Human Molecular Genetics, 2001, Vol. 10, No. 8 835-843
© 2001 Oxford University Press
Mutation of the gene encoding fibrillin-2 results in syndactyly in mice
1School of Biological Sciences and Department of Dental Medicine and Surgery, 3.239 Stopford Building, University of Manchester, Oxford Road, Manchester M13 9PT, UK, 2MRC Institute of Hearing Research, University Park, Nottingham NG7 2RD, UK and 3School of Biological Sciences and School of Medicine, 2.205 Stopford Building, University of Manchester, Oxford Road, Manchester M13 9PT, UK
Received 14 December 2000; Revised and Accepted 15 February 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Fibrillins are large, cysteine-rich glycoproteins that form microfibrils and play a central role in elastic fibrillogenesis. Fibrillin-1 and fibrillin-2, encoded by FBN1 on chromosome 15q21.1 and FBN2 on chromosome 5q23–q31, are highly similar proteins. The finding of mutations in FBN1 and FBN2 in the autosomal dominant microfibrillopathies Marfan syndrome (MFS) and congenital contractural arachnodactyly (CCA), respectively, has highlighted their essential role in the development and homeostasis of elastic fibres. MFS is characterized by cardiovascular, skeletal and ocular abnormalities, and CCA by long, thin, flexed digits, crumpled ears and mild joint contractures. Although mutations arise throughout FBN1, those clustering within exons 24–32 are associated with the most severe form of MFS, so-called neonatal MFS. All the mutations described in CCA occur in the ‘neonatal region’ of FBN2. Both MFS and CCA are thought to arise via a dominant negative mechanism. The analysis of mouse mutations has demonstrated that fibrillin-1 microfibrils are mainly engaged in tissue homeostasis rather than elastic matrix assembly. In the current investigation, we have analysed the classical mouse mutant shaker-with-syndactylism using a positional candidate approach and demonstrated that loss-of-function mutations outside the ‘neonatal region’ of Fbn2 cause syndactyly in mice. These results suggest that phenotypes distinct from CCA may result in man as a consequence of mutations outside the ‘neonatal region’ of FBN2.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Fibrillins are large, cysteine-rich glycoproteins that form the molecular scaffold of a class of extensible beaded microfibrils which act as a structural template for elastin deposition during elastic fibre formation (1,2). Fibrillin-1 and fibrillin-2, which are encoded by FBN1 on human chromosome 15q21.1 (3) and FBN2 on human chromosome 5q23–q31 (4), respectively, are extremely similar proteins. Their primary structures are highly modular being dominated by 47 epidermal growth factor-like (EGF) repeats, 43 of which are associated with calcium binding (cbEGF repeats) (5–7). These domains are interspersed with seven latent transforming growth factor ß1 binding protein-like (TB) modules that are characterized by eight cysteine residues which are predicted to form four intramolecular disulphide bonds (8). A predicted hinge region (proline-rich in fibrillin-1, glycine-rich in fibrillin-2) occurs towards the N-terminus. N- and C-terminal sequences contain furin/paired basic amino acid converting enzyme (PACE) proprotein convertase tetrabasic consensus sequences, and processing at these sites is an important regulatory step in fibrillin deposition (9–11).
On the basis of distinct but overlapping expression patterns, it has been proposed that the two fibrillins have different functional roles. During development, FBN2 transcripts accumulate prior to tissue differentiation, decreasing rapidly thereafter (12). Subsequently, fibrillin-2 is found preferentially in elastic tissues, such as the elastic cartilage, the tunica media layer of the aorta and along the bronchial tree (7). Conversely, FBN1 transcripts increase at a gradual rate during development such that fibrillin-1 is expressed in load bearing structures such as the aortic adventitia, the ciliary zonules and the skin (12). These combined observations have led to the hypotheses that fibrillin-2 has a major functional role during early morphogenesis in directing elastic fibre assembly and in imparting physical stability to embryonic tissues, whereas fibrillin-1 is mainly responsible for the structural function of the microfibrils (12,13). The analysis of mouse mutations has confirmed that fibrillin-1 microfibrils are mainly engaged in tissue homeostasis rather than elastic matrix assembly (14).
The crucial contribution of the fibrillins to normal development and homeostasis is emphasized by the finding of mutations in FBN1 and FBN2 in the autosomal dominant microfibrillopathies Marfan syndrome (MFS) and congenital contractural arachnodactyly (CCA), respectively (15,16). MFS is characterized by severe cardiovascular, skeletal and ocular abnormalities, whereas CCA is characterized by arachnodactyly (long, thin digits), camptodactyly (flexed digits), crumpled ears, mild contractures in the elbows, knees and hips and mild hypoplasia of the calf muscles (17,18). Although mutations have been found throughout FBN1, those clustering in the region encompassing exons 24–32 are associated with the most severe form of MFS, so-called neonatal MFS (19,20). Interestingly, all of the mutations that have been described in CCA occur in the region of FBN2 that is analagous to the ‘neonatal region’ of FBN1 (21). Both MFS and CCA are thought to arise via a dominant negative mechanism (2).
Shaker-with-syndactylism (sy) is a radiation-induced mouse mutation that results in auditory/vestibular defects together with fusion of the digits (syndactyly) and early lethality (22–24). The phenotype results from the deletion of an 
0.7 cM interval of central mouse chromosome 18 that shows conservation of synteny with distal human chromosome 5q. Three additional alleles of sy, all of which have arisen spontaneously, exhibit either hearing loss or syndactyly, but not both. The fused phalanges mutation arose in the strain C3HeB/FeHu (25) and has been designated syfp (26). All syfp homozygous animals show variable fusion of the three central digits of the hindfeet and about half show the same abnormality of the forefeet; however, behaviour is normal (25). More recently, a second spontaneous mutation with the same homozygous phenotype as syfp was shown to be allelic with fused phalanges and consequently given the allele symbol syfp-2J (27). The third spontaneous mutation at the sy locus results in vestibular and hearing defects, but normal digits, and has therefore been termed no syndactylism, syns (28). A series of complementation studies have confirmed that syfp and syfp-2J are alleles of the same gene, but that syns is an allele of a different gene; however, the original sy mutation includes the genes defined by the three spontaneous mutations (28). A combination of deletion mapping studies, mutation analysis and gene targeting has demonstrated that the deafness observed in these mice results from disruption of the Na-K-2Cl co-transporter gene Slc12a2 (29–32). In the current investigation, we have used a positional candidate approach to demonstrate that loss-of-function mutations in the gene encoding fibrillin-2, which are not within the so-called ‘neonatal region‘, cause syndactyly in mice and not CCA as observed in man.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The paws of sy, syfp and syfp-2J mutant mice have been examined after alcian blue/alizarin red staining of skeletal preparations. While no abnormalities were detected in any of the heterozygous animals analysed, the types and severity of the syndactyly observed in the mice homozygous for the different mutations varied on an allele-by-allele basis (Table 1; Fig. 1). In general, the syndactyly associated with all three mutant alleles was found to be more frequently expressed in the hind-paws than in the fore-paws and involved digits 2 and 3, 3 and 4, or 2, 3 and 4; digits 1 and 5 were never involved. sy homozygous mice were found to be the most severely affected with more extensive syndactyly, more hind-paws affected, more cases of 2,3,4 syndactyly observed, more digits with complete fusion along their entire length and more fore-paws showing syndactyly compared with the syfp and syfp-2J mutant mice (Table 1). For example, whereas >50% of sy mutant hind-paws displayed syndactyly involving digits 2, 3 and 4, <20% of syfp-2J mutants exhibited the same phenomenon. Moreover, only sy mutants showed 2,3,4 syndactyly in the fore-paws. In contrast, syfp-2J homozygous mice were found to be the least severely affected with no evidence of fore-paw involvement, less involvement of all three central digits, less severe fusion of the digits that was more commonly limited to the soft tissues rather than bone or cartilaginous elements and more instances of incomplete fusion along the entire length of the digits (Fig. 1). For example, over half of all syfp-2J mutant hind-paws were incompletely fused along the entire length of the digits, whereas this observation was only made in ~10% of cases with the other two alleles. Nevertheless, as each mutation arose, and is maintained on a different genetic background, potential background effects as well as different underlying mutations (see below) may contribute to this phenotypic variation.
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Genetic mapping studies have previously demonstrated that the syfp mutation is located within a region of central mouse chromosome 18 that shows conservation of synteny with human chromosome 5q21–q31 (29,33). STS content analysis has further indicated that the shaker-with-syndactylism phenotype results from an
0.7 cM interstitial deletion encompassing the polymorphic markers D18Mit124, D18Mit181, D18Mit52 and D18Mit205, the flanking markers D18Mit238 and D18Mit28 remaining undeleted (28). Using a combination of deletion mapping studies, mutation analysis and gene targeting, we and others have subsequently shown that the deafness observed in these mice results from disruption of the Na-K-2Cl co-transporter gene Slc12a2 (29–32). These observations have allowed us to identify candidate genes for the syndactyly based on published chromosomal locations. Deletion analysis using sy/sy DNA and a total of 21 STSs (mostly representing genes) revealed that most of them were not deleted, but that Slc12a2, D19296 and part of Fbn2 were deleted (data not shown). PCR amplification of +/sy and sy/sy DNA using an EST derived from the 3' untranslated region (UTR) of Fbn2 in a duplex reaction with primers defining D18Mit123, which is not deleted in sy (28), indicated that Fbn2 is deleted in sy/sy DNA (Fig. 2A). A similar experiment using primers designed from exon 1 indicated that Fbn2 is totally deleted in sy (Fig. 2B). Conversely, no evidence for a gross deletion of Fbn2 in the syfp and syfp-2J alleles was obtained.
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Subsequently, RT–PCR analysis of the coding sequence of Fbn2 in the syfp and syfp-2J alleles of sy was undertaken. Initial analysis of cDNA derived from the heart of C3H, +/syfp and syfp/syfp mice indicated that, under the same amplification conditions, less transcript was produced from the fp allele than from the wild-type allele (Fig. 2C). Sequencing of the region encompassing nucleotides 4891–5621 of Fbn2 in the syfp allele led to the identification of the mutation nt5051 del(A) in exon 39, which causes a frameshift with the introduction of a termination codon after 44 amino acid residues (Fig. 3A). This mutation is not present in the C3H strain on which the syfp mutation arose (25). Similarly, RT–PCR analysis of syfp-2J/syfp-2J mice revealed the presence of an aberrant band in the region of nucleotides 4259–5032 (Fig. 2D). Sequencing of this product revealed the deletion of nucleotides 4859–4927 that comprise exon 38 of Fbn2 and encode the second half of the fourth transforming growth factor ß (TGFß)-binding module (TB4) (Fig. 3B). Sequencing of cDNA from +/syfp-2J mice revealed that both alleles were present confirming that the deletion was not present in the parental strain on which the syfp-2J mutation originally arose (27). As the exon 38-lacking product was not detected in RNA extracted from a panel of control embryonic and adult tissues, this product was likely to be the result of aberrant exon skipping rather than normal alternative splicing (Fig. 2D). To identify the underlying mutation, introns 37 and 38 were amplified from genomic DNA extracted from syfp-2J mutants. Sequencing of these fragments indicated that whereas intron 37 was unaffected, a 21 nucleotide deletion that removed the last 7 nucleotides of exon 38 and 14 nucleotides of intron 38, including the entire splice donor sequence, was detected in syfp-2J/syfp-2J DNA (Fig. 3C). We did not detect this mutation in any of the other mouse strains analysed, including the parental strain.
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SDS–PAGE and western blotting of tissue extracts of littermate pairs of +/sy, sy/sy, +/syfp-2J, syfp-2J/syfp-2J, +/syfp and syfp/syfp mice revealed immunoreactive bands corresponding to fibrillin-2 in all heterozygotes, and in syfp-2J/syfp-2J mice, but not in samples prepared from sy/sy or syfp/syfp mice (Fig. 4A). Thus, in line with the deletion of the Fbn2 gene, no fibrillin-2 protein is detected in sy homozygotes. Fibrillin-2 is also not detected in syfp homozygotes, as predicted by the occurrence of a premature termination codon. Conversely, mutant fibrillin-2 is produced by syfp-2J mice, although the predicted reduction of
3.0 kDa could not be resolved by SDS–PAGE (Fig. 4A). Fibrillin-2 has been proposed to play a central role in elastic tissue formation as the microfibrillar scaffold of elastic fibres (7). We therefore examined whether microfibrils were, in fact, present in elastic fibre-rich tissues of the heterozygous and homozygous mutant mice, and if so, whether they appeared morphologically normal. Extracts prepared from skin and lung of heterozygous and homozygous sy, syfp and syfp-2J mice were examined by rotary shadowing electron microscopy (Fig. 4B–F). Abundant extensive microfibrils with characteristic untensioned beaded periodicity of 56 nm (34) were detected in all extracts. This analysis thus confirmed that morphologically normal, stable microfibrils were present in the elastic tissues of all the mutant mice. Thus, neither the deletion of exon 38 nor the complete absence of fibrillin-2 protein prevented the formation and deposition of apparently functional microfibrils in these elastic tissues, suggesting that these microfibrils contain or comprise fibrillin-1.
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Although the sy and syfp mutations lead to reduced/absent fibrillin-2 production, the functional consequences of the syfp-2J exon-skipping mutation are not clear, the predicted structural effects of the exon 38 deletion were therefore investigated using homology modelling. The TB4 module, which is encoded by exons 37 and 38, is composed of six anti-parallel ß-strands and two
-helices (Fig. 5B and C), held together by four disulphide bonds (all of which are shown in Fig. 5C). The 23 amino acid residues encoded by exon 38 are at the C-terminal end of the domain, and include the second -helix and 16 residues with no defined secondary structure that provide a linker to the following cbEGF domain (Fig. 5B and C). Ordinarily, the N- and C-termini are located at the bottom of the domain, pointing in opposite directions (Fig. 5B). However, based on the homology model, when exon 38 is deleted (sequence highlighted in red in Fig. 5B–D), the new C-terminus (Ser1618) is predicted to be situated at the top of the domain pointing in the same direction as the N-terminus (Fig. 5B and C). One can speculate that loss of TB domain-cbEGF domain linker residues would lead to less conformational flexibility between TB4 and the following cbEGF, and may alter the calcium-binding capability of that domain. Overall, it is possible that the deletion could lead to a kink in the fibrillin-2 molecule around TB4. Loss of the eighth cysteine in TB4 would disrupt the normal disulphide bond formed with the fifth cysteine residue. Finally, loss of an N-glycosylation sequence (NXT to NXD) may disrupt protein–protein interactions (35).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Using a positional candidate approach, we have demonstrated for the first time that mutations of Fbn2 can cause syndactyly in mice. Two of the mutations that are the basis of this syndactyly consist of a deletion of the entire Fbn2 gene (sy) and a frameshift mutation that introduces a termination codon into fibrillin-2 (syfp). The net effect of both mutations is ablation of fibrillin-2 protein. A third mutation is an in-frame exon-skipping mutation that results in absence of the C-terminal sequence of a TB module, which we have suggested could introduce a kink in the molecule, disrupt an N-glycosylation site and affect calcium binding by the following cbEGF thereby possibly increasing proteolytic susceptibility and molecular degradation (36–39). All three mutations, which lie outside the ‘neonatal’ MFS/CCA region (exons 24–34), are recessive in nature and are thus likely to produce their effect by loss of function.
In contrast, a dominant negative model is indicated for the pathogenesis of both MFS and CCA (2). In this model, the product of the mutant allele interferes with the function of that produced by the wild-type allele. Since fibrillin molecules assemble into multimeric beaded microfibrils, which in turn form a template for tropoelastin deposition in elastic fibre formation, incorporation of mutant molecules would perturb the assembly, function or stability of the microfibrils and disrupt elastic fibrillogenesis. mRNAs of alleles with premature termination codons are generally present in reduced amounts, with a corresponding reduction in dominant negative impact. In MFS, fibrillin-1 mutations have been shown to occur throughout the molecule, the majority being missense mutations (72%) mostly affecting the cbEGF-like domains but also TB modules (2). Mutations predicted to cause premature termination codons represent only a small proportion of the mutations. Exon skipping mutations are associated mainly with mutations affecting splicing consensus sequences but cases that are due to nonsense mutations in the skipped exons are relatively common. Usually, exon-skipping mutations cause relatively severe classical MFS.
Neonatal MFS (nMFS) represents the most severe end of the spectrum of MFS and is characterized by mutations in exons 24–27 and 31–32, and a series of manifestations that are rare in classical MFS (19,20). Patients are typically diagnosed at or shortly after birth, and present with symptoms such as mitral or tricuspid valve insufficiency, congestive heart failure, pulmonary emphysema, joint contractures, crumpled ears and loose skin. Death usually occurs within the first year of life. Apart from the heart failure, many features of nMFS are similar to those of CCA. Intriguingly, the 10 different mutations that have been reported to date in CCA are also located, without exception, between exons 24 and 34 of FBN2, which encodes that segment of fibrillin-2 analogous to the neonatal region of fibrillin-1. These mutations consist exclusively of missense changes and exon-skipping mutations; no mutations that introduce a termination codon into fibrillin-2 have been described (16,21,40–44). Interestingly, it has recently been hypothesized that mutations in other regions of FBN2 may cause a different phenotype that is not reminiscent of a Marfan-like disorder (21). We have shown here that, in mice, fusion of the digits is caused by some mutations in the gene encoding fibrillin-2; no such features have been reported in CCA. While this may be due to species-specific differences in development (45), the mutational spectra observed in human CCA and the shaker-with-syndactylism allelic series of mice are clearly different. To our knowledge, humans homozygous for FBN2 mutations have not been reported. Nevertheless, it will be interesting to investigate whether recessive mutations outside the ‘neonatal region’ of FBN2 lead to syndactyly in man and whether FBN2 should be considered as a candidate gene for this condition.
How might a Fbn2 mutation lead to syndactyly? It has previously been noted that establishment of the cartilaginous skeleton of the limb is associated with the transient presence of elastin-rich microfibrillar networks (46). Therefore, the absence or abnormality of fibrillin-2 in sy mutants could directly affect the function of microfibrils in laying down a structural scaffold for later bone deposition. Alternatively, fibrillin-2 might directly, or indirectly through interactions with microfibril-associated latent TGF-ß binding proteins, mediate a specific growth factor binding event at a key developmental stage in digit development. An example of this latter mechanism comes from studies of fibrillin-1 from Tsk mutant mice, which has been shown to have a significantly higher TGFß binding capacity than wild-type fibrillin-1 (47).
Fibrillin-2 is developmentally expressed, and has previously been predicted to play an essential functional role in directing neonatal elastic fibre formation (12). It is, therefore, interesting to note that the absence of fibrillin-2 in the homozygous syfp/syfp mice is compatible with a normal life span, indicating that elastic fibre-rich tissues, such as the cardiovasculature and lungs, are functional. Morphologically normal microfibrils are abundant in tissues from these animals, but are presumably based on fibrillin-1. At any rate, we have demonstrated that assembled beaded microfibrils are present in abundance in the complete absence of fibrillin-2 molecules. Increased fibrillin-1 production may compensate for loss of fibrillin-2 during development in order to underpin elastic fibre formation, but in that case, subtle structural and functional differences reflecting the presence of this alternative isoform, are predicted.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mice
Mice carrying the sy, syfp and syfp-2J mutations were obtained from the Jackson Laboratory and breeding colonies established. sy is maintained on a B6C3Fe background, syfp on a C3HeB/FeHu background and the syfp-2J on a C.B10-Hsb/LiMcdJ background. All experiments were performed in accordance with the Animals (Scientific Procedures) Act, UK, 1986. For skeletal analysis, young adult mice were killed, skinned, eviscerated and fixed in 100% ethanol for 24 h prior to processing as detailed by Wallin et al. (48).
Genetic analysis
DNA was extracted from +/sy, sy/sy, +/syfp, syfp/syfp, +/syfp-2J, syfp-2J/syfp-2J, A/J, AKR/J, BALB/c, C3H, C57BL/6J, CAST/Ei, CBA/J, DBA/2J and SPRET/Ei mice by standard methods. Total RNA was extracted according to the method of Chomczynski and Saachi (49). PCR and RT–PCR analyses were performed and analysed as detailed previously (50). For deletion analysis, oligonucleotide primers representing parts of the following genes, ESTs and STSs: Lox, Adrb2, Csf1R, Pou4f3, Cdo, Pdea, D19292, MHAa29d4.seq, 21.MHAa93f9.seq, Fbn2, as well as the previously reported markers Dtd, Tcof1, Atq1, Slc12a2, Hsst1, Lmnb1, Npybr, Hsp74, Dtn, D18Ertd5e and D18Mit123 were used to amplify +/sy, sy/sy, +/syfp, syfp/syfp, +/syfp-2J, syfp-2J/syfp-2J and wild-type DNA. Where there was no apparent band amplified from sy/sy DNA, the experiment was repeated including control (non-deleted) primers in the homozygote sample to ensure that the PCR had worked. The primer sequences used for the analysis of Fbn2 were: 3' UTR-F 5'-GTG AGC CTT CTT CCT GTT AG-3', 3' UTR-R 5'-CAT TCA GCT CCG CAA GAC TT-3'; exon 1-F 5'-TTG TGT CTC CAG CCC TAC TT-3', exon 1-R 5'-TCG GAG TAT TTC CTG CTG TC-3'; and for RT–PCR analysis of nucleotides 4259–5032; F 5'-CAT AAA CGC CCA GTG TGT CA-3', R 5'-TTC ACA GAT GCG GGT TTC CT-3', and nucleotides 4912–5621; F 5'-GCT TTA GAC CGA ACC CCA TC-3', R 5'-CGT TGG GGG AGA GTT TGA AG-3'.
For sequence analysis, PCR products were cloned into pBluescript and sequenced via the dideoxy chain termination method using dye primer chemistry. At least three independent templates were sequenced for each DNA sample.
SDS–PAGE and western blotting
Skin, heart/lungs and spleen were extracted overnight using a reductive saline protocol (51). Solubilized fractions were dialysed exhaustively against distilled H2O for 72 h, freeze-dried, then taken up in NuPAGE LDA sample buffer (Invitrogen) in the presence of 50 mM dithiothreitol, and boiled for 10 min prior to SDS–PAGE analysis. Samples were spun down at 28 000 g then loaded onto 3–8% Tris-acetate gels (Invitrogen). Gels were blotted onto BIOTRACE NT 45 µM membrane (Pall Gelman Laboratories), then incubated at 4°C overnight with TBST (0.05 M Tris–HCl pH 7.4, containing 0.15 M NaCl and 0.5% Tween-20) supplemented with 5% milk protein. Blots were incubated with primary antibody for 3 h at 20°C, then washed three times with TBST. Secondary antibody incubations were for 45 min at 20°C. Visualization of epitopes was by enhanced chemiluminescence (Amersham Pharmacia Biotech). The polyclonal primary antibody used was raised to a recombinant peptide encoding the glycine-rich region of fibrillin-2 expressed in a bacterial system (gift of Dr R.P. Mecham, St Louis, MO). The secondary antibody used was a goat anti-mouse IgG peroxidase conjugate (DAKO).
Microfibril analysis
Intact fibrillin-rich microfibrils were extracted from skin samples from adult +/syfp-2J and syfp-2J/syfp-2J littermates, from wild-type animals and from lung tissue from +/syfp and syfp/syfp mice. Samples were incubated with purified collagenase in the presence of freshly prepared protease inhibitors (2 mM PMSF, 5 mM N-ethylmaleimide), as described previously (34). Extracts were size fractionated on a Sepharose CL-2B column. The void volume contained abundant, extensive fibrillin-rich microfibrils, which were visualized after rotary shadowing electron microscopy and after negative staining with 1% uranyl acetate pH 4.5.
Domain modelling
The amino acid sequence of the fourth TB module from mouse fibrillin-2 was used to interrogate the Research Collaboratory for Structural Bioinformatics Protein Database (PDB) using the advanced BLAST 2.0 search (52). Sequences resulting from this search included the sixth TB module from human fibrillin-1 (PBD entry 1APJ) with 32% sequence identity and this structure was used as the starting point for homology modelling. All molecular modelling was performed on an R10 000 O2 Silicon Graphics workstation using QUANTA and CHARMm 23.2 programs. The three-dimensional model of the TB4 domain from fibrillin-2 was built based on the co-ordinates of the peptide backbone and completely conserved residues from the TB6 domain of fibrillin-1. The remaining side chains were built in the Protein Design module using the Ponder and Richards rotamer library. There was one insertion in the fibrillin TB4 domain compared with the fibrillin-1 TB6 structure; this was five residues in the loop between two ß-strands and contained an Arg–Gly–Asp (RGD) motif. The fibrillin-2 TB4 domain was energy minimized using steepest descents followed by the conjugate gradient algorithm to convergence removing bad steric and electrostatic contacts. The Protein Health module in QUANTA was used to check the integrity of the model using a Ramachandran plot, and to identify buried hydrophilic or exposed hydrophobic residues and close contacts.
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ACKNOWLEDGEMENTS |
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We thank Ken Johnson and Sue Cook for kindly providing the mutant mice, Bob Mecham for the anti-fibrillin-2 antibody and Amanda Morgan for technical assistance The work was supported by the BBSRC (34/G11908), Wellcome Trust (050591 and 058423), the MRC, European Commission contract CT97-2715, and Defeating Deafness.
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FOOTNOTES |
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+ These authors contributed equally to this work
To whom correspondence should be addressed. Tel: +44 161 275 5620; Fax: +44 161 275 5620; Email: mike.dixon@man.ac.uk
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