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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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