O-GlcNAc Transferase Recognizes Protein Substrates Using an Asparagine Ladder in the Tetratricopeptide Repeat (TPR) Superhelix

Journal of the American Chemical Society
Zebulon G LevineSuzanne Walker

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 developed a protein microarray assay that chemoenzymatically labels de novo sites of glycosylation with biotin, allowing us to simultaneously assess OGT activity across >6000 human proteins. With this assay we examined the contribution to substrate selection of a conserved asparagine ladder within the lumen of OGT's superhelical tetratricopeptide repeat (TPR) domain. When five asparagines were mutated, OGT retained significant activity against short peptides, but showed limited limited glycosylation of protein substrates on the microarray. O-GlcNAcylation of protein substrates in cell extracts was also greatly attenuated. We conclude that OGT recognizes the majority of its substrates by binding them to the asparagine ladder in the TPR lumen proximal to the catalytic domain.

Citations

Sep 11, 2018·Chembiochem : a European Journal of Chemical Biology·Chia-Wei HuJiaoyang Jiang
May 15, 2020·Chembiochem : a European Journal of Chemical Biology·Eun Ju Kim
Apr 4, 2019·Proceedings of the National Academy of Sciences of the United States of America·Xuexia LiWen Yi
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Jan 3, 2020·Neuromolecular Medicine·Jinsu ParkDong-Gyu Jo
Jun 15, 2019·International Journal of Molecular Medicine·Yu LiJianling Du
Jan 10, 2021·Proceedings of the National Academy of Sciences of the United States of America·Zebulon G LevineSuzanne Walker
Dec 18, 2020·International Journal of Biological Macromolecules·Adam KositzkeJiaoyang Jiang
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