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Narayanan B, Zahra F, Reeves RA, Aggarwal A, O'Meally RN, Henry RK, Craven M, Jacobson A, Cole RN, Kohr MJ, Umapathi P, Zachara NE. Differential Detection of O-GlcNAcylated proteins in the heart using antibodies. Analytical biochemistry 2023 678 37507081
Abstract:
Thousands of mammalian intracellular proteins are dynamically modified by O-linked β-N-acetylglucosamine (O-GlcNAc). Global changes in O-GlcNAcylation have been associated with the development of cardiomyopathy, heart failure, hypertension, and neurodegenerative disease. Levels of O-GlcNAc in cells and tissues can be detected using numerous approaches; however, immunoblotting using GlcNAc-specific antibodies and lectins is commonplace. The goal of this study was to optimize the detection of O-GlcNAc in heart lysates by immunoblotting. Using a combination of tissue fractionation, immunoblotting, and galactosyltransferase labeling, as well as hearts from wild-type and O-GlcNAc transferase transgenic mice, we demonstrate that contractile proteins in the heart are differentially detected by two commercially available antibodies (CTD110.6 and RL2). As CTD110.6 displays poor reactivity toward contractile proteins, and as these proteins represent a major fraction of the heart proteome, a better assessment of cardiac O-GlcNAcylation is obtained in total tissue lysates with RL2. The data presented highlight tissue lysis approaches that should aid the assessment of the cardiac O-GlcNAcylation by immunoblotting.
O-GlcNAc proteins:
A0A023T778, A0A075B5P3, A0A075B5P4, A0A075B5P6, A0A075B5T2, A0A075B5T7, A0A087WP81, A0A087WPL5, A0A087WR50, A0A087WS16, A0A087WS46, A0A087WSC6, A0A0A0MQ79, A0A0A0MQA5, A0A0A0MQD2, A0A0A0MQF6, A0A0A0MQJ4, A0A0A0MQM0, A0A0A6YVU8, A0A0A6YW67, A0A0A6YW80, A0A0A6YWP6, A0A0A6YX26, A0A0A6YXF6, A0A0A6YXH3, A0A0A6YXV1, A0A0B4J1E7, A0A0B4J1H7, A0A0F6AIX5, A0A0G2JDI9, A0A0G2JDL9, A0A0G2JDR9, GBP6, A0A0G2JDW2, A0A0G2JE25, A0A0G2JEP0, A0A0G2JEX1, A0A0G2JEY6, A0A0G2JF52, A0A0G2JF85, A0A0G2JG74, A0A0G2JGJ1, A0A0G2JGS0, A0A0J9YU07, A0A0J9YU24, A0A0J9YUZ4, A0A0M3HEQ0, A0A0N4SV40, A0A0N4SV66, A0A0N4SVB1, A0A0N4SVB8, A0A0N4SVK8, A0A0N4SVL9, A0A0N4SW07, A0A0R4J023, A0A0R4J038, A0A0R4J050, A0A0R4J052, A0A0R4J093, A0A0R4J0H8, A0A0R4J0I1, A0A0R4J0K2, A0A0R4J0L5, A0A0R4J0L6, A0A0R4J0S3, A0A0R4J0T5, A0A0R4J0X5, A0A0R4J0Z1, A0A0R4J138, A0A0R4J196, A0A0R4J1E2, A0A0R4J1J3, A0A0R4J1Q6, A0A0R4J1R7, A0A0R4J1W0, A0A0R4J1W7, A0A0R4J260, A0A0R4J275, A0A0U1RPW2, A0A0U1RPY8, A0A140LHG8, A0A140LIU4, A0A140LIZ5, A0A140LJ98, A0A140LJB7, A0A171EBL2, A0A1B0GQU8, A0A1B0GR11, A0A1B0GRP7, A0A1B0GRV0, A0A1B0GRV3, A0A1B0GRW3, A0A1B0GS58, A0A1B0GS68, A0A1B0GS70, A0A1B0GS91, A0A1B0GSG5, A0A1B0GSK8, A0A1B0GSR9, A0A1B0GX27, A0A1C7CYV0, A0A1C7ZN10, A0A1D5RM79, A0A1L1SQ51, A0A1L1SQA8, A0A1L1SS27, A0A1L1SST0, A0A1L1STE6, A0A1L1SV25, A0A1L1SV73, A0A1W2P6E6, A0A1W2P6P1, A0A1W2P768, A0A1W2P7A1, A0A1W2P812, A0A1Y7VJY8, A0A1Y7VKT9, A0A1Y7VKY1, A0A1Y7VLP0, A0A286YCI8, A0A286YCS6, A0A286YD12, A0A286YDA2, A0A2I3BPW0, A0A2I3BQE3, A0A2I3BQF4, A0A2I3BRL8, A0A2K6EDJ7, A0A2R8VHF9, A0A2R8VHX0, A0A338P6D9, A0A338P6G0, A0A338P6N4, A0A338P703, A0A338P731, A0A338P786, A0A338P7D7, A0A338P7F1, A0A338P7G4, A0A3B2W824, A0A3B2WB97, A0A3B2WBC6, A0A3B2WCN9, A0A3Q4EBV4, A0A3Q4EG54, A0A3Q4EH93, A0A3Q4EI56, A0A494B908, A0A494B923, A0A494B955, A0A494B985, A0A494B9D8, A0A494B9P3, A0A494B9W3, A0A494B9X1, A0A494B9Y4, A0A494BA51, A0A494BAJ6, A0A494BB38, A0A494BBC1, A0A494BBG8, A0A571BEC9, A0A571BEG4, A0A571BG59, A0A571BGD8, A0A5F8MPB9, A0A5F8MPD6, A0A5F8MPK9, A0A5F8MPM4, A0A5F8MPN8, A0A5F8MPW1, A0A5F8MPY2, A0A5K1VVQ9, A0A668KL51, A0A668KLD3, A0A6I8MWZ8, A0A6I8MX27, A1BN54, A2A513, A2A5F5, A2A6F8, A2A6Q8, A2A7A7, A2A813, A2A848, TM201, A2A997, A2A9Q2, OBSCN, A2AAW9, A2AE89, A2AEX6, A2AFQ0, A2AFQ2, A2AGJ9, A2AI69, A2AI87, NDUF6, A2AIM4, A2AIW9, A2AK42, A2AKD7, A2AKU9, A2AKV9, A2AL12, A2ALV7, CAVN4, A2AMW0, UBR4, A2AN84, A2APD7, A2AQN4, MYH7B, A2AQR0, A2AQY8, A2AS98, TITIN, A2AT02, A2AU61, A2AUD5, RBGP1, A2BDW0, A2BH06, A2BI12, A2CEK3, A2CES4, A2CG35, A3KFU5, A3KGU5, ARMT1, A6X925, A8DUK4, A8JYK8, CISD3, B1AR28, B1AR93, B1ARA3, B1ASE2, B1ASZ3, B1AT10, B1AU25, B1AUX2, B1AV14, B1AXW5, B1AZ14, B1B0C7, B2C3G8, GNAI1, B2RXT3, B2RY24, B7FAU9, B7ZCI2, B8JJI4, B8JJM5, B8JK32, B9EIZ7, D3YTL5, D3YU39, D3YUG3, D3YUK5, D3YUM1, D3YUP1, D3YVF4, D3YVI6, D3YVV9, D3YW19, D3YWA4, D3YX99, D3YXT0, D3YY36, D3YYE1, D3YYG9, D3YYS6, D3YZ06, D3YZ71, D3Z041, D3Z067, D3Z0G0, D3Z0X5, D3Z0Y2, D3Z1B5, D3Z1V4, D3Z263, D3Z2B3, D3Z2Y8, D3Z4A4, D3Z5K6, D3Z5W3, D3Z627, D3Z636, D3Z6F5, D3Z780, D3Z7A7, D3Z7D5, D3Z7K3, D3Z7U0, D3Z7V3, D5MCW4, D6REH1, D6RFU2, D6RFU9, D6RGM3, D6RH37, E0CXB1, E0CXH2, E0CXN7, E0CXS3, E0CY23, E0CZ90, E0CZE0, E9PUD2, E9PUE8, E9PUM3, E9PUU2, E9PUY9, E9PV12, FIBA, E9PV63, E9PV66, E9PVP0, E9PVU0, E9PWE8, E9PX89, E9PYF1, E9PYI8, E9PZ00, E9PZ88, E9PZC4, E9PZD8, E9PZF0, E9PZI9, E9PZP8, E9Q133, E9Q1J7, E9Q1T9, E9Q1V0, E9Q264, RYR2, E9Q4T8, ACACB, DESP, E9Q5B5, E9Q5I9, E9Q616, E9Q6A9, E9Q6C2, E9Q6W2, E9Q7A5, E9Q800, E9Q933, E9Q9C0, E9Q9T8, E9QJV4, E9QKY4, E9QL13, E9QN70, E9QPD7, E9QPX3, F6QPR1, F6QYE1, F6QYF8, F6RR81, F6VVE6, F6VVY4, F6VY18, F6XI62, F6YCA7, F6ZV59, F7A3N3, F7DBQ0, SKI3, F8VPN4, F8VQJ3, F8WGL3, F8WHU8, F8WIB1, F8WIE5, F8WIT2, F8WIV2, F8WIV5, F8WJ05, G3UVU2, G3UVV4, G3UW30, G3UW85, G3UWN9, G3UX26, G3UXI6, G3UXX3, G3UYR8, G3UZ33, G3UZ48, G3X8R0, G3X8R1, G3X8T3, G3X8T9, QNG1, G3X956, G3X975, G3X977, G3X983, G3X9Q1, G3X9U9, G3XA48, G5DDB7, G5E823, G5E839, G5E883, G5E8R7, H3BIY9, H3BJI5, H3BJP9, H3BJQ7, H3BKH4, H3BKH6, H3BL49, H3BLH2, H7BWZ3, H7BX01, H7BX88, H7BX99, I7HLV2, J3QMG3, J3QPW1, J3QPZ9, J3QQ13, K3W4R2, K3W4S6, K4DI63, L7N451, M0QWU8, CAN2, DPYL2, DLDH, GSTO1, RL21, AMACR, SCRB2, PSMD4, ECH1, ANXA3, C1QBP, TIM44, AL1A7, CAVN1, SYUA, IMPCT, DSG2, NIPS2, AT2A2, PGAM2, PDLI1, AOC3, FHL2, PSA3, DHB12, WDR1, MTX2, ROA2, COMT, BIRC6, IDHC, GNPI1, KBL, AFAM, C1QR1, LANC1, ADH1, COX1, CAH2, CO3, CO4B, HBE, NU1M, NU4M, NU5M, FABP4, MYG, ALDOA, KAPCA, AATC, AATM, TBA1B, CO5, G6PI, MAOX, TTHY, KCRM, ANXA2, ALBU, K2C4, HS90A, CBR2, ENPL, MDHM, PDIA1, NUCL, PGK1, FRIH, SODM, H2AZ, IFI5B, ANXA1, EF1A1, NID1, CATB, THIO, RRAS, H2B1M, H10, LIPL, FABPH, HS90B, DMD, K2C8, ITPR1, TCPA, UCP1, PFKAL, RL7A, GELS, CAH1, GPDA, AT1B1, RS16, RSSA, CALR, HA2B, PSMD3, LMNB1, GLNA, PMGE, CAH3, LEG1, LEG3, CN37, DDX3L, AMPE, ENOA, SBP1, TPIS, CATD, FAS, SERPH, COX41, BIP, PRDX3, VIME, PLMN, ENOB, VTDB, TGM2, AP1G1, EIF3A, CXA1, CRYAB, EST1C, CATA, PPIB, GSTA4, LKHA4, AL1A1, RS2, LYN, TLN1, EZRI, MOES, CTNA1, PTMA, DHE3, PSMD7, FKB1A, PDIA3, ACOHC, ADHX, PGS1, PGS2, NP1L1, ATX10, MUG1, OAT, FKBP4, HMGB2, DESM, MP2K1, SCP2, LA, ANT3, SYWC, MIF, RAB21, HSPB7, TSP1, ODPA, CALX, RL12, PPM1B, TAGL, HMGCL, HSPA9, CAP1, TKT, VP26A, INMT, ECI1, H14, ALDR, ALD2, COF2, ACADM, NSF, VPS4B, PRS7, RB11B, NEDD4, PA24A, ALDH2, GSHR, MK14, PFKAM, RL6, ANXA5, ABCD1, LMNA, CBR1, COX8B, TNNI3, ADT1, HEP2, ROA1, INPP, MCM4, CAV1, SAHH, FMO1, GDIA, CSRP3, VATA, ACADL, DHB4, MLRV, HDGF, ADT2, THTR, PON1, KPYM, NDUS6, CPT2, ODB2, IDHP, RD23A, PUR8, DDX6, ADK, ACYP2, AP4A, CX6B1, UBP5, ATPB, CD38, ERP29, EF2, TPM1, B2L13, RUVB1, EIF3E, PCBP1, NPL4, ACTB, CDC42, IF4A1, UBC12, UBE2N, ARP2, ARF3, RL27, 1433G, RRAS2, RS7, PP1B, RS8, RS15A, 1433E, RS23, SMD3, EF1A2, RS4X, AP2S1, RS6, H4, RAN, RS24, RS26, CYC, RL32, FBX40, PROF1, LIS1, HSP7C, TCTP, CH60, IF4E, 1433Z, RS17, PHB1, ACTC, RACK1, 1433T, TBB4B, 1433F, USP9X, IDHG1, NACAM, F16P2, DCUP, PYRG1, TCPB, TCPE, TCPZ, ARF5, AP2M1, RL19, ISC2A, CSRP1, NRP1, RS3A, CPT1A, WBP2, FUMH, LAMA4, TBB5, G6PD1, APOA1, A1AT5, APOH, TERA, UBA1, PLAK, MYH6, KCRB, CO6A1, FABP5, ATP5I, CBG, MPRI, ACADS, AMBP, CD36, Q14BI5, ACSS3, A1BG, TRI72, Q20BD0, NLR1A, HSDL2, OSBP1, CUL4A, Q3TG45, Q3TGM7, GUAA, TM38A, Q3TXN1, PSMD1, SC31B, SYRM, DDB1, Q3U2G2, Q3U422, Q3U816, Q3U8Y1, Q3UER8, Q3UF75, TM10C, Q3UGX2, HAP28, MYLK3, Q3UJQ9, GPD1L, PP1R7, HDGR2, SC31A, THEM4, Q3UZJ4, ST1D1, Q3V117, HSPB6, D39U1, Q5SQ27, Q5SQB0, Q5SUS9, Q5SWN2, ACACA, MYH4, MYH1, COPD, A1AG1, ODO1, LAMA2, STIP1, VDAC1, COQ8A, ADIPO, 2A5G, LAMA5, HARS1, HCDH, GDIB, HS105, SERA, PZP, MIME, POSTN, PON3, DAG1, MYOM1, SPTB2, TX261, TIF1B, TSN, TFR1, NDUA4, CYTB, CAVN2, BTF3, CH10, ADH7, CP4B1, TOP2B, TPP2, H2A2B, H2A2C, NQO1, VINC, PUR2, CTNA3, CLH1, RFTN1, NOMO1, K2C73, 2ABA, U520, XPO1, KCRS, Q6P8N8, OXSR1, LPPRC, UBR2, CAND1, CAND2, MLEC, RS9, IF2A, KAT3, 2AAA, ATPMK, Q7TNL5, SRCA, ELP1, SESD1, FLNB, PANK4, IREB2, EFTU, MIRO1, SAM50, S2540, SYAC, SYNM, F210A, S2512, F13A, EHD2, ECHM, SYIM, AHSA1, RCC2, IPO5, ODPX, MAON, ODP2, ECHA, PPA6, Q8BRB6, AAPK2, Q8BTS3, CSN7B, PPME1, NUDT9, AOFB, SSDH, THIM, KANK2, VP13C, COMD2, PGM5, NHLC2, GPCP1, ASGL1, LCAP, EXOG, THOP1, Q8C483, EMC1, Q8C845, Q8C8G3, ANK2, VWA8, ARK72, SYEP, LONM, UN45B, BICRL, PGP, FBX4, AL4A1, PDLI5, DI3L2, PYGB, FERM2, COPA, PAK2, HMCS1, ACAD9, MIRO2, AFG32, EIF3B, ACSL5, FIBB, TACO1, PDXK, COQ9, SDHA, FMO2, LZIC, HIBCH, THIL, ERF3A, SUOX, FAHD1, S2542, EIF3C, CMBL, COQ6, Q8R2P8, FABD, GBRL1, CLYBL, TMC2, UBP15, RMC1, MAVS, EST1D, ACSF2, CK068, SYLM, MYH9, PPCS, HNRL1, AT1A1, LMCD1, HNRPU, S25A3, RBM39, FLNC, ETFR1, Q91VB8, NDUS1, HNMT, DDX1, CBR4, EIF3H, CISD1, HEMO, VPS36, RHG35, FAKD4, L2HDH, RPN1, TWF1, MYH7, PCCA, UGPA, SMCA5, HNRLL, ETFD, TRFE, RHEB, MACD1, SYDC, MPI, RT02, CPT1B, DCMC, SMRD2, EIF3M, ECHB, Q99JZ4, RRAGC, VMA5A, ACON, DCTN2, NAMPT, PPIF, DHRS1, 3HIDH, DHRS4, MAT2B, NDUAA, ETFA, DDAH2, RTCB, GRPE1, NDUS5, DNJA3, ASPN, MCCA, PPR3A, RM09, ACS2L, NUDT7, RTN4, GDIR1, PRP8, NDUA5, COX6C, ATP5L, LGUL, AMPL, MYL9, NDUC2, DECR, MTAP, PKP2, NDUA2, SDHB, NDUB4, TM223, COTL1, NDUB9, TXD17, TRAP1, AT5F1, ACO13, 1433B, DPH2, M2OM, CHSP1, NAR4, TPPP3, MIC19, SMC1A, NUD17, PUR9, SNX2, CYGB, ROA0, COQ5, MITOS, MPPB, CYBP, BIEA, QCR1, HS12B, RL15, GLYM, EFTS, AL1B1, CISY, 5NT3A, ODPB, SYRC, EXOS7, CY1, SYTC, HINT2, ILEUA, GAL3A, HHATL, RM53, ODO2, OXSM, CUL2, EFHD1, PLCC, NDUV2, FUND2, F162A, ECHD3, IPYR, QCR7, ITPA, RL4, EF1G, T126A, PHP14, MTU1, ATPO, QCR2, PLIN3, LMAN2, PGAM1, ACDSB, OCTC, RT15, 6PGD, NDUA8, PUR6, GSTK1, MECR, NDUBA, NDUS3, CRIP2, RMD1, ETFB, LACTB, DHB11, MMSA, ERAP1, VPS35, RM46, EHD4, PARVB, M3K20, PSMG2, PYGL, IVD, DYHC1, NQO2, ACTN2, COPB, AK1A1, DDX21, RL38, SH3L1, PROF2, TBA8, PRELP, IQGA1, HYOU1, LDB3, GLTP, PALS2, PPCE, MLRA, DRG2, PLEC, S2513, TOM40, CLIC4, DNJA2, ACOT2, AAKB1, DEST, ACOT9, KAD1, PSA4, PSB3, PSB2, SAE1, BMP10, MYO1C, KAD2, KAD3, CUL1, PDC6I, COR1C, SUCA, KAD4, CATZ, ABEC2, CBPQ, EHD1, S12A7, GRK1, MTNB, GBP2, SYUG, TWF2, NFS1, NDUA7, CLIC1, STK39, MTMR9, SUCB2, SUCB1, PCKGC, PSD10, MCAT, S4R1F2, S4R1N1, S4R242
Species: Mus musculus
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Zaro BW, Yang YY, Hang HC, Pratt MR. Chemical reporters for fluorescent detection and identification of O-GlcNAc-modified proteins reveal glycosylation of the ubiquitin ligase NEDD4-1. Proceedings of the National Academy of Sciences of the United States of America 2011 108(20) 21540332
Abstract:
The dynamic modification of nuclear and cytoplasmic proteins by the monosaccharide N-acetyl-glucosamine (GlcNAc) continues to emerge as an important regulator of many biological processes. Herein we describe the development of an alkynyl-modified GlcNAc analog (GlcNAlk) as a new chemical reporter of O-GlcNAc modification in living cells. This strategy is based on metabolic incorporation of reactive functionality into the GlcNAc biosynthetic pathway. When combined with the Cu(I)-catalyzed [3 + 2] azide-alkyne cycloaddition, this chemical reporter allowed for the robust in-gel fluorescent visualization of O-GlcNAc and affinity enrichment and identification of O-GlcNAc-modified proteins. Using in-gel fluorescence detection, we characterized the metabolic fates of GlcNAlk and the previously reported azido analog, GlcNAz. We confirmed previous results that GlcNAz can be metabolically interconverted to GalNAz, whereas GlcNAlk does not, thereby yielding a more specific metabolic reporter of O-GlcNAc modification. We also used GlcNAlk, in combination with a biotin affinity tag, to identify 374 proteins, 279 of which were not previously reported, and we subsequently confirmed the enrichment of three previously uncharacterized proteins. Finally we confirmed the O-GlcNAc modification of the ubiquitin ligase NEDD4-1, the first reported glycosylation of this protein.
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