O-GlcNAc Engineering of GPCR Peptide-Agonists Improves Their Stability and in Vivo Activity

Paul M. Levine*, Aaron T. Balana, Emmanuel Sturchler, Cassandra Koole, Hiroshi Noda, Barbara Zarzycka, Eileen J. Daley, Tin T. Truong, Vsevolod Katritch, Thomas J. Gardella, Denise Wootten, Patrick M. Sexton, Patricia McDonald, Matthew R. Pratt

*Corresponding author for this work

Research output: Contribution to JournalArticleAcademicpeer-review


Peptide agonists of GPCRs and other receptors are powerful signaling molecules with high potential as biological tools and therapeutics, but they are typically plagued by instability and short half-lives in vivo. Nature uses protein glycosylation to increase the serum stability of secreted proteins. However, these extracellular modifications are complex and heterogeneous in structure, making them an impractical solution. In contrast, intracellular proteins are subjected to a simple version of glycosylation termed O-GlcNAc modification. In our studies of this modification, we found that O-GlcNAcylation inhibits proteolysis, and strikingly, this stabilization occurs despite large distances in primary sequence (10-15 amino acids) between the O-GlcNAc and the site of cleavage. We therefore hypothesized that this "remote stabilization" concept could be useful to engineer the stability and potentially additional properties of peptide or protein therapeutics. Here, we describe the application of O-GlcNAcylation to two clinically important peptides: Glucagon-like peptide-1 (GLP-1) and the parathyroid hormone (PTH), which respectively help control glucose and calcium levels in the blood. For both peptides, we found O-GlcNAcylated analogs that are equipotent to unmodified peptide in cell-based activation assays, while several GLP-1 analogs were biased agonists relative to GLP-1. As we predicted, O-GlcNAcylation can improve the stability of both GLP-1 and PTH in serum despite the fact that the O-GlcNAc can be quite remote from characterized sites of peptide cleavage. The O-GlcNAcylated GLP-1 and PTH also displayed significantly improved in vivo activity. Finally, we employed structure-based molecular modeling and receptor mutagenesis to predict how O-GlcNAcylation can be accommodated by the receptors and the potential interactions that contribute to peptide activity. This approach demonstrates the potential of O-GlcNAcylation for generating analogs of therapeutic peptides with enhanced proteolytic stability.

Original languageEnglish
Pages (from-to)14210-14219
Number of pages10
JournalJournal of the American Chemical Society
Issue number36
Publication statusPublished - 11 Sept 2019
Externally publishedYes


This research was supported by the National Institutes of Health (R01GM114537 to M.R.P and P01-DK11794 and P30-AR066261 to T.J.G.), the University of Southern California, The Scripps Research Institute, and the National Health and Medical Research Council of Australia (NHMRC) (1157539 and 1126857 to D.W. and 1150083 to P.M.S.). Circular dichroism was performed at the USC Nano Biophysics Core Facility. P.M.S. is a NHMRC Senior Principal Research Fellow, and D.W. is a NHMRC Senior Research Fellow.

FundersFunder number
National Institutes of HealthP01-DK11794, P30-AR066261
National Institute of General Medical SciencesR01GM114537
University of Southern California
Center for Outcomes Research and Evaluation, Yale School of Medicine
University of South Carolina
Scripps Research Institute
National Health and Medical Research Council1150083, 1157539, 1126857


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