Project name: O-GlcNAc transferase recognizes protein substrates using an asparagine ladder in the TPR superhelix

Description: Abstract: The essential mammalian enzyme O-GlcNAc Transferase (OGT) is uniquely responsible for transferring N-acetylglucosamine to over a thousand nuclear and cytoplasmic proteins, yet there is no known consensus sequence and it remains unclear how OGT recognizes its substrates. To address this question, we have developed a protein microarray assay that chemoenzymatically labels de novo sites of glycosylation with biotin, allowing us to simultaneously as-sess OGT activity across >6000 human proteins. We used this assay to examine the contribution of a conserved asparagine ladder within the lumen of OGT’s superhelical tetratri-copeptide repeat (TPR) domain to substrate selection. When these residues were mutated, OGT retained full activity against short peptides, but showed low to no activity against most of the OGT substrates on the microarray. O-GlcNAcylation of protein substrates in cell extracts was also greatly attenuated. We conclude that OGT recognizes a majority of its substrates by binding them to the asparagine ladder in the TPR lumen proximal to the catalytic domain.This series contains microarray data both comparing the new chemoenzymatic method to antibody-based detection as well as comparing arrays treated with wild-type OGT, 5N5A mutant OGT, or controls not treated with enzyme. Note: all CTD-stained arrays or control array raw files are contained in GSE107911_RAW.tarOverall 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. Two microarrays were treated with or without OGT and O-GlcNAc was detected by the antibody CTD110.6, 8 microarrays were treated with wild-type OGT and UDP-GlcNAz, followed by chemical labeling with biotin, 7 microarrays were treated with UDP-GlcNAz and biotin but no enzyme as controls, and 3 microarrays were treated with UDP-GlcNAz and an OGT mutant dubbed 5N5A, followed by biotinylation. Individual arrays and treatments are described below in samplesPlease note that raw data files associated with multiple analyses/sample records are available as Series supplementary file and are indicated in the corresponding sample description field.

Data type: Proteome

Sample scope: Multiisolate

Relevance: Medical

Last updated: 2017-12-11