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A disintegrin and metalloproteinase with thrombospondin type I motif (ADAMTS)2, 3, and 14 are collectively named procollagen N-proteinases (pNPs) because of their specific ability to cleave the aminopropeptide of fibrillar procollagens. Several reports also indicate that they could be involved in other biological processes, such as blood coagulation, development, and male fertility, but the potential substrates associated with these activities remain unknown. Using the recently described N-terminal amine isotopic labeling of substrate approach, we analyzed the secretomes of human fibroblasts and identified 8, 17, and 22 candidate substrates for ADAMTS2, 3, and 14, respectively. Among these newly identified substrates, many are components of the extracellular matrix and/or proteins related to cell signaling such as latent TGF-β binding protein 1, TGF-β RIII, and dickkopf-related protein 3. Candidate substrates for the 3 ADAMTS have been biochemically validated in different contexts, and the implication of ADAMTS2 in the control of TGF-β activity has been further demonstrated in human fibroblasts. Finally, the cleavage site specificity was assessed showing a clear and unique preference for nonpolar or slightly hydrophobic amino acids. This work shows that the activities of the pNPs extend far beyond the classically reported processing of the aminopropeptide of fibrillar collagens and that they should now be considered as multilevel regulators of matrix deposition and remodeling.-Bekhouche, M., Leduc, C., Dupont, L., Janssen, L., Delolme, F., Vadon-Le Goff, S., Smargiasso, N., Baiwir, D., Mazzucchelli, G., Zanella-Cleon, I., Dubail, J., De Pauw, E., Nusgens, B., Hulmes, D. J. S., Moali, C., Colige, A. Determination of the substrate repertoire of ADAMTS2, 3, and 14 significantly broadens their functions and identifies extracellular matrix organization and TGF-β signaling as primary targets.

Bone morphogenetic protein-1 (BMP-1)/tolloid-like proteinases, here called BTPs, include the proteases originally identified for their roles in the C-terminal maturation of fibrillar procollagens ("procollagen C-proteinase"). Though numerous other substrates have since been discovered, the BTPs remain the main proteases involved in extracellular matrix assembly with little or no implication in matrix degradation. During the same period however, the BTPs have also become established as important proteases in the activation of growth factors, including TGF-β1, BMP-2/-4, GDF-8/-11 and IGFs, as well as the release of anti-angiogenic fragments from parent proteins. The BTPs are therefore key players in many pathophysiological processes such as morphogenesis, tissue repair and tumor progression. This mini-review summarizes our current knowledge of the functions of BTPs, their substrates and unusual mechanisms of regulation, and discusses their potential as new targets for future therapies.

International audience; Purpose: To characterize the expression of the bone morphogenetic protein-1 (BMP-1)/tolloid-like proteinases (collectively called BTPs), which include BMP-1, mammalian tolloid (mTLD), and mammalian tolloid-like 1 (mTLL-1) and 2 (mTLL-2), as well as the associated proteins procollagen C-proteinase enhancers (PCPE-1 and -2), in corneal scarring. Methods: Using a mouse full-thickness corneal excision model, wound healing was followed for up to 28 days by transmission electron microscopy, immunohistology (BMP-1/mTLD and PCPE-1), and quantitative PCR (Q-PCR: collagen III, BMP-1/mTLD, mTLL-1, mTLL-2, PCPE-1, PCPE-2). Bone morphogenetic protein-1/mTLD and PCPE-1 were also immunolocalized in cases of human corneal scarring following injuries. Results: In the mouse model, throughout the follow-up period, there was a large increase in collagen III mRNA expression in the stroma. By transmission electron microscopy, there was marked cellular infiltration into the wound as well as disorganization of collagen fibrils, but no significant difference in fibril diameter. In control corneas, by Q-PCR, BMP-1/mTLD showed the highest expression, compared to low levels of mTLL-1 and undetectable levels of mTLL-2, in both epithelium and stroma. Following wounding, both BMP-1/mTLD and PCPE-1 mRNA and protein increased, while PCPE-2 mRNA decreased. Finally, by immunofluorescence, BMP-1/mTLD and PCPE-1 were strongly expressed in the scar region in both mouse and human corneas. Conclusions: Bone morphogenetic protein-1/mTLD and PCPE-1 are upregulated in corneal scars. Both proteins may therefore contribute to the process of corneal scarring.

International audience; The type I procollagen carboxyterminal(C-)propeptides are crucial in directing correct assembly of the procollagen heterotrimers. Defects in these domains have anecdotally been reported in patients with Osteogenesis Imperfecta (OI) and few genotype-phenotype correlations have been described. To gain insight in the functional consequences of C-propeptide defects, we performed a systematic review of clinical, molecular, and biochemical findings in all patients in whom we identified a type I procollagen C-propeptide defect, and compared this with literature data. We report 30 unique type I procollagen C-propeptide variants, 24 of which are novel. The outcome of COL1A1 nonsense and frameshift variants depends on the location of the premature termination codon. Those located prior to 50-55 nucleotides upstream of the most 3' exon-exon junction lead to nonsense-mediated mRNA decay (NMD) and cause mild OI. Those located beyond this boundary escape NMD, generally lead to production of stable, overmodified procollagen chains, which may partly be retained intracellularly, and are usually associated with severe-to-lethal OI. Proα1(I)-C-propeptide defects that permit chain association result in more severe phenotypes than those inhibiting chain association. We demonstrate that the crystal structure of the proα1(III)-C-propeptide is a reliable tool to predict phenotypic severity for most COL1A1-C-propeptide missense variants, whereas for COL1A2-C-propeptide variants, the phenotypic outcome is milder than predicted.

International audience; Tight regulation of collagen fibril deposition in the extracellular matrix is essential for normal tissue homeostasis and repair, defects in which are associated with several degenerative or fibrotic disorders. A key regulatory step in collagen fibril assembly is the C-terminal proteolytic processing of soluble procollagen precursors. This step, carried out mainly by bone morphogenetic protein-1/tolloid-like proteinases, is itself subject to regulation by procollagen C-proteinase enhancer proteins (PCPEs) which can dramatically increase bone morphogenetic protein-1/tolloid-like proteinase activity, in a substrate-specific manner. Although it is known that this enhancing activity requires binding of PCPE to the procollagen C-propeptide trimer, identification of the precise binding site has so far remained elusive. Here, use of small-angle X-ray scattering provides structural data on this protein complex indicating that PCPE binds to the stalk region of the procollagen C-propeptide trimer, where the three polypeptide chains associate together, at the junction with the base region. This is supported by site-directed mutagenesis, which identifies two highly conserved, surface-exposed lysine residues in this region of the trimer that are essential for binding, thus revealing structural parallels with the interactions of Complement C1r/C1s, Uegf, BMP-1 (CUB) domain-containing proteins in diverse biological systems such as complement activation, receptor signaling, and transport. Together with detailed kinetics and interaction analysis, these results provide insights into the mechanism of action of PCPEs and suggest clear strategies for the development of novel antifibrotic therapies.

The C-propeptide domains of the fibrillar procollagens, which are present throughout the Metazoa in the form of ∼90 kDa trimers, play crucial roles in both intracellular molecular assembly and extracellular formation of collagen fibrils. The first crystallization of a C-propeptide domain, that from human procollagen III, is described. Following transient expression in mammalian 293T cells of both the native protein and a selenomethionine derivative, two crystal forms of the homotrimer were obtained: an orthorhombic form (P2(1)2(1)2(1)) that diffracted to 1.7 Å resolution and a trigonal form (P321) that diffracted to 3.5 Å resolution. Characterization by MALDI-TOF mass spectrometry allowed the efficiency of selenomethionine incorporation to be determined.

International audience; The proteolytic processing of procollagen V is complex and depends on the activity of several enzymes among which the BMP-1 (bone morphogenetic protein-1)/tolloid metalloproteinase and the furin-like proprotein convertases. Few of these processing interactions could have been predicted by analysing the presence of conserved consensus sequences in the proalpha1(V) chain. In the present study we opted for a cell approach that allows a straightforward identification of processing interactions. A construct encompassing the complete N-terminal end of the proalpha1(V) chain, referred to as Nalpha1, was recombinantly expressed to be used for enzymatic assays and for antibody production. Structural analysis showed that Nalpha1 is a monomer composed of a compact globule and an extended tail, which correspond respectively to the non-collagenous Nalpha1 subdomains, TSPN-1 (thrombospondin-1 N-terminal domain-like) and the variable region. Nalpha1 was efficiently cleaved by BMP-1 indicating that the triple helix is not required for enzyme activity. By mutating residues flanking the cleavage site, we showed that the aspartate residue at position P2' is essential for BMP-1 activity. BMP-1 activity at the C-terminal end of the procollagen V was assessed by generating a furin double mutant (R1584A/R1585A). We showed that, in absence of furin activity, BMP-1 is capable of processing the C-propeptide even though less efficiently than furin. Altogether, our results provide new relevant information on this complex and poorly understood mechanism of enzymatic processing in procollagen V function.The proteolytic processing of procollagen V is complex and depends on the activity of several enzymes among which the BMP-1 (bone morphogenetic protein-1)/tolloid metalloproteinase and the furin-like proprotein convertases. Few of these processing interactions could have been predicted by analysing the presence of conserved consensus sequences in the proalpha1(V) chain. In the present study we opted for a cell approach that allows a straightforward identification of processing interactions. A construct encompassing the complete N-terminal end of the proalpha1(V) chain, referred to as Nalpha1, was recombinantly expressed to be used for enzymatic assays and for antibody production. Structural analysis showed that Nalpha1 is a monomer composed of a compact globule and an extended tail, which correspond respectively to the non-collagenous Nalpha1 subdomains, TSPN-1 (thrombospondin-1 N-terminal domain-like) and the variable region. Nalpha1 was efficiently cleaved by BMP-1 indicating that the triple helix is not required for enzyme activity. By mutating residues flanking the cleavage site, we showed that the aspartate residue at position P2' is essential for BMP-1 activity. BMP-1 activity at the C-terminal end of the procollagen V was assessed by generating a furin double mutant (R1584A/R1585A). We showed that, in absence of furin activity, BMP-1 is capable of processing the C-propeptide even though less efficiently than furin. Altogether, our results provide new relevant information on this complex and poorly understood mechanism of enzymatic processing in procollagen V function.

The creation of 3D scaffolds that mimic the structure of physiological tissue required for normal cell function is a major bio engineering challenge. For corneal stroma reconstruction this necessitates the creation of a stroma-like scaffold consisting of a stack of orthogonally disposed sheets of aligned collagen fibrils. This study demonstrates that such a scaffold can be built up using magnetic alignment. By allowing neutralized acid soluble type I collagen to gel in a horizontal magnetic field (7 T) and by combining a series of gelation-rotation-gelation cycles, a scaffold of orthogonal lamellae composed of aligned collagen fibrils has been formed. Although initially dilute the gels can be concentrated without noticeable loss in orientation. The gels are translucent but their transparency can be greatly improved by the addition of proteoglycans to the gel-forming solution. Keratocytes align by contact guidance along the direction of collagen fibrils and respect the orthogonal design of the collagen template as they penetrate into the bulk of the 3- dimensional matrix. The scaffold is a significant step towards the creation of a corneal substitute with properties resembling those of native corneal stroma.

International audience; The lethal disease anthrax is propagated by spores of Bacillus anthracis, which can penetrate into the mammalian host by inhalation, causing a rapid progression of the disease and a mostly fatal outcome. We have solved the three-dimensional structure of the major surface protein BclA on B. anthracis spores. Surprisingly, the structure resembles C1q, the first component of complement, despite there being no sequence homology. Although most assays for C1q-like activity, including binding to C1q receptors, suggest that BclA does not mimic C1q, we show that BclA, as well as C1q, interacts with components of the lung alveolar surfactant layer. Thus, to better recognize and invade its hosts, this pathogenic soil bacterium may have evolved a surface protein whose structure is strikingly close to a mammalian protein.The lethal disease anthrax is propagated by spores of Bacillus anthracis, which can penetrate into the mammalian host by inhalation, causing a rapid progression of the disease and a mostly fatal outcome. We have solved the three-dimensional structure of the major surface protein BclA on B. anthracis spores. Surprisingly, the structure resembles C1q, the first component of complement, despite there being no sequence homology. Although most assays for C1q-like activity, including binding to C1q receptors, suggest that BclA does not mimic C1q, we show that BclA, as well as C1q, interacts with components of the lung alveolar surfactant layer. Thus, to better recognize and invade its hosts, this pathogenic soil bacterium may have evolved a surface protein whose structure is strikingly close to a mammalian protein.

Procollagen C-proteinase enhancer (PCPE) is an extracellular matrix glycoprotein that binds to the C-propeptide of procollagen I and can enhance the activities of procollagen C-proteinases up to 20-fold. To determine the molecular mechanism of PCPE activity, the interactions of the recombinant protein with the procollagen molecule as well as with its isolated C-propeptide domain were studied using surface plasmon resonance (BIAcore) technology. Binding required the presence of divalent metal cations such as calcium and manganese. By ligand blotting, calcium was found to bind to the C-propeptide domains of procollagens I and III but not to PCPE. By chemical cross-linking, the stoichiometry of the PCPE/C-propeptide interaction was found to be 1:1 in accordance with enzyme kinetic data. The use of a monoclonal antibody directed against the N-terminal region of the C-propeptide suggested that this region is probably not involved in binding to PCPE. Association and dissociation kinetics of the C-propeptide domains of procollagens I and III on immobilized PCPE were rapid. Extrapolation to saturation equilibrium yielded apparent equilibrium dissociation constants in the range 150-400 nM. In contrast, the association/dissociation kinetics of intact procollagen molecules on immobilized PCPE were relatively slow, corresponding to a dissociation constant of 1 nM. Finally, pN-collagen (i.e. procollagen devoid of the C-terminal propeptide domain) was also found to bind to immobilized PCPE, suggesting that PCPE binds to sites on either side of the procollagen cleavage site, thereby facilitating the action of procollagen C-proteinases.Procollagen C-proteinase enhancer (PCPE) is an extracellular matrix glycoprotein that binds to the C-propeptide of procollagen I and can enhance the activities of procollagen C-proteinases up to 20-fold. To determine the molecular mechanism of PCPE activity, the interactions of the recombinant protein with the procollagen molecule as well as with its isolated C-propeptide domain were studied using surface plasmon resonance (BIAcore) technology. Binding required the presence of divalent metal cations such as calcium and manganese. By ligand blotting, calcium was found to bind to the C-propeptide domains of procollagens I and III but not to PCPE. By chemical cross-linking, the stoichiometry of the PCPE/C-propeptide interaction was found to be 1:1 in accordance with enzyme kinetic data. The use of a monoclonal antibody directed against the N-terminal region of the C-propeptide suggested that this region is probably not involved in binding to PCPE. Association and dissociation kinetics of the C-propeptide domains of procollagens I and III on immobilized PCPE were rapid. Extrapolation to saturation equilibrium yielded apparent equilibrium dissociation constants in the range 150-400 nM. In contrast, the association/dissociation kinetics of intact procollagen molecules on immobilized PCPE were relatively slow, corresponding to a dissociation constant of 1 nM. Finally, pN-collagen (i.e. procollagen devoid of the C-terminal propeptide domain) was also found to bind to immobilized PCPE, suggesting that PCPE binds to sites on either side of the procollagen cleavage site, thereby facilitating the action of procollagen C-proteinases.

The effects of strain selection and body weight on proteoglycan metabolism and the onset of degenerative joint disease (DJD) were investigated in avian articular cartilage. Samples from the hock joint (proximal tarsometatarsus, PTM; distal tibiotarsus, DTT) of rapidly growing broiler fowl, fed either ad libitum or on a restricted-diet, were compared with those from a slow growing, light and non-selected strain (J-line). Synthesis and degradation of proteoglycans were investigated by radioactive pulse-chase studies, determination of total sulphated glycosaminoglycans and electrophoretic analysis. By gross morphology, degenerative changes in articular cartilage occurred solely in the DTT from ad libitum-fed broiler fowl, after 13 weeks. Differences in proteoglycan metabolism were also observed, most markedly in the DTT, where the rate of proteoglycan synthesis in the ad libitum-fed group was less than in age-matched J-line cartilage, and the proportions of both newly synthesised and resident proteoglycans released into the culture medium were greater. Results with the feed-restricted group were intermediate between ad libitum-fed and J-line. Electrophoretic analysis of proteoglycans in the culture media showed evidence of degradation solely in the ad libitum-fed group, with earliest onset in the DTT. The results indicate that proteoglycan metabolism in avian articular cartilage is similar to that in mammalian cartilage during the development of DJD, and that the onset of cartilage degeneration is linked with excessive load bearing.The effects of strain selection and body weight on proteoglycan metabolism and the onset of degenerative joint disease (DJD) were investigated in avian articular cartilage. Samples from the hock joint (proximal tarsometatarsus, PTM; distal tibiotarsus, DTT) of rapidly growing broiler fowl, fed either ad libitum or on a restricted-diet, were compared with those from a slow growing, light and non-selected strain (J-line). Synthesis and degradation of proteoglycans were investigated by radioactive pulse-chase studies, determination of total sulphated glycosaminoglycans and electrophoretic analysis. By gross morphology, degenerative changes in articular cartilage occurred solely in the DTT from ad libitum-fed broiler fowl, after 13 weeks. Differences in proteoglycan metabolism were also observed, most markedly in the DTT, where the rate of proteoglycan synthesis in the ad libitum-fed group was less than in age-matched J-line cartilage, and the proportions of both newly synthesised and resident proteoglycans released into the culture medium were greater. Results with the feed-restricted group were intermediate between ad libitum-fed and J-line. Electrophoretic analysis of proteoglycans in the culture media showed evidence of degradation solely in the ad libitum-fed group, with earliest onset in the DTT. The results indicate that proteoglycan metabolism in avian articular cartilage is similar to that in mammalian cartilage during the development of DJD, and that the onset of cartilage degeneration is linked with excessive load bearing.

Procollagen C-proteinase enhancer (PCPE) is a 55 kDa glycoprotein that increases the activity of procollagen C-proteinase (PCP)/bone morphogenetic protein-1 (BMP-1) during C-terminal processing of fibrillar collagen precursors. Here we show that the 36 kDa, active fragment of PCPE enhances the activity of both the short (mouse) and long (chick) forms of PCP/BMP-1. The activity of PCPE is not associated with the formation of sedimentable procollagen aggregates. In addition, PCPE (36 kDa) has no effect in vitro on N-terminal procollagen processing by highly purified procollagen N-proteinase. Finally, when the amount of PCP is adjusted so that the rate of C-terminal processing remains constant, PCPE (36 kDa) has no effect on the assembly of collagen or pN-collagen in vitro following C-terminal processing of the corresponding precursors.Procollagen C-proteinase enhancer (PCPE) is a 55 kDa glycoprotein that increases the activity of procollagen C-proteinase (PCP)/bone morphogenetic protein-1 (BMP-1) during C-terminal processing of fibrillar collagen precursors. Here we show that the 36 kDa, active fragment of PCPE enhances the activity of both the short (mouse) and long (chick) forms of PCP/BMP-1. The activity of PCPE is not associated with the formation of sedimentable procollagen aggregates. In addition, PCPE (36 kDa) has no effect in vitro on N-terminal procollagen processing by highly purified procollagen N-proteinase. Finally, when the amount of PCP is adjusted so that the rate of C-terminal processing remains constant, PCPE (36 kDa) has no effect on the assembly of collagen or pN-collagen in vitro following C-terminal processing of the corresponding precursors.

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International audience

The carboxyl-terminal propeptide of procollagen type I (CPP-I) plays a key role in the regulation of collagen fibrillogenesis. In addition, it has been reported that, after cleavage from procollagen, CPP-I exerts feedback control of collagen biosynthesis. To further elucidate the mechanisms involved in each of these processes, we have investigated the nature of cell surface receptors for CPP-I. CPP-I affinity chromatography, using detergent extracts of iodinated HT1080 cells and EDTA elution, resulted in the isolation of two polypeptides of molecular mass 160 and 110 kDa. Since the migratory behavior of these polypeptides under nonreducing and reducing conditions was characteristic of a subset of integrin receptors, their reactivity with anti-integrin monoclonal antibodies was tested. Antibodies directed against the alpha 2 and beta 1 subunits specifically immunoprecipitated both CPP-I-binding polypeptides, indicating that the CPP-I receptor is the integrin alpha 2 beta 1. CPP-I was found to support the attachment and spreading of HT1080 cells, demonstrating that it can function as an adhesion protein. Two other approaches supported the identification of alpha 2 beta 1 as the CPP-I receptor. First, anti-functional anti-integrin monoclonal antibodies directed against the alpha 2 and beta 1 subunits completely abrogated the adhesive activity of CPP-I and, second, highly purified CPP-I bound specifically to alpha 2 beta 1-containing integrin preparations in a solid-phase receptor-ligand binding assay. These findings have important implications for the function of fibrillar collagen carboxyl-terminal propeptides and for the role played by integrins in the regulation of cellular phenotype.The carboxyl-terminal propeptide of procollagen type I (CPP-I) plays a key role in the regulation of collagen fibrillogenesis. In addition, it has been reported that, after cleavage from procollagen, CPP-I exerts feedback control of collagen biosynthesis. To further elucidate the mechanisms involved in each of these processes, we have investigated the nature of cell surface receptors for CPP-I. CPP-I affinity chromatography, using detergent extracts of iodinated HT1080 cells and EDTA elution, resulted in the isolation of two polypeptides of molecular mass 160 and 110 kDa. Since the migratory behavior of these polypeptides under nonreducing and reducing conditions was characteristic of a subset of integrin receptors, their reactivity with anti-integrin monoclonal antibodies was tested. Antibodies directed against the alpha 2 and beta 1 subunits specifically immunoprecipitated both CPP-I-binding polypeptides, indicating that the CPP-I receptor is the integrin alpha 2 beta 1. CPP-I was found to support the attachment and spreading of HT1080 cells, demonstrating that it can function as an adhesion protein. Two other approaches supported the identification of alpha 2 beta 1 as the CPP-I receptor. First, anti-functional anti-integrin monoclonal antibodies directed against the alpha 2 and beta 1 subunits completely abrogated the adhesive activity of CPP-I and, second, highly purified CPP-I bound specifically to alpha 2 beta 1-containing integrin preparations in a solid-phase receptor-ligand binding assay. These findings have important implications for the function of fibrillar collagen carboxyl-terminal propeptides and for the role played by integrins in the regulation of cellular phenotype.

The interactions of [3H]procollagen I with various phospholipids were studied by density gradient centrifugation. At physiological conditions of pH, ionic strength, and temperature, there was no evidence for procollagen binding to phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, or phosphatidylserine liposomes. In contrast, procollagen I bound strongly to sphingomyelin liposomes in a reversible and saturable manner, with an apparent dissociation constant (Kd) of 2.6 nM. Binding occurred over a range of temperatures (4-37 degrees C) and was relatively unaffected by salt concentrations up to 1.2 M NaCl. Binding was observed in phosphate buffers, but not in the presence of high concentrations of Tris or Hepes. Bovine serum albumin had no effect on procollagen binding to sphingomyelin, and neither did unlabeled type I collagen, with or without the nonhelical telopeptides. Procollagen II and denatured procollagen I also bound to sphingomyelin. Procollagen binding to sphingomyelin at 35 degrees C was considerably reduced when small amounts of phosphatidylcholine were present, although binding was partially restored when the temperature was reduced below the corresponding phase transition temperature. Purified unlabeled procollagen COOH-terminal propeptides successfully competed for binding, and 125I-labeled COOH-terminal propeptides bound to sphingomyelin in the absence of procollagen. Weaker binding to sphingomyelin, mediated by the collagen triple helical region, was also observed; but this was dominated by the sphingomyelin-COOH-terminal propeptide interaction. The data suggest a novel mechanism for matrix vesicle-mediated biomineralization.The interactions of [3H]procollagen I with various phospholipids were studied by density gradient centrifugation. At physiological conditions of pH, ionic strength, and temperature, there was no evidence for procollagen binding to phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, or phosphatidylserine liposomes. In contrast, procollagen I bound strongly to sphingomyelin liposomes in a reversible and saturable manner, with an apparent dissociation constant (Kd) of 2.6 nM. Binding occurred over a range of temperatures (4-37 degrees C) and was relatively unaffected by salt concentrations up to 1.2 M NaCl. Binding was observed in phosphate buffers, but not in the presence of high concentrations of Tris or Hepes. Bovine serum albumin had no effect on procollagen binding to sphingomyelin, and neither did unlabeled type I collagen, with or without the nonhelical telopeptides. Procollagen II and denatured procollagen I also bound to sphingomyelin. Procollagen binding to sphingomyelin at 35 degrees C was considerably reduced when small amounts of phosphatidylcholine were present, although binding was partially restored when the temperature was reduced below the corresponding phase transition temperature. Purified unlabeled procollagen COOH-terminal propeptides successfully competed for binding, and 125I-labeled COOH-terminal propeptides bound to sphingomyelin in the absence of procollagen. Weaker binding to sphingomyelin, mediated by the collagen triple helical region, was also observed; but this was dominated by the sphingomyelin-COOH-terminal propeptide interaction. The data suggest a novel mechanism for matrix vesicle-mediated biomineralization.

International audience; The assembly of type I collagen and type I pN-collagen was studied in vitro using a system for generating these molecules enzymatically from their immediate biosynthetic precursors. Collagen generated by C-proteinase digestion of pC-collagen formed D-periodically banded fibrils that were essentially cylindrical (i.e. circular in cross-section). In contrast, pN-collagen generated by C-proteinase digestion of procollagen formed thin, sheet-like structures that were axially D-periodic in longitudinal section, of varying lateral widths (up to several microns) and uniform in thickness (approximately 8 nm). Mixtures of collagen and pN-collagen assembled to form a variety of pleomorphic fibrils. With increasing pN-collagen content, fibril cross-sections were progressively distorted from circular to lobulated to thin and branched structures. Some of these structures were similar to fibrils observed in certain heritable disorders of connective tissue where N-terminal procollagen processing is defective. The observations are considered in terms of the hypothesis that the N-propeptides are preferentially located on the surface of a growing assembly. The implications for normal diameter control of collagen fibrils in vivo are discussed.The assembly of type I collagen and type I pN-collagen was studied in vitro using a system for generating these molecules enzymatically from their immediate biosynthetic precursors. Collagen generated by C-proteinase digestion of pC-collagen formed D-periodically banded fibrils that were essentially cylindrical (i.e. circular in cross-section). In contrast, pN-collagen generated by C-proteinase digestion of procollagen formed thin, sheet-like structures that were axially D-periodic in longitudinal section, of varying lateral widths (up to several microns) and uniform in thickness (approximately 8 nm). Mixtures of collagen and pN-collagen assembled to form a variety of pleomorphic fibrils. With increasing pN-collagen content, fibril cross-sections were progressively distorted from circular to lobulated to thin and branched structures. Some of these structures were similar to fibrils observed in certain heritable disorders of connective tissue where N-terminal procollagen processing is defective. The observations are considered in terms of the hypothesis that the N-propeptides are preferentially located on the surface of a growing assembly. The implications for normal diameter control of collagen fibrils in vivo are discussed.

The polymorphic forms of ordered collagen aggregation in vitro and in vivo are reviewed. The axially projected structures of a class of fibrils known as fibrous long spacing (FLS) collagen are solved using simulated positively stained banding patterns based on the amino acid sequence. This method is also used to solve the axial projection of a 670 A ( D ) periodic structure with a symmetrical banding pattern (DPS) re-precipitated from skin collagen. The relation between the obliquely striated and 110 A periodic forms of collagen is discussed. The specificity for the formation of FLS, DPS and segment long spacing (SLS) collagen is shown to be in the distributions of various amino acids in the sequence. Different residues are important for each type of structure, their importance being dependent on the chemical conditions and the presence of other macromolecules. The interaction of collagen fibrils with proteoglycans in vivo is discussed in terms of the amino acid sequence. Also the factors which affect collagen morphology in the presence of mucopolysaccharides and proteoglycans in vitro and in vivo are discussed. Some insight is gamed into the principles which govern the self-assembly of molecules into ordered fibrous aggregates.

Small-angle x-ray and neutron diffraction patterns have been obtained from the annulus fibrosus of porcine intervertebral disk. These show that the collagen in this tissue is modified compared with that in tendon.Small-angle x-ray and neutron diffraction patterns have been obtained from the annulus fibrosus of porcine intervertebral disk. These show that the collagen in this tissue is modified compared with that in tendon.

International audience; Initial aggregates formed in collagen self-assembly were visualized by electron microscopy, using formaldehyde to fix the state of aggregation at various points in the turbidimetric lag phase. Measurements of the length distributions of monomers and small oligomers show that the first-formed aggregates are dimeric, with the most prevalent dimer having a maximal (approximately equal to 4D; D = 67 nm) stagger between constituent molecules.Initial aggregates formed in collagen self-assembly were visualized by electron microscopy, using formaldehyde to fix the state of aggregation at various points in the turbidimetric lag phase. Measurements of the length distributions of monomers and small oligomers show that the first-formed aggregates are dimeric, with the most prevalent dimer having a maximal (approximately equal to 4D; D = 67 nm) stagger between constituent molecules.

A model is described that provides a molecular mechanism for the regulation of collagen fibril diameters in vivo by the N-propeptide. The model accounts for the observed 80 A increment in fibril diameters, without the requirement for microfibrillar subunits.A model is described that provides a molecular mechanism for the regulation of collagen fibril diameters in vivo by the N-propeptide. The model accounts for the observed 80 A increment in fibril diameters, without the requirement for microfibrillar subunits.

Although the distribution of hydrophobic residues in the α1 chain collagen sequence has a D ⋍ 670 A periodicity, it is dipoles formed by 100 residues occurring in pairs of unlike charge which are responsible for the 1D stagger between molecules. Sheet models based on the Hodge-Petruska model for the axially projected collagen structure require interactions specifying 1D and 4D staggers. We found no evidence for interactions specifying a strong 4D stagger and, therefore, favour the Smith microfibril model which is specified by 1D stagger interactions alone. Two hydroxylysine residues, 234 residues apart, may form a covalent cross-link stabilising the 1D stagger. Gene duplication does not appear to be responsible for the periodicity in the sequence.

Low angle X-ray diffraction patterns of rat tail tendons with heavy metal stains added were examined to help clarify the effects of fixation and staining on collagen fibrils. Fixing and staining of rat tail tendon fibers gives an X-ray pattern with an intensified 3.8 nm row and the preservation of most equatorial features found in the native pattern. The presence of the native pattern features suggests the value of fixation in preserving native structure before staining. Staining of rat tail tendon fibers without prior fixation led to the disappearance of the native equatorial features and the appearance of a new broad row line corresponding to a spacing of around 10.0--17.5 nm. This observation suggests that some alteration has taken place in the native structure and may be related to electron microscopic observations of units of 10.0--20.0 nm in collagen fibrils under some disruptive or developmental conditions.Low angle X-ray diffraction patterns of rat tail tendons with heavy metal stains added were examined to help clarify the effects of fixation and staining on collagen fibrils. Fixing and staining of rat tail tendon fibers gives an X-ray pattern with an intensified 3.8 nm row and the preservation of most equatorial features found in the native pattern. The presence of the native pattern features suggests the value of fixation in preserving native structure before staining. Staining of rat tail tendon fibers without prior fixation led to the disappearance of the native equatorial features and the appearance of a new broad row line corresponding to a spacing of around 10.0--17.5 nm. This observation suggests that some alteration has taken place in the native structure and may be related to electron microscopic observations of units of 10.0--20.0 nm in collagen fibrils under some disruptive or developmental conditions.

International audience; A new image processing technique, content-dependent anisotropic spatial frequency filtering, has been developed to visualize the location and orientation of crystalline regions in collagen fibril cross-sections. The results show that most crystalline regions are oriented with their approximately 4 nm periodicity directed radially from the fibril centre. This periodicity corresponds to the separation between rows of molecular ends in the quasi-hexagonal molecular packing scheme. The extent of crystallinity increases with radius and frequently the lattice is either continuously distorted or interrupted by sharp discontinuities.A new image processing technique, content-dependent anisotropic spatial frequency filtering, has been developed to visualize the location and orientation of crystalline regions in collagen fibril cross-sections. The results show that most crystalline regions are oriented with their approximately 4 nm periodicity directed radially from the fibril centre. This periodicity corresponds to the separation between rows of molecular ends in the quasi-hexagonal molecular packing scheme. The extent of crystallinity increases with radius and frequently the lattice is either continuously distorted or interrupted by sharp discontinuities.