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.     

The evolution of extracellular fibrillins and their functional domains

Fibrillins constitute the major backbone of multifunctional microfibrils in elastic and non-elastic extracellular matrices, and are known to interact with several binding partners including tropoelastin and integrins. Here, we study the evolution of fibrillin proteins. Following sequence collection from 39 organisms representative of the major evolutionary groups, molecular evolutionary genetics and phylogeny inference software were used to generate a series of evolutionary trees using distance-based and maximum likelihood methods. The resulting trees support the concept of gene duplication as a means of generating the three vertebrate fibrillins. Beginning with a single fibrillin sequence found in invertebrates and jawless fish, a gene duplication event, which coincides with the appearance of elastin, led to the creation of two genes. One of the genes significantly evolved to become the gene for present-day fibrillin-1, while the other underwent evolutionary changes, including a second duplication, to produce present-day fibrillin-2 and fibrillin-3. Detailed analysis of several sequences and domains within the fibrillins reveals distinct similarities and differences across various species. The RGD integrin-binding site in TB4 of all fibrillins is conserved in cephalochordates and vertebrates, while the integrin-binding site within cbEGF18 of fibrillin-3 is a recent evolutionary change. The proline-rich domain in fibrillin-1, glycine-rich domain in fibrillin-2 and proline-/glycine-rich domain in fibrillin-3 are found in all analyzed tetrapod species, whereas it is completely replaced with an EGF-like domain in cnidarians, arthropods, molluscs and urochordates. All collected sequences contain the first 9-cysteine hybrid domain, and the second 8-cysteine hybrid domain with exception of arthropods containing an atypical 10-cysteine hybrid domain 2. Furin cleavage sites within the N- and C-terminal unique domains were found for all analyzed fibrillin sequences, indicating an essential role for processing of the fibrillin pro-proteins. The four cysteines in the unique N-terminus and the two cysteines in the unique C-terminus are also highly conserved.     

Differential expression of fibrillin-3 adds to microfibril variety in human and avian, but not rodent, connective tissues

The human genome contains three fibrillins: FBN1 and FBN2, both well characterized, and FBN3, reported only as a cDNA sequence. Like FBN2, the highest expression levels of FBN3 were found in fetal tissues, with only low levels in postnatal tissues. Immunolocalization demonstrated fibrillin-3 in extracellular microfibrils abundant in developing skeletal elements, skin, lung, kidney, and skeletal muscle. Unlike the other two fibrillins, FBN3 expression is high in brain, and FBN3 is alternatively spliced, removing the exon encoding cbEGF2. Like FBN1, FBN3 contains three alternate exons in the 5' UTR. While FBN3 orthologs were identified in cow and chicken, Fbn3 appears to have been inactivated in the mouse genome, perhaps during chromosome fission events. Located on chromosome 19p13.3-13.2, FBN3 is a candidate gene for Weill-Marchesani syndrome.     

Latent transforming growth factor beta-binding protein 1 interacts with fibrillin and is a microfibril-associated protein

Latent transforming growth factor beta-binding protein 1 (LTBP-1) targets latent complexes of transforming growth factor beta to the extracellular matrix, where the latent cytokine is subsequently activated by several different mechanisms. Fibrillins are extracellular matrix macromolecules whose primary function is architectural: fibrillins assemble into ultrastructurally distinct microfibrils that are ubiquitous in the connective tissue space. LTBPs and fibrillins are highly homologous molecules, and colocalization in the matrix of cultured cells has been reported. To address whether LTBP-1 functions architecturally like fibrillins, microfibrils were extracted from tissues and analyzed immunochemically. In addition, binding studies were conducted to determine whether LTBP-1 interacts with fibrillins. LTBP-1 was not detected in extracted beaded-string microfibrils, suggesting that LTBP-1 is not an integral structural component of microfibrils. However, binding studies demonstrated interactions between LTBP-1 and fibrillins. The binding site was within three domains of the LTBP-1 C terminus, and in fibrillin-1 the site was defined within four domains near the N terminus. Immunolocalization data were consistent with the hypothesis that LTBP-1 is a fibrillin-associated protein present in certain tissues but not in others. In tissues where LTBP-1 is not expressed, LTBP-4 may substitute for LTBP-1, because the C-terminal end of LTBP-4 binds equally well to fibrillin. A model depicting the relationship between LTBP-1 and fibrillin microfibrils is proposed.     

Fibrillin Assembly Requires Fibronectin

Fibrillins constitute the major backbone of multifunctional microfibrils in elastic and nonelastic extracellular matrices. Proper assembly mechanisms are central to the formation and function of these microfibrils, and their properties are often compromised in pathological circumstances such as in Marfan syndrome and in other fibrillinopathies. Here, we have used human dermal fibroblasts to analyze the assembly of fibrillin-1 in dependence of other matrix-forming proteins. siRNA knockdown experiments demonstrated that the assembly of fibrillin-1 is strictly dependent on the presence of extracellular fibronectin fibrils. Immunolabeling performed at the light and electron microscopic level showed colocalization of fibrillin-1 with fibronectin fibrils at the early stages of the assembly process. Protein-binding assays demonstrated interactions of fibronectin with a C-terminal region of fibrillin-1, -2, and -3 and with an N-terminal region of fibrillin-1. The C-terminal half of fibrillin-2 and -3 had propensities to multimerize, as has been previously shown for fibrillin-1. The C-terminal of all three fibrillins interacted strongly with fibronectin as multimers, but not as monomers. Mapping studies revealed that the major binding interaction between fibrillins and fibronectin involves the collagen/gelatin-binding region between domains FNI₆ and FNI₉.     

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.     

The fibrillin-marfan syndrome connection

A few years ago no one would have suspected that the well-known disorder of connective tissue, Marfan syndrome, could be caused by mutations in a recently discovered extracellular component, fibrillin. Likewise, nobody would have predicted that fibrillin represents a small family of proteins that are associated with several pheno-typically overlapping disorders. The fibrillins are integral constituents of the non-collagenous microfibrils, with an average diameter of 10 nm. These aggregates are distributed in the extracellular matrix of virtually every tissue. Microfibrillar bundles provide the external coating to elastin in elastic fibers, and serve an anchoring function in non-elastic tissues. At higher resolution, individual microfibrils have a “beads-on-a-string” appearance resulting from the head-to-tail polymerization of multiple fibrillin aggregates. Structurally, fibrillin contains a series of repeated sequences homologous to the epidermal growth factor calcium-binding motif. Characterization of fibrillin mutations in Marfan syndrome patients, together with the elucidation of the structure of the fibrillin proteins, have provided new insights, and raised new questions, about the function of the 10 nm microfibrils. For example, it is possible that the fibrillins, in addition to serving a structural function, might also be involved in regulating cellular activities and morphogenetic programs. It is fitting that the long search for the Marfan syndrome gene has brought a novel group of proteins to the forefront of extracellular matrix biology.     

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.     

Biogenesis and function of fibrillin assemblies

Fibrillin-1 and fibrillin-2 are large cysteine-rich glycoproteins that serve two key physiological functions: as supporting structures that impart tissue integrity and as regulators of signaling events that instruct cell performance. The structural role of fibrillins is exerted through the temporal and hierarchical assembly of microfibrils and elastic fibers, whereas the instructive role reflects the ability of fibrillins to sequester transforming growth factor β (TGFβ) and bone morphogenetic protein (BMP) complexes in the extracellular matrix. Characterization of fibrillin mutations in human patients and in genetically engineered mice has demonstrated that perturbation of either function manifests in disease. More generally, these studies have indicated that fibrillins are integral components of a broader biological network of extracellular, cell surface, and signaling molecules that orchestrate morphogenetic and homeostatic programs in multiple organ systems. They have also suggested that the relative composition of fibrillin-rich microfibrils imparts contextual specificity to TGFβ and BMP signaling by concentrating the ligands locally so as to regulate cell differentiation within a spatial context during organ formation (positive regulation) and by restricting their bioavailability so as to modulate cell performance in a timely fashion during tissue remodeling/repair (negative regulation). Correlative evidence suggests functional coupling of the cell-directed assembly of microfibrils and targeting of TGFβ and BMP complexes to fibrillins. Hence, the emerging view is that fibrillin-rich microfibrils are molecular integrators of structural and instructive signals, with TGFβ and BMPs as the nodal points that convert extracellular inputs into discrete and context-dependent cellular responses.     

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.     

Fibrillin degradation by matrix metalloproteinases: identification of amino- and carboxy-terminal cleavage sites.

Fibrillin molecules form the structural framework of elastic fibrillin-rich microfibrils of the extracellular matrix. We have investigated the proteolysis of recombinant fibrillin molecules by five matrix metalloproteinases. Cleavage sites were defined at the carboxy-terminal end of the fibrillin-1 proline-rich region and the corresponding fibrillin-2 glycine-rich region (exon 10), and within exon 49 towards the carboxy-terminus of fibrillin-1. Cleavage at these sites is predicted to disrupt the structure and function of the fibrillin-rich microfibrils.     

Material and mechanical properties of bones deficient for fibrillin-1 or fibrillin-2 microfibrils

The contribution of non-collagenous components of the extracellular matrix to bone strength is largely undefined. Here we report that deficiency of fibrillin-1 or fibrillin-2 microfibrils causes distinct changes in bone material and mechanical properties. Morphometric examination of mice with hypomorphic or null mutations in fibrillin-1 or fibrillin-2, respectively, revealed appreciable differences in the postnatal shaping and growth of long bones. Fourier transform infrared imaging spectroscopy indicated that fibrillin-1 plays a predominantly greater role than fibrillin-2 in determining the material properties of bones. Biomechanical tests demonstrated that fibrillin-2 exerts a greater positive influence on the mechanical properties of bone than fibrillin-1 assemblies. Published evidence indirectly supports the notion that the above findings are mostly, if not exclusively, related to the differential control of TGFβ family signaling by fibrillin proteins. Our study therefore advances our understanding of the role that extracellular microfibrils play in bone physiology and implicitly, in the pathogenesis of bone loss in human diseases caused by mutations in fibrillin-1 or -2.     

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.     

Cell adhesion to fibrillin-1 molecules and microfibrils is mediated by alpha 5 beta 1 and alpha v beta 3 integrins

Fibrillins are the major glycoprotein components of microfibrils that form a template for tropoelastin during elastic fibrillogenesis. We have examined cell adhesion to assembled purified microfibrils, and its molecular basis. Human dermal fibroblasts exhibited Arg-Gly-Asp and cation-dependent adhesion to microfibrils and recombinant fibrillin-1 protein fragments. Strong integrin alpha 5 beta 1 interactions with fibrillin ligands were identified, but integrin alpha v beta 3 also contributed to cell adhesion. Fluorescence-activated cell sorting analysis confirmed the presence of abundant alpha 5 beta 1 and some alpha v beta 3 receptors on these cells. Adhesion to microfibrils and to Arg-Gly-Asp containing fibrillin-1 protein fragments induced signaling events that led to cell spreading, altered cytoskeletal organization, and enhanced extracellular fibrillin-1 deposition. Differences in cell shape when plated on fibrillin or fibronectin implied substrate-specific alpha 5 beta 1-mediated cellular responses. An Arg-Gly-Asp-independent cell adhesion sequence was also identified within fibrillin-1. Adhesion and spreading of smooth muscle cells on fibrillin ligands was enhanced by antibody-induced beta1 integrin activation. A375-SM melanoma cells bound Arg-Gly-Asp-containing fibrillin-1 protein fragments mainly through alpha v beta 3, whereas HT1080 cells used mainly alpha 5 beta 1. This study has shown that fibrillin microfibrils mediate cell adhesion, that alpha 5 beta 1 and alpha v beta 3 are both important but cell-specific fibrillin-1 receptors, and that cellular interactions with fibrillin-1 influence cell behavior.     

Microfibril-associated glycoprotein-2 interacts with fibrillin-1 and fibrillin-2 suggesting a role for MAGP-2 in elastic fiber assembly

Elastic fibers are composed of the protein elastin and a network of 10-12 nm microfibrils. The microfibrillar proteins include, among others, the fibrillins and microfibril-associated glycoproteins-1 and -2 (MAGP-1 and MAGP-2). Little is known about how microfibrillar proteins interact to support fiber assembly. We used the C-terminal half of MAGP-2 in a yeast two-hybrid library screen to identify relevant ligands. Six of 13 positive clones encoded known microfibrillar proteins, including fibrillin-1 and -2. Deletion analysis of partial fibrillin-1 and -2 clones revealed a calcium-binding epidermal growth factor repeat-containing region near the C terminus responsible for binding. This region is distinct from the region of fibrillin-1 reported by others to bind MAGP-1. The MAGP-2 bait was unable to interact productively with other epidermal growth factor repeats in fibrillin-1, demonstrating specificity of the interaction. Deletion analysis of the MAGP-2 bait demonstrated that binding occurred in a core region containing 48% identity and 7 conserved cysteine residues with MAGP-1. Immunoprecipitation of MAGP-2 from transfected COS-7 cells resulted in the coprecipitation of fibrillin. These results demonstrate that MAGP-2 specifically interacts with fibrillin-1 and -2 and suggest that MAGP-2 may help regulate microfibrillar assembly. The results also demonstrate the utility of the yeast two-hybrid system to study protein-protein interactions of the extracellular matrix.     

Structure of the integrin binding fragment from fibrillin-1 gives new insights into microfibril organization

Human fibrillin-1, the major structural protein of extracellular matrix (ECM) 10-12 nm microfibrils, is dominated by 43 calcium binding epidermal growth factor-like (cbEGF) and 7 transforming growth factor beta binding protein-like (TB) domains. Crystal structures reveal the integrin binding cbEGF22-TB4-cbEGF23 fragment of human fibrillin-1 to be a Ca(2+)-rigidified tetragonal pyramid. We suggest that other cbEGF-TB pairs within the fibrillins may adopt a similar orientation to cbEGF22-TB4. In addition, we have located a flexible RGD integrin binding loop within TB4. Modeling, cell attachment and spreading assays, immunocytochemistry, and surface plasmon resonance indicate that cbEGF22 bound to TB4 is a requirement for integrin activation and provide insight into the molecular basis of the fibrillin-1 interaction with alphaVbeta3. In light of our data, we propose a novel model for the assembly of the fibrillin microfibril and a mechanism to explain its extensibility.     

The microfibrillar proteins MAGP-1 and fibrillin-1 form a ternary complex with the chondroitin sulfate proteoglycan decorin

MAGP-1 and fibrillin-1, two protein components of extracellular microfibrils, were shown by immunoprecipitation studies to interact with the chondroitin sulfate proteoglycan decorin in the medium of cultured fetal bovine chondrocytes. Decorin interacted with each protein individually and with both proteins together to form a ternary complex. Expression of truncated fibrillin-1 proteins in Chinese hamster ovary cells localized proteoglycan binding to an amino-terminal region near the proline-rich domain. A spatially analogous fibrillin-2 truncated protein did not coprecipitate the same sulfated molecule, suggesting that chondroitin sulfate proteoglycan binding in this region is specific for fibrillin-1. An interaction between fibrillin and MAGP-1 was also observed under culture conditions that abrogated decorin secretion, suggesting that the two microfibrillar proteins can associate in the absence of the proteoglycan. Sulfation of matrix proteins is important for elastic fiber assembly because inhibition of sulfation was shown to prevent microfibrillar protein incorporation into the extracellular matrix of cultured cells.