Selective detection of proteins at the cell surface is a prerequisite for studying the trafficking behavior of membrane proteins, such as CFTR, to and from the plasma membrane and for evaluating therapeutic approaches to diseases of protein folding. Traditionally, this has been accomplished by time- and labor-intensive biochemical labeling methods such as biotinylation and immunofluorescence. By using a genetically encoded FAP reporter, however, we can selectively label CFTR at the cell surface in live cells instantly without the need for incubation or wash steps that may modify the cellular handling of CFTR. The cell-impermeant fluorogen remains dark when free in solution; however, upon binding to the FAP, fluorogen fluorescence increases >15,000-fold. These features provide low background signal, excellent signal-to-noise and a large dynamic range, and they eliminate the need for wash or blocking steps to remove nonspecific signals. Proper trafficking and localization of FAP-tagged CFTR to the cell surface was confirmed using confocal fluorescence microscopy in stable cell lines. Importantly, F508del-CFTR FAP protein was absent from the plasma membrane under normal conditions, but was readily detected by cell-impermeant fluorogen after treatment by currently available correctors.
Immunoblot analysis was used to reveal the glycosylation state of the FAP-CFTR WT and CFTR WT EL4-FAP fusion constructs. The N-terminal FAP-CFTR WT fusion protein migrated as two distinct bands, the core and mature bands characteristic of untagged CFTR WT. The CFTR EL4-FAP construct did not produce a distinct mature band, which suggests that the efficiency of its glycosylation was impaired. The fourth extracellular loop of CFTR is the locus of two asparagine residues (N894 and N900) that acquire N-linked glycosylation and other modifications; therefore, careful consideration was taken to preserve these residues, and the consensus glycosylation sequences (NXS/T) surrounding them, in the FAP fusion construct (29
). The observed incomplete glycosylation could be due to reduced glycan modification or partial oligosac-charide modification at these sites, which may result from impaired glycosylation enzyme recognition and/or accessibility at these sites when the FAP tag is present. Modification of the FAP location within EL4 or by varying the spacer length and/or properties may improve glycosylation. However, in view of the adequacy of the N-terminal reporter, this step has not been pursued.
Importantly, immunoblots showed that corrector treatment was capable of rescuing the maturation of FAP-F508del-CFTR, albeit modestly (). Accumulation of the C band after corrector treatment is quite low with untagged F508del-CFTR and can depend on the cell type examined (13
). To date, C4 is one of the most well-studied correctors for F508del-CFTR rescue. However, the efficiency of rescue for C4 is quite limited across multiple cell types that have been tested (15
). This result is in agreement with data obtained from both F508del-CFTR FAP constructs in HEK293 cells as well as the currents associated with F508del-CFTR endogenously expressed from HBE cells, where functional C4 correction efficacy was poor (). Unlike C4, C18 displayed a more robust rescue in both HEK293 cells expressing FAP-CFTR constructs and in HBE cells expressing endogenous F508del-CFTR. Because rescue by C18 was independent of cellular background, this molecule may interact directly with F508del-CFTR or through a common quality control pathway expressed in both systems. Future studies to determine the exact mechanism of these correctors, especially C18 and related compounds, will increase our understanding of the F508del-CFTR folding defect and can help guide the design of new and more effective correctors or corrector combinations.
Combinations of corrector treatments, particularly CFFT-002 and C18, improved F508del-CFTR trafficking and function over single corrector treatments alone (). The enhanced effects produced from combinations of correctors, acting at their maximally effective concentrations, indicate that these corrector compounds likely have distinct targets and/or mechanisms of action. One possibility is that each corrector interacts with a different structural feature of F508del-CFTR to stabilize their folding or reduce nonproductive folding intermediates. Because the folding defect of F508del involves multiple domain and interdomain interactions, multiple interaction sites may be necessary for optimal correction of the misfolded protein (33
). Alternatively, rescue might be achieved indirectly through the modulation of folding chaperones or by suppressing ER–associated degradation (ERAD) quality control pathways that promote F508del-CFTR ubiquitylation and degradation. In addition, since rescued F508del-CFTR may also be subject to quality control pathways at the cell surface, rescue improvements may arise via actions at different cell loci (28
). Because the development of combination therapies to correct CFTR trafficking may be required to obtain sufficient efficacy, the availability of a method with a broad dynamic range should optimize the detection of a signal that approaches that of WT CFTR.
Alternative tagging strategies were used to study CFTR to minimize artifacts due to the fluorescent reporter position. Although, the N-terminus and EL4 constructs both produced functional CFTR fusion proteins for which behavior was qualitatively similar, there were some important differences between them. In addition to the impaired glycosylation of the EL4 constructs, the plasma membrane density and anion transport of F508del-CFTR EL4-FAP, even after corrector treatment, was substantially diminished compared with the N-terminal fusion. Therefore, the N-terminal fusion appears to be superior because of its more robust expression, glycosylation state and anion transport capabilities.
The pattern of corrector efficacy was strikingly similar across the disparate cell types and assays that we examined, and this has not always been observed (15
). Nevertheless, our results highlight the fidelity of the FAP reporter system to recapitulate the behavior of F508del-CFTR in a system that expresses the mutant untagged protein endogenously. Taken together, these results demonstrate that the FAP reporter system is sensitive enough to elucidate differences in the extent of correction of cell surface expression among different correctors. Moreover, this pattern of corrector action correlates with functional data obtained from differentiated human airway epithelia, a drug development system that has enabled the transition from preclinical data to clinical trials (39