Elucidating the protein substrate recognition of O-GlcNAc transferase (OGT) toward O-GlcNAcase (OGA) using a GlcNAc electrophilic probe

Kositzke, Adam; Fan, Dacheng; Wang, Ao; Li, Hao; Worth, Matthew; Jiang, Jiaoyang

doi:10.1016/j.ijbiomac.2020.12.078

Cited by 15 publications

(15 citation statements)

References 77 publications

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“…Undoubtedly, the full-length OGT is the construct that consistently generates the most detectable number of glycosites on overexpressed and endogenous protein substrates. Extension of efforts to determine how substrates engage the TPR domain of OGT would define the features of the extended TPR that promote this selection [11,12]. Nevertheless, our dataset can shed light on the TPRs that are required to engage certain substrates in a global manner and the particular glycosites affected by TPR truncations.…”

Section: Discussionmentioning

confidence: 99%

“…Due to the essential requirement of OGT for substrate O-GlcNAcylation [10], deciphering the basis of OGT's substrate selection mechanisms has attracted much interest [2,11,12]. The canonical nucleocytoplasmic form of OGT (ncOGT) is composed of two domains: an N-terminal tetratricopeptide repeat (TPR) domain and a C-terminal catalytic domain (Fig.…”

Section: Introductionmentioning

confidence: 99%

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Truncation of the TPR domain of OGT alters substrate and glycosite selection

et al. 2021

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Truncation of the TPR domain of OGT alters substrate and glycosite selection

et al. 2021

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“…Our work, along with a recent study of how glycosylation of O-GlcNAcase (OGA) is affected when lumenal residues in OGT’s TPR domain are altered, ,, is beginning to clarify how different substrates use OGT’s TPR domain to access its active site, paving the way for future work to develop a molecular understanding of substrate recognition. The work on OGA showed that it behaves like TAB1 in using lumenal interactions that involve most of the TPR domain for substrate recognition. ,, Further progress in understanding OGT substrate recognition will require integration of glycosylation assays with quantitative assays to measure substrate binding affinities, which so far do not exist, and ideally with structural information. In the meantime, we note that modulating glycosylation of broad subsets of substrates by changing features of the TPR domain in cells or in animal models may help clarify the function of O-GlcNAcylation in health and disease.…”

mentioning

confidence: 95%

“…23 We speculate that phosphorylation may prevent engagement of CAMKIV with OGT's TPR lumen by introducing a repulsive interaction, providing one possible mechanism to explain "crosstalk" between phosphorylation and glycosylation. 51 Our work, along with a recent study of how glycosylation of O-GlcNAcase (OGA) is affected when lumenal residues in OGT's TPR domain are altered, 21,52,53 is beginning to clarify how different substrates use OGT's TPR domain to access its active site, paving the way for future work to develop a molecular understanding of substrate recognition. The work on OGA showed that it behaves like TAB1 in using lumenal interactions that involve most of the TPR domain for substrate recognition.…”

mentioning

confidence: 99%

“…The work on OGA showed that it behaves like TAB1 in using lumenal interactions that involve most of the TPR domain for substrate recognition. 21,52,53 Further progress in understanding OGT substrate recognition will require integration of glycosylation assays with quantitative assays to measure substrate binding affinities, which so far do not exist, and ideally with structural information. In the meantime, we note that modulating glycosylation of broad subsets of substrates by changing features of the TPR domain in cells or in animal models may help clarify the function of O-GlcNAcylation in health and disease.…”

mentioning

confidence: 99%

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Protein Substrates Engage the Lumen of O-GlcNAc Transferase’s Tetratricopeptide Repeat Domain in Different Ways

et al. 2021

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Glycosylation of nuclear and cytoplasmic proteins is an essential post-translational modification in mammals. O-GlcNAc transferase (OGT), the sole enzyme responsible for this modification, glycosylates more than 1000 unique nuclear and cytoplasmic substrates. How OGT selects its substrates is a fundamental question that must be answered to understand OGT’s unusual biology. OGT contains a long tetratricopeptide repeat (TPR) domain that has been implicated in substrate selection, but there is almost no information about how changes to this domain affect glycosylation of individual substrates. By profiling O-GlcNAc in cell extracts and probing glycosylation of purified substrates, we show here that ladders of asparagines and aspartates that extend the full length of OGT’s TPR lumen control substrate glycosylation. Different substrates are sensitive to changes in different regions of OGT’s TPR lumen. We also found that substrates with glycosylation sites close to the C-terminus bypass lumenal binding. Our findings demonstrate that substrates can engage OGT in a variety of different ways for glycosylation.

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Photoactivatable O‐GlcNAc Transferase Library Enables Covalent Chemical Capture of Solvent‐Exposed TPR Domain Interactions

Joiner,

Glogowski,

NewRingeisen

et al. 2024

ChemBioChem

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O‐linked N‐acetylglucosamine (O‐GlcNAc) transferase (OGT) is an essential, stress‐sensing enzyme responsible for adding the O‐GlcNAc monosaccharide to thousands of nuclear and cytoplasmic proteins to regulate cellular homeostasis. OGT substrates are found in almost all intracellular processes, and perturbations in protein O‐GlcNAc levels have been implicated in proteostatic diseases, such as cancers, metabolic disorders, and neurodegeneration. This broad disease activity makes OGT an attractive therapeutic target; however, the substrate diversity makes pan‐inhibition as a therapeutic strategy unfeasible. Rather, a substrate‐specific approach to targeting is more advantageous, but how OGT chooses its substrates remains poorly understood. Substrate specificity is controlled by the interactions between OGT’s non‐catalytic tetratricopeptide repeat (TPR) domain, rather than its glycosyltransferase domain. OGT’s TPR domain forms a 100 Å superhelical structure, containing a lumenal surface, known as the substrate‐binding surface, and a solvent‐exposed surface. To date, there are no tools to site‐selectively target regions of the domain and differentiate between the two binding surfaces. Here, we developed a library of recombinant OGT constructs containing site‐specifically incorporated photoactivatable unnatural amino acids (UAAs) along the solvent‐exposed surface of the TPR domain to covalently capture and map OGT’s interactome.

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Elucidating the protein substrate recognition of O-GlcNAc transferase (OGT) toward O-GlcNAcase (OGA) using a GlcNAc electrophilic probe

Cited by 15 publications

References 77 publications

Truncation of the TPR domain of OGT alters substrate and glycosite selection

Truncation of the TPR domain of OGT alters substrate and glycosite selection

Protein Substrates Engage the Lumen of O-GlcNAc Transferase’s Tetratricopeptide Repeat Domain in Different Ways

Photoactivatable O‐GlcNAc Transferase Library Enables Covalent Chemical Capture of Solvent‐Exposed TPR Domain Interactions

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