Biochemical Journal 2014-12-01

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

Nordine Helassa, James P Garnett, Matthew Farrant, Faaizah Khan, John C Pickup, Klaus M Hahn, Christopher J MacNevin, Robert Tarran, Deborah L Baines

Index: Biochem. J. 464(2) , 213-20, (2014)

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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/cm(2)) 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.