Technical Notes: CFTR Monoclonal Antibodies

Figure 1. Immunohistochemical staining of CFTR in syncytiotrophoblast in human placenta. Cells were stained with R&D Systems' mouse anti-human CFTR C-terminus (Catalog # MAB25031) using R&D Systems' anti-mouse HRP-AEC Cell and Tissue Staining Kit (red) (Catalog # CTS003) and Haematoxylin (blue) counterstain.	Figure 2. Immunochemical staining of CFTR in decidual cells in human placenta. Cells were stained with R&D Systems' mouse anti-human CFTR R-domain (Catalog # MAB1660) using R&D Systems' anti-mouse HRP-DAB Cell and Tissue Staining Kit (brown) (Catalog # CTS002) and Haematoxylin (blue) counterstain.

The gene implicated in cystic fibrosis encodes the cystic fibrosis transmembrane conductance regulator (CFTR). The CFTR protein consists of 1480 amino acids (aa) with 2 extracellular glycosylation sites, 12 transmembrane sequences and two nucleotide binding folds.¹ CFTR is in a class of proteins that includes the human multidrug resistance P-glycoproteins and the protein encoded by the yeast STE6 gene. CFTR has been described as a cell volume regulator (possibly through regulation of the expression of some water channels, aquaporins), a regulator of other ion transporters [epithelial sodium channels (ENaCs), the inwardly-rectifying potassium channel (ROMK2), outwardly rectifying chloride channels (ORCCs)], and a lyotropically selective anion channel. CFTR may physically interact with the AMP-activated protein kinase, syntaxin 1A, syntaxin 8, Na⁺/H⁺ Exchange Regulatory Factor (NHERF or EBP50), b-COP, as well as other, as yet unidentified, proteins.

The complete characterization of three mouse monoclonal antibodies (R-domain/1, C-term/1 and C-term/2) that recognize epitopes within the human CFTR sequence is shown in Table 1. Epitope determination was performed using the SPOTs synthetic peptide technology, using sequential octapeptides of the immunogen sequence. For each monoclonal antibody, the corresponding aa sequence from all available CFTR species variants was synthesized to determine predicted cross-species reactivity. Potential uses for the antibodies include techniques such as immunoprecipitation,² immunofluorescence,³ immunoblotting,⁴ immunoaffinity chromatography,⁵ confocal microscopy,⁶ immunocytochemistry,⁷ immunohistochemistry,⁸ and ELISA.⁴

The R-domain antibody (i.e., the first published anti-CFTR monoclonal antibody^2,3) binds to a region essential in regulation of the chloride channel, adjacent to one of the major protein kinase A phosphorylation sites.⁹ Many of the protein associations mentioned previously are within the C-terminus of CFTR and investigation of these may be facilitated by the antibody C-term/1. This antibody recognizes an epitope that includes the aa ‘Thr-Arg-Leu’ (TRL), a PDZ domain involved in targeting of proteins to specific subcellular compartments. The third antibody, C-term/2, has a predicted affinity for a wider variety of CFTR species than the other two antibodies. Its epitope does not lie within any identified functional or binding region. The unique identities and characteristics of these antibodies identify them as valuable tools for ongoing CFTR research.

References

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2. Gregory, R. et al. (1990) Nature 347:382.
3. Cheng, S.H. et al. (1990) Cell 63:827.
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5. Scheule, R.K. et al. (1995) Am. J. Resp. Cell Mol. Biol. 13:330.
6. Denning, G.M. et al. (1992) Cell Biology 118:551.
7. Zeng, W. et al. (1997) Am. J. Physiol. (Cell Physiol. 42) 273:C442.
8. Dray-Charier, N. et al. (1995) Lab. Invest. 73:828.
9. Cheng, S.H. et al. (1991) Cell 66:1027.