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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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Alternative NGLY1 transcripts and the impact of their down regulation on fOS generation in HepG2 cells.A. Genomic organization of the human NGLY1 gene and alternative transcripts found in HepG2 cells. The major transcript found in HepG2 cells is indicated in blue and corresponds to the already published NGLY1 mRNA sequence (NM_018297.3). A minor transcript indicated in orange is generated by skipping of exon 11 (NM_001145295.1). B. Partial alignment of homologous Ngly1p sequences (amino acids 522–558) encoded by the minor transcript and comparison with the corresponding region of the major NGLY1 transcript (accession numbers: Macaca mulatta; XP_001092914, Equus caballus; XP_001492093, Canis lupus familiaris; DN746692.1). Note that the isoform encoded by the minor transcript contains 21 amino acids in its COOH terminal which are not found in the peptide sequence encoded by the major transcript. C. HepG2 cells were transiently transfected with the different siRNA duplexes designed to silence either or both NGLY1 transcripts (see Table S1). Total RNA were extracted three days later and the mRNA levels of each specific transcript were analyzed by QPCR. In parallel, HepG2 cells were co transfected with ENG-3 and the different siRNA duplexes targeting the two NGLY1 transcripts. As described in Materials and Methods, because of the variable GC content of the different RNAi duplexes, two negative control siRNA duplexes with either medium (med GC) or low GC (low GC) content were used. After 3 days cells were pulse-radiolabeled with [2-3H]mannose for 30 min. In some experiments, cells transfected with ENG-3 alone or with control RNAi duplexes were treated with either Z-vad-fmk (40 µM) dissolved in DMSO (Z-vad*) or DMSO alone (*) for 45 min prior to, and during the radiolabeling period. Subsequently, [2-3H]fOS, [2-3H]LLO and [2-3H]Glycoproteins were extracted, purified and quantitated, and [2-3H]fOS were analysed by TLC. In order to take into account the differences in total incorporation of radiolabel into cells cultivated under the different conditions, a fraction of total fOS was loaded onto the TLC according to the ratio of the total cellular radioactivity for a given incubation to that recovered from the incubation incorporating least radioactivity. The scanned TLC lanes are from the same fluorograph, but due to uneven migration, the scans were aligned manually to facilitate interpretation of data. The migration positions of standard oligosaccharides are shown to the left of the chromatograms and the abbreviations used are as described in Fig 1. The percent inhibition of the major and minor NGLY1 transcripts provoked by the different RNAi duplexes was calculated using the QPCR data and is indicated underneath the appropriate lanes. This experiment was performed once.
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pone-0011734-g004: Alternative NGLY1 transcripts and the impact of their down regulation on fOS generation in HepG2 cells.A. Genomic organization of the human NGLY1 gene and alternative transcripts found in HepG2 cells. The major transcript found in HepG2 cells is indicated in blue and corresponds to the already published NGLY1 mRNA sequence (NM_018297.3). A minor transcript indicated in orange is generated by skipping of exon 11 (NM_001145295.1). B. Partial alignment of homologous Ngly1p sequences (amino acids 522–558) encoded by the minor transcript and comparison with the corresponding region of the major NGLY1 transcript (accession numbers: Macaca mulatta; XP_001092914, Equus caballus; XP_001492093, Canis lupus familiaris; DN746692.1). Note that the isoform encoded by the minor transcript contains 21 amino acids in its COOH terminal which are not found in the peptide sequence encoded by the major transcript. C. HepG2 cells were transiently transfected with the different siRNA duplexes designed to silence either or both NGLY1 transcripts (see Table S1). Total RNA were extracted three days later and the mRNA levels of each specific transcript were analyzed by QPCR. In parallel, HepG2 cells were co transfected with ENG-3 and the different siRNA duplexes targeting the two NGLY1 transcripts. As described in Materials and Methods, because of the variable GC content of the different RNAi duplexes, two negative control siRNA duplexes with either medium (med GC) or low GC (low GC) content were used. After 3 days cells were pulse-radiolabeled with [2-3H]mannose for 30 min. In some experiments, cells transfected with ENG-3 alone or with control RNAi duplexes were treated with either Z-vad-fmk (40 µM) dissolved in DMSO (Z-vad*) or DMSO alone (*) for 45 min prior to, and during the radiolabeling period. Subsequently, [2-3H]fOS, [2-3H]LLO and [2-3H]Glycoproteins were extracted, purified and quantitated, and [2-3H]fOS were analysed by TLC. In order to take into account the differences in total incorporation of radiolabel into cells cultivated under the different conditions, a fraction of total fOS was loaded onto the TLC according to the ratio of the total cellular radioactivity for a given incubation to that recovered from the incubation incorporating least radioactivity. The scanned TLC lanes are from the same fluorograph, but due to uneven migration, the scans were aligned manually to facilitate interpretation of data. The migration positions of standard oligosaccharides are shown to the left of the chromatograms and the abbreviations used are as described in Fig 1. The percent inhibition of the major and minor NGLY1 transcripts provoked by the different RNAi duplexes was calculated using the QPCR data and is indicated underneath the appropriate lanes. This experiment was performed once.

Mentions: The preceeding results indicate that Engase1p has an important role in conversion of fOSGN2 to fOSGN but only a minor, if any, role in glycoprotein deglycosylation. Next, the role of the NGLY1-encoded cytosolic PNGase in fOS generation was examined in HepG2 cells. First, RNAi duplexes that effectively targeted NGLY1 expression were sought, but searches in silico indicated several EST sequences potentially arising from alternative transcripts. In order to detect these transcripts, primers were designed to span the regions containing sequence differences but after RT-PCR, only the region corresponding to the C-terminus of Ngly1p yielded different amplified products. Indeed, using forward primer NGLY1-10long and the reverse primer NGLY1-8 (Table S1), the major PCR product (339 bp) expected of the published human NGLY1 mRNA sequence [20] and a minor PCR product of 161 bp were detected. Both PCR products were sequenced and found to correspond to the transcripts shown in Fig 4A. The minor transcript was found to correspond to the EST sequence (Gene bank accession NM_001145295.1) in which exon 11 has been skipped, generating a premature STOP codon that in turn shortens the peptide sequence at its C-terminus. The missing region in this transcript comprises most of the C-terminal domain that is required for the binding of Ngly1p to N-linked oligosaccharides [39], suggesting that these two NGLY1 gene products may not have equivalent functions. Furthermore, inspection of the databases revealed the presence of this minor NGLY1 transcript in the sequence data from several mammals (Fig 4B). Next, the impact of these two NGLY1 gene products on fOS generation was examined. siRNA duplexes targeting both common and distinctive regions of the transcripts were designed (Table S1) and the ability of each to silence the two transcripts was assessed by QPCR. Additionally, the ability of the duplexes to block fOSGN2 generation was examined. In order to simplify identification of efficient NGLY1-targeted RNAi duplexes, fOS were examined in a background of Engase1p down regulation where the majority of fOS occur as fOSGN2. Results shown in Fig 4C demonstrate that among the three duplexes which cause greater than 88% silencing of both transcripts, NGLY1-1 and NGLY1-3 provoke a striking reduction in the generation of a fOSGN2 migrating as Glc1Man9GlcNAc2, and a lesser reduction of a fOSGN2 migrating as Man9GlcNAc2, whereas the fOSGN2 migrating as Man8GlcNAc2 appeared to be unaffected. The fOSGN2 profiles obtained with NGLY1-1 and NGLY1-3, but not NGLY1-2, were found to be identical to that obtained when the ENG-3 transfected cells were pretreated with, and radiolabeled in the presence of the PNGase inhibitor Z-vad-fmk (Z-vad) [40] (Fig 4C). Although successful design of an effective duplex specifically targeting the minor transcript was not achieved (Fig 4C), one of the 3 duplexes targeting the major NGLY1 transcript selectively silenced this transcript without silencing the minor transcript (NGLY1 Long-3). The fOS profile obtained with this duplex was similar to those observed with Z-vad and NGLY1-1 and NGLY1-3 duplexes, indicating that the major NGLY1 transcript that encodes Ngly1p is responsible for the observed changes in fOS generation in HepG2 cells (Fig 4C).


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)

Alternative NGLY1 transcripts and the impact of their down regulation on fOS generation in HepG2 cells.A. Genomic organization of the human NGLY1 gene and alternative transcripts found in HepG2 cells. The major transcript found in HepG2 cells is indicated in blue and corresponds to the already published NGLY1 mRNA sequence (NM_018297.3). A minor transcript indicated in orange is generated by skipping of exon 11 (NM_001145295.1). B. Partial alignment of homologous Ngly1p sequences (amino acids 522–558) encoded by the minor transcript and comparison with the corresponding region of the major NGLY1 transcript (accession numbers: Macaca mulatta; XP_001092914, Equus caballus; XP_001492093, Canis lupus familiaris; DN746692.1). Note that the isoform encoded by the minor transcript contains 21 amino acids in its COOH terminal which are not found in the peptide sequence encoded by the major transcript. C. HepG2 cells were transiently transfected with the different siRNA duplexes designed to silence either or both NGLY1 transcripts (see Table S1). Total RNA were extracted three days later and the mRNA levels of each specific transcript were analyzed by QPCR. In parallel, HepG2 cells were co transfected with ENG-3 and the different siRNA duplexes targeting the two NGLY1 transcripts. As described in Materials and Methods, because of the variable GC content of the different RNAi duplexes, two negative control siRNA duplexes with either medium (med GC) or low GC (low GC) content were used. After 3 days cells were pulse-radiolabeled with [2-3H]mannose for 30 min. In some experiments, cells transfected with ENG-3 alone or with control RNAi duplexes were treated with either Z-vad-fmk (40 µM) dissolved in DMSO (Z-vad*) or DMSO alone (*) for 45 min prior to, and during the radiolabeling period. Subsequently, [2-3H]fOS, [2-3H]LLO and [2-3H]Glycoproteins were extracted, purified and quantitated, and [2-3H]fOS were analysed by TLC. In order to take into account the differences in total incorporation of radiolabel into cells cultivated under the different conditions, a fraction of total fOS was loaded onto the TLC according to the ratio of the total cellular radioactivity for a given incubation to that recovered from the incubation incorporating least radioactivity. The scanned TLC lanes are from the same fluorograph, but due to uneven migration, the scans were aligned manually to facilitate interpretation of data. The migration positions of standard oligosaccharides are shown to the left of the chromatograms and the abbreviations used are as described in Fig 1. The percent inhibition of the major and minor NGLY1 transcripts provoked by the different RNAi duplexes was calculated using the QPCR data and is indicated underneath the appropriate lanes. This experiment was performed once.
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Related In: Results  -  Collection

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pone-0011734-g004: Alternative NGLY1 transcripts and the impact of their down regulation on fOS generation in HepG2 cells.A. Genomic organization of the human NGLY1 gene and alternative transcripts found in HepG2 cells. The major transcript found in HepG2 cells is indicated in blue and corresponds to the already published NGLY1 mRNA sequence (NM_018297.3). A minor transcript indicated in orange is generated by skipping of exon 11 (NM_001145295.1). B. Partial alignment of homologous Ngly1p sequences (amino acids 522–558) encoded by the minor transcript and comparison with the corresponding region of the major NGLY1 transcript (accession numbers: Macaca mulatta; XP_001092914, Equus caballus; XP_001492093, Canis lupus familiaris; DN746692.1). Note that the isoform encoded by the minor transcript contains 21 amino acids in its COOH terminal which are not found in the peptide sequence encoded by the major transcript. C. HepG2 cells were transiently transfected with the different siRNA duplexes designed to silence either or both NGLY1 transcripts (see Table S1). Total RNA were extracted three days later and the mRNA levels of each specific transcript were analyzed by QPCR. In parallel, HepG2 cells were co transfected with ENG-3 and the different siRNA duplexes targeting the two NGLY1 transcripts. As described in Materials and Methods, because of the variable GC content of the different RNAi duplexes, two negative control siRNA duplexes with either medium (med GC) or low GC (low GC) content were used. After 3 days cells were pulse-radiolabeled with [2-3H]mannose for 30 min. In some experiments, cells transfected with ENG-3 alone or with control RNAi duplexes were treated with either Z-vad-fmk (40 µM) dissolved in DMSO (Z-vad*) or DMSO alone (*) for 45 min prior to, and during the radiolabeling period. Subsequently, [2-3H]fOS, [2-3H]LLO and [2-3H]Glycoproteins were extracted, purified and quantitated, and [2-3H]fOS were analysed by TLC. In order to take into account the differences in total incorporation of radiolabel into cells cultivated under the different conditions, a fraction of total fOS was loaded onto the TLC according to the ratio of the total cellular radioactivity for a given incubation to that recovered from the incubation incorporating least radioactivity. The scanned TLC lanes are from the same fluorograph, but due to uneven migration, the scans were aligned manually to facilitate interpretation of data. The migration positions of standard oligosaccharides are shown to the left of the chromatograms and the abbreviations used are as described in Fig 1. The percent inhibition of the major and minor NGLY1 transcripts provoked by the different RNAi duplexes was calculated using the QPCR data and is indicated underneath the appropriate lanes. This experiment was performed once.
Mentions: The preceeding results indicate that Engase1p has an important role in conversion of fOSGN2 to fOSGN but only a minor, if any, role in glycoprotein deglycosylation. Next, the role of the NGLY1-encoded cytosolic PNGase in fOS generation was examined in HepG2 cells. First, RNAi duplexes that effectively targeted NGLY1 expression were sought, but searches in silico indicated several EST sequences potentially arising from alternative transcripts. In order to detect these transcripts, primers were designed to span the regions containing sequence differences but after RT-PCR, only the region corresponding to the C-terminus of Ngly1p yielded different amplified products. Indeed, using forward primer NGLY1-10long and the reverse primer NGLY1-8 (Table S1), the major PCR product (339 bp) expected of the published human NGLY1 mRNA sequence [20] and a minor PCR product of 161 bp were detected. Both PCR products were sequenced and found to correspond to the transcripts shown in Fig 4A. The minor transcript was found to correspond to the EST sequence (Gene bank accession NM_001145295.1) in which exon 11 has been skipped, generating a premature STOP codon that in turn shortens the peptide sequence at its C-terminus. The missing region in this transcript comprises most of the C-terminal domain that is required for the binding of Ngly1p to N-linked oligosaccharides [39], suggesting that these two NGLY1 gene products may not have equivalent functions. Furthermore, inspection of the databases revealed the presence of this minor NGLY1 transcript in the sequence data from several mammals (Fig 4B). Next, the impact of these two NGLY1 gene products on fOS generation was examined. siRNA duplexes targeting both common and distinctive regions of the transcripts were designed (Table S1) and the ability of each to silence the two transcripts was assessed by QPCR. Additionally, the ability of the duplexes to block fOSGN2 generation was examined. In order to simplify identification of efficient NGLY1-targeted RNAi duplexes, fOS were examined in a background of Engase1p down regulation where the majority of fOS occur as fOSGN2. Results shown in Fig 4C demonstrate that among the three duplexes which cause greater than 88% silencing of both transcripts, NGLY1-1 and NGLY1-3 provoke a striking reduction in the generation of a fOSGN2 migrating as Glc1Man9GlcNAc2, and a lesser reduction of a fOSGN2 migrating as Man9GlcNAc2, whereas the fOSGN2 migrating as Man8GlcNAc2 appeared to be unaffected. The fOSGN2 profiles obtained with NGLY1-1 and NGLY1-3, but not NGLY1-2, were found to be identical to that obtained when the ENG-3 transfected cells were pretreated with, and radiolabeled in the presence of the PNGase inhibitor Z-vad-fmk (Z-vad) [40] (Fig 4C). Although successful design of an effective duplex specifically targeting the minor transcript was not achieved (Fig 4C), one of the 3 duplexes targeting the major NGLY1 transcript selectively silenced this transcript without silencing the minor transcript (NGLY1 Long-3). The fOS profile obtained with this duplex was similar to those observed with Z-vad and NGLY1-1 and NGLY1-3 duplexes, indicating that the major NGLY1 transcript that encodes Ngly1p is responsible for the observed changes in fOS generation in HepG2 cells (Fig 4C).

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