Günter Kostka

Fibulins: a versatile family of extracellular matrix proteins

Fibulins are a newly recognized family of extracellular matrix proteins. The five known members of the family share an elongated structure and many calcium-binding sites, owing to the presence of tandem arrays of epidermal growth factor-like domains. They have overlapping binding sites for several basement-membrane proteins, tropoelastin, fibrillin, fibronectin and proteoglycans, and they participate in diverse supramolecular structures. New insights into their biological roles are now emerging from studies of transgenic mice and of some inherited human diseases.

Fibulin-1 is a ligand for the C-type lectin domains of aggrecan and versican

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 Ca2+-dependent, withK 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.

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 ∼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.

Perinatal Lethality and Endothelial Cell Abnormalities in Several Vessel Compartments of Fibulin-1-Deficient Mice

ABSTRACT The extracellular matrix protein fibulin-1 is a distinct component of vessel walls and can be associated with other ligands present in basement membranes, microfibrils, and elastic fibers. Its biological role was investigated by the targeted inactivation of the fibulin-1 gene in mice. This led to massive hemorrhages in several tissues starting at midgestation, ultimately resulting in the death of almost all homozygous embryos upon birth. Histological analysis demonstrated dilation and ruptures in the endothelial lining of various small vessels but not in that of larger vessels. Kidneys displayed a distinct malformation of glomeruli and disorganization of podocytes. A delayed development of lung alveoli suggested impairment in lung inflation. Immunohistology demonstrated the absence of fibulin-1 in its typical localizations but no aberrant patterns for several other extracellular matrix proteins. Electron microscopy revealed intact basement membranes but very irregular cytoplasmic processes of capillary endothelial cells in the organs that were most severely affected. Absence of fibulin-1 caused considerable blood loss but did not compromise blood clotting. The data indicate a strong but restricted abnormality in some endothelial compartments which, together with some kidney and lung defects, may be responsible for early death.

Genetic Heterogeneity of Cutis Laxa: A Heterozygous Tandem Duplication within the Fibulin-5 (FBLN5) Gene

Inherited cutis laxa is a connective tissue disorder characterized by loose skin and variable internal organ involvement, resulting from paucity of elastic fibers. Elsewhere, frameshift mutations in the elastin gene have been reported in three families with autosomal dominant inheritance, and a family with autosomal recessive cutis laxa was recently reported to have a homozygous missense mutation in the fibulin-5 gene. In the present study, we analyzed the gene expression of elastin and fibulins 1-5 in fibroblasts from five patients with cutis laxa. One patient was found to express both normal (2.2 kb) and mutant (2.7 kb) fibulin-5 mRNA transcripts. The larger transcript contains an internal duplication of 483 nucleotides, which resulted in the synthesis and secretion of a mutant fibulin-5 protein with four additional tandem calcium-binding epidermal growth factor-like motifs. The mutation arose from a 22-kb tandem gene duplication, encompassing the sequence from intron 4 to exon 9. No fibulin-5 or elastin mutations were detected in the other patients. The results demonstrate that a heterozygous mutation in fibulin-5 can cause cutis laxa and also suggest that fibulin-5 and elastin gene mutations are not the exclusive cause of the disease.

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.

Sequence and expression of a novel member (LTBP-4) of the family of latent transforming growth factor-β binding proteins

Overlapping cDNA clones from human heart and melanoma libraries were used to establish the 1587‐residue sequence of a novel protein (LTBP‐4) belonging to the family of extracellular microfibrillar proteins which also bind transforming growth factor‐β. LTBP‐4 consists of 20 EG modules, 17 of them with a consensus sequence for calcium binding, 4 TB modules with 8 cysteines and several proline‐rich regions. Northern blots demonstrated a single 5 kb mRNA which is highly expressed in heart but also present in skeletal muscle, pancreas, placenta and lung. The modular structure predicts that LTBP‐4 should be a microfibrillar protein which probably also binds TGF‐β.

Defective osteoblast function in ICAP-1-deficient mice

The integrin receptor family plays important roles in cell-to-cell and cell-to-extracellular matrix interactions through the recruitment of accessory molecules. One of them, the integrin cytoplasmic domain-associated protein-1 (ICAP-1; also known as ITGB1BP1), specifically interacts with the cytoplasmic domain of the β1 integrin subunit and negatively regulates its function in vitro. To address the role of ICAP-1 in vivo, we ablated the Icap-1 gene in mice. We report an unexpected role of ICAP-1 in osteoblast function during bone development. Icap-1-deficient mice suffer from reduced osteoblast proliferation and delayed bone mineralization, resulting in the retarded formation of bone sutures. In vitro studies reveal that primary and immortalized Icap-1-null osteoblasts display enhanced adhesion and spreading on extracellular matrix substrates, probably owing to an increase in β1 integrin activation. Finally, we provide evidence that ICAP-1 promotes differentiation of osteoprogenitors by supporting their condensation through modulating the integrin high affinity state.

Differential effects of carboxy-terminal sequence deletions on platelet-derived growth factor receptor signaling activities and interactions with cellular substrates.

Chimeric receptors composed of the human epidermal growth factor receptor (EGF-R) extracellular domain fused to wild-type and truncated platelet-derived growth factor receptor (PDGF-R) intracellular sequences were stably expressed in NIH 3T3 cells devoid of endogenous EGF-Rs. This experimental system allowed us to investigate the biological activity of PDGF-R cytoplasmic-domain mutants in PDGF-R-responsive NIH 3T3 cells by activating PDGF-specific signaling pathways with EGF. Deletion of 74 carboxy-terminal amino acids severely impaired the ability of the PDGF-R cytoplasmic domain to associate with cellular substrates in vitro. This deletion also inhibited receptor and substrate phosphorylation, reduced the receptors mitogenic activity, and completely abolished its oncogenic signaling potential. Surprisingly, removal of only six additional amino acids, including Tyr-989, restored substantial receptor and substrate phosphorylation capacity as well as transforming potential and yielded a receptor with wild-type levels of ligand-induced mitogenic activity. However, the ability of this chimera to bind phospholipase C gamma was severely impaired in comparison with the ability of the wild-type receptor, while the association with other cellular proteins was not affected. Further deletion of 35 residues, including Tyr-977, nearly abolished all PDGF-R cytoplasmic-domain biological signaling activities. None of the three C-terminal truncations completely abolished the mitogenic potential of the receptors or had any influence on ligand binding or receptor down regulation. Together, these data implicate the 80 C-terminal-most residues of the PDGF-R, and possibly Tyr-989, in phospholipase C gamma binding, while receptor sequences upstream from Asp-988 appear to be essential for specific interactions with other cellular polypeptides such as ras GTPase-activating protein and phosphatidylinositol 3-kinase. Thus, the mutants described here allow the separation of distinct PDGF-activated signaling pathways and demonstrate that phospholipase C gamma phosphorylation is not required for mitogenesis and transformation.

Complete exon-intron organization of the mouse fibulin-1 gene and its comparison with the human fibulin-1 gene

Fibulin‐1 is a 90 kDa calcium‐binding protein present in the extracellular matrix and in the blood. Two major variants, C and D, differ in their C‐termini as well as the ability to bind the basement membrane protein nidogen. Here we characterized genomic clones encoding the mouse fibulin‐1 gene, which contains 18 exons spanning at least 75 kb of DNA. The two variants are generated by alternative splicing of exons in the 3′ end. By searching the database we identified most of the exons encoding the human fibulin‐1 gene and showed that its exon‐intron organization is similar to that of the mouse gene.