Human Ehlers-Danlos syndrome type VII C and bovine dermatosparaxis are caused by mutations in the procollagen I N-proteinase gene.

Ehlers-Danlos syndrome (EDS) type VIIC is a recessively inherited connective-tissue disorder, characterized by extreme skin fragility, characteristic facies, joint laxity, droopy skin, umbilical hernia, and blue sclera. Like the animal model dermatosparaxis, EDS type VIIC results from the absence of activity of procollagen I N-proteinase (pNPI), the enzyme that excises the N-propeptide of type I and type II procollagens. The pNPI enzyme is a metalloproteinase containing properdin repeats and a cysteine-rich domain with similarities to the disintegrin domain of reprolysins. We used bovine cDNA to isolate human pNPI. The human enzyme exists in two forms: a long version similar to the bovine enzyme and a short version that contains the Zn++-binding catalytic site but lacks the entire C-terminal domain in which the properdin repeats are located. We have identified the mutations that cause EDS type VIIC in the six known affected human individuals and also in one strain of dermatosparactic calf. Five of the individuals with EDS type VIIC were homozygous for a C-->T transition that results in a premature termination codon, Q225X. Four of these five patients were homozygous at three downstream polymorphic sites. The sixth patient was homozygous for a different transition that results in a premature termination codon, W795X. In the dermatosparactic calf, the mutation is a 17-bp deletion that changes the reading frame of the message. These data provide direct evidence that EDS type VIIC and dermatosparaxis result from mutations in the pNPI gene.     

Regulation of procollagen amino-propeptide processing during mouse embryogenesis by specialization of homologous ADAMTS proteases: insights on collagen biosynthesis and dermatosparaxis

Mutations in ADAMTS2, a procollagen amino-propeptidase, cause severe skin fragility, designated as dermatosparaxis in animals, and a subtype of the Ehlers-Danlos syndrome (dermatosparactic type or VIIC) in humans. Not all collagen-rich tissues are affected to the same degree, which suggests compensation by the ADAMTS2 homologs ADAMTS3 and ADAMTS14. In situ hybridization of Adamts2, Adamts3 and Adamts14, and of the genes encoding the major fibrillar collagens, Col1a1, Col2a1 and Col3a1, during mouse embryogenesis, demonstrated distinct tissue-specific, overlapping expression patterns of the protease and substrate genes. Adamts3, but not Adamts2 or Adamts14, was co-expressed with Col2a1 in cartilage throughout development, and with Col1a1 in bone and musculotendinous tissues. ADAMTS3 induced procollagen I processing in dermatosparactic fibroblasts, suggesting a role in procollagen I processing during musculoskeletal development. Adamts2, but not Adamts3 or Adamts14, was co-expressed with Col3a1 in many tissues including the lungs and aorta, and Adamts2(-/-) mice showed widespread defects in procollagen III processing. Adamts2(-/-) mice had abnormal lungs, characterized by a decreased parenchymal density. However, the aorta and collagen fibrils in the aortic wall appeared normal. Although Adamts14 lacked developmental tissue-specific expression, it was co-expressed with Adamts2 in mature dermis, which possibly explains the presence of some processed skin procollagen in dermatosparaxis. The data show how evolutionarily related proteases with similar substrate preferences may have distinct biological roles owing to tissue-specific gene expression, and provide insights into collagen biosynthesis and the pathobiology of dermatosparaxis.     

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.     

Domains and maturation processes that regulate the activity of ADAMTS-2, a metalloproteinase cleaving the aminopropeptide of fibrillar procollagens types I-III and V

Processing of fibrillar collagens is required to generate collagen monomers able to self-assemble into elongated and cylindrical collagen fibrils. ADAMTS-2 belongs to the "A disintegrin and metalloproteinase with thrombospondin type 1 motifs" (ADAMTS) family. It is responsible for most of the processing of the aminopropeptide of type I procollagen in the skin, and it also cleaves type II and type III procollagens. ADAMTS are complex secreted enzymes that are implicated in various physiological and pathological processes. Despite accumulating evidence indicating that their activity is regulated by ancillary domains, additional information is required for a better understanding of the specific function of each domain. We have generated 17 different recombinant forms of bovine ADAMTS-2 and characterized their processing, activity, and cleavage specificity. The results indicated the following: (i) activation of the ADAMTS-2 zymogen involves several cleavages, by proprotein convertases and C-terminal processing, and generates at least seven distinct processed forms; (ii) the C-terminal domain negatively regulates enzyme activity, whereas two thrombospondin type 1 repeats are enhancer regulators; (iii) the 104-kDa form displays the highest aminoprocollagen peptidase activity on procollagen type I; (iv) ADAMTS-2 processes the aminopropeptide of alpha1 type V procollagen homotrimer at the end of the variable domain; and (v) the cleaved sequence (PA) is different from the previously described sites ((P/A)Q) for ADAMTS-2, redefining its cleavage specificity. This finding and the existence of multiple processed forms of ADAMTS-2 strongly suggest that ADAMTS-2 may be involved in function(s) other than processing of fibrillar procollagen types I-III.     

The NH2-terminal propeptide of type I procollagen acts intracellularly to modulate cell function

The function of the NH(2)-terminal propeptide of type I procollagen (N-propeptide) is poorly understood. We now show that a recombinant trimeric N-propeptide interacts with transforming growth factor-beta1 and BMP2 and exhibits functional effects in stably transfected cells. The synthesis of N-propeptide by COS-7 cells results in an increase in phosphorylation of Akt and Smad3 and is associated with a marked reduction in type I procollagen synthesis and impairment in adhesion. In C2C12 cells, N-propeptide inhibits the osteoblastic differentiation induced by BMP2. Our data suggest that these effects are mediated by the interaction of N-propeptide with an intracellular receptor in the secretory pathway, because they are not observed when recombinant N-propeptide is added to the culture medium of either COS-7 or C2C12 cells. Both the binding of N-propeptide to cytokines and its functional properties are entirely dependent on the exon 2-encoded globular domain, and a mutation that substitutes a serine for a highly conserved cysteine in exon 2 abolishes its function. Our findings suggest that N-propeptide performs an important feedback regulatory function and provides a rationale for the prominence of a homotrimeric form of type I procollagen (alpha1 trimer) during vertebrate development.     

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.     

Partial purification and characterization of a neutral protease which cleaves the N-terminal propeptides from procollagen

A rapid assay procedure was developed for cleavage of the N-terminal propeptides of procollagen. With the assay a neutral procollagen N-protease was purified about 300-fold from chick embryo tendon extract. The enzyme had an apparent molecular weight of 260 000 and a pH optimum of 7.4. Ca2+ was required for enzymic activity but this requirement was partially replaced by Mg2+ or Mn2+. The enzyme was bound to concanavalin A-agarose and therefore was presumably a glycoprotein. The N-propeptides released from type I procollagen were of about 23 000 and 11 000 daltons as estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The partially purified enzyme was also found to cleave type II procollagen and the N-propeptide obtained was about 18 000 daltons. Heat denaturation of either type I or type II procollagen decreased the rate at which the proteins were cleaved by the N-protease.     

Assessment of procollagen processing defects by fibroblasts cultured in the presence of dextran sulphate.

The culture of skin fibroblasts in the presence of 0.01% (w/v) dextran sulphate results in complete proteolytic processing of procollagen to collagen. Processing occurs predominantly via a pN-collagen intermediate, suggesting that C-propeptide cleavage occurs early during the processing pathway. The processed collagen is associated with the cell-layer fraction. This method of inducing procollagen processing was evaluated for use in detecting procollagen processing abnormalities in heritable connective-tissue diseases. Abnormal type I procollagen processing was clearly demonstrated in two cases with known defects of pN-propeptide cleavage. In one, the cleavage deficiency was due to diminished N-proteinase activity (dermatosparaxis) and in the other case (Ehler's-Danlos syndrome type VIIA) the cleavage site was deleted. In a case of osteogenesis imperfecta (type II) the slow electrophoretic migration of type I collagen alpha-chains due to over-modification of lysine was readily demonstrated. Inefficient procollagen processing was also evident in this patient, as had been previously reported [de Wet, Pihlanjaniemi, Myers, Kelly & Prockop (1983) J. Biol. Chem. 258, 7721-7728]. Thus this method of culture in the presence of dextran sulphate provides a simple and rapid procedure for the detection of procollagen processing defects and electrophoretic abnormalities.     

Organization of the exons coding for pro alpha 1(II) collagen N-propeptide confirms a distinct evolutionary history of this domain of the fibrillar collagen genes

The organization of the exons coding for the N-terminal portion of human type II procollagen has been determined. Aside from inferring the previously unknown primary structure of type II N-propeptide, this study has revealed that this coding domain of the gene exhibits an organization uniquely distinct from those of type I and type III collagens. This finding substantiates the notion that the N-propeptide coding domains of the fibrillar collagen genes evolved under less stringent selection than those encoding the C-propeptide and triple helical regions.     

Inhibiting effect of procollagen peptides on collagen biosynthesis in fibroblast cultures.

NH2-terminal extension peptides of type I and type III procollagens were isolated from dermatosparactic and normal fetal calfskin, respectively. Cell culture experiments showed that the globular domains of the tested procollagen peptides were biologically active but that peptides from the helical region of collagen had no effect. The peptides were added to the incubation medium of calf fibroblasts along with radioactive precursor amino acids, and the amount of newly synthesized collagen was determined. The experiments indicated that procollagen peptides exerted a feedback-like inhibitory effect specific for the synthesis of collagen. Neither degradation of collagen, hydroxylation of collagen alpha chains, nor synthesis of noncollagenous proteins were affected. Synthesis of type II collagen by calf chondrocytes was not reduced. In addition, it was shown that procollagen peptides from calf were equally effective when added to human fibroblast cultures, an observation that could be of considerable medical interest.     

Assembly of collagen types II, IX and XI into nascent hetero-fibrils by a rat chondrocyte cell line.

The cell line, RCS-LTC (derived from the Swarm rat chondrosarcoma), deposits a copious extracellular matrix in which the collagen component is primarily a polymer of partially processed type II N-procollagen molecules. Transmission electron microscopy of the matrix shows no obvious fibrils, only a mass of thin unbanded filaments. We have used this cell system to show that the type II N-procollagen polymer nevertheless is stabilized by pyridinoline cross-links at molecular sites (mediated by N- and C-telopeptide domains) found in collagen II fibrils processed normally. Retention of the N-propeptide therefore does not appear to interfere with the interactions needed to form cross-links and mature them into trivalent pyridinoline residues. In addition, using antibodies that recognize specific cross-linking domains, it was shown that types IX and XI collagens, also abundantly deposited into the matrix by this cell line, become covalently cross-linked to the type II N-procollagen. The results indicate that the assembly and intertype cross-linking of the cartilage type II collagen heteropolymer is an integral, early process in fibril assembly and can occur efficiently prior to the removal of the collagen II N-propeptides.     

BMP-6 enhances chondrogenesis in a subpopulation of human marrow stromal cells

Marrow stromal cells (MSCs) can differentiate into several mesenchymal lineages. MSCs were recently shown to form cartilage in micromass cultures with serum-free medium containing TGF-beta and dexamethasone. Here we found that addition of BMP-6 increased the weight of the pellets about 10-fold and they stained more extensively for proteoglycans. mRNAs for type II procollagen and type X collagen were detected at 1 week and the levels were increased at 3 weeks. We also compared two subpopulation of cultures of MSCs: Small and rapidly self-renewing cells (RS cells) and the large, more mature and slowly replicating cells (mMSCs). The cartilage pellets prepared from cultures enriched for RS cells were about 2.5-fold larger, stained more extensively for proteoglycans, and had levels of mRNA for type II procollagen that were 1.6-fold higher. Also, RS cells retained more of their chondrogenic potential as the cells were passaged.