Identification of binding partners interacting with the α1-N-propeptide of type V collagen

The predominant form of type V collagen is the [α1(V)]?α2(V) heterotrimer. Mutations in COL5A1 or COL5A2, encoding respectively the α1(V)- and α2(V)-collagen chain, cause classic EDS (Ehlers-Danlos syndrome), a heritable connective tissue disorder, characterized by fragile hyperextensible skin and joint hypermobility. Approximately half of the classic EDS cases remain unexplained. Type V collagen controls collagen fibrillogenesis through its conserved α1(V)-N-propeptide domain. To gain an insight into the role of this domain, a yeast two-hybrid screen among proteins expressed in human dermal fibroblasts was performed utilizing the N-propeptide as a bait. We identified 12 interacting proteins, including extracellular matrix proteins and proteins involved in collagen biosynthesis. Eleven interactions were confirmed by surface plasmon resonance and/or co-immunoprecipitation: α1(I)- and α2(I)-collagen chains, α1(VI)-, α2(VI)- and α3(VI)-collagen chains, tenascin-C, fibronectin, PCPE-1 (procollagen C-proteinase enhancer-1), TIMP-1 (tissue inhibitor of metalloproteinases-1), MMP-2 (matrix metalloproteinase 2) and TGF-β1 (transforming growth factor β1). Solid-phase binding assays confirmed the involvement of the α1(V)-N-propeptide in the interaction between native type V collagen and type VI collagen, suggesting a bridging function of this protein complex in the cell-matrix environment. Enzymatic studies showed that processing of the α1(V)-N-propeptide by BMP-1 (bone morphogenetic protein 1)/procollagen C-proteinase is enhanced by PCPE-1. These interactions are likely to be involved in extracellular matrix homoeostasis and their disruption could explain the pathogenetic mechanism in unresolved classic EDS cases.     

Differential ultrastructural aberrations of collagen fibrils in Ehlers-Danlos syndrome types I–IV as a means of diagnostics and classification

Among the different subtypes of Ehlers-Danlos syndrome (EDS), the dominant types I–III have, so far, been uninformative biochemically and molecular genetically, and diagnostic problems with subgroup boundaries often arise. We have investigated the ultrastructural pattern of connective tissue macromolecules in skin biopsy specimens of some 85 patients aged 4 months-54 years who exhibit clinical symptoms or the suspicion of EDS I–IV. Based on the differential features of collagen fibrils and ground substance material, four distinct groups could be established. Group I (clinically EDS type I) showed disorganized collagen bundles and dense aggregations of collagen fibrils with bizarre shapes. Group II (clinically varying from EDS types I–III) revealed collagen bundles that regularly contained numerous “composite collagen fibrils” with enlarged “flower-like” cross-sections and rope-like longitudinal sections, often associated with increased amounts of matrix substances in the form of electron-dense irregular strands and filaments in a branched network. Group III (clinically EDS types II–III) presented smaller isolated collagen flowers and ropes associated with excessive filamentous ground substance material and flocculent material. Group IV (with clinical symptoms of EDS type IV) had a dermis thinned to one third of the normal and a reduced number of collagen bundles with small diameter fibrils. In 13 patients, the abnormal ultrastructural dermal architecture did not coincide with any of these four groups or with the pattern of any other inherited connective tissue disorder. In 16 additional patients with mostly mild clinical symptoms, such as muscle weakness and small joint hyperlaxity, no ultrastructural aberrations could be found. Even though the primary defects underlying the respective aberration of the collagen fibrils are still unknown, the differential ultrastructural changes of the collagen fibrils together with clinical symptoms should, as in other heterogeneous genetic disorders, facilitate the (provisional?) classification of EDS and permit the diagnosis of individual cases.     

Reduced type I collagen utilization: a pathogenic mechanism in COL5A1 haplo-insufficient Ehlers-Danlos syndrome

To examine mechanisms by which reduced type V collagen causes weakened connective tissues in the Ehlers-Danlos syndrome (EDS), we examined matrix deposition and collagen fibril morphology in long-term dermal fibroblast cultures. EDS cells with COL5A1 haplo-insufficiency deposited less than one-half of hydroxyproline as collagen compared to control fibroblasts, though total collagen synthesis rates are near-normal because type V collagen represents a small fraction of collagen synthesized. Cells from patients with osteogenesis imperfecta (OI) and haplo-insufficiency for proalpha1(I) chains of type I collagen also incorporated about one-half the collagen as controls, but this amount was proportional to their reduced rates of total collagen synthesis. Collagen fibril diameter was inversely proportional to type V/type I collagen ratios (EDS > control > OI). However, a reduction of type V collagen, in the EDS derived cells, was associated with the assembly of significantly fewer fibrils compared to control and OI cells. These data indicate that in cell culture, the quantity of collagen fibrils deposited in matrix is highly sensitive to reduction in type V collagen, far out of proportion to type V collagen's contribution to collagen mass.     

Mutations in the COL5A1 gene are causal in the Ehlers-Danlos syndromes I and II.

The Ehlers-Danlos syndrome (EDS) is a heterogeneous connective-tissue disorder of which at least nine subtypes are recognized. Considerable clinical overlap exists between the EDS I and II subtypes, suggesting that both are allelic disorders. Recent evidence based on linkage and transgenic mice studies suggest that collagen V is causally involved in human EDS. Collagen V forms heterotypic fibrils with collagen I in many tissues and plays an important role in collagen I fibrillogenesis. We have identified a mutation in COL5A1, the gene encoding the pro(alpha)1(V) collagen chain, segregating with EDS I in a four-generation family. The mutation causes the substitution of the most 5' cysteine residue by a serine within a highly conserved sequence of the pro(alpha)1(V) C-propeptide domain and causes reduction of collagen V by preventing incorporation of the mutant pro(alpha)1(V) chains in the collagen V trimers. In addition, we have detected splicing defects in the COL5A1 gene in a patient with EDS I and in a family with EDS II. These findings confirm the causal role of collagen V in at least a subgroup of EDS I, prove that EDS I and II are allelic conditions, and represent a, so far, unique example of a human collagen disorder caused by substitution of a highly conserved cysteine residue in the C-propeptide domain of a fibrillar collagen.     

Clinical and ultrastructural heterogeneity of type IV Ehlers-Danlos syndrome

Summary Ehlers-Danlos syndrome (EDS) type IV is a clinically and genetically heterogeneous disorder characterized by thin skin, prominent venous vascular markings, markedly increased bruising, and an increased likelihood of large bowel and large artery rupture. We studied two type IV EDS patients. Both have decreased amounts of type III collagen in skin, but ultrastructural examination of dermis showed massive dilation of rough endoplasmic reticulum in dermal fibroblasts in one, but not the other. Both had a major population of collagen fibrils of small diameter. Although previous studies suggested absent synthesis of type III collagen as the hallmark of one type of EDS IV, several abnormalities in metabolism of that type of collagen may be responsible for the phenotype in these disorders. Such disorders are likely to provide better understanding of the function of specific collagens in tissues.     

Classical Ehlers-Danlos syndrome caused by a mutation in type I collagen

Classical Ehlers-Danlos syndrome (EDS) is characterized by skin hyperelasticity, joint hypermobility, increased tendency to bruise, and abnormal scarring. Mutations in type V collagen, a regulator of type I collagen fibrillogenesis, have been shown to underlie this type of EDS. However, to date, mutations have been found in only a limited number of patients, which suggests genetic heterogeneity. In this article, we report two unrelated patients with typical features of classical EDS, including excessive skin fragility, in whom we found an identical arginine-->cysteine substitution in type I collagen, localized at position 134 of the alpha1(I) collagen chain. The arginine residue is highly conserved and localized in the X position of the Gly-X-Y triplet. As a consequence, intermolecular disulfide bridges are formed, resulting in type I collagen aggregates, which are retained in the cells. Whereas substitutions of glycine residues in type I collagen invariably result in osteogenesis imperfecta, substitutions of nonglycine residues in type I collagen have not yet been associated with a human disease. In contrast, arginine-->cysteine substitutions in type II collagen have been identified in a variety of chondrodysplasias. Our findings show that mutations in other fibrillar collagens can be causally involved in classical EDS and point to genetic heterogeneity of this disorder.     

The pro-alpha3(V) collagen chain. Complete primary structure, expression domains in adult and developing tissues, and comparison to the structures and expression domains of the other types V and XI procollagen chains

The low abundance fibrillar collagen type V is widely distributed in tissues as an alpha1(V)(2)alpha2(V) heterotrimer that helps regulate the diameters of fibrils of the abundant collagen type I. Mutations in the alpha1(V) and alpha2(V) chain genes have been identified in some cases of classical Ehlers-Danlos syndrome (EDS), in which aberrant collagen fibrils are associated with connective tissue fragility, particularly in skin and joints. Type V collagen also exists as an alpha1(V)alpha2(V)alpha3(V) heterotrimer that has remained poorly characterized chiefly due to inability to obtain the complete primary structure or nucleic acid probes for the alpha3(V) chain or its biosynthetic precursor, pro-alpha3(V). Here we provide human and mouse full-length pro-alpha3(V) sequences. Pro-alpha3(V) is shown to be closely related to the alpha1(V) precursor, pro-alpha1(V), but with marked differences in N-propeptide sequences, and collagenous domain features that provide insights into the low melting temperature of alpha1(V)alpha2(V)alpha3(V) heterotrimers, lack of heparin binding by alpha3(V) chains and the possibility that alpha1(V)alpha2(V)alpha3(V) heterotrimers are incorporated into heterotypic fibrils. In situ hybridization of mouse embryos detects alpha3(V) expression primarily in the epimysial sheaths of developing muscles and within nascent ligaments adjacent to forming bones and in joints. This distribution, and the association of alpha1(V), alpha2(V), and alpha3(V) chains in heterotrimers, suggests the human alpha3(V) gene COL5A3 as a candidate locus for at least some cases of classical EDS in which the alpha1(V) and alpha2(V) genes have been excluded, and for at least some cases of the hypermobility type of EDS, a condition marked by gross joint laxity and chronic musculoskeletal pain. COL5A3 is mapped to 19p13.2 near a polymorphic marker that should be useful in analyzing linkage with EDS and other disease phenotypes.     

Phenotypic effects of Ehlers�CDanlos syndrome-associated mutation on the FnIII domain of tenascin-X

Tenascin‐X (TNX) is an extracellular matrix (ECM) protein and interacts with a wide variety of molecules in the ECM as well as on the membrane. Deficiency of TNX causes a recessive form of Ehlers–Danlos syndrome (EDS) characterized by hyperelastic and fragile skin, easy bruising, and hypermobile joints. Three point mutations in TNX gene were found to be associated with hypermobility type EDS and one of such mutations is the V1195M mutation at the 7th fibronectin Type III domain (TNXfn7). To help elucidate the underlying molecular mechanism connecting this mutation to EDS, here we combined homology modeling, chemical denaturation, single molecule atomic force microscopy, and molecular dynamics (MD) simulation techniques to investigate the phenotypic effects of V1195M on TNXfn7. We found that the V1195M mutation does not alter the three‐dimensional structure of TNXfn7 and had only mild destabilization effects on the thermodynamic and mechanical stability of TNXfn7. However, MD simulations revealed that the mutation V1195M significantly alters the flexibility of the C′E loop of TNXfn7. As loops play important roles in protein–protein and protein–ligand interactions, we hypothesize that the decreased loop flexibility by V1195M mutation may affect the binding of TNX to ECM molecules and thus adversely affect collagen deposition and fibrillogenesis. Our results may provide new insights in understanding the molecular basis for the pathogenesis of V1195M‐resulted EDS.     

Development of a functional skin matrix requires deposition of collagen V heterotrimers

Collagen V is a minor component of the heterotypic I/III/V collagen fibrils and the defective product in most cases of classical Ehlers Danlos syndrome (EDS). The present study was undertaken to elucidate the impact of collagen V mutations on skin development, the most severely affected EDS tissues, using mice harboring a targeted deletion of the alpha2(V) collagen gene (Col5a2). Contrary to the original report, our studies indicate that the Col5a2 deletion (a.k.a. the pN allele) represents a functionally null mutation that affects matrix assembly through a complex sequence of events. First the mutation impairs assembly and/or secretion of the alpha1(V)(2)alpha2(V) heterotrimer with the result that the alpha1(V) homotrimer is the predominant species deposited into the matrix. Second, the alpha1(V) homotrimer is excluded from incorporation into the heterotypic collagen fibrils and this in turn severely impairs matrix organization. Third, the mutant matrix stimulates a compensatory loop by the alpha1(V) collagen gene that leads to additional deposition of alpha1(V) homotrimers. These data therefore underscore the importance of the collagen V heterotrimer in dermal fibrillogenesis. Furthermore, reduced thickness of the basement membranes underlying the epidermis and increased apoptosis of the stromal fibroblasts in pN/pN skin strongly indicate additional roles of collagen V in the development of a functional skin matrix.     

Murine model of the Ehlers-Danlos syndrome. col5a1 haploinsufficiency disrupts collagen fibril assembly at multiple stages

The most commonly identified mutations causing Ehlers-Danlos syndrome (EDS) classic type result in haploinsufficiency of proalpha1(V) chains of type V collagen, a quantitatively minor collagen that co-assembles with type I collagen as heterotypic fibrils. To determine the role(s) of type I/V collagen interactions in fibrillogenesis and elucidate the mechanism whereby half-reduction of type V collagen causes abnormal connective tissue biogenesis observed in EDS, we analyzed mice heterozygous for a targeted inactivating mutation in col5a1 that caused 50% reduction in col5a1 mRNA and collagen V. Comparable with EDS patients, they had decreased aortic stiffness and tensile strength and hyperextensible skin with decreased tensile strength of both normal and wounded skin. In dermis, 50% fewer fibrils were assembled with two subpopulations: relatively normal fibrils with periodic immunoreactivity for collagen V where type I/V interactions regulate nucleation of fibril assembly and abnormal fibrils, lacking collagen V, generated by unregulated sequestration of type I collagen. The presence of the aberrant fibril subpopulation disrupts the normal linear and lateral growth mediated by fibril fusion. Therefore, abnormal fibril nucleation and dysfunctional fibril growth with potential disruption of cell-directed fibril organization leads to the connective tissue dysfunction associated with EDS.     

Exclusion of COL1A1, COL1A2, and COL3A1 genes as candidate genes for Ehlers-Danlos syndrome type I in one large family

Summary Ehlers-Danlos syndrome (EDS) type I is a generalized connective tissue disorder, the major manifestations of which are soft, velvety hyperextensible skin and moderately severe joint hypermobility. The gene defect or defects causing EDS type I have not yet been defined, but previous observations suggested that the syndrome may be caused by mutations in the genes for type-I collagen (COL1A1 and COL1A2) or type-III collagen (COL3A1). Here, we performed linkage studies for these three genes in large Azerbaijanian family with EDS type I. Three polymorphisms in the COL3A1 gene, two in the COL1A1 gene, and one in the COL1A2 gene were tested using the polymerase chain reaction. The data obtained excluded linkage of any of the three genes to EDS type I in the family.On leave of absence from Institute of Human Genetics, National Research Center of Medical Genetics, Moskvorechie St., 1. Moscow 115478, USSR     

Mutations near amino end of alpha1(I) collagen cause combined osteogenesis imperfecta/Ehlers-Danlos syndrome by interference with N-propeptide processing

Patients with OI/EDS form a distinct subset of osteogenesis imperfecta (OI) patients. In addition to skeletal fragility, they have characteristics of Ehlers-Danlos syndrome (EDS). We identified 7 children with types III or IV OI, plus severe large and small joint laxity and early progressive scoliosis. In each child with OI/EDS, we identified a mutation in the first 90 residues of the helical region of alpha1(I) collagen. These mutations prevent or delay removal of the procollagen N-propeptide by purified N-proteinase (ADAMTS-2) in vitro and in pericellular assays. The mutant pN-collagen which results is efficiently incorporated into matrix by cultured fibroblasts and osteoblasts and is prominently present in newly incorporated and immaturely cross-linked collagen. Dermal collagen fibrils have significantly reduced cross-sectional diameters, corroborating incorporation of pN-collagen into fibrils in vivo. Differential scanning calorimetry revealed that these mutant collagens are less stable than the corresponding procollagens, which is not seen with other type I collagen helical mutations. These mutations disrupt a distinct folding region of high thermal stability in the first 90 residues at the amino end of type I collagen and alter the secondary structure of the adjacent N-proteinase cleavage site. Thus, these OI/EDS collagen mutations are directly responsible for the bone fragility of OI and indirectly responsible for EDS symptoms, by interference with N-propeptide removal.     

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.     

Type V collagen: molecular structure and fibrillar organization of the chicken alpha 1(V) NH2-terminal domain, a putative regulator of corneal fibrillogenesis

Previous work from our laboratories has demonstrated that: (a) the striated collagen fibrils of the corneal stroma are heterotypic structures composed of type V collagen molecules coassembled along with those of type I collagen, (b) the high content of type V collagen within the corneal collagen fibrils is one factor responsible for the small, uniform fibrillar diameter (25 nm) characteristic of this tissue, (c) the completely processed form of type V collagen found within tissues retains a large noncollagenous region, termed the NH2- terminal domain, at the amino end of its alpha 1 chain, and (d) the NH2- terminal domain may contain at least some of the information for the observed regulation of fibril diameters. In the present investigation we have employed polyclonal antibodies against the retained NH2- terminal domain of the alpha 1(V) chain for immunohistochemical studies of embryonic avian corneas and for immunoscreening a chicken cDNA library. When combined with cDNA sequencing and molecular rotary shadowing, these approaches provide information on the molecular structure of the retained NH2-terminal domain as well as how this domain might function in the regulation of fibrillar structure. In immunofluorescence and immunoelectron microscopy analyses, the antibodies against the NH2-terminal domain react with type V molecules present within mature heterotypic fibrils of the corneal stroma. Thus, epitopes within at least a portion of this domain are exposed on the fibril surface. This is in marked contrast to mAbs which we have previously characterized as being directed against epitopes located in the major triple helical domain of the type V molecule. The helical epitopes recognized by these antibodies are antigenically masked on type V molecules that have been assembled into fibrils. Sequencing of the isolated cDNA clones has provided the conceptual amino acid sequence of the entire amino end of the alpha 1(V) procollagen chain. The sequence shows the location of what appear to be potential propeptidase cleavage sites. One of these, if preferentially used during processing of the type V procollagen molecule, can provide an explanation for the retention of the NH2-terminal domain in the completely processed molecule. The sequencing data also suggest that the NH2-terminal domain consists of several regions, providing a structure which fits well with that of the completely processed type V molecule as visualized by rotary shadowing.     

Human collagen gene COL5A1 maps to the q34.2----q34.3 region of chromosome 9, near the locus for nail-patella syndrome.

Type V collagen is a fibrillar collagen that is widely distributed in tissues as a minor component of extracellular matrix and is usually composed of one pro alpha 2 (V) and two pro alpha 1 (V) chains. In this report, recently isolated cDNA and genomic clones, which encode the pro alpha 1 (V) chain, are used as probes for hybridization to filter-bound DNA from a panel of human-mouse hybrid cell lines and for in situ hybridization to metaphase chromosomes. These studies establish the chromosomal location of the COL5A1 gene, which encodes the pro alpha 1 (V) chain, within segment 9q34.2----q34.3. These findings add to the previously characterized dispersion of collagen genes in the human genome, as this is the first example of a collagen locus on chromosome 9. In addition, these studies place COL5A1 near the locus for the genetic disorder, nail-patella syndrome (hereditary osteo-onychodysplasia), which also maps to 9q34.     

Mutations in FKBP14 cause a variant of Ehlers-Danlos syndrome with progressive kyphoscoliosis, myopathy, and hearing loss

We report on an autosomal-recessive variant of Ehlers-Danlos syndrome (EDS) characterized by severe muscle hypotonia at birth, progressive scoliosis, joint hypermobility, hyperelastic skin, myopathy, sensorineural hearing impairment, and normal pyridinoline excretion in urine. Clinically, the disorder shares many features with the kyphoscoliotic type of EDS (EDS VIA) and Ullrich congenital muscular dystrophy. Linkage analysis in a large Tyrolean kindred identified a homozygous frameshift mutation in FKBP14 in two affected individuals. Based on the cardinal clinical characteristics of the disorder, four additional individuals originating from different European countries were identified who carried either homozygous or compound heterozygous mutations in FKBP14. FKBP14 belongs to the family of FK506-binding peptidyl-prolyl cis-trans isomerases (PPIases). ER-resident FKBPs have been suggested to act as folding catalysts by accelerating cis-trans isomerization of peptidyl-prolyl bonds and to act occasionally also as chaperones. We demonstrate that FKBP14 is localized in the endoplasmic reticulum (ER) and that deficiency of FKBP14 leads to enlarged ER cisterns in dermal fibroblasts in vivo. Furthermore, indirect immunofluorescence of FKBP14-deficient fibroblasts indicated an altered assembly of the extracellular matrix in vitro. These findings suggest that a disturbance of protein folding in the ER affecting one or more components of the extracellular matrix might cause the generalized connective tissue involvement in this disorder. FKBP14 mutation analysis should be considered in all individuals with apparent kyphoscoliotic type of EDS and normal urinary pyridinoline excretion, in particular in conjunction with sensorineural hearing impairment.