Defining the enzymatic pathway for polymorphic O-glycosylation of the pneumococcal serine-rich repeat protein PsrP

Abstract

Protein O-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 the O-glycosylation pathway of a mucin-like serine-rich repeat protein PsrP from the human pathogen Streptococcus pneumoniae TIGR4. 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 use in vivo and in vitro glycosylation 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.

Keywords: Streptococcus pneumoniae; bacterial pathogenesis; crystal structure; enzyme; glycosylation; glycosyltransferase; polymorphism; serine-rich repeat protein.

Figure 1.

The SRRP loci. A, SRRP genes are colored in red, whereas gtfA and gtfB are colored in blue. Arrows indicate the direction of transcription. B, domain organization of PsrP. The BR represents the ligand-binding region of PsrP. The sequence of SRR1 is shown, and the residues for glycosylation assays in this study are colored in red. C, a scheme for the previously identified glycosylation pathway of PsrP. D, domain organizations of the glycosyltransferases encoded by the psrP gene cluster.

Figure 2.

The hydrolytic activities toward UDP-Glc and UDP-Gal of putative glycosyltransferases encoded by the psrP gene cluster. The reaction lasted for 60 min at 37 °C in the presence of 10 μm enzyme and 1 mm UDP-Glc or UDP-Gal. The velocities were calculated by determining the production of UDP (μm) per minute. Data are presented as the means ± S.D. from three independent assays. Two-tailed Student's t test was used for the comparison of statistical significance. p values of <0.05 and <0.01 are indicated with *, and **, respectively.

Figure 3.

The third-step glycosylation: glycosylation of SRR1-GlcNAc-Glc. The glycosyltransferase activity assays of putative enzymes and mutants using UDP-[³H]glucose or UDP-[³H]galactose as the sugar donor are shown in A–D, respectively. Separation of the reaction mixture was performed by SDS-PAGE (upper column), which was further detected by ³H autoradiography (lower column). GST-SRR1 was labeled, whereas the other bonds in the SDS-PAGE correspond to different glycosyltransferases. Augmentation of hydrolytic activities in the presence of SRR1-GlcNAc or SRR1-GlcNAc-Glc toward UDP-Glc (E) or UDP-Gal (F).

Figure 4.

The fourth-step glycosylation of PsrP. The glycosyltransferase activities were performed using the substrates SRR1-GlcNAc-Glc-Glc and UDP-[³H]glucose (A) or UDP-[³H]galactose (B) or SRR1-GlcNAc-Glc-Gal, and UDP-[³H]glucose (C) or UDP-[³H]galactose (D). Augmentation of hydrolytic activities in the presence of trisaccharide-modified SRR1 toward UDP-Glc (E) or UDP-Gal (F).

Figure 5.

The fourth-step glycosylation of PsrP using the mixed acceptors of trisaccharide-modified SRR1 (SRR1-GlcNAc-Glc-Glc and SRR1-GlcNAc-Glc-Gal) in the presence of UDP-[³H]glucose (A) or UDP-[³H]galactose (B).

Figure 6.

Overall structure and active-site pocket of GlyE. A, schematic representation of GlyE with the secondary structural elements labeled sequentially. The UDP molecule is shown as sticks and Mn²⁺ is presented as a sphere. The GT8 domain is colored in cyan, whereas the add-on domain is colored in red. B, the substrate-binding pocket. The UDP-binding residues are shown as sticks, and the putative acceptor-binding groove is indicated as a dotted black line. C, the binding site of UDP. The UDP molecule and UDP-binding residues are shown as sticks, whereas the Mn²⁺ is shown as a sphere. The polar interactions are indicated as dashed lines. D, the hydrolytic activities of the wild-type GlyE and mutants from the UDP-binding pocket. E, structural comparison of the add-on domain of GlyE (red) against the C-terminal Rossmann-fold domain of GtfB (light blue). The putative acceptor-binding residues of GlyE and GtfB are shown as sticks. F, the glycosyltransferase activities of the wild-type GlyE and mutants of acceptor-binding residues in the presence of SRR1-GlcNAc-Glc. The p values of < 0.01 and 0.001 are indicated with ** and ***, respectively. G, structure-based sequence alignment of the shared add-on domains within the GT8 glycosyltransferases and GtfB. The secondary structural elements of GlyE and Gtf3 are labeled on the top and at the bottom, respectively. The putative acceptor-binding residues are marked with red spheres.

Figure 7.

A proposed pathway for the heavy O-glycosylation of PsrP.