A heterozygous collagen defect in a variant of the Ehlers-Danlos syndrome type VII. Evidence for a deleted amino-telopeptide domain in the pro-alpha 2(I) chain

A structural defect in the alpha 2(I) chain of type I collagen was characterized in a new case of the Ehlers-Danlos syndrome type VII. The patient's skin, fascia, and bone collagens all showed an abnormal additional chain, pN-alpha 2(I)s, running slower than the alpha 2(I) chain on electrophoresis. The extension was shown to be on the amino-terminal fragment of pN-alpha (I)s by cleavage with human collagenase, but pepsin was unable to convert pN-alpha 2(I)s to alpha 2(I). Skin collagen was 4-fold more extractable and contained fewer beta-dimers and a lower concentration of cross-linking amino acids than control skin collagen. Electron micrographs of both dermis and bone showed markedly irregular ragged outlines of the collagen fibrils in cross-section, although the patient had no clinical signs of bone disease. Procollagen secreted by her skin fibroblasts in culture showed equal amounts of the normal and abnormal alpha 2(I) chains on pepsin digestion. Before pepsin, the pN-alpha 2(I) component ran as a doublet on electrophoresis; pepsin removed only the normal slower chain. The suspected deletion in pN-alpha 2(I)s was traced by CNBr peptide analysis to the N-propeptide fragment, which behaved on electrophoresis about 15-20 residues smaller than that from the normal pN-alpha 2(I) chain. The simplest genetic explanation is a spontaneous heterozygote in which one normal and one abnormal allele for the pro-alpha 2(I) gene are expressed, the protein defect being a deletion of the junction domain that spans the N-propeptidase cleavage site and the N-telopeptide cross-linking sequence.     

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.     

Deletion of 24 amino acids from the pro-alpha 1(I) chain of type I procollagen in a patient with the Ehlers-Danlos syndrome type VII

A child with the type VII form of the Ehlers-Danlos syndrome was shown to have a structural defect in the amino terminus of the pro-alpha 1(I) chain of type I procollagen. Normal and mutant amino-terminal cyanogen bromide peptides (pN-alpha 1(I) CB0,1 peptides) were purified from the medium of the patient's cultured fibroblasts. Amino acid sequencing of tryptic peptides derived from the mutant pN-alpha 1(I) CB0,1 peptide showed that an expected sequence of 24 amino acids (positions 136-159 of the normal pN-alpha 1(I) CB0,1 peptide) was deleted. The segment deleted from the mutant pro-alpha 1(I) chain contains the small globular region of the NH2-propeptide, the procollagen N-proteinase cleavage site, the NH2-telopeptide, and first triplet of the helix of the alpha I(I) collagen chain (Chu, M.-L., de Wet, W., Bernard, M., Ding, J.F., Morabito, M., Myers, J., Williams, C., and Ramirez, F. (1984) Nature 310, 337-340). Loss of the procollagen N-proteinase cleavage site from the mutant pro-alpha 1(I) chain accounted for the persistence of its NH2-propeptide despite normal production of the N-proteinase by cultured mutant fibroblasts. Collagen production by mutant fibroblasts was doubled possibly due to reduced feedback inhibition by the NH2-propeptides. The child appeared to be heterozygous for the peptide deletion and, as the parents did not show any evidence of the deletion, it is likely that the child had a new mutation of one allele of the pro-alpha 1(I) gene. The deleted peptide corresponds precisely to the sequence coded by exon 46 of the normal pro-alpha 1(I) gene (Chu, M.-L., de Wet, W., Bernard, M., Ding, J.F., Morabito, M., Myers, J., Williams, C., and Ramirez, F. (1984) Nature 310, 337-340).     

Ehlers-Danlos syndrome type VIIB. Deletion of 18 amino acids comprising the N-telopeptide region of a pro-alpha 2(I) chain

A patient with Ehlers-Danlos syndrome Type VIIB was found to have an interstitial deletion of 18 amino acids in approximately half of the pro-alpha 2(I) chains of Type I procollagen. Analysis of pepsin-solubilized tissue and fibroblast collagen revealed an abnormal additional chain, alpha 2(I)', which migrated in sodium dodecyl sulfate-5% polyacrylamide gel electrophoresis between the normal alpha 1(I) and alpha 2(I) chains. The apparent ratio of normal alpha 1(I):mutant alpha 2(I)':normal alpha 2(I) was 4:1:1. Procollagen studies and enzyme digestion studies of native mutant collagen suggested defective removal of the amino propeptide. Sieve chromatography of CNBr peptides from purified alpha 2(I)' chains revealed the absence of the normal amino telopeptide fragment CB 1 and the appearance of a larger new peptide of approximately 60 residues (CB X). Compositional and sequencing studies of this peptide identified normal amino propeptide sequences. However, the most carboxyl-terminal tryptic peptide of CB X differed substantially in composition and sequence from the expected and was found to have an interstitial deletion of 18 amino acids corresponding to the N-telopeptide of the pro-alpha 2(I) chain. This deletion removes the normal sites of cleavage of the N-proteinase and also removes a critical cross-linking lysine residue. The 18 amino acids deleted correspond exactly to the residues encoded by exon 6 of the pro-alpha 2(I) collagen gene (COL 1 A2), and, therefore, the protein defect may be due to a genomic deletion, or alternatively, an RNA splicing defect.     

Molecular mechanism of alpha 1(I)-osteogenesis imperfecta/Ehlers-Danlos syndrome: unfolding of an N-anchor domain at the N-terminal end of the type I collagen triple helix

We demonstrate that 85 N-terminal amino acids of the alpha1(I) chain participate in a highly stable folding domain, acting as the stabilizing anchor for the amino end of the type I collagen triple helix. This anchor region is bordered by a microunfolding region, 15 amino acids in each chain, which include no proline or hydroxyproline residues and contain a chymotrypsin cleavage site. Glycine substitutions and amino acid deletions within the N-anchor domain induce its reversible unfolding above 34 degrees C. The overall triple helix denaturation temperature is reduced by 5-6 degrees C, similar to complete N-anchor removal. N-propeptide partially restores the stability of mutant procollagen but not sufficiently to prevent N-anchor unfolding and a conformational change at the N-propeptide cleavage site. The ensuing failure of N-proteinase to cleave at the misfolded site leads to incorporation of pN-collagen into fibrils. Similar, but weaker, effects are caused by G88E substitution in the adjacent triplet, which appears to alter N-anchor structure as well. As in Ehlers-Danlos syndrome (EDS) VIIA/B, fibrils containing pN-collagen are thinner and weaker causing EDS-like laxity of large and small joints and paraspinal ligaments. However, distinct structural consequences of N-anchor destabilization result in a distinct alpha1(I)-osteogenesis imperfecta (OI)/EDS phenotype.     

A targeted mutation at the known collagenase cleavage site in mouse type I collagen impairs tissue remodeling

Degradation of type I collagen, the most abundant collagen, is initiated by collagenase cleavage at a highly conserved site between Gly775 and Ile776 of the alpha 1 (I) chain. Mutations at or around this site render type I collagen resistant to collagenase digestion in vitro. We show here that mice carrying a collagenase-resistant mutant Col1a-1 transgene die late in embryo-genesis, ascribable to overexpression of the transgene, since the same mutation introduced into the endogenous Col1a-1 gene by gene targeting permitted normal development of mutant mice to young adulthood. With increasing age, animals carrying the targeted mutation developed marked fibrosis of the dermis similar to that in human scleroderma. Postpartum involution of the uterus in the mutant mice was also impaired, with persistence of collagenous nodules in the uterine wall. Although type I collagen from the homozygous mutant mice was resistant to cleavage by human or rat fibroblast collagenases at the helical site, only the rat collagenase cleaved collagen trimers at an additional, novel site in the nonhelical N-telopeptide domain. Our results suggest that cleavage by murine collagenase at the N-telopeptide site could account for resorption of type I collagen during embryonic and early adult life. During intense collagen resorption, however, such as in the immediate postpartum uterus and in the dermis later in life, cleavage at the helical site is essential for normal collagen turnover. Thus, type I collagen is degraded by at least two differentially controlled mechanisms involving collagenases with distinct, but overlapping, substrate specificities.     

Type I procollagens containing substitutions of aspartate, arginine, and cysteine for glycine in the pro alpha 1 (I) chain are cleaved slowly by N-proteinase, but only the cysteine substitution introduces a kink in the molecule.

Type I procollagen was purified from the medium of dermal fibroblasts cultured from four individuals with osteogenesis imperfecta (OI) type II who had mutations in the COL1A1 gene of type I procollagen. The procollagens were mixtures of normal molecules and molecules that contained substitutions of aspartate for glycine 97, arginine for glycine 550, cysteine for glycine 718, and aspartate for glycine 883 in one or both of the alpha 1 (I) chains of the molecule. The procollagens were cleaved more slowly than control type I procollagen by procollagen N-proteinase. Double-reciprocal plots of initial relative velocities and initial substrate concentrations indicated that the OI procollagens were all cleaved slowly by N-proteinase because of decreased Vmax, rather than increased Km. This suggested that slow cleavage of the OI procollagens by N-proteinase was the result of slow conversion of the N-proteinase-procollagen complex. Further experiments showed that the vertebrate collagenase A fragment of the aspartate for glycine alpha 1(I) 883 OI procollagen that contained the N-proteinase cleavage site but not the site of the substitution was also cleaved more slowly by N-proteinase than the normal vertebrate collagenase A fragments in the samples. These data show, for the first time, that an altered triple-helical structure is propagated from the site of a substitution of a bulky residue for glycine to the amino-terminal end of the procollagen molecule and disrupts the conformation of the N-proteinase cleavage site. Rotary shadowing electron microscopy of molecules in the preparation of cysteine for glycine alpha 1(I)-718 showed the presence of a kink in approximately 5% of a population of molecules in which 60% were abnormal and 20% contained a disulfide bond. In contrast, procollagens containing aspartate and arginine for glycine were indistinguishable by rotary shadowing electron microscopy from those in control samples. The results here confirm previous suggestions that substitution of cysteine for glycine in the alpha 1(I) chain of type I collagen can introduce a kink near the site of the substitution. However, the presence of a kink is not a prerequisite for delayed cleavage of abnormal procollagens by N-proteinase.     

A base substitution at a splice site in the COL3A1 gene causes exon skipping and generates abnormal type III procollagen in a patient with Ehlers-Danlos syndrome type IV

The dermis of a child with Ehlers-Danlos syndrome type IV (EDS-IV) contained about 11% of the normal amount of type III collagen and cultured dermal fibroblasts produced a reduced amount of type III procollagen which was secreted poorly. Type III collagen produced by these cells contained normal and abnormal alpha-chains and cyanogen bromide peptides. The site of the structural defect in the abnormal alpha 1 (III) chains was localized to the region of Met797, which is at the junction of the two carboxyl-terminal CB5 and CB9 cyanogen bromide peptides. Chemical cleavage of heteroduplexes formed between EDS-IV mRNA and a normal cDNA clone covering the CB5 and CB9 region showed that about 100 nucleotides were mismatched. Sequencing of amplified and cloned cDNA spanning the mutant region revealed a 108 nucleotide deletion corresponding to amino acid residues Gly775 to Lys810. The deleted nucleotide sequence corresponded to sequences that, by analogy to the organization of the type I collagen genes, should be precisely encoded by exon 41 of the COL3A1 gene. Sequencing of amplified genomic DNA, prepared using disimilar amounts of primers specific for exons 41 and 42, displayed a base substitution (G-to-A) in the highly conserved GT dinucleotide of the 5' splice site of intron 41. Normal sequences were also obtained from the normal allele. It is likely that the GT-to-AT transition at the splice donor site of intron 41 generated an abnormally spliced mRNA in which sequences of exon 40 and 42 were joined together with maintenance of the reading frame. The corresponding peptide deletion included the cyanogen bromide cleavage site Met797-Pro798 and the mammalian collagenase cleavage site at Gly781-Ile782. These losses account for the resistance of EDS-IV collagen to cyanogen bromide and mammalian collagenase digestion. Cultured fibroblasts produced normal homotrimer, mutant homotrimer, and mixed heterotrimer type III collagen molecules. The mutant homotrimer molecules were the major pepsin-resistant species and about 69% of the alpha 1(III) mRNA was in the mutant form.     

Deletion of the pro-alpha 1(I) N-propeptide affects secretion of type I collagen in Chinese hamster lung cells but not in Mov-13 mouse cells.

We have introduced two mutations into a full-length human pro-alpha 1(I) cDNA that delete 114 amino acids or the entire 139 amino acids of the N-propeptide domain. Wild-type and mutated versions of the cDNA were introduced into cultured Chinese hamster lung (CHL) cells, which do not produce endogenous type I collagen, and into Mov-13 mouse cells, which produce endogenous pro-alpha 2(I) chains but not pro-alpha 1(I) chains. As judged by resistance to proteases, neither mutation impaired intracellular triple helical assembly of human alpha 1(I) homotrimers in CHL cells, or of chimeric type I collagen comprised of human alpha 1(I) and mouse alpha 2(I) chains in Mov-13 cells. Thus, the N-propeptide is not necessary for intracellular assembly of the main helical collagen domain of type I collagen. In CHL cells the rate of secretion of the mutant homotrimers was greatly reduced as compared to wild type homotrimers, and by immunofluorescence and immunoelectron microscopy, the mutant chains were shown to be accumulated in large vesicular expansions of the rough endoplasmic reticulum. When such cells were retransfected with cDNA encoding wild-type human alpha 2(I) chains, mutant alpha 1(I) chains were not rescued and heterotrimers containing the mutant chains were also retained in the intracellular vesicles. By contrast, deletion of the N-propeptide did not affect secretion of heterotrimers containing mutant chains from Mov-13 cells. Thus, an intact N-propeptide appears necessary for efficient secretion of type I collagen from some but not all cell types.     

Mutations that alter the primary structure of type I procollagen have long-range effects on its cleavage by procollagen N-proteinase

Type I/II procollagen N-proteinase was partially purified from chick embryos and used to examine the rate of cleavage of a series of purified type I procollagens synthesized by fibroblasts from probands with heritable disorders of connective tissue. The rate of cleavage was normal with procollagen from a proband with osteogenesis imperfecta that was overmodified by posttranslational enzymes. Therefore, posttranslational overmodification of the protein does not in itself alter the rate of cleavage under the conditions of the assay employed. Cleavage of the procollagen, however, was altered in several procollagens with known mutations in primary structure. Two of the procollagens had in-frame deletions of 18 amino acids encoded by exons 11 and 33 of the pro alpha 2(I) gene. In both procollagens, both the pro alpha 1(I) and the pro alpha 2(I) chains were totally resistant to cleavage. With a procollagen in which glycine-907 of the alpha 2(I) chain domain was substituted with aspartate, both pro alpha chains were cleaved but at a markedly decreased rate. The results, therefore, establish that mutations that alter the primary structure of the pro alpha chains of procollagen at sites far removed from the N-proteinase cleavage site can make the protein resistant to cleavage by the enzyme. The long-range effects of in-frame deletions or other changes in amino acid sequence are probably explained by their disruption of the hairpin structure that is formed by each of the three pro alpha chains in the region containing the cleavage site and that is essential for cleavage of the procollagen molecule by N-proteinase.     

Ehlers Danlos syndrome type VIIB. Incomplete cleavage of abnormal type I procollagen by N-proteinase in vitro results in the formation of copolymers of collagen and partially cleaved pNcollagen that are near circular in cross-section.

We have shown that a child with Ehlers Danlos syndrome (EDS) type VII has a G to A transition at the first nucleotide of intron 6 in one of her COL1A2 alleles. Half of the cDNA clones prepared from the proband's pro alpha 2(I) mRNA lacked exon 6. The type I procollagen secreted by the proband's dermal fibroblasts in culture was purified, and collagen fibrils were generated in vitro by cleavage of the procollagen with the procollagen N- and C-proteinases. Incubation of the procollagen with N-proteinase resulted in a 1:1 mixture of pCcollagen and uncleaved procollagen. Incubation of this mixture with C-proteinase generated collagen and abnormal pNcollagen (pNcollagen-ex6) that readily copolymerized into fibrils. By electron microscopy these fibrils resembled the hieroglyphic fibrils seen in the N-proteinase-deficient skin of dermatosparactic animals and humans and were distinct from the near circular cross-section fibrils seen in the tissues of individuals with EDS type VII. Further incubation of the hieroglyphic fibrils with N-proteinase resulted in partial cleavage of the pNcollagen-ex6 in which the abnormal pN alpha 2(I) chains remained intact. These fibrils were not hieroglyphic but were near circular in cross-section. Fibrils formed from collagen and pNcollagen-ex6 that had been partially cleaved with elevated amounts of N-proteinase prior to fibril formation were also near circular in cross-section. The results are consistent with a model of collagen fibril formation in which the intact N-propeptides are located exclusively at the surface of the hieroglyphic fibrils. Partial cleavage of the pNcollagen-ex6 by N-proteinase allows the N-propeptides to be incorporated within the body of the fibrils. The model provides an explanation for the morphology and molecular composition of collagen fibrils in the tissues of patients with EDS type VII.     

Human and chick alpha 2(I) collagen mRNA: comparison of the 5' end in osteoblasts and fibroblasts

Type I collagen, a heterotrimer of two alpha 1(I) chains and one alpha 2(I) chain, is the major structural protein of bone, skin, and tendon. The collagen of patients with bone diseases has been studied in skin fibroblasts instead of osteoblasts because the genes for type I collagen are single-copy genes. While these studies should detect structural changes in the gene, they might fail to detect defects in processes which are dependent on tissue-specific expression. The studies reported here sought to determine whether the expression of type I collagen in skin and bone was characterized by the use of alternate promoters or alternative splicing in the N-propeptide region. Primer extension and nuclease S1 protection experiments were used to analyze the structure of the alpha 2(I) mRNA from the 5' end of the gene through the N-telopeptide coding region (exons 1-6) in human and chick osteoblasts and fibroblasts. The protection and primer extension experiments using human and chick mRNA demonstrated identical routes of splicing in skin and bone at the first five splice junctions. These studies provide reassurance that information obtained from the study of type I collagen in fibroblasts may be extrapolated to bone.     

A tripeptide deletion in the triple-helical domain of the pro alpha 1(I) chain of type I procollagen in a patient with lethal osteogenesis imperfecta does not alter cleavage of the molecule by N-proteinase.

Dermal fibroblasts from a fetus with perinatal lethal osteogenesis imperfecta synthesized normal and abnormal type I procollagen molecules. The abnormal molecules contained one or two pro alpha 1(I) chains in which glycine, alanine, and hydroxyproline at positions 874, 875, and 876 in the triple-helical region were deleted as the result of a 9-base pair genomic deletion. Molecules that contained abnormal chains were overmodified from the site of the deletion toward the amino-terminal region of the molecule. Secretion of the overmodified molecules was impaired. The thermal stability of molecules containing abnormal chains was lower than that of normally modified molecules. After cleavage of molecules with vertebrate collagenase, the temperature of thermal denaturation of the overmodified A fragments was greater than that of the fragments from the normal molecules. The rates of cleavage of the normal and the abnormal molecules by N-proteinase were indistinguishable. Our findings suggest that the tripeptide deletion introduces a shift in the phase of the chains in the triple helix. This structural change is propagated from the site of the deletion toward the amino terminus of the molecule, but the subsequent alteration in the structure of the N-proteinase cleavage site is not sufficient to cause a decrease in the rate of cleavage by the enzyme.     

Temperature-induced post-translational over-modification of type I procollagen. Effects of over-modification of the protein on the rate of cleavage by procollagen N-proteinase and on self-assembly of collagen into fibrils.

Previous observations suggested that incubating fibroblasts at elevated temperature caused over-modification of type I procollagen by post-translational enzymes because of a delay in folding of the collagen triple helix. Here, human skin fibroblasts were incubated at 40.5 instead of 37 degrees C, and the type I procollagen secreted into the medium was isolated. Analysis of the protein indicated that there was an increase of about 5 residues of hydroxylysine/alpha chain and about 1 residue of glycosylated hydroxylysine/alpha chain. Assays with procollagen N-proteinase indicated that the N-propeptide of the over-modified collagen was cleaved at a decreased rate, apparently because the over-modification altered the conformation-dependent cleavage site for the enzyme. Assays in a system for assembly of collagen into fibrils demonstrated that the over-modified protein had a higher critical concentration for self-assembly. Also, the fibrils formed from the over-modified collagen at 31 and 29 degrees C had smaller diameters than fibrils formed from normal type I collagen. The results provide direct evidence for earlier suggestions that post-translational over-modification of a fibrillar collagen can alter the morphology of the fibrils formed. The results also indicate that some of the biological consequences of the mutations in type I procollagen causing heritable disorders must be ascribed to the effects of post-translational over-modifications that frequently occur as secondary consequences of changes in the primary structure of the protein.     

Production in mammalian cells of chimeric human/sea urchin procollagen molecules displaying distinct versions of the minor triple helix

One of the mechanisms involved in the regulation of the fibril diameter is the retention of the N-propeptide. In sea urchin embryo, thin collagen fibrils harbor numerous extensions at their surface, which we have suggested correspond to the large N-propeptide of the 2alpha collagen chain. To investigate the function of the N-propeptide during fibrillogenesis, we engineered constructs coding for the globular region of the 2alpha N-propeptide. To obtain homotrimeric molecules, the N-telopeptide, the central triple helix and the C-propeptide of the 2alpha chain were replaced by human domains of the proalpha1(I) chain. A single restriction site allowed insertion of distinct versions of the minor triple helix of the N-propeptide. Several human cell lines were transfected, and with one of them we were able to produce intact homotrimeric procollagen molecules. Rotary shadowing of these purified molecules indicates the presence of three large 2alpha N-propeptides that are similar to the extensions present at the surface of the sea urchin thin fibrils. This cassette-vector will be useful in determining the respective contributions of the globular and minor triple helical domains of the N-propeptide in the regulation of fibril diameter.     

Human dermatosparaxis: a form of Ehlers-Danlos syndrome that results from failure to remove the amino-terminal propeptide of type I procollagen.

Dermatosparaxis is a recessively inherited connective-tissue disorder that results from lack of the activity of type I procollagen N-proteinase, the enzyme that removes the amino-terminal propeptides from type I procollagen. Initially identified in cattle more than 20 years ago, the disorder was subsequently characterized in sheep, cats, and dogs. Affected animals have fragile skin, lax joints, and often die prematurely because of sepsis following avulsion of portions of skin. We recently identified two children with soft, lax, and fragile skin, which, when examined by transmission electron microscopy, contained the twisted, ribbon-like collagen fibrils characteristic of dermatosparaxis. Skin extracts from one child contained collagen precursors with amino-terminal extensions. Cultured fibroblasts from both children failed to cleave the amino-terminal propeptides from the pro alpha 1(I) and pro alpha 2(I) chains in type I procollagen molecules. Extracts of normal cells cleaved to collagen, the type I procollagen synthesized by cells from both children, demonstrating that the enzyme, not the substrate, was defective. These findings distinguish dermatosparaxis from Ehlers-Danlos syndrome type VII, which results from substrate mutations that prevent proteolytic processing of type I procollagen molecules.     

A critical crosslink region in human-bone-derived collagen type I. Specific cleavage site at residue Leu95

Collagen was extracted from human adult bone by limited pepsin digestion and collagen types were purified by consecutive salt precipitation first under neutral and then under acid conditions. In SDS/PAGE, all collagen type I preparations showed a protein band [alpha 1s(I)] migrating between alpha 1(I) and alpha 2(I) as well as a band [alpha 2s(I)] migrating in front of alpha 2(I). The collagenous nature of the pepsin-stable alpha 1s(I) protein was clearly demonstrated by digestion with human-leucocyte-derived collagenase, immunoblotting with antibodies against collagen type I and amino acid analysis. Partial amino acid sequencing of alpha 1(I) and alpha 1s(I) identified alpha 1s(I) as a shortened alpha 1(I) chain due to a specific cleavage site between residues Leu95 and Asp96 which is in close vicinity to the hydroxylysine-derived crosslink at position 87. In circular dichroism, the proportion of thermally labile collagen molecules was proportional to the amount of shortened alpha 1(I) and alpha 2(I) chains, respectively. The melting temperature was found to be 36 +/- 0.5 degrees C as judged from circular dichroism and susceptibility to proteolysis. Our data provide clear evidence that a shortened alpha 1-derived collagen chain can be extracted from human adult bone whereas it is hardly found in human skin. The unique cleavage site might provide important information about the collagen I molecule embedded in the calcified matrix of human bone.     

Processing of type II procollagen amino propeptide by matrix metalloproteinases.

In many embryonic tissues, type IIA procollagen is synthesized and deposited into the extracellular matrix containing the NH(2)-propeptide, the cysteine-rich domain of which binds to bone morphogenic proteins. To investigate whether matrix metalloproteinases (MMPs) synthesized during development and disease can cleave the NH(2) terminus of type II procollagens, we tested eight types of enzymes. Recombinant trimeric type IIA collagen NH(2)-propeptide encoded by exons 1-8 fused to the lectin domain of rat surfactant protein D was used as a substrate. The latter allowed trimerization of the propeptide domain and permitted isolation by saccharide affinity chromatography. Although MMPs 1, 2, and 8 did not show cleavage, MMPs 3, 7, 9, 13, and 14 cleaved the recombinant protein both at the telopeptide region and at the procollagen N-proteinase cleavage site. MMPs 7 and 13 demonstrated other cleavage sites in the type II collagen-specific region of the N-propeptide; MMP-7 had another cleavage site close to the COOH terminus of the cysteine-rich domain. To prove that an MMP can cleave the native type IIA procollagen in situ, we demonstrated that MMP-7 removes the NH(2)-propeptide from collagen fibrils in the extracellular matrix of fetal cartilage and identified the cleavage products. Because the N-proteinase and telopeptidase cleavage sites are present in both type IIA and type IIB procollagens and the telopeptide cleavage site is retained in the mature collagen fibril, this processing could be important to type IIB procollagen and to mature collagen fibrils as well.     

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.     

Type I collagen triplet duplication mutation in lethal osteogenesis imperfecta shifts register of alpha chains throughout the helix and disrupts incorporation of mutant helices into fibrils and extracellular matrix

The majority of collagen mutations causing osteogenesis imperfecta (OI) are glycine substitutions that disrupt formation of the triple helix. A rare type of collagen mutation consists of a duplication or deletion of one or two Gly-X-Y triplets. These mutations shift the register of collagen chains with respect to each other in the helix but do not interrupt the triplet sequence, yet they have severe clinical consequences. We investigated the effect of shifting the register of the collagen helix by a single Gly-X-Y triplet on collagen assembly, stability, and incorporation into fibrils and matrix. These studies utilized a triplet duplication in COL1A1 exon 44 that occurred in the cDNA and gDNA of two siblings with lethal OI. The normal allele encodes three identical Gly-Ala-Hyp triplets at aa 868-876, whereas the mutant allele encodes four. The register shift delays helix formation, causing overmodification. Differential scanning calorimetry yielded a decrease in T(m) of 2 degrees C for helices with one mutant chain and a 6 degrees C decrease in helices with two mutant chains. An in vitro binary co-processing assay of N-proteinase cleavage demonstrated that procollagen with the triplet duplication has slower N-propeptide cleavage than in normal controls or procollagen with proalpha1(I) G832S, G898S, or G997S substitutions, showing that the register shift persists through the entire helix. The register shift disrupts incorporation of mutant collagen into fibrils and matrix. Proband fibrils formed inefficiently in vitro and contained only normal helices and helices with a single mutant chain. Helices with two mutant chains and a significant portion of helices with one mutant chain did not form fibrils. In matrix deposited by proband fibroblasts, mutant chains were abundant in the immaturely cross-linked fraction but constituted a minor fraction of maturely cross-linked chains. The profound effects of shifting the collagen triplet register on chain interactions in the helix and on fibril formation correlate with the severe clinical consequences.