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Xu S, Zheng J, Xiao H, Wu R. Simultaneously Identifying and Distinguishing Glycoproteins with O-GlcNAc and O-GalNAc (the Tn Antigen) in Human Cancer Cells. Analytical chemistry 2022 94(7) 35132862
Abstract:
Glycoproteins with diverse glycans are essential to human cells, and subtle differences in glycan structures may result in entirely different functions. One typical example is proteins modified with O-linked β-N-acetylglucosamine (O-GlcNAc) and O-linked α-N-acetylgalactosamine (O-GalNAc) (the Tn antigen), in which the two glycans have very similar structures and identical chemical compositions, making them extraordinarily challenging to be distinguished. Here, we developed an effective method benefiting from selective enrichment and the enzymatic specificity to simultaneously identify and distinguish glycoproteins with O-GlcNAc and O-GalNAc. Metabolic labeling was combined with bioorthogonal chemistry for enriching glycoproteins modified with O-GlcNAc and O-GalNAc. Then, the enzymatic reaction with galactose oxidase was utilized to specifically oxidize O-GalNAc, but not O-GlcNAc, generating the different tags between glycopeptides with O-GlcNAc and O-GalNAc that can be easily distinguishable by mass spectrometry (MS). Among O-GlcNAcylated proteins commonly identified in three types of human cells, those related to transcription and RNA binding are highly enriched. Cell-specific features are also revealed. Among glycoproteins exclusively in Jurkat cells, those involved in human T-lymphotropic virus type 1 (HTLV-1) infection are overrepresented, which is consistent with the cell line source and suggests that protein O-GlcNAcylation participated in the response to the virus infection. Furthermore, glycoproteins with the Tn antigen have different subcellular distributions in different cells, which may be attributed to the distinct mechanisms for the formation of protein O-GalNAcylation.
O-GlcNAc proteins:
RBM47, E2F8, SBNO1, CNOT1, HMX3, ABTB3, RHG32, P121C, PDLI1, SNP23, PSMD9, TAF4, ARI1A, ABLM1, STX16, HGS, MYPT1, SC16A, SR140, SET1A, FYB1, TIF1A, PPM1G, SHIP2, EIF3D, NUP42, KDM6A, TET3, SI1L1, DC1L2, HNRPR, PRPF3, TPD54, E41L2, ZN207, BUB3, AKAP8, ZNRD2, MYPT2, GANP, HNRPQ, DIAP1, PLIN3, MAFK, TBL1X, MITF, N4BP1, ZC11A, T22D2, PP6R2, ANR17, BCAS1, NCOR1, SPAG7, TIPRL, SPF30, TOX4, TOX, PCF11, AGFG2, ZFPL1, KIF4A, SC24A, SC24B, CNOT4, ASML, M4K4, BPNT1, PX11B, CHK2, LMNA, GLPA, TFR1, ALDOA, GCR, HSPB1, GNAI2, RLA1, RLA2, RLA0, K1C18, K2C8, RB, CATD, SYEP, PTPRC, VIME, GSTP1, HMGB1, ROA1, ATX1L, DERPC, ZN865, TPR, LAMP2, EF2, PLSL, PLST, GLU2B, HCLS1, PO2F1, RAC2, ATF2, ZEP1, TFE2, MUC1, CREB1, JUNB, ATF7, PTN2, DDX5, SON, ATF1, CSK22, NFKB1, FLNA, PUR2, RFX1, CBL, COF1, PTBP1, ARNT, DCK, PYR1, MAP4, CALX, 3MG, PRDX6, CDC27, AMRP, CLIP1, ZEP2, HNRH1, 1433S, ELF1, LSP1, PTN7, IRS1, ADDA, NU214, CUX1, TXLNA, MLH1, ECHA, IF2G, HNF4A, LAP2B, GPDM, RANG, KI67, CRKL, CAPZB, RFX5, SOX2, CAMLG, NASP, FAS, CDK8, SRP09, YLPM1, NU153, RBP2, TAF6, EMD, LRBA, PAPOA, HCFC1, HDGF, AGFG1, HNRPF, HXK2, NUP98, ATX1, RD23B, AF10, AF17, DSRAD, FOXA1, HNRH2, NU107, TPIS, PSME3, TPM4, F193A, GTF2I, PHC1, PRKDC, MAP1A, SARNP, FOXK1, FBLN2, FAM3A, EM55, NFKB2, HNRPU, SPTB2, FOXK2, RUNX1, FLI1, SATB1, SP2, MP2K1, NUCB1, KMT2A, IF4G1, TLE3, TLE4, KPCT, PSME1, GABPA, PRDX1, ACK1, AHNK, IFFO1, GALT2, SRBP2, TROAP, BPTF, TP53B, CBX3, NFAC2, PICAL, CUL4B, ASPP2, NFYC, CDK13, VEZF1, UBP2L, SRC8, CAPR1, LAGE3, PUM1, MDC1, EPN4, RRP1B, NCOA6, GSE1, UBP10, 2A5D, MEF2D, LASP1, NUMA1, CND1, TEBP, PCBP1, RBMS2, SF3A1, TSN, SF01, MED1, TRIP6, ELF2, TAB1, ZFHX3, ZYX, ADRM1, DPYL2, TAF9, MAPK3, CSPP1, PDS5A, QSER1, AAK1, LRRF1, VP26B, ACSF3, TPRN, CRTC2, PAN3, YIF1B, PRC2B, CEP78, ZN362, FKB15, LRIF1, CAF17, UBAP2, NT5D1, AHDC1, LYRM7, RPRD2, ZN318, TASO2, TBC9B, ARID2, C19L1, ABLM2, TWF2, GRHL2, CPZIP, NIPBL, LIN54, ZCHC8, C2D1A, SCYL2, NFRKB, RSBNL, MDEAS, ZC3HE, LARP1, SAMD1, FIP1, CRTC3, SAS6, MCAF1, BCOR, GGYF2, NBEL2, CO039, SRCAP, UBN2, TM1L2, ASXL2, SPT6H, MEPCE, BOP, KDM3B, ERMP1, TRM1L, ZCCHV, KANL1, POGZ, ZFY16, NUFP2, MAVS, EMSY, RAI1, I2BP2, SRGP1, RHG30, SH3R1, HUWE1, YTHD3, GALT7, LYRIC, BCL9L, CASZ1, TSYL5, DDX42, CACL1, P66A, I2BP1, VRK3, FOXP4, ARI3B, TEX2, MGAP, ANKH1, SUGP1, MILK2, ERF3B, K2013, PHAR4, XRN1, ZN687, FNBP4, ARFG1, ENAH, NHLC2, AVL9, XXLT1, GOLM1, TXND5, SERB1, CHSTE, SLAI1, TNR6A, PHC3, SP20H, VP37A, KMT2C, ARI1B, KNL1, NEDD1, ALMS1, PREX1, DLG5, GEMI5, PIGO, UBS3B, WIPF2, FRS2, PDC6I, ZFN2B, TPC12, SEN15, PCNP, LMO7, ATX2L, CSKI2, PSPC1, P66B, GBF1, SMG7, RTF1, TOPB1, PHF3, MAML1, TTC9A, PRCC, RREB1, CBP, DDX17, SEM4D, ARHG1, GPKOW, FUBP2, LPP, TTC28, PF21A, FAF2, ESS2, EDC3, A7L3B, P121A, PDLI5, FUBP3, VCIP1, PDLI2, Z512B, ZFR, EP400, PRRC1, NOL4L, RBM14, PURB, NACC1, CIC, MED15, NUDC1, SIN3A, AEDO, MINT, HTF4, CNN2, RGPD5, ATX2, HCD2, S29A1, ARI3A, SH3G1, TRIR, DPH2, MGME1, ERP44, ESYT1, CCM2, CNPY3, WAC, DIDO1, HGH1, MMTA2, PAXX, NTM1A, RBM4, SGPP1, HEMGN, HDHD5, YTHD1, FTO, CEP44, BC11B, PITH1, SP130, BRD8, RGAP1, I2BPL, ADNP, DHX36, FOXP1, CENPH, WNK1, E41L1, ZHX3, YTDC2, RANB3, PHAX, ECT2, CNO10, MLXIP, PKHA5, PKHA1, RC3H2, LY9, RDH14, TAF9B, NCOA5, TANC2, TNR6C, CHD8, SDF2L, ARFG3, UBN1, RTN4, PDLI7, CHSTC, STRN4, PNO1, BMP2K, RBM12, STAU2, TXLNG, PNPO, CARF, TAB2, TMOD3, CDK12, F120A, HPBP1, ITSN2, CNOT2, CHMP5, VAPA, CAMP3, RBM27, KANL3, RERE, ZN219, SE1L1, STAP2, LIMD1, TCF20, SEPT9, UBQL2, TRPS1, S30BP, NRBP, EI2BD, SIX4, APC7, TASOR, GMEB2, PARP4, MA1B1, ACINU, ZHX1, CDV3, MRTFB, ZBT21, YETS2, HECD1, ZMYD8, SCAF8, PP6R1, TRI33, TNR6B, ZC3H4, SHAN2, SRRM2, CTND2, SCML2, ZN148, T3JAM, VDAC3, AKAP2, DDX52, NOP58, GIT1, ZN281, SIT1, SALL2, ARIP4, CRBG1, HYOU1, KLF12, PRC2C, YTHD2, CD2AP, TNPO3, SRPRB, TSSC4, NUBP2, HCFC2, FHOD1, NCOR2, GMEB1, NCOA3, S23IP
Species: Homo sapiens
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Xiang J, Chen C, Liu R, Gou D, Chang L, Deng H, Gao Q, Zhang W, Tuo L, Pan X, Liang L, Xia J, Huang L, Yao K, Wang B, Hu Z, Huang A, Wang K, Tang N. Gluconeogenic enzyme PCK1 deficiency promotes CHK2 O-GlcNAcylation and hepatocellular carcinoma growth upon glucose deprivation. The Journal of clinical investigation 2021 131(8) 33690219
Abstract:
Although cancer cells are frequently faced with a nutrient- and oxygen-poor microenvironment, elevated hexosamine-biosynthesis pathway (HBP) activity and protein O-GlcNAcylation (a nutrient sensor) contribute to rapid growth of tumor and are emerging hallmarks of cancer. Inhibiting O-GlcNAcylation could be a promising anticancer strategy. The gluconeogenic enzyme phosphoenolpyruvate carboxykinase 1 (PCK1) is downregulated in hepatocellular carcinoma (HCC). However, little is known about the potential role of PCK1 in enhanced HBP activity and HCC carcinogenesis under glucose-limited conditions. In this study, PCK1 knockout markedly enhanced the global O-GlcNAcylation levels under low-glucose conditions. Mechanistically, metabolic reprogramming in PCK1-loss hepatoma cells led to oxaloacetate accumulation and increased de novo uridine triphosphate synthesis contributing to uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) biosynthesis. Meanwhile, deletion of PCK1 also resulted in AMPK-GFAT1 axis inactivation, promoting UDP-GlcNAc synthesis for elevated O-GlcNAcylation. Notably, lower expression of PCK1 promoted CHK2 threonine 378 O-GlcNAcylation, counteracting its stability and dimer formation, increasing CHK2-dependent Rb phosphorylation and HCC cell proliferation. Moreover, aminooxyacetic acid hemihydrochloride and 6-diazo-5-oxo-L-norleucine blocked HBP-mediated O-GlcNAcylation and suppressed tumor progression in liver-specific Pck1-knockout mice. We reveal a link between PCK1 depletion and hyper-O-GlcNAcylation that underlies HCC oncogenesis and suggest therapeutic targets for HCC that act by inhibiting O-GlcNAcylation.
O-GlcNAc proteins:
CHK2
Species: Homo sapiens
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Hahne H, Sobotzki N, Nyberg T, Helm D, Borodkin VS, van Aalten DM, Agnew B, Kuster B. Proteome wide purification and identification of O-GlcNAc-modified proteins using click chemistry and mass spectrometry. Journal of proteome research 2013 12(2) 23301498
Abstract:
The post-translational modification of proteins with N-acetylglucosamine (O-GlcNAc) is involved in the regulation of a wide variety of cellular processes and associated with a number of chronic diseases. Despite its emerging biological significance, the systematic identification of O-GlcNAc proteins is still challenging. In the present study, we demonstrate a significantly improved O-GlcNAc protein enrichment procedure, which exploits metabolic labeling of cells by azide-modified GlcNAc and copper-mediated Click chemistry for purification of modified proteins on an alkyne-resin. On-resin proteolysis using trypsin followed by LC-MS/MS afforded the identification of around 1500 O-GlcNAc proteins from a single cell line. Subsequent elution of covalently resin bound O-GlcNAc peptides using selective β-elimination enabled the identification of 185 O-GlcNAc modification sites on 80 proteins. To demonstrate the practical utility of the developed approach, we studied the global effects of the O-GlcNAcase inhibitor GlcNAcstatin G on the level of O-GlcNAc modification of cellular proteins. About 200 proteins including several key players involved in the hexosamine signaling pathway showed significantly increased O-GlcNAcylation levels in response to the drug, which further strengthens the link of O-GlcNAc protein modification to cellular nutrient sensing and response.
O-GlcNAc proteins:
UBA6, SHOT1, MEX3A, TPC13, CNOT1, PGP, RIMC1, SMHD1, CC85C, I4E1B, ZBBX, B2R8K8, B2RB32, B3KNS4, B3KUR4, B4DF70, B4DIH0, B4DIR9, B4DTN6, B8XCX8, DX39A, BACH, GTPB1, SMAP, AP3B1, PSD12, PSMD9, ATOX1, PGRC1, SPT5H, TAF4, DFFA, CLIC1, EIF3F, WASL, IPO5, EF2K, PLOD2, MEIS1, PSDE, IMA4, BCL9, SDCB1, NOP56, IMA3, ARI1A, UBFD1, CCS, DVL2, KMT2D, ANM5, TNPO2, GEMI2, HAT1, MYPT1, GAK, XPO1, ZN609, SC16A, SR140, PUR4, NKRF, ZN185, CASC3, OGT1, PMM2, HMGB3, PPM1G, SHIP2, NVL, NUP42, R113A, KDM6A, DHX15, MCES, RRP8, SERA, DC1L2, ZW10, M3K7, RIPK2, WDR62, TXNL1, IF4G3, E41L2, FOXO3, DENR, XPOT, BUB3, ACTN4, HTSF1, SGTA, SYNC, LANC1, DHX16, ZNRD2, KPRB, AQR, GANP, HNRPQ, BUB1B, DIAP1, PLIN3, ANM3, CTND1, EIF1B, USO1, IF2P, NBN, SRGP2, N4BP1, ROCK2, CLAP2, CPNE3, BRE1B, ZC11A, CLU, T22D2, ANR17, GGCT, HMMR, VATG1, FLNB, NCOR1, SPAG7, PR40A, GGYF1, PSIP1, NDUS3, SRS10, KHDR3, SF3B1, CSDE1, PRKRA, KS6A5, NPM3, LYPA1, U520, TIPRL, OFD1, WDHD1, CD123, EIF3G, PSD10, SPF27, CIAO1, RMP, TOX4, SC24D, ST65G, UBXN7, PLPHP, NFAT5, SOGA1, UBP19, WDR47, SC31A, HEXI1, UBR5, SCAF4, UBE4B, ELP1, ZRAB2, KIF4A, VAPB, SNAPN, 6PGL, SMC2, IPO7, AGM1, ZFYV9, SVIL, BAG4, AHSA1, PARN, PSMG1, SC24A, SC24B, AGRL2, TYDP2, PCNT, YETS4, STABP, ASML, EYA4, CEP43, HS74L, WIZ, STAU1, BAG2, BAG3, BPNT1, ECD, MBD3, BCL10, MOC2B, TOM40, CHK2, GSHR, PNPH, HPRT, ADA, CYTB, OAT, KITH, CPNS1, P53, HSPB1, TYSY, RPN1, RLA1, JUN, LA, NPM, NFL, NFM, ANXA2, SYEP, HS90A, HNRPC, SP1, 4F2, HS90B, ASNS, VIME, GSTP1, LEG1, HMGB1, UCHL1, LKHA4, H2A1, UBC, GLI2, ODP2, LAMP1, G6PD, ADHX, CDK4, SRF, PCNA, HARS1, ADT3, IMDH2, ACTN1, PEPD, XRCC5, LAMP2, RINI, EF2, PLST, ACPH, KAP2, CD99, GLU2B, AK1A1, KPYM, PO2F1, SYDC, ALDR, ERF3A, GNS, ZEP1, DESP, CREB1, GCFC2, UBF1, ATF7, CAN2, PYRG1, TCPA, SON, NELFE,