ABCMdb

PMID: 14623323

Mendes F, Roxo Rosa M, Dragomir A, Farinha CM, Roomans GM, Amaral MD, Penque D
Unusually common cystic fibrosis mutation in Portugal encodes a misprocessed protein.
Biochem Biophys Res Commun. 2003 Nov 21;311(3):665-71., [PubMed]

Sentences

No. Mutations Sentence Comment

0 ABCC7 p.Ala561Glu Unusually common cystic fibrosis mutation in Portugal encodes a misprocessed protein Filipa Mendes,a M o onica Roxo Rosa,a,c Anca Dragomir,b Carlos M. Farinha,a,c Godfried M. Roomans,b Margarida D. Amaral,a,c and Deborah Penquea,* a Centro de Gen e etica Humana, Instituto Nacional de Sa u ude Dr Ricardo Jorge, Lisboa 1649-016, Portugal b Department of Medical Cell Biology, University of Uppsala, Uppsala, Sweden c Departamento de Qu mica e Bioqu mica, Faculdade de Ci^ e encias, Universidade de Lisboa, Portugal Received 8 October 2003 Abstract A561E, a novel cystic fibrosis (CF) associated mutation in the first nucleotide binding domain of CFTR, is the second most common CF mutation in Portugal. Login to comment 1 ABCC7 p.Ala561Glu Properties of the A561E-CFTR protein were studied by immunoblotting, pulse-chase, immunocytochemistry, and MQAE halide-efflux assay in stably transfected BHK cells. Login to comment 2 ABCC7 p.Ala561Glu Altogether, results presented here suggest that A561E causes protein mislocalization in the endoplasmic reticulum where the mutant protein must be trapped by the quality control mechanism. Login to comment 3 ABCC7 p.Ala561Glu We conclude that A561E originates a protein trafficking defect, thus belonging to class II of CFTR mutations. Login to comment 4 ABCC7 p.Ala561Glu ABCC7 p.Ala561Glu As it is the case for F508del-CFTR (the most common CF mutant), low temperature treatment partially rescues a functional A561E-CFTR channel, suggesting that substitution of glutamic acid for alanine at position 561 does not completely abolish CFTR function. Login to comment 5 ABCC7 p.Ala561Glu Pharmacological strategies previously reported for treatment of CF patients with the F508del mutation could thus be also effective in CF patients bearing the A561E mutation. Login to comment 7 ABCC7 p.Ala561Glu Keywords: Cystic fibrosis; CFTR; A561E mutation; Classes of CFTR mutations Cystic fibrosis (CF) is a common autosomal recessive inherited disorder in the Caucasian population [1,2]. Login to comment 12 ABCC7 p.Ala561Glu One of them is the novel CF missense mutation A561E, in which alanine is replaced by glutamic acid at position 561 of the CFTR polypeptide (http://www.genet.sickkids.on.ca/cftr). Login to comment 13 ABCC7 p.Ala561Glu The A561E mutation accounts for 3% of Portuguese CF genes, being the second most frequent CF mutation in Portugal. Login to comment 26 ABCC7 p.Ala561Glu The aim of the present work was to classify the A561E mutation into one of the functional defect classes of CFTR mutations. Login to comment 27 ABCC7 p.Ala561Glu ABCC7 p.Ala561Glu Therefore, we have stably overexpressed A561E CFTR in a heterologous expression system, baby hamster kidney (BHK) cell lines, and analysed them by immunoblotting, pulse-chase, halide sensitive fluorescent dye assay, and immunocytochemistry in order to characterize the molecular mechanism of the A561E mutation. Login to comment 28 ABCC7 p.Ala561Glu The classification of A561E mutation into one of the classes of CFTR mutations will allow a better understanding of the relationship between the functional alterations and disease phenotype and pave the way for designing appropriate pharmacological interventions in CF patients bearing this particular mutation. Login to comment 31 ABCC7 p.Ala561Glu A561E mutation was created in pBQ 4.7 using Muta-gene phagemid in vitro mutagenesis Kit (BioRad Laboratories, Hercules, CA, USA) according to manufacturer`s recommendations. Login to comment 32 ABCC7 p.Ala561Glu The oligonucleotide 50 -CTTTAGCAAGAGAAG TATACAAAGATGC-30 was used to produce A561E mutation and the mutants were subsequently confirmed by DNA sequence analysis. Login to comment 33 ABCC7 p.Ala561Glu Full-length fragment cut off from A561E CFTR cDNA/pBQ4.7 was subcloned into the eukaryotic expression vector pNUT (kindly provided by J. Riordan, Scottsdale, USA). Login to comment 35 ABCC7 p.Ala561Glu Baby hamster kidney (BHK) cells were stably transfected with A561E-CFTR pNUT recombinant vector by using DOTAP (Boehringer Mannheim GmbH, Mannheim, Germany) following the manufacturer`s recommendations. Login to comment 38 ABCC7 p.Ala561Glu BHK cells stably expressing wild-type (wt)-, F508del-CFTR (both kindly provided by G. Lukacs, Toronto, Canada) or A561E-CFTR were cultivated as described [9,10]. Login to comment 72 ABCC7 p.Ala561Glu ABCC7 p.Ala561Glu Results A561E-CFTR is a misprocessed protein The A561E mutation was subcloned into the higher eukaryotic expression vector pNUT and the recombinant vector was stably expressed in BHK cell lines. Login to comment 74 ABCC7 p.Ala561Glu Fig. 1A shows the Western blotting of a total protein extract from one of these BHK cell clones stably expressing A561E analysed in parallel with extracts of BHK cells expressing wt- or F508del-CFTR as controls. Login to comment 75 ABCC7 p.Ala561Glu Like F508del-CFTR (Fig. 1A, lane 3), A561E-CFTR is only detected as an immature, ER core-glycosylated form of CFTR (band B), of 150 kDa in this heterologous expression cell system (Fig. 1A, lane 4). Login to comment 76 ABCC7 p.Ala561Glu No mature complex-glycosylated forms (band C) of 170-180 kDa were detected for A561E-CFTR. Login to comment 78 ABCC7 p.Ala561Glu Immunolocalization studies revealed that in contrast to the wt-CFTR, which is essentially detected in the cell membrane (Fig. 2A), A561E-CFTR shows the same prominent ER localization (Fig. 2C) as F508del-CFTR (Fig. 2B). Login to comment 79 ABCC7 p.Ala561Glu To assess whether a small amount of A561E-CFTR, below the biochemical and immunocytochemical detection limits, traverses the Golgi to the plasma membrane, we employed the more sensitive single-cell membrane halide permeability assay using the Cl indicator MQAE. Login to comment 81 ABCC7 p.Ala561Glu By contrast, no measurable cAMP-stimulated Cl channel activity was detected in cells expressing A561E- or F508del-CFTR (Fig. 3A, middle and lower panels, respectively). Login to comment 82 ABCC7 p.Ala561Glu Taken together, these data demonstrate that A561E-CFTR is misprocessed and retained intracellularly, thus failing both to localize correctly and to function at the plasma membrane. Login to comment 83 ABCC7 p.Ala561Glu Like F508del, the A561E should thus be included into class II of defective processing CFTR mutations. Login to comment 85 ABCC7 p.Ala561Glu Western-blotting analysis of BHK cells stably expressing wt-, F508del- or A561E-CFTR. Login to comment 87 ABCC7 p.Ala561Glu Cell lysates were prepared (see Materials and methods) from BHK cells non-transfected (50 lg, lane 1), or stably transfected with wt- (30 lg, lane 2), F508del- (50 lg, lane 2) or A561E-CFTR cDNA cloned into pNUT vector (50 lg, lane 4), and resolved on a 6% SDS-polyacrylamide gel before electrophoretic transfer to nitrocellulose for immunodetection of CFTR, using M3A7 anti-CFTR antibody (1:1000). Login to comment 88 ABCC7 p.Ala561Glu The complex-glycosylated forms of CFTR (band C, see arrow) are absent in cells expressing A561E or F508del-CFTR. Login to comment 89 ABCC7 p.Ala561Glu (B) Effect of low temperature on the trafficking defect of A561E- and F508del-CFTR. Login to comment 90 ABCC7 p.Ala561Glu ABCC7 p.Ala561Glu BHK cells stably expressing F508del- or A561E-CFTR were incubated at 26 &#b0;C for 24 h (100 lg/per lane, lanes 3 and 6, respectively) or for 48 h (100 lg/lane, lanes 4 and 7, respectively) prior to evaluation by Western-blotting (as described in (A)), for the presence of mature or immature A561E- or F508del-CFTR. Login to comment 92 ABCC7 p.Ala561Glu As controls, cell lysates from BHK cells expressing wt- (30 lg, lane 1), F508del- (50 lg, lane 2) or A561E-CFTR (50 lg, lane 5) grown at 37 &#b0;C were also analysed in parallel. Login to comment 93 ABCC7 p.Ala561Glu The abundance of complex-glycosylated forms of wt-, F508del-, and A561E-CFTR persisting in the cell (indicated by arrows) was calculated from densitometry of immunoblots. Login to comment 95 ABCC7 p.Ala561Glu Immunolocalization of CFTR in BHK cells expressing wt-, F508del-, or A561E-CFTR. Login to comment 97 ABCC7 p.Ala561Glu At normal temperature of cell culture (37 &#b0;C), A561E-CFTR (C) is detected strictly in the area around the nuclei of the cells like F508del-CFTR (B); while wt-CFTR (A,D) is predominantly located at the plasma membrane. Login to comment 98 ABCC7 p.Ala561Glu At low temperature (26 &#b0;C), some A561E- and F508del-CFTR are rescued to the cell surface (see arrows head in (E) and (F)). Login to comment 101 ABCC7 p.Ala561Glu (A) Characteristic records of MQAE fluorescence (thin lines) and corresponding intracellular Cl concentration (thick lines) in BHK cells stably expressing wt-, F508del- or A561E-CFTR. Login to comment 104 ABCC7 p.Ala561Glu (B) Effect of low temperature on Cl concentration and efflux in BHK cells stably expressing wt-, F508del- or A561E-CFTR. Login to comment 110 ABCC7 p.Ala561Glu ABCC7 p.Ala561Glu The turnover rates of A561E-CFTR are similar to those of F508del-CFTR To study whether A561E-CFTR and F508del-CFTR have the same biogenesis and kinetics degradation, we analysed these two mutants and the wt-protein by pulse-chase technique. Login to comment 113 ABCC7 p.Ala561Glu The newly synthesized immature form of A561E-CFTR migrates with the same molecular mass of wtand F508del-CFTR (Fig. 4A). Login to comment 117 ABCC7 p.Ala561Glu Here, we observed the same phenomenon for A561E-CFTR. Login to comment 118 ABCC7 p.Ala561Glu Multiple pulse-chase experiments (n &#bc; 3) indicate that the t1=2 of immature core-glycosylated A561E-CFTR was very similar to that of F508del-CFTR, around 40 min, which is slightly lower than that of the wt CFTR in BHK cells as described by Luckas et al. [11,14] (Fig. 4C). Login to comment 119 ABCC7 p.Ala561Glu As the immature A561E-CFTR is not converted into band C, its turnover corresponds solely to protein degradation. Login to comment 120 ABCC7 p.Ala561Glu Therefore, we suggest that the A561E mutation does not significantly alter the susceptibility to degradation of the newly synthesized protein. Login to comment 121 ABCC7 p.Ala561Glu Low temperature partially restores A561E-CFTR trafficking defect Reduced temperature is thought to partially revert the folding defect of F508del-CFTR and thus promote the traffic of functional channels to the cell surface [15-18]. Login to comment 122 ABCC7 p.Ala561Glu To examine whether the processing defect of A561E-CFTR can be overcome by low temperature treatment, similar to F508del-CFTR, BHK cells overexpressing this mutant were incubated at 26 &#b0;C for 24 or 48 h and analysed by Western blotting (Fig. 1B). Login to comment 124 ABCC7 p.Ala561Glu Fig. 1B shows that mature forms of A561E and F508del-CFTR (band C) were readily detected in cells incubated at 26 &#b0;C for 24 and 48 h (lanes 6/7 and 3/4, respectively). Login to comment 125 ABCC7 p.Ala561Glu Densitometric analysis revealed that the amount of rescued band C of either A561E or F508del does not increase upon longer incubation (48 h) at 26 &#b0;C. Login to comment 127 ABCC7 p.Ala561Glu At low temperature, some A561E-CFTR and F508del-CFTR were also detectable by immunocyto- Fig. 4. Login to comment 128 ABCC7 p.Ala561Glu Turnover rates of Wt, F508del- and A561E-CFTR in BHK cells. Login to comment 129 ABCC7 p.Ala561Glu (A) Pulse-chase experiments followed by immunoprecipitation of wt-, F508del-, and A561E-CFTR. Login to comment 130 ABCC7 p.Ala561Glu F508del- and A561E-CFTR proteins are synthesized as 140 kDa precursors that are very rapidly (t1=2 < 30 min) degraded with no apparent conversion to mature forms (fully glycosylated) in contrast to wt-CFTR. Login to comment 131 ABCC7 p.Ala561Glu (B) Quantification of fluorographic data of F508del-CFTR and A561E-CFTR experiments (n &#bc; 3/each). Login to comment 133 ABCC7 p.Ala561Glu No significant differences are found between the turnover kinetics of immature forms of both F508del- and A561E-CFTR. Login to comment 135 ABCC7 p.Ala561Glu Additionally, the MQAE fluorescence assay also demonstrates that cells expressing A561E- or F508del-CFTR generate cAMP-stimulated Cl efflux after incubation at low temperature for 24 or 48 h (Fig. 3B), albeit to a lesser extent than that observed for wt-CFTR (Fig. 3C). Login to comment 137 ABCC7 p.Ala561Glu However, the difference in the amplitude of response to cAMP agonists between the cells expressing wt-CFTR and cells expressing CFTR mutants remains significant, even following growth at low temperature suggesting that low thermal treatment only partially corrects the trafficking defect of both A561E- and F508del-CFTR. Login to comment 138 ABCC7 p.Ala561Glu Taken together all data strongly indicate that A561E-CFTR is a temperature-sensitive traffic mutant. Login to comment 139 ABCC7 p.Ala561Glu Functional cAMP-stimulated A561E-CFTR Cl channel activity in the plasma membrane can be promoted by reduced temperature treatment. Login to comment 140 ABCC7 p.Ala561Glu The substitution of glutamic acid for alanine at position 561 of CFTR does not completely abolish CFTR function once the trafficking defect is corrected. Login to comment 145 ABCC7 p.Ala561Glu The novel missense mutation A561E, located in exon 12, is part of a cluster of missense mutations that affect the highly conserved amino acid residues, between the signature (C) and Walker B motifs within the NBD1 of CFTR (Fig. 5). Login to comment 146 ABCC7 p.Ala561Glu In Portugal, A561E is the second most frequent CF mutation, accounting for 3% of CF alleles. Login to comment 147 ABCC7 p.Gly542* ABCC7 p.Ala561Glu ABCC7 p.Ala561Glu To date, 14 patients carrying A561E were identified in Portugal; nine are compound heterozygotes with F508del, one with G542X and four are homozygous for A561E mutation (Pacheco et al., personal communication and manuscript in preparation). Login to comment 149 ABCC7 p.Ala561Glu ABCC7 p.Ala561Glu Functional assessment of native colonic epithelia of patients, including one homozygote for A561E and another compound heterozygote for A561E and F508del, showed that CFTR-mediated Cl secretion is absent in the colon of these patients (Hirtz et al. 2003, submitted for publication) [21]. Login to comment 150 ABCC7 p.Ala561Glu ABCC7 p.Ala561Glu In order to explore the molecular mechanism that leads to defective Cl transport in A561E patients we have studied the processing, localization, and function of A561E-CFTR stably overexpressed in BHK cell lines. Login to comment 151 ABCC7 p.Ala561Glu The results presented here clearly demonstrate that A561E has a trafficking defect when heterologously expressed in these cells. Login to comment 152 ABCC7 p.Ala561Glu Like F508del, A561E-CFTR is not processed correctly and, as a consequence, is not delivered to the plasma membrane of these cells. Login to comment 153 ABCC7 p.Ala561Glu Therefore, A561E mutation belongs to the class II of CFTR mutations. Login to comment 154 ABCC7 p.Ala561Glu In BHK cells, A561E-CFTR must not acquire its fully folded native conformation. Login to comment 155 ABCC7 p.Ala561Glu Indeed, placing the A561 residue in the homology model of CFTR NBD1, Dorwart et al. [22] recently suggested that this residue is deeply buried in this domain and that the A561E mutation probably disrupts its efficient folding. Login to comment 156 ABCC7 p.Ala561Glu The core-glycosylated immature form of A561E-CFTR must thus be retained in the ER by its quality control from where it is degraded. Login to comment 157 ABCC7 p.Ala561Glu The properties of A561E-CFTR revert towards those of wt-CFTR as the incubation temperature is reduced to 26 &#b0;C. Login to comment 158 ABCC7 p.Ala561Glu When the processing defect is (partially) corrected, functional cAMP-regulated Cl channels appear in the plasma membrane, indicating that A561E mutation does not completely abolish CFTR function. Login to comment 159 ABCC7 p.Ala561Glu Based on the results reported here, we hypothesize that A561E-CFTR can be also re-directed to the normal protein trafficking pathway by manipulation of chaperone protein/CFTR interactions, with chemical chaperones or other drugs that affect gene regulation such as genistein and xanthine derivatives [20]. Login to comment 160 ABCC7 p.Ala561Glu Thus, the pharmacological therapies that have been tried for CF patients bearing F508del could be also useful to those with the A561E mutation. Login to comment 162 ABCC7 p.Ala561Glu F508del- and A561E-CFTR mutations in NBD1. Login to comment