title: Defining the enzymatic pathway for polymorphicO-glycosylation of the pneumococcal serine-rich repeat protein PsrP.
abstract: ProteinO-glycosylation is an important post-translational modification in all organisms, but deciphering the specific functions of these glycans is difficult due to their structural complexity. Understanding the glycosylation of mucin-like proteins presents a particular challenge as they are modified numerous times with both the enzymes involved and the glycosylation patterns being poorly understood. Here we systematically explored theO-glycosylation pathway of a mucin-like serine-rich repeat protein PsrP from the human pathogenStreptococcus pneumoniaeTIGR4. Previous works have assigned the function of 3 of the 10 glycosyltransferases thought to modify PsrP, GtfA/B, and Gtf3 as catalyzing the first two reactions to form a unified disaccharide core structure. We now usein vivoandin vitroglycosylation assays combined with hydrolytic activity assays to identify the glycosyltransferases capable of decorating this core structure in the third and fourth steps of glycosylation. Specifically, the full-length GlyE and GlyG proteins and the GlyD DUF1792 domain participate in both steps, whereas full-length GlyA and the GlyD GT8 domain catalyze only the fourth step. Incorporation of different sugars to the disaccharide core structure at multiple sites along the serine-rich repeats results in a highly polymorphic product. Furthermore, crystal structures of apo- and UDP-complexed GlyE combined with structural analyses reveal a novel Rossmann-fold "add-on" domain that we speculate to function as a universal module shared by GlyD, GlyE, and GlyA to forward the peptide acceptor from one enzyme to another. These findings define the complete glycosylation pathway of a bacterial glycoprotein and offer a testable hypothesis of how glycosyltransferase coordination facilitates glycan assembly.
authors.affiliation: From the Hefei National Laboratory for Physical Sciences at the Microscale and School of Life Sciences, University of Science and Technology of China and.|Key Laboratory of Structural Biology, Chinese Academy of Science, Hefei, Anhui 230027, China.
authors.forename: Yong-Liang
authors.initials: YL
authors.lastname: Jiang
authors.affiliation: From the Hefei National Laboratory for Physical Sciences at the Microscale and School of Life Sciences, University of Science and Technology of China and.|Key Laboratory of Structural Biology, Chinese Academy of Science, Hefei, Anhui 230027, China.
authors.forename: Hua
authors.initials: H
authors.lastname: Jin
authors.affiliation: From the Hefei National Laboratory for Physical Sciences at the Microscale and School of Life Sciences, University of Science and Technology of China and.|Key Laboratory of Structural Biology, Chinese Academy of Science, Hefei, Anhui 230027, China.
authors.forename: Hong-Bo
authors.initials: HB
authors.lastname: Yang
authors.affiliation: From the Hefei National Laboratory for Physical Sciences at the Microscale and School of Life Sciences, University of Science and Technology of China and.|Key Laboratory of Structural Biology, Chinese Academy of Science, Hefei, Anhui 230027, China.
authors.forename: Rong-Li
authors.initials: RL
authors.lastname: Zhao
authors.affiliation: From the Hefei National Laboratory for Physical Sciences at the Microscale and School of Life Sciences, University of Science and Technology of China and.
authors.forename: Shiliang
authors.initials: S
authors.lastname: Wang
authors.affiliation: From the Hefei National Laboratory for Physical Sciences at the Microscale and School of Life Sciences, University of Science and Technology of China and cyxing@ustc.edu.cn.|Key Laboratory of Structural Biology, Chinese Academy of Science, Hefei, Anhui 230027, China.
authors.forename: Yuxing
authors.initials: Y
authors.lastname: Chen
authors.affiliation: From the Hefei National Laboratory for Physical Sciences at the Microscale and School of Life Sciences, University of Science and Technology of China and zcz@ustc.edu.cn.|Key Laboratory of Structural Biology, Chinese Academy of Science, Hefei, Anhui 230027, China.
authors.forename: Cong-Zhao
authors.initials: CZ
authors.lastname: Zhou
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D000072417:Protein Domains; D013296:Streptococcus pneumoniae / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D006023:Glycoproteins; D016695:Glycosyltransferases
keywords: Streptococcus pneumoniae; bacterial pathogenesis; crystal structure; enzyme; glycosylation; glycosyltransferase; polymorphism; serine-rich repeat protein