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

Abstract: O-GlcNAc is an abundant post-translational modification found on nuclear and cytoplasmic proteins in all metazoans. This modification regulates a wide variety of cellular processes, and elevated O-GlcNAc levels have been implicated in cancer progression. A single essential enzyme, O-GlcNAc transferase (OGT), is responsible for all nucleocytoplasmic O-GlcNAcylation. Understanding how this enzyme chooses its substrates is critical for understanding, and potentially manipulating, its functions. Here we use protein microarray technology and proteome-wide glycosylation profiling to show that conserved aspartate residues in the tetratricopeptide repeat (TPR) lumen of OGT drive substrate selection. Changing these residues to alanines alters substrate selectivity and unexpectedly increases rates of protein glycosylation. Our findings support a model where sites of glycosylation for many OGT substrates are determined by TPR domain contacts to substrate side chains five to fifteen residues C-terminal to the glycosite. In addition to guiding design of inhibitors that target OGT's TPR domain, this information will inform efforts to engineer substrates to explore biological functions. Overall design: In this study, Invitrogen's Human ProtoArray protein microarray was used as a platform for high-throughput enzyme activity profiling. Multiple batches of microarrays were used over the course of the study, thus the platform represents a composite of the proteins on all microarray batches. 3 microarrays each were treated with wild-type OGT, the mutant dubbed D2A, or treated with reagents without enzyme. Enzyme microarrays were normalized 2 different ways; both modes of normalization are represented here.