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Lookup NU author(s): Dr James Garnett
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Both lung disease and elevation of blood glucose are associated with increased glucose concentration (from 0.4 to ~4.0 mM) in the airway surface liquid (ASL). This perturbation of ASL glucose makes the airway more susceptible to infection by respiratory pathogens. ASL is minute (~1 μl/cm2) and the measurement of glucose concentration in the small volume ASL is extremely difficult. Therefore, we sought to develop a fluorescent biosensor with sufficient sensitivity to determine glucose concentrations in ASL in situ. We coupled a range of environmentally sensitive fluorophores to mutated forms of a glucose/galactose-binding protein (GBP) including H152C and H152C/A213R and determined their equilibrium binding properties. Of these, GBP H152C/A213R–BADAN (Kd 0.86±0.01 mM, Fmax/F0 3.6) was optimal for glucose sensing and in ASL increased fluorescence when basolateral glucose concentration was raised from 1 to 20 mM. Moreover, interpolation of the data showed that the glucose concentration in ASL was increased, with results similar to that using glucose oxidase analysis. The fluorescence of GBP H152C/A213R–BADAN in native ASL from human airway epithelial cultures in situ was significantly increased over time when basolateral glucose was increased from 5 to 20 mM. Overall our data indicate that this GBP is a useful tool to monitor glucose homoeostasis in the lung.
Author(s): Helassa N, Garnett JP, Farrant M, Khan F, Pickup JC, Hahn KM, MacNevin CJ, Tarran R, Baines DL
Publication type: Article
Publication status: Published
Journal: Biochemical Journal
Year: 2014
Volume: 464
Issue: 2
Pages: 213-220
Print publication date: 01/12/2014
Online publication date: 15/09/2014
Acceptance date: 15/09/2014
ISSN (print): 0264-6021
ISSN (electronic): 1470-8728
Publisher: Portland Press
URL: http://dx.doi.org/10.1042/BJ20141041
DOI: 10.1042/BJ20141041
PubMed id: 25220254
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