Characterization of EHD4, an EH domain-containing protein expressed in the extracellular matrix.

To identify proteins that promote assembly of type VI collagen tetramers or stabilize type VI collagen filaments, a two-hybrid screen of a human placenta library was used and a new extracellular protein discovered. The cDNA sequence of the new protein encodes 541 amino acid residues. This cDNA sequence is identical to EHD4, a recently described member of the EH domain family of proteins. Two mRNAs of 4.4 and 3.0 kilobases were present in human skin fibroblasts and most tissues tested but were most prevalent in the heart. The chromosomal localization of the gene for this new protein was determined to be at 15q14-q15. Three polyclonal peptide antibodies were made against synthetic EHD4 peptides. The affinity-purified antibodies were used in immunofluorescent staining of developing limbs and matrices produced by human skin fibroblasts and mouse NIH3T3 fibroblasts in culture. Embryonic rat limb cartilage was strongly stained throughout development, and cultured fibroblasts deposited an extracellular filamentous network containing EHD4. In non-denaturing extracts of fetal bovine cartilage and in human skin fibroblast culture media, two components of approximately 220 and 158 kDa were observed, which, after reduction, migrated as a 56-kDa component on SDS-polyacrylamide gel electrophoresis. EHD4 is the first extracellular matrix protein described that contains an EH domain.

Fibrillin containing elastic microfibrils support platelet adhesion under dynamic shear conditions.

The vascular subendothelium contains macromolecular structures called microfibrils. Type VI collagen is one protein found in microfibrils that supports platelet adhesion and aggregation and we have previously evaluated the roles of platelet receptors and vWf involved in these processes under physiological shear conditions. Here we investigate the ability of fibrillin containing elastic microfibrils to support mural thrombus formation. Our results show that elastic microfibril surfaces support platelet adhesion under low shear conditions at a level similar to collagen VI tetramers. However, the degree of aggregation on the elastic microfibril surface is much higher. Both adhesion and aggregation were shown to be mediated by the GPIIb-IIIa platelet receptor. Elastic microfibrils do not support the formation of mural thrombi under high shear conditions. These results suggest roles for both collagen VI and fibrillin containing elastic microfibrils in modulating the platelet response to blood vessel injury.

Type VI collagen anchors endothelial basement membranes by interacting with type IV collagen.

Type VI collagen filaments are found associated with interstitial collagen fibers, around cells, and in contact with endothelial basement membranes. To identify type VI collagen binding proteins, the amino-terminal domains of the alpha1(VI) and alpha2(VI) chains and a part of the carboxyl-terminal domain of the alpha3(VI) chain were used as bait in a yeast two-hybrid system to screen a human placenta library. Eight persistently positive clones were identified, two coding the known matrix proteins fibronectin and basement membrane type IV collagen and the rest coding new proteins. The amino-terminal domain of alpha1(VI) was shown to interact with the carboxyl-terminal globular domain of type IV collagen. The specificity of this interaction was further studied using the yeast two-hybrid system in a one-on-one format and confirmed by using isolated protein domains in immunoprecipitation, affinity blots, and enzyme-linked immunosorbent assay-based binding studies. Co-distribution of type VI and type IV collagens in human muscle was demonstrated using double labeling immunofluorescent microscopy and immunoelectron microscopy. The strong interaction of type VI collagen filaments with basement membrane collagen provided a possible molecular pathogenesis for the heritable disorder Bethlem myopathy.

The significance of subendothelial von Willebrand factor.

von Willebrand factor (vWf) serves to bridge between receptors on the platelet cytoplasmic membrane and the extracellular matrix. In addition to circulating in plasma, vWf is deposited into the extracellular matrix of the subendothelium where it is associated with type VI collagen microfibrils, but not with the elastin-associated microfibrils which are present in the deepest portion of the subendothelium at the zone of the internal elastic lamina. The reaction of platelets to type VI collagen in flow systems is qualitatively different from the shear rate dependent adhesion and aggregation response which is observed with fibrillar type I collagen, exhibiting a response only at low shear rates. The adhesion response to type VI collagen is dependent upon vWf, GP Ib and the GP IIb-IIIa complex. Platelets exposed to purified fibrillin-containing elastin-associated microfibrils adhere and aggregate at low shear rates; this response appears to involve GP IIb-IIIa but not GP Ib. The data are consistent with the hypothesis that type VI collagen is a physiologically relevant binding site for vWf in subendothelium.

Alignment of fibrillin molecules in elastic microfibrils is defined by transglutaminase-derived cross-links.

Microfibrils were extracted from human amnion in the form of a beaded filament and analyzed for the presence of transglutaminase-derived cross-links using acrylonitrile derivatization. The cross-link structure was isolated from protease hydrolysates of beaded filaments and identified as a phenylthiocarbamyl amino acid derivative by comparison to a standard. Acid hydrolysis of the isolated cross-link gave the expected lysine and glutamic acid in a 1:1 ratio. The beaded filaments were also treated with trypsin to produce a fraction that contained the bead structure and a fraction containing fragments of the interbead filaments. Cross-links were detected in the interbead filaments but not in the beads. A large tryptic peptide that contained a cross-link was isolated and sequenced. The two amino acid sequences obtained identified both of the cross-linked molecules as fibrillin-1 and enabled the approximate localization of the cross-link sites within the molecule. The locations of cross-link sites on two adjacent molecules fixed the relative positions of fibrillin monomers within the microfibrils, providing insight into the spatial organization of fibrillin within the elastic microfibrils.

Morphological relationships of von Willebrand factor, type VI collagen, and fibrillin in human vascular subendothelium.

von Willebrand factor (vWF) plays an important role in the process of platelet adhesion after endothelial injury by serving as a bridge between constituents of the vascular subendothelium and platelet membrane receptors. We previously presented evidence that type VI collagen microfibrils serve as a binding site for vWF in human vascular subendothelium. However, others have proposed that vWF is not associated with type VI collagen but rather with the thicker elastin-associated microfibrils, which contain several proteins including fibrillin. We therefore investigated the relationships among vWF, type VI collagen, and fibrillin in human vascular subendothelium by immunoelectron microscopy using single- and double-labeling immunogold localization techniques. In addition, we observed the three-dimensional ultrastructure of vWF-microfibril complexes by stereo paired micrographs and stereo viewer. We found that vWF co-localizes only with the type VI collagen microfibrils in subendothelium but not with fibrillin microfibrils or striated collagen. The vWF is present in subendothelium in the form of electron-dense aggregates having diameters varying between 65 and 80 nm that are closely associated with, and enmesh, the type VI collagen microfibrils and have structural similarities to intracellular Weibel-Palade bodies. The occasional co-localization of type VI collagen and fibrillin adjacent to internal elastic lamina was observed. These results are consistent with the hypothesis that type VI collagen, but not fibrillin-containing microfibrils, serves as a physiologically relevant binding site for vWF in the vascular subendothelium, where the type VI collagen-vWF complex may play an important role modulating the hemostatic response to vascular injury.

The macromolecular structure of type-VI collagen. Formation and stability of filaments.

Type-VI collagen microfilaments were directly isolated from human amnion without using strong denaturing reagents. The microfilaments were characterized by electron microscopy, SDS/PAGE and immunoprecipitation. There was no evidence of other components bound to the isolated filaments and no covalent bonds between adjacent tetramers. The association between tetramers was further analyzed by studying the affinity between globular domains and the helix of type-VI collagen. Solid-phase-binding assays and conventional column chromatography showed that the globular domains have a high affinity for each other and for the helices of type-VI collagen, indicating that filaments may be assembled and stabilized in the absence of additional components. Hyaluronan did not stabilize the filaments nor facilitate the assembly of tetramers into filaments. The interaction between domains was also studied after modifying the sugar moieties of type-VI collagen globular domains and monomeric triple-helical domains. The oligosaccharides are involved in helix-helix interactions but not in interactions of the globular domains with each other or with the triple helix.

Reduced thrombogenicity of type VI collagen as compared to type I collagen.

Type VI collagen has been recently identified as a major constituent of vascular subendothelium where it serves as a binding site for von Willebrand factor. The present study compares the functional characteristics of type VI collagen with those of type I collagen with respect to platelet aggregating and secretory activities. The differences between the two collagens in platelet aggregation and serotonin and beta-thromboglobulin release were found to be highly significant (p < 0.001, p < 0.0007, p < 0.005 respectively). Our results indicate that under in vitro conditions, type VI collagen stimulates a significantly lesser platelet activation and aggregation response than collagen I, suggesting that type VI collagen may play a role in vivo to limit the platelet thrombotic response following injury to the vascular subendothelium.

Calcium binding, hydroxylation, and glycosylation of the precursor epidermal growth factor-like domains of fibrillin-1, the Marfan gene protein.

The extracellular matrix protein fibrillin-1 is a major component of elastic microfibrils, which are complex assemblies of several proteins and are found in most connective tissues, frequently associated with elastin. Fibrillin-1 contains 43 precursor epidermal growth factor-like (pEGF) domains that have a consensus sequence for calcium binding. The calcium binding potential of a fibrillin-1 pepsin fragment (PF2) was quantitatively analyzed using microvolume equilibrium dialysis. Peptide sequence data and pepsin fragment size determination indicate that PF2 contains seven pEGF domains, each with the calcium binding consensus sequence. Scatchard plot analysis of the calcium binding data shows that PF2 has six to seven high affinity binding sites with a Kd = 250 microM at pH 7.5. There is a second overlapping consensus sequence in the pEGF domains for beta-hydroxylation of a specific Asp/Asn residue. Five partially hydroxylated Asn residues have been identified by protein sequence analysis of fibrillin-1 fragments. This is the first demonstration of this modification in a connective tissue protein. The calcium binding consensus sequence also contains a conserved Ser residue with an apparently novel modification, which causes the Ser residue to behave like an Asp residue during protein sequencing. Marfan syndrome, a heritable disorder of connective tissue, is known to be associated with mutations in the FBN1 gene. Most of these mutations have been found in pEGF domains, frequently substituting Cys for another amino acid, destroying the pEGF motif secondary structure along with its calcium binding potential. Other mutations cause the substitution of single amino acids in the calcium binding consensus sequence, which could affect calcium binding but also the hydroxylation of Asp/Asn residues or the modification of Ser residues.

Neural crest cell interaction with type VI collagen is mediated by multiple cooperative binding sites within triple-helix and globular domains.

Collagen type VI (Col VI) is a primary constituent of the extracellular matrix encountered by migrating avian neural crest cells in situ and is effective in promoting attachment and motility of these cells in vitro. In this study, we have explored the molecular mechanisms of neural crest-Col VI interaction by using quantitative assays for cell attachment and migration in vitro, proteolytic fragments of the collagen, and a panel of domain-specific monoclonal antibodies. Removal of the predominant portion of the amino-terminal globular domains of Col VI tetramers by pepsin digestion (P6 fragment) resulted in a > fivefold decrease in their cell adhesion and motility-promoting activity. Further digestion of P6 with bacterial collagenase, which causes a complete loss of the amino-terminal domains plus an adjacent triple-helical segment, did not affect adhesion but reduced migration down to 40% of that seen on undigested P6. Untreated and pepsin-digested Col VI monomers were significantly less effective than their tetrameric counterparts and a M(r) 200,000 fragment, generated from pepsin-digested monomers by a second pepsin treatment, only retained 40% of the motility-promoting activity while preserving the adhesive capacity. A mixture of amino- and carboxyl-terminal globular domains supported both cell attachment and migration. While neural crest cells adhered equally well to the individual intact alpha 1 (VI)/alpha 2(VI) and alpha 3(VI) chains, they migrated most extensively on the alpha 3(VI) chain. Conversely, pepsin-digested individual alpha chains were significantly less effective in promoting cell adhesion and locomotion. Selective preincubation of Col VI microfilaments and isolated tetramers with a panel of monoclonal antibodies against triple helix, carboxyl-terminal, and amino-terminal epitopes of the different constituent chains differentially perturbed neural crest cell attachment and migration. Sites differentially involved in neural crest cell attachment and migration seemed to be present at the carboxyl termini of the alpha 1(VI) and alpha 2(VI) chains and at the amino-terminus of the alpha 3(VI) chain. The results suggest that neural crest cells interact with Col VI through multiple and cooperative binding sites present within its triple-helical and globular domains. The differential involvement and efficiency of these sites in stimulating neural crest cell adhesion and migration is strongly determined by the supramolecular organization of the collagen and requires inter- and intramolecular structural integrity. Since neural crest cell attachment and migration on Col VI was completely inhibited by anti-beta 1 integrin antibodies, there is evidence that this class of integrins is essential for the neural crest cell--Col VI interaction.

Collagen type VI in neural crest development: distribution in situ and interaction with cells in vitro.

We have examined the spatio-temporal distribution of collagen type VI (Col VI) during neural crest development in vivo and its ability to promote neural crest cell attachment and migration in vitro. An affinity purified antiserum and chain-specific monoclonal antibodies against chicken Col VI were employed to immunolocalize the collagen in tissue sections and by immunoblotting. At stages of initial neural crest cell migration, the alpha 1(VI) and alpha 2(VI) chains were immunolocalized in apposition with basement membranes of the neural tube, somites, notochord and ectoderm, whereas no immunoreactivity was seen for the alpha 3(VI) chain. Immunoblotting analysis confirmed the expression of alpha 1(VI) and alpha 2(VI) chains and the lack of detectable immunoreactivity for the alpha 3(VI) chain at these early phases of neural crest development. Conversely, at advanced phases of migration and following gangliogenesis, expression of alpha 3(VI) chain coincided with that of alpha 1(VI) and alpha 2(VI) chains in apposition with basement membranes, around the dorsal root ganglia, and in fibrillar arrangements within the developing dermis and ventral sclerotome. The ability of Col VI to promote neural crest cell attachment and migration was tested in vitro using quantitative assays for these processes. Both native microfilaments and isolated tetramers of Col VI strongly promoted neural crest cell attachment and migration. Optimal stimulation of neural crest cell adhesion and migration was dependent upon structural integrity of Col VI since unfolded and disassembled alpha chains only weakly promoted cell attachment and were virtually inactive in supporting cell movement. The importance of a native macromolecular organization of Col VI further was analyzed in experiments in which dissociated tetramers were reassociated by Ca(2+)- and temperature-dependent self-aggregation. In contrast to native microfilaments, these oligomeric complexes were less effective in promoting neural crest cell movement, but still retained the ability to stimulate maximal cell attachment. The results indicate that Col VI is a primary component of the extracellular matrix deposited along neural crest migratory pathways, where it may participate in the regulation of cell movement by functioning as a migratory substrate. The ability of Col VI to promote neural crest cell adhesion and motility is highly dependent upon maintainance of a native macromolecular arrangement.

The molecular basis of Marfan syndrome.

The Marfan syndrome is an inherited, autosomal dominant disorder that affects the skeletal, ocular, and cardiovascular systems. Recent biochemical and genetic studies have demonstrated that this deadly genetic disorder arises from defects in the connective tissue protein fibrillin. Fibrillin is a component of microfibrils, structures found in the extracellular matrices of most tissues, including those affected in Marfan patients. The appearance of microfibrils in the matrix produced by Marfan patient fibroblasts is different from that of normal cells. Genetic linkage between the fibrillin gene and the Marfan phenotype has been established and the gene mapped to the same chromosomal position as the disease locus. In several instances, the disease has been associated with mutations in the fibrillin gene, confirming that defects in fibrillin cause the Marfan syndrome.

A cysteine for glycine substitution at position 175 in an alpha 1 (I) chain of type I collagen produces a clinically heterogeneous form of osteogenesis imperfecta.

The molecular basis for Osteogenesis Imperfecta in a large kindred with a highly variable phenotype was identified by sequencing the mutant pro alpha 1 (I) protein, cDNA and genomic DNA from the proband. Fibroblasts from different affected individuals all synthesize both normal Type I procollagen molecules and abnormal Type I procollagen molecules in which one or both pro alpha 1 (I) chain(s) contain a cysteine residue within the triple helical domain. Protein studies of the proband localized the mutant cysteine residue to the alpha 1 (I) CB 8 peptide. We now report that cysteine has replaced glycine at triple helical residue 175 disrupting the invariant Gly-X-Y structural motif required for perfect triple helix formation. The consequences include post-translational overmodification, decreased thermal stability, and delayed secretion of mutant molecules. The highly variable phenotype in the present kindred cannot be explained solely on the basis of the cysteine for glycine substitution but will require further exploration.

Purification and partial characterization of fibrillin, a cysteine-rich structural component of connective tissue microfibrils.

Fibrillin, a connective tissue macromolecule (Mr = 350,000) which is normally insoluble in its tissue form, has been purified from the medium of human skin fibroblast and ligament cells in culture. Analysis of the amino acid composition indicates that fibrillin contains approximately 14% cysteine, of which one-third appears to be in the free reactive sulfhydryl form. Electron microscopic images of fibrillin reveal an extended, flexible molecule approximately 148 nm long and 2.2 nm wide. These length measurements are consistent with shape calculations based upon velocity sedimentation data. It is likely that the material we have purified from cell culture medium represents monomeric fibrillin consisting of a single polypeptide chain. Additional ultrastructural immunohistochemical data presented here suggest a model for the parallel, head-to-tail alignment of fibrillin molecules in microfibrils.

Partial sequence of a candidate gene for the Marfan syndrome.

Fibrillin is a large (relative molecular mass 350,000) glycoprotein which can be isolated from fibroblast cell cultures and is a component of the microfibrils that are ubiquitous in the connective tissue space. The microfibrils of the suspensory ligament of the lens as well as the elastic fibre microfibrils of the blood vessel wall are composed of fibrillin. The ocular and cardiovascular manifestations of the Marfan syndrome are consistent with a defect in the gene coding for a structural constituent of these connective tissues. Immunohistological experiments have recently implicated fibrillin microfibrils in the pathogenesis of the Marfan syndrome. Genetic linkage data localizing the Marfan gene to chromosome 15 and the in situ hybridization of fibrillin complementary DNA to 15q21.1 together support fibrillin as a candidate Marfan gene. As a first step towards investigating the function of fibrillin in the architecture and development of connective tissues and its relationship to the Marfan syndrome, we report the cloning and partial sequencing of fibrillin cDNA.

Extraction of extendable beaded structures and their identification as fibrillin-containing extracellular matrix microfibrils.

High molecular weight aggregates were extracted from human amnion using buffers containing 6 M guanidine hydrochloride. Rotary shadowed preparations and negatively stained samples examined by electron microscopy showed that each aggregate appeared to be a string of globular structures joined by fine filaments, giving the appearance of beads on a string. The periodicity of the beads was variable. A mouse monoclonal antibody directed against a previously characterized pepsin fragment of fibrillin was used with gold-conjugated secondary antibody and immunoelectron microscopy to show that the aggregates contained fibrillin. Similar structures were found in non-denaturing homogenates of skin, tongue, ligament, ciliary zonule, cartilage, and vitreous humor. When immunogold-labeled beaded structures were prepared for electron microscopy in the same manner as tissue, the beaded structures could no longer be seen. Instead, gold-labeled microfibrils were found which appeared to be the same as the fibrillin-containing matrix microfibrils observed in connective tissues and often associated with elastin. Thus, standard TEM protocols including fixation, dehydration, and embedding alter the ultrastructural appearance of microfibrils as compared with negative stain or rotary shadowing techniques. When skin was stretched and prepared for electron microscopy while still under tension, beaded filaments were seen in the tissue sections, but were not visible in non-stretched controls. In addition, when stretched ligament was immunolabeled with antibody directed against fibrillin while still under tension, the periodicity of antibodies along the microfibrils increased compared with non-stretched controls. We propose that microfibrils contain globular structures connected by fine filaments composed at lease in part of highly ordered, periodically distributed fibrillin molecules, whose periodicity is subject to change dependent on the tensional forces applied to the tissue in which they are contained.

In vivo and in vitro noncovalent association of excised alpha 1 (I) amino-terminal propeptides with mutant pN alpha 2(I) collagen chains in native mutant collagen in a case of Ehlers-Danlos syndrome, type VII.

The cause of the Ehlers-Danlos syndrome Type VII (EDS VII) is considered to be defective removal of the amino-terminal propeptide (N-propeptide) of Type I procollagen due to deficiency of procollagen N-proteinase, the enzyme responsible for the normal proteolytic excision of this precursor-specific domain. Molecules retaining the N-propeptide (pN-collagen molecules) are thought to cause defective fibrillogenesis and cross-linking which eventuate in dramatic joint laxity and joint dislocations, the clinical hallmark of this variety of EDS. Recent studies demonstrate that some EDS VII patients harbor small deletions of either the pro-alpha 1(I) or pro-alpha 2(I) chain of Type I procollagen. We have found an 18-amino acid deletion (due to exon outsplicing) in a mutant pro-alpha 2(I) chain from such a patient. The deleted peptide is the junctional segment (N-telopeptide) linking the alpha 2(I) N-propeptide and major triple helical domains; loss of this short segment results in union of these latter domains and produces a shortened pN alpha 2(I) chain. Directly extracted tissue collagen and pepsin-digested fibroblast collagen contain this mutant pN alpha 2(I) chain and normal alpha 1(I) chains, but not pN alpha 1(I) chains, indicating that the relatively larger alpha 1(I) N-propeptide is excised from the related alpha 1(I) chains. The fate of this alpha 1(I) N-propeptide was unclear and therefore whether or not the intact N-propeptide was, in fact, retained in native mutant collagen was also unclear. In this paper, we describe morphologic, chemical, and immunochemical studies which indicate that the alpha 1(I) N-propeptide is retained in noncovalent association with the mutant pN alpha 2(I) chain in native mutant collagen molecules both in vivo and in vitro. In both instances, the alpha 1(I) N-propeptides are proteolytically cleaved from the related alpha 1(I) chains. These data suggest that retention of a partially cleaved, but essentially intact N-propeptide in mutant collagen may play a role in the pathogenesis of this disease.