Journal of the American Chemical Society, Vol.139, No.33, 11421-11426, 2017
Substrate Recognition of Glycoprotein Folding Sensor UGGT Analyzed by Site-Specifically N-15-Labeled Glycopeptide and Small Glycopeptide Library Prepared by Parallel Native Chemical Ligation
UDP-glucose:glycoprotein glucosyltransferase (UGGT) distinguishes glycoproteins in non-native conformations from those in native conformations and glucosylates from only non-native glycoproteins. To analyze how UGGT recognizes non-native glycoproteins, we chemically synthesized site-specifically N-15-labeled interleukin 8 (IL-8) C-terminal (34-72) glycopeptides bearing a Man(9)GlcNAc(2) (M9) oligosaccharide. Chemical shift perturbation mapping NMR experiments suggested that Phe65 of the glycopeptide specifically interacts with UGGT. To analyze this interaction, we constructed a glycopeptide library by varying Phe65 with 10 other natural amino acids, via parallel native chemical ligation between a glycopeptide-alpha-thioester and a peptide library consisting of 11 peptides. UGGT assay against the glycopeptide library revealed that, although less hydrophobic glycopeptides could be used as substrates for UGGT, hydrophobic glycopeptides are preferred.