantitation by scintillation counting, inhibition of total fOS appearance with respect to the control was calculated and is shown under the appropriate TLC lanes. The percentage inhibitions of individual oligosaccharides (Glc1Man9GlcNAc2: G1M9, Man9GlcNAc2: M9, Man8GlcNAc2: M8) observed in the ENG + NGLY and ENG + Z-vad conditions with respect to the ENG condition are shown to the right of the chromatograms. The scanned TLC lanes are from the same fluorograph, but due to uneven migration, the scans were aligned manually to facilitate interpretation of data. This experiment was repeated 4 times and the error bars represent the standard deviation.

Mentions: Next the size of the Ngly1p-dependent fOS pool was calculated. Accordingly, pulse-chase studies were performed in control RNAi-, or ENG-3-transfected cells in which Ngly1p was either co-down regulated with NGLY1-3, or inhibited with Z-vad. In 30 min pulse radiolabeling incubations, Z-vad provokes about 25% total fOS inhibition whereas Ngly1p down regulation leads to about 12% fOS inhibition (Fig 6, Pulse, left panel). Down-regulation of Engase1p alone did not provoke a significant inhibition of total fOS, and comparing the Engase1p down regulation condition with the Engase1p and Ngly1p compromised conditions, similar total fOS inhibitions were noted (Fig 6, Pulse, left panel). In the latter conditions, where quantitation of Man8GlcNAc2 is more meaningful due to reduced fOSGN2-to-fOSGN conversion, substantial inhibitions of the appearance of fOS migrating as Glc1Man9GlcNAc2 and Man9GlcNAc2 were noted whereas lesser inhibition of Man8GlcNAc2 was observed (Fig 6, Pulse, right panel). Overall, however, NGLY1-3-, or Z-vad-mediated fOS inhibitions were similar irrespective of whether or not Engase1p was co-down regulated. In cells that were pulse radiolabeled and chased for 1 h (Fig 6, lower panels) the situation changed such that Engase1p down regulation alone caused a small (5–10%) but significant inhibition of total fOS generation (Fig 6, Chase, left panel). This inhibition of the appearance of fOS mediated by Engase1p down regulation alone was also reflected by increases in fOS inhibition observed when both Engase1p and Ngly1p are compromised compared to when the latter activity is reduced alone (Fig 6, Chase, left panel). Indeed, when both enzyme activities are reduced, ∼35% total fOS inhibition is noted in 1 h chase incubations. In addition it can be seen that, in the presence of ENG-3, the inhibitions of the appearance of fOS migrating as Glc1Man9GlcNAc2 Man9GlcNAc2 and Man8GlcNAc2 caused by Z-vad and NGLY1-3 were higher than those noted for the pulse incubations (Fig 6, compare upper and lower right panels). Although Z-vad and NGLY1-3 provoked small inhibitions of the appearance of Man8GlcNAc2, this inhibition may be overestimated because of incomplete Engase1p down regulation leading to contamination of this component with Glc1Man9GlcNAc and Man9GlcNAc whose appearance is inhibited by Z-vad and NGLY1-3. To summarise, taking into account that ENG-3 itself causes a small but significant inhibition in the appearance of fOS, it can be concluded that the Ngly1p-dependent fOS pool corresponds to ∼30% of total fOS.


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)

Quantitation of Ngly1p-dependent and -independent fOS pools in HepG2 cells.Cells were transfected with 50 pmoles of control siRNA (Ctrl and Z-vad), 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, where indicated, cells were preincubated for 30 min with 40 µM Z-vad-fmk (Z-vad). Cells were then pulse-radiolabeled in either the absence or presence of Z-vad with [2-3H]mannose for 30 min (Pulse, left panels) or pulse-radiolabeled for 30 min prior to conducting a 1 h chase incubation (Chase, right panels). [2-3H]lipid-linked oligosaccharides, [2-3H]N-glycans released by EndoH from both cellular and medium glycoproteins and [2-3H]fOS were prepared and quantitated by scintillation counting. After summing the radioactivity associated with the above described fractions to generate total radioactivity incorporation into cells. [2-3H]fOS were examined by TLC. In order to take into account the differences in total incorporation of radiolabel into cells cultivated under the different conditions, the fraction of total fOS that was loaded onto the TLC was adjusted to take into account the ratio of the total cellular radioactivity for a given incubation to that recovered from the incubation incorporating least radioactivity. The migration positions of standard oligosaccharides are shown to the left of the chromatograms and the abbreviations used are as described in Fig 2. After elution of the oligosaccharide components from TLC plates and quantitation by scintillation counting, inhibition of total fOS appearance with respect to the control was calculated and is shown under the appropriate TLC lanes. The percentage inhibitions of individual oligosaccharides (Glc1Man9GlcNAc2: G1M9, Man9GlcNAc2: M9, Man8GlcNAc2: M8) observed in the ENG + NGLY and ENG + Z-vad conditions with respect to the ENG condition are shown to the right of the chromatograms. The scanned TLC lanes are from the same fluorograph, but due to uneven migration, the scans were aligned manually to facilitate interpretation of data. This experiment was repeated 4 times and the error bars represent the standard deviation.
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Related In: Results  -  Collection

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

pone-0011734-g006: Quantitation of Ngly1p-dependent and -independent fOS pools in HepG2 cells.Cells were transfected with 50 pmoles of control siRNA (Ctrl and Z-vad), 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, where indicated, cells were preincubated for 30 min with 40 µM Z-vad-fmk (Z-vad). Cells were then pulse-radiolabeled in either the absence or presence of Z-vad with [2-3H]mannose for 30 min (Pulse, left panels) or pulse-radiolabeled for 30 min prior to conducting a 1 h chase incubation (Chase, right panels). [2-3H]lipid-linked oligosaccharides, [2-3H]N-glycans released by EndoH from both cellular and medium glycoproteins and [2-3H]fOS were prepared and quantitated by scintillation counting. After summing the radioactivity associated with the above described fractions to generate total radioactivity incorporation into cells. [2-3H]fOS were examined by TLC. In order to take into account the differences in total incorporation of radiolabel into cells cultivated under the different conditions, the fraction of total fOS that was loaded onto the TLC was adjusted to take into account the ratio of the total cellular radioactivity for a given incubation to that recovered from the incubation incorporating least radioactivity. The migration positions of standard oligosaccharides are shown to the left of the chromatograms and the abbreviations used are as described in Fig 2. After elution of the oligosaccharide components from TLC plates and quantitation by scintillation counting, inhibition of total fOS appearance with respect to the control was calculated and is shown under the appropriate TLC lanes. The percentage inhibitions of individual oligosaccharides (Glc1Man9GlcNAc2: G1M9, Man9GlcNAc2: M9, Man8GlcNAc2: M8) observed in the ENG + NGLY and ENG + Z-vad conditions with respect to the ENG condition are shown to the right of the chromatograms. The scanned TLC lanes are from the same fluorograph, but due to uneven migration, the scans were aligned manually to facilitate interpretation of data. This experiment was repeated 4 times and the error bars represent the standard deviation.
Mentions: Next the size of the Ngly1p-dependent fOS pool was calculated. Accordingly, pulse-chase studies were performed in control RNAi-, or ENG-3-transfected cells in which Ngly1p was either co-down regulated with NGLY1-3, or inhibited with Z-vad. In 30 min pulse radiolabeling incubations, Z-vad provokes about 25% total fOS inhibition whereas Ngly1p down regulation leads to about 12% fOS inhibition (Fig 6, Pulse, left panel). Down-regulation of Engase1p alone did not provoke a significant inhibition of total fOS, and comparing the Engase1p down regulation condition with the Engase1p and Ngly1p compromised conditions, similar total fOS inhibitions were noted (Fig 6, Pulse, left panel). In the latter conditions, where quantitation of Man8GlcNAc2 is more meaningful due to reduced fOSGN2-to-fOSGN conversion, substantial inhibitions of the appearance of fOS migrating as Glc1Man9GlcNAc2 and Man9GlcNAc2 were noted whereas lesser inhibition of Man8GlcNAc2 was observed (Fig 6, Pulse, right panel). Overall, however, NGLY1-3-, or Z-vad-mediated fOS inhibitions were similar irrespective of whether or not Engase1p was co-down regulated. In cells that were pulse radiolabeled and chased for 1 h (Fig 6, lower panels) the situation changed such that Engase1p down regulation alone caused a small (5–10%) but significant inhibition of total fOS generation (Fig 6, Chase, left panel). This inhibition of the appearance of fOS mediated by Engase1p down regulation alone was also reflected by increases in fOS inhibition observed when both Engase1p and Ngly1p are compromised compared to when the latter activity is reduced alone (Fig 6, Chase, left panel). Indeed, when both enzyme activities are reduced, ∼35% total fOS inhibition is noted in 1 h chase incubations. In addition it can be seen that, in the presence of ENG-3, the inhibitions of the appearance of fOS migrating as Glc1Man9GlcNAc2 Man9GlcNAc2 and Man8GlcNAc2 caused by Z-vad and NGLY1-3 were higher than those noted for the pulse incubations (Fig 6, compare upper and lower right panels). Although Z-vad and NGLY1-3 provoked small inhibitions of the appearance of Man8GlcNAc2, this inhibition may be overestimated because of incomplete Engase1p down regulation leading to contamination of this component with Glc1Man9GlcNAc and Man9GlcNAc whose appearance is inhibited by Z-vad and NGLY1-3. To summarise, taking into account that ENG-3 itself causes a small but significant inhibition in the appearance of fOS, it can be concluded that the Ngly1p-dependent fOS pool corresponds to ∼30% of total fOS.

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