Study of the N-linked protein glycosylation pathway in Campylobacter species

• Author / Creator

  Dwivedi, Ritika

• Campylobacter jejuni was the first Gram- negative bacterial species demonstrated to possess an N-linked protein glycosylation pathway (Pgl), however it is now well established that all known Campylobacter species and a few other bacterial species possess this pathway. In Campylobacter, the pathway is responsible for the synthesis of species specific oligosaccharides that are covalently attached to asparagine residues on multiple proteins to form N-glycosylated proteins, and also releases free oligosaccharides (fOS) into the periplasmic space. The central enzyme, the membrane bound oligosaccharyltransferase (OTase), PglB, is responsible for the formation of both major products and is extensively studied due to its application in the production of glycoconjugate vaccines and therapeutics. N-glycosylated proteins and fOS were discovered in C. jejuni several years ago, however further studies are important to understand their generation, abundance and specific role(s). In addition, the mechanism of protein N-glycosylation and fOS generation by PglB are not well understood. This PhD thesis focuses on developing tools to study the generation, abundance and structural diversity of fOS. In addition, new tools were developed to characterize and understand the fOS generation and protein glycosylation activity of PglB in greater detail. Efficient and sensitive fOS purification, quantitation and analysis methods were successfully developed in this thesis. These methods are based on thin layer chromatography, porous graphitized carbon purification, high performance anion exchange chromatography, mass spectrometry (MS) and nuclear magnetic resonance. The amount of fOS purified by these methods is 120 times more compared to previously published methods. This method also allows determination of molar quantities of fOS compared to previously published semi-quantitative methods and is applicable to the structurally diverse fOS generated by different Campylobacter species. fOS from selected species were quantitated and found to range from 49.8 0.5 nMoles in C. fetus fetus to 7.8 0.8 nMoles in C. lari. In addition, novel phosphorylated fOS structures were also discovered in C. lari, that were not detected by previous methods. The methods established in this project are more sensitive, significantly faster and more efficient compared to previously published methods that were time-consuming and produced lower yields of fOS. Studies focused on understanding the fOS generation activity of PglB revealed that N-glycosylation of PglB itself may affect both its ability to N-glycosylate other cellular proteins and generate fOS in C. jejuni. Bioinformatic analysis showed that the N-glycosylation site is conserved in the majority of the PglB homologues in Campylobacters. Significantly lower fOS levels were detected in C. jejuni cells expressing unglycosylated PglB compared to wild-type suggesting a role of N-glycosylation in the fOS generation activity of PglB. In addition, overall N-glycoprotein profiles were different between the two strains as determined by Western blot analysis with anti-N-glycan antibodies. This is the first report of the N-glycosylation of an OTase enzyme possibly affecting its own enzymatic activities. In order to better understand the N-glycosylation activity of PglB, a fluorescence resonance energy transfer (FRET) assay was developed. This method is much faster compared to the other gel electrophoresis or ELISA based assays currently used to assess OTase activity. The FRET assay utilizes a peptide labeled with fluorophore/quencher at each end, along with a glycosylation acceptor sequon and a Factor Xa cleavage site within the peptide sequence. After incubation with the OTase enzyme, the peptide is exposed to the Factor Xa protease. The glycosylated peptide is protected from protease mediated cleavage due to the glycan modification, whereas the unglycosylated peptide gets cleaved resulting in fluorescence that is measured in a plate reader. In addition, MS based methods were successfully established to identify the peptide composition following glycosylation. This assay can be adapted to a 96-well plate based high-throughput assay that allows quick analysis of OTase activity. This study has made important contributions to understanding the generation of both major products of the N-linked protein glycosylation pathway in Campylobacter species and provided efficient and faster tools to further characterize the pathway in Campylobacters and other bacterial species that possess an N-linked protein glycosylation pathway.

• Subjects / Keywords

  • Free oligosaccharides
  • Campylobacter
  • Protein glycosylation
  • N-linked
  • PglB

• Graduation date

  Fall 2015

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3V11W111

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.