Abstract
Glycosylation is increasingly recognised as a common protein modification within bacterial proteomes. While great strides have been made in identifying species that contain glycosylation systems, our understanding of the proteins and sites targeted by these systems is far more limited. Within this work we explore the conservation of glycoproteins and glycosylation sites across the pan-Burkholderia glycoproteome. Using a multi-protease glycoproteomic approach, we generate high-confidence glycoproteomes in two widely utilized B. cenocepacia strains, K56-2 and H111. This resource reveals glycosylation occurs exclusively at Serine residues and that glycoproteins/glycosylation sites are highly conserved across B. cenocepacia isolates. This preference for glycosylation at Serine residues is observed across at least 9 Burkholderia glycoproteomes, supporting that Serine is the dominant residue targeted by PglL-mediated glycosylation across the Burkholderia genus. Combined, this work demonstrates that PglL enzymes of the Burkholderia genus are Serine-preferring oligosaccharyltransferases that target conserved and shared protein substrates.
Highlights
Glycosylation is increasingly recognised as a common protein modification within bacterial proteomes
We noted that the physiochemical properties of peptides heavily influences the ability of ZIC-HILIC enrichment to isolate bacterial glycopeptides[49]. This observation suggests numerous glycoproteins have likely been overlooked when previous studies used Trypsin alone to assess the glycoproteome of B. cenocepacia[13,29]
Glycopeptide enrichments of Trypsin, Thermolysin, and Pepsin digested samples enabled the identification of 584 and 666 unique glycopeptides from K56-2 and H111 strains respectively (Fig. 1a, Supplementary Data 1 and 2). These glycopeptide datasets identified more glycoproteins than identified within previously published studies using the ET12 B. cenocepacia strain J2315 (Supplementary Fig. 1a13,29), the majority of identified glycoproteins were unique to a single strain (Supplementary Fig. 1b)
Summary
Glycosylation is increasingly recognised as a common protein modification within bacterial proteomes. Using a multi-protease glycoproteomic approach, we generate high-confidence glycoproteomes in two widely utilized B. cenocepacia strains, K56-2 and H111 This resource reveals glycosylation occurs exclusively at Serine residues and that glycoproteins/glycosylation sites are highly conserved across B. cenocepacia isolates. With the aid of mass spectrometry-based proteomics, a range of bacterial glycosylation systems have been identified with many of these demonstrated to be conserved across genera[1,2,10,11] Within these systems follow up studies have mostly focused on understanding the glycans used for protein glycosylation, defining their diversity[12,13,14] and elucidating their biosynthetic pathways[15,16,17]. This limited understanding of bacterial O-linked glycosylation sites is largely driven by technical limitations associated with the analysis of O-linked glycopeptides, where the chemical nature of O-linked glycosylation requires approaches such as electron-transfer dissociation (ETD) or Electron-transfer/
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