Institut National de la Santé et de la Recherche Médicale EMI 01 15, Etablissement Français du SangBretagne, Universite de Bretagne Occidentale, and Centre Hospitalier Universitaire, Brest, France;
School of Biology, University of Saint Andrews, Saint Andrews, Fife, Scotland;
Laboratoire de Biochimie-Biologie Moleculaire, CHU Angers, Angers, France;
Observatoire Océanologique, Université Pierre et Marie Curie/Centre National de la Recherche Scientifique, Banyuls-sur-Mer, France;
Institut National de la Santé et de la Recherche Médicale U458, Hôpital Robert Debré, Paris, France;
Service de Systématique Moléculaire, Museum National d'Histoire Naturelle, Paris, France
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Abstract |
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Introduction |
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The cloning of CFTR (Riordan et al. 1989
) has promoted extensive research activities in two interrelated aspects: first, identification of CFTR mutations in classic and atypical CF patients, and second, elucidation of the functional role of CFTR in both the normal and the diseased states during the past decade. However, while screening for CFTR mutations has become a purely molecular pursuit (currently, nearly 1,000 DNA variants of the gene are deposited in the CF Mutation Data Base: http://www.genet.sickkids.on.ca/cftr), the principal challenge is interpretation of these events in terms of their likely medical consequences. This is especially the case with the vast number of missense mutations (
40%), most of which are rare or even "private." The formidable nature of this situation lies in the large size and complexity of the gene and, more importantly, in the complex multidomain structure of the protein.
Although functional analysis, which unfortunately is still not routinely available, will continue to be used to bridge this gap, it can be complemented with other strategies. In this regard, the impact of comparative sequence analysis has been tremendous. Even the originally proposed CFTR structure, which has proved to be remarkably prescient, was largely based on a comparison of human CFTR with a number of other ATP-binding cassette (ABC) family members (Riordan et al. 1989
). Furthermore, cross-species sequence comparisons between the small number of CFTR homologs available in the early 1990s highlighted that identified mutations had occurred in evolutionary conserved residues (Diamond et al. 1991
; Tucker, Tannahill, and Higgins 1992
). These early but necessarily limited analyses clearly demonstrated the importance of evolutionary information in interpreting the effects of mutations and have also frequently been taken into account in the design of functional studies. Therefore, such a large number of missense mutations identified in the human CFTR and the abundance of structural and functional analysis data accumulated over the past decade, if evaluated together in the context of a sequence comparison of all of the currently available CFTR orthologs, will allow a significantly improved refinement of the domain structure of CFTR. This will, in turn, facilitate interpretation of the identified mutations. The central thesis of this work represents such an attempt, and, towards that end, the already large number of CFTR orthologs has been expanded by the determination of the sequences of two full-length CFTR orthologous isoforms from a single teleost fish species, the Atlantic salmon (Salmo salar).
The availability of this newly expanded number of CFTR sequences also offered an opportunity to reevaluate the use of mouse models for CF studies and, from a phylogenetic point of view, to check whether the paraphyly of the Glires (Lagomorpha and Rodentia), determined using CFTR sequences, was reliable or simply the result of a long-branch attraction artifact between the long rodent branch and the very divergent outgroup (Xenopus) (Vuillaumier et al. 1997
). In this regard, a partial coding sequence from the eastern gray kangaroo (Macropus giganteus; a marsupial mammal whose divergence time with placental mammals is about 120 Myr) was also produced in order to determine whether such an outgroup long-branch attraction artifact existed.
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Materials and Methods |
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Three steps were taken to isolate the salmon CFTR cDNAs by polymerase chain reaction (PCR). First, three pairs of inosine (I)containing degenerate oligonucleotide primers (sense 5'-TT(T/C) GG(I/C) (C/T)T(I/C) CA(I/C) C(A/G)(C/T) (A/C)T(I/C) GG(I/C) ATG CA(A/G) ATG (A/C)G-3', antisense 5'-(A/G)TC (I/C)A(A/G) (I/C)A(A/G) (A/G)TA (I/C)A(A/G) (A/G)TC (I/C)GC (A/G)TC (C/T) TT (A/G)TA-3'; sense 5'-GC(I/C) (I/C)T(I/C) TA(T/C) AA(A/G) GA(T/C) GC(I/C) GA(T/C) (C/T)T(I/C) TA(T/C) (C/T)T-3', antisense 5'-TGG TTC CT(C/T) TTC CGC (A/T)(G/T)(C/T) GAC ATI (A/C)TC TT 3'; and sense 5' TA(T/C) TT(T/C) GA(A/G) AC(I/C) (T/C)T(I/C) TT(T/C) CA(T/C) AA(A/G) GC-3', antisense 5'-A(A/G)(A/G) CAC AT(I/C) A(A/G)(T/C) TG(T/C) TT(A/G) TG(I/C) CC(A/G) T-3') which targeted highly conserved regions of the CFTR gene based on published CFTR sequences were synthesized and used to generate candidate PCR fragments. Second, when a specific CFTR fragment was obtained, new specific primers were designed and further PCR fragments were amplified. These efforts generated two sets of three overlapping clones covering most of the salmon CFTR-coding sequence. Finally, the remaining translated and the 5' and 3' untranslated regions (UTRs) of the two cDNAs were obtained using a 5'/3' Rapid Amplification of cDNA Ends (RACE) Kit (Boehringer Mannheim, Mannheim, Germany) or a similar Marathon cDNA cloning kit (Clontech) utilizing isoform-specific primers.
Cloning of CFTR Exon 13Coding Sequence from the Eastern Gray Kangaroo (M. giganteus)
To determine the possible presence of a long-branch attraction artifact between the long rodent branch and the very divergent outgroup (Xenopus) (Vuillaumier et al. 1997
), and also to maximize the refinement of the boundaries between NBD1 and the R domain, the cloning of the coding sequence of exon 13 (the largest exon in the CFTR gene) from the eastern gray kangaroo was undertaken. A pair of degenerate oligonucleotide primers, sense 5'-TGT IT(C/T) TG(C/T) AAA (C/T)T(G/A) ATG G(C/T)I A(A/G)C AAA AC-3' and antisense 5'-C(T/C)T (T/C)A(G/A) (A/G)TC (T/C)TC TTC (A/G)TT (A/T)AT ITC TTC-3', which targeted the highly conserved sequences between the published CFTR homologs at each end of exon 13, respectively, were synthesized. Genomic DNA was used for PCR amplification.
DNA Sequencing
PCR products were purified by Centricon-100 (Millipore, Bedford, Mass.) and cloned into the pGEM-T vector (Promega, Madison, Wis.). Double-stranded DNA was sequenced using a combination of specific primers and the vector-specific T7/SP6 primers from both strands using the ABI PRISM BigDye Terminator Cycle Sequencing Kit (PE Applied Biosystems, Foster City, Calif.) on an ABI 310 sequencer.
Multiple-Sequence Alignments
Full-length CFTR sequences were collated from GenBank or SwissProt. In order to refine the boundaries of NBD1, several partial sequences were also collated. Multiple-sequence alignments were first performed with CLUSTAL W (http://www2.ebi.ac.uk). Then, the alignment was corrected by eye using MUST (Philippe 1993
) in order to take into account similarities in chemical properties of amino acid side chains.
Missense Mutations and Single-Amino-Acid-Deletion Mutations
All of the missense mutations and single-amino-acid deletions are from the CF Mutation Data Base (http://www.genet.sickkids.on.ca/cftr).
Phylogenetic Analysis
Two CFTR sequence data sets were utilized for phylogenetic analysis. One used 17 exon 13coding nucleotide sequences that provided 543 positions, of which 343 were informative for parsimony. The other contained 16 full-length CFTR amino acid sequences, with 1,544 positions, among which 771 were informative for parsimony. Phylogenetic analysis was performed principally using the branch-and-bound search of PAUP (Swofford 1999
). To check the impact of a suspected artifact on the tree reconstruction method, trees were also reconstructed using uncorrected distances and corrected distances (Kimura 1983
) through the neighbor-joining approach (Saitou and Nei 1987
) using the MUST package (Philippe 1993
). Moreover, absolute mutational saturation was tested by plotting pairwise observed differences against pairwise inferred substitutions in the most parsimonious tree using PAUP and MUST. No saturation was detected, either from the nucleotide or from the amino acid data set. For statistical robustness of nodes, 1,000 bootstrap replicates were generated, and Bremer (1994)
supports were calculated using PAUP.
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Results and Discussion |
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Candidate PCR fragments were generated from salmon gill tissue using degenerate oligonucleotide primers. After many rounds of cloning, sequencing, and sequence aligning (which was particularly complicated by the unexpected presence of two highly homologous but clearly different sequences), two CFTR transcripts were finally obtained. Sequence accuracy of the two transcripts was ensured by isoform-specific PCR amplifications using cDNA samples prepared from individual salmon. The general properties of the two transcripts, which were designated sCFTR-I (GenBank accession number AF155237) and sCFTR-II (GenBank accession number AF161070), respectively, are summarized in table 1 . Although there is no guarantee that the 5' UTR of each sequence is complete, the 3' UTR of each sequence is considered complete due to the presence of a polyA addition signal sequence 16 bp (for sCFTR-I) or 12 bp (for sCFTR-II) upstream of the polyA tail.
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The new definition of the NH2 terminus of NBD1 combines the following considerations. The principal evidence comes from evolutionary data. As illustrated in figure 2
, there is a sharp increase in sequence conservation beginning at P439 in comparison with the immediately preceding sequence. The first six amino acids of the originally defined NBD1 (i.e., F433 to T438) are quite divergent (even to the extent of deletions having occurred in several species), and the upstream region is a divergent sequence track of 20 amino acids. In contrast, downstream of P439, not only is there a stringently conserved three-amino-acid motif (i.e., P439-V440-L441), but also most of the nonstringently conserved residues from K442 to A457 have changes mainly involving substitutions between conservative amino acids, and this region is then followed by the highly conserved Walker A motif (GSTGAGKT, amino acid 458 to amino acid 465). A similar transition from divergence to sequence conservation is also present in the equivalent location of the aligned sequences of other ABC transporter family members, as illustrated in a sequence comparison of the 17 bacterial membrane proteins that form the periplasmic permease gene family (Mimura, Holbrook, and Ames 1991
). Second, this definition is consistent with most of the structural models of human CFTR, where the NH2 terminus of NBD1 was regarded as T438 (Higgins 1992
), L441 (Bianchet et al. 1997
), and around V440 (Hoedemaeker, Davidson, and Rose 1998
), respectively. This definition also appears to correlate reasonably well with the three-dimensional crystal structure of HisP (the ATP-binding subunit of the bacterial histidine transporter), where P439 would be predicted to occur at the very beginning of the second ß strand of HisP (Hung et al. 1998
). Third, this definition falls within amino acids N432 to E449the functionally defined NH2 terminus of NBD1 (Chan et al. 2000)
. Finally, this definition is given further support by the mutational pattern in this transitional region (fig. 2
).
However, one may argue that the new definition of the NH2 terminus of NBD1 might have inappropriately excluded the sequences aligned to the first ß strand of HisP (Hung et al. 1998
). Of particular relevance to this point is residue F433, an aromatic amino acid that corresponds to residue Y16 of the HisP. The structure of HisP has been considered a good model for the binding of ATP to an NBD, in which the aromatic Y16 was confirmed to play a critical role (as determined by mutational studies by Shyamala et al. [1991
]). Moreover, the structures of the other two prokaryotic NBD proteins, LivF and the amino-terminal NBD of RbsA, show a very similar binding interaction between the NBD and ATP, that is, the side chain of an aromatic residue (analogous to Y16 in HisP) interacts with the adenine ring of ATP to stabilize nucleotide binding. Furthermore, an aromatic residue 2030 amino acids upstream of the start of the Walker A motif (in a similar position relative to Y16 in HisP) is conserved in other ABC transporter NBDs (summarized in Berger and Welsh 2000)
. By inference, F433 in the human CFTR may participate in ATP binding. However, a recent functional analysis has refuted such a possibility and highlighted the unique structure of CFTR NBD compared with other NBD structures (Berger and Welsh 2000)
. Undoubtedly, this finding adds further weight to the newly defined NBD1.
We previously defined the NH2 terminus of the R domain as beginning at C647 (Chen, Scotet, and Ferec 2000)
. Consequently, G646 could be considered the COOH terminus of NBD1. However, the concurrent functional approach defined the COOH terminus of NBD1 as occurring between G622 and Q634 (Chan et al. 2000)
. Nevertheless, G646 seems to be a more logical positioning of the true boundary linking NBD1 and the R domain in CFTR. In addition to the previous arguments (Chen, Scotet, and Ferec 2000)
, further supporting evidence also exists. Again, the principal evidence comes from evolutionary data. As indicated in figure 2
, the sequence from S623 to G646 is as well conserved as that from F587 to G622; the latter is the common part of the extended NBD1 in the two studies (Chan et al. 2000; Chen, Scotet, and Ferec 2000)
. Perhaps a more important indication comes from a sequence alignment with other ABC transporter NBDs; for example, HisP ends exactly at residue G646 of the human CFTR homolog (see fig. 5 in Hung et al. 1998
). Moreover, it is important to note that the functional assembly of paired CFTR segments severed at L633/Q634 is not as good as that of those cleaved at N432/F433 (Chan et al. 2000)
. In this regard, it will be interesting to compare paired CFTR fragments cleaved at the newly defined NBD1/R domain boundary (i.e., G646/C647) with those cleaved at L633/Q634.
Refinement of the Boundaries of NBD2
The original location in the amino acid sequence of NBD2 is from Y1219 to R1386 (Riordan et al. 1989
; fig. 2). Historically, NBD2 has received much less attention than NBD1. Now, with the boundaries of NBD1 redefined, those of NBD2 can be similarly refined. Accordingly, the NH2 terminus of NBD2 could be defined as going from A1225 by reference to the relative position of the highly conserved Walker A motif, and this also correlates fairly well with the mutational pattern (the appearance of missense mutations clustering around the Walker A motif: GRTGSGKS, at amino acid 1244 to amino acid 1251) in the transitional region in NBD2 (fig. 2
). However, when the COOH terminus of NBD2 is extended to P1443 by reference to the redefined NBD1 (see also fig. 5 in Hung et al. 1998
), the very COOH-terminal portion of this "new NBD2" shows poor sequence conservation. Yet, this is not so surprising if it is considered in the context of the ABC transporter family, where for example, its counterpart region in the 17 bacterial periplasmic permeases is similarly divergent (Mimura, Holbrook, and Ames 1991
). Thus, it seems clear that the very COOH-terminal portions of NBDs are, in general, less functionally restrained throughout the ABC transporter family, although this is in stark contrast to the NBD1 of CFTR, where the very COOH-terminal portion is highly conserved (fig. 2
). While it is not clear whether this sequence conservation is related to the immediately following R domain (which is unique to CFTR), it lends further weight to the unique structure of CFTR NBD1.
Although P1443 could represent the COOH terminus of a "new NBD2," this should not be accepted without any reservations. The significant sequence divergence of the very COOH-terminal portion of this "new NBD2," the different structural requirements of a CFTR NBD compared with other ABC transporter NBDs in general (Berger and Welsh 2000)
, and the differential roles between the CFTR NBD1 and NBD2 in particular (discussed below) argue for an alternative definition. Here again, it is reassuring how well the evolutionary data, clinical observations, and functional studies concur. The initial location of sequence divergence in this transitional region was clearly situated at E1418 (fig. 2
). In addition, the shortest truncation that is associated with CF disease (with pancreatic insufficiency and recurrent pulmonary infection) terminates at Q1412 (http://www.genet.sickkids.on.ca/cftr). Furthermore, a review of several functional studies clearly points to the fact that CFTR truncated at/or upstream of V1415 significantly affects the biogenesis, maturation, and stability of the protein, but those molecules truncated at or downstream of N1419 do not (Rich et al. 1993
; Zhang et al. 1998
; Haardt et al. 1999
; Gentzsch and Riordan 2001
). These three lines of observation, together with the mutational pattern in this region, strongly suggest that residue E1417 can be confidently assigned as the COOH terminus of NBD2. This new definition is also further validated by the analysis which has redefined the C-tail of the CFTR (discussed below).
Functional Delineation of the C-Tail
The COOH terminus of NBD2 could naturally be anticipated to be the NH2 boundary of the C-tail, and this appears to best delineate its functional role in the CFTR. On the one hand, the newly defined C-tail is "nonessential," as the effects on the biogenesis, processing, and stability of the CFTR previously assigned to the original C-tail have now been relocated to within the newly defined NBD2. Moreover, even the two trafficking signals thought to reside in the newly defined C-terminal tail, at Y1424 and the dileucine pair at L1430-L1431, have been demonstrated not to alter the steady-state amount of nascent or mature CFTR in recent functional studies (Gentzsch and Riordan 2001)
. On the other hand, the newly defined C-tail does appear to be "essential," as the well-conserved motif involving the final three amino acids (TRL) represents a PDZ-interacting domain which is required for the polarization of CFTR to the apical plasma membrane in epithelial cells (Moyer et al. 2000)
. Even so, it is important to note that this PDZ-binding motif alone is not sufficient for the polarized distribution of CFTR (Milewski et al. 2001)
. Therefore, the C-tail as a whole would play a minor role in the pathogenesis of CF disease. Indeed, neither S1455X (Mickle et al. 1998
) nor Q1476X (http://www.genet.sickkids.on.ca/cftr) caused a severe CF phenotype. By inference, any single missense mutations identified in the C-tail may not have a significant functional consequence. Consistent with this hypothesis, none of the three missense mutations identified in this region (fig. 2 ) can be confidently assigned as causal for CF disease; for example, R1422W was identified in a CF patient who also carried the mutations F508del and D993Y, and R1453W was identified in a patient with diffuse panbronchiolitis (http://www.genet.sickkids.on.ca/cftr). Finally, attention may be drawn to the well conserved, negatively charged motif (E1469 to E1474) in the C-tail.
Further Insights into the Differential Function of NBD1 and NBD2
That the two NBDs are not functionally equivalent was immediately noticed in the early 1990s. Not only were CF-associated mutations found predominantly in CFTR's NBD1 as opposed to NBD2, but also mutations of homologous amino acid residues within NBD1 or NBD2 resulted in different effects on CFTR maturation and function (Cheng et al. 1990
; Gregory et al. 1991
). More recently, the different roles of NBD1 and NBD2 in the CFTR chloride channel and the regulation of other ion channels have been further investigated through a variety of approaches (e.g., Szabo et al. 1999
; Cahill et al. 2000
; Mickle et al. 2000
; Aleksandrov et al. 2001
).
Apart from the fact that there are more disease-associated mutations in NBD1 than in NBD2, and more than half of the most common missense mutations or single-amino-acid deletions occur in NBD1, figure 2
reveals an additional interesting observation regarding mutational patterns in the well-conserved motifs in the NBDs. While there are 17 missense mutations clustered in the LSGGQ motif (LSGGQ, amino acids 548552) and the Walker B motif (RAVYKDADLYLLD, amino acids 560572) in NBD1, there are only four missense mutations in the equivalent regions (LSHGH [amino acids 13461350] and RSVLSKAKILLLD [amino acids 13581370]) of NBD2. Conversely, while there is only one missense mutation in the Walker A motif (GSTGAGKT, amino acids 458465) in NBD1, there are five mutations in the corresponding motif (GRTGSGKS, amino acids 12441251) in NBD2. This finding is intriguing since, on the one hand, although Walker A and Walker B are presumably both involved in ATP binding and hydrolysis, the exact mechanisms remain largely unknown. On the other hand, while it is generally believed that ATP hydrolysis at NBD1 and NBD2 may drive channel opening and closing, respectively (reviewed by Nagel 1999
), recent biochemical analysis suggests that opening and closing may occur as a consequence of nucleotide binding and dissociation or hydrolysis at a functional unit involving both domains (Aleksandrov et al. 2001)
. Therefore, it is tempting to ask whether the distinct mutational patterns in these important motifs are linked intrinsically to functional differences of the Walker A and Walker B sites within the two NBDs. Elucidation of this question would improve our understanding of the basic physiology of CFTR, as well as that of other ABC transporters. Nevertheless, this observation gives further support to the idea that NBD1 and NBD2 make different contributions to the gating of CFTR channels.
Further Insights into the Differential Role of MSD1 (F81 to V350) and MSD2 (I860 to S1150)
Each MSD was predicted to contain six transmembrane segments (TMs), three extracellular loops (ECLs), and two intracellular loops (ICLs). As in the case of the two NBDs, functional differences have also been found for the two MSDs (e.g., Yue, Devidas, and Guggino 2000)
. A comparison of the stringently conserved residues in the TMs of the two MSDs reveals that the TMs in MSD1 are generally more conserved than those in MSD2 (data not shown; see also fig. 2
); TM6 (12 out of 21 residues; 57.1%) and TM12 (11 out of 22 residues; 50.0%) are the most conserved motifs in the two MSDs, respectively, and TM6 is the most conserved of all. The evolutionary conservation of these regions correlates perfectly with analysis that suggests that (1) TM6 and TM12 line the CFTR pore, (2) TM6 is the main determinant of the pore properties of CFTR, and (3) MSD1 and MSD2 each contribute differently to CFTR pore properties (Sheppard and Welsh 1999
).
A defining feature of ion channels is their selectivity, the ability to pass certain ions at a high rate while at the same time effectively excluding others. While discrimination between different cations in certain cation channels is known to occur over a short region of the pore known as the selectivity filter, the existence of a discrete anion selectivity filter in the pores of Cl- channels is controversial (Linsdell, Evagelidis, and Hanrahan 2000)
. Nevertheless, based on the above-mentioned evolutionary data, such a selectivity filter in the CFTR, if it indeed exists, would most likely be located in the most highly conserved TMTM6. Not surprisingly, the anion selectivity (Br- > Cl- > I-) of the CFTR pore was found to be mainly determined by the first half of the protein (Yue, Devidas, and Guggino 2000)
, and mutations in several residues in TM6, including K335 (Anderson et al. 1991
), F337 (Linsdell, Evagelidis, and Hanrahan 2000)
, T338 (Linsdell, Zheng, and Hanrahan 1998
), and R347 (Cheung and Akabas 1997
), were shown to significantly affect anion selectivity. Moreover, the stringently conserved motif T351-R352-Q353 immediately downstream of TM6 has also been suggested to loop back into the channel, narrowing the lumen and thereby forming both the major resistance to current flow and the anion selectivity filter (Cheung and Akabas 1997
). Assuming that an anion selectivity filter resides in the vicinity of TM6, it is interesting to note the presence of another highly conserved motif also in this vicinity (Q359-T360-W361-Y362-D363). Furthermore, there is also a similarly stringently conserved motif downstream of TM12 in MSD2, L1156-M1157-R1158-S1159-V1160.
Inspection of figure 2
also reveals that the four ICLs are generally more conserved than the TM and ECL subdomains in the MSDs. To date, most of the missense mutations identified in each ICL and a 19-amino-acid deletion concerning ICL2 have been investigated by functional analysis (reviewed in Sheppard and Welsh 1999
). These studies strongly indicated that the ICLs were critical for correct protein processing. Among the four ICLs, ICL4 (amino acid Q1035 to amino acid R1102, with a sequence homology as high as 72% across the 15 CFTR species) turns out to be the most conserved subdomain in the entire CFTR sequence. This stringent sequence conservation, coupled with a cluster of missense mutations identified in this subdomain (among which two homozygosities, H1085R [Yoshimura et al. 1999
] and R1066C [Casals et al. 1997
], cause a severe CF phenotype), undoubtedly underlies its functional significance within CFTR. While a large fraction of the analyzed ICL4 missense mutations disrupted the biosynthetic processing of CFTR, this was not universally the case. Most importantly, those processed mutants had no discernible effect on the channel's pore properties, but they did alter gating behavior, the response to increasing concentrations of ATP, and stimulation in response to pyrophosphate (Cotten et al. 1996
; Seibert et al. 1996
). Thus, ICL4, and probably the other three ICLs, may actively participate in interdomain interactions; for example, they may link the NBDs to MSDs, thereby coupling the activity of NBDs to the gating of the channel.
Possible Motifs Implicated in the Interdomain Interaction Between the NH2-Terminal Tail (N-tail; M1 to R80) and the R Domain
An eliciting finding is that the N-tail controls CFTR channel gating through a physical interaction with the R domain. Not surprisingly, this positive regulatory activity was mapped to a cluster of acidic residues, D47, E51, E54, and D58, within the highly charged and conserved motif D44 to E60 (fig. 2
). Since no effects of any of the N-tail mutations on steady-state phosphorylation in vitro or in vivo were detected, the N-tail most likely modulates channel activity by controlling access of the phosphorylated R domain to inhibitory or stimulatory sites within the channel (Naren et al. 1999
).
The fact that a cluster of stringently conserved acidic residues in the N-tail appears to bind to part of the R domain (most probably the region involving amino acids 708740 and the nearby surrounding sequences; Naren et al. 1999
) is significant. As illustrated in figure 2
, there are three highly conserved, positively charged motifs (K696-R697-K698, R709-K710, and R764-R765-R766) in this region. Although the conservation of these motifs may be explained by the fact that they potentially participate in phosphorylation of the R domain, the possibility that all, or at least some, of them serve as counterparts to the acidic residues in the N-tail cannot be excluded, particularly if the fact that none of them appear to be phosphyorylated by PKA in vivo is taken into account (Cheng et al. 1991
). More recently, a relatively small portion of the R domain (residues 760783) that encompasses the stringently conserved motif (R765-R766-Q767-S768-V769-L760) was found to be critical for the prevention of constitutive activity of the CFTR (Baldursson et al. 2001)
. This further highlights the functional importance of these motifs when it is considered that they are conserved even within the most evolutionarily divergent R domain sequences. Future studies focusing on these motifs may result in the revelation of further novel molecular mechanisms.
How Many Missense Mutations Are Functional?
Unlike splicing and truncating mutations, it is always difficult to assign a definitive role to missense mutations, especially when they are detected in single cases. Functional analysis provides important data, but extrapolation to clinical effects may not be straightforward. This can be seen in the conflicting results that often occur in some aspects of CF research, exemplified by the two different models for salt and water transport across epithelia (reviewed by Wine 1999
), as well as the disparity among results on the intracellular localization of F508del-CFTR (reviewed by Drumm 1999
). There is ongoing effort to tackle this issue (e.g., the CF European Network: the Second Consensus Meeting Towards Validation of CFTR Gene Expression and Functional Assays, Estoril, March 30 April 1, 2001).
We believe that a combined analysis of the currently available data can complement this effort and enhance our understanding of the identified mutations, particularly those that are missense. Taking advantage of the previously redefined R domain, a systemic evaluation of the missense mutations occurring in this region was produced, and as a result, some of these mutations, such as F693L, V754M, and T760M, were identified as being likely to represent neutral polymorphisms (Chen, Scotet, and Ferec 2000)
. Similarly, the missense mutations occurring in evolutionarily divergent regions, for example, N418S, G424S, Q890R, and K1177R, may also represent neutral polymorphisms. At the other extreme, for example, the mutations I506M and F508C, which occur in stringently conserved residues in the functionally important NBD1, have also been confirmed to be nonfunctional variants (Kobayashi et al. 1990
; Kalin, Dork, and Tummler 1992
; Will et al. 1992
). The assignment of mutations as in these two extreme cases would raise a serious concern that a certain fraction of the identified missense mutations in classic and atypical CF patients may in fact merely represent polymorphisms rather than mutations associated with the disease. If this assumption is correct, it is not unreasonable to infer that the truly causal mutation event may remain unidentified in a certain fraction of patients in whom the disease has been incorrectly assumed to be caused by identified "missense mutations."
The above assumption is further supported by the following considerations. The large size of the CFTR gene makes it difficult to analyze the entire coding sequence, not to mention the intronic sequence. Consequently, initial identification of one DNA variant may discourage further screening efforts, thereby ignoring a second variant in cis in the gene. Even if the whole CFTR gene were analyzed, certain variants, such as large deletions within the gene (which are being detected at an increasing rate; http://www.genet.sickkids.on.ca/cftr), are still not readily detected by common mutational screening techniques (Girodon-Boulandet, Cazeneuve, and Goossens 2000)
. In addition, the promoter sequence (Romey et al. 1999
) and the upstream regulatory region (Nuthall et al. 1999
) of the CFTR gene may be targets for extended mutational screening. Moreover, mutations in other genes in certain CF-like disease subjects should be considered (Mekus et al. 1998
).
Taken together, it is likely that a certain fraction of the missense mutations identified in the CFTR gene may be ultimately confirmed to be nonpathological polymorphisms. It is even possible that some of them may turn out to be modifiers of another variant in the CFTR gene or even in another gene. An answer to this question will significantly improve our understanding of the disease, as well as that of the protein itself. Nevertheless, this vast number of missense mutations represents an invaluable resource for exploring the subtlety of the CFTR structure-function relationship, and figure 2 could serve as an important complement to ongoing mutation detection and functional analysis efforts in the CF field.
A Comprehensive Phylogenetic Analysis: Implications for Animal Models of CF
An additional dividend of the availability of a large number of CFTR sequences is their benefit concerning the use of animal models in CF studies. We had previously suggested that the rabbit could be a better CF model than the current murine model, mainly based on (1) anatomical and developmental data, (2) comparison of CFTR promotor sequences and partial coding sequences, and (3) phylogenetic analysis of those sequences (Vuillaumier et al. 1997
). Although sequence comparisons clearly showed a higher similarity between rabbit and primate sequences than between murine and primate sequences or even between rabbit and murine sequences, the phylogenetic implications were ambiguous.
A long-branch attraction artifact (Felsenstein 1978
) of the murine lineage toward the outgroup was suspected (Vuillaumier et al. 1997
). This classical artifact of phylogenetics concerns all tree reconstruction methods. It occurs when rates of character change vary among lineages, provoking in trees the grouping of lineages sharing close rates, whatever the evolutionary relationships. To solve this ambiguity, additional sequence data (particularly from further species) were required for a successful analysis. When all of the full-length CFTR amino acid sequences were analyzed in this study (fig. 1
), the murine lineage was the most basal of the placental mammals, a strange result possibly due to a long-branch attraction. Moreover, the position of the rabbit was ambiguous. In the most parsimonious tree (fig. 1
) and in the neighbor-joining tree from corrected distances (data not shown), the rabbit is a sister group to the bovids + primates clade. In the neighbor-joining tree based on uncorrected distances, the rabbit is the sister group to the primates as in our previous report (Vuillaumier et al. 1997
). When an extended set of exon 13coding sequences including a nonplacental mammal (the kangaroo) and another nonmurid rodent (guinea pig) were analyzed (fig. 3
), the guinea pig represented a sister group to the rabbit corresponding to the Glires (node E), while the murids were still the most basal of the placental mammals. Assuming that rodents are a monophyletic group (Novacek and Wyss 1986
; Luckett and Hartenberger 1993
; Philippe 1997
), it appears that the murids are artifactually basal in the placental tree, as they should be a sister group to the guinea pig within clade E. Thus, the high level of divergence of the mouse sequence not only makes the mouse an inappropriate model for CF studies, but also provokes a phylogenetic artifact. Unfortunately, the addition of the kangaroo sequence did not correct this artifact, as it was added as a taxon closer to placental mammals than the frog is, in order to break a long branch that could have reduced the possibility of the murid long-branch attraction. Nevertheless, the conclusions from the phylogenetic analysis do not challenge the proposition of the rabbit as a better CF model whose sequence is more closely related to humans (than murine species are). Additionally, the guinea pig appears to be an equally good model candidate.
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1 Keywords: cystic fibrosis transmembrane conductance regulator
missense mutation
structure and disease models
phylogeny
Atlantic salmon
rabbit
2 Address for correspondence and reprints: Claude Férec, Etablissement Français du SangBretagne, Universite de Bretagne Occidentale, Centre Hospitalier Universitaire, 46 rue Félix Le Dantec, 29275 Brest, France. claude.ferec{at}univ-brest.fr
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