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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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Related in: MedlinePlus

Phenotypic analysis of Acinetobacter baylyi strain ADP1 candidate PglL O-OTase mutants.Western blot analysis using α-ComP antibody of wild-type (ADP1), pglL mutant (ADP1pglL::kan), pglL mutant complemented with plasmid-borne pglL (ADP1pglL::kan pWH1266-pglL) and pglL mutant with empty vector (ADP1pglL::kan pWH1266).
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pone-0062768-g003: Phenotypic analysis of Acinetobacter baylyi strain ADP1 candidate PglL O-OTase mutants.Western blot analysis using α-ComP antibody of wild-type (ADP1), pglL mutant (ADP1pglL::kan), pglL mutant complemented with plasmid-borne pglL (ADP1pglL::kan pWH1266-pglL) and pglL mutant with empty vector (ADP1pglL::kan pWH1266).

Mentions: We tested the hypothesis that the pilin homologs closely associated with the PglL O-OTase candidates were the cognate target glycoproteins. In A. baylyi strain ADP1, the pglL gene (accession number ACIAD3337) is adjacent to the comP gene (ACIAD3338) which encodes a pilin-like protein which is essential for natural transformation and has previously been shown to be glycosylated [20]. However, the mechanism of glycosylation of ComP has not previously been investigated. We created a knockout mutant in the pglL gene of A. baylyi strain ADP1, and complemented this mutant strain with expression of plasmid-borne native pglL. Western blot analysis of extracts from the wild-type ADP1 and ADP1pglL::kan mutant strains using an α-ComP antibody indicated the presence of the 20 kDa glycosylated ComP protein in the wild-type strain and a shift in MW to the 18 kDa non-glycosylated form of ComP in the mutant strain (Fig. 3), consistent with the loss of glycosylation of this protein. This glycosylation could be partially rescued by complementation with native pglL, but not with empty vector. This validated the role of the PglL homolog in glycosylation of the ComP pilin-like protein in strain ADP1.


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)

Phenotypic analysis of Acinetobacter baylyi strain ADP1 candidate PglL O-OTase mutants.Western blot analysis using α-ComP antibody of wild-type (ADP1), pglL mutant (ADP1pglL::kan), pglL mutant complemented with plasmid-borne pglL (ADP1pglL::kan pWH1266-pglL) and pglL mutant with empty vector (ADP1pglL::kan pWH1266).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0062768-g003: Phenotypic analysis of Acinetobacter baylyi strain ADP1 candidate PglL O-OTase mutants.Western blot analysis using α-ComP antibody of wild-type (ADP1), pglL mutant (ADP1pglL::kan), pglL mutant complemented with plasmid-borne pglL (ADP1pglL::kan pWH1266-pglL) and pglL mutant with empty vector (ADP1pglL::kan pWH1266).
Mentions: We tested the hypothesis that the pilin homologs closely associated with the PglL O-OTase candidates were the cognate target glycoproteins. In A. baylyi strain ADP1, the pglL gene (accession number ACIAD3337) is adjacent to the comP gene (ACIAD3338) which encodes a pilin-like protein which is essential for natural transformation and has previously been shown to be glycosylated [20]. However, the mechanism of glycosylation of ComP has not previously been investigated. We created a knockout mutant in the pglL gene of A. baylyi strain ADP1, and complemented this mutant strain with expression of plasmid-borne native pglL. Western blot analysis of extracts from the wild-type ADP1 and ADP1pglL::kan mutant strains using an α-ComP antibody indicated the presence of the 20 kDa glycosylated ComP protein in the wild-type strain and a shift in MW to the 18 kDa non-glycosylated form of ComP in the mutant strain (Fig. 3), consistent with the loss of glycosylation of this protein. This glycosylation could be partially rescued by complementation with native pglL, but not with empty vector. This validated the role of the PglL homolog in glycosylation of the ComP pilin-like protein in strain ADP1.

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