Functional expression of O-linked GlcNAc transferase. Domain structure and substrate specificity

Area of Science:

• Biochemistry
• Molecular Biology
• Cellular Signaling

Background:

• O-GlcNAc transferase (OGT) is a key enzyme involved in post-translational modification of proteins.
• Dysregulation of OGT is implicated in metabolic disorders like insulin resistance.
• Understanding OGT's structure-function relationship is crucial for therapeutic development.

Purpose of the Study:

• To produce and characterize human OGT in a heterologous system for detailed analysis.
• To investigate the role of specific domains, particularly the tetratricopeptide repeat (TPR) domain, in OGT activity.
• To identify OGT's kinase substrates involved in glucose-sensitive signaling pathways.

Main Methods:

• Expression and purification of 103-kDa human OGT in Escherichia coli.
• Kinetic analysis of purified recombinant OGT using Nup 62 and UDP-GlcNAc.
• Site-directed mutagenesis to assess the function of C-terminal deletions and N-terminal TPR domains.
• In vitro glycosylation assays using various kinase substrates.

Main Results:

• Purified recombinant human OGT exhibited kinetic parameters similar to native mammalian OGT.
• The C-terminal region is essential for OGT activity, while the N-terminal TPR domain is critical for macromolecular substrate recognition.
• Removal of the first three TPRs reduced activity on macromolecular substrates but not synthetic peptides, indicating domain-specific roles.
• OGT was found to glycosylate glycogen synthase kinase-3 and casein kinase II, enzymes critical for glycogen synthesis.

Conclusions:

• The recombinant OGT system provides a valuable tool for studying OGT structure-function relationships.
• Specific domains of OGT play distinct roles in substrate binding and catalytic activity.
• OGT directly modifies key kinases in glucose metabolism, highlighting its role in insulin signaling and potential therapeutic targeting.