Mutations in fibrillin-1 cause congenital scleroderma: stiff skin syndrome.

The predisposition for scleroderma, defined as fibrosis and hardening of the skin, is poorly understood. We report that stiff skin syndrome (SSS), an autosomal dominant congenital form of scleroderma, is caused by mutations in the sole Arg-Gly-Asp sequence-encoding domain of fibrillin-1 that mediates integrin binding. Ordered polymers of fibrillin-1 (termed microfibrils) initiate elastic fiber assembly and bind to and regulate the activation of the profibrotic cytokine transforming growth factor-beta (TGFbeta). Altered cell-matrix interactions in SSS accompany excessive microfibrillar deposition, impaired elastogenesis, and increased TGFbeta concentration and signaling in the dermis. The observation of similar findings in systemic sclerosis, a more common acquired form of scleroderma, suggests broad pathogenic relevance.

The molecular genetics of Marfan syndrome and related disorders.

Marfan syndrome (MFS), a relatively common autosomal dominant hereditary disorder of connective tissue with prominent manifestations in the skeletal, ocular, and cardiovascular systems, is caused by mutations in the gene for fibrillin-1 (FBN1). The leading cause of premature death in untreated individuals with MFS is acute aortic dissection, which often follows a period of progressive dilatation of the ascending aorta. Recent research on the molecular physiology of fibrillin and the pathophysiology of MFS and related disorders has changed our understanding of this disorder by demonstrating changes in growth factor signalling and in matrix-cell interactions. The purpose of this review is to provide a comprehensive overview of recent advances in the molecular biology of fibrillin and fibrillin-rich microfibrils. Mutations in FBN1 and other genes found in MFS and related disorders will be discussed, and novel concepts concerning the complex and multiple mechanisms of the pathogenesis of MFS will be explained.

Marfan syndrome caused by a mutation in FBN1 that gives rise to cryptic splicing and a 33 nucleotide insertion in the coding sequence.

We have studied a patient with Marfan syndrome whose mutation was not detected by heteroduplex analysis. Primary cultured patient fibroblasts were metabolically labelled and found to secrete fibrillin-1 defectively when compared with an age-matched control. Sequencing of patient cDNA, isolated by reverse transcription-polymerase chain reaction of patient fibroblast RNA, detected a 33-bp insertion. The reading frame of the mutant allele was maintained and predicted the insertion of 11 amino acids at the beginning of calcium-binding epidermal growth factor-like domain 29. Direct sequencing of genomic DNA detected a heterozygous G+1-->A transversion in intron 46 of FBN1. The 11 amino acid insertion was the consequence of the usage of a cryptic splice site 33-bp downstream of the mutation. This is the first reported case of a splicing defect in FBN1 leading to the production of a full-length fibrillin-1 transcript containing a large amino acid insertion.

Solution structure of the LDL receptor EGF-AB pair: a paradigm for the assembly of tandem calcium binding EGF domains.

BACKGROUND: From the observed structure and sequence of a pair of calcium binding (cb) epidermal growth factor-like (EGF) domains from human fibrillin-1, we proposed that many tandem cbEGF domains adopt a conserved relative conformation. The low-density lipoprotein receptor (LDLR), which is functionally unrelated to fibrillin-1, contains a single pair of EGF domains that was chosen for study in the validation of this hypothesis. The LDLR is the protein that is defective in familial hypercholesterolaemia, a common genetic disorder that predisposes individuals to cardiovascular complications and premature death. RESULTS: Here, we present the solution structure of the first two EGF domains from the LDL receptor, determined using conventional NMR restraints and residual dipolar couplings. The cbEGF domains have an elongated, rod-like arrangement, as predicted. The new structure allows a detailed assessment of the consequences of mutations associated with familial hypercholesterolaemia to be made. CONCLUSIONS: The validation of the conserved arrangement of EGF domains in functionally distinct proteins has important implications for structural genomics, since multiple tandem cbEGF pairs have been identified in many essential proteins that are implicated in human disease. Our results provide the means to use homology modeling to probe structure-function relationships in this diverse family of proteins and may hold the potential for the design of novel diagnostics and therapies in the future.

Molecular analysis of the epidermal growth factor-like short consensus repeat domain-mediated protein-protein interactions: dissection of the CD97-CD55 complex.

Epidermal growth factor-like (EGF) and short consensus repeat (SCR) domains are commonly found in cell surface and soluble proteins that mediate specific protein-protein recognition events. Unlike the immunoglobulin (Ig) superfamily, very little is known about the general properties of intermolecular interactions encoded by these common modules, and in particular, how specificity of binding is achieved. We have dissected the binding of CD97 (a member of the EGF-TM7 family) to the complement regulator CD55, two cell surface modular proteins that contain EGF and SCR domains, respectively. We demonstrate that the interaction is mediated solely by these domains and is characterized by a low affinity (86 microm) and rapid off-rate (at least 0.6 s(-1)). The interaction is Ca(2+) -dependent but is unaffected by glycosylation of the EGF domains. Using biotinylated multimerized peptides in cell binding assays and surface plasmon resonance, we show that a CD97-related EGF-TM7 molecule (termed EMR2), differing by only three amino acids within the EGF domains, binds CD55 with a K(D) at least an order of magnitude weaker than that of CD97. These results suggest that low affinity cell-cell interactions may be a general feature of highly expressed cell surface proteins and that specificity of SCR-EGF binding can be finely tuned by a small number of amino acid changes on the EGF module surface.

A G1127S change in calcium-binding epidermal growth factor-like domain 13 of human fibrillin-1 causes short range conformational effects.

Human fibrillin-1, an extracellular matrix glycoprotein, has a modular organization that includes 43 calcium-binding epidermal growth factor-like (cbEGF) domains arranged as multiple tandem repeats. A missense mutation that changes a highly conserved glycine to serine (G1127S) has been identified in cbEGF13, which results in a variant of Marfan syndrome, a connective tissue disease. Previous experiments on isolated cbEGF13 and a cbEGF13-14 pair indicated that the G1127S mutation caused defective folding of cbEGF13 but not cbEGF14. We have used limited proteolysis methods and two-dimensional NMR spectroscopy to identify the structural consequences of this mutation in a covalently linked cbEGF12-13 pair and a cbEGF12-14 triple domain construct. Protease digestion studies of the cbEGF12-13 G1127S mutant pair indicated that both cbEGF12 and 13 retained similar calcium binding properties and thus tertiary structure to the normal domain pair, because all identified cleavage sites showed calcium-dependent protection from proteolysis. However, small changes in the conformation of cbEGF13 G1127S, revealed by the presence of a new protease-sensitive site and comparative two-dimensional NOESY data, suggested that the fold of the mutant domain was not identical to the wild-type, but was native-like. Additional cleavage sites identified in cbEGF12-14 G1127S indicated further subtle changes within the mutant domain but not the flanking domains. We have concluded the following in this study. (i) Covalent linkage of cbEGF12 preserves the native-like fold of cbEGF13 G1127S and (ii) conformational effects introduced by G1127S are localized to cbEGF13. This study demonstrates that missense mutations in fibrillin-1 cbEGF domains can cause short range structural effects in addition to long range effects previously observed with a E1073K mutation in cbEGF12.

Fibrillin-1, a calcium binding protein of extracellular matrix.

Fibrillin-1 is a large extracellular matrix glycoprotein which assembles to form 10-12 nm microfibrils in extracellular matrix. Mutations in the human fibrillin-1 gene (FBN-1) cause the connective tissue disease Marfan syndrome and related disorders, which are characterised by defects in the skeletal, cardiovascular and ocular systems of the body. Fibrillin-1 has a striking modular organisation which is dominated by multiple tandem repeats of the calcium binding epidermal growth factor-like (cbEGF) domain. This review focuses on recent studies which have investigated the structural and functional role of calcium binding to cbEGF domains in fibrillin-1 and 10-12 nm microfibrils.

Fibrillin: from domain structure to supramolecular assembly.

In the last 5 years, significant progress has been made in understanding the structure and function of all the major domains composing the fibrillins. A previous review [Meth. Enzymol. 245 (1994), 29] focused on the isolation of fibrillin monomers and fibrillin-containing polymers (microfibrils). In this article, information gained from recent studies which have further elucidated molecular structure and investigated effects of mutations on structural and functional properties will be summarized. In addition, studies of functional domains in fibrillins which may be important in assembling microfibrils will be discussed. Throughout this review, the authors have attempted to identify areas of research which have been controversial. In the conclusion, we raise important questions which remain unresolved.

Molecular effects of calcium binding mutations in Marfan syndrome depend on domain context.

Mutations in the human fibrillin-1 (FBN-1) gene cause Marfan syndrome (MFS), an autosomal dominant disease of connective tissue. Fibrillin-1, a 350 kDa extracellular calcium binding protein, is a major structural component of 10-12 nm microfibrils and consists predominantly of two repeated module types: the calcium binding epidermal growth factor-like (cbEGF) domain and the transforming growth factor beta1 binding protein-like (TB) domain. A group of reported FBN-1 mutations is predicted to reduce calcium binding to cbEGF domains by removal of a side chain ligand for calcium. These mutations occur in two protein domain contexts, either in a cbEGF preceded by a TB domain or in a cbEGF preceded by another cbEGF domain. In this study we have used three proteases to probe structural changes caused by an N2144S MFS calcium binding mutation in a TB6-cbEGF32 and a cbEGF32-33 domain pair, and an N2183S mutation in the cbEGF32-33 pair. N-terminal sequence analysis of domain pairs digested in the presence and absence of calcium show that: (i) domain interactions between TB6 and cbEGF32 are calcium independent, despite the presence of a calcium binding site in cbEGF32; (ii) domain interactions between cbEGF32 and cbEGF33 are calcium dependent; and (iii) an N-->S mutation causes increased proteolytic susceptibility only when located in cbEGF33, consistent with a key role for interdomain calcium binding in rigidifying cbEGF domain linkages. These data demonstrate for the first time that the structural consequences of calcium binding mutations in fibrillin-1 cbEGF domains can be influenced by domain context.

Characterisation of fibrillin-1 cDNA clones in a human fibroblast cell line that assembles microfibrils.

Fibrillin-1 is a large extracellular glycoprotein which is a major structural component of 10-12 nm microfibrils. Defects in human fibrillin-1 give rise to the autosomal dominant connective tissue disease the Marfan syndrome and related disorders. Previous studies examining the biosynthesis and secretion of recombinant fibrillin-1 fragments have been performed in cell lines which do not assemble fibrillin into extracellular 10-12 nm microfibrils. Conflicting data have been obtained regarding N-terminal processing. In this study we have characterised a human fibroblast cell line MSU-1.1 which shows a similar endogenous fibrillin-1 pulse chase profile to primary human dermal fibroblasts and produces microfibrils. Expression of a approximately 50 kDa N-terminal recombinant peptide in MSU-1.1 resulted in efficient secretion of this peptide into conditioned media, N-terminal sequence analysis of the purified peptide identified 2 protease cleavage sites and a presumed signal peptidase site. Together these data identify the natural leader sequence of fibrillin-1 and the presence of two processing sites in the N-terminus of fibrillin-1. The identification of an N-terminal processing site in recombinant fibrillin-1 similar to that obtained in a previous study which used an HT1080 fibrosarcoma host cell line excludes defective N-terminal processing as the cause of the assembly defect in this cell line. A full length normal and mutant fibrillin cDNA (approximately 8.6 kb) was constructed and stable integration of each into MSU1.1 led to RNA transcription at approximately 5% of endogenous levels. This is the first report of transcription from the full length fibrillin-1 cDNA. The low levels of transcription achieved, suggest that additional upstream and downstream DNA sequence elements will be required for high levels of full length fibrillin-1 cDNA expression.

Backbone dynamics of a cbEGF domain pair in the presence of calcium.

Calcium binding (cb) epidermal growth factor-like (EGF) domains are found in a wide variety of extracellular proteins with diverse functions. In several proteins, including the fibrillins (1 and 2), the low-density lipoprotein receptor, the Notch receptor and related molecules, these domains are organised as multiple tandem repeats. The functional importance of calcium-binding by EGF domains has been underscored by the identification of missense mutations associated with defective calcium-binding, which have been linked to human diseases. Here, we present (15)N backbone relaxation data for a pair of cbEGF domains from fibrillin-1, the defective protein in the Marfan syndrome. The data were best fit using a symmetric top model, confirming the extended conformation of the cbEGF domain pair. Our data demonstrate that calcium plays a key role in stabilising the rigidity of the domain pair on the pico- to millisecond time-scale. Strikingly, the most dynamically stable region of the construct is centred about the domain interface. These results provide important insight into the properties of intact fibrillin-1, the consequences of Marfan syndrome causing mutations, and the ultrastructure of fibrillins and other extracellular matrix proteins.

A molecular phylogeny of warbling-finches (Poospiza): paraphyly in a neotropical Emberizid genus.

We investigated the phylogenetic relationships of 12 species within a single genus of neotropical passerine (Poospiza) using 849 bp (283 codons) of the cytochrome b mitochondrial gene. We further explored evolutionary affinities of these taxa using sequence from an additional 47 thraupine (tanagers) and 7 emberizine (sparrows and buntings) genera, members of the predominantly New World family Emberizidae. Poospiza have traditionally been considered part of the emberizine radiation. However, our analyses suggest that members of this genus are more closely related to some thraupine lineages than they are to the other neotropical emberizine genera included in our study (Atlapetes, Embernagra, Melopyrrha, Phrygilus, Saltatricula, Tiaris). Although member taxa are closely related, the genus Poospiza appears to be paraphyletic with representatives of 6 thraupine genera (Cnemoscopus, Cypsnagra, Hemispingus, Nephelornis, Pyrrhocoma, Thylpopsis) interspersed among four well-supported Poospiza clades. The majority of species within this Poospiza-thraupine clade have geographic ranges that are exclusive to, or partially overlap with, the Andes Mountains. It is probable that these mountains have played an important role in driving cladogenesis within this group. Sequence divergence (transversions only; mean 4.7+/-1.3%) within the clade suggests that much of this diversification occurred within the late Miocene and Pliocene, a period coincident with major orogenic activity in central-western South America.

EGF-like domain calcium affinity modulated by N-terminal domain linkage in human fibrillin-1.

Calcium binding epidermal growth factor-like domains (cbEGFs) are present in many extracellular proteins, including fibrillin-1, Notch-3, protein S, factor IX and the low density lipoprotein (LDL) receptor, which perform a diverse range of functions. Genetic mutations that cause amino acid changes within these proteins have been linked to the Marfan syndrome (MFS), CADASIL, protein S deficiency, haemophilia B and familial hypercholesterolaemia, respectively. A number of these mutations disrupt calcium binding to cbEGFs, emphasising the critical functional role of calcium in these proteins. We have determined the calcium binding affinity of two sites within a cbEGF pair (cbEGF12-13) from human fibrillin-1 using two-dimensional nuclear magnetic resonance (NMR) and fluorescence techniques. Fibrillin-1 is a mosaic protein containing 43 cbEGF domains, mainly arranged as tandem repeats. Our results show that the cbEGF13 site in the cbEGF12-13 pair possesses the highest calcium affinity of any cbEGF investigated from fibrillin-1. A comparative analysis of these and previously reported calcium binding data from fibrillin-1 demonstrate that the affinity of cbEGF13 is enhanced more than 70-fold by the linkage of an N-terminal cbEGF domain. In contrast, comparison of calcium binding by cbEGF32 in isolation relative to when linked to a transforming growth factor beta-binding protein-like domain (TB6-cbEGF32) reveals that the same enhancement is not observed for this heterologous domain pair. Taken together, these results indicate that fibrillin-1 cbEGF Ca2+ affinity can be significantly modulated by the type of domain which is linked to its N terminus. The cbEGF12-13 pair is located within the longest contiguous section of cbEGFs in fibrillin-1, and a number of mutations in this region are associated with the most severe neonatal form of MFS. The affinities of cbEGF domains 13 and 14 in this region are substantially higher than in the C-terminal region of fibrillin-1. This increased affinity may be important for fibrillin assembly into 10-12 nm connective tissue microfibrils and/or may contribute to the biomechanical properties of the microfibrillar network.

Defective calcium binding to fibrillin-1: consequence of an N2144S change for fibrillin-1 structure and function.

Fibrillin-1 is a major structural component of 10-12 nm connective tissue microfibrils and has a modular organisation that includes 43 calcium binding epidermal growth factor-like (cbEGF) domains and seven transforming growth factor beta-binding protein-like (TB) domains. Mutations in the fibrillin-1 (FBN1) gene cause the Marfan syndrome (MFS) and related connective tissue disorders. We have previously investigated an N2144S change, identified in a MFS patient, which removes one of the key calcium binding ligands within cbEGF domain 32. In this study the structural consequences of the N2144S amino acid change for the folding and calcium binding properties of mutant and wild-type TB6-cbEGF32 and cbEGF32-33 domain pairs have been analysed by nuclear magnetic resonance. The presence of an N2144S substitution does not alter the native fold of either the TB6 domain, or cbEGF domains 32 and 33. Comparison of calcium dissociation constants measured for the wild-type and mutant pairs shows that: (i) the affinity of cbEGF32 is weakly enhanced by N-terminal linkage of TB6 relative to cbEGF32 in isolation; (ii) the affinity of cbEGF32 is approximately ninefold decreased by the N2144S substitution in the TB-cbEGF pair; and (iii) reduced affinity of cbEGF32 does not result in lower affinity of cbEGF33 for calcium. Together, these data suggest that the TB6-cbEGF32 linkage is flexible and the structural effect of the mutation is localised to the interdomain linkage. We have also investigated the effect of defective calcium binding to cbEGF32 on fibrillin-1 produced by N2144S MFS fibroblasts. 35S-pulse-chase analysis shows that the N2144S substitution does not detectably affect fibrillin-1 biosynthesis, rate of secretion or processing. Deposition of reducible fibrillin-1 into the extracellular matrix was also unaffected. The implications of these results for the assembly and properties of the microfibril are discussed.

Molecular analysis of eight mutations in FBN1.

Mutations in the gene encoding extracellular glycoprotein fibrillin-1 (FBN1) cause Marfan syndrome (MFS) and other related connective tissue disorders. In this study, eight mutations have been detected in MFS patients by heteroduplex analysis. These comprise two missense mutations, C1835Y and C2258Y in calcium-binding epidermal growth factor-like domains, two nonsense mutations, R1541X and R2394X in transforming growth factor beta1-binding protein-like domains, one splice site mutation, which has been detected previously, and three small insertions or deletions resulting in a frameshift. Fibroblast cells have been established from seven of the MFS patients and the biochemical effects of the mutations on fibrillin-1 synthesis and secretion assessed by pulse-chase analysis. Each cysteine mutation resulted in the delayed secretion of fibrillin-1 and both nonsense and frameshift mutations caused reduced levels of synthesis and/or deposition of fibrillin-1. Indirect immunofluorescence and rotary shadowing electron microscopy analysis of fibrillin microfibrils revealed no major differences between normal and patient samples. We discuss the relative merits of the biochemical techniques used in this study.

Effects of proline cis-trans isomerization on TB domain secondary structure.

The transforming growth factor beta (TGF-beta) binding protein-like (TB) domain is found principally in proteins localized to extracellular matrix fibrils, including human fibrillin-1, the defective protein in the Marfan syndrome. Analysis of the nuclear magnetic resonance (NMR) data for the sixth TB module from human fibrillin-1 has revealed the existence of two stable conformers that differ in the isomerization states of two proline residues. Unusually, the two isoforms do not readily interconvert and are stable on the time scale of milliseconds. We have computed independent structures of the major and minor conformers of TB6 to assess how the domain fold adjusts to incorporate alternatively cis- or trans-prolines. Based on previous observations, it has been suggested that multiple conformers can only be accommodated in flexible regions of protein structure. In contrast, P22, which exists in trans in the major form and cis in the minor form of TB6, is in a rigid region of the domain, which is confirmed by backbone dynamics measurements. Overall, the structures of the major and minor conformers are similar. However, the secondary structure topologies of the two forms differ as a direct consequence of the changes in proline conformation.

Metal ion dependency of microfibrils supports a rod-like conformation for fibrillin-1 calcium-binding epidermal growth factor-like domains.

The effects of the removal and replacement of divalent cations on the ultrastructure of 10 to 12 nm fibrillin-1-containing microfibrils have been studied, in order to investigate the conformation of fibrillin-1 calcium-binding epidermal growth factor-like (cbEGF-like) domains within the microfibril. The NMR structure of a covalently linked pair of cbEGF-like domains from fibrillin-1 recently identified a rigid rod-like conformation for the domain pair stabilised by interdomain calcium binding. This suggested that tandem arrays of fibrillin-1 cbEGF-like domains may adopt an extended conformation within a microfibril. If correct, then removal of bound calcium from fibrillin-1 would be expected to increase the flexibility of each cbEGF-like interdomain linkage, resulting in a decrease in the length of the interbead region of the microfibril (and thus a decrease in bead to bead periodicity), a concomitant increase in its diameter, and an overall increase in the flexibility of the microfibril. Our results show that removal of calcium by treatment with EGTA causes a large alteration of the microfibril structure, resulting in microfibrils with a reduced beaded periodicity, a disrupted interbead region and an increased overall flexibility. These effects are readily reversible by the re-addition of calcium (in the form of CaCl2), but not by the addition of magnesium (MgCl2). This is consistent with conformational changes in cbEGF-like domains causing the major structural effects on the microfibril. These results provide the first direct experimental evidence to support an extended rod-like conformation for multiple tandem repeats of fibrillin-1 cbEGF-like domains within the microfibril, as predicted by the NMR structure of an isolated fibrillin-1 cbEGF-like domain pair.

A Gly --> Ser change causes defective folding in vitro of calcium-binding epidermal growth factor-like domains from factor IX and fibrillin-1.

The calcium-binding epidermal growth factor-like (cbEGF) domain is a common motif found in extracellular proteins. A mutation that changes a highly conserved Gly residue to Ser in this domain has been identified both in the factor IX (FIX) and fibrillin-1 genes, where it is associated with relatively mild variants of hemophilia B and Marfan syndrome, respectively. We have investigated the structural consequences in vitro of this amino acid change when introduced into single cbEGF domains from human FIX (G60S) and human fibrillin-1 (G1127S), and a covalently linked pair of cbEGF domains from fibrillin-1. High pressure liquid chromatography analysis, mass spectrometry, and 1H NMR analysis demonstrate that wild-type cbEGF domains purified in the reduced form and refolded in vitro adopt the native fold. In contrast, the Gly --> Ser change causes defective folding of FIX and fibrillin-1 cbEGF domains. However, in the case of the factor IX mutant domain, a Ca2+-dependent change in conformation, identified by NMR in a proportion of the refolded material, suggests that some material refolds to a native-like structure. This is consistent with enzyme-linked immunosorbent assay analysis of FIX G60S from a hemophilia B patient Oxford d2, which demonstrates that the mutant protein is partially recognized by a monoclonal antibody specific for this region of FIX. NMR analysis of a covalently linked pair of fibrillin cbEGF domains demonstrates that the C-terminal domain adopts the native epidermal growth factor fold, despite the fact that the adjacent mutant domain is misfolded. The implications of these results for disease pathogenesis are discussed.

NMR analysis of cbEGF domains gives new insights into the structural consequences of a P1148A substitution in fibrillin-1.

Fibrillin-1 is a modular glycoprotein and a major component of the 10-12 nm microfibrils of the extracellular matrix. Mutations in the fibrillin-1 (FBN 1) gene result in the connective tissue disease the Marfan syndrome (MFS) and related disorders. The calcium binding EGF-like (cbEGF) domain is the predominant structural motif of the protein and >70% of mutations leading to MFS disrupt this domain. A missense mutation which changes a proline to alanine (P1148A) in cbEGF domain 13 has been associated with a number of fibrillin disorders including MFS and Shprintzen-Goldberg syndrome. However, it has also been described as a polymorphism. In this study comparative NMR analyses on wild-type and mutant forms of covalently-linked fibrillin cbEGF domain pairs have been performed to investigate the structural consequences of this substitution. A comparison of the two-dimensional NOESY spectra of the wild-type and mutant forms of cbEGF domains 12 & 13 and cbEGF domains 13 & 14 indicated that the proline to alanine amino acid change does not introduce a significant structural defect into cbEGF domain 13 or the adjacent domains and most likely represents a polymorphism. These results demonstrate how, in the case of a protein with a well defined domain organisation such as fibrillin-1, comparative NMR analyses can be used to substantiate genetic evidence for the polymorphic status of an amino acid.