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Identification of bacterial protein O-oligosaccharyltransferases and their glycoprotein substrates.

Schulz BL, Jen FE, Power PM, Jones CE, Fox KL, Ku SC, Blanchfield JT, Jennings MP - PLoS ONE (2013)

Bottom Line: We show that in the general glycosylation system of N. meningitidis, efficient glycosylation of additional protein substrates requires local structural similarity to the pilin acceptor site.For some Neisserial PglL substrates identified by sensitive analytical approaches, only a small fraction of the total protein pool is modified in the native organism, whereas others are completely glycosylated.Our results show that bacterial protein O-glycosylation is common, and that substrate selection in the general Neisserial system is dominated by recognition of structural homology.

View Article: PubMed Central - PubMed

Affiliation: School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland, Australia.

ABSTRACT
O-glycosylation of proteins in Neisseria meningitidis is catalyzed by PglL, which belongs to a protein family including WaaL O-antigen ligases. We developed two hidden Markov models that identify 31 novel candidate PglL homologs in diverse bacterial species, and describe several conserved sequence and structural features. Most of these genes are adjacent to possible novel target proteins for glycosylation. We show that in the general glycosylation system of N. meningitidis, efficient glycosylation of additional protein substrates requires local structural similarity to the pilin acceptor site. For some Neisserial PglL substrates identified by sensitive analytical approaches, only a small fraction of the total protein pool is modified in the native organism, whereas others are completely glycosylated. Our results show that bacterial protein O-glycosylation is common, and that substrate selection in the general Neisserial system is dominated by recognition of structural homology.

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Glycosylation status of purified AniA-FLAG variants.Extracted ion chromatograms corresponding to the un- (full) mono- (dashed) and di- (dotted) glycosylated versions of the Leu358-Lys387 tryptic peptide containing the AniA glycosylation sites: (A) wild type, (B) S373A, (C) S378A, (D) S373A, S378A, (E) S373A, S378A, S385P. Corresponding variant sequences are shown in Table S6. (F) Proportion of un- (white) mono- (gray) or di- (black) glycosylated versions of each variant shown in (A)–(E), as determined by integration of extracted ion chromatograms. Values are mean, error bars show s.e.m. *, P = 0.01 2-sided Mann-Whitney test.
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pone-0062768-g005: Glycosylation status of purified AniA-FLAG variants.Extracted ion chromatograms corresponding to the un- (full) mono- (dashed) and di- (dotted) glycosylated versions of the Leu358-Lys387 tryptic peptide containing the AniA glycosylation sites: (A) wild type, (B) S373A, (C) S378A, (D) S373A, S378A, (E) S373A, S378A, S385P. Corresponding variant sequences are shown in Table S6. (F) Proportion of un- (white) mono- (gray) or di- (black) glycosylated versions of each variant shown in (A)–(E), as determined by integration of extracted ion chromatograms. Values are mean, error bars show s.e.m. *, P = 0.01 2-sided Mann-Whitney test.

Mentions: We identified the precise sites of glycosylation in the Leu358-Lys387 glycopeptide by site-directed mutagenesis and LC-ESI-MS/MS analysis of peptides and glycopeptides from purified variant glycoproteins. The extent of glycosylation is likely under-estimated by this MS analysis due to reduced ionisation efficiency of the glycosylated peptides relative to their unglycosylated forms. Nonetheless, relative quantification of glycosylation occupancy is possible with this analysis [26], [27]. Up to two sites of glycosylation were detected in wild type AniA (Fig. 5A, Fig. S4 and [3]). As this sequence included two identical repeats of the local sequence Ala-Ala-Ser-Ala-Pro, encompassing Ser373 and Ser378, we created AniA variants with each of these Ser residues individually mutated to Ala (Table S6). LC-ESI-MS/MS analysis of both of these variants showed loss of a single efficiently modified glycosylation site, as these variants showed very low levels of di-glycosylated peptide (Fig. 5B,C,F). Further, a variant with both Ser373Ala and Ser378Ala mutations showed loss of both efficiently used glycosylation sites, as essentially only un-glycosylated peptide was identified (Fig. 5D,F). This confirmed that the two Ser residues present in the local sequence Ala-Ala-Ser-Ala-Pro (Ser373 and Ser378) were efficiently glycosylated by PglL. Several other Ser residues are present in the Leu358-Lys387 glycopeptide, and of particular note were the residues present in an imperfect repeat reminiscent of the efficiently glycosylated sites, Ala-Ala-Ser383-Ala-Ser385-Glu. The local sequence context of Ser383 differed from the efficiently glycosylated Ser373 and Ser378 only by having the sequence Ser383-Ala-Ser rather than Ser373/8-Ala-Pro. We tested if this local sequence influenced glycosylation by creating a Ser385Pro AniA variant in a Ser373Ala, Ser378Ala background. Indeed, this AniA-Ser373Ala, Ser378Ala, Ser385Pro variant showed significantly increased glycosylation compared with the Ser373Ala, Ser378Ala control, with substantial mono-glycosylated peptide detected (2-sided Mann-Whitney test, P = 0.01, Fig. 5E,F).


Identification of bacterial protein O-oligosaccharyltransferases and their glycoprotein substrates.

Schulz BL, Jen FE, Power PM, Jones CE, Fox KL, Ku SC, Blanchfield JT, Jennings MP - PLoS ONE (2013)

Glycosylation status of purified AniA-FLAG variants.Extracted ion chromatograms corresponding to the un- (full) mono- (dashed) and di- (dotted) glycosylated versions of the Leu358-Lys387 tryptic peptide containing the AniA glycosylation sites: (A) wild type, (B) S373A, (C) S378A, (D) S373A, S378A, (E) S373A, S378A, S385P. Corresponding variant sequences are shown in Table S6. (F) Proportion of un- (white) mono- (gray) or di- (black) glycosylated versions of each variant shown in (A)–(E), as determined by integration of extracted ion chromatograms. Values are mean, error bars show s.e.m. *, P = 0.01 2-sided Mann-Whitney test.
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Related In: Results  -  Collection

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pone-0062768-g005: Glycosylation status of purified AniA-FLAG variants.Extracted ion chromatograms corresponding to the un- (full) mono- (dashed) and di- (dotted) glycosylated versions of the Leu358-Lys387 tryptic peptide containing the AniA glycosylation sites: (A) wild type, (B) S373A, (C) S378A, (D) S373A, S378A, (E) S373A, S378A, S385P. Corresponding variant sequences are shown in Table S6. (F) Proportion of un- (white) mono- (gray) or di- (black) glycosylated versions of each variant shown in (A)–(E), as determined by integration of extracted ion chromatograms. Values are mean, error bars show s.e.m. *, P = 0.01 2-sided Mann-Whitney test.
Mentions: We identified the precise sites of glycosylation in the Leu358-Lys387 glycopeptide by site-directed mutagenesis and LC-ESI-MS/MS analysis of peptides and glycopeptides from purified variant glycoproteins. The extent of glycosylation is likely under-estimated by this MS analysis due to reduced ionisation efficiency of the glycosylated peptides relative to their unglycosylated forms. Nonetheless, relative quantification of glycosylation occupancy is possible with this analysis [26], [27]. Up to two sites of glycosylation were detected in wild type AniA (Fig. 5A, Fig. S4 and [3]). As this sequence included two identical repeats of the local sequence Ala-Ala-Ser-Ala-Pro, encompassing Ser373 and Ser378, we created AniA variants with each of these Ser residues individually mutated to Ala (Table S6). LC-ESI-MS/MS analysis of both of these variants showed loss of a single efficiently modified glycosylation site, as these variants showed very low levels of di-glycosylated peptide (Fig. 5B,C,F). Further, a variant with both Ser373Ala and Ser378Ala mutations showed loss of both efficiently used glycosylation sites, as essentially only un-glycosylated peptide was identified (Fig. 5D,F). This confirmed that the two Ser residues present in the local sequence Ala-Ala-Ser-Ala-Pro (Ser373 and Ser378) were efficiently glycosylated by PglL. Several other Ser residues are present in the Leu358-Lys387 glycopeptide, and of particular note were the residues present in an imperfect repeat reminiscent of the efficiently glycosylated sites, Ala-Ala-Ser383-Ala-Ser385-Glu. The local sequence context of Ser383 differed from the efficiently glycosylated Ser373 and Ser378 only by having the sequence Ser383-Ala-Ser rather than Ser373/8-Ala-Pro. We tested if this local sequence influenced glycosylation by creating a Ser385Pro AniA variant in a Ser373Ala, Ser378Ala background. Indeed, this AniA-Ser373Ala, Ser378Ala, Ser385Pro variant showed significantly increased glycosylation compared with the Ser373Ala, Ser378Ala control, with substantial mono-glycosylated peptide detected (2-sided Mann-Whitney test, P = 0.01, Fig. 5E,F).

Bottom Line: We show that in the general glycosylation system of N. meningitidis, efficient glycosylation of additional protein substrates requires local structural similarity to the pilin acceptor site.For some Neisserial PglL substrates identified by sensitive analytical approaches, only a small fraction of the total protein pool is modified in the native organism, whereas others are completely glycosylated.Our results show that bacterial protein O-glycosylation is common, and that substrate selection in the general Neisserial system is dominated by recognition of structural homology.

View Article: PubMed Central - PubMed

Affiliation: School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland, Australia.

ABSTRACT
O-glycosylation of proteins in Neisseria meningitidis is catalyzed by PglL, which belongs to a protein family including WaaL O-antigen ligases. We developed two hidden Markov models that identify 31 novel candidate PglL homologs in diverse bacterial species, and describe several conserved sequence and structural features. Most of these genes are adjacent to possible novel target proteins for glycosylation. We show that in the general glycosylation system of N. meningitidis, efficient glycosylation of additional protein substrates requires local structural similarity to the pilin acceptor site. For some Neisserial PglL substrates identified by sensitive analytical approaches, only a small fraction of the total protein pool is modified in the native organism, whereas others are completely glycosylated. Our results show that bacterial protein O-glycosylation is common, and that substrate selection in the general Neisserial system is dominated by recognition of structural homology.

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