Microenvironmental regulation by fibrillin-1

Fibrillin-1 is a ubiquitous extracellular matrix molecule that sequesters latent growth factor complexes. A role for fibrillin-1 in specifying tissue microenvironments has not been elucidated, even though the concept that fibrillin-1 provides extracellular control of growth factor signaling is currently appreciated. Mutations in FBN1 are mainly responsible for the Marfan syndrome (MFS), recognized by its pleiotropic clinical features including tall stature and arachnodactyly, aortic dilatation and dissection, and ectopia lentis. Each of the many different mutations in FBN1 known to cause MFS must lead to similar clinical features through common mechanisms, proceeding principally through the activation of TGFβ signaling. Here we show that a novel FBN1 mutation in a family with Weill-Marchesani syndrome (WMS) causes thick skin, short stature, and brachydactyly when replicated in mice. WMS mice confirm that this mutation does not cause MFS. The mutation deletes three domains in fibrillin-1, abolishing a binding site utilized by ADAMTSLIKE-2, -3, -6, and papilin. Our results place these ADAMTSLIKE proteins in a molecular pathway involving fibrillin-1 and ADAMTS-10. Investigations of microfibril ultrastructure in WMS humans and mice demonstrate that modulation of the fibrillin microfibril scaffold can influence local tissue microenvironments and link fibrillin-1 function to skin homeostasis and the regulation of dermal collagen production. Hence, pathogenetic mechanisms caused by dysregulated WMS microenvironments diverge from Marfan pathogenetic mechanisms, which lead to broad activation of TGFβ signaling in multiple tissues. We conclude that local tissue-specific microenvironments, affected in WMS, are maintained by a fibrillin-1 microfibril scaffold, modulated by ADAMTSLIKE proteins in concert with ADAMTS enzymes.     

ADAMTS proteins as modulators of microfibril formation and function

The ADAMTS (a disintegrin-like and metalloproteinase domain with thrombospondin-type 1 motifs) protein superfamily includes 19 secreted metalloproteases and 7 secreted ADAMTS-like (ADAMTSL) glycoproteins. The possibility of functional linkage between ADAMTS proteins and fibrillin microfibrils was first revealed by a human genetic consilience, in which mutations in ADAMTS10, ADAMTS17, ADAMTSL2 and ADAMTSL4 were found to phenocopy rare genetic disorders caused by mutations affecting fibrillin-1 (FBN1), the major microfibril component in adults. The manifestations of these ADAMTS gene disorders in humans and animals suggested that they participated in the structural and regulatory roles of microfibrils. Whereas two such disorders, Weill–Marchesani syndrome 1 and Weill–Marchesani-like syndrome involve proteases (ADAMTS10 and ADAMTS17, respectively), geleophysic dysplasia and isolated ectopia lentis in humans involve ADAMTSL2 and ADAMTSL4, respectively, which are not proteases. In addition to broadly similar dysmorphology, individuals affected by Weill–Marchesani syndrome 1, Weill–Marchesani-like syndrome or geleophysic dysplasia each show characteristic anomalies suggesting molecule-, tissue-, or context-specific functions for the respective ADAMTS proteins. Ectopia lentis occurs in each of these conditions except geleophysic dysplasia, and is due to a defect in the ciliary zonule, which is predominantly composed of FBN1 microfibrils. Together, this strongly suggests that ADAMTS proteins are involved either in microfibril assembly, stability, and anchorage, or the formation of function-specific supramolecular networks having microfibrils as their foundation. Here, the genetics and molecular biology of this subset of ADAMTS proteins is discussed from the perspective of how they might contribute to fully functional or function-specific microfibrils.     

In Vivo Studies of Mutant Fibrillin-1 Microfibrils

In humans, mutations in fibrillin-1 result in a variety of genetic disorders with distinct clinical phenotypes. While most of the known mutations in fibrillin-1 cause Marfan syndrome, a number of other mutations lead to clinical features unrelated to Marfan syndrome. Pathogenesis of Marfan syndrome is currently thought to be driven by mechanisms due to haploinsufficiency of wild-type fibrillin-1. However, haploinsufficiency-driven mechanisms cannot explain the distinct phenotypes found in other fibrillinopathies. To test the hypothesis that mutations in fibrillin-1 cause disorders through primary effects on microfibril structure, two different mutations were generated in Fbn1 in mice. One mutation leads to a truncated fibrillin-1 molecule that is tagged with green fluorescent protein, allowing visualization of mutant fibrillin-1 incorporated into microfibrils. In heterozygosity, these mutant mice demonstrate progressive fragmentation of the aortic elastic lamellae and also display fragmentation of microfibrils in other tissues. Fibrillin-2 epitopes are also progressively revealed in these mice, suggesting that fibrillin-2 immunoreactivity can serve as a marker for microfibril degradation. In contrast, a second mutation (in-frame deletion of the first hybrid domain) in fibrillin-1 results in stable microfibrils, demonstrating that fibrillin-1 molecules are not required to be in perfect register for microfibril structure and function and that the first hybrid domain is dispensable for microfibril assembly. Taken together, these results suggest that perturbation of microfibril structure may underlie one of the major features of the Marfan syndrome: fragmentation of aortic elastic lamellae.     

Fibrillin-1 Mutations Causing Weill-Marchesani Syndrome and Acromicric and Geleophysic Dysplasias Disrupt Heparan Sulfate Interactions

The extracellular glycoprotein fibrillin-1 forms microfibrils that act as the template for elastic fibers. Most mutations in fibrillin-1 cause Marfan syndrome with severe cardiovascular and ocular symptoms, and tall stature. This is in contrast to mutations within a heparin-binding TB domain (TB5), which is downstream of the arg-gly-asp cell adhesion domain, which can cause Weill-Marchesani syndrome (WMS) or Acromicric (AD) and Geleophysic Dysplasias (GD). WMS is characterized by short limbs, joint stiffness and ocular defects, whilst fibrillin-1 AD and GD have severe short stature, joint defects and thickened skin. We previously showed that TB5 binds heparin. Here, we show that the corresponding region of fibrillin-2 binds heparin very poorly, highlighting a novel functional difference between the two isoforms. This finding enabled us to map heparin/heparan sulfate binding to two sites on fibrillin-1 TB5 using a mutagenesis approach. Once these sites were mapped, we were able to investigate whether disease-causing mutations in this domain disrupt binding to HS. We show that a WMS deletion mutant, and five AD and GD point mutants all have disrupted heparin binding to TB5. These data provide insights into the biology of fibrillins and the pathologies of WMS, AD and GD.     

Microfibril Structure Masks Fibrillin-2 in Postnatal Tissues

Fibrillin microfibrils are polymeric structures present in connective tissues. The importance of fibrillin microfibrils to connective tissue function has been demonstrated by the multiple genetic disorders caused by mutations in fibrillins and in microfibril-associated molecules. However, knowledge of microfibril structure is limited, largely due to their insolubility. Most previous studies have focused on how fibrillin-1 is organized within microfibril polymers. In this study, an immunochemical approach was used to circumvent the insolubility of microfibrils to determine the role of fibrillin-2 in postnatal microfibril structure. Results obtained from studies of wild type and fibrillin-1 null tissues, using monoclonal and polyclonal antibodies with defined epitopes, demonstrated that N-terminal fibrillin-2 epitopes are masked in postnatal microfibrils and can be revealed by enzymatic digestion or by genetic ablation of Fbn1. From these studies, we conclude that fetal fibrillin polymers form an inner core within postnatal microfibrils and that microfibril structure evolves as growth and development proceed into the postnatal period. Furthermore, documentation of a novel cryptic site present in EGF4 in fibrillin-1 underscores the molecular complexity and tissue-specific differences in microfibril structure.     

The fibrillin microfibril scaffold: A niche for growth factors and mechanosensation?

The fibrillins, large extracellular matrix molecules, are polymerized to form “microfibrils.” The fibrillin microfibril scaffold is populated by microfibril-associated proteins and by growth factors, which are likely to be latent. The scaffold, associated proteins, and bound growth factors, together with cellular receptors that can sense the microfibril matrix, constitute the fibrillin microenvironment. Activation of TGFβ signaling is associated with the Marfan syndrome, which is caused by mutations in fibrillin-1. Today we know that mutations in fibrillin-1 cause the Marfan syndrome as well as Weill-Marchesani syndrome (and other acromelic dysplasias) and result in opposite clinical phenotypes: tall or short stature; arachnodactyly or brachydactyly; joint hypermobility or stiff joints; hypomuscularity or hypermuscularity. We also know that these different syndromes are associated with different structural abnormalities in the fibrillin microfibril scaffold and perhaps with specific cellular receptors (mechanosensors). How does the microenvironment, framed by the microfibril scaffold and populated by latent growth factors, work? We must await future investigations for the molecular and cellular mechanisms that will answer this question. However, today we can appreciate the importance of the fibrillin microfibril niche as a contextual environment for growth factor signaling and potentially for mechanosensation.     

Fibrillin-rich microfibrils: Structural determinants of morphogenetic and homeostatic events

Fibrillin-rich microfibrils are specialized extracellular matrix assemblies that endow connective tissues with mechanical stability and elastic properties, and that participate in the regulation of organ formation, growth and homeostasis. Their physiological importance is underscored by the complex spectrum of clinical manifestations associated with mutations of fibrillin-1 and fibrillin-2 in Marfan syndrome (MFS) and congenital contractural arachnodactyly, respectively. Early evidence suggested that fibrillin-1 mutations in MFS lead to loss of tissue integrity by perturbing microfibril assembly and function. Recent studies in genetically targeted mice have however revealed that fibrillin-1 and fibrillin-2 mutations perturb signaling events mediated by TGF-beta superfamily members. As such, these studies have established a new biological paradigm whereby fibrillin-rich microfibrils are structural networks that specify the local concentration and timely release of signaling molecules during morphogenesis and tissue remodeling. This review summarizes our current understanding of the role of fibrillin-rich microfibrils in development and disease, as well as exciting new applications in the clinical management of MFS and related connective tissue disorders.     

Homo- and heterotypic fibrillin-1 and -2 interactions constitute the basis for the assembly of microfibrils

Fibrillin-1 and fibrillin-2 constitute the backbone of extracellular filaments, called microfibrils. Fibrillin assembly involves complex multistep mechanisms to result in a periodical head-to-tail alignment in microfibrils. Impaired assembly potentially plays a role in the molecular pathogenesis of genetic disorders caused by mutations in fibrillin-1 (Marfan syndrome) and fibrillin-2 (congenital contractural arachnodactyly). Presently, the basic molecular interactions involved in fibrillin assembly are obscure. Here, we have generated recombinant full-length human fibrillin-1, and two overlapping recombinant polypeptides spanning the entire human fibrillin-2 in a mammalian expression system. Characterization by gel electrophoresis, electron microscopy after rotary shadowing, and reactivity with antibodies demonstrated correct folding of these recombinant polypeptides. Analyses of homotypic and heterotypic interaction repertoires showed N- to C-terminal binding of fibrillin-1, and of fibrillin-1 with fibrillin-2. The interactions were of high affinity with dissociation constants in the low nanomolar range. However, the N- and C-terminal fibrillin-2 polypeptides did not interact with each other. These results demonstrate that fibrillins can directly interact in an N- to C-terminal fashion to form homotypic fibrillin-1 or heterotypic fibrillin-1/fibrillin-2 microfibrils. This conclusion was further strengthened by double immunofluorescence labeling of microfibrils. In addition, the binding epitopes as well as the entire fibrillin molecules displayed very stable properties.     

A novel ADAMTS17 variant that causes Weill-Marchesani syndrome 4 alters fibrillin-1 and collagen type I deposition in the extracellular matrix

Weill-Marchesani syndrome (WMS) is a rare genetic disorder that affects the musculoskeletal system, the eye, and the cardiovascular system. Individuals with WMS present with short stature, joint contractures, thick skin, microspherophakia, small and dislocated lenses, and cardiac valve anomalies. WMS can be caused by recessive mutations in ADAMTS10 (WMS 1), ADAMTS17 (WMS 4), or LTBP2 (WMS 3), or by dominant mutations in fibrillin-1 (FBN1) (WMS 2); all genes encode secreted extracellular matrix (ECM) proteins. Individuals with WMS 4 due to ADAMTS17 mutations appear to have less severe cardiac involvement and present predominantly with the musculoskeletal and ocular features of WMS. ADAMTS17 is a member of the ADAMTS family of secreted proteases and directly binds to fibrillins. Here we report a novel pathogenic variant in ADAMTS17 that causes WMS 4 in an individual with short stature, brachydactyly, and small, spherical, and dislocated lenses. We provide biochemical and cell biological insights in the pathomechanisms of WMS 4, which also suggest potential biological functions for ADAMTS17. We show that the variant in ADAMTS17 prevents its secretion and we found intracellular accumulation of fibrillin-1 and collagen type I in patient-derived skin fibroblasts. In accordance, transmission electron microscopy revealed elastic fiber abnormalities, decreased collagen fibril diameters, and intracellular collagen accumulation in the dermis of the proband. Together, the data indicate a possible role for ADAMTS17 in the secretion of fibrillin-1 and collagen type I or in their early assembly in the pericellular matrix or the ECM.     

Proteomic analysis of fibrillin-rich microfibrils

MS has been used to investigate the composition of fibrillin-rich microfibrils from non-elastic and elastic tissues, and to compare fibrillin-1 tryptic fingerprints derived from whole zonules, microfibrils and recombinant fibrillin-1. In all microfibril preparations, fibrillin-1 was abundant and the only fibrillin isoform. MAGP-1 was the only other microfibril-associated molecule. gamma-Crystallin co-purified with zonular microfibrils, so this association may contribute to ciliary zonule anchorage to lens. Recombinant fibrillin-1 tryptic peptides mapped throughout the molecule and included virtually all predicted peptides except for those larger than 4.5 kDa, smaller than 600 Da or post-translationally modified. In contrast, fewer microfibril tryptic fibrillin-1 peptides were detected, although they were derived from domains throughout the molecule and included two peptides after the C-terminal furin processing site. Several microfibril-derived N- and C-terminal domains never yielded any peptides, while tryptic peptides from other domains yielded numerous peptides, suggesting that some tissue microfibril features are retained after trypsinisation. This first MS analysis of a purified extracellular matrix assembly has provided new insights into microfibril composition and fibrillin-1 organisation within them.     

Effects of fibrillin-1 degradation on microfibril ultrastructure

Current models of the elastic properties and structural organization of fibrillin-containing microfibrils are based primarily on microscopic analyses of microfibrils liberated from connective tissues after digestion with crude collagenase. Results presented here demonstrate that this digestion resulted in the cleavage of fibrillin-1 and loss of specific immunoreactive epitopes. The proline-rich region and regions near the second 8-cysteine domain in fibrillin-1 were easily cleaved by crude collagenase. Other sites that may also be cleaved during microfibril digestion and extraction were identified. In contrast to collagenase-digested microfibrils, guanidine-extracted microfibrils contained all fibrillin-1 epitopes recognized by available antibodies. The ultrastructure of guanidine-extracted microfibrils differed markedly from that of collagenase-digested microfibrils. Fibrillin-1 filaments splayed out, extending beyond the width of the periodic globular beads. Both guanidine-extracted and collagenase-digested microfibrils were subjected to extensive digestion by crude collagenase. Collagenase digestion of guanidine-extracted microfibrils removed the outer filaments, revealing a core structure. In contrast to microfibrils extracted from tissues, cell culture microfibrils could be digested into short units containing just a few beads. These data suggest that additional cross-links stabilize the long beaded microfibrils in tissues. Based on the microfibril morphologies observed after these experiments, on the crude collagenase cleavage sites identified in fibrillin-1, and on known antibody binding sites in fibrillin-1, a model is proposed in which fibrillin-1 molecules are staggered in microfibrils. This model further suggests that the N-terminal half of fibrillin-1 is asymmetrically exposed in the outer filaments, whereas the C-terminal half of fibrillin-1 is present in the interior of the microfibril.     

Versican interacts with fibrillin-1 and links extracellular microfibrils to other connective tissue networks.

Fibrillin-containing microfibrils are polymeric structures that are difficult to extract from connective tissues. Proteolytic digestion of tissues has been utilized to release microfibrils for study. Few of the molecules that connect microfibrils to other elements in the matrix have been identified. In this study, electron microscopic immunolocalization of anti-versican antibodies in tissues and in extracted microfibrils demonstrated that the C-terminal region of versican is found associated with fibrillin microfibrils. Extraction of microfibrils followed by treatment of microfibrils under dissociating conditions suggested that the versican C terminus is covalently bound to microfibrils. Binding assays using recombinant fibrillin-1 polypeptides and recombinant lectican lectin domains indicated that the versican lectin domain binds to specific fibrillin-1 polypeptides. The versican lectin domain also bound to molecules comigrating with authentic fibrillin-1 monomers in an assay using cell culture medium. In assays using microfibrils, the versican lectin domain demonstrated preferential binding compared with other lecticans. Binding was calcium-dependent. The binding site for versican in microfibrils is most likely within a region of fibrillin-1 between calcium-binding epidermal growth factor-like domains 11 and 21. Human mutations in this region can result in severe forms of the Marfan syndrome ("neonatal" Marfan syndrome). The connection between versican and fibrillin microfibrils may be functionally significant, particularly in cardiovascular tissues.     

MAGP-2 has multiple binding regions on fibrillins and has covalent periodic association with fibrillin-containing microfibrils

The interactions of microfibril-associated glycoprotein (MAGP)-2 have been investigated with fibrillins and fibrillin-containing microfibrils. Solid phase binding assays were conducted with recombinant fragments covering fibrillin-1 and most of fibrillin-2. MAGP-2, and its structure relative MAGP-1, were found to bind two fragments spanning the N-terminal half of fibrillin-1 and an N-terminal fragment of fibrillin-2. Blocking experiments indicated that MAGP-2 had a binding site(s) close to the N terminus of the fibrillin-1 molecule that was distinct from that for MAGP-1 and an additional, more central binding site(s) that may be shared by the two MAGPs. Immunogold labeling of developing nuchal ligament tissue showed that MAGP-2 had regular covalent and periodic (about 56 nm) association with fibrillin-containing microfibrils of elastic fibers in this tissue. Further analysis of isolated microfibrils indicated that MAGP-2 was attached at two points along the microfibril substructure, "site 1" on the "beads" and "site 2" at the "shoulder" of the interbead region close to where the two "arms" fuse. In contrast, MAGP-1 was located only on the beads. Comparison of the MAGP-2 binding data with known fibrillin epitope maps of the microfibrils showed that site 1 correlated with the N-terminal MAGP-2 binding region, and site 2 correlated with the second, more central, MAGP-2 binding region on the fibrillin-1 molecule. Of particular note, immunolabeling at site 2 was markedly decreased, relative to that at site 1, on extended microfibrils with bead-to-bead periods over 90 nm, suggesting that site 2 may move toward the beads when the microfibril is stretched. The study points to MAGP-2 being an integral component of some populations of fibrillin-containing microfibrils. Moreover, the identification of multiple MAGP-binding sequences on fibrillins supports the concept that MAGPs may function as molecular cross-linkers, stabilizing fibrillin monomers in folded conformation within or between the microfibrils, and thus MAGPs may be implicated in the modulation of the elasticity of these structures.     

Classical and neonatal Marfan syndrome mutations in fibrillin-1 cause differential protease susceptibilities and protein function

Mutations in fibrillin-1 give rise to Marfan syndrome (MFS) characterized by vascular, skeletal, and ocular abnormalities. Fibrillins form the backbone of extracellular matrix microfibrils in tissues including blood vessels, bone, and skin. They are crucial for regulating elastic fiber biogenesis and growth factor bioavailability. To compare the molecular consequences of mutations causing the severe neonatal MFS with mutations causing the milder classical MFS, we introduced representative point mutations from each group in a recombinant human fibrillin-1 fragment. Structural effects were analyzed by circular dichroism spectroscopy and analytical gel filtration chromatography. Proteolytic susceptibility was probed with non-physiological and physiological proteases, including plasmin, thrombin, matrix metalloproteinases, and cathepsins. All mutant proteins showed a similar gross secondary structure and no differences in heat stability as compared with the wild-type protein. Proteins harboring neonatal mutations were typically more susceptible to proteolytic cleavage compared with those with classical mutations and the wild-type protein. Proteolytic neo-cleavage sites were found both in close proximity and distant to the mutations, indicating small but significant structural changes exposing cryptic cleavage sites. We also report for the first time that cathepsin K and V cleave non-mutated fibrillin-1 at several domain boundaries. Compared with the classical mutations and the wild type, the group of neonatal mutations more severely affected the ability of fibrillin-1 to interact with heparin/heparan sulfate, which plays a role in microfibril assembly. These results suggest differential molecular pathogenetic concepts for neonatal and classical MFS including enhanced proteolytic susceptibility for physiologically relevant enzymes and loss of function for heparin binding.     

Molecular basis of elastic fiber formation. Critical interactions and a tropoelastin-fibrillin-1 cross-link

We have investigated the molecular basis of elastic fiber formation on fibrillin microfibrils. Binding assays revealed high affinity calcium-independent binding of two overlapping fibrillin-1 fragments (encoded by central exons 18-25 and 24-30) to tropoelastin, which, in microfibrils, map to an exposed "arms" feature adjacent to the beads. A further binding site within an adjacent fragment (encoded by exons 9-17) was within an eight-cysteine motif designated TB2 (encoded by exons 16 and 17). Binding to TB2 was ablated by the presence of N-terminal domains (encoded by exons 1-8) and reduced after deleting the proline-rich region. A novel transglutaminase cross-link between tropoelastin and fibrillin-1 fragment (encoded by exons 9-17) was localized by mass spectrometry to a sequence encoded by exon 17. The high affinity binding and cross-linking of tropoelastin to a central fibrillin-1 sequence confirm that this association is fundamental to elastic fiber formation. Microfibril-associated glycoprotein-1 showed calcium-dependent binding of moderate affinity to fibrillin-1 N-terminal fragment (encoded by exons 1-8), which localize to the beads. Microfibril-associated glycoprotein-1 thus contributes to microfibril organization but may also form secondary interactions with adjacent microfibril-bound tropoelastin.     

Microfibrils at basement membrane zones interact with perlecan via fibrillin-1

Mutational defects in fibrillin-rich microfibrils give rise to a number of heritable connective tissue disorders, generally termed microfibrillopathies. To understand the pathogenesis of these microfibrillopathies, it is important to elucidate the supramolecular composition of microfibrils and their interaction properties with extracellular matrix components. Here we demonstrate that the proteoglycan perlecan is an associated component of microfibrils typically close to basement membrane zones. Double immunofluorescence studies demonstrate colocalization of fibrillin-1, the major backbone component of microfibrils, with perlecan in fibroblast cultures as well as in dermal and ocular tissues. Double immunogold labeling further confirms colocalization of perlecan to microfibrils in various tissues at the ultrastructural level. Extraction studies revealed that perlecan is not covalently associated with microfibrils. High affinity interactions between fibrillin-1 and perlecan were found by kinetic binding studies with dissociation constants in the low nanomolar range. A detailed mapping study of the interaction epitopes by solid phase binding assays primarily revealed interactions of perlecan domains I and II with a central region of fibrillin-1. Analysis of perlecan null embryos showed less microfibrils at the dermal-epidermal junction as compared with wild-type littermates. The data presented indicate a functional significance for perlecan in anchoring microfibrils to basement membranes and in the biogenesis of microfibrils.     

Fibrillin-1 interactions with heparin. Implications for microfibril and elastic fiber assembly

Fibrillin-1 assembly into microfibrils and elastic fiber formation involves interactions with glycosaminoglycans. We have used BIAcore technology to investigate fibrillin-1 interactions with heparin and with heparin saccharides that are analogous to S-domains of heparan sulfate. We have identified four high affinity heparin-binding sites on fibrillin-1, localized three of these sites, and defined their binding kinetics. Heparin binding to the fibrillin-1 N terminus has particularly rapid kinetics. Hyaluronan and chondroitin sulfate did not interact significantly with fibrillin-1. Heparin saccharides with more than 12 monosaccharide units bound strongly to all four fibrillin-1 sites. Heparin did not inhibit fibrillin-1 N- and C-terminal interactions or RGD-dependent cell attachment, but heparin and MAGP-1 competed for binding to the fibrillin-1 N terminus, and heparin and tropoelastin competed for binding to a central fibrillin-1 sequence. By regulating these key interactions, heparin can profoundly influence microfibril and elastic fiber assembly.     

1H, 13C and 15N assignments of the four N-terminal domains of human fibrillin-1

Fibrillins are extracellular, disulphide-rich glycoproteins that form 10–12 nm diameter microfibrils in connective tissues. They are found in the majority of higher animals, from jellyfish to humans. Fibrillin microfibrils confer properties of elasticity and strength on connective tissue and regulate growth factor availability in the extracellular matrix (ECM). Mutations in FBN1, the human gene encoding the fibrillin-1 isoform, are linked to several inherited connective tissue disorders. The fibrillin-1 N-terminus forms many functionally-important interactions, both with other fibrillin molecules and various ECM components. In particular, the first four domains, the fibrillin unique N-terminal (FUN) and three epidermal growth factor (EGF)-like domains (FUN-EGF3), are implicated in microfibril assembly and growth factor sequestration. The structure of these domains, which comprise 134 residues, is unknown. We have produced a recombinant fragment corresponding to this region of human fibrillin-1. Here, we report ¹H, ¹³C and ¹⁵N resonance assignments of the FUN-EGF3 fragment. Assignments will facilitate structure determination, analysis of interdomain dynamics and the mapping of interaction surfaces.     

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.