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• W2140161331 abstract "Impairment of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel causes cystic fibrosis, a fatal genetic disease. Here, to gain insight into CFTR structure and function, we exploited interspecies differences between CFTR homologues using human (h)-murine (m) CFTR chimeras containing murine nucleotide-binding domains (NBDs) or regulatory domain on an hCFTR backbone. Among 15 hmCFTR chimeras analyzed, all but two were correctly processed, one containing part of mNBD1 and another containing part of mNBD2. Based on physicochemical distance analysis of divergent residues between human and murine CFTR in the two misprocessed hmCFTR chimeras, we generated point mutations for analysis of respective CFTR processing and functional properties. We identified one amino acid substitution (K584E-CFTR) that disrupts CFTR processing in NBD1. No single mutation was identified in NBD2 that disrupts protein processing. However, a number of NBD2 mutants altered channel function. Analysis of structural models of CFTR identified that although Lys584 interacts with residue Leu581 in human CFTR Glu584 interacts with Phe581 in mouse CFTR. Introduction of the murine residue (Phe581) in cis with K584E in human CFTR rescued the processing and trafficking defects of K584E-CFTR. Our data demonstrate that human-murine CFTR chimeras may be used to validate structural models of full-length CFTR. We also conclude that hmCFTR chimeras are a valuable tool to elucidate interactions between different domains of CFTR. Impairment of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel causes cystic fibrosis, a fatal genetic disease. Here, to gain insight into CFTR structure and function, we exploited interspecies differences between CFTR homologues using human (h)-murine (m) CFTR chimeras containing murine nucleotide-binding domains (NBDs) or regulatory domain on an hCFTR backbone. Among 15 hmCFTR chimeras analyzed, all but two were correctly processed, one containing part of mNBD1 and another containing part of mNBD2. Based on physicochemical distance analysis of divergent residues between human and murine CFTR in the two misprocessed hmCFTR chimeras, we generated point mutations for analysis of respective CFTR processing and functional properties. We identified one amino acid substitution (K584E-CFTR) that disrupts CFTR processing in NBD1. No single mutation was identified in NBD2 that disrupts protein processing. However, a number of NBD2 mutants altered channel function. Analysis of structural models of CFTR identified that although Lys584 interacts with residue Leu581 in human CFTR Glu584 interacts with Phe581 in mouse CFTR. Introduction of the murine residue (Phe581) in cis with K584E in human CFTR rescued the processing and trafficking defects of K584E-CFTR. Our data demonstrate that human-murine CFTR chimeras may be used to validate structural models of full-length CFTR. We also conclude that hmCFTR chimeras are a valuable tool to elucidate interactions between different domains of CFTR. Cystic fibrosis (CF) 4The abbreviations used are: CFcystic fibrosisCFTRcystic fibrosis transmembrane conductance regulatorNBDnucleotide-binding domainRDregulatory domainWBWestern blotABCATP-binding cassetteERendoplasmic reticulumERQCER quality controlmmurinehhumanBHKbaby hamster kidneyTESN-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acidPoopen probability. is the most common lethal genetic disease in the Caucasian population, resulting from the dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) (1Welsh M. Ramsey B. Accurso F. Cutting G. Scriver C. Beaudet A. Sly W. The Metabolic and Molecular Bases of Inherited Disease. 8th Ed. McGraw Hill, New York2001: 5121-5188Google Scholar). CFTR is a multidomain protein containing 1480 amino acid residues located at the apical membrane of epithelial cells where it functions as a chloride (Cl−) ion channel regulated by cAMP-dependent phosphorylation and cycles of ATP binding and hydrolysis (2Sheppard D.N. Welsh M.J. Physiol. Rev. 1999; 79: S23-S45Crossref PubMed Scopus (813) Google Scholar, 3Gadsby D.C. Vergani P. Csanády L. Nature. 2006; 440: 477-483Crossref PubMed Scopus (545) Google Scholar). Based on its structure and function, CFTR (ABCC7) is a member of the ATP-binding cassette (ABC) transporter superfamily. It contains two membrane-spanning domains, two nucleotide-binding domains (NBDs), and a unique regulatory domain (RD) with multiple consensus phosphorylation sites and many charged amino acids (2Sheppard D.N. Welsh M.J. Physiol. Rev. 1999; 79: S23-S45Crossref PubMed Scopus (813) Google Scholar, 4Riordan J.R. Rommens J.M. Kerem B. Alon N. Rozmahel R. Grzelczak Z. Zielenski J. Lok S. Plavsic N. Chou J.L. Science. 1989; 245: 1066-1073Crossref PubMed Scopus (5977) Google Scholar). The most common disease-causing mutation, occurring in ∼90% of CF patients worldwide on at least one CFTR allele, is deletion of phenylalanine 508 (F508del) located in NBD1. cystic fibrosis cystic fibrosis transmembrane conductance regulator nucleotide-binding domain regulatory domain Western blot ATP-binding cassette endoplasmic reticulum ER quality control murine human baby hamster kidney N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid open probability. A critical aspect of CF research is to understand how the F508del mutation disrupts CFTR function at the molecular and cellular levels. Depending on the cell type examined, the maturation efficiency of wild-type (WT)-CFTR protein is 25–70% (5Lukacs G.L. Chang X.B. Bear C. Kartner N. Mohamed A. Riordan J.R. Grinstein S. J. Biol. Chem. 1993; 268: 21592-21598Abstract Full Text PDF PubMed Google Scholar, 6Ward C.L. Kopito R.R. J. Biol. Chem. 1994; 269: 25710-25718Abstract Full Text PDF PubMed Google Scholar, 7Varga K. Jurkuvenaite A. Wakefield J. Hong J.S. Guimbellot J.S. Venglarik C.J. Niraj A. Mazur M. Sorscher E.J. Collawn J.F. Bebök Z. J. Biol. Chem. 2004; 279: 22578-22584Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). By contrast, very little or virtually no functional F508del-CFTR reaches the cell surface because it is retained in the endoplasmic reticulum (ER) by the ER quality control (ERQC) machinery and targeted for degradation (8Amaral M.D. J. Mol. Neurosci. 2004; 23: 41-48Crossref PubMed Google Scholar). The exact nature of the structural divergence imposed on CFTR by absence of F508del, which is recognized by the ERQC (9Lukacs G.L. Mohamed A. Kartner N. Chang X.B. Riordan J.R. Grinstein S. EMBO J. 1994; 13: 6076-6086Crossref PubMed Scopus (342) Google Scholar, 10Qu B.H. Thomas P.J. J. Biol. Chem. 1996; 271: 7261-7264Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), is currently unknown. To understand better how F508del perturbs CFTR architecture, high resolution structures of murine (m) and human (h) NBD1 as well as of human F508del-NBD1 (11Lewis H.A. Buchanan S.G. Burley S.K. Conners K. Dickey M. Dorwart M. Fowler R. Gao X. Guggino W.B. Hendrickson W.A. Hunt J.F. Kearins M.C. Lorimer D. Maloney P.C. Post K.W. Rajashankar K.R. Rutter M.E. Sauder J.M. Shriver S. Thibodeau P.H. Thomas P.J. Zhang M. Zhao X. Emtage S. EMBO J. 2004; 23: 282-293Crossref PubMed Scopus (338) Google Scholar, 12Lewis H.A. Zhao X. Wang C. Sauder J.M. Rooney I. Noland B.W. Lorimer D. Kearins M.C. Conners K. Condon B. Maloney P.C. Guggino W.B. Hunt J.F. Emtage S. J. Biol. Chem. 2005; 280: 1346-1353Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar) have been resolved. However, the structure of human NBD1 incorporates a series of mutations, which rescue in vivo the cell surface expression and function of F508del-CFTR (13Pissarra L.S. Farinha C.M. Xu Z. Schmidt A. Thibodeau P.H. Cai Z. Thomas P.J. Sheppard D.N. Amaral M.D. Chem. Biol. 2008; 15: 62-69Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Thus, the available crystal structure of F508del-NBD1 plausibly corresponds to a partially corrected conformation of this domain. Based on these NBD1 structures and atomic resolution structures of other ABC transporters (14Hollenstein K. Frei D.C. Locher K.P. Nature. 2007; 446: 213-216Crossref PubMed Scopus (405) Google Scholar), molecular models of the CFTR protein have been developed (15Serohijos A.W. Hegedus T. Aleksandrov A.A. He L. Cui L. Dokholyan N.V. Riordan J.R. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 3256-3261Crossref PubMed Scopus (313) Google Scholar, 16Mornon J.P. Lehn P. Callebaut I. Cell. Mol. Life Sci. 2008; 65: 2594-2612Crossref PubMed Scopus (133) Google Scholar). These molecular models are a valuable guide for studies of CFTR structure and function. Another powerful approach to investigate the structure and function of hCFTR is to examine interspecies differences to identify conserved and divergent regions. Since the identification and cloning of the hCFTR gene (4Riordan J.R. Rommens J.M. Kerem B. Alon N. Rozmahel R. Grzelczak Z. Zielenski J. Lok S. Plavsic N. Chou J.L. Science. 1989; 245: 1066-1073Crossref PubMed Scopus (5977) Google Scholar), various homologues have been isolated from different species (e.g. mouse, sheep, pig, Xenopus, macaque, rabbit, bovine, killifish, and salmon); all possess a domain organization similar to that of hCFTR (17Chen J.M. Cutler C. Jacques C. Boeuf G. Denamur E. Lecointre G. Mercier B. Cramb G. Férec C. Mol. Biol. Evol. 2001; 18: 1771-1788Crossref PubMed Scopus (61) Google Scholar). Nevertheless, significant interspecies differences in CFTR processing and function have been reported previously. For example, human and murine CFTR Cl− channels exhibit different patterns of channel gating (18Lansdell K.A. Delaney S.J. Lunn D.P. Thomson S.A. Sheppard D.N. Wainwright B.J. J. Physiol. 1998; 508: 379-392Crossref PubMed Scopus (47) Google Scholar, 19Lansdell K.A. Kidd J.F. Delaney S.J. Wainwright B.J. Sheppard D.N. J. Physiol. 1998; 512: 751-764Crossref PubMed Scopus (43) Google Scholar), which are specified, in part, by amino acid sequence differences in the NBDs (20Scott-Ward T.S. Cai Z. Dawson E.S. Doherty A. Da Paula A.C. Davidson H. Porteous D.J. Wainwright B.J. Amaral M.D. Sheppard D.N. Boyd A.C. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 16365-16370Crossref PubMed Scopus (38) Google Scholar). Moreover, Ostedgaard et al. (21Ostedgaard L.S. Rogers C.S. Dong Q. Randak C.O. Vermeer D.W. Rokhlina T. Karp P.H. Welsh M.J. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 15370-15375Crossref PubMed Scopus (107) Google Scholar) identified differences in the processing of the F508del mutation between human, porcine, and murine CFTR. In that study, both porcine and murine F508del-CFTR proteins were found to be at least partially processed like their WT counterparts, and this processing was unaffected by the origin of the cell line where the mutant proteins were expressed. Thus, cross-species CFTR chimeras appear to be an excellent resource to investigate the folding and functional consequences of interdomain interactions in CFTR. Indeed, insights into the biochemical properties of mCFTR subdomains in the context of hCFTR, such as those provided by the present study, will help to further understanding of the structural and functional effects of disease-causing mutations in both NBDs and validate existing structural models (15Serohijos A.W. Hegedus T. Aleksandrov A.A. He L. Cui L. Dokholyan N.V. Riordan J.R. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 3256-3261Crossref PubMed Scopus (313) Google Scholar, 16Mornon J.P. Lehn P. Callebaut I. Cell. Mol. Life Sci. 2008; 65: 2594-2612Crossref PubMed Scopus (133) Google Scholar, 22Rosenberg M.F. Kamis A.B. Aleksandrov L.A. Ford R.C. Riordan J.R. J. Biol. Chem. 2004; 279: 39051-39057Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). In the present work, we aimed to identify the structural features of hCFTR (mis)folding using a human-murine comparative approach by investigating whether CFTR domains (NBDs and RD) are structurally interchangeable between human and murine CFTR. Our data reveal that two chimeras (one in NBD1 and another in NBD2) failed to be processed into their mature forms. Systematic mutagenesis of the divergent murine residues present in these chimeras followed by biochemical and functional studies revealed that K584E is responsible for the maturation defect of the hmNBD1 chimera. Furthermore, replacement of Leu581 (interacting with Lys584 in the structure of hNBD1) by the corresponding murine amino acid (Phe581) rescued the maturation of K584E-CFTR. Thus, our results demonstrate that hmCFTR chimeras can be used to identify critical residues responsible for both structural and functional differences between human and murine CFTR. Furthermore, the CFTR mutants generated here constitute a valuable resource to characterize the possible binding sites and mechanism of action of small molecules used in CFTR assist therapies (23Amaral M.D. Curr. Drug Targets. 2010; (in press)Google Scholar). Human-murine CFTR chimeras containing sequences from murine NBD1, NBD2, and RD were constructed by homologous recombination as described previously (20Scott-Ward T.S. Cai Z. Dawson E.S. Doherty A. Da Paula A.C. Davidson H. Porteous D.J. Wainwright B.J. Amaral M.D. Sheppard D.N. Boyd A.C. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 16365-16370Crossref PubMed Scopus (38) Google Scholar). The QuikChange® mutagenesis kit (Stratagene) was used to introduce mutations into the pNUT WT-CFTR cDNA. Each mutation was verified by sequencing. For a list of primers used, see supplemental Table 2. We transiently expressed wild-type and chimeric CFTRs in HEK-293 cells using Lipofectin® (Invitrogen). Cells were transfected with 1 μg of cDNA for all CFTR variants. 48 h after transfection, we extracted total protein from HEK-293 cells expressing wild-type and chimeric CFTRs and assayed for CFTR expression by Western blotting. To generate cell lines stably expressing high levels of CFTR variants, baby hamster kidney (BHK) cells were transfected with CFTR cDNAs (3 μg) using Lipofectin (Invitrogen) and selected for stable transfectants using methotrexate (500 μm). For each CFTR variant, 10 BHK clones were analyzed by Western blotting. Of these, the clone expressing the highest level of CFTR protein was selected for further analyses, ensuring that CFTR expression levels among the different CFTR variants were equivalent. Cells were cultured, seeded, and used as described previously (24Roxo-Rosa M. Xu Z. Schmidt A. Neto M. Cai Z. Soares C.M. Sheppard D.N. Amaral M.D. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 17891-17896Crossref PubMed Scopus (89) Google Scholar). In some experiments (see figure legends), cell surface expression of F508del-CFTR was enhanced by incubating cells at 26 °C for 24 h. To assay for CFTR protein expression by Western blot (WB), cells expressing CFTR variants were lysed, and extracts were analyzed as described (25Farinha C.M. Mendes F. Roxo-Rosa M. Penque D. Amaral M.D. Mol. Cell. Probes. 2004; 18: 235-242Crossref PubMed Scopus (30) Google Scholar) using the anti-CFTR antibody 596 (CF Foundation). Densitometry was performed as described previously (25Farinha C.M. Mendes F. Roxo-Rosa M. Penque D. Amaral M.D. Mol. Cell. Probes. 2004; 18: 235-242Crossref PubMed Scopus (30) Google Scholar). Cells were starved for 30 min in methionine-free α-modified Eagle's medium (Invitrogen) before being radiolabeled for 25 min in the same medium supplemented with 150 μCi/ml [35S]methionine (ICN Biomedicals). For the chase (0, 0.5, 1, 2, and 3 h), the labeling medium was replaced by α-modified Eagle's medium supplemented with fetal bovine serum (8%, v/v; Invitrogen) and non-radioactive methionine (1 mm; Sigma-Aldrich). Cells were then lysed in radioimmunoprecipitation assay buffer (1 ml) containing deoxycholic acid (1%, w/v; Sigma-Aldrich), Triton X-100 (1%, v/v; GE Healthcare Bio-Sciences), SDS (0.1%, w/v; Invitrogen), Tris (50 mm, pH 7.4; Sigma-Aldrich), and NaCl (150 mm). CFTR protein was immunoprecipitated as described by Farinha et al. (26Farinha C.M. Penque D. Roxo-Rosa M. Lukacs G. Dormer R. McPherson M. Pereira M. Bot A.G. Jorna H. Willemsen R. Dejonge H. Heda G.D. Marino C.R. Fanen P. Hinzpeter A. Lipecka J. Fritsch J. Gentzsch M. Edelman A. Amaral M.D. J. Cyst. Fibros. 2004; 3: 73-77Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) after centrifugation of samples at 14,000 × g for 30 min at 4 °C. To detect specifically CFTR, the supernatant was incubated overnight with 1.5 μg of the anti-CFTR monoclonal antibody M3A7, which recognizes NBD2 and the C terminus of CFTR (residues 1197–1480; Chemicon), at 4 °C, and then protein G-agarose beads (25 μg; Roche Applied Science) were added for a further 4 h at 4% (v/v). Beads were washed four times using radioimmunoprecipitation assay buffer (1 ml), and protein was eluted for 1 h at room temperature (RT) with cracking buffer (80 μl) containing dithiothreitol (0.5 mm; Sigma-Aldrich), bromphenol blue (0.001%, w/v), glycerol (5%, v/v), SDS (1.5%, w/v), and Tris (31.25 mm), pH 6.8. Samples were separated electrophoretically on 7% (v/v) polyacrylamide gels. Then, gels were prefixed (methanol (30%, v/v) and acetic acid (10%, v/v)) for 30 min, washed thoroughly in water, and soaked in salicylic acid (1 m) for 1 h. After drying at 80 °C under vacuum for 2 h, gels were exposed to x-ray film (Fujifilm Medical Systems). Fluorograms of gels were digitized (Sharp JX-330, Sharp Europe), and integrated peak areas were determined using ImageMaster® software (GE Healthcare). CFTR-mediated iodide efflux was measured at room temperature as described (19Lansdell K.A. Kidd J.F. Delaney S.J. Wainwright B.J. Sheppard D.N. J. Physiol. 1998; 512: 751-764Crossref PubMed Scopus (43) Google Scholar) using the cAMP agonist forskolin (10 μm) and the CFTR potentiator genistein (50 μm; Sigma-Aldrich). Prior to commencing experiments, BHK cells expressing CFTR variants were incubated for 1 h in loading buffer containing 136 mm NaI, 3 mm KNO3, 2 mm Ca(NO3)2, 20 mm Hepes, and 11 mm glucose, pH 7.4 with 1 m NaOH and then washed thoroughly with efflux buffer (136 mm NaNO3 replacing 136 mm NaI in the loading buffer). The amount of iodide in each sample of efflux buffer was determined using an iodide-selective electrode (MP225, Thermo Electron Corp., Waltham, MA). BHK cells were loaded, and experiments were performed at RT (∼23 °C). Immunocytochemistry was performed as described previously (27Mendes F. Wakefield J. Bachhuber T. Barroso M. Bebok Z. Penque D. Kunzelmann K. Amaral M.D. Cell. Physiol. Biochem. 2005; 16: 281-290Crossref PubMed Scopus (15) Google Scholar). In brief, BHK cells expressing CFTR variants were rinsed twice with cold PBS and fixed with ice-cold 4% p-formaldehyde (Fluka BioChemica) in PBS for 20 min. After four washes with cold PBS at RT, nonspecific staining was prevented by blocking with BSA (1%, w/v) in PBS for 30 min. Cells were stained with anti-wheat germ agglutinin for 1 h, coupled with Texas Red, and then permeabilized with Triton (0.2%, w/v) for 20 min. Finally, cells were washed three times with PBS (10 min each at RT) and incubated with (i) monoclonal anti-CFTR antibody 570 and then (ii) secondary antibody, Alexa Fluor 488 anti-mouse, for 1 h each. Cells were again washed three times with PBS (10 min each at RT), and slides were mounted with Vectashield (Vector Laboratories) containing 4′,6-diamidino-2-phenylindole dihydrochloride (Sigma-Aldrich) to stain nuclei and covered with a glass coverslip. Immunofluorescence staining was investigated using a confocal microscope (Leica TCS SPE). No background staining or autofluorescence was observed with untransfected BHK cells (data not shown). CFTR Cl− currents were recorded in excised inside-out membrane patches using an Axopatch 200B patch clamp amplifier (MDS Analytical Technologies) and pCLAMP data acquisition and analysis software (versions 6.0.4 and 9.2, MDS Analytical Technologies) as described previously (18Lansdell K.A. Delaney S.J. Lunn D.P. Thomson S.A. Sheppard D.N. Wainwright B.J. J. Physiol. 1998; 508: 379-392Crossref PubMed Scopus (47) Google Scholar). The pipette (extracellular) solution contained 140 mm N-methyl-d-glucamine, 140 mm aspartic acid, 5 mm CaCl2, 2 mm MgSO4, and 10 mm TES, pH 7.3 with Tris ([Cl−], 10 mm). The bath (intracellular) solution contained 140 mm N-methyl-d-glucamine, 3 mm MgCl2, 1 mm CsEGTA, and 10 mm TES, pH 7.3 with HCl ([Cl−], 147 mm; [Ca2+]free, <10−8 m) and was maintained at 37 °C. After their excision, membrane patches were voltage-clamped at −50 mV, and CFTR Cl− channels were activated by the addition of ATP (1 mm; Sigma-Aldrich) and the catalytic subunit of protein kinase A (75 nm; Promega UK) to the intracellular solution within 5 min of patch excision. In this study, we used membrane patches containing small numbers of active channels (≤5). We determined the number of channels in a membrane patch from the maximum number of simultaneous channel openings observed during the course of an experiment. To minimize errors when counting the number of active channels, we used the strategies described by Cai et al. (28Cai Z. Taddei A. Sheppard D.N. J. Biol. Chem. 2006; 281: 1970-1977Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). We recorded, filtered, and digitized data as described (18Lansdell K.A. Delaney S.J. Lunn D.P. Thomson S.A. Sheppard D.N. Wainwright B.J. J. Physiol. 1998; 508: 379-392Crossref PubMed Scopus (47) Google Scholar). To measure single channel current amplitude (i), Gaussian distributions were fit to current amplitude histograms. For open probability (Po) analyses, lists of open and closed times were created using a half-amplitude crossing criterion for event detection. Transitions <1 ms in duration were excluded from the analyses, and Po was calculated as described previously (28Cai Z. Taddei A. Sheppard D.N. J. Biol. Chem. 2006; 281: 1970-1977Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Results are expressed as means ± S.E. of n observations. To compare sets of data, we used Student's t test for iodide efflux and single channel data. Pulse-chase data were analyzed by comparing degradation rates (slopes of regression lines) by Student's t test. On a regression modeling procedure, the slope is known to follow a t distribution (29Kleinbaum D.G. Kupper L.L. Muller K.E. Applied Regression Analysis and Other Multivariate Methods.4th Ed. Duxbury Press, Pacific Grove, CA2008: 114-138Google Scholar, 30Helliwell P.S. Jackson S. Ann. Rheum. Dis. 1994; 53: 726-728Crossref PubMed Scopus (51) Google Scholar). Therefore, slopes of two straight lines can be compared using a t distribution with n1 + n2 − 4 degrees of freedom where n1 and n2 are the number of points used on the regression procedure in groups 1 and 2, respectively. Differences between groups of data were considered statistically significant when p < 0.05. A previously described recombination strategy was used to generate hmCFTR chimeras (20Scott-Ward T.S. Cai Z. Dawson E.S. Doherty A. Da Paula A.C. Davidson H. Porteous D.J. Wainwright B.J. Amaral M.D. Sheppard D.N. Boyd A.C. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 16365-16370Crossref PubMed Scopus (38) Google Scholar). Because of the high degree of homology between hCFTR and mCFTR sequences, homologous recombination frequently occurred within the murine domains rather than at the chimeric junction defined by the PCR primers. From the library of chimeras generated by Scott-Ward et al. (20Scott-Ward T.S. Cai Z. Dawson E.S. Doherty A. Da Paula A.C. Davidson H. Porteous D.J. Wainwright B.J. Amaral M.D. Sheppard D.N. Boyd A.C. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 16365-16370Crossref PubMed Scopus (38) Google Scholar), in the present work, we focused on the following constructs (Fig. 1A): 562c-NBD1 (limits of inserted mCFTR sequence, Leu433–Val586), 12b-NBD1 (Lys518–Val586), 323c-NBD2 (Met1260–Ser1419), 114c-NBD2 (Met1260–Arg1412), 64a-RD (Ser654–Leu834), and NBD1+2 (Lys518–Val586; Met1260–Ser1419). The latter NBD1+2 refers to chimera 12b+323c, which is not the same as that previously described (20Scott-Ward T.S. Cai Z. Dawson E.S. Doherty A. Da Paula A.C. Davidson H. Porteous D.J. Wainwright B.J. Amaral M.D. Sheppard D.N. Boyd A.C. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 16365-16370Crossref PubMed Scopus (38) Google Scholar); hence, we term it hereafter 12b-NBD1+323c-NBD2. As controls, we also analyzed WT- and F508del-hCFTR as well as mCFTR. The traffic of CFTR protein to the cell membrane can be assessed indirectly by its maturation. Indeed, WT-CFTR synthesized in the ER as a core-glycosylated, immature form undergoes additional glycosylation in the Golgi apparatus to generate its mature form (band C; ∼170–190 kDa) (9Lukacs G.L. Mohamed A. Kartner N. Chang X.B. Riordan J.R. Grinstein S. EMBO J. 1994; 13: 6076-6086Crossref PubMed Scopus (342) Google Scholar, 31Cheng S.H. Gregory R.J. Marshall J. Paul S. Souza D.W. White G.A. O'Riordan C.R. Smith A.E. Cell. 1990; 63: 827-834Abstract Full Text PDF PubMed Scopus (1427) Google Scholar). By contrast, due to its ER retention, F508del-CFTR can only be detected as the core-glycosylated, ER-specific immature form of CFTR (band B; ∼150 kDa) (31Cheng S.H. Gregory R.J. Marshall J. Paul S. Souza D.W. White G.A. O'Riordan C.R. Smith A.E. Cell. 1990; 63: 827-834Abstract Full Text PDF PubMed Scopus (1427) Google Scholar). Quantification of these immature and mature forms at steady state by WB provides a measure of the extent of maturation of a given CFTR variant and also, indirectly, of whether its in vivo folding has been achieved by ERQC protein conformation criteria. Thus, such data can also be interpreted as indicative of structural divergence between WT- and mutant CFTR. When we evaluated the maturation efficiency of the hmCFTR chimeras by assessing their production of immature and mature CFTR protein at steady state by WB analysis in the same cell line, we found that two chimeras failed to generate the mature form of CFTR (band C): clone 12b-NBD1 (Fig. 1B, lane 9) and clone 114c-NBD2 (Fig. 1B, lane 7). Indeed, both these chimeras were only detected as their immature core-glycosylated ER-specific forms (150 kDa; band B) (31Cheng S.H. Gregory R.J. Marshall J. Paul S. Souza D.W. White G.A. O'Riordan C.R. Smith A.E. Cell. 1990; 63: 827-834Abstract Full Text PDF PubMed Scopus (1427) Google Scholar). These results contrast with all the other chimeras analyzed: 12b-NBD1+323c-NBD2 (Fig. 1B, lane 2), 323c-NBD2 (lane 3), 562c-NBD1 (lane 4), 64a-RD (lane 5), human CFTR (lane 1), and murine CFTR (lane 8). For each of these CFTR constructs, the mature form was detected, albeit at varying levels. After quantification of independent experiments (n = 10), the rank order of processing efficiency was as follows (WT-CFTR taken as 100%): 323c-NBD2 (129 ± 6%) ≫ murine (86 ± 5%) = human (83 ± 3%) = 562c-NBD1 (80 ± 4%) = 64a-RD (77 ± 4%) > 12b-NBD1+323c-NBD2 (67 ± 5%). Additional chimeric constructs analyzed by WB in transiently transfected HEK-293 cells included the following: 20b-NBD1 (Thr531–Tyr647), 966c-NBD1 (Ile481–Val546), 158a-NBD2 (Met1220–Asp1409), 189b-NBD2 (Met1220–Tyr1381), 207c-NBD2 (Val1226–Asn1420), 51a-RD (Phe653–Met837), 52a-RD (Thr654–Val693), 71a-RD (Thr654–Leu834), and 85a-RD (Thr654–Val837). Like hCFTR and mCFTR, each of these hmCFTR chimeras was processed normally (data not shown). When we compared the murine amino acid sequences of the two non-processed chimeric proteins (12b-NBD1 and 114c-NBD2) with their corresponding regions in human CFTR, we found that 12 and 27 amino acid residues diverge between the human and murine sequences of 12b-NBD1 and 114c-NBD2, respectively (see also supplemental Fig. 1). To identify which of these residues could account for the failure of these chimeric proteins to mature, we selected as most probable those residues with greatest sequence divergence between human and murine CFTR based on their values for physicochemical distance (32Grantham R. Science. 1974; 185: 862-864Crossref PubMed Scopus (1718) Google Scholar). Thus, we identified six residues in 12b-NBD1 (E527Q, E528Q, S531T, K536Q, I539T, and K584E) and 12 residues in 114c-NBD2 (T1263I, P1290T, K1302Q, Y1307N, Q1309K, S1311K, R1325K, V1338T, C1344Y, L1367I, D1394G, and E1409D) (see supplemental Fig. 1 and supplemental Table 1, A and B). The above six NBD1 and 12 NBD2 point mutants (mCFTR residues) were introduced into full-length human CFTR, the constructs were used to generate stable BHK cell lines, and CFTR protein expression was assessed by WB analysis (Fig. 2). With one exception (K584E; Fig. 2A, lane 8), all point mutants derived from 12b-NBD1 were processed (lanes 3–7) similarly to WT-hCFTR (Fig. 2A, lane 1). Like F508del (Fig. 2A, lane 2), K584E (lane 8) only generated immature CFTR protein (band B; 150-kDa). By contrast, all point mutants derived from 114c-NBD2 were processed to the fully glycosylated (band C; 170–190-kDa) form (Fig. 2B, lanes 1–6 and 9–14) like WT-hCFTR (lane 7). These data suggest that all CFTR constructs except K584E are delivered to the cell surface. Results from quantification of the mature form (as percentage of total expressed) for each of the CFTR point mutants studied (Table 1) show that physicochemical distance (supplemental Table 1, A and B) is not inversely correlated with extent of protein processing.TABLE 1Summary information of CFTR point mutants analyzed in present studyCFTR variantsClinical dataaData from the CFTR mutation database.Band C/band BbPercentage of processing given by (band C/band B) × 100. (±S.E., n = 5)ProcessingcPercentage of processing given by (band C/(band B + band C) × 100).Normalized processingdRatio of the “percent band C” value for each mutant (from previous column) over the same “percentage for WT-CFTR.”Normalized iodide efflux functioneIodide efflux expressed as percentage of peak int" @default.
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• W2140161331 title "Folding and Rescue of a Cystic Fibrosis Transmembrane Conductance Regulator Trafficking Mutant Identified Using Human-Murine Chimeric Proteins" @default.
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