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He J, Fan Z, Tian Y, Yang W, Zhou Y, Zhu Q, Zhang W, Qin W, Yi W. Spatiotemporal Activation of Protein O-GlcNAcylation in Living Cells. Journal of the American Chemical Society 2022 144(10) 35138101
Abstract:
O-linked N-acetylglucosamine (O-GlcNAc) is a prevalent protein modification that plays fundamental roles in both cell physiology and pathology. O-GlcNAc is catalyzed solely by O-GlcNAc transferase (OGT). The study of protein O-GlcNAc function is limited by the lack of tools to control OGT activity with spatiotemporal resolution in cells. Here, we report light control of OGT activity in cells by replacing a catalytically essential lysine residue with a genetically encoded photocaged lysine. This enables the expression of a transiently inactivated form of OGT, which can be rapidly reactivated by photo-decaging. We demonstrate the activation of OGT activity by monitoring the time-dependent increase of cellular O-GlcNAc and profile glycoproteins using mass-spectrometry-based quantitative proteomics. We further apply this activation strategy to control the morphological contraction of fibroblasts. Furthermore, we achieved spatial activation of OGT activity predominantly in the cytosol. Thus, our approach provides a valuable chemical tool to control cellular O-GlcNAc with much needed spatiotemporal precision, which aids in a better understanding of O-GlcNAc function.
O-GlcNAc proteins:
SBNO1, CNOT1, BACH, PSD11, PSD12, TAF4, CLIC1, EIF3F, IPO5, IF2B3, ARI1A, KMT2D, ANM5, PSA7, HAT1, HGS, MYPT1, XPO1, SC16A, SR140, SET1A, PUR4, NPC1, OGT1, HMGB3, PPM1G, EIF3D, EIF3H, P4HA2, SERA, PSMD3, PAPS1, MSI1H, IF4G3, E41L2, FOXO3, ZN207, BUB3, ACTN4, SYNC, SAHH2, KPRB, GANP, PEPL, OGA, PLOD3, IMA7, IF2P, DNJA2, MITF, CPNE3, CLU, PP6R2, CREST, ANR17, NCOR1, VP26A, CLN5, CSDE1, IDHC, SRP72, MTA2, TOX4, SC24D, PCF11, NFAT5, SC31A, AGFG2, SCAF4, SMC2, IPO7, PSMG1, SC24A, SC24B, EYA4, HS74L, TOM40, LDHA, PNPH, HPRT, PGK1, CAH2, ALDOA, ANXA1, G3P, IF2A, RLA1, RLA2, RLA0, JUN, LA, AGAL, KCRM, ENOA, PYGL, G6PI, LDHB, H10, ANXA2, TBB5, PROF1, APT, SYEP, HS90A, LAMB1, SP1, ANXA6, DAF, PFKAM, HS90B, ASNS, RS17, ANXA5, RSSA, GSTP1, HMGB1, PARP1, LKHA4, ALDOC, ATX1L, HS71B, RO60, PTPRF, THIO, HSP7C, EPB41, UMPS, G6PD, C1TC, ADHX, SRF, PRPS2, PABP1, PCNA, IMDH2, KCRB, PEPD, XRCC6, XRCC5, RINI, EF2, P4HA1, PLST, ACPH, GYS1, KPYM, PO2F1, SYDC, PLAK, ERF3A, NDKA, RS2, CBR1, CREB1, HSP76, PYRG1, DDX5, PFKAL, TCPA, RL35A, ARF4, RL7, RL17, PGAM1, DNLI1, NUCL, SPEE, CSK22, PSB1, FLNA, PIMT, PUR2, PUR6, UBA1, NDKB, RFX1, CBL, RS3, NFYA, SAHH, COF1, EF1B, MCM3, RS12, BRD2, PSA1, PSA2, PSA3, PSA4, MOES, DDX6, DNMT1, PAX6, U2AF2, RL13, SYTC, SYVC, EF1G, 1433T, ARNT, RL10, RFA1, APEX1, PYR1, MAP4, PSB6, PSB5, AMPL, TKT, RBMS1, EF1D, PRDX6, RL12, PEBP1, 2AAA, CDC27, NMT1, PURA2, PUR8, METK2, DNJA1, PUR9, 1433B, STIP1, PRDX2, ELF1, CGL, RL9, KINH, MCM4, MCM5, MCM7, HSP74, RL22, CBS, MYH9, MYH10, COPB2, FUS, DEK, PRS7, RL4, SRP14, TALDO, RS19, RL3, TCPZ, RL13A, MDHC, IF2G, CSK, GARS, SYIC, RS27, RANG, BAG6, NSF, RL27A, RL5, RL21, RL28, RS9, RS10, SYQ, RL29, ATPO, PPCE, COPD, TCPE, PIPNB, AL9A1, NASP, FAS, TCPG, SYAC, SYSC, PSB3, MCM2, YLPM1, RBM25, HINT1, GSK3A, GUAA, DNLI3, GDIB, SERPH, F10A1, RL14, TCPQ, TCPD, ANX11, PAPOA, SMCA4, HCFC1, SSDH, 6PGD, IMA1, AGFG1, HNRPF, THOP1, PPP5, ACLY, COPB, COPA, SC24C, SYRC, ATN1, SYYC, RD23B, ANAG, XPO2, TERA, NP1L1, PSA, EIF3B, ATPK, SYMC, TPIS, EIF3E, IF4A1, RS20, PRPS1, PSA6, CDC42, UBC12, UBE2N, ARP3, ARP2, ACTZ, CSN2, ABCE1, RS3A, RL26, RL15, RL27, 1433G, RS7, PRS8, RS8, RS15A, RS16, 1433E, RS23, RS18, RS13, RS11, RUXE, PRS10, RL7A, ERF1, RS4X, RL23A, RS6, RAN, RL23, UB2D2, RS24, RS25, RS26, RL30, RL10A, RL32, RL11, RL8, PPIA, RS27A, RAC1, AP2B1, 1433Z, RSMN, SUMO1, RL38, IF5A1, RACK1, YBOX1, EF1A1, TBA1B, CSK21, F193A, IF4G2, PHC1, TCPB, GSTO1, RL24, RL36A, ARF1, RL19, FOXK1, RBM10, CYC, CLH1, SPTB2, SET, FOXK2, CAP1, OTUD4, EWS, SP3, RL18A, FKBP4, RL6, KMT2A, IF4G1, TLE3, TLE4, 1433F, SRS11, EF1A2, GFPT1, EXOS9, SUH, GABPA, PRDX1, RL18, SRSF1, SSRP1, RBBP4, EP300, AP1B1, SFSWA, FOXC1, ACACA, CSN1, AIMP2, PSMD2, G3BP1, PABP4, EIF3I, SF3B2, PICAL, ULA1, CUL4B, FHL1, NACA, SPTN1, NFYC, CKAP5, EIF3A, UBP2L, TTL12, DYHC1, RCN2, CAPR1, RBM39, PUM1, EPN4, NCOA6, GSE1, MEF2D, ZN638, IMB1, NOLC1, NUMA1, PSMD6, SEPT2, R3HD1, BRD3, PA1B3, IPYR, TEBP, RCN1, PCBP1, PCBP2, SC23A, SF3A1, NCOA2, SF01, MED1, JHD2C, ELF2, TAB1, TBCE, VAS1, ZYX, SEPT7, ADRM1, CCDC6, PKN2, DDB1, CDC37, NRF1, FSCN1, RFX7, QSER1, QRIC1, TBB8, LARP7, TB10B, AMOT, TGO1, PRC2B, UBAP2, QSPP, RBM26, RPRD2, TASO2, TSH3, ARID2, LIN54, EDC4, SCYL2, NFRKB, ZC3HE, FIP1, MCAF1, BCOR, UBN2, LARP4, SPT6H, SND1, DDX46, CYFP1, KDM3B, ZCCHV, NUFP2, PLGT3, RAI1, RBBP6, SH3R1, HUWE1, YTHD3, CENPV, KAISO, KTN1, CAND1, RTTN, CARM1, PRSR1, P66A, SPA12, Z3H7A, ANKH1, SUGP1, CCAR1, PHC2, SMAP1, PHAR4, DCP1B, FNBP4, CPSF7, ARFG1, ENAH, SUMF2, PGLT1, SERB1, LS14A, TNR6A, ABCF1, NEDD1, WDR36, SMRC2, PO210, PDC6I, ATX2L, P66B, DDX1, SMG7, MAML1, HS105, LAR4B, GCN1, AN32B, TFG, CBP, RENT1, SMRC1, FUBP2, TNPO1, USP9X, NCLN, FERM2, FKB10, P5CR2, ISOC1, NMD3, EDC3, OTUB1, PDLI5, FUBP3, ZC3HA, EP400, PRRC1, RBM14, VPS35, CIC, MED15, SEC62, PSMD1, PARK7, EYA3, VAT1, SCAFB, EIF3C, ATX2, TS101, TCPH, ANM1, RNZ2, TBA1C, CNPY3, WAC, DIDO1, AN32E, TBB6, HNRL1, TBB2B, GNL3, THIC, RBM4, NAA15, YTHD1, WNK3, UNK, UBA5, BRD8, LMA2L, FOXP1, NELFA, PTN23, WNK1, AMPB, RPF2, GORS2, LRC40, MLXIP, MYG1, RISC, CYBP, RC3H2, TAF9B, NCOA5, CHD8, CELR2, DCP1A, PDLI7, SAR1A, SHLB2, MBNL1, SALL1, SYFB, PDS5B, OLA1, RBM12, DD19A, FANCI, LYAR, CARF, TAB2, UGGG1, CDK12, IF2B1, ITSN2, BICRA, CNOT2, RCC2, SYLC, RBM27, KANL3, ATX10, SAE1, SAE2, SUN2, SRP68, CHRD1, UBQL2, S30BP, PUF60, DACH1, SIX4, HOOK1, MRT4, NUP50, MRTFB, ZMIZ1, YETS2, HECD1, MYO6, PRP19, UBQL1, G3BP2, MAGD2, CSN3, SCAF8, TRI33, SRRM2, PA2G4, RUVB2, EIF3L, DRG1, OFUT2, E41L3, R3HD2, RRP44, NOP58, ZN281, LC7L2, SBDS, STRAP, RTCB, SALL2, TLN1, ARIP4, HYOU1, KLF12, ARI1, PRC2C, YTHD2, SP16H, SERC, GMEB1, ZHX2, S23IP
Species: Homo sapiens
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Song J, Liu C, Wang X, Xu B, Liu X, Li Y, Xia J, Li Y, Zhang C, Li D, Sun H. O-GlcNAcylation Quantification of Certain Protein by the Proximity Ligation Assay and Clostridium perfringen OGA(D298N)(CpOGA(D298N)). ACS chemical biology 2021 16(6) 34105348
Abstract:
O-GlcNAcylation is an O-linked β-N-acetyl-glucosamine (O-GlcNAc)-monosaccharide modification of serine or threonine in proteins that plays a vital role in many critical cellular processes. Owing to its low molecular weight, uncharged property, and difficulty in distinguishing from β-N-acetyl-galactosamine (GalNAc), the lack of high specificity and avidity tools and sophisticated quantification methods have always been the bottleneck in analyzing O-GlcNAc functions. Here, we compared glycan array data of the mutant of Clostridium perfringen OGA (CpOGAD298N), O-GlcNAc antibody CTD110.6, and several lectins. We found that CpOGAD298N can effectively distinguish GlcNAc from GalNAc. Glycan array analysis and isothermal titration calorimetry (ITC) show that CpOGAD298N has a GlcNAc specific binding characteristic. CpOGAD298N could be used in far-western, flow cytometry analysis, and confocal imaging to demonstrate the existence of O-GlcNAc proteins. Using the CpOGAD298N affinity column, we identified 84 highly confident O-GlcNAc modified peptides from 82 proteins in the MCF-7 cell line and 33 highly confident peptides in 33 proteins from mouse liver tissue; most of them are novel O-GlcNAc proteins and could not bind with wheat germ agglutinin (WGA). Besides being used as a facile enrichment tool, a combination of CpOGAD298N with the proximity ligation assay (PLA) is successfully used to quantify O-GlcNAc modified histone H2B, which is as low as femtomoles in MCF-7 cell lysate. These results suggest that CpOGAD298N is a specific tool for detection (far-western, flow cytometry analysis, and confocal imaging) and enrichment of O-GlcNAcylated proteins and peptides, and the CpOGAD298N-PLA method is useful for quantifying certain O-GlcNAc protein.
O-GlcNAc proteins:
FSIP2, DYTN, P121C, ANM5, RGRF2, MSI1H, SNX2, IDHC, BAZ1A, AT10B, DDAH2, ADH1, ASSY, APOB, PPBT, SRC, K1C18, INVO, TRY1, A1AT1, O5AL1, PYC, ITPR1, GLU2B, ASSY, SON, DRD4, H2B1O, CP2DA, CXCR1, DHE3, MAP4, TRY3, BASI, LMNA, NU153, BRCA2, HCFC1, H2B1D, NAL11, CH60, ACTC, PI42B, HRH2, FKBP4, ANK3, ATM, UBP2L, ADRM1, TEN4, PAN3, FSIP2, TGO1, COA6, FRY, MYOME, SHE, HCFC1, ZN148, Q63800, SBP2, DYH17, A2MG, IQCE, AEDO, FBX38, QSOX2, NRAP, CNDG2, THIM, LONM, CLC10, MFS6L, DSEL, HMCS1, TACO1, CNTRB, NUP93, S2541, KCD17, MB213, CFA43, STAB1, SMRC2, DTX3L, GANC, GRK7, TRIM7, F227B, ADAD1, ADCYA, ERP44, FANCJ, TMM59, TBB2B, SPIT4, AP2B1, NUD12, I2BPL, CARD9, PABP3, ABHGB, WDCP, TDRD3, SUCHY, DCA13, KI21B, HEBP1, CTNL1, UBQL2, CAD22, TNIK, WDR3, RBM7
Species: Homo sapiens, Mus musculus, Rattus sp
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Qin W, Qin K, Fan X, Peng L, Hong W, Zhu Y, Lv P, Du Y, Huang R, Han M, Cheng B, Liu Y, Zhou W, Wang C, Chen X. Artificial Cysteine S-Glycosylation Induced by Per-O-Acetylated Unnatural Monosaccharides during Metabolic Glycan Labeling. Angewandte Chemie (International ed. in English) 2018 57(7) 29237092
Abstract:
The unexpected, non-enzymatic S-glycosylation of cysteine residues in various proteins by per-O-acetylated monosaccharides is described. This artificial S-glycosylation greatly compromises the specificity and validity of metabolic glycan labeling in living cells by per-O-acetylated azido and alkynyl sugars, which has been overlooked in the field for decades. It is demonstrated that the use of unacetylated unnatural sugars can avoid the artifact formation and a corrected list of O-GlcNAcylated proteins and O-GlcNAc sites in HeLa cells has been assembled by using N-azidoacetylgalactosamine (GalNAz).
O-GlcNAc proteins:
RBM47, SBNO1, CNOT1, PDLI1, TAF4, AGRIN, ABLM1, CSKP, MYPT1, ZN609, SC16A, NUP42, KDM6A, MSI1H, HNRPR, TPD54, SYNJ1, KLF4, RA51C, ZN207, NPA1P, DIAP1, PLIN3, MAFK, ANR17, LIPA2, NCOR1, SC24B, CNOT4, TXD12, EGFR, LMNA, GCR, HSPB1, RLA2, K1C18, VIME, ROS1, SNRPA, ROA1, IFIT2, ATX1L, SP9, TPR, MYH1, XRCC6, GLU2B, PO2F1,