Request ID: cfg_rRequest_1952
Status
:
Approved
Project Description
:
Continued studies - to sample more broadly examine the extent of the specificity of FimH-glycan receptor interaction, because the latter receptor(s) are constitutively or incidentally present on a much wide range of cell types.
CFG Member
:
Not Member
Requester First Name
:
Julie
Requester Last Name
:
bouckaert
Head of Lab First Name
:
lode
Head of Lab Last Name
:
wyns
Assigned First Name
:
Lode
Assigned Last Name
:
Wyns
Requester Email
:
bouckaej@vub.ac.be
Requester Interest
:
Information not entered/not applicable.
Request Date [yyyy-mm-dd]
:
2010-02-03
Institution
:
Department of Molecular and Cellular Interactions
Vrije Universiteit Brussel (VUB) and
Flanders Interuniversity Institute for Biotechnology (VIB)
Vrije Universiteit Brussel (VUB) and
Flanders Interuniversity Institute for Biotechnology (VIB)
Department of Molecular and Cellular Interactions
Vrije Universiteit Brussel (VUB) and
Flanders Interuniversity Institute for Biotechnology (VIB)
Shipping Address
:
Structural Biology Brussels, Building E 4.18
Vrije Universiteit Brussel
Pleinlaan 2, 1050 Brussel, Belgium
Tel. 32-2-629-1988, Fax 32-2-6291963
Comments
:
Information not entered/not applicable.
CFG Core
:
H
Resource Type
:
Glycoarray
Amount Requested
:
Information not entered/not applicable.
Date that your RNA/GBP samples will be sent to the core [yyyy-mm-dd]
:
2010-08-02
Experiment to be conducted
:
FimH is the type-1 fimbrial tip adhesin and invasin of Escherichia coli, the most prevalent causative agent of urinary tract infection or cystitis. Upon uptake in the superficial bladder cell linings, E. coli grows in dynamic intracellular bacterial communities or resides quiescently in underlaying bladder cell layers. Inhibition of FimH binding completely abolishes adhesion and thus also bacterial invasion.
The physiological and differentiation status of the superficial uroepithelial cells largely determines the conditions for uropathogenic E. coli to adhere by means of FimH, as it is clearly shown in EM pictures that not all uropeithelial cells are as easily colonized.
We previously analysed the glycan specificities of the E. coli adhesin FimH on a natural glycan microarray prepared from the glycoprotein fraction of human urinary epithelial cells. Although the fluorescence intensities detected in the natural glycan microarray assay, concerning the spots presenting natural glycans, were low compared to other glycans presented on the array, the analysis showed that Fim H specifically binds to oligomannosidic N-glycans of urinary cells (unpublished). This confirmed previous findings of FimH recognising with high affinity paucimannosidic glycans, that are shortened high-mannose glycans (such as oligomannose-3 and oligomannose-5 (Bouckaert,J. et al.., Mol Microbiol 2005, 55, 441-455). High-mannose glycans in the bladder epithelium occur on the abundant membrane glycoproteins uroplakin Ia and on integrins.
Low glycan branching and low glycan multilipicities of receptor membrane proteins lead to increased endocytosis and decreased retention of mammalian cell envelope glycoproteins (Lau,K.S. et al., Cell 2007, 129, 123-134). The activities of these glycoproteins, such as the FimH adhesin receptor integrin β1, depend on chaperones, typical for the endoplasmic reticulum such as calreticulin, that patrol just under at the plasma membrane ( Watts,J.C. et al., PLoS. Pathog. 2009, 5, e1000608).
We want to sample more broadly, using the synthetic glycan arrays from the consortium, the extent of the specificity of FimH-glycan receptor interaction, because the latter receptor(s) are constitutively or incidentally present on a much wide range of cell types, such as M cells (Hase,K. et al, Nature 2009, 462, 226-230) and its secreted natural inhibitory glycoproteins such as Tamm horsefall glycoprotein. Unravelling general principles that govern protein-glycan interactions at host-pathogen cell surfaces is necessary in order to understand how to prevent pathogenic fungi or bacteria from establishing resilient infections.
Within Scope of Consortium
:
Y
If yes, indicate the person responsible for inputing data into core B
:
Julie Bouckaert
GBP being Addressed
:
urified Fim H protein and an IgG rabbit-anti-FimH antibody will be provided by Julie Bouckaert. Blocking can happen with 4% BSA and 50 mmol/L ethanolamine in PBS for 60 min at RT. A secondary, fluorescently labeled antibody is goat-anti-rabbit IgG Alexa 555, Invitrogen.
Specifc aims being addressed
:
Define the specificity and affinity for carbohydrate ligands. Establish the cell types involved in communication. Identify the ligand(s) that mediate GBP binding. Determine how GBP-ligand interactions mediate cell communication. Determine the structures of selected GBPs. Identify the glycosyltransferases that synthesize carbohydrate ligand(s). Determine whether regulation of glycosylation modulates GBP function.