Structure of an O-GlcNAc transferase homolog provides insight into intracellular glycosylation

Abstract

N-Acetylglucosamine (O-GlcNAc) modification of proteins provides a mechanism for the control of diverse cellular processes through a dynamic interplay with phosphorylation. UDP-GlcNAc:polypeptidyl transferase (OGT) catalyzes O-GlcNAc addition. The structure of an intact OGT homolog and kinetic analysis of human OGT variants reveal a contiguous superhelical groove that directs substrates to the active site.

Drosophila O -GlcNAc transferase (OGT) is encoded by the Polycomb group (PcG) gene, super sex combs ( sxc )

“…In sxc 1 /sxc 1 and sxc 5 /sxc 5 embryos, we identified nonsense mutations in Ogt that would result in expression of truncated enzyme with a resulting loss of function, while sxc 4 /sxc 4 embryos showed a missense mutation of a highly conserved N residue to I, which is located close to the active site (16). In sxc NC130 /sxc NC130 embryos we found a splice acceptor mutation (AG to AA) that should result in an aberrant transcript and production of truncated enzyme.…”

Insights into O-Linked N-Acetylglucosamine ([0-9]O-GlcNAc) Processing and Dynamics through Kinetic Analysis of O-GlcNAc Transferase and O-GlcNAcase Activity on Protein Substrates

“…The gene encoding Nup62 and the region of the pMAL-c2X vector encoding the MBP tag (Nup62-MBP) were then subcloned into a pET28a vector to give higher levels of Nup62 expression with the desired antibiotic resistance for the co-expression system (see below). The plasmid encoding hOGT, which has been described previously (28), was subcloned into the pMAL-c2X vector using the primers listed in supplemental Table 1. The plasmids encoding hOGA (and mutants) and Bacteroides thetaiotaomicron OGA (BtGH84) have been described previously (31,32).…”

“…Human OGT Kinetics on TAB1, CaMKIV, CARM1, Nup62, and Tau-We next performed hOGT kinetics on the protein substrates we had examined with hOGA using an established radioactive hOGT assay (28). First, an appropriate hOGT concentration to use and an assay period over which hOGT activity remained linear needed to be established; we found that linear rates were observed when using hOGT concentrations of between 100 and 500 nM over an assay time of 60 min (supplemental Fig.…”

“…Indeed, quantitative kinetic studies of the activity of OGT on protein substrates have been limited to investigating the modification of only one protein, Rattus norvegicus nucleoporin 62 (Nup62) (18,28,29). With respect to OGA, there is a greater paucity of kinetic studies.…”

Dynamic O-GlcNAcylation and its roles in the cellular stress response and homeostasis

“…Two functional domains characterize OGT: an N-terminal tetratricopeptide repeat (TPR domain) and a C-terminal catalytic domain belonging to the glycogen phosphorylase superfamily (Kreppel et al 1997;Lubas et al 1997;Wrabl and Grishin 2001). Recently, the structure of OGT and the TPR domain have been solved (Lazarus et al 2011;Martinez-Fleites et al 2008;Jinek et al 2004). The TPR domain forms an extended alpha-helix similar to importin-α (Jinek et al 2004), and is important for mediating protein-protein interactions and enzyme activity (Kreppel and Hart 1999;Lubas and Hanover 2000).…”