Comparative Proteomics and Glycoproteomics Reveal Increased N-Linked Glycosylation and Relaxed Sequon Specificity in Campylobacter jejuni NCTC11168 O

Nichollas E. Scott, N. Bishara Marzook, Joel A. Cain, Nestor Solis, Morten Thaysen-Andersen, Steven P. Djordjevic, Nicolle H. Packer, Martin Røssel Larsen, Stuart J. Cordwell

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Resumé

Campylobacter jejuni is a major cause of bacterial gastroenteritis. C. jejuni encodes a protein glycosylation (Pgl) locus responsible for the N-glycosylation of membrane-associated proteins. We examined two variants of the genome sequenced strain NCTC11168: O, a representative of the original clinical isolate, and GS, a laboratory-adapted relative of O. Comparative proteomics by iTRAQ and two-dimensional liquid chromatography coupled to tandem mass spectrometry (2D-LC-MS/MS) allowed the confident identification of 1214 proteins (73.9% of the predicted C. jejuni proteome), of which 187 were present at statistically significant altered levels of abundance between variants. Proteins associated with the O variant included adhesins (CadF and FlpA), proteases, capsule biosynthesis, and cell shape determinants as well as six proteins encoded by the Pgl system, including the PglK flippase and PglB oligosaccharyltransferase. Lectin blotting highlighted specific glycoproteins more abundant in NCTC11168 O, whereas others remained unaltered. Hydrophilic interaction liquid chromatography (HILIC) and LC-MS/MS identified 30 completely novel glycosites from 15 proteins. A novel glycopeptide from a 14 kDa membrane protein (Cj0455c) was identified that did not contain the C. jejuni N-linked sequon D/E-X-N-X-S/T (X ≠ Pro) but that instead contained a sequon with leucine at the -2 position. Occupied atypical sequons were also observed in Cj0958c (OxaA; Gln at the -2 position) and Cj0152c (Ala at the +2 position). The relative O and GS abundances of 30 glycopeptides were determined by label-free quantitation, which revealed a >100-fold increase in the atypical glycopeptide from Cj0455c in isolate O. Our data provide further evidence for the importance of the Pgl system in C. jejuni.

OriginalsprogEngelsk
TidsskriftJournal of Proteome Research
Vol/bind13
Udgave nummer11
Sider (fra-til)5136-50
Antal sider15
ISSN1535-3893
DOI
StatusUdgivet - 7. nov. 2014

Fingeraftryk

Glycosylation
Campylobacter jejuni
Glycopeptides
Proteins
Liquid Chromatography
Membrane Proteins
Liquid chromatography
Cell Shape
Gastroenteritis
Proteome
Tandem Mass Spectrometry
Leucine
Peptide Hydrolases
Biosynthesis
Proteomics
Lectins
Capsules
Mass spectrometry
Labels
Glycoproteins

Citer dette

Scott, N. E., Marzook, N. B., Cain, J. A., Solis, N., Thaysen-Andersen, M., Djordjevic, S. P., ... Cordwell, S. J. (2014). Comparative Proteomics and Glycoproteomics Reveal Increased N-Linked Glycosylation and Relaxed Sequon Specificity in Campylobacter jejuni NCTC11168 O. Journal of Proteome Research, 13(11), 5136-50. https://doi.org/10.1021/pr5005554
Scott, Nichollas E. ; Marzook, N. Bishara ; Cain, Joel A. ; Solis, Nestor ; Thaysen-Andersen, Morten ; Djordjevic, Steven P. ; Packer, Nicolle H. ; Larsen, Martin Røssel ; Cordwell, Stuart J. / Comparative Proteomics and Glycoproteomics Reveal Increased N-Linked Glycosylation and Relaxed Sequon Specificity in Campylobacter jejuni NCTC11168 O. I: Journal of Proteome Research. 2014 ; Bind 13, Nr. 11. s. 5136-50.
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title = "Comparative Proteomics and Glycoproteomics Reveal Increased N-Linked Glycosylation and Relaxed Sequon Specificity in Campylobacter jejuni NCTC11168 O",
abstract = "Campylobacter jejuni is a major cause of bacterial gastroenteritis. C. jejuni encodes a protein glycosylation (Pgl) locus responsible for the N-glycosylation of membrane-associated proteins. We examined two variants of the genome sequenced strain NCTC11168: O, a representative of the original clinical isolate, and GS, a laboratory-adapted relative of O. Comparative proteomics by iTRAQ and two-dimensional liquid chromatography coupled to tandem mass spectrometry (2D-LC-MS/MS) allowed the confident identification of 1214 proteins (73.9{\%} of the predicted C. jejuni proteome), of which 187 were present at statistically significant altered levels of abundance between variants. Proteins associated with the O variant included adhesins (CadF and FlpA), proteases, capsule biosynthesis, and cell shape determinants as well as six proteins encoded by the Pgl system, including the PglK flippase and PglB oligosaccharyltransferase. Lectin blotting highlighted specific glycoproteins more abundant in NCTC11168 O, whereas others remained unaltered. Hydrophilic interaction liquid chromatography (HILIC) and LC-MS/MS identified 30 completely novel glycosites from 15 proteins. A novel glycopeptide from a 14 kDa membrane protein (Cj0455c) was identified that did not contain the C. jejuni N-linked sequon D/E-X-N-X-S/T (X ≠ Pro) but that instead contained a sequon with leucine at the -2 position. Occupied atypical sequons were also observed in Cj0958c (OxaA; Gln at the -2 position) and Cj0152c (Ala at the +2 position). The relative O and GS abundances of 30 glycopeptides were determined by label-free quantitation, which revealed a >100-fold increase in the atypical glycopeptide from Cj0455c in isolate O. Our data provide further evidence for the importance of the Pgl system in C. jejuni.",
author = "Scott, {Nichollas E.} and Marzook, {N. Bishara} and Cain, {Joel A.} and Nestor Solis and Morten Thaysen-Andersen and Djordjevic, {Steven P.} and Packer, {Nicolle H.} and Larsen, {Martin R{\o}ssel} and Cordwell, {Stuart J.}",
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Scott, NE, Marzook, NB, Cain, JA, Solis, N, Thaysen-Andersen, M, Djordjevic, SP, Packer, NH, Larsen, MR & Cordwell, SJ 2014, 'Comparative Proteomics and Glycoproteomics Reveal Increased N-Linked Glycosylation and Relaxed Sequon Specificity in Campylobacter jejuni NCTC11168 O', Journal of Proteome Research, bind 13, nr. 11, s. 5136-50. https://doi.org/10.1021/pr5005554

Comparative Proteomics and Glycoproteomics Reveal Increased N-Linked Glycosylation and Relaxed Sequon Specificity in Campylobacter jejuni NCTC11168 O. / Scott, Nichollas E.; Marzook, N. Bishara; Cain, Joel A.; Solis, Nestor; Thaysen-Andersen, Morten; Djordjevic, Steven P.; Packer, Nicolle H.; Larsen, Martin Røssel; Cordwell, Stuart J.

I: Journal of Proteome Research, Bind 13, Nr. 11, 07.11.2014, s. 5136-50.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - Comparative Proteomics and Glycoproteomics Reveal Increased N-Linked Glycosylation and Relaxed Sequon Specificity in Campylobacter jejuni NCTC11168 O

AU - Scott, Nichollas E.

AU - Marzook, N. Bishara

AU - Cain, Joel A.

AU - Solis, Nestor

AU - Thaysen-Andersen, Morten

AU - Djordjevic, Steven P.

AU - Packer, Nicolle H.

AU - Larsen, Martin Røssel

AU - Cordwell, Stuart J.

PY - 2014/11/7

Y1 - 2014/11/7

N2 - Campylobacter jejuni is a major cause of bacterial gastroenteritis. C. jejuni encodes a protein glycosylation (Pgl) locus responsible for the N-glycosylation of membrane-associated proteins. We examined two variants of the genome sequenced strain NCTC11168: O, a representative of the original clinical isolate, and GS, a laboratory-adapted relative of O. Comparative proteomics by iTRAQ and two-dimensional liquid chromatography coupled to tandem mass spectrometry (2D-LC-MS/MS) allowed the confident identification of 1214 proteins (73.9% of the predicted C. jejuni proteome), of which 187 were present at statistically significant altered levels of abundance between variants. Proteins associated with the O variant included adhesins (CadF and FlpA), proteases, capsule biosynthesis, and cell shape determinants as well as six proteins encoded by the Pgl system, including the PglK flippase and PglB oligosaccharyltransferase. Lectin blotting highlighted specific glycoproteins more abundant in NCTC11168 O, whereas others remained unaltered. Hydrophilic interaction liquid chromatography (HILIC) and LC-MS/MS identified 30 completely novel glycosites from 15 proteins. A novel glycopeptide from a 14 kDa membrane protein (Cj0455c) was identified that did not contain the C. jejuni N-linked sequon D/E-X-N-X-S/T (X ≠ Pro) but that instead contained a sequon with leucine at the -2 position. Occupied atypical sequons were also observed in Cj0958c (OxaA; Gln at the -2 position) and Cj0152c (Ala at the +2 position). The relative O and GS abundances of 30 glycopeptides were determined by label-free quantitation, which revealed a >100-fold increase in the atypical glycopeptide from Cj0455c in isolate O. Our data provide further evidence for the importance of the Pgl system in C. jejuni.

AB - Campylobacter jejuni is a major cause of bacterial gastroenteritis. C. jejuni encodes a protein glycosylation (Pgl) locus responsible for the N-glycosylation of membrane-associated proteins. We examined two variants of the genome sequenced strain NCTC11168: O, a representative of the original clinical isolate, and GS, a laboratory-adapted relative of O. Comparative proteomics by iTRAQ and two-dimensional liquid chromatography coupled to tandem mass spectrometry (2D-LC-MS/MS) allowed the confident identification of 1214 proteins (73.9% of the predicted C. jejuni proteome), of which 187 were present at statistically significant altered levels of abundance between variants. Proteins associated with the O variant included adhesins (CadF and FlpA), proteases, capsule biosynthesis, and cell shape determinants as well as six proteins encoded by the Pgl system, including the PglK flippase and PglB oligosaccharyltransferase. Lectin blotting highlighted specific glycoproteins more abundant in NCTC11168 O, whereas others remained unaltered. Hydrophilic interaction liquid chromatography (HILIC) and LC-MS/MS identified 30 completely novel glycosites from 15 proteins. A novel glycopeptide from a 14 kDa membrane protein (Cj0455c) was identified that did not contain the C. jejuni N-linked sequon D/E-X-N-X-S/T (X ≠ Pro) but that instead contained a sequon with leucine at the -2 position. Occupied atypical sequons were also observed in Cj0958c (OxaA; Gln at the -2 position) and Cj0152c (Ala at the +2 position). The relative O and GS abundances of 30 glycopeptides were determined by label-free quantitation, which revealed a >100-fold increase in the atypical glycopeptide from Cj0455c in isolate O. Our data provide further evidence for the importance of the Pgl system in C. jejuni.

U2 - 10.1021/pr5005554

DO - 10.1021/pr5005554

M3 - Journal article

VL - 13

SP - 5136

EP - 5150

JO - Journal of Proteome Research

JF - Journal of Proteome Research

SN - 1535-3893

IS - 11

ER -