PMC

Validation of the approach. (a) Distinct antibodies detect the same glycosylation stoichiometry on OGT (AL-28 and DM-17), CREB (Chemicon 5432 and Upstate 06-863), and MeCP2 (Hu and Upstate 07-013). p110-OGT was expressed in Sf9 cells, endogenous CREB was from rat liver, and MeCP2 was co-expressed with OGT in Sf9 cells. (b) As little as 0.8% of glycosylated OGT is readily detected. Sf9 cell lysate containing over-expressed p110-OGT was chemoezymatically labeled with PEG derivative 3 and diluted with unlabeled lysate to generate standards with varying percentages of label incorporation. The lysate was resolved by SDS-PAGE and immunoblotted with the anti-OGT antibody DM-17. The limit of detection was defined as the lowest stoichiometry value within 10% of the linear fit. See for details. (c) Detection of PEG incorporation into ketogalactose-labeled Nup62 is linear across a wide range of stoichiometries (0–100%). 293T cell lysate was labeled with UDP-ketogalactose 1 and then diluted with varying amounts of unlabeled lysate to simulate different levels of glycosylation. Each mixture was reacted with 2, resolved by SDS-PAGE, and immunoblotted for Nup62. Full-length blots are presented in .

Dissecting the interplay between O-GlcNAc and phosphorylation. Detection of four subpopulations of CREB or MeCP2: glycosylated (1), nonglycosylated (2), glycosylated and phosphorylated (3), and nonglycosylated and phosphorylated (4). (a) CREB can be simultaneously glycosylated and phosphorylated. Mass-tagged lysates from 293T cells treated with Fsk, PUGNAc or vehicle were immunoblotted with a pS133-specific or general CREB antibody. (b) Fsk stimulates pS133 phosphorylation of glycosylated and non-glycosylated CREB similarly (n = 6). (c) Inhibition of OGA increases CREB glycosylation levels on phosphorylated and total CREB similarly (n = 4). Neither Fsk nor PUGNAc affected overall CREB glycosylation or pS133 phosphorylation levels, respectively (). (d) Complex interplay between MeCP2 glycosylation and phosphorylation. Mass-tagged lysates from neurons treated with GlcN or vehicle were immunoblotted with a pS80-specific or general MeCP2 antibody (n = 6). (e) GlcN increases MeCP2 glycosylation levels predominantly on the pS80 subpopulation. (f) GlcN decreases pS80 levels on the nonglycosylated subpopulation and increases pS80 levels on the glycosylated subpopulation. (g) Reverse yin-yang relationship on MeCP2 under physiological conditions. Mass-tagged nuclear extracts from synchronously depolarized or non-depolarized neurons were immunoblotted with a pS80-specific or general MeCP2 antibody (n = 8). (h) Neuronal depolarization induces pS80 levels on glycosylated MeCP2. (i) Depolarization increases the glycosylation level of pS80 MeCP2, while decreasing that of total MeCP2. UDP ketogalactose 1 was excluded as a control. Data represent mean ± s.e.m. Statistics were analyzed by Student’s t-test. * P = 0.0005, ** P = 0.00007 for (b). * P = 0.02, ** P = 0.0007 for (c). * P = 0.04, ** P = 0.008 for (e). * P = 0.01, ** P = 0.04 for (f). * P = 0.01, ** P = 0.001 for (h). * P = 0.04, ** P = 0.001 for (i). Full-length blots are presented in .

Monitoring O-GlcNAc glycosylation levels on proteins across various tissues and after cellular stimulation. (a) CREB is O-GlcNAc glycosylated at higher levels in the rat cerebellum (CE) and hippocampus (HP) than in the liver (LV). Cell lysates from the indicated tissues were chemoenzymatically labeled, resolved by SDS-PAGE, and immunoblotted for CREB. A representative immunoblot from one animal is shown. Plotted are the glycosylation stoichiometries of CREB averaged across multiple animals (n = 4–6). * P = 0.0003, ** P = 0.03. (b) O-GlcNAc glycosylation levels of CREB and GRASP55 decreased in rat embryonic neuronal cultures upon DON treatment (D) and increased upon GlcN treatment (G) relative to untreated neurons (U). Cell lysates were chemoenzymatically labeled, resolved by SDS-PAGE, and immunoblotted for the indicated proteins. A representative immunoblot is shown. Plotted are the glycosylation stoichiometries averaged across n = 3 experiments. * P = 0.002, ** P = 0.006, *** P = 0.001. (c) The rate constant for monoglycosylation of CREB is approximately threefold higher than that for diglycosylation of CREB. Neuro2a cells were treated with 10 mM GlcN for the indicated times. Crude nuclear protein lysates were chemoezymatically labeled, resolved by SDS-PAGE, and immunoblotted for CREB. Formation of monoglycosylated or diglycosylated CREB was plotted as a function of time for n = 3 experiments. See for details. Data represent mean ± s.e.m. Statistical analyses were performed using the Student’s t-test (two-tailed, paired). Full-length blots are presented in .

Mass-tagging strategy for quantifying O-GlcNAc glycosylation levels on specific proteins. (a) Schematic of the approach. O-GlcNAc-modified proteins are chemoenzymatically labeled using the UDP-ketogalactose analogue 1 and an engineered GalT enzyme, followed by reaction with an aminooxy-functionalized PEG mass tag (2 or 3). This approach enables facile visualization of O-GlcNAc-glycosylated species upon SDS-PAGE and immunoblotting with antibodies against proteins of interest. (b) Validation of the approach using CREB_mono and CREB_co. CREB_mono was 28.2% glycosylated and existed primarily in the mono-glycosylated state, whereas CREB_co was 88.8% glycosylated and present in multiply glycosylated forms. (c) In vivo O-GlcNAc stoichiometries vary significantly even among proteins with similar functions. Cell lysates (100 μg) from 293T cells (source of Sp1), adult rat brain (source of MeCP2, Nup62, and synapsin Ia (upper band) and IIa (lower band)), embryonic neuronal cultures (source of CREB, and OGA) or purified p75-OGT (0.43 μg) from Sf9 cells were subjected to chemoenzymatic labeling, SDS-PAGE, and immunoblotting with antibodies against the indicated proteins. See Methods for details. In all cases, 1 was excluded as a control for selectivity. The indicated glycosylation stoichiometries were determined by measuring the relative intensities of the glycosylated and nonglycosylated bands. (….) denotes the nonglycosylated protein fraction, (→) denotes O-GlcNAc glycosylated protein fraction shifted with 2 or 3. Full-length blots are presented in .