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Escobar EE, Seeley EH, Serrano-Negrón JE, Vocadlo DJ, Brodbelt JS. In Situ Imaging of O-Linked β-N-Acetylglucosamine Using On-Tissue Hydrolysis and MALDI Mass Spectrometry. Cancers 2023 15(4) 36831567
Abstract:
Post-translational O-glycosylation of proteins via the addition of N-acetylglucosamine (O-GlcNAc) is a regulator of many aspects of cellular physiology. Processes driven by perturbed dynamics of O-GlcNAcylation modification have been implicated in cancer development. Variability in O-GlcNAcylation is emerging as a metabolic biomarker of many cancers. Here, we evaluate the use of MALDI-mass spectrometry imaging (MSI) to visualize the location of O-GlcNAcylated proteins in tissue sections by mapping GlcNAc that has been released by the enzymatic hydrolysis of glycoproteins using an O-GlcNAc hydrolase. We use this strategy to monitor O-GlcNAc within hepatic VX2 tumor tissue. We show that increased O-GlcNAc is found within both viable tumor and tumor margin regions, implicating GlcNAc in tumor progression.
O-GlcNAc proteins:
A0A5F9C1Y1, A0A5F9CI43, A0A5F9CL80, A0A5F9D539, A0A5F9DAJ2, A0A5F9DU78, G1T8N1, G1U460, HBA, HBB, HBG, ADH1
Species: Oryctolagus cuniculus
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González-Cuesta M, Sidhu P, Ashmus RA, Males A, Proceviat C, Madden Z, Rogalski JC, Busmann JA, Foster LJ, García Fernández JM, Davies GJ, Ortiz Mellet C, Vocadlo DJ. Bicyclic Picomolar OGA Inhibitors Enable Chemoproteomic Mapping of Its Endogenous Post-translational Modifications. Journal of the American Chemical Society 2022 144(2) 34985906
Abstract:
Owing to its roles in human health and disease, the modification of nuclear, cytoplasmic, and mitochondrial proteins with O-linked N-acetylglucosamine residues (O-GlcNAc) has emerged as a topic of great interest. Despite the presence of O-GlcNAc on hundreds of proteins within cells, only two enzymes regulate this modification. One of these enzymes is O-GlcNAcase (OGA), a dimeric glycoside hydrolase that has a deep active site cleft in which diverse substrates are accommodated. Chemical tools to control OGA are emerging as essential resources for helping to decode the biochemical and cellular functions of the O-GlcNAc pathway. Here we describe rationally designed bicyclic thiazolidine inhibitors that exhibit superb selectivity and picomolar inhibition of human OGA. Structures of these inhibitors in complex with human OGA reveal the basis for their exceptional potency and show that they extend out of the enzyme active site cleft. Leveraging this structure, we create a high affinity chemoproteomic probe that enables simple one-step purification of endogenous OGA from brain and targeted proteomic mapping of its post-translational modifications. These data uncover a range of new modifications, including some that are less-known, such as O-ubiquitination and N-formylation. We expect that these inhibitors and chemoproteomics probes will prove useful as fundamental tools to decipher the mechanisms by which OGA is regulated and directed to its diverse cellular substrates. Moreover, the inhibitors and structures described here lay out a blueprint that will enable the creation of chemical probes and tools to interrogate OGA and other carbohydrate active enzymes.
O-GlcNAc proteins:
E1BQ16
Species: Bos taurus
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Meek RW, Blaza JN, Busmann JA, Alteen MG, Vocadlo DJ, Davies GJ. Cryo-EM structure provides insights into the dimer arrangement of the O-linked β-N-acetylglucosamine transferase OGT. Nature communications 2021 12(1) 34764280
Abstract:
The O-linked β-N-acetylglucosamine modification is a core signalling mechanism, with erroneous patterns leading to cancer and neurodegeneration. Although thousands of proteins are subject to this modification, only a single essential glycosyltransferase catalyses its installation, the O-GlcNAc transferase, OGT. Previous studies have provided truncated structures of OGT through X-ray crystallography, but the full-length protein has never been observed. Here, we report a 5.3 Å cryo-EM model of OGT. We show OGT is a dimer, providing a structural basis for how some X-linked intellectual disability mutations at the interface may contribute to disease. We observe that the catalytic section of OGT abuts a 13.5 tetratricopeptide repeat unit region and find the relative positioning of these sections deviate from the previously proposed, X-ray crystallography-based model. We also note that OGT exhibits considerable heterogeneity in tetratricopeptide repeat units N-terminal to the dimer interface with repercussions for how OGT binds protein ligands and partners.
O-GlcNAc proteins:
TAB1
Species: Homo sapiens
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Zhu Y, Willems LI, Salas D, Cecioni S, Wu WB, Foster LJ, Vocadlo DJ. Tandem Bioorthogonal Labeling Uncovers Endogenous Cotranslationally O-GlcNAc Modified Nascent Proteins. Journal of the American Chemical Society 2020 142(37) 32870666
Abstract:
Hundreds of nuclear, cytoplasmic, and mitochondrial proteins within multicellular eukaryotes have hydroxyl groups of specific serine and threonine residues modified by the monosaccharide N-acetylglucosamine (GlcNAc). This modification, known as O-GlcNAc, has emerged as a central regulator of both cell physiology and human health. A key emerging function of O-GlcNAc appears to be to regulate cellular protein homeostasis. We previously showed, using overexpressed model proteins, that O-GlcNAc modification can occur cotranslationally and that this process prevents premature degradation of such nascent polypeptide chains. Here, we use tandem metabolic engineering strategies to label endogenously occurring nascent polypeptide chains within cells using O-propargyl-puromycin (OPP) and target the specific subset of nascent chains that are cotranslationally glycosylated with O-GlcNAc by metabolic saccharide engineering using tetra-O-acetyl-2-N-azidoacetyl-2-deoxy-d-galactopyranose (Ac4GalNAz). Using various combinations of sequential chemoselective ligation strategies, we go on to tag these analytes with a series of labels, allowing us to define conditions that enable their robust labeling. Two-step enrichment of these glycosylated nascent chains, combined with shotgun proteomics, allows us to identify a set of endogenous cotranslationally O-GlcNAc modified proteins. Using alternative targeted methods, we examine three of these identified proteins and further validate their cotranslational O-GlcNAcylation. These findings detail strategies to enable isolation and identification of extremely low abundance endogenous analytes present within complex protein mixtures. Moreover, this work opens the way to studies directed at understanding the roles of O-GlcNAc and other cotranslational protein modifications and should stimulate an improved understanding of the role of O-GlcNAc in cytoplasmic protein quality control and proteostasis.
O-GlcNAc proteins:
P121C, PSD11, PSD12, PGRC1, DDX3X, IRS4, PUR4, ARPC3, PSMD3, IF4G3, ACTN4, CPNE3, NDUS7, SC22B, MPPB, RL1D1, SC31A, 6PGL, GNAS3, HS74L, NB5R3, LMNA, OAT, RPN1, JUN, TPM3, PDIA1, SYEP, ANXA6, ASNS, PABP1, HARS1, IMDH2, PLSL, ENPL, NDKA, PYRG1, TCPA, GSTM3, PIMT, PUR2, SFPQ, PPIB, RS12, PSA3, PSA4, SYTC, RL10, MAP4, CALX, PRDX6, PRDX3, METK2, HXA10, DNJA1, QCR1, PUR9, HNRH3, HNRH1, 1433B, STIP1, RL9, MCM7, PROF2, RL22, MYH9, COPB2, NU214, PRS7, NUP62, TALDO, TCPZ, RFC3, IF2G, GARS, ECI1, LPPRC, MATR3, RAGP1, RL5, CAPZB, SYQ, SYAC, NU153, TAF6, GUAA, TCPQ, ANX11, HCFC1, HNRPM, NUP98, COPA, SC24C, SYRC, AT1B3, RD23B, P5CS, XPO2, DSRAD, PSA, EIF3E, DEST, UBE2N, ABCE1, 1433G, PRS8, RS18, PRS10, RL23, GNAS2, IF4G2, KRT85, GSTO1, PRKDC, SRSF3, ALEX, EWS, MEF2A, IF4G1, SRSF1, SF3A3, CSN1, AIMP2, CBX3, STF1, CUL4B, DX39B, MCM6, GANAB, PDIA6, IPYR, SF01, ZYX, ADRM1, CDC37, AMOT, GNAS1, HP1B3, UBAP2, RBM26, SND1, POGZ, PTOV1, CCAR1, CISD2, NNRE, PDC6I, ATX2L, NDRG1, HS105, TFG, DDX17, FUBP2, FKB10, PDLI5, ZFR, RBM14, VPS35, PHB2, TS101, TCPH, NUP58, NAA15, PTN23, AMPB, PDLI7, DDX18, IF2B1, HPBP1, VAPA, SYLC, ATX10, SRP68, COR1C, VDAC3, DRG1, RRP44, PRC2C, S23IP
Species: Homo sapiens
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Wong KKL, Liu TW, Parker JM, Sinclair DAR, Chen YY, Khoo KH, Vocadlo DJ, Verheyen EM. The nutrient sensor OGT regulates Hipk stability and tumorigenic-like activities in Drosophila. Proceedings of the National Academy of Sciences of the United States of America 2020 117(4) 31932432
Abstract:
Environmental cues such as nutrients alter cellular behaviors by acting on a wide array of molecular sensors inside cells. Of emerging interest is the link observed between effects of dietary sugars on cancer proliferation. Here, we identify the requirements of hexosamine biosynthetic pathway (HBP) and O-GlcNAc transferase (OGT) for Drosophila homeodomain-interacting protein kinase (Hipk)-induced growth abnormalities in response to a high sugar diet. On a normal diet, OGT is both necessary and sufficient for inducing Hipk-mediated tumor-like growth. We further show that OGT maintains Hipk protein stability by blocking its proteasomal degradation and that Hipk is O-GlcNAcylated by OGT. In mammalian cells, human HIPK2 proteins accumulate posttranscriptionally upon OGT overexpression. Mass spectrometry analyses reveal that HIPK2 is at least O-GlcNAc modified at S852, T1009, and S1147 residues. Mutations of these residues reduce HIPK2 O-GlcNAcylation and stability. Together, our data demonstrate a conserved role of OGT in positively regulating the protein stability of HIPKs (fly Hipk and human HIPK2), which likely permits the nutritional responsiveness of HIPKs.
O-GlcNAc proteins:
HIPK2
Species: Homo sapiens
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Escobar EE, King DT, Serrano-Negrón JE, Alteen MG, Vocadlo DJ, Brodbelt JS. Precision Mapping of O-Linked N-Acetylglucosamine Sites in Proteins Using Ultraviolet Photodissociation Mass Spectrometry. Journal of the American Chemical Society 2020 142(26) 32510947
Abstract:
Despite its central importance as a regulator of cellular physiology, identification and precise mapping of O-linked N-acetylglucosamine (O-GlcNAc) post-translational modification (PTM) sites in proteins by mass spectrometry (MS) remains a considerable technical challenge. This is due in part to cleavage of the glycosidic bond occurring prior to the peptide backbone during collisionally activated dissociation (CAD), which leads to generation of characteristic oxocarbenium ions and impairs glycosite localization. Herein, we leverage CAD-induced oxocarbenium ion generation to trigger ultraviolet photodissociation (UVPD), an alternate high-energy deposition method that offers extensive fragmentation of peptides while leaving the glycosite intact. Upon activation using UV laser pulses, efficient photodissociation of glycopeptides is achieved with production of multiple sequence ions that enable robust and precise localization of O-GlcNAc sites. Application of this method to tryptic peptides originating from O-GlcNAcylated proteins TAB1 and Polyhomeotic confirmed previously reported O-GlcNAc sites in TAB1 (S395 and S396) and uncovered new sites within both proteins. We expect this strategy will complement existing MS/MS methods and be broadly useful for mapping O-GlcNAcylated residues of both proteins and proteomes.
O-GlcNAc proteins:
PHP, TAB1
Species: Homo sapiens, Drosophila melanogaster
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Alteen MG, Gros C, Meek RW, Cardoso DA, Busmann JA, Sangouard G, Deen MC, Tan HY, Shen DL, Russell CC, Davies GJ, Robinson PJ, McCluskey A, Vocadlo DJ. A Direct Fluorescent Activity Assay for Glycosyltransferases Enables Convenient High-Throughput Screening: Application to O-GlcNAc Transferase. Angewandte Chemie (International ed. in English) 2020 59(24) 32092778
Abstract:
Glycosyltransferases carry out important cellular functions in species ranging from bacteria to humans. Despite their essential roles in biology, simple and robust activity assays that can be easily applied to high-throughput screening for inhibitors of these enzymes have been challenging to develop. Herein, we report a bead-based strategy to measure the group-transfer activity of glycosyltransferases sensitively using simple fluorescence measurements, without the need for coupled enzymes or secondary reactions. We validate the performance and accuracy of the assay using O-GlcNAc transferase (OGT) as a model system through detailed Michaelis-Menten kinetic analysis of various substrates and inhibitors. Optimization of this assay and application to high-throughput screening enabled screening for inhibitors of OGT, leading to a novel inhibitory scaffold. We believe this assay will prove valuable not only for the study of OGT, but also more widely as a general approach for the screening of glycosyltransferases and other group-transfer enzymes.
O-GlcNAc proteins:
HCFC1, CSK21, TAB1
Species: Homo sapiens
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Tan HY, Eskandari R, Shen D, Zhu Y, Liu TW, Willems LI, Alteen MG, Madden Z, Vocadlo DJ. Direct One-Step Fluorescent Labeling of O-GlcNAc-Modified Proteins in Live Cells Using Metabolic Intermediates. Journal of the American Chemical Society 2018 140(45) 30296064
Abstract:
The modification of proteins with O-linked N-acetylglucosamine ( O-GlcNAc) by the enzyme O-GlcNAc transferase (OGT) has emerged as an important regulator of cellular physiology. Metabolic labeling strategies to monitor O-GlcNAcylation in cells have proven of great value for uncovering the molecular roles of O-GlcNAc. These strategies rely on two-step labeling procedures, which limits the scope of experiments that can be performed. Here, we report on the creation of fluorescent uridine 5'-diphospho- N-acetylglucosamine (UDP-GlcNAc) analogues in which the N-acyl group of glucosamine is modified with a suitable linker and fluorophore. Using human OGT, we show these donor sugar substrates permit direct monitoring of OGT activity on protein substrates in vitro. We show that feeding cells with a corresponding fluorescent metabolic precursor for the last step of the hexosamine biosynthetic pathway (HBP) leads to its metabolic assimilation and labeling of O-GlcNAcylated proteins within live cells. This one-step metabolic feeding strategy permits labeling of O-GlcNAcylated proteins with a fluorescent glucosamine-nitrobenzoxadiazole (GlcN-NBD) conjugate that accumulates in a time- and dose-dependent manner. Because no genetic engineering of cells is required, we anticipate this strategy should be generally amenable to studying the roles of O-GlcNAc in cellular physiology as well as to gain an improved understanding of the regulation of OGT within cells. The further expansion of this one-step in-cell labeling strategy should enable performing a range of experiments including two-color pulse chase experiments and monitoring OGT activity on specific protein substrates in live cells.
O-GlcNAc proteins:
NU214, NUP62, NU153, KCC4
Species: Homo sapiens
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Liu TW, Myschyshyn M, Sinclair DA, Cecioni S, Beja K, Honda BM, Morin RD, Vocadlo DJ. Genome-wide chemical mapping of O-GlcNAcylated proteins in Drosophila melanogaster. Nature chemical biology 2017 13(2) 27918560
Abstract:
N-Acetylglucosamine β-O-linked to nucleocytoplasmic proteins (O-GlcNAc) is implicated in the regulation of gene expression in organisms, from humans to Drosophila melanogaster. Within Drosophila, O-GlcNAc transferase (OGT) is one of the Polycomb group proteins (PcGs) that act through Polycomb group response elements (PREs) to silence homeotic (HOX) and other PcG target genes. Using Drosophila, we identify new O-GlcNAcylated PcG proteins and develop an antibody-free metabolic feeding approach to chemoselectively map genomic loci enriched in O-GlcNAc using next-generation sequencing. We find that O-GlcNAc is distributed to specific genomic loci both in cells and in vivo. Many of these loci overlap with PREs, but O-GlcNAc is also present at other loci lacking PREs. Loss of OGT leads to altered gene expression not only at loci containing PREs but also at loci lacking PREs, including several heterochromatic genes. These data suggest that O-GlcNAc acts through multiple mechanisms to regulate gene expression in Drosophila.
O-GlcNAc proteins:
PHO, HCF
Species: Drosophila melanogaster
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Shen DL, Liu TW, Zandberg W, Clark T, Eskandari R, Alteen MG, Tan HY, Zhu Y, Cecioni S, Vocadlo D. Catalytic Promiscuity of O-GlcNAc Transferase Enables Unexpected Metabolic Engineering of Cytoplasmic Proteins with 2-Azido-2-deoxy-glucose. ACS chemical biology 2017 12(1) 27935279
Abstract:
O-GlcNAc transferase (OGT) catalyzes the installation of N-acetylglucosamine (GlcNAc) O-linked to nucleocytoplasmic proteins (O-GlcNAc) within multicellular eukaryotes. OGT shows surprising tolerance for structural changes in the sugar component of its nucleotide sugar donor substrate, uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). Here, we find that OGT uses UDP-glucose to install O-linked glucose (O-Glc) onto proteins only 25-fold less efficiently than O-GlcNAc. Spurred by this observation, we show that OGT transfers 2-azido-2-deoxy-d-glucose (GlcAz) in vitro from UDP-GlcAz to proteins. Further, feeding cells with per-O-acetyl GlcAz (AcGlcAz), in combination with inhibition or inducible knockout of OGT, shows OGT-dependent modification of nuclear and cytoplasmic proteins with O-GlcAz as detected using microscopy, immunoblot, and proteomics. We find that O-GlcAz is reversible within cells, and an unidentified cellular enzyme exists to cleave O-Glc that can also process O-GlcAz. We anticipate that AcGlcAz will prove to be a useful tool to study the O-GlcNAc modification. We also speculate that, given the high concentration of UDP-Glc within certain mammalian tissues, O-Glc may exist within mammals and serve as a physiologically relevant modification.
O-GlcNAc proteins:
A0A0D9R5K0, A0A0D9SE53, NUP62, TAB1, KCC4
Species: Homo sapiens, Chlorocebus sabaeus
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Hastings NB, Wang X, Song L, Butts BD, Grotz D, Hargreaves R, Fred Hess J, Hong KK, Huang CR, Hyde L, Laverty M, Lee J, Levitan D, Lu SX, Maguire M, Mahadomrongkul V, McEachern EJ, Ouyang X, Rosahl TW, Selnick H, Stanton M, Terracina G, Vocadlo DJ, Wang G, Duffy JL, Parker EM, Zhang L. Inhibition of O-GlcNAcase leads to elevation of O-GlcNAc tau and reduction of tauopathy and cerebrospinal fluid tau in rTg4510 mice. Molecular neurodegeneration 2017 12(1) 28521765
Abstract:
Hyperphosphorylation of microtubule-associated protein tau is a distinct feature of neurofibrillary tangles (NFTs) that are the hallmark of neurodegenerative tauopathies. O-GlcNAcylation is a lesser known post-translational modification of tau that involves the addition of N-acetylglucosamine onto serine and threonine residues. Inhibition of O-GlcNAcase (OGA), the enzyme responsible for the removal of O-GlcNAc modification, has been shown to reduce tau pathology in several transgenic models. Clarifying the underlying mechanism by which OGA inhibition leads to the reduction of pathological tau and identifying translatable measures to guide human dosing and efficacy determination would significantly facilitate the clinical development of OGA inhibitors for the treatment of tauopathies.
O-GlcNAc proteins:
TAU
Species: Homo sapiens
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Roth C, Chan S, Offen WA, Hemsworth GR, Willems LI, King DT, Varghese V, Britton R, Vocadlo DJ, Davies GJ. Structural and functional insight into human O-GlcNAcase. Nature chemical biology 2017 13(6) 28346405
Abstract:
O-GlcNAc hydrolase (OGA) removes O-linked N-acetylglucosamine (O-GlcNAc) from a myriad of nucleocytoplasmic proteins. Through co-expression and assembly of OGA fragments, we determined the three-dimensional structure of human OGA, revealing an unusual helix-exchanged dimer that lays a structural foundation for an improved understanding of substrate recognition and regulation of OGA. Structures of OGA in complex with a series of inhibitors define a precise blueprint for the design of inhibitors that have clinical value.
O-GlcNAc proteins:
TAB1
Species: Homo sapiens
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Zhu Y, Liu TW, Madden Z, Yuzwa SA, Murray K, Cecioni S, Zachara N, Vocadlo DJ. Post-translational O-GlcNAcylation is essential for nuclear pore integrity and maintenance of the pore selectivity filter. Journal of molecular cell biology 2016 8(1) 26031751
Abstract:
O-glycosylation of the nuclear pore complex (NPC) by O-linked N-acetylglucosamine (O-GlcNAc) is conserved within metazoans. Many nucleoporins (Nups) comprising the NPC are constitutively O-GlcNAcylated, but the functional role of this modification remains enigmatic. We show that loss of O-GlcNAc, induced by either inhibition of O-GlcNAc transferase (OGT) or deletion of the gene encoding OGT, leads to decreased cellular levels of a number of natively O-GlcNAcylated Nups. Loss of O-GlcNAc enables increased ubiquitination of these Nups and their increased proteasomal degradation. The decreased half-life of these deglycosylated Nups manifests in their gradual loss from the NPC and a downstream malfunction of the nuclear pore selective permeability barrier in both dividing and post-mitotic cells. These findings define a critical role of O-GlcNAc modification of the NPC in maintaining its composition and the function of the selectivity filter. The results implicate NPC glycosylation as a regulator of NPC function and reveal the role of conserved glycos