OGT Binding Peptide-Tagged Strategy Increases Protein O-GlcNAcylation Level in E. coli

Abstract

O-GlcNAcylation is a single glycosylation of GlcNAc mediated by OGT, which regulates the function of substrate proteins and is closely related to many diseases. However, a large number of O-GlcNAc-modified target proteins are costly, inefficient, and complicated to prepare. In this study, an OGT binding peptide (OBP)-tagged strategy for improving the proportion of O-GlcNAc modification was established successfully in E. coli. OBP (P1, P2, or P3) was fused with target protein Tau as tagged Tau. Tau or tagged Tau was co-constructed with OGT into a vector expressed in E. coli. Compared with Tau, the O-GlcNAc level of P1Tau and TauP1 increased 4~6-fold. Moreover, the P1Tau and TauP1 increased the O-GlcNAc-modified homogeneity. The high O-GlcNAcylation on P1Tau resulted in a significantly slower aggregation rate than Tau in vitro. This strategy was also used successfully to increase the O-GlcNAc level of c-Myc and H2B. These results indicated that the OBP-tagged strategy was a successful approach to improve the O-GlcNAcylation of a target protein for further functional research.

Regulation of Glycogen Synthase Kinase-3β by Phosphorylation and O-β-Linked N-Acetylglucosaminylation: Implications on Tau Protein Phosphorylation

“…Together with the limited impact on overall O-GlcNAc levels, this means that the OBP tag does not improve the levels of site-specific O-GlcNAc modifications nor the O-GlcNAcylation homogeneity in conditions involving purified OGT in solution in contrast with what was successfully described for coexpression of target proteins with OGT in E. coli. 72 Accordingly, we have observed a significant increase of GSK3β O-GlcNAcylation level upon coexpression with OGT in bacteria (Figure S4). Therefore, it is likely that the OBP tag could favor the formation of O-GlcNAcylation complexes with OGT and target proteins by recruiting regulatory proteins.…”

“…Indeed, this strategy was shown to promote O-GlcNAcylation of tau and other target proteins coexpressed with OGT in E. coli and improve the homogeneity of O-GlcNAc modifications to further explore O-GlcNAc functional implications . The OBP-GSK3β (noted o G) and GSK3β-OBP (noted G o ) proteins in which an OBP-tag was fused either at the N- or C-terminus of GSK3β, respectively, were expressed in E.…”

“…Indeed, this strategy was shown to promote O-GlcNAcylation of tau and other target proteins coexpressed with OGT in E. coli and improve the homogeneity of O-GlcNAc modifications to further explore O-GlcNAc functional implications. 72 The OBP-GSK3β (noted oG) and GSK3β-OBP (noted Go) proteins in which an OBP-tag was fused either at the N-or C-terminus of GSK3β, respectively, were expressed in E. coli as C-terminal polyhistidine tagged proteins and purified in the same way than non-OBP tagged GSK3β (Figure S2A−C). In a kinase assay with OBP GSK3β fusion proteins, we first validated that the OBP tag had no significant impact on GSK3β kinase activity (Figure S2D).…”

Exploiting O-GlcNAc transferase promiscuity to dissect site-specific O-GlcNAcylation

“…O-GlcNAcylation modulates diverse cellular and developmental processes, from stem cell pluripotency through to bioenergetics (Jang, Kim et al 2012, Zhu, Zhou et al 2022, Co-expression of OGT with protein substrates has previously been used to study the effects of O-GlcNAc on Tau and malate dehydrogenase 1, among other targets (Gao, Shi et al 2018, Zhu, Zhou et al 2022, Li, Yang et al 2023. This method provides a considerable simplicity over the most demanding and comparatively expensive chemical alternatives only affordable by a handful of very specialised laboratories worldwide (Dadová, Galan et al 2018, Balana, Levine et al 2021).…”

Exploiting O-GlcNAc transferase promiscuity to dissect site-specific O-GlcNAcylation

“…The use of bacterial systems to co-express OGT with recombinant protein substrates has also been explored (Shen, Gloster et al 2012, Gao, Shi et al 2018, Li, Yang et al 2023. This approach takes advantage of the lack of OGT in the E. coli genome and the absence of bacterial OGT substrates (Lubas, Frank et al 1997).…”