Recent studies suggest that immature, core-glycosylated
DeltaF508-CFTR [the predominant mutant form of the CFTR (
cystic fibrosis transmembrane conductance regulator)] can reach the plasma membrane under some conditions. In the present study we investigated this possibility since it has implications for understanding how
therapeutics rescue the trafficking of mutant CFTR and perhaps other misfolded
proteins. Core-glycosylated CFTR was labelled and pulled down on
streptavidin beads after exposure to
sulfo-NHS-SS-
biotin [
biotin attached to a reactive NHS (
N-hydroxysuccinimide)
ester with a
disulfide spacer; molecular mass=606.7 Da]; however, intracellular
proteins were also detected in the precipitates. When the R domain of CFTR was expressed in the cytosol of BHK (baby-hamster kidney) cells as a soluble
polypeptide it was also labelled after surface biotinylation and pulled down on
streptavidin beads. Intracellular biotinylation was reduced when cells were treated with
sulfo-NHS-LC-biotin (
biotin attached to a reactive NHS
ester with an
aminocaproic acid spacer) or sulfo-NHS-PEO(12)-biotin [
biotin attached to a reactive NHS
ester with a poly(
ethylene glycol) spacer], but the reduction could be explained by the lower reactivity of these
reagents. The R domain was detected on Western blots after loading <0.25% of the pulldown sample ( approximately 0.01% of total lysate
protein), a fraction that could be ascribed to cells that were permeable to
ethidium homodimer-1 (molecular mass=856.8 Da) and
propidium iodide (molecular mass=668.6 Da). When BHK cells were incubated at 29 degrees C to rescue
DeltaF508-CFTR trafficking, and then biotinylated and sorted to remove permeable cells, labelling of core-glycosylated
DeltaF508-CFTR was no longer detected although a weak signal was still observed using CFBE (
cystic fibrosis bronchial epithelial) cells. These results suggest that there is weak surface expression of immature
DeltaF508-CFTR on airway epithelial cells and demonstrate the need to remove permeable cells when studying CFTR glycoforms by surface biotinylation.