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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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Western blot analysis of putative Neisserial glycoproteins.N. gonorrhoeae MS11pglL::kan, N. gonorrhoeae MS11, N. meningitidis C311pglL::kan and N. meningitidis C311 whole cell extracts were separated by SDS-PAGE, blotted to nitrocellulose membrane and probed with (A) α-AniA, (B) α-Laz, (C) α-MetQ, (D) α-Sco, (E) α-Mip or (F) α-CcoP antisera.
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pone-0062768-g004: Western blot analysis of putative Neisserial glycoproteins.N. gonorrhoeae MS11pglL::kan, N. gonorrhoeae MS11, N. meningitidis C311pglL::kan and N. meningitidis C311 whole cell extracts were separated by SDS-PAGE, blotted to nitrocellulose membrane and probed with (A) α-AniA, (B) α-Laz, (C) α-MetQ, (D) α-Sco, (E) α-Mip or (F) α-CcoP antisera.

Mentions: Several PglL substrate glycoproteins in addition to PilE and AniA have been reported in N. gonorrhoeae[4]. However, Western blotting using our α-glycan antisera failed to detect bands in addition to AniA and PilE in N. meningitidis C311 whole cell extracts [3]. To investigate if other glycoproteins were also present in N. meningitidis C311, we performed IP of whole cell extracts using α-glycan antisera, and identified eluted proteins with mass spectrometry. α-Glycan co-IP identified three proteins: PilE, azurin and MetQ (Fig. S1, S2 and S3; Tables S2, S3, S4 and S5). These proteins were not identified by negative control IP with unrelated rabbit antisera, suggesting that they were glycoproteins. To validate the glycosylation status in N. meningitidis of azurin and MetQ, as well as selected other reported N. gonorrhoeae glycoproteins [4], we performed western blotting with protein-specific antisera for each candidate glycoprotein. This showed that AniA, Sco, CcoP and Mip were glycosylated in N. meningitidis C311 and N. gonorrhoeae MS11, as they displayed clear MW shifts upon genomic deletion of the PglL O-OTase (Fig. 4A,D,E,F). However, MetQ and Laz failed to show clear changes in MW in the presence and absence of glycosylation (Fig. 4B,C). This phenotype was also observed in N. meningitidis MC58, and in N. gonorrhoeae 1291 and O1G1370. Homologs of all of these proteins had been identified as glycoproteins in N. gonorrhoeae[4]. Together with our α-glycan IP results, this suggests that although glycosylated forms of MetQ and Laz can be detected by MS [4], the major fraction of these proteins in the cell under the conditions tested is not in fact glycosylated.


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)

Western blot analysis of putative Neisserial glycoproteins.N. gonorrhoeae MS11pglL::kan, N. gonorrhoeae MS11, N. meningitidis C311pglL::kan and N. meningitidis C311 whole cell extracts were separated by SDS-PAGE, blotted to nitrocellulose membrane and probed with (A) α-AniA, (B) α-Laz, (C) α-MetQ, (D) α-Sco, (E) α-Mip or (F) α-CcoP antisera.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3643930&req=5

pone-0062768-g004: Western blot analysis of putative Neisserial glycoproteins.N. gonorrhoeae MS11pglL::kan, N. gonorrhoeae MS11, N. meningitidis C311pglL::kan and N. meningitidis C311 whole cell extracts were separated by SDS-PAGE, blotted to nitrocellulose membrane and probed with (A) α-AniA, (B) α-Laz, (C) α-MetQ, (D) α-Sco, (E) α-Mip or (F) α-CcoP antisera.
Mentions: Several PglL substrate glycoproteins in addition to PilE and AniA have been reported in N. gonorrhoeae[4]. However, Western blotting using our α-glycan antisera failed to detect bands in addition to AniA and PilE in N. meningitidis C311 whole cell extracts [3]. To investigate if other glycoproteins were also present in N. meningitidis C311, we performed IP of whole cell extracts using α-glycan antisera, and identified eluted proteins with mass spectrometry. α-Glycan co-IP identified three proteins: PilE, azurin and MetQ (Fig. S1, S2 and S3; Tables S2, S3, S4 and S5). These proteins were not identified by negative control IP with unrelated rabbit antisera, suggesting that they were glycoproteins. To validate the glycosylation status in N. meningitidis of azurin and MetQ, as well as selected other reported N. gonorrhoeae glycoproteins [4], we performed western blotting with protein-specific antisera for each candidate glycoprotein. This showed that AniA, Sco, CcoP and Mip were glycosylated in N. meningitidis C311 and N. gonorrhoeae MS11, as they displayed clear MW shifts upon genomic deletion of the PglL O-OTase (Fig. 4A,D,E,F). However, MetQ and Laz failed to show clear changes in MW in the presence and absence of glycosylation (Fig. 4B,C). This phenotype was also observed in N. meningitidis MC58, and in N. gonorrhoeae 1291 and O1G1370. Homologs of all of these proteins had been identified as glycoproteins in N. gonorrhoeae[4]. Together with our α-glycan IP results, this suggests that although glycosylated forms of MetQ and Laz can be detected by MS [4], the major fraction of these proteins in the cell under the conditions tested is not in fact glycosylated.

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.

Show MeSH
Related in: MedlinePlus