title: A novel fluorescent sensor protein for detecting changes in airway surface liquid glucose concentration.
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.
authors.affiliation: *Institute of Cardiovascular and Cell Science, St George's, University of London, London SW17 0RE, U.K.
authors.forename: Nordine
authors.initials: N
authors.lastname: Helassa
authors.affiliation: †Institute for Infection and Immunity, St George's, University of London, London SW17 0RE, U.K.
authors.forename: James P
authors.initials: JP
authors.lastname: Garnett
authors.affiliation: †Institute for Infection and Immunity, St George's, University of London, London SW17 0RE, U.K.
authors.forename: Matthew
authors.initials: M
authors.lastname: Farrant
authors.affiliation: ‡Diabetes Research Group, King's College London, Guy's Hospital Campus, London SE1 1UL, U.K.
authors.forename: Faaizah
authors.initials: F
authors.lastname: Khan
authors.affiliation: ‡Diabetes Research Group, King's College London, Guy's Hospital Campus, London SE1 1UL, U.K.
authors.forename: John C
authors.initials: JC
authors.lastname: Pickup
authors.affiliation: §Department of Pharmacology/Cell Biology & Physiology, University of North Carolina, Chapel Hill, NC, 27599 U.S.A.
authors.forename: Klaus M
authors.initials: KM
authors.lastname: Hahn
authors.affiliation: §Department of Pharmacology/Cell Biology & Physiology, University of North Carolina, Chapel Hill, NC, 27599 U.S.A.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: MacNevin
authors.affiliation: §Department of Pharmacology/Cell Biology & Physiology, University of North Carolina, Chapel Hill, NC, 27599 U.S.A.
authors.forename: Robert
authors.initials: R
authors.lastname: Tarran
authors.affiliation: †Institute for Infection and Immunity, St George's, University of London, London SW17 0RE, U.K.
authors.forename: Deborah L
authors.initials: DL
authors.lastname: Baines
mesh_terms: D015081:2-Naphthylamine / Q000031:analogs & derivatives / Q000737:chemistry; D015374:Biosensing Techniques / Q000379:methods*; D001786:Blood Glucose / Q000737:chemistry / Q000302:isolation & purification*; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D018929:Cell Culture Techniques; D004847:Epithelial Cells / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D006706:Homeostasis; D006801:Humans; D008168:Lung / Q000166:cytology / Q000378:metabolism; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000235:genetics; D009154:Mutation; D033902:Periplasmic Binding Proteins / Q000737:chemistry* / Q000235:genetics
chemical_list: C122155:6-bromoacetyl-2-dimethylaminonaphthalene; D001786:Blood Glucose; D002135:Calcium-Binding Proteins; D005456:Fluorescent Dyes; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; C023550:galactose-binding protein; D015081:2-Naphthylamine