Proc. Nadl. Acad. Sci. USA Vol. 88, pp. 9262-9266, October 1991 Cell Biology
Immunocytochemical localization of the cystic fibrosis gene product CFTR (epithelium/apical membrane/chloride ions/lung/pancreas)
ISABELLE CRAWFORD*, PETER C. MALONEY*, PAMELA L. ZEITLINt, WILLIAM B. GUGGINO*t, STEPHEN C. HYDE§, HELEN TURLEY¶, KEVIN C. GATTER¶,, ANN HARRIS", AND CHRISTOPHER F. HIGGINS§ Departments of *Physiology and tPediatrics, The Johns Hopkins University School of Medicine, Baltimore, MD 21205; SICRF Laboratories and IlPaediatric Molecular Genetics, Institute of Molecular Medicine, and IDepartment of Pathology, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom
Communicated by David Weatherall, July 3, 1991 (received for review May 1, 1991)
ABSTRACT Antisera against two peptides, corresponding to different domains of the cystic fibrosis gene product CFTR, have been raised and extensively characterized. Both antisera recognize CFTR as a 165-kDa polypeptide in Western analysis of cells transfected with CFTR cDNA as well as in epithellal cell lines. The cell and tissue distribution of CFTR has been studied by immunocytochemistry. CFTR is abundant in epithelial cells, including those lining sweat ducts, small pancreatic ducts, and intestinal crypts. Unexpectedly, the level of CFTR in lung epithelia is relatively low, while it is abundant in the epithelia of kidney tubules. The protein appears to be restricted to the apical, rather than basolateral, regions of epithelial cells and at least a proportion is associated with the plasma membrane. The cell and tissue distributions of CFTR are consistent with a function for this protein as a chloride channel or as a regulator of channel activity.
MATERIALS AND METHODS
Cystic fibrosis (CF) is the most common lethal autosomal recessive disease among Caucasians. An important clinical symptom in CF is the accumulation of mucus in the lung. However, pathologies associated with other epithelia are common and can include pancreatic insufficiency, altered sweat electrolytes, and intestinal abnormalities such as meconium ileus. The most intensively studied biochemical difference between normal and CF cells is a defect in epithelial chloride ion channel activity (1-4). The CF gene encodes a predicted 168-kDa protein, designated the CF transmembrane conductance regulator (CFTR) (5). The gene can correct chloride channel defects when expressed in cell lines derived from CF patients (6, 7) and overexpression of CFTR in mammalian and insect cells can cause the appearance of a new chloride channel activity (8, 9). The latter observations support the notion that CFTR is a chloride channel. However, the similarities in sequence and domain organization between CFTR and the ABC superfamily of ATP-linked transporters suggest that CFTR may be more than a conventional channel (10). In addition, the mechanisms by which a defect in this protein leads to the many biochemical and clinical symptoms of CF remain to be elucidated. A more complete understanding of the biochemical function of CFTR, and its role in the clinical pathology of CF, requires a detailed analysis of its cell and tissue distribution. It is known that CFTR mRNA is present in epithelial cells and in tissues that are affected by the disease (5). This paper describes the isolation and extensive characterization of antibodies against CFTR and their use in immunocytochemical localization of the protein.
Anti-CFTR Antibodies. Antibodies against peptides comprising amino acids 724-746 (from exon 13 of the R domain of CFTR) and amino acids 415-427 (from exon 9 preceding the first nucleotide-binding domain) were raised as follows. The synthetic peptides were crosslinked to bovine serum albumin with 1% glutaraldehyde (11). The efficiency of cross-linking was virtually complete as assessed by SDS/ PAGE. Immunogen, 200 gg, in Freund's complete adjuvant was injected intradermally into New Zealand White rabbits, followed by biweekly booster injections of 100-200 ,.g of immunogen in Freund's incomplete adjuvant. The antibodies were designated 169 (anti-R domain) and 181 (antiprenucleotide-binding fold; anti-pre-NBF). Expression of the CFIR R Domain in Escherichia coli. The R domain of CFTR was expressed in E. coli as follows. Exon 13 (nucleotides 1915-2616) of the CF gene was amplified from HeLa cell DNA by the polymerase chain reaction. The primers used (5 '-GCTTGTAAACGCATGCTGAACAAAACTAGG-3' and 5 '-CGGATCCTTAGTCTTCTTCGTTAATTTC-3') placed restriction sites at either end of the amplified fragment to facilitate direct cloning into the Sph I and BamHI sites of plasmid pJLA504 (12). This placed the R domain under control of the temperature-inducible APL and APR promoters and the atpE translation initiation signal. The resulting plasmid, pIMS5105, was sequenced and shown to contain the correct DNA insert except for a single difference (A to C at nucleotide 1990) from the published sequence (the published sequence contains an error at this point; L.-C. Tsui, personal communication). pIMS5105 was transformed into the Alon htpR::TnlO protease-deficient strain CH1790 (13). Cells were grown to midexponential phase at 300C in LB, and expression of the R domain from the APL and APR promoters was induced by rapidly shifting the temperature to 420C and incubating the cells for a further 2 h at this temperature; uninduced cells were retained at 300C. Gel Electrophoresis and Western Blotting. Proteins were separated by electrophoresis on SDS/5% or 15% polyacrylamide gels (14) as indicated. The proteins were then transferred to nitrocellulose (15) and probed with a 1:1000 dilution of antibody. Cross-reacting proteins were detected by the enhanced chemiluminescence procedure (15-60 sec exposure; Amersham). Cell Culture and Transfection. Plasmid DNA was transfected into COS-1 cells by the DEAE-dextran method (16), and the cells were harvested after 72 h. For transfections, full-length CFTR cDNA (6) was cloned into the expression plasmid pSGM3X to generate pCOF-1 (25). pCOF-1 and the
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviations: CF, cystic fibrosis; CFTR, CF transmembrane ductance regulator; NBF, nucleotide-binding fold. tTo whom reprint requests should be addressed.
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Cell Biology: Crawford et al. vector pSGM3X were transfected, separately, into COS cells. Proteins from transfected cells were extracted by adding 10% SDS at 1000C, followed by an equal volume of buffer (250 mM sucrose/5 mM Mops/i mM EDTA, pH 7.5) and a mixture of protease inhibitors. Approximately 50 pg of total protein was loaded into each gel track. Immunocytochemistry. Unfixed, fresh human tissue samples were snap-frozen in liquid nitrogen immediately after collection. Cryostat sections (5-8 um) were dried at room temperature, overnight, fixed in acetone for 10 min, and stained by the alkaline phosphatase/anti-alkaline phosphatase method (pink staining is positive; ref. 17). Antibodies (1:1000) were added to the sections in a moist chamber for 30 min. The alkaline phosphatase was developed with naphthol AS-BI phosphate (Sigma) and new fuchsin (Merck) for 15-20 min. The sections were then washed, counterstained with hematoxylin, and mounted in suitable aqueous mounting medium. For sections in which endogenous phosphatases cannot easily be eliminated, a three-stage immunoperoxidase technique was used with diaminobenzidine as chromogen (brown staining is positive; ref. 18).
RESULTS Antl-CFTR Antibodies. Polyclonal antibodies were raised against two synthetic peptides, one corresponding to sequences within the R domain ofCFTR (antibody 169) and one corresponding to sequences between the first transmembrane domain and the first nucleotide-binding domain (antibody 181). These peptides were selected because oftheir predicted immunogenicity and because they spanned potential phosphorylation sites. In addition, each was targeted to a different domain of CFTR and, because the R domain appears to be unique to CFTR, the anti-R-domain antibody might be expected to recognize CFTR specifically. Several lines of evidence demonstrate that both these antibodies recognize CFTR specifically, yet do not cross-react significantly with other cellular proteins. Recognition of the R Domain Expressed in E. coHl. Fig. LA shows that the anti-R-domain antibodies (antibody 169) gave a strong, specific signal against the R domain of CFTR expressed in E. coli. A cross-reacting polypeptide of the predicted size (27 kDa) was detected after temperature induction of cells harboring the R-domain expression plasmid pIMS5105 (lane 4). This cross-reacting protein was absent from uninduced cells (lane 3) and from cells, induced or uninduced, harboring the vector alone (lanes 1 and 2). The specificity of this reaction was verified by preincubation of the antibodies with the peptide immunogen that blocked recognition of the R domain (Fig. 1B, compare lane 3 with lane 2). The expressed R domain was not recognized by preimmune serum (lane 1). As expected, antibodies raised against sequences from the pre-NBF region of CFTR (antibody 181) failed to cross-react with the expressed R domain (lane 4), confirming that the two antisera recognize different epitopes. Recognition of CFFR as a 165-kDa Polypeptide. To demonstrate that the anti-peptide antibodies recognize CFTR itself, COS-1 cells were transfected with full-length cDNA encoding CFTR. PCR amplification confirmed that CFTR mRNA was significantly increased in COS-1 cells transfected with the CFTR expression plasmid, compared with the vector alone (data not shown). Proteins were extracted from transfected and mock-transfected cells and separated by electrophoresis; Western transfers of the proteins were stained separately with the two anti-peptide antibodies (antibodies 169 and 181). Fig. 2 shows the result of such an experiment using the anti-pre-NBF antibodies (antibody 181). Similar results were obtained when the blot was reprobed with the anti-R-domain
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FIG. 1. Detection of the R domain of CF1R by Western blotting. Total cell protein from E. coli strain CH1790 harboring the vector pJLAS04 (lanes 1 and 2) or the R-domain expression plasmid pIMS5105 (lanes 3 and 4) was separated by electrophoresis on a SDS/15% polyacrylamide gel, transferred to nitrocellulose, and probed with a 1:1000 dilution of the appropriate antibody (antibody 169). Cross-reacting proteins were detected by chemiluminescence. (A) Cells were uninduced (lanes 1 and 3) or induced for R-domain expression (lanes 2 and 4) by temperature shift to 4rC. The blot was probed with the anti-R-domain antibodies (antibody 169). The crossreactive R domain is indicated by the arrow. The two smaller cross-reacting bands in lane 4 are proteolytic products of the R domain expressed in E. coli. The -65-kDa polypeptide detected in all four lanes is GroEL, an E. coli molecular chaperone that is recognized by many immunoglobulin preparations. The mobility of molecular mass markers (kDa) is indicated. (B) Total cell proteins from E. coli strain CH1790 harboring the R-domain expression plasmid pIMS5105, temperature induced for expression of the R domain, were separated and transferred to nitrocellulose as described in A. The nitrocellulose filter was cut into strips and probed with different antisera. Lanes: 1, preimmune serum; 2, anti-Rdomain antiserum (antibody 169); 3, anti-R-domain antiserum (antibody 169) preincubated with the peptide immunogen (1 mg-ml-') at 4°C for 16 h; 4, anti-pre-NBF antiserum (antibody 181). The R domain is indicated by the arrow. The 65-kDa polypeptide detected in all lanes is GroEL (see above). The mobility of molecular mass markers (kDa) is indicated.
antibodies (antibody 169; data not shown). In CFTRtransfected COS-1 cells, strong cross-reaction with a polypeptide of apparent mobility 165 kDa was obtained (lane 3); this is close to the molecular size predicted for CFTR from the cDNA sequence (5). The amount of this protein was significantly elevated after transfection of COS-1 cells with CFTlR cDNA, although low levels of the protein were detected in mock-transfected COS-1 cells (lane 1) and in COS-1 cells transfected with the vector alone (lane 2). A number of smaller polypeptides were also detected in CFTR-expressing cells, which are presumed to be degradation products. Several lines of evidence confirm that the 165-kDa protein is indeed CFTR. First, a protein of indistinguishable mobility was detected in Western blots of proteins from two different human epithelial cell lines, T84 and FHTE (lanes 4 and 5), but was absent from extracts of an endothelial cell line (lane 6). The T84 proteins loaded in lane 4 were derived from membrane preparations (isolated as described elsewhere; ref. 19) demonstrating that at least some CFTR in these cells is on the membrane. Second, a protein of identical mobility (165 kDa) was detected with the same antibodies in Xenopus oocytes transfected with CFTR cDNA (20). Third, a well-characterized monoclonal antibody against CFTR, raised previously by another group (21), identified the same 165-kDa protein in CFTR-transfected COS-1 cells (data not shown). Finally, and most importantly, both antibodies 169 and 181 recognized the same 165-kDa polypeptide despite being directed against
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FIG. 2. Detection of the CFTR protein by Western blotting. Cellular proteins were separated on a SDS/5% polyacrylamide gel, transferred to nitrocellulose, and probed with anti-CFTR antibodies (antibody 181). The gel was loaded as follows. Lanes: 1, mocktransfected COS-1 cells; 2, COS-1 cells transfected with the vector pSGM3X; 3, COS-1 cells transfected with the CFTR expression plasmid pCOF-1; 4, membranes from T84 colon carcinoma cells; 5, FHTE fetal tracheal epithelial cells; 6, bronchial pulmonary artery endothelial cells. The 165-kDa CFTR polypeptide is indicated by the arrow. The mobility of molecular mass markers (kDa) is indicated.
peptide sequences derived from different domains of CFTR and despite the demonstration (Fig. 1B; see above) that they recognize different epitopes. Thus, the 165-kDa protein must contain both epitopes, giving a high degree of confidence to its assignment as CFTR. The CFTR protein routinely migrated as a relatively tight band of -165 kDa in SDS/polyacrylamide gels, yet Gregory et al. (21) reported that CFTR migrates as a diffuse band of -150 kDa. In our hands, the band detected with our antibodies was indistinguishable from that identified by the antibody of Gregory et al. (ref. 21; purchased from Genzyme; data not shown). Thus, there is no fundamental discrepancy between the two data sets, and the apparent differences in electrophoretic mobility must reflect local assay conditions. Specificity of Immunocytochemical Staining. To use the two antibodies for tissue localization of CFTR, they must be shown to be proficient and specific for immunocytochemical staining. Fig. 3 shows that the anti-R-domain antibodies (antibody 169) stain E. coli cells expressing the R domain of
CFTR (Fig. 3A) but not control cells (Fig. 3B). Similarly, the antibodies stained COS-1 cells transfected with full-length CFTR cDNA (Fig. 3C); untransfected cells and cells transfected with the vector control (Fig. 3D) stained very weakly, consistent with the observation (see above; Fig. 2) that COS-1 cells express CFTR at a low level. Similar results for transfected COS-1 cells were also obtained with antibody 181 (data not shown). Specificity was confirmed by showing that tissue staining was blocked by preincubation of the antiserum with the corresponding peptide immunogen. Thus, under the conditions used for immunocytochemical staining, the antibodies recognize CFTR and do not appear to recognize any other protein expressed in these cells. Fig. 3 also shows that the antibodies recognize CFTR in tissue sections. Fig. 3E shows staining of a section of pancreas. Positive staining was restricted to epithelial cells lining the small ducts (exocrine canniculi) (see Fig. 4 for further details). A similar pattern of staining was observed with antibodies 181 (data not shown). The tightness of this staining pattern and the fact that two antibodies raised against different immunogens gave similar staining patterns are strong evidence of specificity. Confirmation that staining is not due to endogenous phosphatases was obtained by showing that preincubation of the antibody with peptide immunogen inhibited staining (Fig. 3F); staining was not inhibited by preincubation with a heterologous peptide. Tissue Distribution of CFTR. Fig. 4 shows the cell and tissue distribution of CFTR using the anti-R-domain antibodies (antibody 169). Similar staining patterns were obtained when the anti-pre-NBF antibodies (antibody 181) were used (data not shown). The fact that these antibodies recognize different domains of the protein is strong evidence that the staining patterns accurately reflect the distribution of CFTR. CFTR was detected in many epithelial cells. In the pancreas (Fig. 4 A-D; see also Fig. 3D), CFTR is restricted to epithelial cells lining the small secretory ducts (exocrine canniculi). No staining of the acinar cells themselves, or of the islets of Langerhans (indicated by an asterisk in A), was observed. CFTR was not present at detectable levels in the epithelia of larger pancreatic ducts (Fig. 3E, arrow). At high magnification (Fig. 4 B and D), it is clear that CFTR is not evenly distributed throughout the epithelial cells lining the small pancreatic ducts but that it is restricted to the luminal (apical) regions of each cell. Little or no protein was detected in basolateral regions of the cells. In the jejunum (Fig. 4E), the mucosal epithelial cells were positive for CFTR; less protein was present in the epithelia lining the crypts of the
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Immunocytochemical localization of the cystic fibrosis gene product CFTR (epithelium/apical membrane/chloride ions/lung/pancreas)
ISABELLE CRAWFORD*, PETER C. MALONEY*, PAMELA L. ZEITLINt, WILLIAM B. GUGGINO*t, STEPHEN C. HYDE§, HELEN TURLEY¶, KEVIN C. GATTER¶,, ANN HARRIS", AND CHRISTOPHER F. HIGGINS§ Departments of *Physiology and tPediatrics, The Johns Hopkins University School of Medicine, Baltimore, MD 21205; SICRF Laboratories and IlPaediatric Molecular Genetics, Institute of Molecular Medicine, and IDepartment of Pathology, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom
Communicated by David Weatherall, July 3, 1991 (received for review May 1, 1991)
ABSTRACT Antisera against two peptides, corresponding to different domains of the cystic fibrosis gene product CFTR, have been raised and extensively characterized. Both antisera recognize CFTR as a 165-kDa polypeptide in Western analysis of cells transfected with CFTR cDNA as well as in epithellal cell lines. The cell and tissue distribution of CFTR has been studied by immunocytochemistry. CFTR is abundant in epithelial cells, including those lining sweat ducts, small pancreatic ducts, and intestinal crypts. Unexpectedly, the level of CFTR in lung epithelia is relatively low, while it is abundant in the epithelia of kidney tubules. The protein appears to be restricted to the apical, rather than basolateral, regions of epithelial cells and at least a proportion is associated with the plasma membrane. The cell and tissue distributions of CFTR are consistent with a function for this protein as a chloride channel or as a regulator of channel activity.
MATERIALS AND METHODS
Cystic fibrosis (CF) is the most common lethal autosomal recessive disease among Caucasians. An important clinical symptom in CF is the accumulation of mucus in the lung. However, pathologies associated with other epithelia are common and can include pancreatic insufficiency, altered sweat electrolytes, and intestinal abnormalities such as meconium ileus. The most intensively studied biochemical difference between normal and CF cells is a defect in epithelial chloride ion channel activity (1-4). The CF gene encodes a predicted 168-kDa protein, designated the CF transmembrane conductance regulator (CFTR) (5). The gene can correct chloride channel defects when expressed in cell lines derived from CF patients (6, 7) and overexpression of CFTR in mammalian and insect cells can cause the appearance of a new chloride channel activity (8, 9). The latter observations support the notion that CFTR is a chloride channel. However, the similarities in sequence and domain organization between CFTR and the ABC superfamily of ATP-linked transporters suggest that CFTR may be more than a conventional channel (10). In addition, the mechanisms by which a defect in this protein leads to the many biochemical and clinical symptoms of CF remain to be elucidated. A more complete understanding of the biochemical function of CFTR, and its role in the clinical pathology of CF, requires a detailed analysis of its cell and tissue distribution. It is known that CFTR mRNA is present in epithelial cells and in tissues that are affected by the disease (5). This paper describes the isolation and extensive characterization of antibodies against CFTR and their use in immunocytochemical localization of the protein.
Anti-CFTR Antibodies. Antibodies against peptides comprising amino acids 724-746 (from exon 13 of the R domain of CFTR) and amino acids 415-427 (from exon 9 preceding the first nucleotide-binding domain) were raised as follows. The synthetic peptides were crosslinked to bovine serum albumin with 1% glutaraldehyde (11). The efficiency of cross-linking was virtually complete as assessed by SDS/ PAGE. Immunogen, 200 gg, in Freund's complete adjuvant was injected intradermally into New Zealand White rabbits, followed by biweekly booster injections of 100-200 ,.g of immunogen in Freund's incomplete adjuvant. The antibodies were designated 169 (anti-R domain) and 181 (antiprenucleotide-binding fold; anti-pre-NBF). Expression of the CFIR R Domain in Escherichia coli. The R domain of CFTR was expressed in E. coli as follows. Exon 13 (nucleotides 1915-2616) of the CF gene was amplified from HeLa cell DNA by the polymerase chain reaction. The primers used (5 '-GCTTGTAAACGCATGCTGAACAAAACTAGG-3' and 5 '-CGGATCCTTAGTCTTCTTCGTTAATTTC-3') placed restriction sites at either end of the amplified fragment to facilitate direct cloning into the Sph I and BamHI sites of plasmid pJLA504 (12). This placed the R domain under control of the temperature-inducible APL and APR promoters and the atpE translation initiation signal. The resulting plasmid, pIMS5105, was sequenced and shown to contain the correct DNA insert except for a single difference (A to C at nucleotide 1990) from the published sequence (the published sequence contains an error at this point; L.-C. Tsui, personal communication). pIMS5105 was transformed into the Alon htpR::TnlO protease-deficient strain CH1790 (13). Cells were grown to midexponential phase at 300C in LB, and expression of the R domain from the APL and APR promoters was induced by rapidly shifting the temperature to 420C and incubating the cells for a further 2 h at this temperature; uninduced cells were retained at 300C. Gel Electrophoresis and Western Blotting. Proteins were separated by electrophoresis on SDS/5% or 15% polyacrylamide gels (14) as indicated. The proteins were then transferred to nitrocellulose (15) and probed with a 1:1000 dilution of antibody. Cross-reacting proteins were detected by the enhanced chemiluminescence procedure (15-60 sec exposure; Amersham). Cell Culture and Transfection. Plasmid DNA was transfected into COS-1 cells by the DEAE-dextran method (16), and the cells were harvested after 72 h. For transfections, full-length CFTR cDNA (6) was cloned into the expression plasmid pSGM3X to generate pCOF-1 (25). pCOF-1 and the
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviations: CF, cystic fibrosis; CFTR, CF transmembrane ductance regulator; NBF, nucleotide-binding fold. tTo whom reprint requests should be addressed.
9262
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Cell Biology: Crawford et al. vector pSGM3X were transfected, separately, into COS cells. Proteins from transfected cells were extracted by adding 10% SDS at 1000C, followed by an equal volume of buffer (250 mM sucrose/5 mM Mops/i mM EDTA, pH 7.5) and a mixture of protease inhibitors. Approximately 50 pg of total protein was loaded into each gel track. Immunocytochemistry. Unfixed, fresh human tissue samples were snap-frozen in liquid nitrogen immediately after collection. Cryostat sections (5-8 um) were dried at room temperature, overnight, fixed in acetone for 10 min, and stained by the alkaline phosphatase/anti-alkaline phosphatase method (pink staining is positive; ref. 17). Antibodies (1:1000) were added to the sections in a moist chamber for 30 min. The alkaline phosphatase was developed with naphthol AS-BI phosphate (Sigma) and new fuchsin (Merck) for 15-20 min. The sections were then washed, counterstained with hematoxylin, and mounted in suitable aqueous mounting medium. For sections in which endogenous phosphatases cannot easily be eliminated, a three-stage immunoperoxidase technique was used with diaminobenzidine as chromogen (brown staining is positive; ref. 18).
RESULTS Antl-CFTR Antibodies. Polyclonal antibodies were raised against two synthetic peptides, one corresponding to sequences within the R domain ofCFTR (antibody 169) and one corresponding to sequences between the first transmembrane domain and the first nucleotide-binding domain (antibody 181). These peptides were selected because oftheir predicted immunogenicity and because they spanned potential phosphorylation sites. In addition, each was targeted to a different domain of CFTR and, because the R domain appears to be unique to CFTR, the anti-R-domain antibody might be expected to recognize CFTR specifically. Several lines of evidence demonstrate that both these antibodies recognize CFTR specifically, yet do not cross-react significantly with other cellular proteins. Recognition of the R Domain Expressed in E. coHl. Fig. LA shows that the anti-R-domain antibodies (antibody 169) gave a strong, specific signal against the R domain of CFTR expressed in E. coli. A cross-reacting polypeptide of the predicted size (27 kDa) was detected after temperature induction of cells harboring the R-domain expression plasmid pIMS5105 (lane 4). This cross-reacting protein was absent from uninduced cells (lane 3) and from cells, induced or uninduced, harboring the vector alone (lanes 1 and 2). The specificity of this reaction was verified by preincubation of the antibodies with the peptide immunogen that blocked recognition of the R domain (Fig. 1B, compare lane 3 with lane 2). The expressed R domain was not recognized by preimmune serum (lane 1). As expected, antibodies raised against sequences from the pre-NBF region of CFTR (antibody 181) failed to cross-react with the expressed R domain (lane 4), confirming that the two antisera recognize different epitopes. Recognition of CFFR as a 165-kDa Polypeptide. To demonstrate that the anti-peptide antibodies recognize CFTR itself, COS-1 cells were transfected with full-length cDNA encoding CFTR. PCR amplification confirmed that CFTR mRNA was significantly increased in COS-1 cells transfected with the CFTR expression plasmid, compared with the vector alone (data not shown). Proteins were extracted from transfected and mock-transfected cells and separated by electrophoresis; Western transfers of the proteins were stained separately with the two anti-peptide antibodies (antibodies 169 and 181). Fig. 2 shows the result of such an experiment using the anti-pre-NBF antibodies (antibody 181). Similar results were obtained when the blot was reprobed with the anti-R-domain
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FIG. 1. Detection of the R domain of CF1R by Western blotting. Total cell protein from E. coli strain CH1790 harboring the vector pJLAS04 (lanes 1 and 2) or the R-domain expression plasmid pIMS5105 (lanes 3 and 4) was separated by electrophoresis on a SDS/15% polyacrylamide gel, transferred to nitrocellulose, and probed with a 1:1000 dilution of the appropriate antibody (antibody 169). Cross-reacting proteins were detected by chemiluminescence. (A) Cells were uninduced (lanes 1 and 3) or induced for R-domain expression (lanes 2 and 4) by temperature shift to 4rC. The blot was probed with the anti-R-domain antibodies (antibody 169). The crossreactive R domain is indicated by the arrow. The two smaller cross-reacting bands in lane 4 are proteolytic products of the R domain expressed in E. coli. The -65-kDa polypeptide detected in all four lanes is GroEL, an E. coli molecular chaperone that is recognized by many immunoglobulin preparations. The mobility of molecular mass markers (kDa) is indicated. (B) Total cell proteins from E. coli strain CH1790 harboring the R-domain expression plasmid pIMS5105, temperature induced for expression of the R domain, were separated and transferred to nitrocellulose as described in A. The nitrocellulose filter was cut into strips and probed with different antisera. Lanes: 1, preimmune serum; 2, anti-Rdomain antiserum (antibody 169); 3, anti-R-domain antiserum (antibody 169) preincubated with the peptide immunogen (1 mg-ml-') at 4°C for 16 h; 4, anti-pre-NBF antiserum (antibody 181). The R domain is indicated by the arrow. The 65-kDa polypeptide detected in all lanes is GroEL (see above). The mobility of molecular mass markers (kDa) is indicated.
antibodies (antibody 169; data not shown). In CFTRtransfected COS-1 cells, strong cross-reaction with a polypeptide of apparent mobility 165 kDa was obtained (lane 3); this is close to the molecular size predicted for CFTR from the cDNA sequence (5). The amount of this protein was significantly elevated after transfection of COS-1 cells with CFTlR cDNA, although low levels of the protein were detected in mock-transfected COS-1 cells (lane 1) and in COS-1 cells transfected with the vector alone (lane 2). A number of smaller polypeptides were also detected in CFTR-expressing cells, which are presumed to be degradation products. Several lines of evidence confirm that the 165-kDa protein is indeed CFTR. First, a protein of indistinguishable mobility was detected in Western blots of proteins from two different human epithelial cell lines, T84 and FHTE (lanes 4 and 5), but was absent from extracts of an endothelial cell line (lane 6). The T84 proteins loaded in lane 4 were derived from membrane preparations (isolated as described elsewhere; ref. 19) demonstrating that at least some CFTR in these cells is on the membrane. Second, a protein of identical mobility (165 kDa) was detected with the same antibodies in Xenopus oocytes transfected with CFTR cDNA (20). Third, a well-characterized monoclonal antibody against CFTR, raised previously by another group (21), identified the same 165-kDa protein in CFTR-transfected COS-1 cells (data not shown). Finally, and most importantly, both antibodies 169 and 181 recognized the same 165-kDa polypeptide despite being directed against
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FIG. 2. Detection of the CFTR protein by Western blotting. Cellular proteins were separated on a SDS/5% polyacrylamide gel, transferred to nitrocellulose, and probed with anti-CFTR antibodies (antibody 181). The gel was loaded as follows. Lanes: 1, mocktransfected COS-1 cells; 2, COS-1 cells transfected with the vector pSGM3X; 3, COS-1 cells transfected with the CFTR expression plasmid pCOF-1; 4, membranes from T84 colon carcinoma cells; 5, FHTE fetal tracheal epithelial cells; 6, bronchial pulmonary artery endothelial cells. The 165-kDa CFTR polypeptide is indicated by the arrow. The mobility of molecular mass markers (kDa) is indicated.
peptide sequences derived from different domains of CFTR and despite the demonstration (Fig. 1B; see above) that they recognize different epitopes. Thus, the 165-kDa protein must contain both epitopes, giving a high degree of confidence to its assignment as CFTR. The CFTR protein routinely migrated as a relatively tight band of -165 kDa in SDS/polyacrylamide gels, yet Gregory et al. (21) reported that CFTR migrates as a diffuse band of -150 kDa. In our hands, the band detected with our antibodies was indistinguishable from that identified by the antibody of Gregory et al. (ref. 21; purchased from Genzyme; data not shown). Thus, there is no fundamental discrepancy between the two data sets, and the apparent differences in electrophoretic mobility must reflect local assay conditions. Specificity of Immunocytochemical Staining. To use the two antibodies for tissue localization of CFTR, they must be shown to be proficient and specific for immunocytochemical staining. Fig. 3 shows that the anti-R-domain antibodies (antibody 169) stain E. coli cells expressing the R domain of
CFTR (Fig. 3A) but not control cells (Fig. 3B). Similarly, the antibodies stained COS-1 cells transfected with full-length CFTR cDNA (Fig. 3C); untransfected cells and cells transfected with the vector control (Fig. 3D) stained very weakly, consistent with the observation (see above; Fig. 2) that COS-1 cells express CFTR at a low level. Similar results for transfected COS-1 cells were also obtained with antibody 181 (data not shown). Specificity was confirmed by showing that tissue staining was blocked by preincubation of the antiserum with the corresponding peptide immunogen. Thus, under the conditions used for immunocytochemical staining, the antibodies recognize CFTR and do not appear to recognize any other protein expressed in these cells. Fig. 3 also shows that the antibodies recognize CFTR in tissue sections. Fig. 3E shows staining of a section of pancreas. Positive staining was restricted to epithelial cells lining the small ducts (exocrine canniculi) (see Fig. 4 for further details). A similar pattern of staining was observed with antibodies 181 (data not shown). The tightness of this staining pattern and the fact that two antibodies raised against different immunogens gave similar staining patterns are strong evidence of specificity. Confirmation that staining is not due to endogenous phosphatases was obtained by showing that preincubation of the antibody with peptide immunogen inhibited staining (Fig. 3F); staining was not inhibited by preincubation with a heterologous peptide. Tissue Distribution of CFTR. Fig. 4 shows the cell and tissue distribution of CFTR using the anti-R-domain antibodies (antibody 169). Similar staining patterns were obtained when the anti-pre-NBF antibodies (antibody 181) were used (data not shown). The fact that these antibodies recognize different domains of the protein is strong evidence that the staining patterns accurately reflect the distribution of CFTR. CFTR was detected in many epithelial cells. In the pancreas (Fig. 4 A-D; see also Fig. 3D), CFTR is restricted to epithelial cells lining the small secretory ducts (exocrine canniculi). No staining of the acinar cells themselves, or of the islets of Langerhans (indicated by an asterisk in A), was observed. CFTR was not present at detectable levels in the epithelia of larger pancreatic ducts (Fig. 3E, arrow). At high magnification (Fig. 4 B and D), it is clear that CFTR is not evenly distributed throughout the epithelial cells lining the small pancreatic ducts but that it is restricted to the luminal (apical) regions of each cell. Little or no protein was detected in basolateral regions of the cells. In the jejunum (Fig. 4E), the mucosal epithelial cells were positive for CFTR; less protein was present in the epithelia lining the crypts of the
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