article published online: 16 march 2015 | doi: 10.1038/nchembio.1774
O-GlcNAc occurs cotranslationally to stabilize nascent polypeptide chains
© 2015 Nature America, Inc. All rights reserved.
Yanping Zhu1,2, Ta-Wei Liu1,2, Samy Cecioni2, Razieh Eskandari2, Wesley F Zandberg2 & David J Vocadlo1,2* Nucleocytoplasmic glycosylation of proteins with O-linked N-acetylglucosamine residues (O-GlcNAc) is recognized as a conserved post-translational modification found in all metazoans. O-GlcNAc has been proposed to regulate diverse cellular processes. Impaired cellular O-GlcNAcylation has been found to lead to decreases in the levels of various proteins, which is one mechanism by which O-GlcNAc seems to exert its varied physiological effects. Here we show that O-GlcNAcylation also occurs cotranslationally. This process protects nascent polypeptide chains from premature degradation by decreasing cotranslational ubiquitylation. Given that hundreds of proteins are O-GlcNAcylated within cells, our findings suggest that cotranslational O-GlcNAcylation may be a phenomenon regulating proteostasis of an array of nucleocytoplasmic proteins. These findings set the stage to assess whether O-GlcNAcylation has a role in protein quality control in a manner that bears similarity with the role played by N-glycosylation within the secretory pathway.
H
undreds of nuclear and cytoplasmic proteins are posttranslationally modified with N-acetylglucosamine mono saccharide units O-linked to serine or threonine residues of proteins. This modification, known as O-GlcNAc1, is conserved among multicellular eukaryotes, where it regulates diverse cellular processes including, for example, epigenetic regulation of gene expression2–4, stress response5,6 and circadian rhythm7,8. Remarkably, O-GlcNAc executes its physiological functions in part by regulating the levels of various key proteins8–12. In some cases it has emerged that O-GlcNAc influences protein ubiquitylation8–12, but knowledge of the various molecular processes by which O-GlcNAc regulates protein levels remains limited. Protein O-GlcNAcylation is regulated by only two enzymes: O-GlcNAc transferase (OGT) installs O-GlcNAc using uridine 5′-diphosphate-N-acetylglucosamine (UDP-GlcNAc) as a donor substrate13,14, and O-GlcNAcase (OGA) removes O-GlcNAc15. These two enzymes act together to dynamically modulate the levels of O-GlcNAc on proteins within cells. Notably, no consensus sequence governing which residues are O-GlcNAcylated has been found, and structures of human OGT reveal a long binding cleft, suggesting that only extended polypeptide substrates can be O-GlcNAcylated16. Given these data, as well as the observation that OGT stably interacts with actively translating ribosomes17, we considered whether nascent chains emerging from the ribosome might be cotranslationally O-GlcNAcylated. Although O-GlcNAc is accepted as a post-translational modification, we found this hypothesis intriguing given the pleiotropic effects associated with decreased O-GlcNAcylation. Further, considering recent reports that have shown a large fraction of nascent polypeptides are cotranslationally degraded, a process that contributes both to protein quality control and proteostasis18–20, we speculated that if cotranslational O-GlcNAcylation occurs it could have an important role in governing the fate of nascent polypeptides of a subset of nucleocytoplasmic proteins. Using the known O-GlcNAcylated model proteins specificity protein (Sp1) and nuclear pore protein p62 (Nup62), we report that O-GlcNAcylation occurs cotranslationally within both a cell-free expression system and cells. We further find that cotranslational O-GlcNAcylation protects nascent polypeptide chains of Sp1 and Nup62 from premature proteasomal degradation by decreasing cotranslational ubiquitylation. Considering that
hundreds of proteins are O-GlcNAcylated within cells, our data indicate that cotranslational O-GlcNAcylation may be a common phenomenon regulating proteostasis of a subset of O-GlcNAcylated nucleocytoplasmic proteins.
RESULTS Sp1 is cotranslationally O-GlcNAcylated in vitro
To first test whether OGT can cotranslationally O-GlcNAcylate proteins, we selected proteins known to be constitutively O-GlcNAcylated as extensive glycosylation reasonably suggests a functionally important role. We therefore chose Sp1 as it is highly O-GlcNAcylated, and its glycosylation status regulates its cellular levels21,22. Six sites of O-GlcNAcylation are known on Sp123,24 that are mostly in regions of intrinsic disorder. We used rabbit recticulocyte (RR) lysate as a cell-free expression system that contains all the cellular machinery for protein production and processing and is also capable of producing O-GlcNAcylated proteins25. To sensitively monitor O-GlcNAcylation on nascent polypeptides, we used uridine 5′-diphosphate-N-azidoacetylglucosamine (UDP-GlcNAz), a close analog of UDP-GlcNAc that has a small pendent azide functionality. OGT accepts UDP-GlcNAz as a substrate, leading to formation of O-GlcNAz–modified proteins26. Using the Staudinger ligation, a highly chemoselective reaction27, the azide group can be exclusively tagged within RR lysates by using a biotin-modified triarylphosphine substituted at one aryl ring with an ortho-substituted methyl ester. For these studies, we synthesized a phosphine probe containing a cleavable linker (biotin-diazo-phosphine probe 1; Fig. 1a; see Supplementary Results, Supplementary Note for details of the synthesis and characterization). Using these tools, we supplemented lysates with polymerase, UDP-GlcNAz and a plasmid encoding N-terminally Flag-tagged Sp1 and then incubated these samples for various times. By using cetrimonium bromide (CTAB) fractionation, we separated peptidyl-tRNAs from mature proteins released from the ribosome (Fig. 1b). This method permitted us to detect nascent Sp1 peptidyl-tRNAs in the precipitates, as expected (Fig. 1c), whereas mature full-length Sp1 protein was only detected in the supernatants (Supplementary Fig. 1a), indicating that the fractionation method was effective. By tagging glycosylated proteins obtained from CTAB precipitates using the Staudinger ligation, we were able to observe time-dependent changes in the mass of
Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada. 2Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada. *e-mail: [email protected] 1
nature CHEMICAL BIOLOGY | Advance online publication | www.nature.com/naturechemicalbiology
1
article
Nature chemical biology doi: 10.1038/nchembio.1774
other proteins that are not O-GlcNAcylated, we assessed the expression in RR lysates of the O N PPh cytoplasmic protein FBXO22 and Clusterin, HO O O HN NH a protein expressed in the secretory pathN N H H H H way, neither of which are O-GlcNAcylated. N N O O O S We found that the levels of these proteins O O 1 produced were unchanged by OGT inhibib tion (Supplementary Fig. 1c), indicating that 5′ 5′ 40S 40S OGT inhibition does not influence the general tRNA 60S 60S G Staudinger transcriptional or translational machinery 3′ 3′ G OGT ligation G CTAB-PPT within RR lysates responsible for the producG G Biotin Nascent UDP-GlcNAz Phosphine tion of these proteins. To determine whether G O-GlcNAz polypeptide biotin probe (1) these observations stemmed from indirect effects of OGT inhibition leading to decreased O-GlcNAc on components of the ubiquitinCTAB-nascent Flag-Sp1 chain precipitates proteasome system30, we blocked O-GlcNAc c cycling in both directions by simultaneously Time (min): 0 15 30 60 90 0 15 30 60 90 0 15 30 60 90 adding high concentrations of both OGA and 170 130 OGT inhibitors before initiating translation. * * 100 * Upon inhibiting both OGA and OGT within 70 lysates, we found no change in the amount 55 of mature Sp1 produced relative to that pro40 duced with OGT inhibition alone, indicating 35 that only OGT-catalyzed O-GlcNAcylation of 25 newly synthesized polypeptides influences Sp1 stability (Supplementary Fig. 1d). Together, 15 these results indicate that Sp1 is either rapidly MW (kDa) Flag Strvn Merge degraded upon release from the ribosome or that Sp1 nascent polypeptide chains are Figure 1 | Sp1 is cotranslationally O-GlcNAcylated in a cell-free expression system. (a) Structure cotranslationally degraded. On examination of the biotin-diazo-phosphine probe 1. (b) Schematic of the experimental design. (c) Immunoblot of immunoblots, we noted that a range of analysis of CTAB precipitates using anti-Flag antibody and fluorescent streptavidin (Strvn). Sp1 lower-abundance Sp1 polypeptides of smaller expression in RR lysates was performed by incubating plasmid, T7 polymerase and UDP-GlcNAz mass (
O-GlcNAc occurs cotranslationally to stabilize nascent polypeptide chains
© 2015 Nature America, Inc. All rights reserved.
Yanping Zhu1,2, Ta-Wei Liu1,2, Samy Cecioni2, Razieh Eskandari2, Wesley F Zandberg2 & David J Vocadlo1,2* Nucleocytoplasmic glycosylation of proteins with O-linked N-acetylglucosamine residues (O-GlcNAc) is recognized as a conserved post-translational modification found in all metazoans. O-GlcNAc has been proposed to regulate diverse cellular processes. Impaired cellular O-GlcNAcylation has been found to lead to decreases in the levels of various proteins, which is one mechanism by which O-GlcNAc seems to exert its varied physiological effects. Here we show that O-GlcNAcylation also occurs cotranslationally. This process protects nascent polypeptide chains from premature degradation by decreasing cotranslational ubiquitylation. Given that hundreds of proteins are O-GlcNAcylated within cells, our findings suggest that cotranslational O-GlcNAcylation may be a phenomenon regulating proteostasis of an array of nucleocytoplasmic proteins. These findings set the stage to assess whether O-GlcNAcylation has a role in protein quality control in a manner that bears similarity with the role played by N-glycosylation within the secretory pathway.
H
undreds of nuclear and cytoplasmic proteins are posttranslationally modified with N-acetylglucosamine mono saccharide units O-linked to serine or threonine residues of proteins. This modification, known as O-GlcNAc1, is conserved among multicellular eukaryotes, where it regulates diverse cellular processes including, for example, epigenetic regulation of gene expression2–4, stress response5,6 and circadian rhythm7,8. Remarkably, O-GlcNAc executes its physiological functions in part by regulating the levels of various key proteins8–12. In some cases it has emerged that O-GlcNAc influences protein ubiquitylation8–12, but knowledge of the various molecular processes by which O-GlcNAc regulates protein levels remains limited. Protein O-GlcNAcylation is regulated by only two enzymes: O-GlcNAc transferase (OGT) installs O-GlcNAc using uridine 5′-diphosphate-N-acetylglucosamine (UDP-GlcNAc) as a donor substrate13,14, and O-GlcNAcase (OGA) removes O-GlcNAc15. These two enzymes act together to dynamically modulate the levels of O-GlcNAc on proteins within cells. Notably, no consensus sequence governing which residues are O-GlcNAcylated has been found, and structures of human OGT reveal a long binding cleft, suggesting that only extended polypeptide substrates can be O-GlcNAcylated16. Given these data, as well as the observation that OGT stably interacts with actively translating ribosomes17, we considered whether nascent chains emerging from the ribosome might be cotranslationally O-GlcNAcylated. Although O-GlcNAc is accepted as a post-translational modification, we found this hypothesis intriguing given the pleiotropic effects associated with decreased O-GlcNAcylation. Further, considering recent reports that have shown a large fraction of nascent polypeptides are cotranslationally degraded, a process that contributes both to protein quality control and proteostasis18–20, we speculated that if cotranslational O-GlcNAcylation occurs it could have an important role in governing the fate of nascent polypeptides of a subset of nucleocytoplasmic proteins. Using the known O-GlcNAcylated model proteins specificity protein (Sp1) and nuclear pore protein p62 (Nup62), we report that O-GlcNAcylation occurs cotranslationally within both a cell-free expression system and cells. We further find that cotranslational O-GlcNAcylation protects nascent polypeptide chains of Sp1 and Nup62 from premature proteasomal degradation by decreasing cotranslational ubiquitylation. Considering that
hundreds of proteins are O-GlcNAcylated within cells, our data indicate that cotranslational O-GlcNAcylation may be a common phenomenon regulating proteostasis of a subset of O-GlcNAcylated nucleocytoplasmic proteins.
RESULTS Sp1 is cotranslationally O-GlcNAcylated in vitro
To first test whether OGT can cotranslationally O-GlcNAcylate proteins, we selected proteins known to be constitutively O-GlcNAcylated as extensive glycosylation reasonably suggests a functionally important role. We therefore chose Sp1 as it is highly O-GlcNAcylated, and its glycosylation status regulates its cellular levels21,22. Six sites of O-GlcNAcylation are known on Sp123,24 that are mostly in regions of intrinsic disorder. We used rabbit recticulocyte (RR) lysate as a cell-free expression system that contains all the cellular machinery for protein production and processing and is also capable of producing O-GlcNAcylated proteins25. To sensitively monitor O-GlcNAcylation on nascent polypeptides, we used uridine 5′-diphosphate-N-azidoacetylglucosamine (UDP-GlcNAz), a close analog of UDP-GlcNAc that has a small pendent azide functionality. OGT accepts UDP-GlcNAz as a substrate, leading to formation of O-GlcNAz–modified proteins26. Using the Staudinger ligation, a highly chemoselective reaction27, the azide group can be exclusively tagged within RR lysates by using a biotin-modified triarylphosphine substituted at one aryl ring with an ortho-substituted methyl ester. For these studies, we synthesized a phosphine probe containing a cleavable linker (biotin-diazo-phosphine probe 1; Fig. 1a; see Supplementary Results, Supplementary Note for details of the synthesis and characterization). Using these tools, we supplemented lysates with polymerase, UDP-GlcNAz and a plasmid encoding N-terminally Flag-tagged Sp1 and then incubated these samples for various times. By using cetrimonium bromide (CTAB) fractionation, we separated peptidyl-tRNAs from mature proteins released from the ribosome (Fig. 1b). This method permitted us to detect nascent Sp1 peptidyl-tRNAs in the precipitates, as expected (Fig. 1c), whereas mature full-length Sp1 protein was only detected in the supernatants (Supplementary Fig. 1a), indicating that the fractionation method was effective. By tagging glycosylated proteins obtained from CTAB precipitates using the Staudinger ligation, we were able to observe time-dependent changes in the mass of
Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada. 2Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada. *e-mail: [email protected] 1
nature CHEMICAL BIOLOGY | Advance online publication | www.nature.com/naturechemicalbiology
1
article
Nature chemical biology doi: 10.1038/nchembio.1774
other proteins that are not O-GlcNAcylated, we assessed the expression in RR lysates of the O N PPh cytoplasmic protein FBXO22 and Clusterin, HO O O HN NH a protein expressed in the secretory pathN N H H H H way, neither of which are O-GlcNAcylated. N N O O O S We found that the levels of these proteins O O 1 produced were unchanged by OGT inhibib tion (Supplementary Fig. 1c), indicating that 5′ 5′ 40S 40S OGT inhibition does not influence the general tRNA 60S 60S G Staudinger transcriptional or translational machinery 3′ 3′ G OGT ligation G CTAB-PPT within RR lysates responsible for the producG G Biotin Nascent UDP-GlcNAz Phosphine tion of these proteins. To determine whether G O-GlcNAz polypeptide biotin probe (1) these observations stemmed from indirect effects of OGT inhibition leading to decreased O-GlcNAc on components of the ubiquitinCTAB-nascent Flag-Sp1 chain precipitates proteasome system30, we blocked O-GlcNAc c cycling in both directions by simultaneously Time (min): 0 15 30 60 90 0 15 30 60 90 0 15 30 60 90 adding high concentrations of both OGA and 170 130 OGT inhibitors before initiating translation. * * 100 * Upon inhibiting both OGA and OGT within 70 lysates, we found no change in the amount 55 of mature Sp1 produced relative to that pro40 duced with OGT inhibition alone, indicating 35 that only OGT-catalyzed O-GlcNAcylation of 25 newly synthesized polypeptides influences Sp1 stability (Supplementary Fig. 1d). Together, 15 these results indicate that Sp1 is either rapidly MW (kDa) Flag Strvn Merge degraded upon release from the ribosome or that Sp1 nascent polypeptide chains are Figure 1 | Sp1 is cotranslationally O-GlcNAcylated in a cell-free expression system. (a) Structure cotranslationally degraded. On examination of the biotin-diazo-phosphine probe 1. (b) Schematic of the experimental design. (c) Immunoblot of immunoblots, we noted that a range of analysis of CTAB precipitates using anti-Flag antibody and fluorescent streptavidin (Strvn). Sp1 lower-abundance Sp1 polypeptides of smaller expression in RR lysates was performed by incubating plasmid, T7 polymerase and UDP-GlcNAz mass (
