Date of Award
2010
Degree Type
Thesis
Degree Name
Master of Science
Program
Microbiology and Immunology
Supervisor
Dr. Carole Creuzenet
Abstract
The glycosylation of the flagellins of Helicobacter pylori with pseudaminic acid (Pse) is absolutely required for the motility and virulence of this gastric pathogen. We hypothesize that protein glycosylation extends to proteins other than flagellins. Soluble and membrane proteins from wild type H. pylori and from Pse biosynthesis pathway mutants were separated by SDS- PAGE. Glycosylated proteins were detected via chemical labeling of the sugar groups by DIG- hydrazide, showing that several proteins aside from the flagellins are glycosylated, and not dependant on the Pse glycosylation pathway. Soluble proteins were fractionated by anion and cation exchange chromatography and the fractions were separated by SDS-PAGE and DIG- labeled, demonstrating the enrichment of several glycoproteins. Free monosaccharides were released from the glycoproteins by acid hydrolysis in 2M trifluoroacetic acid (TFA). The free carbohydrates were separated from the intact proteins by ultrafiltration, and the protein-free carbohydrate mixtures were analyzed by high performance anion exchange chromatography with pulsed amperometric detection, revealing characteristic monosaccharide peaks in these fractions. The carbohydrate mixtures were then analyzed by LC-ESI MS to identify the monosaccharide, revealing a number of sugar matches. Among the identified sugars, the presence of 5-acetamidino-7-acetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid (Pse5Am7Ac), not previousy identified in H.pylori, was confirmed by MS/MS. The initial list of sugar matches was used to prepare a list for subsequent MS/MS analysis to confirm the sugar .
identities. Tryptic digests of the proteins of interest chemically deglycosylated by TFA hydrolysis were compared to untreated tryptic peptides by RP-HPLC, revealing a number of TFA-sensitive glycopeptides. The observed glycopeptides are being analyzed by MS/MS to specifically map
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the glycosylation site. The results presented here demonstrate that the glycosylation of flagellins with Pse is not the only glycosylation that occurs in H. pylori, and identifies some of the sugars present in these glycans. The glycan sugar composition and protein glycosylation
map will allow us to address the mechanism and function of glycosylation in H. pylori.
Recommended Citation
Hopf, Patrick Stefan Chabot, "Protein glycosylation in Helicobacter pylori" (2010). Digitized Theses. 4593.
https://ir.lib.uwo.ca/digitizedtheses/4593