Aspartate Residues Far from the Active Site Drive O-GlcNAc Transferase Substrate Selection

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• O-GlcNAc is a crucial post-translational modification regulating numerous cellular processes.
• Elevated O-GlcNAc levels are linked to cancer progression.
• O-GlcNAc transferase (OGT) is the sole enzyme responsible for nucleocytoplasmic O-GlcNAcylation.

Purpose of the Study:

• To elucidate the mechanism of substrate selection by the OGT enzyme.
• To identify specific residues within OGT that dictate substrate specificity.
• To understand how OGT's structure influences protein glycosylation.

Main Methods:

• Utilized protein microarray technology for high-throughput screening.
• Performed proteome-wide glycosylation profiling.
• Employed site-directed mutagenesis to alter conserved aspartate residues in OGT's TPR domain.

Main Results:

• Identified conserved aspartate residues within the tetratricopeptide repeat (TPR) lumen of OGT as critical for substrate selection.
• Demonstrated that mutating these aspartate residues to alanines alters OGT's substrate selectivity.
• Observed an unexpected increase in protein glycosylation rates upon mutation of these residues.

Conclusions:

• Conserved aspartate residues in the OGT TPR domain are key drivers of substrate recognition.
• A model is proposed where TPR domain contacts determine glycosylation sites on substrates.
• Findings provide a basis for designing OGT inhibitors and engineering substrates for functional studies.