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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, PLAK2, 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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Liu J, Hao Y, Wang C, Jin Y, Yang Y, Gu J, Chen X. An Optimized Isotopic Photocleavable Tagging Strategy for Site-Specific and Quantitative Profiling of Protein O-GlcNAcylation in Colorectal Cancer Metastasis. ACS chemical biology 2022 17(3) 35254053
Abstract:
O-linked-β-N-acetylglucosamine (O-GlcNAc) glycosylation is a ubiquitous protein post-translational modification of the emerging importance in metazoans. Of the thousands of O-GlcNAcylated proteins identified, many carry multiple modification sites with varied stoichiometry. To better match the scale of O-GlcNAc sites and their dynamic nature, we herein report an optimized strategy, termed isotopic photocleavable tagging for O-GlcNAc profiling (isoPTOP), which enables quantitative and site-specific profiling of O-GlcNAcylation with excellent specificity and sensitivity. In HeLa cells, ∼1500 O-GlcNAcylation sites were identified with the optimized procedures, which led to quantification of ∼1000 O-GlcNAcylation sites with isoPTOP. Furthermore, we apply isoPTOP to probe the O-GlcNAcylation dynamics in a pair of colorectal cancer (CRC) cell lines, SW480 and SW620 cells, which represent primary carcinoma and metastatic cells, representatively. The stoichiometric differences of 625 O-GlcNAcylation sites are quantified. Of these quantified sites, many occur on important regulators involved in tumor progression and metastasis. Our results provide a valuable database for understanding the functional role of O-GlcNAc in CRC. IsoPTOP should be applicable for investigating O-GlcNAcylation dynamics in various pathophysiological processes.
O-GlcNAc proteins:
A0A0B4J203, A0A0C4DFX4, RBM47, E2F8, WDR27, SBNO1, CNOT1, P121B, P121C, H0YAE9, H0YHG0, H7C469, K7ELQ4, M0QZ24, PDLI1, HAX1, TAF4, BCL9, CAC1A, DDX3X, NFIB, PPP6, MA2B1, ARI1A, SOCS7, ABLM1, KMT2D, GBRD, RGRF2, TX1B3, HGS, MYPT1, SYN3, ZN609, TRI66, PDZD2, MAST4, SC16A, SET1A, CASC3, FOXP2, MOT4, P4HA2, ARPC5, CLOCK, MAFG, PER1, KDM6A, TET3, SI1L1, TGFI1, M3K7, MCA3, PRPF3, TPD54, SYNJ1, IF4G3, E41L2, WIPF1, FOXO3, TGM5, RNF13, SPY2, PLRG1, ZN207, AKAP8, CALU, ORC5, MYPT2, GANP, OGA, CCNT1, BUB1B, PLOD3, PLIN3, MOT2, MAFK, PQBP1, BRD4, TBL1X, PP1RB, NBN, MITF, SRGP2, N4BP1, ROCK2, PP6R2, CNOT3, ANR17, FLNB, NCOR1, SF3B1, REM1, CREG1, CRTAP, SYUG, CYTF, TOX4, TOX, SUN1, PCF11, AGFG2, UBE4B, CAC1H, SVIL, SC24A, SC24B, CNOT4, EYA4, ZMYM6, BAG3, LATS1, DDAH2, TXD12, ONEC2, CLPT1, ABL1, CRYAB, LMNA, TFR1, CATA, GBA1, FUCO, ALDOA, GCR, G3P, CPNS1, HSPB1, RLA2, RLA0, ITB1, K1C18, NPM, CATL1, CATB, MCR, BGLR, ITA5, NFIC, VIME, SNRPA, FGR, ATX1L, DERPC, ZN865, GLI2, MYBB, CLUS, PPAL, MPRI, PABP1, TPR, BMP3, SKIL, ENPL, PO2F1, PLAK, ATF2, ZEP1, RS2, TFE2, F261, ITB4, ZNF23, ZNF25, JUNB, ATF7, TPH1, DDX5, EGR1, SON, NELFE, ATF1, ATF6A, CADH2, ICAL, CSRP1, FLNA, RFX1, CBL, SFPQ, COF1, IF4B, GATA2, APC, DDX6, ARNT, MAP4, LYOX, HXD9, MZF1, CLIP1, 5HT1F, HXA11, ZEP2, ELF1, CTNB1, FBN1, ADDA, BASI, NU214, VGFR2, SRP14, NUP62, SYUA, VATA, CUX1, TXLNA, STAT3, LAP2A, EPS15, HELZ, MATR3, SSRA, SSRB, KI67, ATRX, MAP1B, YAP1, UTRN, STT3A, SC6A8, RFX5, SOX2, PRC2A, HSP13, NR2C2, NASP, CDK8, DHE4, YLPM1, NU153, RBP2, TAF6, MRE11, EMD, MXI1, MAP2, TOB1, PPT1, TCPQ, PAPOA, HCFC1, GDS1, AGFG1, CRIP2, NUP98, SMTN, SC24C, HIRA, ATX1, ATN1, AFAD, AF10, AF17, DSRAD, SEC13, NU107, ZN445, CSN2, RL37, WDR5, TIM10, F193A, RBM6, PITX1, IF4G2, PHC1, ADA17, KGD4, RL19, FOXK1, DAB2, RHG04, RBM10, HNRPU, SPTB2, FOXK2, RUNX1, MEF2A, SP2, SP3, PLOD1, KMT2A, TF65, IF4G1, NOTC2, TLE3, TLE4, PTN12, CALD1, MEF2C, P5F1B, GABPA, ZO1, ACK1, EP300, AHNK, FCHO2, HMGX3, SRBP2, FOXO1, ASPH, TROAP, BPTF, FSTL1, NFIA, DPYD, TP53B, FOXC1, ECH1, ROA0, DDX10, TBX2, GPS2, G3BP1, PABP4, ADAM9, PICAL, NAB1, SERC3, RIPK1, IQGA2, STIM1, CUL4B, ASPP2, CAC1S, RUNX2, NFYC, CDK13, TOB2, VEZF1, UBP2L, GIT2, SRC8, CAPR1, LAGE3, PUM1, MDC1, EPN4, TTLL4, RRP1B, NCOA6, GSE1, MEF2D, LASP1, MYPC3, ZN638, NUMA1, SART3, CND1, R3HD1, KIF14, WDR43, PLCL1, PLEC, NOMO1, NONO, RCN1, RYR3, KS6A1, RBMS2, TAF1C, SF01, MED1, JHD2C, TRIP6, T22D1, ELF2, TAB1, HERC1, NCOA1, VAS1, ZFHX3, ZYX, ADRM1, SYPL1, TAF9, DREB, DGKD, CGT, GEN, LY6K, RFX7, QSER1, AAK1, PRSR3, QRIC1, MA7D1, WDR72, TBRG1, TB10B, TPRN, FIL1L, SVEP1, AMOT, EPC2, CRTC2, PAN3, HS904, YIF1B, AG10A, IGS11, ZN628, BCORL, FIGN, BRD10, SH319, TGO1, PRC2B, TOIP1, CEP78, P4R3B, HP1B3, CE170, ZN362, FKB15, AKND1, ZEP3, LRIF1, SWT1, RHG21, UBAP2, RBM26, DEP1A, OGRL1, AHDC1, F222A, RPRD2, RN220, ZN318, TASO2, ZMYM4, PAPD7, TENS2, KANK2, ARID2, USF3, RHG17, CYTSA, ANR40, BICRL, JADE1, PKHA7, NIPBL, LIN54, TET2, RINT1, CRCDL, ZNT6, TTC41, RHGBA, NFRKB, RSBNL, KCD18, NCEH1, MDEAS, ZC3HE, LARP1, NHS, CRTC3, SAS6, MCAF1, BCOR, MPRIP, DNMBP, GGYF2, THADA, BNC2, NFXL1, NBEL2, CO039, SRCAP, CBAR2, UBN2, XIRP1, RAPH1, LARP4, HAKAI, ASXL2, SPT6H, KDM3B, ZCCHV, KANL1, RGPD4, POGZ, ZFY16, NUFP2, MAVS, CLAP1, EMSY, I2BP2, SRGP1, RBBP6, SH3R1, HUWE1, YTHD3, NPM2, ILDR1, KAISO, MYPN, LDB1, LYRIC, BCL9L, LUZP1, NRAP, RTTN, PRSR1, DDX42, CEP57, CD20B, CACL1, P66A, HIPK1, KCC1D, RN135, MY18B, AHNK2, FOXP4, NAV3, NAV2, MISP, ARI3B, IPRI, TEX2, MGAP, CC28A, Z3H7A, ANKH1, SUGP1, RPAP2, MILK2, SRRM1, ZZZ3, GAR4, PHAR4, RTKN2, DCP1B, XRN1, PELP1, CKLF8, TENS4, SPART, RPTOR, NUP93, ZN687, DOCK4, RHG24, RUSC2, SYNPO, FNBP4, D2HDH, RP25L, ATPF2, CPSF7, ARFG1, ENAH, SPOT1, SUMF1, KCNH5, SLAI1, TNR6A, PHC3, DRC6, CBPC3, NAV1, VP37A, KMT2C, ZMIZ2, BD1L1, ARI1B, FLCN, NUP35, TOIP2, TNIP2, KNL1, OR2L2, PUM2, CC110, TBC15, STT3B, ZN507, ALMS1, DLG5, KCNV2, BRX1, DOT1L, GEMI5, PARD3, ZN384, SMAP2, IASPP, TM263, ZFN2B, NUDC2, PCNP, TRUB1, LMO7, ATX2L, PALLD, P66B, BBX, ZCH14, GBF1, SMG7, RTF1, NICA, PHF3, MAML1, ZN592, LAR4B, TFG, TAF4B, RREB1, SC65, CBP, SYMPK, DDX17, GPKOW, FUBP2, UBP7, LPP, LSM10, NCLN, MRTFA, FUBP1, TTC17, PBIP1, TTC28, TOM6, PF21A, INT12, REPS1, ESS2, MBD6, ELP4, SGF29, RBM33, ZN503, P121A, TONSL, PDLI5, ERO1A, DOCK6, FUBP3, RSRC1, ZN594, VCIP1, ZN462, LCOR, PDLI2, CLP1L, Z512B, ZFR, EP400, MRFL, H6ST2, TIGD1, NOL4L, DOCK7, RPR1A, RBM14, ADCYA, QKI, LENG8, TRNT1, PP1RA, PHF12, CIC, MED15, ERBIN, HMCN1, LMF1, PIGS, WRIP1, SIN3A, MINT, HTF4, EYA3, POP1, TEAD3, TTC1, CSN8, ATX2, ARI3A, ANM1, PKP2, TEP1, DPH2, WAC, DIDO1, HNRL1, RBM4, SSBP4, PRR14, SSBP3, YTHD1, KPCD2, ZCHC2, TB182, AMRA1, CE295, TANC1, ZC12C, CEP44, STRAB, SP130, BRD8, RGAP1, SMG9, APC1, I2BPL, TMX4, KI13A, WDR13, EPC1, ADNP, ZN106, TM245, FOXP1, PABP3, WNK1, E41L1, ZHX3, BICC1, PEAK1, PPR3E, ZN703, PKHA5, CLSPN, BCDO1, RC3H2, ZFYV1, TAF9B, EMAL4, ZBT20, NCOA5, TANC2, ZN532, NCK5L, TNR6C, CHD8, FBSL, APMAP, DMAP1, UBN1, DCP1A, INCE, ANLN, GEPH, PDLI7, TULP4, HOME2, SLX9, DIAP3, BMP2K, RBM12, STAU2, DDX28, CWC25, CARF, ETAA1, ABI2, HXC10, BCLF1, TAB2, CELR3, CDK12, GRHL1, SACS, ITSN2, BICRA, CNOT2, TMEM9, CAC1I, CAMP3, DAPLE, RCC2, DIP2B, MBD5, CT2NL, F135A, KANL3, RERE, SE1L1, TRM7, YM012, KDM5B, LIMD1, TCF20, SUN2, LIMA1, SEPT9, UBQL2, TRPS1, S30BP, NRBP, BAZ2B, SIX4, HOOK1, CDC23, TASOR, GMEB2, TNIK, PARP4, NUP50, ZHX1, CDV3, MCTS1, KCNH3, LRFN2, MRTFB, ZBT21, PRR12, YETS2, HECD1, ZMYD8, NOTC3, SPAT2, SOX13, G3BP2, MAGD2, MINP1, MACF1, CP131, SCAF8, TRI33, PHF8, LIMC1, TNR6B, SRRM2, SCML2, ZN148, POLH, INVS, ICE1, R3HD2, MAN1, TR150, WBP11, ZN281, STA13, WNK2, HBS1L, ARIP4, MTCL1, DCAF1, RPGF2, IRS2, CRBG1, HYOU1, SAM50, PRC2C, YTHD2, NCOR2, GMEB1, DC1L1, EPN1, NCOA3, ZHX2, S23IP, U3KPZ7, V9GYH0
Species: Homo sapiens
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Kim DK, Lee JS, Lee EY, Jang H, Han S, Kim HY, Hwang IY, Choi JW, Shin HM, You HJ, Youn HD, Jang H. O-GlcNAcylation of Sox2 at threonine 258 regulates the self-renewal and early cell fate of embryonic stem cells. Experimental & molecular medicine 2021 53(11) 34819616
Abstract:
Sox2 is a core transcription factor in embryonic stem cells (ESCs), and O-GlcNAcylation is a type of post-translational modification of nuclear-cytoplasmic proteins. Although both factors play important roles in the maintenance and differentiation of ESCs and the serine 248 (S248) and threonine 258 (T258) residues of Sox2 are modified by O-GlcNAcylation, the function of Sox2 O-GlcNAcylation is unclear. Here, we show that O-GlcNAcylation of Sox2 at T258 regulates mouse ESC self-renewal and early cell fate. ESCs in which wild-type Sox2 was replaced with the Sox2 T258A mutant exhibited reduced self-renewal, whereas ESCs with the Sox2 S248A point mutation did not. ESCs with the Sox2 T258A mutation heterologously introduced using the CRISPR/Cas9 system, designated E14-Sox2TA/WT, also exhibited reduced self-renewal. RNA sequencing analysis under self-renewal conditions showed that upregulated expression of early differentiation genes, rather than a downregulated expression of self-renewal genes, was responsible for the reduced self-renewal of E14-Sox2TA/WT cells. There was a significant decrease in ectodermal tissue and a marked increase in cartilage tissue in E14-Sox2TA/WT-derived teratomas compared with normal E14 ESC-derived teratomas. RNA sequencing of teratomas revealed that genes related to brain development had generally downregulated expression in the E14-Sox2TA/WT-derived teratomas. Our findings using the Sox2 T258A mutant suggest that Sox2 T258 O-GlcNAc has a positive effect on ESC self-renewal and plays an important role in the proper development of ectodermal lineage cells. Overall, our study directly links O-GlcNAcylation and early cell fate decisions.
O-GlcNAc proteins:
SOX2
Species: Homo sapiens
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Xu S, Sun F, Wu R. A Chemoenzymatic Method Based on Easily Accessible Enzymes for Profiling Protein O-GlcNAcylation. Analytical chemistry 2020 92(14) 32574038
Abstract:
O-GlcNAcylation has gradually been recognized as a critically important protein post-translational modification in mammalian cells. Besides regulation of gene expression, its crosstalk with protein phosphorylation is vital for cell signaling. Despite its importance, comprehensive analysis of O-GlcNAcylation is extraordinarily challenging due to the low abundances of many O-GlcNAcylated proteins and the complexity of biological samples. Here, we developed a novel chemoenzymatic method based on a wild-type galactosyltransferase and uridine diphosphate galactose (UDP-Gal) for global and site-specific analysis of protein O-GlcNAcylation. This method integrates enzymatic reactions and hydrazide chemistry to enrich O-GlcNAcylated peptides. All reagents used are more easily accessible and cost-effective as compared to the engineered enzyme and click chemistry reagents. Biological triplicate experiments were performed to validate the effectiveness and the reproducibility of this method, and the results are comparable with the previous chemoenzymatic method using the engineered enzyme and click chemistry. Moreover, because of the promiscuity of the galactosyltransferase, 18 unique O-glucosylated peptides were identified on the EGF domain from nine proteins. Considering that effective and approachable methods are critical to advance glycoscience research, the current method without any sample restrictions can be widely applied for global analysis of protein O-GlcNAcylation in different samples.
O-GlcNAc proteins:
SBNO1, CNOT1, SWAHB, P121C, PDLI1, TAF4, RNT2, PODXL, KMT2D, MYPT1, ZN609, SC16A, SET1A, ZN185, TNC18, PRPF3, TPD54, SYNJ1, PLIN3, MAFK, BRD4, N4BP1, ICOSL, ANR17, ZN217, NCOR1, ATRN, TOX4, ERLN2, AGFG2, VAPB, SC24A, SC24B, CNOT4, BAG3, LMNA, GCR, HSPB1, IF2A, K1C18, K2C8, K1C19, ROA1, TACD2, ATX1L, LYAG, PPAL, TPR, K1C13, ZEP1, SDC1, ATF1, CBL, GATA3, ARNT, MAP4, CLIP1, HXC9, NU214, MP2K2, CUX1, PBX2, MLH1, STAT3, LAP2A, KI67, RFX5, SOX2, NU153, RBP2, TAF6, HCFC1, AFF3, AGFG1, ATX1, AF17, DSRAD, FOXA1, NU107, FOXK1, SPTB2, TFAP4, EWS, SP2, KMT2A, IF4G1, NOTC2, TLE3, TLE4, REL, ACK1, LG3BP, AHNK, ARHG5, FOXO1, BPTF, RIPK1, NFYC, CDK13, UBP2L, LAGE3, MDC1, EPN4, RRP1B, NCOA6, GSE1, MEF2D, NUMA1, R3HD1, JHD2C, TRIP6, ELF2, TAB1, ZFHX3, ZYX, ADRM1, TAF9, RFX7, QSER1, QRIC1, TB10B, CRTC2, PRC2B, ZN362, UBAP2, RPRD2, ZN318, TASO2, ARID2, ANR40, BICRL, ABLM2, GRHL2, NIPBL, LIN54, TET2, NFRKB, KCD18, MDEAS, ZC3HE, FIP1, SAS6, MCAF1, BCOR, HAKAI, SPT6H, KDM3B, POGZ, MAVS, EMSY, RAI1, SRGP1, SH3R1, YTHD3, CASZ1, P66A, I2BP1, RB6I2, FOXP4, NAV2, GID4, MGAP, CDAN1, SUGP1, MILK2, NUP93, ZN687, FNBP4, ARFG1, ENAH, PHC3, SP20H, KMT2C, STT3B, DLG5, WIPF2, ZFN2B, LMO7, ATX2L, CSKI2, P66B, SMG7, CBP, SEM4D, FUBP2, LPP, PF21A, INT12, CERS2, GWL, PDLI5, CHAP1, ANCHR, Z512B, ZFR, EP400, RBM14, CIC, MINT, S29A1, DPH2, WAC, DIDO1, HNRL1, YTHD1, CEP44, SP130, I2BPL, FOXP1, WNK1, E41L1, ZHX3, GORS2, PKHA5, RC3H2, TAF9B, NCOA5, TANC2, CELR2, UBN1, PDLI7, RBM12, CARF, TAB2, CNOT2, KANL3, STAP2, TCF20, UBQL2, S30BP, SIX4, TASOR, GMEB2, ZHX1, YETS2, ZMYD8, NOTC3, TRI33, SRRM2, CHM2B, SCML2, POLH, R3HD2, ZN281, WNK2, PRC2C, NCOR2, GMEB1, ZHX2
Species: Homo sapiens
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Li J, Li Z, Duan X, Qin K, Dang L, Sun S, Cai L, Hsieh-Wilson LC, Wu L, Yi W. An Isotope-Coded Photocleavable Probe for Quantitative Profiling of Protein O-GlcNAcylation. ACS chemical biology 2019 14(1) 30620550
Abstract:
O-linked N-acetylglucosamine ( O-GlcNAc) is a ubiquitous post-translational modification of proteins and is essential for cell function. Quantifying the dynamics of O-GlcNAcylation in a proteome-wide level is critical for uncovering cellular mechanisms and functional roles of O-GlcNAcylation in cells. Here, we develop an isotope-coded photocleavable probe for profiling protein O-GlcNAcylation dynamics using quantitative mass spectrometry-based proteomics. This probe enables selective tagging and isotopic labeling of O-GlcNAcylated proteins in one step from complex cellular mixtures. We demonstrate the application of the probe to quantitatively profile O-GlcNAcylation sites in 293T cells upon chemical induction of O-GlcNAc levels. We further applied the probe to quantitatively analyze the stoichiometry of O-GlcNAcylation between sorafenib-sensitive and sorafenib-resistant liver cancer cells, which lays the foundation for mechanistic investigation of O-GlcNAcylation in regulating cancer chemoresistance. Thus, this probe provides a powerful tool to profile O-GlcNAcylation dynamics in cells.
O-GlcNAc proteins:
A0A0B4J203, A0A0C4DFX4, SBNO1, P121B, CX028, RGPD3, AN36A, P121C, GG6L6, S31C2, E9PCH4, H0YAE9, H0YHG0, GG6LV, H3BMH7, PDLI1, TAF4, BCL9, ABLM1, CHD2, KMT2D, RGPD8, OPLA, HGS, MYPT1, ZN609, SC16A, SET1A, TIF1A, EIF3H, TET3, M3K7, PRPF3, TPD54, IF4G3, E41L2, AKAP8, TM11D, MYPT2, GANP, PLIN3, MAFK, BRD4, MITF, N4BP1, ATG13, PP6R2, ANR17, NCOR1, SPAG7, SRS10, SF3B1, CSDE1, TOX4, PCF11, AGFG2, SMC2, M3K6, SC24B, ZBT11, CNOT4, EYA4, OXSR1, ZMYM6, CCNE2, ANGT, LMNA, ALDOA, GCR, HSPB1, F13B, RLA2, K1C18, K2C8, ZFY, SRPRA, RU17, VIME, RU2A, ATX1L, RGPD1, S31C1, GLI3, LYAG, PABP1, COBA1, CO6A3, MYH7, ENPL, ZEP1, RS2, ZFX, ZNF30, ANPRC, ATF7, EGR1, SON, RCC1, ATF1, ATF6A, HXA5, ROA2, CBL, IF4B, GATA2, RIR1, RAE1, APC, ATPA, ARNT, MAP4, HXD9, HLAF, CLIP1, ZEP2, MYH10, TIE1, NU214, DEK, PDE6B, SRP14, CUX1, LPPRC, GATA4, KI67, YAP1, RFX5, SOX2, PRC2A, NASP, CDK8, NU153, RBP2, TAF6, EMD, PAPOA, HCFC1, NEK4, AGFG1, NUP98, INHBC, CADH6, F193A, KGD4, RT34, SARNP, LACTB, COG7, FOXK1, DAB2, PLIN5, SPTB2, SP2, NRG1, IF4G1, K1C17, TLE1, TLE3, TLE4, UBE3A, ACK1, AHNK, FCHO2, FOXO1, TROAP, BPTF, IRAG2, BFSP1, FOXC1, PRDM2, DDX10, G3BP1, PABP4, GRB10, PPIG, MADCA, PICAL, MAMD1, CUL4B, ASPP2, SPTN1, CDK13, CYLC2, DSG2, UBP2L, SRC8, ITPR3, PUM1, MDC1, EPN4, RRP1B, NCOA6, RRP5, RFTN1, R3HD1, WDR43, EEA1, MTFR1, SF3B3, RYR3, SF01, JHD2C, ELF2, MYLK, TAB1, ZYX, ADRM1, QSER1, CL16A, RHG31, AAK1, TMM44, AMOT, IF44L, YIF1B, AG10A, CD048, FSIP2, ESCO1, S2553, BCORL, AN36C, MTUS2, PRC2B, CEP78, SAMD9, TSBP1, LRIF1, CXG2, SKT, ZN648, UBAP2, RBM26, RC3H1, EFCB6, CE350, RPRD2, S31A6, TASO2, ECM29, RN123, PLCX3, ARID2, DEN2C, K0930, LIN54, M18BP, SCYL2, NFRKB, KLH35, ZC3HE, ANR11, FIP1, SBSN, S49A3, FAT4, MCAF1, BCOR, DUSTY, GGYF2, BNC2, CO039, SRCAP, UBN2, FOXNB, UBP54, HAKAI, ASXL2, KNDC1, SPT6H, TAOK1, KDM3B, RGPD4, POGZ, NUFP2, EMSY, I2BP2, SH3R1, HUWE1, YTHD3, FLIP1, KAISO, MYPN, TTC6, LDB1, TM135, TBC26, ZFHX4, ANGL5, SPAS2, DZIP1, P66A, AHNK2, FMNL3, NAV2, ARI3B, MGAP, RP1L1, CC28A, Z3H7A, CDAN1, ANKH1, SUGP1, PHAR4, KMT2E, XRN1, SPART, NUP93, ZN687, CMTR1, THMS1, AN36B, TMTC2, SYNPO, FNBP4, GG6L1, ENAH, GG6L2, SLAI1, PHC3, BD1L1, NUP35, DDX55, NEIL3, GSDMB, ALMS1, STK35, GEMI5, RPGF6, SMCR8, WIPF2, TM171, RN133, TEKT4, LMO7, CKAP2, ATX2L, ACO11, P66B, DAAF4, BBX, FIG4, ZN516, RREB1, FUBP2, LPP, E2F7, TTC28, TOM6, ASTRA, OTUB2, PGBD4, LEG12, ELP4, RBM33, MYEOV, SMRD1, DDX27, P121A, TONSL, PDLI5, THOC3, VCIP1, LRIQ1, ZFR, EP400, CBPC4, RBM14, IPP2L, QKI, PLIN4, JMJD8, RBM15, MINT, SEC62, AGAP2, RGPD5, ATX2, MYD88, ARI3A, SPI2A, SPI2B, DPH2, MCMBP, TMPSD, YTHD1, WNK3, PP12C, TB182, TANC1, CEP44, SENP6, BRD8, RGAP1, ALX4, KI13A, KCNH6, ZN106, FOXP1, PABP3, SMOC2, WNK1, ZHX3, CP095, REEP4, DOCK5, ZN703, GORS2, MLXIP, PKHA5, FOH1B, RC3H2, TANC2, TRPM3, SYTL2, CP4FC, GAK5, JPH1, APMAP, DMAP1, GP108, KMT5A, GPR84, DUOX2, DUOX1, PCDBG, MDN1, NALP2, CARF, HXC10, TAB2, CDK12, ADA2, ITSN2, F135A, SI1L2, RBM27, KANL3, ZN219, DYH17, AFF4, NB5R1, S30BP, NRBP, BAZ2A, SIX4, HOOK1, TASOR, GMEB2, ZHX1, TAOK2, CFA92, MRTFB, ZBT21, PRR12, YETS2, HECD1, MYO6, ICAM5, MAGD2, SCAF8, TRAK1, SHAN2, SRRM2, EXO1, SCML2, POK19, POLH, NCKP1, AT11B, NOP58, ZN281, UB2J1, GRIP1, SALL2, ARIP4, RPGF2, HYOU1, TTLL3, PRC2C, PCDB4, NCOR2, CP46A, 5MP1, CABIN, NCOA3, S23IP, U3KPZ7
Species: Homo sapiens
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Sharma NS, Gupta VK, Dauer P, Kesh K, Hadad R, Giri B, Chandra A, Dudeja V, Slawson C, Banerjee S, Vickers SM, Saluja A, Banerjee S. O-GlcNAc modification of Sox2 regulates self-renewal in pancreatic cancer by promoting its stability. Theranostics 2019 9(12) 31281487
Abstract:
Pancreatic adenocarcinoma (PDAC) claims more than 90% of the patients diagnosed with the disease owing to its aggressive biology that is manifested by high rate of tumor recurrence. Aberrant upregulation in the transcriptional activity of proteins involved in self-renewal like Sox2, Oct4 and Nanog is instrumental in these recurrence phenomena. In cancer, Sox2 is aberrantly "turned-on" leading to activation of downstream genes those results in relapse of the tumor. Molecular mechanisms that regulate the activity of Sox2 in PDAC are not known. In the current study, we have studied the how glycosylation of Sox2 by O-GlcNAc transferase (OGT) can affect its transcriptional activity and thus regulate self-renewal in cancer. Methods: RNA-Seq analysis of CRISPR-OGTi PDAC cells indicated a deregulation of differentiation and self-renewal pathways in PDAC. Pancreatic tumor burden following inhibition of OGT in vivo was done by using small molecule inhibitor, OSMI, on subcutaneous implantation of PDAC cells. Sox2 activity assay was performed by Dual Luciferase Reporter Assay kit. Results: Our study shows for the first time that in PDAC, glycosylation of Sox2 by OGT stabilizes it in the nucleus. Site directed mutagenesis of this site (S246A) prevents this modification. We further show that inhibition of OGT delayed initiation of pancreatic tumors by inhibition of Sox2. We also show that targeting OGT in vivo with a small molecule-inhibitor OSMI, results in decreased tumor burden in PDAC. Conclusion: Understanding this mechanism of SOX2 regulation by its glycosylation is expected to pave the way for development of novel therapy that has the potential to eradicate the cells responsible for tumor-recurrence.
O-GlcNAc proteins:
SOX2
Species: Homo sapiens
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Hahne H, Gholami AM, Kuster B. Discovery of O-GlcNAc-modified proteins in published large-scale proteome data. Molecular & cellular proteomics : MCP 2012 11(10) 22661428
Abstract:
The attachment of N-acetylglucosamine to serine or threonine residues (O-GlcNAc) is a post-translational modification on nuclear and cytoplasmic proteins with emerging roles in numerous cellular processes, such as signal transduction, transcription, and translation. It is further presumed that O-GlcNAc can exhibit a site-specific, dynamic and possibly functional interplay with phosphorylation. O-GlcNAc proteins are commonly identified by tandem mass spectrometry following some form of biochemical enrichment. In the present study, we assessed if, and to which extent, O-GlcNAc-modified proteins can be discovered from existing large-scale proteome data sets. To this end, we conceived a straightforward O-GlcNAc identification strategy based on our recently developed Oscore software that automatically analyzes tandem mass spectra for the presence and intensity of O-GlcNAc diagnostic fragment ions. Using the Oscore, we discovered hundreds of O-GlcNAc peptides not initially identified in these studies, and most of which have not been described before. Merely re-searching this data extended the number of known O-GlcNAc proteins by almost 100 suggesting that this modification exists even more widely than previously anticipated and the modification is often sufficiently abundant to be detected without enrichment. However, a comparison of O-GlcNAc and phospho-identifications from the very same data indicates that the O-GlcNAc modification is considerably less abundant than phosphorylation. The discovery of numerous doubly modified peptides (i.e. peptides with one or multiple O-GlcNAc or phosphate moieties), suggests that O-GlcNAc and phosphorylation are not necessarily mutually exclusive, but can occur simultaneously at adjacent sites.
O-GlcNAc proteins:
MA2B1, HGS, MYPT1, PLXB2, IF4G3, MAFK, ANR17, CREG1, SYUG, ERLN2, SC24B, AACT, LMNA, FIBG, TFR1, FUCO, K2C1, HSPB1, RLA2, ITB1, CATD, SAP, CATL1, CD3E, BGLR, LYAG, BIP, TPR, CD99, GLU2B, SON, AL5AP, MAP4, CALX, GRN, AMRP, BASI, NU214, STAT3, SOX3, LAP2A, LAP2B, SOX2, TMEDA, NU153, RBP2, SERPH, HCFC1, RD23B, FOXK1, SPTB2, NUCB1, AHNK, BPTF, PICAL, UBP2L, RCN1, CNN3, JHD2C, TAB1, ADRM1, QSER1, TGO1, PRC2B, TOIP1, UBAP2, LITD1, RPRD2, NFRKB, CREL2, IKIP, KDM3B, POGZ, NUFP2, YTHD3, ARI3B, ANKH1, GT251, TXND5, MCFD2, MZB1, NUDC2, ATX2L, SMG7, FKB10, PF21A, PDLI5, ZFR, EP400, PRRC1, RBM14, SIN3A, COCA1, S29A1, H2B1L, ERP44, CNPY3, YTHD1, LACRT, SP130, CPVL, WNK1, SDF2L, APMAP, DCP1A, PDLI7, RBM12, CARF, FKB11, CDK12, S30BP, SALL4, GMEB2, ZMYD8, PRP19, MINP1, SCAF8, ERGI3, SALL2, HYOU1, PRC2C
Species: Homo sapiens
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