Year-end Sale % OFF 
Abstract
Serine-rich repeat glycoproteins (SRRPs) conserved in streptococci and staphylococci are important for bacterial colonization and pathogenesis. Fap1, a well studied SRRP is a major surface constituent of Streptococcus parasanguinis and is required for bacterial adhesion and biofilm formation. Biogenesis of Fap1 is a multistep process that involves both glycosylation and secretion. A series of glycosyltransferases catalyze sequential glycosylation of Fap1. We have identified a unique hybrid protein dGT1 (dual glycosyltransferase 1) that contains two distinct domains. N-terminal DUF1792 is a novel GT-D-type glycosyltransferase, transferring Glc residues to Glc-GlcNAc-modified Fap1. C-terminal dGT1 (CgT) is predicted to possess a typical GT-A-type glycosyltransferase, however, the activity remains unknown. In this study, we determine that CgT is a distinct glycosyltransferase, transferring GlcNAc residues to Glc-Glc-GlcNAc-modified Fap1. A 2.4-Å x-ray crystal structure reveals that CgT has a unique binding domain consisting of three α helices in addition to a typical GT-A-type glycosyltransferase domain. The helical domain is crucial for the oligomerization of CgT. Structural and biochemical studies revealed that the helix domain is required for the protein-protein interaction and crucial for the glycosyltransferase activity of CgT in vitro and in vivo. As the helix domain presents a novel structural fold, we conclude that CgT represents a new member of GT-A-type glycosyltransferases.
Highlights
Protein glycosylation is one of the most common post-translational modifications
RFap1R1 was co-expressed with N-terminal DUF1792, C-terminal dGT1 (CgT), or the fulllength dGT1 in addition to three glycosyltransferases, Gtf1, Gtf2, and Gtf3
DGT1 with deletion of a helical motif failed to Glycosylation catalyzed by a series of glycosyltransferases is crucial for biogenesis of SRRP adhesins in Gram-positive bacteria [7]. dGT1 is a unique bifunctional protein involved in the biosynthesis of an SRRP adhesin Fap1 of S. parasanguinis
Summary
CgT Plays an Important Role in the Fap Glycosylation— dGT1 contains an N-terminal unknown function domain (DUF1792) and C-terminal putative glycosyltransferase domain (CgT). When only the full-length dGT1 (Fig. 1, lane 4) was used the recombinant Fap was further super-shifted to two higher molecular weight positions, indicating that the glycosylation of rFap1RI by DUF1792 is a prerequisite for the subsequent modification by CgT. Previous and current studies indicate that DUF1792-modified rFap1R1 (Fig. 2A) carries a trisaccharide with a sequence of Glc-Glc-GlcNAc and that rFap1R1 can only carry Glc as the hexose and GlcNAc as the HexNAc. the presence of an additional HexNAc in the dGT1-modified glycan suggests that the C-terminal dGT1 catalyzes the transfer of GlcNAc to the Gtf123- and DUF1792-modified Fap, so we hypothesized that CgT has the activity to transfer GlcNAc. To test this, we performed an in vitro glycosyltransferase assay using the UDP-Glo method (Promega) [29]. Total reflections Unique reflections Multiplicity Completeness (%) Mean I/ (I) Wilson B-factor CC1/2 Rmerge
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
