In this study, we demonstrate a powerful new approach for visualizing the O-GlcNAc-glycosylated protein subpopulation in complex biological samples. Through the selective, chemoenzymatic attachment of PEG mass tags to O-GlcNAc modification sites, in vivo glycosylation stoichiometries can be readily quantified on endogenous proteins, without the need for protein purification, advanced instrumentation, or expensive radiolabels. In addition, the approach allows for direct interrogation of proteins of interest by immunoblotting, without requiring O-GlcNAc site identification or O-GlcNAc site-specific antibodies. This feature is critical given that glycosylation sites have been mapped for only a small fraction of the O-GlcNAc proteome, and comprehensive analyses of all glycosylation sites within a given protein are lacking, even for many well-studied proteins.
Our mass-tagging approach yields information that cannot be obtained using current methods. Because glycosylation levels can be rapidly monitored by immunoblotting in parallel, O-GlcNAc glycosylation stoichiometries can be readily profiled across the proteome. We quantified the stoichiometries of a broad range of proteins, including proteins that eluded detection by the O-GlcNAc antibodies RL-2 and CTD110.6. Even among proteins with similar functions, we observed a wide range of O-GlcNAc stoichiometries in vivo. For example, both the transcriptional repressor MeCP2 and transcription factor CREB were mono-glycosylated at moderate levels in neurons (15.3% and 33.0%, respectively), while the transcription factor Sp1 was 100% glycosylated at multiple sites in vivo. Similarly, the long form of OGT exhibited very low levels of glycosylation in Sf9 cells (3.3%), whereas the short form of OGT had a significantly higher extent of glycosylation in these cells (39.7%). Unexpectedly, OGT was primarily mono-glycosylated despite having multiple known glycosylation sites, which suggests a high potential for the transient regulation of OGT activity by O-GlcNAc in response to cellular stimuli. These findings underscore the complementarity between our mass-tagging strategy and mass spectrometry approaches. Although mass spectrometry provides key information about the identity and number of O-GlcNAc sites, it cannot readily determine their relative occupancy or interrelationship within the same molecule of protein. In contrast, our mass-tagging approach provides a direct read-out of glycosylation stoichiometry and state (e.g., mono-, di-, tri-, etc.), and when applied in conjunction with site-directed mutagenesis, it may reveal O-GlcNAc stoichiometries at specific amino acid sites.
The ability to monitor glycosylation stoichiometries and states provides unique insights into the cellular functions and regulation of O
-GlcNAc glycosylation. For example, we probed the glycosylation level of CREB across different tissues, cells and organisms in response to a variety of perturbations. Our results revealed tissue-specific differences and tight regulatory control over the levels of CREB glycosylation in vivo
. Moreover, we found that the glycosylation state of CREB influenced its kinetics of glycosylation: upon activation of the hexosamine biosynthesis pathway, unglycosylated CREB was glycosylated three-times faster than mono-glycosylated CREB, suggesting that the presence of one GlcNAc sugar on CREB decreases the rate of deposition of another sugar. Although the presence of only one OGT gene may suggest uniform regulation of O
-GlcNAc substrates, evidence from quantitative mass spectrometry analyses indicates that OGT regulates its substrates more discretely29
. Our approach allows this phenomenon to be explored in detail across and within proteins of interest in response to specific stimuli. Such information would be difficult, if not impossible, to obtain using current methods.
Elucidating the interplay of O-GlcNAc with other post-translational modifications is another powerful application of the mass-tagging approach. For example, we demonstrated that the mass tag enables rapid visualization of distinct post-translationally modified subpopulations, which are distinguished by their glycosylation or phosphorylation status. As such, the approach provides a direct read-out of whether the two modifications are mutually exclusive on proteins of interest (i.e. yin-yang) or whether they can co-exist on the same molecule. We found that glycosylation and phosphorylation occur independently in the case of CREB under the stimuli tested. In addition, we discovered a surprising reverse yin-yang relationship on MeCP2, which was undetectable by traditional methods and revealed only by the mass-tagging approach. A yin-yang relationship on MeCP2 was observed by stimulating O-GlcNAc levels with GlcN and monitoring phosphorylation levels on the total MeCP2 population. However, our mass-tagging strategy enabled changes in glycosylation to be monitored specifically on the phosphorylated subpopulation and vice versa. We found that neuronal activity or stimulation of the hexosamine biosynthesis pathway induced O-GlcNAc glycosylation selectively on the S80-phosphorylated subpopulation of MeCP2 – the opposite of a yin-yang relationship. One possibility is that glycosylation may mark a specific subset of MeCP2-regulated genes and render them less susceptible to activity-dependent derepression. Our results provide strong evidence for the close coupling of glycosylation and phosphorylation on MeCP2 and, more broadly, we find that the net change in glycosylation or phosphorylation on the global protein population can be the opposite of changes occurring on specific modified subpopulations, an observation with significant implications for studying the interplay between modifications. The complexities observed with MeCP2 underscore the importance of carefully dissecting the intricate interplay between post-translational modifications on a molecular level and of developing new methods to address these questions.
In conclusion, we have developed a new mass-tagging strategy to advance our understanding of the stoichiometry, complex regulation, and cellular dynamics of O-GlcNAc glycosylation. In the future, we anticipate extending this approach to explore the complex ‘codes’ or networks of post-translational modifications on O-GlcNAc-modified proteins through the use of antibodies against other modifications. Moreover, we envision that this general strategy of tagging modifications with resolvable PEG mass tags will prove valuable for the study of other post-translational modifications and poorly understood glycosylation motifs.