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Selvan N, George S, Serajee FJ, Shaw M, Hobson L, Kalscheuer V, Prasad N, Levy SE, Taylor J, Aftimos S, Schwartz CE, Huq AM, Gecz J, Wells L. O-GlcNAc transferase missense mutations linked to X-linked intellectual disability deregulate genes involved in cell fate determination and signaling. The Journal of biological chemistry 2018 293(27) 29769320
Abstract:
It is estimated that ∼1% of the world's population has intellectual disability, with males affected more often than females. OGT is an X-linked gene encoding for the enzyme O-GlcNAc transferase (OGT), which carries out the reversible addition of N-acetylglucosamine (GlcNAc) to Ser/Thr residues of its intracellular substrates. Three missense mutations in the tetratricopeptide (TPR) repeats of OGT have recently been reported to cause X-linked intellectual disability (XLID). Here, we report the discovery of two additional novel missense mutations (c.775 G>A, p.A259T, and c.1016 A>G, p.E339G) in the TPR domain of OGT that segregate with XLID in affected families. Characterization of all five of these XLID missense variants of OGT demonstrates modest declines in thermodynamic stability and/or activities of the variants. We engineered each of the mutations into a male human embryonic stem cell line using CRISPR/Cas9. Investigation of the global O-GlcNAc profile as well as OGT and O-GlcNAc hydrolase levels by Western blotting showed no gross changes in steady-state levels in the engineered lines. However, analyses of the differential transcriptomes of the OGT variant-expressing stem cells revealed shared deregulation of genes involved in cell fate determination and liver X receptor/retinoid X receptor signaling, which has been implicated in neuronal development. Thus, here we reveal two additional mutations encoding residues in the TPR regions of OGT that appear causal for XLID and provide evidence that the relatively stable and active TPR variants may share a common, unelucidated mechanism of altering gene expression profiles in human embryonic stem cells.
O-GlcNAc proteins:
CSK21
Species: Homo sapiens
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Constable S, Lim JM, Vaidyanathan K, Wells L. O-GlcNAc transferase regulates transcriptional activity of human Oct4. Glycobiology 2017 27(10) 28922739
Abstract:
O-linked β-N-acetylglucosamine (O-GlcNAc) is a single sugar modification found on many different classes of nuclear and cytoplasmic proteins. Addition of this modification, by the enzyme O-linked N-acetylglucosamine transferase (OGT), is dynamic and inducible. One major class of proteins modified by O-GlcNAc is transcription factors. O-GlcNAc regulates transcription factor properties through a variety of different mechanisms including localization, stability and transcriptional activation. Maintenance of embryonic stem (ES) cell pluripotency requires tight regulation of several key transcription factors, many of which are modified by O-GlcNAc. Octamer-binding protein 4 (Oct4) is one of the key transcription factors required for pluripotency of ES cells and more recently, the generation of induced pluripotent stem (iPS) cells. The action of Oct4 is modulated by the addition of several post-translational modifications, including O-GlcNAc. Previous studies in mice found a single site of O-GlcNAc addition responsible for transcriptional regulation. This study was designed to determine if this mechanism is conserved in humans. We mapped 10 novel sites of O-GlcNAc attachment on human Oct4, and confirmed a role for OGT in transcriptional activation of Oct4 at a site distinct from that found in mouse that allows distinction between different Oct4 target promoters. Additionally, we uncovered a potential new role for OGT that does not include its catalytic function. These results confirm that human Oct4 activity is being regulated by OGT by a mechanism that is distinct from mouse Oct4.
O-GlcNAc proteins:
PO5F1
Species: Homo sapiens
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Zhao P, Schulz TC, Sherrer ES, Weatherly DB, Robins AJ, Wells L. The human embryonic stem cell proteome revealed by multidimensional fractionation followed by tandem mass spectrometry. Proteomics 2015 15(2-3) 25367160
Abstract:
Human embryonic stem cells (hESCs) have received considerable attention due to their therapeutic potential and usefulness in understanding early development and cell fate commitment. In order to appreciate the unique properties of these pluripotent, self-renewing cells, we have performed an in-depth multidimensional fractionation followed by LC-MS/MS analysis of the hESCs harvested from defined media to elucidate expressed, phosphorylated, O-linked β-N-acetylglucosamine (O-GlcNAc) modified, and secreted proteins. From the triplicate analysis, we were able to assign more than 3000 proteins with less than 1% false-discovery rate. This analysis also allowed us to identify nearly 500 phosphorylation sites and 68 sites of O-GlcNAc modification with the same high confidence. Investigation of the phosphorylation sites allowed us to deduce the set of kinases that are likely active in these cells. We also identified more than 100 secreted proteins of hESCs that likely play a role in extracellular matrix formation and remodeling, as well as autocrine signaling for self-renewal and maintenance of the undifferentiated state. Finally, by performing in-depth analysis in triplicate, spectral counts were obtained for these proteins and posttranslationally modified peptides, which will allow us to perform relative quantitative analysis between these cells and any derived cell type in the future.
O-GlcNAc proteins:
PSA7, MYPT1, UGDH, BRD4, NU155, EIF3G, SRP72, PRP6, LSM8, NB5R3, LMNA, GNAI2, H2AZ, ODPB, PCNA, TPR, EF2, ENPL, NUCL, MCM3, PTBP1, PURA2, MAP1B, FAS, RM19, RBP2, TCPD, MSH6, RAB2A, ERF1, RL32, GTF2I, NU160, ARHG7, NOM1, OBSCN, ECM29, SPT6H, TAOK1, NUTM1, ATLA2, SYNE2, TITIN, RT27, UBP7, MIA2, RBM15, MBB1A, DIDO1, DDX21, SEP11, SLTM, TEX10, SYLC, SEPT9, TLN1
Species: Homo sapiens
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Wollaston-Hayden EE, Harris RB, Liu B, Bridger R, Xu Y, Wells L. Global O-GlcNAc Levels Modulate Transcription of the Adipocyte Secretome during Chronic Insulin Resistance. Frontiers in endocrinology 2014 5 25657638
Abstract:
Increased flux through the hexosamine biosynthetic pathway and the corresponding increase in intracellular glycosylation of proteins via O-linked β-N-acetylglucosamine (O-GlcNAc) is sufficient to induce insulin resistance (IR) in multiple systems. Previously, our group used shotgun proteomics to identify multiple rodent adipocytokines and secreted proteins whose levels are modulated upon the induction of IR by indirectly and directly modulating O-GlcNAc levels. We have validated the relative levels of several of these factors using immunoblotting. Since adipocytokines levels are regulated primarily at the level of transcription and O-GlcNAc alters the function of many transcription factors, we hypothesized that elevated O-GlcNAc levels on key transcription factors are modulating secreted protein expression. Here, we show that upon the elevation of O-GlcNAc levels and the induction of IR in mature 3T3-F442a adipocytes, the transcript levels of multiple secreted proteins reflect the modulation observed at the protein level. We validate the transcript levels in male mouse models of diabetes. Using inguinal fat pads from the severely IR db/db mouse model and the mildly IR diet-induced mouse model, we have confirmed that the secreted proteins regulated by O-GlcNAc modulation in cell culture are likewise modulated in the whole animal upon a shift to IR. By comparing the promoters of similarly regulated genes, we determine that Sp1 is a common cis-acting element. Furthermore, we show that the LPL and SPARC promoters are enriched for Sp1 and O-GlcNAc modified proteins during insulin resistance in adipocytes. Thus, the O-GlcNAc modification of proteins bound to promoters, including Sp1, is linked to adipocytokine transcription during insulin resistance.
O-GlcNAc proteins:
SP1
Species: Mus musculus
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Teo CF, Wells L. Monitoring protein O-linked β-N-acetylglucosamine status via metabolic labeling and copper-free click chemistry. Analytical biochemistry 2014 464 24995865
Abstract:
O-Linked β-N-acetylglucosamine (O-GlcNAc) modification found on the serine and threonine residues of intracellular proteins is an inducible post-translational modification that regulates numerous biological processes. In combination with other cell biological and biochemical approaches, a robust and streamlined strategy for detecting the number and stoichiometry of O-GlcNAc modification can provide valuable insights for decoding the functions of O-GlcNAc at the molecular level. Here, we report an optimized workflow for evaluating the O-GlcNAc status of proteins using a combination of metabolic labeling and click chemistry-based mass tagging. This method is strategically complementary to the chemoenzymatic-based mass-tagging method.
O-GlcNAc proteins:
OGA, SP1, BAG6
Species: Homo sapiens
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Kang J, Shen Z, Lim JM, Handa H, Wells L, Tantin D. Regulation of Oct1/Pou2f1 transcription activity by O-GlcNAcylation. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2013 27(7) 23580612
Abstract:
The Oct1 transcription factor is a potent regulator of stress responses, metabolism, and tumorigenicity. Although Oct1 is regulated by phosphorylation and ubiquitination, the presence and importance of other modifications is unknown. Here we show that Oct1 is modified by O-linked β-N-acetylglucosamine (O-GlcNAc) moieties. We map two sites of O-GlcNAcylation at positions T255 and S728 within human Oct1. Under anchorage-independent overgrowth conditions, Oct1 associates 3-fold more strongly with the Gadd45a promoter and mediates transcriptional repression. Increased binding correlates with quantitative reductions in Oct1 nuclear periphery-associated puncta, and a reduced association with lamin B1. The O-GlcNAc modification sites are important for both Gadd45a repression and anchorage-independent survival. In contrast to chronic overgrowth conditions, following acute nutrient starvation Oct1 mediates Gadd45a activation. The O-GlcNAc sites are also important for Gadd45a activation under these conditions. We also, for the first time, identify specific Oct1 ubiquitination sites. The findings suggest that Oct1 integrates metabolic and stress signals via O-GlcNAc modification to regulate target gene activity.
O-GlcNAc proteins:
PO2F1, PO2F1
Species: Homo sapiens, Mus musculus
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Fong JJ, Nguyen BL, Bridger R, Medrano EE, Wells L, Pan S, Sifers RN. β-N-Acetylglucosamine (O-GlcNAc) is a novel regulator of mitosis-specific phosphorylations on histone H3. The Journal of biological chemistry 2012 287(15) 22371497
Abstract:
O-Linked β-N-acetylglucosamine, or O-GlcNAc, is a dynamic post-translational modification that cycles on and off serine and threonine residues of nucleocytoplasmic proteins. The O-GlcNAc modification shares a complex relationship with phosphorylation, as both modifications are capable of mutually inhibiting the occupation of each other on the same or nearby amino acid residue. In addition to diabetes, cancer, and neurodegenerative diseases, O-GlcNAc appears to play a significant role in cell growth and cell cycle progression, although the precise mechanisms are still not well understood. A recent study also found that all four core nucleosomal histones (H2A, H2B, H3, and H4) are modified with O-GlcNAc, although no specific sites on H3 were reported. Here, we describe that histone H3, a protein highly phosphorylated during mitosis, is modified with O-GlcNAc. Several biochemical assays were used to validate that H3 is modified with O-GlcNAc. Mass spectrometry analysis identified threonine 32 as a novel O-GlcNAc site. O-GlcNAc was detected at higher levels on H3 during interphase than mitosis, which inversely correlated with phosphorylation. Furthermore, increased O-GlcNAcylation was observed to reduce mitosis-specific phosphorylation at serine 10, serine 28, and threonine 32. Finally, inhibiting OGA, the enzyme responsible for removing O-GlcNAc, hindered the transition from G2 to M phase of the cell cycle, displaying a phenotype similar to preventing mitosis-specific phosphorylation on H3. Taken together, these data indicate that O-GlcNAcylation regulates mitosis-specific phosphorylations on H3, providing a mechanistic switch that orchestrates the G2-M transition of the cell cycle.
O-GlcNAc proteins:
H31
Species: Homo sapiens
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Zhao P, Viner R, Teo CF, Boons GJ, Horn D, Wells L. Combining high-energy C-trap dissociation and electron transfer dissociation for protein O-GlcNAc modification site assignment. Journal of proteome research 2011 10(9) 21740066
Abstract:
Mass spectrometry-based studies of proteins that are post-translationally modified by O-linked β-N-acetylglucosamine (O-GlcNAc) are challenged in effectively identifying the sites of modification while simultaneously sequencing the peptides. Here we tested the hypothesis that a combination of high-energy C-trap dissociation (HCD) and electron transfer dissociation (ETD) could specifically target the O-GlcNAc modified peptides and elucidate the amino acid sequence while preserving the attached GlcNAc residue for accurate site assignment. By taking advantage of the recently characterized O-GlcNAc-specific IgG monoclonal antibodies and the combination of HCD and ETD fragmentation techniques, O-GlcNAc modified proteins were enriched from HEK293T cells and subsequently characterized using the LTQ Orbitrap Velos ETD (Thermo Fisher Scientific) mass spectrometer. In our data set, 83 sites of O-GlcNAc modification are reported with high confidence confirming that the HCD/ETD combined approach is amenable to the detection and site assignment of O-GlcNAc modified peptides. Realizing HCD triggered ETD fragmentation on a linear ion trap/Orbitrap platform for more in-depth analysis and application of this technique to other post-translationally modified proteins are currently underway. Furthermore, this report illustrates that the O-GlcNAc transferase appears to demonstrate promiscuity with regards to the hydroxyl-containing amino acid modified in short stretches of primary sequence of the glycosylated polypeptides.
O-GlcNAc proteins:
P121C, MCRI1, ARI1A, TCRG1, MYPT1, HNRDL, SET1A, OGT1, HNRPR, ZN207, DNJA2, ANR17, EIF3G, EIF3J, SC31A, SC24B, TXD12, TBB4A, G3P, S10A8, RLA2, ENOA, NPM, PROF1, SYEP, HNRPC, SP1, VIME, GSTP1, ROA1, H14, THIO, HSP7C, PABP1, ENOB, PO2F1, HNRPL, RS2, RL17, NUCL, RO52, PIMT, SFPQ, EF1B, RS12, DDX6, RL13, ERP29, RL12, PPIF, DNJA1, HSP74, RL22, FUS, NU214, SRP14, NUP62, RBMX, GRP75, RANG, RL27A, RL5, RL21, RL28, RL29, RL34, SRP09, NU153, TCPD, HCFC1, ROA3, AGFG1, NUP98, SC24C, SEC13, TPIS, RS20, RL26, WDR5, RS7, RS14, RS23, RS11, RL23A, RS6, RAN, RL23, RL31, RL11, RL8, PPIA, YBOX1, EF1A1, TBA1B, RAE1L, RL36A, HNRPU, RL18A, KMT2A, C1QBP, KHDR1, AIMP1, ILF3, ROA0, CBX3, CIRBP, VEZF1, UBP2L, NOLC1, SYK, RBBP5, SC23A, SF01, JHD2C, ELAV1, QSER1, LSM12, SNUT2, UBAP2, RBM26, LIN54, NFRKB, NUP54, NUFP2, EMSY, CARM1, ANKH1, CCAR1, SACA3, ASXL1, BAP18, SERB1, FA98A, SMRC2, ATX2L, DDX1, HS105, TFG, DDX17, SMRC1, FUBP2, ZC3HA, CIC, NUP88, ATX2, ROAA, DIDO1, NUP58, WNK3, TBL1R, WNK1, DMAP1, DDX21, BCLF1, CDK12, KANL3, ASH2L, NUP50, YETS2, RTRAF, RUVB2, RUVB1, AT8A1, STRAP, RTCB, WNK2, PRC2C, S23IP
Species: Homo sapiens
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