2026-09-22T02:21:12Z https://www.ideals.illinois.edu/oai-pmh

oai:www.ideals.illinois.edu:2142/49596 2023-07-11 col_2142_5131 col_2142_14826 com_2142_5130 com_2142_8913 com_2142_8903

Blanke, Steven R. Smith, Lucas 2014-05-30T16:51:41Z 2014-05-30T16:51:41Z 2014-05 2014-05-30T16:51:41Z 2014-05 The main determinants in toxin cell specificity are often interactions with surface receptors. It has recently been determined that the membrane phospholipid sphingomyelin (SM) is the primary receptor for the Helicobacter pylori vacuolating cytotoxin (VacA). While the cellular receptor has been identified, the molecular basis of SM recognition remains unknown. Molecular modeling of unrelated SM-binding proteins suggests that specific binding occurs through tryptophan rich aromatic clusters located in solvent exposed peptide loops between β-sheet domains. Structural comparison of these proteins with VacA led to the identification of a similar tryptophan containing aromatic cluster in the mid-region of the p55 subunit of VacA. Additionally, sequence alignment of this putative binding site exhibited sequence homology with the phosphorylcholine binding site of another SM-binding toxin. In this study we tested the effects of mutating each of the 11 tryptophan residues in VacA. While mutating each of the 8 tryptophan residues present in the auto-transporter p33 domain did not cause substantial attenuation in toxin activity, alanine substitution of 2 tryptophan residues within the putative binding site led to significant attenuation. Further, tryptophan to alanine mutation of W603, a residue present in the center of the putative binding site, was found to be attenuated in cell binding to human-derived AZ-521 gastric epithelial cells. Interestingly, a W603A mutation is also present within a vacA allele associated with a lessened propensity for gastric disease, decreased toxin binding, and differences in cell specificity. This thesis proposes a novel VacA-SM binding site and identifies a possible cause of allele associated differences in VacA pathogenicity. Item withdrawn by Laura Spradlin (lspradl2@illinois.edu) on 2014-04-08T19:22:35Z Item was in collections: University of Illinois Theses & Dissertations (ID: 1) No. of bitstreams: 4 Final Master Thesis.docx: 9128789 bytes, checksum: 9c2a33fea9adbe37834262b8a3981a95 (MD5) Masters thesis abstract and table of contents.docx: 44381 bytes, checksum: 0894980f45608158548fe5be967e9e98 (MD5) Thesis Title Page.docx: 36735 bytes, checksum: 467c6ef865710a03d4e88f8f6bd38368 (MD5) Masters Thesis for Final Formatting Review.pdf: 2610612 bytes, checksum: dcc82ab2e94c437f533ec081d8e09a0e (MD5) Made available in DSpace on 2014-05-30T16:51:41Z (GMT). No. of bitstreams: 2 Lucas_Smith.pdf: 2609960 bytes, checksum: 7dcc9ce19ee60717f9a1c3ab339ff4bb (MD5) license.txt: 4060 bytes, checksum: 73c100cc243ce29aaaadbe6e68531900 (MD5) http://hdl.handle.net/2142/49596 en Copyright 2014 Lucas Smith Stomach Cancer Gastric Adenocarcinoma H.pylori Helicobacter pylori Vacuolating Cytotoxin (VacA) Toxin Sphingomyelin Molecular characterization of a sphingomyelin binding site in the vacuolating cytotoxin of H. pylori text School of Integrative Biology 1383 Biology 0314 University of Illinois at Urbana-Champaign Thesis M.S. MS:Biology -UIUC 10KS0314MS