Substrate Recognition of Glycoprotein Folding Sensor UGGT Analyzed by Site-Specifically 15N-Labeled Glycopeptide and Small Glycopeptide Library Prepared by Parallel Native Chemical Ligation
Substrate Recognition of Glycoprotein Folding Sensor UGGT Analyzed by Site-Specifically 15N-Labeled Glycopeptide and Small Glycopeptide Library Prepared by Parallel Native Chemical Ligation
复制标题
DOI：
10.1021/jacs.7b03277
复制
发表时间：
2017-08-23
影响因子：
15
通讯作者：
Kajihara, Yasuhiro
中科院分区：
文献类型：
--
作者：
Izumi, Masayuki;Kuruma, Rie;Kajihara, Yasuhiro
UDP-葡萄糖：糖蛋白葡萄糖基转移酶 (UGGT) 可区分非天然构象的糖蛋白和天然构象的糖蛋白，以及仅区分非天然糖蛋白的葡萄糖基化物。为了分析 UGGT 如何识别非天然糖蛋白，我们化学合成了位点特异性 N-15 标记的白细胞介素 8 (IL-8) C 末端 (34-72) 糖肽，其带有 Man(9)GlcNAc(2) (M9) 寡糖。化学位移微扰图谱NMR实验表明糖肽的Phe65与UGGT特异性相互作用。为了分析这种相互作用，我们通过糖肽-α-硫酯和由 11 种肽组成的肽库之间的平行天然化学连接，通过改变 Phe65 与 10 个其他天然氨基酸来构建糖肽库。针对糖肽文库的 UGGT 测定表明，尽管疏水性较低的糖肽可用作 UGGT 的底物，但疏水性糖肽是优选的。
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.