A comparative analysis of the fibulin protein family. Biochemical characterization, binding interactions, and tissue localization

Fibulins are a family of five extracellular matrix proteins characterized by tandem arrays of epidermal growth factor-like domains and a C-terminal fibulin-type module. They are widely distributed and often associated with vasculature and elastic tissues. In this study, we expressed the three more recently identified family members, fibulin-3, fibulin-4, and fibulin-5, as recombinant proteins in mammalian cells. The purified proteins showed short rod structures of approximately 20 nm with a globule at one end, after rotary shadowing and electron microscopy. Two forms of mouse fibulin-3 were purified, and the O-glycan profiles of the larger form were characterized. Polyclonal antibodies raised against the purified proteins did not show any cross-reactivity with other family members and were used to assess the levels and localization of the fibulins in mouse tissues. Their binding interactions, cell adhesive properties, and tissue localization were analyzed in parallel with the previously characterized fibulin-1 and -2. Binding to tropoelastin was strong for fibulin-2 and -5, moderate for fibulin-4 and -1, and relatively weak for fibulin-3. Fibulin-4, but not fibulin-3 and -5, exhibited distinct interactions with collagen IV and nidogen-2 and moderate binding to the endostatin domain from collagen XV. Cell adhesive activities were not observed for all fibulins, except mouse fibulin-2, with various cell lines tested. All five fibulins were found in perichondrium and various regions of the lungs. Immunoelectron microscopy localized fibulin-4 and -5 to fibrillin microfibrils at distinct locations. Our studies suggest there are unique and redundant functions shared by these structurally related proteins.     

Sequence, recombinant expression and tissue localization of two novel extracellular matrix proteins, fibulin-3 and fibulin-4.

Fibulin-1 and fibulin-2 have previously been identified as basement membrane and microfibrillar proteins with a broad binding repertoire for other extracellular ligands. Here we report on the cloning and sequence analysis of human fibulin-3 (487 residues), also known as protein S1-5, and fibulin-4 (443 residues). These novel members of this protein family are most closely related to fibulin-1C. They consist of a C-terminal globular domain III, also shared by the fibrillins, a central rod-like element composed of five calcium-binding epidermal growth factor-like (EG) modules (domain II) and an N-terminal interrupted EG module (domain I) which replaces the anaphylatoxin-like modules of the other fibulins. This predicted domain structure was supported by electron microscopy of fibulin-4, which demonstrated short rods. Northern blots showed that both novel fibulins are expressed in several human tissues to a variable extent and that they are up-regulated in quiescent fibroblasts. Specific antibodies which were raised against each of the novel fibulins did not cross-react with fibulin-1. Immunohistology of adult mouse tissues showed that fibulin-3, fibulin-4 and fibulin-1 have overlapping but distinct extracellular tissue localizations. A particularly prominent feature was the staining of variable sets of large and small blood vessels.     

Fibulin-4 and fibulin-5 in elastogenesis and beyond: Insights from mouse and human studies

The fibulin family of extracellular matrix/matricellular proteins is composed of long fibulins (fibulin-1, -2, -6) and short fibulins (fibulin-3, -4, -5, -7) and is involved in protein–protein interaction with the components of basement membrane and extracellular matrix proteins. Fibulin-1, -2, -3, -4, and -5 bind the monomeric form of elastin (tropoelastin) in vitro and fibulin-2, -3, -4, and -5 are shown to be involved in various aspects of elastic fiber development in vivo. In particular, fibulin-4 and -5 are critical molecules for elastic fiber assembly and play a non-redundant role during elastic fiber formation. Despite manifestation of systemic elastic fiber defects in all elastogenic tissues, fibulin-5 null (Fbln5^−/−) mice have a normal lifespan. In contrast, fibulin-4 null (Fbln4^−/−) mice die during the perinatal period due to rupture of aortic aneurysms, indicating differential functions of fibulin-4 and fibulin-5 in normal development. In this review, we will update biochemical characterization of fibulin-4 and fibulin-5 and discuss their roles in elastogenesis and outside of elastogenesis based on knowledge obtained from loss-of-function studies in mouse and in human patients with FBLN4 or FBLN5 mutations. Finally, we will evaluate therapeutic options for matrix-related diseases.     

Fibulin-3, -4, and -5 Are Highly Susceptible to Proteolysis,Interact with Cells and Heparin,and Form Multimers

Extracellular short fibulins, fibulin-3, -4, and -5, are components of the elastic fiber/microfibril system and are implicated in the formation and homeostasis of elastic tissues. In this study, we report new structural and functional properties of the short fibulins. Full-length human short fibulins were recombinantly expressed in human embryonic kidney cells and purified by immobilized metal ion affinity chromatography. All three fibulins showed various levels of degradation after the purification procedure. N-terminal sequencing revealed that all three fibulins are highly susceptible to proteolysis within the N-terminal linker region of the first calcium-binding epidermal growth factor domain. Proteolytic susceptibility of the linker correlated with its length. Exposure of these fibulins to matrix metalloproteinase (MMP)-1, -2, -3, -7, -9, and -12 resulted in similar proteolytic fragments with MMP-7 and -12 being the most potent proteases. Fibulin-3 proteolysis was almost completely inhibited in cell culture by the addition of 25 μm doxycycline (a broad spectrum MMP inhibitor). Reducible fibulin-4 dimerization and multimerization were consistently observed by SDS-PAGE, Western blotting, and mass spectrometry. Atomic force microscopy identified monomers, dimers, and multimers in purified fibulin-4 preparations with sizes of ∼10–15, ∼20–25, and ∼30–50 nm, respectively. All short fibulins strongly adhered to human fibroblasts and smooth muscle cells. Although only fibulin-5 has an RGD integrin binding site, all short fibulins adhere at a similar level to the respective cells. Solid phase binding assays detected strong calcium-dependent binding of the short fibulins to immobilized heparin, suggesting that these fibulins may bind cell surface-located heparan sulfate.     

Extracellular matrix protein 1 interacts with the domain III of fibulin-1C and 1D variants through its central tandem repeat 2

Extracellular matrix protein 1 (ECM1), a widely expressed glycoprotein, has been shown to harbor mutations in lipoid proteinosis (LP), an autosomal recessive disorder characterized by profound alterations in the extracellular matrix of connective tissue. The biological function of ECM1 and its role in the pathomechanisms of LP are unknown. Fibulins comprise a family of extracellular matrix components, and the prototype of this family, fibulin-1, is expressed in various connective tissues and plays a role in developmental and pathologic processes. In this study, we demonstrate that ECM1, and specifically the second tandem repeat domain which is alternatively spliced, interacts with the C-terminal segments of fibulins 1C and 1D splice variants which differ in their C-terminal domain III. The interactions were detected by yeast two-hybrid genetic system and confirmed by co-immunoprecipitations. Kinetics of the binding between ECM1 and fibulin-1D, measured by biosensor assay, revealed a K(d) of 5.71 x 10(-8) M, indicating a strong protein-protein interaction. Since distinct splice variants of ECM1 and fibulin-1 have been shown to be co-expressed in tissues affected in LP, we propose that altered ECM1/fibulin-1 interactions may play a role in the pathogenesis of this disease as well as in a number of processes involving the extracellular matrix of connective tissues.     

The extracellular matrix proteins fibulin-1 and fibulin-2 in the early human embryo

Summary Fibulin-1 and fibulin-2, two recently identified extracellular matrix proteins with a homologous domain structure, are known to bind various extracellular ligands and calcium. In this study, they have been localized at the light microscopical level in human embryos of gestational weeks 4–10, using polyclonal antibodies. Identical localization patterns were observed for the two fibulins in most of the tissues. In the heart, the endocardial cushion tissue and endocardium, but not the myocardium, were stained, as were the basement membrane zones and adventitia of blood vessels. Staining was also observed in the perichondrium and calcifying regions of developing bones. Moreover, reactions occurred with the gut subepithelium and epithelial basement membranes of the skin. Differences in staining patterns, however, were observed in various neural structures. Fibulin-1 was prominent in the matrix of the leptomeningeal anlage, in basement membranes of the neuroepithelium and the perineurium of peripheral nerves. Fibulin-2 was detected primarily within the neuropithelium, spinal ganglia and peripheral nerves. The early embryonic expression of both fibulins indicates specific roles during organ development and, in particular, involvement in the differentiation of heart, skeletal and neuronal structures.     

The proteoglycans aggrecan and Versican form networks with fibulin-2 through their lectin domain binding

Aggrecan, versican, neurocan, and brevican are important components of the extracellular matrix in various tissues. Their amino-terminal globular domains bind to hyaluronan, but the function of their carboxyl-terminal globular domains has long remained elusive. A picture is now emerging where the C-type lectin motif of this domain mediates binding to other extracellular matrix proteins. We here demonstrate that aggrecan, versican, and brevican lectin domains bind fibulin-2, whereas neurocan does not. As expected for a C-type lectin, the interactions are calcium-dependent, with K(D) values in the nanomolar range as measured by surface plasmon resonance. Solid phase competition assays with previously identified ligands demonstrated that fibulin-2 and tenascin-R bind the same site on the proteoglycan lectin domains. Fibulin-1 has affinity for the common site on versican but may bind to a different site on the aggrecan lectin domain. By using deletion mutants, the interaction sites for aggrecan and versican lectin domains were mapped to epidermal growth factor-like repeats in domain II of fibulin-2. Affinity chromatography and solid phase assays confirmed that also native full-length aggrecan and versican bind the lectin domain ligands. Electron microscopy confirmed the mapping and demonstrated that hyaluronan-aggrecan complexes can be cross-linked by the fibulins.     

Tropoelastin binding to fibulins, nidogen-2 and other extracellular matrix proteins.

Elastic fibers in vessel walls and other tissues consist of cross-linked tropoelastin in association with several microfibrillar proteins. In order to understand the molecular basis of these structures, we examined the binding of recombinant human tropoelastin to other extracellular matrix ligands in solid phase binding and surface plasmon resonance assays. These studies demonstrated a particularly high affinity (K(d) about 1 nM) of tropoelastin for microfibrillar fibulin-2 and the recently described nidogen-2 isoform. More moderate affinities were observed for fibulin-1, laminin-1 and perlecan, while several other ligands such as collagens, nidogen-1, fibronectin and BM-40 showed little or no binding. In immunogold staining of mouse aortic media, elastic fibers were heavily decorated with tropoelastin, fibulin-2 and nidogen-2, while the reaction with fibulin-1 was lower. The colocalization of these proteins emphasizes the potential for in vivo interactions.     

Fibrillin-1 interactions with fibulins depend on the first hybrid domain and provide an adaptor function to tropoelastin

Fibrillin-containing microfibrils in elastic and nonelastic extracellular matrices play important structural and functional roles in various tissues, including blood vessels, lung, skin, and bone. Microfibrils are supramolecular aggregates of several protein and nonprotein components. Recently, a large region in the N-terminal portion of fibrillin-1 was characterized as a multifunctional protein interaction site, including binding sites for fibulin-2 and -5 among others. Using a panel of recombinant fibrillin-1 swapped domain and deletion fragments, we demonstrate here that the conserved first hybrid domain in fibrillin-1 is essential for binding to fibulin-2, -4, and -5. Fibulin-3 and various isoforms of fibulin-1 did not interact with fibrillin-1. Although the first hybrid domain in fibrillin-1 is located in close vicinity to the self-assembly epitope, binding of fibulin-2, -4, and -5 did not interfere with self-assembly. However, these fibulins can associate with microfibrils at various levels of maturity. Formation of ternary complexes between fibrillin-1, fibulins, and tropoelastin demonstrated that fibulin-2 and -5 but much less fibulin-4, are able to act as molecular adaptors between fibrillin-1 and tropoelastin.     

Recombinant domains of mouse nidogen-1 and their binding to basement membrane proteins and monoclonal antibodies.

The basement membrane protein, nidogen-1, was previously shown to consist of three globular domains, G1 to G3, and two connecting segments. Nidogen-1 is a major mediator in the formation of ternary complexes with laminins, collagen IV, perlecan and fibulins. In the present study, we have produced recombinant proteins of these predicted domains in mammalian cells and used these proteins for crystallographic and binding epitope analyses. These fragments included G1, G2, the rod domain and a slightly larger G3 structure; all were obtained in good yields and were shown to be properly folded using electron microscopy. Surface plasmon resonance assays demonstrated high affinity binding (Kd = 3-9 nM) of domain G2 for collagen IV, perlecan domain IV-1 and fibulin-2, and a more moderate Kd for fibulin-1C. Domain G3 contained high affinity binding sites for the laminin gamma1 chain and collagen IV (Kd = 1 nM) and weaker binding sites for fibulin-1C and fibulin-2. A moderate binding affinity was also observed between domain G1 and fibulin-2, while no activity could be detected for the nidogen rod domain. Together, these data indicate the potential of nidogen-1 for multiple interactions within basement membranes. A similar binding repertoire was also identified for seven rat monoclonal antibodies that bound with Kd = 2-30 nM to either G1, G1-G2, G2, the rod domain or G3. Three of the antibodies showed strongly reduced binding to G2 and G3 after complex formation with either a perlecan domain or laminin-1.     

Distinct regions within fibulin-1D modulate interactions with hemicentin

Fibulins are evolutionarily conserved extracellular matrix (ECM) proteins that assemble in elastic fibers and basement membranes. Caenorhabditis elegans has a single fibulin gene that produces orthologs of vertebrate fibulin-1 C and D splice forms. In a structure-function analysis of fibulin-1 domains, a series of deletion constructs show that EGF repeats 4 and 5 are required for the hemicentin-dependent assembly and function of fibulin-1D in native locations. In contrast, constructs missing the second EGF repeat of fibulin-1D (EGF2D) assemble in ectopic locations in a hemicentin dependent manner. Constructs that contain EGF2D are cleaved into two fragments, but constructs with EGF2D missing are not, suggesting that a protease binds and/or cleaves fibulin-1D at a site that is likely within EGF2D. Together, the data suggests that EGF repeats 4 and 5 promote interaction with hemicentin while a region within EGF2D suppresses ectopic interactions with hemicentin and this suppression may be protease dependent.     

Latent Transforming Growth Factor ��-binding Proteins and Fibulins Compete for Fibrillin-1 and Exhibit Exquisite Specificities in Binding Sites

Latent transforming growth factor (TGF) β-binding proteins (LTBPs) interact with fibrillin-1. This interaction is important for proper sequestration and extracellular control of TGFβ. Surface plasmon resonance interaction studies show that residues within the first hybrid domain (Hyb1) of fibrillin-1 contribute to interactions with LTBP-1 and LTBP-4. Modulation of binding affinities by fibrillin-1 polypeptides in which residues in the third epidermal growth factor-like domain (EGF3) are mutated demonstrates that the binding sites for LTBP-1 and LTBP-4 are different and suggests that EGF3 may also contribute residues to the binding site for LTBP-4. In addition, fibulin-2, fibulin-4, and fibulin-5 bind to residues contained within EGF3/Hyb1, but mutated polypeptides again indicate differences in their binding sites in fibrillin-1. Results demonstrate that these protein-protein interactions exhibit “exquisite specificities,” a phrase commonly used to describe monoclonal antibody interactions. Despite these differences, interactions between LTBP-1 and fibrillin-1 compete for interactions between fibrillin-1 and these fibulins. All of these proteins have been immunolocalized to microfibrils. However, in fibrillin-1 (Fbn1) null fibroblast cultures, LTBP-1 and LTBP-4 are not incorporated into microfibrils. In contrast, in fibulin-2 (Fbln2) null or fibulin-4 (Fbln4) null cultures, fibrillin-1, LTBP-1, and LTBP-4 are incorporated into microfibrils. These data show for the first time that fibrillin-1, but not fibulin-2 or fibulin-4, is required for appropriate matrix assembly of LTBPs. These studies also suggest that the fibulins may affect matrix sequestration of LTBPs, because in vitro interactions between these proteins are competitive.Fibrillin microfibrils are ubiquitous structural elements in the connective tissue. Fibrillin microfibrils provide organs with tissue-specific architectural frameworks designed to support the mature functional integrity of the particular organ. In addition, fibrillin microfibrils contribute to proper developmental patterning of organs by targeting growth factors to the right location in the extracellular matrix (1, 2).Molecules of fibrillin-1 (3), fibrillin-2 (4, 5), and fibrillin-3 (6) polymerize to form the backbone structure of microfibrils. Latent TGFβ-binding protein (LTBP)³-1 associates with fibrillin microfibrils in the perichondrium and in osteoblast cultures (7, 8), and LTBP-1 and LTBP-4 interact with fibrillin (9). Other proteins associated with fibrillin microfibrils include the fibulins (10, 11), microfibril-associated glycoprotein-1 and -2 (12, 13), decorin (14), biglycan (15), versican (16), and perlecan (17). It is likely that one function of these associated extracellular matrix molecules is to connect the fibrillin microfibril scaffold to other architectural elements in tissue- and organ-specific patterns.In addition to performing architectural functions, fibrillins bind directly to prodomains of bone morphogenetic proteins and growth and differentiation factors (18, 19) and LTBPs bring with them the small latent TGFβ complex (20), suggesting that the microfibril scaffold may position, concentrate, and control growth factor signaling. Studies of fibrillin-1 (Fbn1) and fibrillin-2 (Fbn2) mutant mice demonstrate that loss of fibrillins results in phenotypes associated with dysregulated TGFβ (21–23) or bone morphogenetic protein (24) signaling. Microfibril-associated glycoprotein-1 (Magp-1) null mice reveal phenotypes that may also be related to abnormal TGFβ signaling (25).In a previous study (9), we determined that the binding site for LTBP-1 and -4 is contained within a specific four-domain region of fibrillin-1. In this study, we performed additional experiments to more precisely define the LTBP binding site. At the same time, we compared binding of fibulins to fibrillin, because the region in fibrillin-1 that was suggested to contain the fibulin binding site (11) was very close to our region of interest for LTBP binding. Our results demonstrate that LTBPs and fibulins compete for binding to fibrillin-1. However, the proteins tested (LTBP-1, LTBP-4, fibulin-2, fibulin-4, and fibulin-5) displayed “exquisite specificities” in their interactions with fibrillin-1.To test the potential significance of these interactions with fibrillin-1, we investigated matrix incorporation of LTBPs in cell cultures obtained from wild type, Fbn1 null, Fbn2 null, fibulin-2 (Fbln-2) null, and fibulin-4 (Fbln-4) null mice. In addition, we examined the distribution of LTBPs in Fbn1 null and Fbn2 null mice.     

Extracellular matrix alterations in brains lacking four of its components

The organization of the brain extracellular matrix appears to be based on aggregates of hyaluronan and proteoglycans, connected by oligomeric glycoproteins. Mild phenotypical consequences were reported from several mouse strains lacking components of this matrix such as neurocan, brevican, tenascin-R, and tenascin-C. To further challenge the flexibility of the extracellular matrix network of the brain, mice lacking all four brain extracellular matrix molecules were generated, which were found to be viable and fertile. Analysis of the brains of 1-month-old quadruple KO mice revealed increased protein levels of fibulin-1 and fibulin-2. Histochemical analysis showed an unusual parenchymal deposition of these fibulins. The quadruple KO mice also displayed obvious changes in the pattern of deposition of hyaluronan. Further, an almost quadruple knockout like extracellular environment was noticed in the brains of triple knockout mice lacking both tenascins and brevican, since these brains had strongly reduced levels of neurocan.     

Basement membrane derived fibulin-1 and fibulin-5 function as angiogenesis inhibitors and suppress tumor growth

The fibulins are a family of secreted glycoproteins that are characterized by repeated epidermal-growth-factor-like domains and a unique C-terminus structure. Fibulins modulate cell morphology, growth, adhesion, and motility. Our initial basement membrane degradome screen using Cathepsin D, a tumor microenvironment-associated protease, contained fragments of fibulin-1 and full length fibulin-5. In this report, we evaluate the antiangiogenic activity of fibulin-1 and fibulin-5. Tumor studies demonstrate that both fibulin-1 and fibulin-5 suppress HT1080 tumor growth. CD31 labeling and TUNEL assay further reveal that fibulin-1 suppression of HT1080 tumor growth is associated with diminished angiogenesis and also enhanced apoptosis of endothelial cells and tumor cells. In contrast, fibulin-5 inhibits tumor angiogenesis with a minimal anti-apoptotic affect. Cathepsin D digestion of fibulin-1 produces a fragment with nearly the same molecular weight as fibulin-5, and this fragment (named Neostatin) inhibits endothelial cell proliferation. Additionally, degradation of basement membrane by cathepsin D liberates both fibulin-1 fragments and fibulin-5, which function to inhibit angiogenesis.     

Yeast two-hybrid identification of prostatic proteins interacting with human sex hormone-binding globulin

Yeast two-hybrid (Y2H) screening of a prostate cDNA library with the cDNA for sex hormone-binding globulin (SHBG) has been used to identify proteins through which SHBG may exert autocrine or paracrine effects on sex steroid target tissues. The library screen gave 230 positive interactions of which around 60 have been sequenced. Of the proteins identified to date from database (BLAST) searches of these sequences, SHBG is one of those occurring most frequently. Amongst the proteins of interest are the membrane-associated proteins flotillin-1 and PRV-1, the enzymes cathepsin D, kallikrein 4 and acid phosphatase, various metallothioneins and translation elongation factor 1 alpha. The significance of the interaction of SHBG with these proteins is discussed.     

A novel intracellular fibulin-1D variant binds to the cytoplasmic domain of integrin beta 1 subunit

Fibulin-1 is a member of a growing family of proteins that includes eight members and is involved in cellular functions such as adhesion, migration and differentiation. Fibulin-1 has also been implicated in embryonic development of the heart and neural crest-derived structures. It is an integral part of the extracellular matrix (ECM) and has been shown to bind to a multitude of ECM proteins. However, fibulin-1 was first identified as a protein purified from placental extracts that binds to the cytoplasmic domain of integrin β1. Human fibulin-1 is alternatively spliced into four different isoforms namely A–D. These isoforms share a common N-terminus sequence that contains a secretion sequence but differ in their carboxy-terminal fibulin-1 module. In this report we identify a new splice variant of fibulin-1 that differs from all other fibulin-1 variants in the N-terminus sequence and has a similar carboxy-terminus sequence as fibulin-1D. This variant that we named fibulin-1D prime (fibulin-1D′) lacks a secretion sequence and the anaphlatoxin region of fibulin-1 variants. The protein has an apparent molecular weight of 70.5 kDa. Herein we show that fibulin-1D′ binds to the intracellular domain of integrin β1 as well as to integrin α5β1. The protein was localized intracellularly in CHO cells transfected with a pEF4 plasmid containing full-length coding sequence of fibulin-1D′. We also localized the protein in human placenta. We propose that the fibulin-1D′ variant might play a role in early embryo development as well as in modulating integrin β1 functions including adhesion and motility.