Use of Recombinant Mucin Glycoprotein to Assess the Interaction of the Gastric Pathogen Helicobacter pylori with the Secreted Human Mucin MUC5AC
There is intense interest in how bacteria interact with mucin glycoproteins in order to colonise mucosal surfaces. In this study, we have assessed the feasibility of using recombinant mucin glycoproteins to study the interaction of the gastric pathogen Helicobacter pylori with MUC5AC, a mucin which...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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MDPI
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5590460/ https://www.ncbi.nlm.nih.gov/pubmed/28952513 http://dx.doi.org/10.3390/bioengineering4020034 |
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author | Dunne, Ciara McDermott, Anthony Anjan, Kumar Ryan, Aindrias Reid, Colm Clyne, Marguerite |
author_facet | Dunne, Ciara McDermott, Anthony Anjan, Kumar Ryan, Aindrias Reid, Colm Clyne, Marguerite |
author_sort | Dunne, Ciara |
collection | PubMed |
description | There is intense interest in how bacteria interact with mucin glycoproteins in order to colonise mucosal surfaces. In this study, we have assessed the feasibility of using recombinant mucin glycoproteins to study the interaction of the gastric pathogen Helicobacter pylori with MUC5AC, a mucin which the organism exhibits a distinct tropism for. Stable clonal populations of cells expressing a construct encoding for a truncated version of MUC5AC containing N- and C-termini interspersed with two native tandem repeat sequences (N + 2TR + C) were generated. Binding of H. pylori to protein immunoprecipitated from cell lysates and supernatants was assessed. High molecular weight mucin could be detected in both cell lysates and supernatants of transfected cells. Recombinant protein formed high molecular weight oligomers, was both N and O glycosylated, underwent cleavage similar to native MUC5AC and was secreted from the cell. H. pylori bound better to secreted mucin than intracellular mucin suggesting that modifications on extracellular MUC5AC promoted binding. Lectin analysis demonstrated that secreted mucin was differentially glycosylated compared to intracellular mucin. H. pylori also bound to a recombinant C-terminus MUC5AC protein, but binding to this protein did not inhibit binding to the N + 2TR + C protein. This study demonstrates the feasibility of using recombinant mucins containing tandem repeat sequences to assess microbial mucin interactions. |
format | Online Article Text |
id | pubmed-5590460 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-55904602017-09-21 Use of Recombinant Mucin Glycoprotein to Assess the Interaction of the Gastric Pathogen Helicobacter pylori with the Secreted Human Mucin MUC5AC Dunne, Ciara McDermott, Anthony Anjan, Kumar Ryan, Aindrias Reid, Colm Clyne, Marguerite Bioengineering (Basel) Article There is intense interest in how bacteria interact with mucin glycoproteins in order to colonise mucosal surfaces. In this study, we have assessed the feasibility of using recombinant mucin glycoproteins to study the interaction of the gastric pathogen Helicobacter pylori with MUC5AC, a mucin which the organism exhibits a distinct tropism for. Stable clonal populations of cells expressing a construct encoding for a truncated version of MUC5AC containing N- and C-termini interspersed with two native tandem repeat sequences (N + 2TR + C) were generated. Binding of H. pylori to protein immunoprecipitated from cell lysates and supernatants was assessed. High molecular weight mucin could be detected in both cell lysates and supernatants of transfected cells. Recombinant protein formed high molecular weight oligomers, was both N and O glycosylated, underwent cleavage similar to native MUC5AC and was secreted from the cell. H. pylori bound better to secreted mucin than intracellular mucin suggesting that modifications on extracellular MUC5AC promoted binding. Lectin analysis demonstrated that secreted mucin was differentially glycosylated compared to intracellular mucin. H. pylori also bound to a recombinant C-terminus MUC5AC protein, but binding to this protein did not inhibit binding to the N + 2TR + C protein. This study demonstrates the feasibility of using recombinant mucins containing tandem repeat sequences to assess microbial mucin interactions. MDPI 2017-04-15 /pmc/articles/PMC5590460/ /pubmed/28952513 http://dx.doi.org/10.3390/bioengineering4020034 Text en © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Dunne, Ciara McDermott, Anthony Anjan, Kumar Ryan, Aindrias Reid, Colm Clyne, Marguerite Use of Recombinant Mucin Glycoprotein to Assess the Interaction of the Gastric Pathogen Helicobacter pylori with the Secreted Human Mucin MUC5AC |
title | Use of Recombinant Mucin Glycoprotein to Assess the Interaction of the Gastric Pathogen Helicobacter pylori with the Secreted Human Mucin MUC5AC |
title_full | Use of Recombinant Mucin Glycoprotein to Assess the Interaction of the Gastric Pathogen Helicobacter pylori with the Secreted Human Mucin MUC5AC |
title_fullStr | Use of Recombinant Mucin Glycoprotein to Assess the Interaction of the Gastric Pathogen Helicobacter pylori with the Secreted Human Mucin MUC5AC |
title_full_unstemmed | Use of Recombinant Mucin Glycoprotein to Assess the Interaction of the Gastric Pathogen Helicobacter pylori with the Secreted Human Mucin MUC5AC |
title_short | Use of Recombinant Mucin Glycoprotein to Assess the Interaction of the Gastric Pathogen Helicobacter pylori with the Secreted Human Mucin MUC5AC |
title_sort | use of recombinant mucin glycoprotein to assess the interaction of the gastric pathogen helicobacter pylori with the secreted human mucin muc5ac |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5590460/ https://www.ncbi.nlm.nih.gov/pubmed/28952513 http://dx.doi.org/10.3390/bioengineering4020034 |
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