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Helicobacter pylori cell surface interactions with glycosaminoglycans. Identification and characterisation of proteins binding to heparin/heparan sulphate.

Utt, Meeme LU (2002)
Abstract (Swedish)
Popular Abstract in Swedish

Helicobacter pylori är en bakterie som orsakar såväl magkatarr, magsår som magsäckscancer. H. pylori producerar olika virulensfaktorer såsom enzymet ureas, ett cell vakuolbildande toxin (VacA), andra cytotoxiska proteiner (cagA eller pathogenicity island-associated proteins), flageller och adhesiner. För etablering av H. pylori infektion, måste dessa virulensfaktorer interagera med värdcellens ytmolekyler (sialylerade glycoproteiner, glycolipider, muciner) i magsäckens cellytlager. Glycosaminoglycaner (GAGs) är molekyler som finns representerade i de flesta humana celler och ett huvudsyfte med denna studie har varit att analysera glycosaminoglykanernas interaktion med olika H. pylori stammar.... (More)
Popular Abstract in Swedish

Helicobacter pylori är en bakterie som orsakar såväl magkatarr, magsår som magsäckscancer. H. pylori producerar olika virulensfaktorer såsom enzymet ureas, ett cell vakuolbildande toxin (VacA), andra cytotoxiska proteiner (cagA eller pathogenicity island-associated proteins), flageller och adhesiner. För etablering av H. pylori infektion, måste dessa virulensfaktorer interagera med värdcellens ytmolekyler (sialylerade glycoproteiner, glycolipider, muciner) i magsäckens cellytlager. Glycosaminoglycaner (GAGs) är molekyler som finns representerade i de flesta humana celler och ett huvudsyfte med denna studie har varit att analysera glycosaminoglykanernas interaktion med olika H. pylori stammar. Bindningen är pH-beroende och kunde i våra studier inhiberas med sulfaterade GAGs and andra sulfaterade molekyler såsom dextransulfat och fuciodan. VacA-toxinets bindning till heparin och heparansulfat studerades i en biosensor apparat (BIAcore, Uppsala, Sweden). Vi syntetiserade specifika peptider som band till heparansulfat med moderat affinitet och vi kunde också visa att nativt toxin interagerar med heparin. Heparansulfat förmodas vara en receptor/co-receptor för VacA toxinets inbindning till värdcellens yta. I nästa publikation studerades hur ureas binder till heparin och heparansulfat. Bindningsegenskaperna mättes i en biosensor apparat (BIAcore) på ett liknande sätt som för VacA-peptider och befanns vara tusen gånger högre vid pH 5.5 jämfört med pH 6.5. Sekvenser med bindande förmåga identifierades med mass-spektroskopi SELDI-TOF MS teknik. Fyra immunogena heparin-bindande H. pylori proteiner karakteriserades med s.k. "proteom" metodik. Med 2-dimensionell gelelektrofores, immunoblotting följt av mass spektroskopisk sekvenering identifierades "cell bindande faktor 2", ureas, ett ytter-membran protein samt ett hypotetiskt protein. Tre av dessa proteiner var inte kända som heparin-bindande tidigare. Cell bindande faktor 2 proteinet visades ha hög specificitet och kan därför komma att användas som ett antigen i framtida serologiska tester. Att förstå GAGs biologiska funktioner i förhållande till patogena mikroorganismer kommer att möjliggöra fördjupade kunskaper om hur anti-mikrobiella komponenter, som t.ex. sulfaterade polysackarider, kan användas. (Less)
Abstract
Helicobacter pylori is a gastric pathogen which cause chronic type B gastritis and peptic ulcer disease. H. pylori produces virulence factors such as urease, vacuolating cytotoxin VacA, cag pathogenicity island-associated proteins, flagella and adhesins. These proteins interact with several host cell molecules such as sialylated cell surface glycoproteins, some of glycolipids and mucins. Glycosaminoglycans are widely distributed molecules in human cells and extracellular matrix and interact with H. pylori cell surface proteins. Heparan sulphate shows a pH-dependent binding to all strains, which is inhibited by sulphated glycosaminoglycans and other polysulphated molecules such as dextran sulphate and fucoidan. Using bioinformatics methods,... (More)
Helicobacter pylori is a gastric pathogen which cause chronic type B gastritis and peptic ulcer disease. H. pylori produces virulence factors such as urease, vacuolating cytotoxin VacA, cag pathogenicity island-associated proteins, flagella and adhesins. These proteins interact with several host cell molecules such as sialylated cell surface glycoproteins, some of glycolipids and mucins. Glycosaminoglycans are widely distributed molecules in human cells and extracellular matrix and interact with H. pylori cell surface proteins. Heparan sulphate shows a pH-dependent binding to all strains, which is inhibited by sulphated glycosaminoglycans and other polysulphated molecules such as dextran sulphate and fucoidan. Using bioinformatics methods, the binding of H. pylori VacA cytotoxin to heparin and heparan sulphate was predicted. Specific peptides were synthesised and their interactions with surface-immobilised heparan sulphate studied in a BIAcore biosensor. Synthetic peptides bound to heparan sulphate with a moderate affinity. It was shown that native toxin interacts with immobilised heparin. Heparan sulphate was proposed as a receptor/co-receptor for VacA cytotoxin binding to host cell surfaces. H. pylori urease binds to surface-immobilised heparin and heparan sulphate. The interaction was characterised using a BIAcore biosensor. The binding was a thousand-fold higher at pH 5.5 compared to pH 6.5. The binding epitopes were identified by SELDI-TOF MS technology. Using a proteomic technology, four immunogenic heparin-binding proteins were identified: cell binding factor 2, urease, one outer membrane protein and one hypothetical protein. The binding of three of these proteins to heparin was demonstrated for the first time. The cell binding factor was identified as a specific and highly immunogenic H. pylori surface protein. 2-DE and 2-D immunoblotting allow efficient separation and identification of immunogenic proteins. Understanding how GAGs interact with pathogens, will allow the design of new experimental putative anti-microbial compounds based on sulphated polysaccharide structures. (Less)
Please use this url to cite or link to this publication:
author
opponent
  • Professor Magnusson, Karl-Eric, Faculty of Health Sciences, Division of Medical Microbiology, University of Linköping, Sweden
organization
publishing date
type
Thesis
publication status
published
subject
keywords
SELDI-TOF MS, bioinformatics, proteomics, immunoblotting, 2-DE, biosensor, heparan sulphate, heparin, binding, Helicobacter pylori, cell surface, bakteriologi, Mikrobiologi, mycology, bacteriology, Microbiology, mykologi, virologi, virology
pages
120 pages
publisher
Meeme Utt, MMDI, University of Lund, Sölvegatan 23, S-223 62 Lund, Sweden,
defense location
Rune Grubb-Salen, BMC, Lund
defense date
2002-04-03 13:15
ISBN
91-628-5158-6
language
English
LU publication?
yes
id
4a6998ed-a333-422e-aa71-e6c66572f9ea (old id 464443)
date added to LUP
2007-10-01 15:00:55
date last changed
2016-09-19 08:45:13
@misc{4a6998ed-a333-422e-aa71-e6c66572f9ea,
  abstract     = {Helicobacter pylori is a gastric pathogen which cause chronic type B gastritis and peptic ulcer disease. H. pylori produces virulence factors such as urease, vacuolating cytotoxin VacA, cag pathogenicity island-associated proteins, flagella and adhesins. These proteins interact with several host cell molecules such as sialylated cell surface glycoproteins, some of glycolipids and mucins. Glycosaminoglycans are widely distributed molecules in human cells and extracellular matrix and interact with H. pylori cell surface proteins. Heparan sulphate shows a pH-dependent binding to all strains, which is inhibited by sulphated glycosaminoglycans and other polysulphated molecules such as dextran sulphate and fucoidan. Using bioinformatics methods, the binding of H. pylori VacA cytotoxin to heparin and heparan sulphate was predicted. Specific peptides were synthesised and their interactions with surface-immobilised heparan sulphate studied in a BIAcore biosensor. Synthetic peptides bound to heparan sulphate with a moderate affinity. It was shown that native toxin interacts with immobilised heparin. Heparan sulphate was proposed as a receptor/co-receptor for VacA cytotoxin binding to host cell surfaces. H. pylori urease binds to surface-immobilised heparin and heparan sulphate. The interaction was characterised using a BIAcore biosensor. The binding was a thousand-fold higher at pH 5.5 compared to pH 6.5. The binding epitopes were identified by SELDI-TOF MS technology. Using a proteomic technology, four immunogenic heparin-binding proteins were identified: cell binding factor 2, urease, one outer membrane protein and one hypothetical protein. The binding of three of these proteins to heparin was demonstrated for the first time. The cell binding factor was identified as a specific and highly immunogenic H. pylori surface protein. 2-DE and 2-D immunoblotting allow efficient separation and identification of immunogenic proteins. Understanding how GAGs interact with pathogens, will allow the design of new experimental putative anti-microbial compounds based on sulphated polysaccharide structures.},
  author       = {Utt, Meeme},
  isbn         = {91-628-5158-6},
  keyword      = {SELDI-TOF MS,bioinformatics,proteomics,immunoblotting,2-DE,biosensor,heparan sulphate,heparin,binding,Helicobacter pylori,cell surface,bakteriologi,Mikrobiologi,mycology,bacteriology,Microbiology,mykologi,virologi,virology},
  language     = {eng},
  pages        = {120},
  publisher    = {ARRAY(0xc56a5c0)},
  title        = {Helicobacter pylori cell surface interactions with glycosaminoglycans. Identification and characterisation of proteins binding to heparin/heparan sulphate.},
  year         = {2002},
}