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Ramirez DH, Aonbangkhen C, Wu HY, Naftaly JA, Tang S, O'Meara TR, Woo CM. Engineering a Proximity-Directed O-GlcNAc Transferase for Selective Protein O-GlcNAcylation in Cells. ACS chemical biology 2020 15(4) 32119511
Abstract:
O-Linked β-N-acetylglucosamine (O-GlcNAc) is a monosaccharide that plays an essential role in cellular signaling throughout the nucleocytoplasmic proteome of eukaryotic cells. Strategies for selectively increasing O-GlcNAc levels on a target protein in cells would accelerate studies of this essential modification. Here, we report a generalizable strategy for introducing O-GlcNAc into selected target proteins in cells using a nanobody as a proximity-directing agent fused to O-GlcNAc transferase (OGT). Fusion of a nanobody that recognizes GFP (nGFP) or a nanobody that recognizes the four-amino acid sequence EPEA (nEPEA) to OGT yielded nanobody-OGT constructs that selectively delivered O-GlcNAc to a series of tagged target proteins (e.g., JunB, cJun, and Nup62). Truncation of the tetratricopeptide repeat domain as in OGT(4) increased selectivity for the target protein through the nanobody by reducing global elevation of O-GlcNAc levels in the cell. Quantitative chemical proteomics confirmed the increase in O-GlcNAc to the target protein by nanobody-OGT(4). Glycoproteomics revealed that nanobody-OGT(4) or full-length OGT produced a similar glycosite profile on the target protein JunB and Nup62. Finally, we demonstrate the ability to selectively target endogenous α-synuclein for O-GlcNAcylation in HEK293T cells. These first proximity-directed OGT constructs provide a flexible strategy for targeting additional proteins and a template for further engineering of OGT and the O-GlcNAc proteome in the future. The use of a nanobody to redirect OGT substrate selection for glycosylation of desired proteins in cells may further constitute a generalizable strategy for controlling a broader array of post-translational modifications in cells.
O-GlcNAc proteins:
SBNO1, CNOT1, P121C, DX39A, GTPB1, AP3B1, PGRC1, TAF4, EIF3F, IPO5, IF2B3, NOP56, DDX3X, ARI1A, IRS4, ANM5, TCRG1, PSA7, HGS, MYPT1, HNRDL, XPO1, SET1A, PUR4, NPC1, TIF1A, NKRF, OGT1, PPM1G, EIF3D, EIF3H, DHX15, SERA, HNRPR, IF4G3, E41L2, ZN207, BUB3, ACTN4, HTSF1, AP1G1, SYNC, AKAP8, CALU, SMCA5, JIP4, OGA, HNRPQ, DIAP1, TSN3, SNX2, DKC1, CLAP2, CPNE3, PHF2, ANR17, H2AY, FLNB, NCOR1, CISY, PR40A, SF3B1, CSDE1, U520, EIF3G, PRAF3, SRP72, MTA2, TOX4, SC24D, SC31A, SCAF4, ZRAB2, LC7L3, VAPB, IPO7, SC24B, ACSL3, AP2A1, AIFM1, LDHA, COX2, HPRT, AATM, PGK1, LMNA, TFR1, ALDOA, OAT, G3P, RPN1, RPN2, AT1A1, ADT2, PCCA, IF2A, RLA0, ITB1, ATPB, ENOA, PYGL, G6PI, NPM, LDHB, PDIA1, H10, TBB5, HEXB, PROF1, SYEP, HS90A, HNRPC, 4F2, HS90B, ASNS, ODPA, RU17, RSSA, SNRPA, GSTP1, HMGB1, DLDH, ROA1, PARP1, LKHA4, HS71B, H14, ODP2, THIO, CH60, BIP, HSP7C, EPB41, ODPB, LAMP1, ACADM, TOP1, TOP2A, PYC, C1TC, MPRI, PRPS2, PABP1, PCNA, HARS1, IMDH2, TPR, KCRB, XRCC6, XRCC5, EF2, PDIA4, PLST, GLU2B, KPYM, ENPL, PO2F1, HNRPL, SYDC, PLAK, ALDR, EZRI, GNS, RS2, CREB1, H12, AT2A2, JUNB, PYRG1, DDX5, PRS6A, TCPA, RL35A, RL7, VINC, SON, RCC1, NUCL, HXK1, E2AK2, SPEE, IF2B, ANXA7, LMNB1, FLNA, VDAC1, FBRL, PUR2, PUR6, UBA1, NDKB, ROA2, RFX1, TCEA1, SFPQ, PPIB, RS3, NFYA, SAHH, COF1, IF4B, EF1B, MCM3, BRD2, ATPA, PSA1, PSA3, PSA4, PAX6, U2AF2, RL13, PTBP1, SYTC, SYVC, EF1G, RFA1, APEX1, PYR1, CALR, MAP4, CALX, PSB5, TKT, PRDX6, PRDX5, PRDX3, RL12, PEBP1, PDIA3, 2AAA, CDC27, AMRP, SDHA, QCR1, PUR9, HNRH1, STIP1, PRDX2, RL9, CSTF2, MCM4, MCM5, MCM7, GLYM, HSP74, PHB, MYH9, COPB2, ADDA, BASI, FUS, NU214, DEK, MP2K2, ATPG, RL4, SRP14, NUP62, RBMX, GRP75, IF4A3, RS19, RL3, TXLNA, TCPZ, MDHC, MDHM, ECHA, IF2G, GARS, SYIC, LAP2A, LAP2B, MTREX, RS27, LPPRC, MATR3, RANG, VDAC2, UBP5, KI67, RAGP1, NOP2, CRKL, BAG6, RL27A, RL5, RL21, RL28, RS9, STT3A, COPD, PRC2A, TCPE, AL9A1, RL34, NASP, FAS, TCPG, EFTU, SYAC, SYSC, PSB3, MCM2, YLPM1, TMEDA, RBM25, NU153, RBP2, GSK3A, TAF6, GUAA, MRE11, GDIB, EMD, F10A1, LRBA, RL14, TCPQ, TCPD, ANX11, PAPOA, RAB7A, SMCA4, HCFC1, DHB4, ROA3, 6PGD, HNRPM, IMA1, AGFG1, HNRPF, MSH6, RBM5, NUP98, ACLY, COPB, COPA, MOT1, SC24C, SYRC, SYYC, AT1B3, RD23B, P5CS, IF5, XPO2, TERA, AFAD, DSRAD, PSA, SYMC, CTBP2, NU107, TPIS, ACTB, IF4A1, PSA6, ARF3, ABCE1, RAP1B, RS3A, RL26, RL15, S61A1, HNRPK, RS7, RS8, 1433E, RS14, RS23, RS11, SMD1, RL7A, RS4X, H4, RAN, RL23, GBB2, RL10A, RL11, RL8, PPIA, RL40, TRA2B, AP2B1, IF5A1, RACK1, YBOX1, EF1A1, TBB4B, CSK21, IF4G2, GTF2I, TCPB, PRKDC, RL24, RL19, SRSF3, FOXK1, RBM10, MPCP, CLH1, HNRPU, SPTB2, FOXK2, CAP1, LAT1, EXOSX, EWS, RL18A, FKBP4, RL6, TOP2B, KMT2A, LMNB2, TF65, IF4G1, TLE3, SRS11, PUR1, SUH, GABPA, PRDX1, RL18, C1QBP, KHDR1, SRSF1, DHX9, CD47, SSRP1, RBBP4, AHNK, AP1B1, NU160, BPTF, TP53B, AIMP1, ILF2, ILF3, LMAN2, TRAP1, PP1R8, ACACA, ROA0, PRDX4, CBX3, PSMD2, SRSF6, TIF1B, PTK7, PABP4, EIF3I, TCOF, SF3B2, TMED1, PICAL, RIPK1, HDAC1, CUL4B, CD166, IDI1, NFYC, CKAP5, HNRPD, SCRB2, DSG2, EIF3A, UBP2L, SCRIB, TTL12, DCTN1, DYHC1, SRC8, CAPR1, RBM39, MCM6, MDC1, EPN4, SMC1A, RRP1B, UBP10, GANAB, LBR, ZN638, IMB1, NUMA1, SEPT2, SART3, U5S1, SYK, BRD3, PDIA6, IPYR, TEBP, NONO, PWP2, RCN1, PCBP1, PCBP2, SF3B3, SAFB1, SF3A1, NCOA2, SF01, MARE1, NSDHL, TAB1, AAAT, VAS1, ZYX, CCDC6, PKN2, DDB1, CDC37, SRSF7, CPSF6, NRF1, H31T, QSER1, QRIC1, P3H1, TB10B, AMOT, DHB12, PRC2B, H2B2F, HP1B3, CE170, ZC3HD, RBM26, RIF1, RPRD2, ZN318, ECM29, ZMYM4, MAP1S, LIN54, EDC4, PRP8, SCYL2, NFRKB, ZC3HE, LARP1, FIP1, MCAF1, GGYF2, SPT6H, SND1, DHX30, KDM3B, ZCCHV, NUP54, POGZ, NUFP2, MAVS, I2BP2, RBBP6, HUWE1, YTHD3, CENPV, LYRIC, ZN598, GP180, CAND1, CARM1, DDX42, P66A, ARI3B, MGAP, PHF6, CHERP, ANKH1, SUGP1, CCAR1, SPB1, PHAR4, SPART, CCAR2, NUP93, S11IP, FNBP4, CPSF7, ARFG1, ENAH, AFG2H, TXND5, LS14A, Z280C, TNR6A, SMRC2, TBC15, PNPT1, HM13, PO210, GEMI5, ZN384, SMAP2, NU133, PDC6I, PCNP, CKAP2, ATX2L, P66B, ELYS, DDX1, GBF1, NICA, UBXN4, HS105, LAR4B, NU205, AKAP1, TFG, CBP, DDX17, CELF1, RENT1, SMRC1, FUBP2, TNPO1, UBP7, NCLN, FUBP1, FKB10, KBP, PDLI5, FUBP3, CHAP1, Z512B, ZFR, PRRC1, DOCK7, RBM14, VPS35, CIC, EFGM, SIN3A, MINT, CDC5L, PSMD1, EYA3, ATX2, HCD2, ACON, TS101, TCPH, ANM1, SH3G1, COR1B, DIDO1, HNRL1, DDX23, TMED9, NUP58, RBM4, NAA15, B2L13, YTHD1, UNK, ILKAP, SP130, BRD8, I2BPL, SLK, S6A15, PININ, NELFA, PTN23, WNK1, AMPB, GORS2, CYBP, TAF9B, GLOD4, CBX8, NCOA5, CHD8, APMAP, DCP1A, RTN4, ANLN, GEPH, PDLI7, DDX21, SYFB, SYIM, SMC4, RBM12, DDX18, CARF, UGGG1, CDK12, TECR, IF2B1, HPBP1, ITSN2, CNOT2, HACD3, RCC2, SYLC, SUCB1, UBQL2, PCYOX, S30BP, PUF60, NRBP, DACH1, BAZ2A, BAZ1B, CDC23, TASOR, ACINU, CDV3, MRTFB, YETS2, HECD1, PKCB1, DD19B, PRP19, MAGD2, FAF1, TRI33, SRRM2, PA2G4, RUVB2, RUVB1, VDAC3, E41L3, TR150, NOP58, SHLB1, LC7L2, TMED7, STRAP, RTCB, HBS1L, TLN1, HYOU1, PRC2C, SP16H, COPG1, DC1L1, S23IP
Species: Homo sapiens
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Liu Y, Chen Q, Zhang N, Zhang K, Dou T, Cao Y, Liu Y, Li K, Hao X, Xie X, Li W, Ren Y, Zhang J. Proteomic profiling and genome-wide mapping of O-GlcNAc chromatin-associated proteins reveal an O-GlcNAc-regulated genotoxic stress response. Nature communications 2020 11(1) 33214551
Abstract:
O-GlcNAc modification plays critical roles in regulating the stress response program and cellular homeostasis. However, systematic and multi-omics studies on the O-GlcNAc regulated mechanism have been limited. Here, comprehensive data are obtained by a chemical reporter-based method to survey O-GlcNAc function in human breast cancer cells stimulated with the genotoxic agent adriamycin. We identify 875 genotoxic stress-induced O-GlcNAc chromatin-associated proteins (OCPs), including 88 O-GlcNAc chromatin-associated transcription factors and cofactors (OCTFs), subsequently map their genomic loci, and construct a comprehensive transcriptional reprogramming network. Notably, genotoxicity-induced O-GlcNAc enhances the genome-wide interactions of OCPs with chromatin. The dynamic binding switch of hundreds of OCPs from enhancers to promoters is identified as a crucial feature in the specific transcriptional activation of genes involved in the adaptation of cancer cells to genotoxic stress. The OCTF nuclear factor erythroid 2-related factor-1 (NRF1) is found to be a key response regulator in O-GlcNAc-modulated cellular homeostasis. These results provide a valuable clue suggesting that OCPs act as stress sensors by regulating the expression of various genes to protect cancer cells from genotoxic stress.
O-GlcNAc proteins:
RBM47, SBNO1, CNOT1, RGPD3, P121C, PDLI1, MYO1C, AIP, PSD11, PGRC1, TAF4, CLIC1, IPO5, IF2B3, AGRIN, PLOD2, HMGN4, IMA4, PESC, NOP56, DDX3X, PODXL, IMA3, NFIB, ARI1A, G3PT, PDCD5, TCRG1, PSA7, SCAM3, HGS, MYPT1, HNRDL, XPO1, ZN609, SC16A, SR140, SET1A, NPC1, TBX3, ARC1B, TIF1A, PGRC2, PFD6, NKRF, ZN185, OGT1, HMGB3, PPM1G, EIF3D, IPO8, RPA34, NUP42, DHX15, PRP4, SERA, PSMD3, RFOX2, PAPS1, MCA3, HNRPR, PRPF3, TPD54, IF4G3, KLF4, E41L2, DENR, XPOT, PRC1, ZN207, GET3, BUB3, ACTN4, BUD23, SYNC, KRT86, CPSF5, U3IP2, CALU, SAHH2, MED14, SMCA5, ZN862, GANP, KDM1A, ACSL4, SNX3, OGA, HNRPQ, PLOD3, MAFK, IMA7, UGDH, PQBP1, DKC1, IF2P, EDF1, DNJA2, BRD4, PFD1, WDR1, CPNE3, ZC11A, CLU, T22D2, PP6R2, CREST, ANR17, PDCD6, TBCA, H2AY, FLNB, NCOR1, SC22B, PR40A, PSIP1, SRS10, SF3B1, CSDE1, NPM3, U520, NU155, WDHD1, CRTAP, IDHC, CCNK, PIAS2, SPF27, DNJC8, RL1D1, SRP72, MTA2, TOM70, TOX4, SC24D, SUN1, NFAT5, AP2A2, SC31A, SEM3D, AGFG2, ZRAB2, LC7L3, LYPD3, FKBP9, SMC2, IPO7, AHSA1, PSMG1, SC24A, SC24B, CNOT4, OXSR1, HS74L, AP2A1, BAG3, CLPT1, ACL6A, LDHA, AATM, EGFR, PGK1, ASSY, LDLR, K1C14, LMNA, APOA2, FINC, ALBU, TFR1, PROC, ALDOA, CYTB, ANXA1, GCR, KITH, THY1, K2C1, G3P, HSPB1, RPN1, GNAI2, AT1A1, AT1B1, ADT2, IF2A, HMGN2, ICAM1, RLA2, JUN, LA, ITB1, K1C18, K2C8, CDK1, ATPB, S10A6, ENOA, PYGL, G6PI, NPM, TPM3, ITAV, ACBP, LDHB, PDIA1, H10, CATD, ANXA2, TBB5, PROF1, SYEP, HS90A, HNRPC, TSP1, SP1, ANXA6, RHOC, DAF, MDR1, 4F2, HS90B, SRPRA, ASNS, CY1, RU17, ITA5, NFIC, VIME, RS17, K2C7, ANXA5, K1C16, RSSA, SNRPA, GSTP1, LEG1, HMGB1, TPM1, ROA1, RU2A, PARP1, PPBI, UCHL1, ALDOC, ATX1L, HS71B, CALM3, RO60, H14, PTPRF, THIO, ESTD, CH60, BIP, HSP7C, LAMP1, TOP1, TOP2A, PYC, C1TC, MPRI, ADHX, PABP1, PCNA, HARS1, IMDH2, TPR, KCRB, ACTN1, XRCC6, XRCC5, RINI, EF2, K1C10, K2C5, PDIA4, P4HA1, PLST, T2FB, CD59, MIF, GLU2B, CBPM, AK1A1, KPYM, ENPL, CCNB1, PO2F1, HNRPL, SYDC, PLAK, ALDR, AMPN, ERF3A, EZRI, FOSL1, FOSL2, MCP, NQO1, GNS, ZEP1, RS2, DESP, MUC1, CD44, CBR1, CREB1, H15, H13, H12, NCPR, AT2A2, CD36, STMN1, HSP76, HMGA1, JUNB, UBF1, JUND, ATF7, CEBPB, PYRG1, DDX5, PFKAL, LEG3, TCPA, PTN1, RL35A, RL7, VINC, SON, RL17, PGAM1, RCC1, ATF1, ML12A, NUCL, SPEE, RXRA, NFKB1, IF2B, ANXA7, BTF3, PSB1, MPRD, LMNB1, CSRP1, FLNA, 5NTD, VDAC1, CD9, TGM2, PIMT, FBRL, PUR2, PUR6, UBA1, NDKB, ROA2, RFX1, CBL, TCEA1, ITA6, SFPQ, PPIB, SYWC, RS3, NFYA, SAHH, COF1, IF4B, KTHY, EF1B, PPAC, CDK2, MCM3, RS12, BRD2, DNJB1, ATPA, PSA1, PSA3, PSA4, S100P, ITA3, MOES, DDX6, DNMT1, PAX6, U2AF2, RL13, S10A4, HMGB2, PTBP1, SYTC, SYVC, EF1G, STOM, 1433T, ARNT, RL10, RFA1, APEX1, PYR1, CALR, MAP4, CALX, TEAD1, GRN, EPHA2, 3MG, TKT, RBMS1, PML, EF1D, ERP29, PRDX6, RL12, KCY, PEBP1, PDIA3, 2AAA, NMT1, PURA2, UFO, SORCN, ILEU, RPB2, METK2, TIA1, ZEP2, DNJA1, PUR9, HNRH3, HNRH1, 1433B, 1433S, STIP1, S10AB, L1CAM, PRDX2, CDD, ELF1, RL9, CD70, KINH, CSTF2, MCM4, MCM5, MCM7, GLYM, HSP74, PROF2, PHB, SPB6, RFC4, RL22, K1C9, MYH9, MYH10, COPB2, BASI, FUS, NU214, DEK, K22E, PRS7, ATPG, RL4, PP1G, GNL1, SRP14, NUP62, TAGL2, TALDO, RBMX, VKGC, GRP75, IF4A3, RS19, RL3, OST48, FEN1, CAPG, TXLNA, TCPZ, RL13A, STAT3, MDHC, MDHM, IF2G, GARS, SYIC, LAP2A, LAP2B, STAT1, MTREX, RS27, LPPRC, RL35, CDN2A, ECE1, LIS1, MUC18, MATR3, MSH2, SSRA, RANG, VDAC2, CBX5, UBP5, KI67, RAGP1, RECQ1, NOP2, BAG6, NOTC1, RL27A, RL5, RL21, RL28, RS9, RS5, RS10, IQGA1, CAPZB, IF1AX, RL29, SOX9, COPD, GSH0, PSMD8, PRC2A, TCPE, PTSS1, K2C6C, AGRE5, PAXI, RL34, LMAN1, NASP, FAS, CDK8, TCPG, EFTU, SYAC, SYSC, MCM2, ACADV, YLPM1, TMEDA, RBM25, HINT1, NU153, RBP2, TAF6, GUAA, CRIP1, GDIB, EMD, SERPH, F10A1, MAP2, RL14, TCPQ, TCPD, ANX11, PAPOA, FXR1, FXR2, RAB7A, SMCA4, SSRD, HCFC1, HDGF, ROA3, 6PGD, HNRPM, IMA1, GDIR1, AGFG1, HNRPF, MSH6, CAZA1, CRIP2, NUP98, ACLY, COPA, SC24C, TCP4, SYRC, ATX1, ATN1, SYYC, UBP14, AT1B3, RD23B, SNAA, IF5, PSMD4, XPO2, TERA, AF10, AF17, NP1L1, ADK, DSRAD, SEC13, NH2L1, PSA, EIF3B, SYMC, IF6, CTBP2, TMM33, NU107, EPIPL, TPIS, EIF3E, SC61B, MYL6, ACTB, IF4A1, RS20, PRPS1, PSA6, S10AA, CDC42, DEST, RAB10, UBC12, UBE2N, ARP3, ABCE1, RS3A, RL26, PSME3, RL15, RL27, RL37A, S61A1, PFD3, B2MG, DAD1, SUMO2, WDR5, NTF2, HNRPK, 1433G, RS7, PP1A, PP1B, RS8, RS15A, RS16, 1433E, RS14, RS23, RS18, RS29, RS13, RS11, RUXE, SMD1, SMD2, SMD3, PRS10, RL7A, ERF1, CNBP, RS4X, RL23A, RS6, H4, RAN, RL23, RAP1A, RS24, RS25, RS26, RS30, GBB2, RL30, RL31, RL10A, RL32, RL11, RL8, PPIA, FKB1A, RS27A, TRA2B, AP2B1, 1433Z, RSMN, SUMO1, DYL1, RL38, RS21, RACK1, UBC9, YBOX1, CSK2B, TPM4, EF1A1, ACTS, TBA1B, TBA4A, TBB4B, CSK21, PA1B2, HBB, HBA, PITX1, GTF2I, PHC1, TCPB, RAE1L, PRKDC, SARNP, RL24, ARF1, ERH, RL19, SRSF3, FOXK1, DAB2, EFNB1, RBM10, RBM3, CYC, MPCP, VIGLN, CLH1, FKBP3, HNRPU, U2AF1, SPTB2, TIAR, SRSF2, FOXK2, RUNX1, FABP5, LAT1, TFAP4, OTUD4, PFKAP, XPC, EWS, MEF2A, SP3, H11, RL18A, FKBP4, PLOD1, RL6, M2OM, DYST, KMT2A, LMNB2, TF65, UBXN1, GLGB, IF4G1, K1C17, TLE3, REL, 1433F, PLP2, CSTF1, SRS11, EF1A2, SUH, GABPA, PAX8, FMR1, PRDX1, RL18, CKAP4, KHDR1, LRP1, SRSF1, DHX9, LG3BP, PPID, SSRP1, NSUN2, RBBP4, EP300, AHNK, HSP7E, GALT2, BST2, NU160, TBL3, ASPH, TROAP, BPTF, NFIA, SF3A3, AIMP1, ILF2, ILF3, LMAN2, TRAP1, FOXC1, MYO1E, CSTF3, ECH1, ACACA, CAF1B, RED, MTAP, TADBP, ROA0, PRDX4, CBX3, PSMD2, GPS2, SRSF9, SRSF5, SRSF6, TIF1B, G3BP1, PTK7, PABP4, EIF3I, TCOF, SF3B2, HAP28, FKBP5, SMAD4, PICAL, TBB3, PRP4B, PIN1, RIPK1, HDAC1, DCTN2, SNW1, TRA2A, CUL4B, DYR1A, TPBG, FHL1, MOGS, CD166, SPTN1, DX39B, TBB2A, KLF5, BYST, RUNX2, CDK13, CKAP5, CIRBP, HNRPD, SCRB2, DAG1, VEZF1, DSG2, EIF3A, UBP2L, SCRIB, TTL12, FHL2, DPYL3, DYHC1, IF4A2, SRC8, TRI25, FLNC, FA50A, CAPR1, RBM39, MCM6, ITPR1, PUM1, MDC1, EPN4, SMC1A, RRP1B, NCOA6, GSE1, UBP10, GANAB, LBR, MEF2D, CHD4, LASP1, ZN638, IMB1, NOLC1, NUMA1, SEPT2, SART3, CND1, ACAP1, U5S1, SYK, IF4H, PDIA6, PLEC, NOMO1, PON2, IPYR, TEBP, NONO, PWP2, RNPS1, PCBP1, PCBP2, SF3B3, KS6A1, SAFB1, SF3A2, RBMS2, SC23A, SC23B, SF3A1, SSXT, NCOA2, TRAM1, SF01, MED1, HMGN3, JHD2C, TRIP6, MARE1, ELAV1, ELF2, TAB1, AAAT, TOM34, UB2V2, NEDD8, ZYX, SEPT7, ADRM1, UAP1, PSMD5, DDB1, CDC37, DPYL2, RBBP7, TAF9, SRSF7, CPSF6, NRF1, FSCN1, IF16, KYNU, H2A2C, H2B2E, TRXR1, HNRL2, PDS5A, QSER1, TSR1, SMU1, P3H1, LSM12, CRTC2, GLE1, H90B2, ZN326, BCORL, TGO1, PRC2B, RRP12, TOIP1, PCID2, NU188, HP1B3, CE170, ZN362, ZC3HD, LRIF1, UBR4, UBAP2, KPRP, RBM26, AHDC1, CROCC, RPRD2, ECM29, MBNL2, ZMYM4, AR6P4, STEA4, ARID2, BICRL, CPIN1, LIN54, TM214, CAVN1, CDC73, EDC4, PRP8, SCYL2, GOLM2, NFRKB, NCEH1, MDEAS, ZC3HE, LARP1, FIP1, CRTC3, SAS6, CSPG4, WDR82, MCAF1, PACS1, SRCAP, RIPR1, UBN2, FBX50, IKIP, H32, LARP4, H2AV, RS27L, HAKAI, SPT6H, SND1, DDX46, BZW1, CYFP1, KDM3B, PHF5A, ZCCHV, NUP54, POGZ, NUFP2, HEAT3, EMSY, RAI1, I2BP2, RBBP6, SH3R1, HUWE1, YTHD3, KHDC4, CENPV, KAISO, MYPN, PEG10, PABP2, KTN1, THOC4, GP180, CAND1, CARM1, PRSR1, DDX42, DAAF5, P66A, RB6I2, CHERP, ANKH1, CCAR1, RAVR1, SPB1, SMAP1, PHAR4, MAML2, PORIM, CCAR2, NUP93, LRC47, MT1M, FNBP4, CPSF7, PR38A, GT251, TXND5, PAIRB, FA98A, TNR6A, PHC3, ABCF1, VP37A, NUP43, NUP37, NUP35, RLA0L, THOC2, WDR36, SMRC2, PUM2, SPP2A, ALMS1, C99L2, DDX54, DLG5, BRX1, DOT1L, PO210, GEMI5, ZN384, SCRB1, ZC3HF, NU133, PDC6I, NUDC2, GLMP, LMO7, ATX2L, PALLD, PSPC1, P66B, DNJC9, ELYS, DDX1, H1X, NICA, TM131, MAML1, HS105, CNOT9, ZN592, LAR4B, GCN1, NU205, TFG, TAF4B, STAM1, CBP, RBP56, DDX17, CELF1, OSTF1, RENT1, SMRC1, SMRD2, FUBP2, TNPO1, NEP1, UBP7, STMN2, LPP, MED12, H2A1C, SMCE1, RL36L, NCLN, FERM2, FUBP1, LRC59, FKB10, PF21A, INT12, FWCH2, AP2M1, REPS1, CMBL, SNR40, SEH1, DAZP1, RBM33, S10AG, OTUB1, HMCES, DDX27, P121A, PDLI5, FUBP3, WNK4, CHAP1, ZC3HA, CLP1L, CNDP2, ZFR, EP400, PRRC1, NOL4L, RBM14, QKI, PLIN4, S38A2, VPS35, CIC, MED15, MCCA, WRIP1, STRBP, TM209, SIN3A, MINT, UHRF1, HTF4, CDC5L, PFD5, RING2, EYA3, NUP88, POP1, MNT, SCAFB, EIF3C, DNJC7, PHB2, ATX2, ROAA, NP1L4, TS101, CPNE1, TCPH, EBP2, ANM1, H2B1M, RNZ2, TBA1C, MBB1A, TXD17, ERP44, ESYT1, WAC, DIDO1, AN32E, TMM43, TBB6, HNRL1, DDX23, TBB2B, TM109, TMED9, NUP58, GNL3, KIFC1, NUP85, RBM4, NAA15, SRRT, PDIP3, YTHD1, WNK3, GTPB4, UNK, SLIRP, IF5A2, NAT10, ILKAP, XRN2, SP130, RGAP1, DDX47, I2BPL, PININ, BOLA2, PTN23, WNK1, FA83D, ZHX3, STEEP, RPF2, ZN703, GORS2, JUPI2, MLXIP, CYBP, RC3H2, TENS1, EMAL4, NCOA5, TNR6C, CHD8, APMAP, ENY2, CD320, UBN1, DCP1A, LUC7L, RTN4, RPR1B, NIT2, ANLN, PDLI7, DDX21, SIAS, SHLB2, MBNL1, OSTC, RAB6B, PHP14, SYFB, RBM12, DD19A, LYAR, CARF, BCLF1, TAB2, TMOD3, CDK12, IF2B1, MAT2B, MYOF, ITSN2, BICRA, CNOT2, SEP10, RCC2, BCCIP, RRBP1, RBM27, KANL3, ATX10, MRC2, SAE2, NXF1, ABCF2, TES, TCF20, SUN2, LIMA1, CHRD1, SEPT9, UBQL2, S30BP, MED13, PFD2, PUF60, XPO7, SIX4, DDX41, CCNL1, JUPI1, MRT4, GPTC8, NUDT5, RALY, ACINU, AGO2, NUP50, ZHX1, CDV3, TRPC5, MRTFB, ZMIZ1, YETS2, HECD1, PKCB1, COR1C, TPX2, AKP8L, PRP19, UBQL1, G3BP2, CHIP, PACN2, SSRG, EPCR, SCAF8, TRI33, SRRM2, PA2G4, CD11A, SMC3, RTRAF, RUVB2, EIF3L, RUVB1, NUDC, SYFA, DRG1, E41L3, RRP44, TR150, WBP11, NOP58, ZN281, SGT1, NOSIP, LSM2, LC7L2, SBDS, STRAP, RTCB, NOC2L, RL36, CHTOP, TLN1, ARIP4, HYOU1, PRC2C, PPME1, YTHD2, SP16H, TNPO3, SRPRB, RBM8A, ZN706, NCOR2, NPTN, COPG1, CLIC4, MD1L1, BZW2, IF2B2, SCC4, ZHX2, S23IP
Species: Homo sapiens
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Woo CM, Lund PJ, Huang AC, Davis MM, Bertozzi CR, Pitteri SJ. Mapping and Quantification of Over 2000 O-linked Glycopeptides in Activated Human T Cells with Isotope-Targeted Glycoproteomics (Isotag). Molecular & cellular proteomics : MCP 2018 17(4) 29351928
Abstract:
Post-translational modifications (PTMs) on proteins often function to regulate signaling cascades, with the activation of T cells during an adaptive immune response being a classic example. Mounting evidence indicates that the modification of proteins by O-linked N-acetylglucosamine (O-GlcNAc), the only mammalian glycan found on nuclear and cytoplasmic proteins, helps regulate T cell activation. Yet, a mechanistic understanding of how O-GlcNAc functions in T cell activation remains elusive, partly because of the difficulties in mapping and quantifying O-GlcNAc sites. Thus, to advance insight into the role of O-GlcNAc in T cell activation, we performed glycosite mapping studies via direct glycopeptide measurement on resting and activated primary human T cells with a technique termed Isotope Targeted Glycoproteomics. This approach led to the identification of 2219 intact O-linked glycopeptides across 1045 glycoproteins. A significant proportion (>45%) of the identified O-GlcNAc sites lie near or coincide with a known phosphorylation site, supporting the potential for PTM crosstalk. Consistent with other studies, we find that O-GlcNAc sites in T cells lack a strict consensus sequence. To validate our results, we employed gel shift assays based on conjugating mass tags to O-GlcNAc groups. Notably, we observed that the transcription factors c-JUN and JUNB show higher levels of O-GlcNAc glycosylation and higher levels of expression in activated T cells. Overall, our findings provide a quantitative characterization of O-GlcNAc glycoproteins and their corresponding modification sites in primary human T cells, which will facilitate mechanistic studies into the function of O-GlcNAc in T cell activation.
O-GlcNAc proteins:
UBA6, ESYT2, HACL2, DEND3, SBNO1, XIRP2, CNOT1, PINLY, MT21E, SWAHB, P121B, TCAF2, MET15, F177B, P121C, GNAT3, MYO1G, SPT5H, TAF4, PK3CD, DNM1L, P3C2A, BT3A1, PSDE, BIN1, PITM1, DDX3X, RNT2, ARI1A, NCKP5, TRAD1, RHG33, ABLM1, KMT2D, IFIT3, HGS, MYPT1, S27A2, GAK, SC16A, SET1A, KDM6B, ARHGB, FYB1, ATX7, SHIP2, EIF3D, EIF3H, TOX3, NUP42, MEFV, DHX15, ZZEF1, PHF1, ZW10, PRPF3, TPD54, EMC8, SYNJ1, IF4G3, E41L2, WIPF1, LAT, OX1R, PLRG1, ZN207, ST1B1, LANC1, AKAP8, PLIN1, ZN292, AQR, GANP, HBP1, LY75, OGA, DIAP1, MAFK, HCN1, CCD22, BRD4, PP1RB, ABCB7, KI21B, LRP4, N4BP1, CPNE3, OBSL1, BRE1B, CAND2, T22D2, PP6R2, ANR17, H2AY, FLNB, NCOR1, PR40A, LRCH4, MPPB, PSIP1, NDUS3, KS6A5, MYCB2, U520, CCNK, CBPD, CYTF, LTN1, TOX4, PHF14, SUN1, PCF11, FRYL, TRI37, SC31A, CE152, AGFG2, SCAF4, SPN1, RTN3, APOL3, ATE1, CELF2, 6PGL, IPO7, CD2B2, ABCA1, SC24A, SC24B, PCNT, CNOT4, HERC2, HS74L, DDX58, M4K4, AIFM1, TXD12, LDHA, COX1, A1AT, FOS, LDLR, LMNA, ALBU, CYTB, GCR, HG2A, K2C1, G3P, HLAA, CPNS1, RPN1, RPN2, GNAI2, AT1A1, RLA2, JUN, ATPB, CD2, NPM, ANXA2, SYEP, TSP1, SP1, ANXA6, MDR1, HS90B, INHBA, ODPA, PTPRC, RU2B, HCK, VIME, GNAI3, ADA2A, HMGB1, ROA1, LKHA4, DERPC, F231L, GLI2, GRAB, RO60, RARB, HSP7C, EGR2, ODPB, LAMP1, SRF, FA5, IMDH2, TPR, SKI, ACTN1, K1C10, CEAM1, PLSL, GLU2B, HCLS1, PO2F1, RAC2, ATF2, FOSL2, PGCA, LEUK, CREB1, GDC, PECA1, MGMT, ZNF25, JUNB, UBF1, JUND, ATF7, PTN2, DDX5, EGR1, PTPRA, SON, RCC1, ATF1, ML12A, PLCG1, NUCL, NFKB1, LMNB1, CAN3, HNF1A, FLNA, TNAP3, PIMT, UBA1, ROA2, RFX1, CBL, QCR2, MAOM, SP100, NFYA, IF4B, AT2B4, RPB1, BRD2, ATPA, DDX6, PTBP1, ARNT, RFA1, APEX1, PYR1, CALR, MAP4, ERCC5, PTN6, SPB3, PDIA3, 2AAA, HLAF, HMOX2, CLIP1, RPB2, COR1A, ZEP2, HNRH3, HNRH1, STIP1, ELF1, KINH, LSP1, H2B1B, PHB, PTN7, RFC4, MYH9, MYH10, COPB2, ACTN2, SOAT1, ADDA, FUS, NU214, ATP7B, MYH11, GLRX1, PPM1A, K22E, MP2K2, NUP62, GRP75, IF4A3, COIA1, STAT3, MDHM, ECHA, IF2G, PERI, ELK3, LAP2A, LAP2B, STAT1, RHG25, DPP6, HD, MATR3, GPDM, ZAP70, TNR4, VDAC2, MP2K4, NOP2, NOTC1, UTRN, IQGA1, STT3A, NPBW1, COPD, AGRE5, NASP, FAS, EFTU, CENPF, MA2A2, YLPM1, CLK1, NU153, RBP2, TAF6, GUAA, IDH3A, EMD, LRBA, AT1A2, MECP2, HCFC1, CCR3, KS6A3, LUM, ROA3, GDIR2, AGFG1, STAT2, TF2AA, CAZA1, NUP98, FOSB, SUCA, COPA, ITA8, SC24C, ATX1, UBP14, RD23B, EPHB3, AF17, CASP6, DSRAD, PSA, TPIS, SC61B, ACTB, ARF3, HNRPK, RS16, ACTA, GBB1, PPIA, RS27A, AP2B1, 1433Z, IF5A1, RACK1, ACTG, ACTS, TBA1B, TBA4A, PHC1, PRKDC, BTG2, SSBP2, ATL3, TXN4A, FOXK1, RHG04, NFKB2, SPTB2, FOXK2, RUNX1, AMPD2, CAP1, FLI1, OTUD4, PFKAP, SATB1, EWS, MEF2A, SP2, RHAG, SP4, SP3, RL18A, NUCB1, DYST, CREM, KMT2A, TF65, IF4G1, TLE3, TLE4, REL, UBE3A, GABPA, GABP1, CD69, ZO1, TLE5, DHX9, GOGA3, SLFN5, S38AB, RBBP4, NCBP1, AHNK, MN1, FOXO1, TBL3, TF3C1, AKP13, BPTF, NFIA, CHD3, TP53B, ANK3, PP1R8, AKAP6, ROA0, PAK2, TBX2, M3K1, ATM, DC1I2, IKZF1, TCOF, ROCK1, NFAC2, SMAD4, PICAL, PRP4B, SNW1, IQGA2, MTMR1, MTMR3, CUL4A, CUL4B, RUNX3, NFYC, KGP1, CDK13, IL16, CKAP5, CO4A6, VEZF1, MORC3, UBP2L, SCRIB, GIT2, DYHC1, ELOA1, FLNC, CAPR1, CASL, SCN5A, SEM3A, ITPR2, PLSI, LAGE3, PUM1, EPN4, RRP1B, NCOA6, LBR, STAT4, MEF2D, LASP1, NUMA1, GAPD1, SPCS2, SUZ12, ACAP1, R3HD1, SYK, ARHG6, ACAP2, BRD3, PLEC, L2GL1, EPHA7, SF3B3, RYR3, TAF5, MARE2, TSN, SF01, MED1, JHD2C, T22D1, ELF2, NAB2, TAB1, SPEG, USF2, ZFHX3, ZYX, SEPT7, ADRM1, PKN1, DDB1, TAF9, OBF1, NRF1, PTPRO, ZN827, EX3L4, HNRL2, AAK1, CCD57, QRIC1, PRTG, CEA16, TM249, FR1L6, LRRF1, EMAL3, UAP1L, GON4L, LARP7, EPC2, CRTC2, PAR10, TYW2, RHG15, H90B3, BCORL, ZN831, TGO1, DOC11, PRC2B, TOIP1, CEP78, CD158, TDIF2, KMCP1, ZN362, FKB15, ZEP3, ODAD2, MPP7, LRIF1, UBR4, UBAP2, GNTK, RBM26, CE350, RPRD2, AGAP9, MYOME, TASO2, RN213, GL8D1, PDPK2, BICRL, OTU7B, RGPA1, TWF2, SDE2, NIPBL, LIN54, ZN544, PPR18, ZCHC8, CDC73, ARMX5, SCYL2, NFRKB, LMOD2, LEG1H, TMM81, PDXD1, RSBNL, MDEAS, ZC3HE, LARP1, SCND3, POTEE, ZN322, ANR11, SPIT4, AFTIN, FIP1, CRTC3, MCAF1, PACS1, BCOR, DJC14, DG2L6, LR74B, OTOG, RHG36, YJ005, RHG27, TMTC3, UN13D, HAKAI, NOL8, HECW1, SPT6H, SND1, KDM3B, S26A9, DYM, PRS41, APTX, ZCCHV, SETX, NUP54, GLUCM, POGZ, MYH14, NUFP2, MAVS, HDGR2, EMSY, I2BP2, AB12B, DHB13, CMKMT, SRGP1, RBBP6, RHG30, NRARP, TCPR1, HUWE1, YTHD3, CENPV, ATL2, YRDC, GPAT4, ZFHX4, ABCAD, BCL9L, KIF27, LRRT3, IQGA3, VS10L, CEP57, FRAS1, CACL1, P66A, I2BP1, CRLF3, CRERF, DYH10, GID4, ARI3B, WDR75, MGAP, ANKH1, SUGP1, SUGP2, CCAR1, BAP18, PLPL6, CMIP, TIGD4, YAF2, IHO1, SRRM1, FANCM, CC116, A16A1, DCP1B, PELP1, WDFY3, ABCA7, LGI4, NUP93, LRC47, ABD12, FNBP4, GALT4, RN175, CARME, AF1L2, TAB3, CPSF7, EFNMT, MAGB6, LRTM2, KRI1, TTC29, POC5, LR75A, S43A3, SUMF2, NETO2, NF2IP, LS14A, MISSL, CA131, TNR6A, PHC3, SRFB1, SP20H, VP37A, PCAT1, DOCK8, SYNE1, ARI1B, ENASE, TET1, MYRIP, OR6K3, CFA61, THOC2, WDR36, GABP2, MARH1, ALMS1, PREX1, PKHO2, DYH3, DSCL1, DTX3L, NETO1, NEK7, MICA1, ATS18, RN128, SNX29, SMCR8, ZN384, HASP, SMAP2, SCFD1, LMO7, ATX2L, PHIP, RUFY2, CSKI1, MADD, AGRV1, SYNE2, MUC16, P66B, AUTS2, BBX, TITIN, CTTB2, GBF1, SMG7, SNX19, PHF3, HS105, ZN592, HMHA1, TFG, TAF4B, CBP, KAT6A, SYMPK, SHIP1, DDX17, TANK, RAD50, CELF1, SMRD2, RAB8B, FUBP2, DVL3, LPP, TATD2, AT2A3, MRTFA, PLPL2, SH3K1, PF21A, DOC10, INT12, ACSF2, GCP3, SLAF6, RSPRY, MTEF3, SIR1, THA11, GLT14, CERS2, SYMM, PDLI5, FUBP3, PP16A, COG3, VCIP1, CHAP1, PDLI2, ANCHR, UBP47, Z512B, ZFR, EP400, CNO6L, CA074, PRRC1, ZN512, CNT3B, LRRC7, ARAP1, AGRA2, INP4A, RBM14, NED4L, LENG8, TRNT1, MCCA, PCX1, CCNL2, SIN3A, SEBP2, MINT, HTF4, CDC5L, EYA3, LGMN, MNT, SCAFB, TTC1, OSMR, ATX2, METH, ACON, CPNE1, TBA1C, MBB1A, GPTC1, ERP44, ESYT1, CCM2, FUZZY, DIDO1, MCMBP, CABL2, NDC1, PAXX, HNRL1, NUP58, RIOK2, THIC, RBM4, NADAP, SSBP3, NAA15, AP1M1, M10L1, YTHD1, BACH2, PANK2, PC11Y, ASPC1, UCK2, TRI31, UNK, FTO, AMRA1, CE295, DRC3, SP130, BRD8, CSTFT, ZCPW1, LMA2L, CK054, SLIK2, CSRN2, I2BPL, VPS16, EPC1, ADNP, IPYR2, FOXP1, PTN23, WNK1, AMPB, E41L1, GSX1, ELOV6, CH033, VISTA, SFR19, GORS2, LN28A, MLXIP, GBB4, PKHA1, RISC, TAF9B, MRM3, ZBT20, NCOA5, TANC2, TNR6C, CHD8, AT131, VTA1, SYSM, UBN1, DCP1A, KI13B, PRD10, XPP1, PDLI7, DDX21, MBNL1, SIR7, TULP4, ABCBA, LATS2, UBQL4, THSD1, CENPM, PDS5B, RBM12, MED9, SLTM, MIC19, NSMA3, THUM1, CARF, SNTG2, MTMR4, TE2IP, TAB2, CDK12, GGA3, ITSN2, BICRA, CNOT2, TMOD2, THYN1, PDP1, VAPA, TEN3, CHD7, DYH1, SYLC, KLH42, KANL3, RERE, HDC, TRPC4, MALT1, ADDG, TCF20, NDRG3, SUN2, NDOR1, UBQL2, S30BP, RPGFL, AGRE2, NRBP, BAZ2A, HOOK1, CMC2, TASOR, AKA11, GMEB2, PARP4, C8AP2, IKZF2, ACINU, CNO11, AT7L1, K1210, YETS2, HECD1, NOTC3, PRP19, UBQL1, FAF1, PPIE, DIM1, MACF1, SCAF8, SET1B, JIP3, ZC3H4, SRRM2, CLCA2, SMC3, ZN148, MTMR6, ENTP4, SAC2, MAST1, FYV1, WDR37, TR150, ZN281, FBX7, 3BP1, OR2W1, NOC2L, SAMH1, DMXL1, ARIP4, MTCL1, RIPR2, PKHM1, RPGF2, CRBG1, PRC2C, YTHD2, SP16H, ANGL3, PCDG3, SNX13, NUBP2, NCOR2, COPG1, GMEB1, DC1L1, ROBO1, NCOA3, M3K4, PCLO, CAN7, SCC4, ZHX2, S23IP
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.