Ultrastructural localization of microfibrillar fibulin-1 and fibulin-2 during heart development indicates a switch in molecular associations

Miosge, Nicolai; Sasaki, Takako; Chu, Mon Li; Herken, Rainer; Timpl, Rupert

doi:10.1007/s000180050188

Cited by 31 publications

(21 citation statements)

References 39 publications

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“…One of those ligands, fibulin-1, is a component of elastic fibers and binds to the lectin-like domain of G3 of both aggrecan and versican. 4,31 In vivo, versican is closely associated with elastic networks. 32 Our recent work has shown that overexpressed V3 is secreted by ASMCs and deposited in the extracellular matrix in close association with elastin (M.G.…”

Section: Discussionmentioning

confidence: 99%

Retrovirally Mediated Overexpression of Versican V3 by Arterial Smooth Muscle Cells Induces Tropoelastin Synthesis and Elastic Fiber Formation In Vitro and In Neointima After Vascular Injury

Merrilees

Lemire

Fischer

et al. 2002

Circulation Research

100

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Abstract-Versican is an extracellular matrix (ECM) proteoglycan that is synthesized as multiple splice variants. In a recent study, we demonstrated that retroviral-mediated overexpression of the variant V3, which lacks chondroitin sulfate (CS) chains, altered arterial smooth muscle cell (ASMC) phenotype in short-term cell culture. We now report that V3-overexpressing ASMCs exhibit significantly increased expression of tropoelastin and increased formation of elastic fibers in long-term cell cultures. In addition, V3-overexpressing ASMCs seeded into ballooned rat carotid arteries continued to overexpress V3 and, at 4 weeks after seeding, produced a highly structured neointima significantly enriched in elastic fiber lamellae. In contrast to the hydrated, myxoid neointima produced by rounded or stellate vector-alonetransduced cells, V3-expressing cells produced a compact and highly ordered neointima, which contained elongated ASMCs that were arranged in parallel arrays and separated by densely packed collagen bundles and elastic fibers. These results indicate that a variant of versican is involved in elastic fiber assembly and may represent a novel therapeutic approach to facilitate the formation of elastic fibers. T he extracellular matrix (ECM) proteoglycan versican, the major chondroitin sulfate proteoglycan of vessel wall, 1 is distinguished by a central extended region with attached glycosaminoglycan (GAG) chains by an amino-terminal globular domain (G1) that binds hyaluronan (HA) and by a carboxy-terminal selectin-like domain (G3) that binds to other matrix components including tenascin-R and fibulin-1. [2][3][4][5][6] The mRNA that codes the GAG attachment region, which in the full-length form has 2 domains, can undergo differential splicing to produce 4 variants: V0, with 2 (␣ and ␤) GAG binding regions; V1 (with the ␤ GAG exon); V2 (with the ␣ GAG exon); and V3 with neither GAG exon and formed from the G1 and G3 domains only. 7-9 V3 is thus predicted to be a glycoprotein and not a proteoglycan. V3 mRNA has been demonstrated in aortic tissue as well as cultures of ASMCs by Northern blot 10 and has been detected, by polymerase chain reaction (PCR), from cDNA libraries for various tissues including brain, stomach, and liver. 9 Recently, we reported that retroviral insertion and overexpression of the gene for V3 into cultured Fischer rat ASMCs induces a number of phenotypic changes. 11 Compared with vector-alone-transduced cells, V3 ASMCs are more flattened and spread, show a large increase in area of close contacts and in the size of peripheral focal contacts, increased resistance to trypsin detachment, reduced pericellular coats, and decreased rates of growth and migration. The mechanism by which V3 effects these changes is not clear, although the morphological features, the reduced growth and migration, and the reduced cell coat, raise the possibility that V3 may affect the accumulation of components of the extracellular matrix. For example, Evanko et al 12 showed recently that the V1 isoform of versican in ...

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Section: Discussionmentioning

confidence: 99%

Retrovirally Mediated Overexpression of Versican V3 by Arterial Smooth Muscle Cells Induces Tropoelastin Synthesis and Elastic Fiber Formation In Vitro and In Neointima After Vascular Injury

Merrilees

Lemire

Fischer

et al. 2002

Circulation Research

100

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“…Striking examples are the strong expression of versican and both fibulins in the endocardial cushion tissue during heart development (26,31,32,34,46) and the prominent expression of aggrecan and both fibulins in newly formed cartilage and bone. Indeed, treatment of endocardial cushion tissue sections with hyaluronidase to degrade hyaluronic acid led to removal of both versican and fibulins, whereas fibronectin staining remains unaffected (26). Furthermore, disruption of the versican gene results in heart malformation and early embryonic death (24,25).…”

Section: Discussionmentioning

confidence: 99%

“…In the heart defect (Hdf) mice, truncation of the versican gene by insertion of a transgene resulted in heart malformation and embryonic lethality (24,25). Interestingly, fibulin-1 and -2 are produced together with versican and hyaluronan in the endocardial cushion tissue (26), suggesting involvement of the fibulins in organization of the hyaluronan-versican complexes of the developing heart extracellular matrix.…”

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confidence: 99%

“…Interactions between domains N and II give the fibulin-2 dimers a polymorph shape with X-, Y-, or rod-shaped dimers (30). During development, fibulins are broadly expressed (31-33) with particularly high levels in the developing heart valves (26,34). In the adult, both fibulin-1 and -2 are found in microfibrils and in many other tissue structures, notably in basement membranes and vessel walls (28,35,36).…”

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confidence: 99%

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The Proteoglycans Aggrecan and Versican Form Networks with Fibulin-2 through Their Lectin Domain Binding

Olin

Mörgelin

Sasaki

et al. 2001

Journal of Biological Chemistry

191

143

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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 carboxylterminal 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.The large aggregating proteoglycans referred to as lecticans (1) or hyalectans (2) are ubiquitous extracellular matrix components. The family consists of aggrecan, versican, brevican, and neurocan. Aggrecan is expressed by chondrocytes in cartilage. Versican is found in many tissues, e.g. blood vessels and dermis expressed by smooth muscle cells and fibroblasts. Brevican and neurocan are found in the central nervous system, expressed by astrocytes and neuronal cells, respectively. These proteoglycans have important functions in many tissues. Aggrecan, for example, with its many glycosaminoglycan side chains and thus high fixed charge density gives rise to a pronounced osmotic swelling pressure that is crucial for the biomechanical properties of cartilage (3, 4).The core proteins of these proteoglycans are organized with a central elongated glycosaminoglycan-carrying part of variable length, flanked by globular domains mediating interactions with other matrix molecules (5-9). The amino-terminal globular G1 domains bind to hyaluronan in interactions stabilized by link protein (10 -13). The carboxyl-terminal globular G3 domain consists of one or two EGF repeats, a C-type lectinlike domain (CLD), 1 and a sushi repeat. It has recently been shown that the CLDs function in binding. So far four ligands have been identified as follows: the brain-specific extracellular matrix protein tenascin-R (14, 15), tenascin-C (16), sulfated glycolipids (17), and fibulin-1 (18).The CLDs of the lecticans are highly conserved; the amino acid sequence of the chick versican CLD, for example, is 96% identical to the human homologue. This high degree of conservation suggests that the C...

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“…An interaction between the molecules is supported by immunogold electron microscopy colocalization in the endocardial cushion tissue. Indeed, treatment of sections with hyaluronidase to degrade hyaluronic acid leads to removal of both versican and fibulin whereas fibronectin staining remains unaffected (42). Interestingly, disruption of the versican gene results in early embryonic death due to failure to form the endocardial cushion and the right cardiac chamber (43,44).…”

Section: Discussionmentioning

confidence: 99%

Fibulin-1 Is a Ligand for the C-type Lectin Domains of Aggrecan and Versican

Aspberg

Adam

Kostka

et al. 1999

Journal of Biological Chemistry

195

154

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The aggregating proteoglycans (aggrecan, versican, neurocan, and brevican) are important components of many extracellular matrices. Their N-terminal globular domain binds to hyaluronan, but the function of their C-terminal region containing a C-type lectin domain is less clear. We now report that a 90-kDa protein copurifies with recombinant lectin domains from aggrecan and versican, but not from the brain-specific neurocan and brevican. Amino acid sequencing of tryptic peptides from this protein identified it as fibulin-1. This extracellular matrix glycoprotein is strongly expressed in tissues where versican is expressed (blood vessels, skin, and developing heart), and also expressed in developing cartilage and bone. It is thus likely to interact with these proteoglycans in vivo. Surface plasmon resonance measurements confirmed that aggrecan and versican lectin domains bind fibulin-1, whereas brevican and neurocan do not. As expected for a C-type lectin, the interactions with fibulin-1 are Ca 2؉ -dependent, with K D values in the low nanomolar range. Using various deletion mutants, the binding site for aggrecan and versican lectin domains was mapped to the epidermal growth factor-like repeats in domain II of fibulin-1. No difference in affinity was found for deglycosylated fibulin-1, indicating that the proteoglycan C-type lectin domains bind to the protein part of fibulin-1.The lectican family of large aggregating proteoglycans consists of versican, aggrecan, neurocan, and brevican (1). Their core proteins are organized with a central, extended, glycosaminoglycan-carrying part of variable length, flanked by globular domains (2-6). It has long been known that these proteoglycans are anchored to the hyaluronan meshwork through their N-terminal globular domains, in link protein-stabilized complexes (7-10). We have recently shown that the C-terminal globular domain also functions as a binding domain, in that it binds the brain-specific extracellular matrix protein tenascin-R (11, 12). Indeed, all these proteoglycans are expressed in the brain (1, 13). Although neurocan and brevican expression is restricted to the central nervous system, aggrecan is prominent in cartilage only (14), while versican is widely expressed in the extracellular matrix of many tissues, notably blood vessels and skin (15). Until now, no extra-neuronal ligand for the C-terminal globular domains of aggrecan and versican has been identified.We now report that the fibrillar extracellular matrix protein fibulin-1 is such a ligand. Fibulin-1 was previously shown to be an elongated protein which consists of three domains (I-III) with different molecular structures. The C-terminal domain III can be varied by splicing (variants A-D) and this was shown to affect its binding properties to nidogen-1 (16 -18). Here we show that fibulin-1 binds to the C-type lectin domains of versican and aggrecan with high affinity, but shows no binding to the brain-specific proteoglycans brevican and neurocan. We also map the binding site on the fibulin-1 molecule to th...

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Ultrastructural localization of microfibrillar fibulin-1 and fibulin-2 during heart development indicates a switch in molecular associations

Cited by 31 publications

References 39 publications

Retrovirally Mediated Overexpression of Versican V3 by Arterial Smooth Muscle Cells Induces Tropoelastin Synthesis and Elastic Fiber Formation In Vitro and In Neointima After Vascular Injury

Retrovirally Mediated Overexpression of Versican V3 by Arterial Smooth Muscle Cells Induces Tropoelastin Synthesis and Elastic Fiber Formation In Vitro and In Neointima After Vascular Injury

The Proteoglycans Aggrecan and Versican Form Networks with Fibulin-2 through Their Lectin Domain Binding

Fibulin-1 Is a Ligand for the C-type Lectin Domains of Aggrecan and Versican

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