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Wong YK, Wang J, Lim TK, Lin Q, Yap CT, Shen HM. O-GlcNAcylation promotes fatty acid synthase activity under nutritional stress as a pro-survival mechanism in cancer cells. Proteomics 2022 35083852
Abstract:
Protein O-GlcNAcylation is a specific form of protein glycosylation that targets a wide range of proteins with important functions. O-GlcNAcylation is known to be deregulated in cancer and has been linked to multiple aspects of cancer pathology. Despite its ubiquity and importance, the current understanding of the role of O-GlcNAcylation in the stress response remains limited. In this study, we performed a quantitative chemical proteomics-based open study of the O-GlcNAcome in HeLa cells, and identified 163 differentially-glycosylated proteins under starvation, involving multiple metabolic pathways. Among them, fatty acid metabolism was found to be targeted and subsequent analysis confirmed that fatty acid synthase (FASN) is O-GlcNAcylated. O-GlcNAcylation led to enhanced de novo fatty acid synthesis activity, and fatty acids contributed to the cytoprotective effects of O-GlcNAcylation under starvation. Moreover, dual inhibition of O-GlcNAcylation and FASN displayed a strong synergistic effect in vitro in inducing cell death in cancer cells. Together, the results from this study provide novel insights into the role of O-GlcNAcylation in the nutritional stress response and suggest the potential of combining inhibition of O-GlcNAcylation and fatty acid synthesis in cancer therapy. This article is protected by copyright. All rights reserved.
O-GlcNAc proteins:
RUXGL, ADAS, DX39A, MYO1C, IPO5, PESC, NOP56, DDX3X, SCD, MGST3, HNRDL, XPO1, SURF4, OGT1, PPM1G, MOT4, DHX15, CYB5B, SERA, HNRPR, BUB3, ACTN4, MYO1B, GANP, HNRPQ, NDUS7, MPU1, H2AY, FLNB, SC22B, SF3B1, U520, UTP20, NU155, ATP5H, RL1D1, MTA2, RTN3, VAPB, IPO7, ACSL3, BAG2, TOM40, LDHA, DHE3, AATM, PGK1, ASSY, LMNA, TFR1, ALDOA, K2C1, G3P, HSPB1, RPN1, AT1A1, ADT2, PCCA, RLA1, RLA0, LA, K1C18, K2C8, ATPB, ENOA, NPM, TPM3, LDHB, PDIA1, ANXA2, TBB5, TRY1, PROF1, SYEP, HS90A, HNRPC, DAF, 4F2, HS90B, ODPA, RU17, VIME, RS17, K2C7, GNAI3, RSSA, LEG1, ROA1, PARP1, PRS56, HS71B, ODP2, THIO, MGST1, CH60, BIP, HSP7C, GTR1, TOP2A, PYC, PABP1, PCNA, ADT3, IMDH2, KCRU, XRCC6, XRCC5, EF2, K1C10, K2C5, PDIA4, PLST, ETFA, MIF, KPYM, ENPL, HNRPL, PLAK, EZRI, NDKA, RS2, DESP, H13, NCPR, AT2A2, DDX5, TCPA, PTN1, ARF4, RL7, RL17, NUCL, GSTM3, FLNA, FBRL, PUR6, UBA1, ROA2, QCR2, SFPQ, PPIB, RS3, SAHH, COF1, MCM3, RS12, ATPA, U2AF2, RL13, S10A4, PTBP1, SYVC, EF1G, STOM, RL10, APEX1, PYR1, CALX, TKT, ERP29, PRDX6, PRDX5, PRDX3, RL12, PDIA3, CPSM, HNRH1, STIP1, L1CAM, PRDX2, P5CR1, DUT, MCM7, GLYM, HSP74, PHB1, RL22, MYH9, SOAT1, DEK, K22E, RL4, LONM, NUP62, GRP75, IF4A3, RL3, RL13A, ARL1, STAT3, MDHM, RFC3, ECHA, SYIC, LAP2A, LPPRC, MATR3, MSH2, GPDM, VDAC2, KI67, BAG6, RL27A, RL5, RS9, STT3A, CAPZB, SYQ, RL29, AT5G3, TCPE, RL34, FAS, TCPG, EFTU, ACADV, TMEDA, NU153, RBP2, CPT1A, SERPH, RL14, TCPQ, TCPD, FXR1, RAB5C, RAB7A, HCFC1, ROA3, 6PGD, HNRPM, IMA1, HNRPF, MSH6, TXTP, ACLY, COPA, MOT1, SYRC, KAD2, P5CS, XPO2, TERA, NP1L1, DSRAD, ATPK, TMM33, TPIS, MYL6, IF4A1, RS20, S10AA, RAP1B, RL15, RL37A, HNRPK, RS8, RS16, 1433E, RS14, RS23, RS11, RUXE, RL7A, RS4X, RS6, H4, RAB1A, RAN, RL23, RS25, RS26, RL10A, RL11, RL8, PPIA, RS27A, RSMN, RACK1, ACTG, UBC9, TBA1B, TBB4B, GTF2I, TCPB, PRKDC, RL24, ARF5, RL19, SRSF3, MPCP, CLH1, HNRPU, SPTB2, EXOSX, RL18A, RL6, IF4G1, K1C17, PRDX1, RL18, C1QBP, KHDR1, DHX9, NCBP1, AHNK, NU160, SF3A3, ILF3, ACACA, PRDX4, CBX3, TIF1B, SPTN1, HNRPD, SAFB2, TTL12, CAPR1, ITPR1, RRP1B, GANAB, LBR, GOGB1, IMB1, NUMA1, SUZ12, U5S1, RRS1, PDIA6, PLEC, TEBP, NONO, PCBP1, PCBP2, DHC24, SF3B3, SF3A1, TRAM1, ELAV1, AAAT, RBBP7, H31T, PDS5A, TSR1, IF2GL, RRP12, NU188, HP1B3, EF1A3, PPR18, PRP8, C1TM, DHX30, CAND1, MISP, SPB1, PELP1, RDH10, CCAR2, TXND5, STT3B, BRX1, PO210, GEMI5, RT27, HS105, GCN1, NU205, AKAP1, AN32B, RBP56, DDX17, FUBP2, TNPO1, UBP7, UTP4, LRC59, PGAM5, FUBP3, MBOA7, MCCA, WRIP1, UHRF1, POP1, HCD2, ROAA, TM9S2, TCPH, ANM1, H2B1L, RNZ2, MEP50, MBB1A, ESYT1, H2AJ, GNL3, HDHD5, GTPB4, API5, RPF2, SFXN1, RDH14, ABCB6, DDX21, MDN1, DCA13, ATD3A, DDX18, MIC19, TEX10, TECR, MYOF, THYN1, HACD3, RRBP1, ABC3B, RLP24, ACINU, OGDHL, COR1C, PRP19, SSRG, TRI33, EIF3L, RUVB1, VDAC3, PDIP2, NOP58, SF3B6, RTCB, RL36, LAS1L, SRPRB, COPG1, MTCH2, CEPT1, ZNT1
Species: Homo sapiens
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Ramirez DH, Yang B, D'Souza AK, Shen D, Woo CM. Truncation of the TPR domain of OGT alters substrate and glycosite selection. Analytical and bioanalytical chemistry 2021 34725712
Abstract:
O-GlcNAc transferase (OGT) is an essential enzyme that installs O-linked N-acetylglucosamine (O-GlcNAc) to thousands of protein substrates. OGT and its isoforms select from these substrates through the tetratricopeptide repeat (TPR) domain, yet the impact of truncations to the TPR domain on substrate and glycosite selection is unresolved. Here, we report the effects of iterative truncations to the TPR domain of OGT on substrate and glycosite selection with the model protein GFP-JunB and the surrounding O-GlcNAc proteome in U2OS cells. Iterative truncation of the TPR domain of OGT maintains glycosyltransferase activity but alters subcellular localization of OGT in cells. The glycoproteome and glycosites modified by four OGT TPR isoforms were examined on the whole proteome and a single target protein, GFP-JunB. We found the greatest changes in O-GlcNAc on proteins associated with mRNA splicing processes and that the first four TPRs of the canonical nucleocytoplasmic OGT had the broadest substrate scope. Subsequent glycosite analysis revealed that alteration to the last four TPRs corresponded to the greatest shift in the resulting O-GlcNAc consensus sequence. This dataset provides a foundation to analyze how perturbations to the TPR domain and expression of OGT isoforms affect the glycosylation of substrates, which will be critical for future efforts in protein engineering of OGT, the biology of OGT isoforms, and diseases associated with the TPR domain of OGT.
O-GlcNAc proteins:
UBA6, SBNO1, CNOT1, SMHD1, ADAS, DX39A, BACH, MYO1C, PSD11, PSD12, TAF4, CLIC1, EIF3F, IPO5, DNM1L, AGRIN, PLOD2, IMA4, NOP56, DDX3X, PDXK, ANM5, TCRG1, PSA7, SCAM3, HAT1, HGS, MYPT1, HNRDL, XPO1, PUR4, NPC1, SCAM2, ARC1B, ARPC2, ARPC3, RPAC1, GNPAT, ZN185, SURF4, OGT1, PPM1G, EIF3D, EIF3H, P4HA2, MAGB2, NUP42, DHX15, MCES, SERA, PSMD3, PAPS1, ZW10, HNRPR, TXNL1, E41L2, XPOT, TIM44, PLRG1, ZN207, GET3, BUB3, ACTN4, AP1G1, SNG1, SNG2, SYNC, MYO1B, NRDC, CALU, EDIL3, SAHH2, DHX16, SMCA5, KDM1A, OGA, HNRPQ, PLOD3, DIAP1, TSN3, PLIN3, CTND1, MAGC1, USO1, DKC1, IF2P, DNJA2, WDR1, CPNE3, T22D2, ANR17, H2AY, FLNB, CISY, SC22B, PR40A, ERLN1, SF3B1, CSDE1, U520, NU155, EIF3G, FACE1, IDHC, ATRN, STAM2, PRAF3, FLOT1, RL1D1, WFS1, MTA2, TOX4, SC24D, SUN1, FRYL, NFAT5, GLSK, SC31A, UBR5, LC7L3, FKBP9, SMC2, IPO7, AHSA1, SGPL1, SC24B, ACSL3, CDS2, AP2A1, BAG2, AIFM1, FADS2, CLPT1, TOM40, ACL6A, LDHA, COX2, PNPH, AATM, PGK1, ASSY, FOS, LMNA, FINC, TFR1, NU4M, NU5M, GLCM, FUCO, ALDOA, ANXA1, OAT, TBB4A, G3P, CPNS1, HSPB1, TYSY, RPN1, RPN2, GNAI2, AT1A1, AT1B1, ALDH2, ADT2, PCCA, IF2A, RLA2, RLA0, LA, ITB1, ATPB, IF4E, ENOA, G6PI, NPM, ITAV, HYEP, LDHB, PDIA1, H10, CATD, ANXA2, CAN1, TBB5, TRY1, SAP, PROF1, SYEP, CATB, HS90A, HNRPC, LAMB1, YES, FUMH, TSP1, ANXA6, MDR1, 4F2, PFKAM, HS90B, SRPRA, ASNS, ODPA, RU17, GNAI3, ANXA5, RSSA, CD63, SNRPA, GSTP1, LEG1, CN37, DLDH, ROA1, PARP1, LKHA4, ALDOC, GPHRB, HS71B, LYAG, RRAS, H14, ODP2, THIO, ESTD, CH60, CLUS, BIP, LAMC1, PPAL, HSP7C, GTR1, GTR3, ODPB, PYGB, LAMP1, TOP1, TOP2A, G6PD, PYC, C1TC, MPRI, ADHX, PABP1, PCNA, HARS1, IMDH2, TPR, KCRB, ACTN1, PEPD, XRCC6, XRCC5, LAMP2, RINI, EF2, PDIA4, P4HA1, TCTP, ACPH, ETFA, GLU2B, FPPS, KPYM, ENPL, IDE, HNRPL, SYDC, ALDR, B4GT1, EZRI, FOSL2, NDKA, GNS, RS2, RFA2, BGAL, H12, AT2A2, FAAA, AATC, JUNB, ITA2, CAN2, PYRG1, DDX5, PFKAL, PRS6A, TCPA, RL35A, ARF4, RL7, VINC, SON, RL17, PGAM1, DNLI1, PLCG1, NUCL, HXK1, RPB3, E2AK2, TFE3, SPEE, IF2B, ANXA7, PSB1, MPRD, LMNB1, VATB2, FLNA, VDAC1, TGM2, FBRL, PUR2, PUR6, UBA1, NDKB, ADRO, ROA2, RFX1, QCR2, SFPQ, PPIB, SYWC, RS3, SAHH, COF1, IF4B, PPAC, RPB1, MCM3, RS12, DNJB1, ATPA, PSA1, PSA2, PSA3, PSA4, ITA3, MOES, DDX6, DNMT1, U2AF2, RL13, PTBP1, SYTC, SYVC, EF1G, STOM, RL10, RFA1, APEX1, CD82, PYR1, CALR, MAP4, CALX, PSB8, PSA5, PSB4, PSB6, PSB5, DPOD1, TEAD1, MK01, AMPL, IMPA1, EPHA2, TKT, EF1D, PRDX6, BLVRB, PRDX5, PRDX3, RL12, ECHM, PEBP1, PDIA3, 2AAA, CDC27, PURA2, AMRP, PUR8, CLIP1, SL7A1, RPB2, SDHA, GDIA, METK2, DNJA1, QCR1, PUR9, HNRH3, HNRH1, 1433B, 1433S, STIP1, PRDX2, RL9, KINH, MRP1, MCM4, MCM5, MCM7, GLYM, HSP74, CTNA1, CTNB1, PHB1, RADI, RFC4, RL22, FBN2, GDE, MYH9, MYH10, COPB2, SOAT1, ADDA, BASI, FUS, NU214, PRS7, MP2K2, ATPG, HEM6, RL4, PGM1, ODO2, SRP14, NUP62, FDFT, TAGL2, TALDO, RBMX, VKGC, VATA, GRP75, IF4A3, RS19, RL3, OST48, AN32A, CAP2, TCPZ, RL13A, STAT3, MDHC, MDHM, ECHA, IF2G, KDM5C, GARS, SYIC, STAT1, CASP3, LPPRC, SATT, LIS1, MUC18, MATR3, MSH2, MAGA4, RANG, NAMPT, PRS6B, PPIC, VDAC2, UBP5, KI67, RAGP1, RECQ1, NOP2, CRKL, BAG6, NSF, RL27A, RL5, RL21, RL28, RS9, RS5, MAP1B, UTRN, IQGA1, STT3A, CAPZB, SYQ, UCRI, PPCE, COPD, LSS, CD151, PSMD8, TCPE, NEST, HSP13, PIPNB, PAXI, AL9A1, RL34, LMAN1, NASP, FAS, TCPG, EFTU, AL7A1, SYAC, SYCC, SYSC, PRI2, PSB3, PSB2, MCM2, ACADV, YLPM1, TMEDA, RBM25, NU153, RBP2, NDUV1, GSK3A, TAF6, GUAA, GDIB, EMD, SERPH, VASP, TCPQ, TCPD, ANX11, PAPOA, FXR1, RAB5C, RAB7A, RT29, SMCA4, BAP31, HCFC1, DHB4, HDGF, ROA3, 6PGD, HNRPM, IMA1, GDIR1, AGFG1, HNRPF, MSH6, RBM5, SPSY, CAZA1, NUP98, BIEA, PPP5, ACLY, MAP11, COPB, COPA, SC5A3, SMTN, MOT1, IST1, SC24C, SYRC, SYYC, UBP14, BCAT1, AT1B3, RD23B, P5CS, IF5, S12A2, XPO2, TERA, ECHB, AFAD, NP1L1, DSRAD, SEC13, PSA, HNRH2, EIF3B, ATPK, SYMC, IF6, CTBP2, TMM33, GEMI4, CORO7, NU107, ARPC4, TPIS, EIF3E, PP4C, IF4A1, PSA6, CDC42, DEST, GMFB, SRP54, RAB10, UBC12, ARP3, ARP2, ABCE1, RAP2B, RS3A, RL15, RL37A, S61A1, DAD1, NPC2, HNRPK, 1433G, RS7, PRS4, PRS8, RS8, RS15A, RS16, 1433E, RS14, RS23, RS13, RS11, SMD2, SMD3, PRS10, RL7A, ERF1, CNBP, RS4X, PP2AB, RS6, H4, RAB1A, RAN, RL23, RS25, RS28, RL30, RL31, RL10A, RL32, RL11, RL8, PPIA, TRA2B, AP2B1, 1433Z, IF5A1, RACK1, YBOX1, UB2L3, TBA4A, IF4G2, GTF2I, TCPB, PRKDC, RL24, SRSF3, FOXK1, DAB2, RBM10, MPCP, VIGLN, CLH1, FKBP3, HNRPU, U2AF1, SPTB2, AMPD2, CAP1, HMCS1, LAT1, EXOSX, PFKAP, EWS, PLCB3, ODO1, RL18A, MP2K1, FKBP4, PLOD1, RL6, TOP2B, DYST, KMT2A, LMNB2, CEBPZ, TF65, IF4G1, LGUL, 1433F, DYN1, EF1A2, PTN11, PUR1, GFPT1, EXOS9, SUH, GABPA, PRDX1, RL18, C1QBP, CKAP4, KHDR1, LRP1, SRSF1, PP2BA, DHX9, GOGA2, LG3BP, CD47, SSRP1, NSUN2, NCBP1, EP300, AHNK, MGAT2, GALT2, AP1B1, MPPA, SCRN1, NU160, SCAP, TBL3, TF3C1, ASPH, TROP, BPTF, AIMP1, ILF2, ILF3, LMAN2, TRAP1, FLII, ACACA, CSN1, MTAP, EI2BE, TADBP, ROA0, PRDX4, PSMD2, SRSF6, TIF1B, DHRS2, G3BP1, MTA1, EIF3I, UB2V1, DC1I2, NNTM, TCOF, TMED1, VPP3, PICAL, RIPK1, CUL2, CUL4A, CUL4B, FCL, MOGS, CD166, SPTN1, DX39B, BLMH, CKAP5, COTL1, HNRPD, SCRB2, EIF3A, UBP2L, SCRIB, FHL2, DPYL3, DCTN1, DYHC1, SRC8, FLOT2, TRI25, FLNC, GALE, CAPR1, RBM39, MCM6, TRIPC, PUM1, EPN4, RRP5, BMS1, UBP10, GANAB, LBR, MVP, MEF2D, CHD4, LASP1, GPNMB, ZN638, IMB1, NUMA1, CND2, SPCS2, PSMD6, ABRX2, SEPT2, CND1, U5S1, SYK, WDR43, PDIA6, PA1B3, IPYR, TEBP, NONO, PWP2, RCN1, L2GL1, TMED2, PCBP1, PCBP2, SF3B3, SC23A, SF3A1, TSN, SF01, PCH2, MED1, MARE1, ELAV1, NSDHL, TAB1, AAAT, MLF2, VAS1, ZYX, SEPT7, ADRM1, CCDC6, UAP1, PSMD5, PKN2, DDB1, DPYL2, KCC4, SRSF7, DREB, NRF1, FSCN1, IF16, PCKGM, UPP1, UGPA, TRXR1, HNRL2, INF2, PDS5A, QSER1, QRIC1, SMU1, P3H1, K2C71, TIM50, GXLT1, TB10B, SNUT2, HERC4, P4R3B, EOGT, CE170, UBR4, SKT, UBAP2, RBM26, RPRD2, ZMYM4, TWF2, LIN54, RHBD2, MBOA5, EDC4, PRP8, SCYL2, NFRKB, TTC37, NCEH1, BRAT1, ZC3HE, C1TM, SBSN, CREL2, PACS1, GGYF2, SRCAP, TMTC3, IKIP, SPT6H, SND1, DDX46, BZW1, DHX30, EIF3M, CYFP1, KDM3B, RIR2B, ERMP1, ZCCHV, NUP54, POGZ, MYH14, CLAP1, EMSY, SH3R1, HUWE1, TMED4, SYVN1, ZN598, PHLB1, LUZP1, CAND1, TSYL5, EZHIP, CARM1, DDX42, P66A, C2CD5, I2BP1, RB6I2, PLD3, P3H3, WDR75, Z3H7A, ANKH1, CCAR1, DJC10, SMAP1, SPART, RPTOR, NUP93, ZN687, SYNPO, FNBP4, CPSF7, EMC1, ENAH, S43A3, GT251, SUMF2, PGLT1, TXND5, PAIRB, LS14A, ABCF1, FBX22, NEUA, CADM4, ATLA2, PLBL2, WDR36, SMRC2, NPL4, S35B2, UBA3, TBC15, CIP2A, STT3B, PNPT1, HM13, DTX3L, PO210, GEMI5, PARD3, IPO4, SCRB1, RAB2B, NU133, PDC6I, S20A1, ATX2L, PALLD, DDX1, GBF1, NICA, TM9S4, HS105, GCN1, PRP16, NU205, PXDN, TFG, ARC1A, HDAC2, CBP, SYMPK, DDX17, CELF1, UFD1, GSLG1, RENT1, TNPO1, GLMN, USP9X, UBP7, SCAM4, SMCE1, MYDGF, PIGT, NCLN, EXOC4, FERM2, FUBP1, FKB10, APH1A, PPWD1, AP2M1, RCN3, ELP4, SEH1, DNJA3, CYFP2, PGM2, PDLI5, ERO1A, FUBP3, ITCH, TMX3, CNDP2, ZFR, EP400, SNX27, PRRC1, ZN512, MBOA7, DOCK7, ARAP1, IPO9, RBM14, NED4L, VPS35, CIC, MED15, EFGM, MCCA, MYADM, SIN3A, MINT, PNKP, NIBA2, TBCB, PSB7, CNN2, PSMD1, EYA3, SORT, NUP88, P2RX4, DNJC7, PHB2, KIF2C, ATX2, HCD2, ROAA, NP1L4, ACON, TM9S2, S29A1, TS101, TCPH, ANM1, CND3, MBB1A, COR1B, GCP2, ESYT1, DIDO1, MCMBP, TBCD, TBB6, RPC3, HNRL1, HTAI2, TMED9, NUP58, SFXN3, OSB10, NAA15, S12A9, SRRT, EIF2A, WDR11, FTO, TTYH3, WDR12, YIPF3, PITH1, PAIP1, NAT10, XRN2, TOLIP, SP130, BRD8, DDX47, MAGT1, RAB1B, LMA2L, I2BPL, GHITM, TMX1, PTN23, OFUT1, PIGU, WNK1, AMPB, HEAT1, NOL6, WDR26, AT133, ACAD9, GORS2, MARH7, SFXN1, PKHA5, XPO5, MYG1, EMAL4, TM9S3, APMAP, ARFG3, RTN4, ANLN, XPP1, PDLI7, DDX21, EI2BG, MBNL1, HEBP1, SERC1, F1142, SYFB, SYIM, SMC4, OLA1, RBM12, T106B, CYRIB, DCA13, CC50A, SEP11, FANCI, ARL8B, DDX18, TM160, MIO, CARF, DPP3, RM39, UGGG1, ERAP1, HPBP1, MAT2B, MYOF, ITSN2, HACD3, SEP10, RCC2, DIP2B, CT2NL, RRBP1, SYLC, RBM27, SUCB1, MRC2, DHCR7, DJB11, SAE2, NXF1, SUN2, SRP68, SEPT9, UBQL2, PCYOX, CEIP2, S30BP, MED13, PUF60, NRBP, VATH, IPO11, XPO7, BAZ2A, GGT7, NAGK, DBNL, CDC23, STML2, PARP4, NUDT5, TF3C4, ACINU, RAB21, DNPEP, ASAP1, MRTFB, MED23, HECD1, PKCB1, COR1C, MYO6, PRP19, MAGD2, PSD13, MINP1, NOVA2, WDR3, S12A4, MACF1, SCAF8, TRI33, SMAG1, XCT, SRRM2, PA2G4, SMC3, RUVB2, EIF3L, PLAP, RUVB1, VDAC3, ERGI3, SC5A6, DRG1, NCKP1, AT11B, E41L3, CHSP1, NOP58, ZN281, SYYM, TMX2, SHLB1, SBDS, TMED7, STRAP, RTCB, FBX7, RBGP1, NOC2L, TLN1, DCAF1, HYOU1, PRC2C, YTHD2, SP16H, TNPO3, MAGD1, SNX8, TRUA, NPTN, S12A7, COPG1, BIG1, BZW2, DC1L1, IF2B2, ZNT1, S4A7, 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, PHB1, 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,