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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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Structures of PglL acceptor substrate proteins.(A) CD Spectrum of AniA C-terminal peptide (NGAAPAASAPAASAPAASASEKSVY). Peptide was analysed in 50 mM KH2PO4 with 0–50% of TFE. (B) The difference in CD spectra between the peptide in 50% TFE and no TFE. (C) Modelling of N. meningitidis PilE glycosylation site structure. Peptide corresponding to the glycosylated region of C311 PilE (57WPGNNTS(Gal(β1–4)Gal(α1–3)2,4-diacetimido-2,4,6-trideoxyhexose)AGVASSSTIK73) constrained as in the structure of N. gonorrhoeae PilE was modelled.
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pone-0062768-g006: Structures of PglL acceptor substrate proteins.(A) CD Spectrum of AniA C-terminal peptide (NGAAPAASAPAASAPAASASEKSVY). Peptide was analysed in 50 mM KH2PO4 with 0–50% of TFE. (B) The difference in CD spectra between the peptide in 50% TFE and no TFE. (C) Modelling of N. meningitidis PilE glycosylation site structure. Peptide corresponding to the glycosylated region of C311 PilE (57WPGNNTS(Gal(β1–4)Gal(α1–3)2,4-diacetimido-2,4,6-trideoxyhexose)AGVASSSTIK73) constrained as in the structure of N. gonorrhoeae PilE was modelled.

Mentions: To investigate the structure of the AniA glycosylation acceptor sites, we performed circular dichroism (CD) spectroscopy to characterize the secondary structure of a synthesized peptide corresponding to the unglycosylated AniA glycopeptide. The CD spectrum of AniA in phosphate buffer showed substantial negative ellipticity centred at 198 nm (Fig. 6A), indicative of an unstructured conformation. The glycosylation site in PilE (NTS63(glycan)AG) is part of a short α-helix located in the so called ‘ab loop’ [23], so to investigate if the AniA peptide in solution samples an energy landscape that contains transiently structured conformations we obtained CD spectra in the presence of increasing concentrations of TFE, which allows peptide intramolecular hydrogen bonds to form by limiting competing bond formation with solvent water. The CD spectra showed that increasing TFE caused loss of negative ellipticity at 198 nm with a corresponding increase in negative ellipticity around 225 nm (Fig. 6). Subtraction of the spectrum of the AniA peptide in 0% TFE from that in 50% TFE resulted in a spectrum with positive ellipticity at 195 nm and a broad negative peak centred on 220 nm (Fig. 6B). These features are suggestive of a helical conformation, but we note that even in 50% TFE the AniA peptide was still predominantly unstructured. NMR analysis (Fig. S5) of the AniA peptide in the absence of TFE showed that most amide protons had chemical shifts clustered between 8.1 ppm and 8.3 ppm; Val24 and Tyr25 were shifted upfield due to Tyr ring current effects. In agreement with the CD results, the NMR result was indicative of an unstructured conformation. The presence of increasing concentrations of TFE showed a corresponding increase in the dispersion of amide chemical shifts (both upfield and downfield shifts were observed) suggesting that some residues adopted a structured conformation.


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)

Structures of PglL acceptor substrate proteins.(A) CD Spectrum of AniA C-terminal peptide (NGAAPAASAPAASAPAASASEKSVY). Peptide was analysed in 50 mM KH2PO4 with 0–50% of TFE. (B) The difference in CD spectra between the peptide in 50% TFE and no TFE. (C) Modelling of N. meningitidis PilE glycosylation site structure. Peptide corresponding to the glycosylated region of C311 PilE (57WPGNNTS(Gal(β1–4)Gal(α1–3)2,4-diacetimido-2,4,6-trideoxyhexose)AGVASSSTIK73) constrained as in the structure of N. gonorrhoeae PilE was modelled.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3643930&req=5

pone-0062768-g006: Structures of PglL acceptor substrate proteins.(A) CD Spectrum of AniA C-terminal peptide (NGAAPAASAPAASAPAASASEKSVY). Peptide was analysed in 50 mM KH2PO4 with 0–50% of TFE. (B) The difference in CD spectra between the peptide in 50% TFE and no TFE. (C) Modelling of N. meningitidis PilE glycosylation site structure. Peptide corresponding to the glycosylated region of C311 PilE (57WPGNNTS(Gal(β1–4)Gal(α1–3)2,4-diacetimido-2,4,6-trideoxyhexose)AGVASSSTIK73) constrained as in the structure of N. gonorrhoeae PilE was modelled.
Mentions: To investigate the structure of the AniA glycosylation acceptor sites, we performed circular dichroism (CD) spectroscopy to characterize the secondary structure of a synthesized peptide corresponding to the unglycosylated AniA glycopeptide. The CD spectrum of AniA in phosphate buffer showed substantial negative ellipticity centred at 198 nm (Fig. 6A), indicative of an unstructured conformation. The glycosylation site in PilE (NTS63(glycan)AG) is part of a short α-helix located in the so called ‘ab loop’ [23], so to investigate if the AniA peptide in solution samples an energy landscape that contains transiently structured conformations we obtained CD spectra in the presence of increasing concentrations of TFE, which allows peptide intramolecular hydrogen bonds to form by limiting competing bond formation with solvent water. The CD spectra showed that increasing TFE caused loss of negative ellipticity at 198 nm with a corresponding increase in negative ellipticity around 225 nm (Fig. 6). Subtraction of the spectrum of the AniA peptide in 0% TFE from that in 50% TFE resulted in a spectrum with positive ellipticity at 195 nm and a broad negative peak centred on 220 nm (Fig. 6B). These features are suggestive of a helical conformation, but we note that even in 50% TFE the AniA peptide was still predominantly unstructured. NMR analysis (Fig. S5) of the AniA peptide in the absence of TFE showed that most amide protons had chemical shifts clustered between 8.1 ppm and 8.3 ppm; Val24 and Tyr25 were shifted upfield due to Tyr ring current effects. In agreement with the CD results, the NMR result was indicative of an unstructured conformation. The presence of increasing concentrations of TFE showed a corresponding increase in the dispersion of amide chemical shifts (both upfield and downfield shifts were observed) suggesting that some residues adopted a structured conformation.

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