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A novel fluorescent sensor protein for detecting changes in airway surface liquid glucose concentration

Lookup NU author(s): Dr James Garnett

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Abstract

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 (K_d 0.86±0.01 mM, F_max/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.

Publication metadata

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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