Lysyl oxidase-like 2 (LOXL2)-mediated cross-linking of tropoelastin.

Lysyl oxidases (LOXs) play a central role in extracellular matrix remodeling during development and tumor growth and fibrosis through cross-linking of collagens and elastin. We have limited knowledge of the structure and substrate specificity of these secreted enzymes. LOXs share a conserved C-terminal catalytic domain but differ in their N-terminal region, which is composed of 4 repeats of scavenger receptor cysteine-rich (SRCR) domains in LOX-like (LOXL) 2. We investigated by X-ray scattering and electron microscopy the low-resolution structure of the full-length enzyme and the structure of a shorter form lacking the catalytic domain. Our data demonstrate that LOXL2 has a rod-like structure with a stalk composed of the SRCR domains and the catalytic domain at its tip. We detected direct interaction between LOXL2 and tropoelastin (TE) and also LOXL2-mediated deamination of TE. Using proteomics, we identified several allysines together with cross-linked TE peptides. The elastin-like material generated was resistant to trypsin proteolysis and displayed mechanical properties similar to mature elastin. Finally, we detected the codistribution of LOXL2 and elastin in the vascular wall. Altogether, these data suggest that LOXL2 could participate in elastogenesis in vivo and could be used as a means of cross-linking TE in vitro for biomimetic and cell-compatible tissue engineering purposes.-Schmelzer, C. E. H., Heinz, A., Troilo, H., Lockhart-Cairns, M.-P., Jowitt, T. A., Marchand, M. F., Bidault, L., Bignon, M., Hedtke, T., Barret, A., McConnell, J. C., Sherratt, M. J., Germain, S., Hulmes, D. J. S., Baldock, C., Muller, L. Lysyl oxidase-like 2 (LOXL2)-mediated cross-linking of tropoelastin.

Atypical COL3A1 variants (glutamic acid to lysine) cause vascular Ehlers-Danlos syndrome with a consistent phenotype of tissue fragility and skin hyperextensibility.

PURPOSE: The Ehlers-Danlos syndromes (EDS) are a group of rare inherited connective tissue disorders. Vascular EDS (vEDS) is caused by pathogenic variants in COL3A1, most frequently glycine substitutions. We describe the phenotype of the largest series of vEDS patients with glutamic acid to lysine substitutions (Glu>Lys) in COL3A1, which were all previously considered to be variants of unknown significance. METHODS: Clinical and molecular data for seven families with three different Glu>Lys substitutions in COL3A1 were analyzed. RESULTS: These Glu>Lys variants were reclassified from variants of unknown significance to either pathogenic or likely pathogenic in accordance with American College of Medical Genetics and Genomics guidelines. All individuals with these atypical variants exhibited skin hyperextensibility as seen in individuals with classical EDS and classical-like EDS and evidence of tissue fragility as seen in individuals with vEDS. CONCLUSION: The clinical data demonstrate the overlap between the different EDS subtypes and underline the importance of next-generation sequencing gene panel analysis. The three different Glu>Lys variants point toward a new variant type in COL3A1 causative of vEDS, which has consistent clinical features. This is important knowledge for COL3A1 variant interpretation. Further follow-up data are required to establish the severity of tissue fragility complications compared with patients with other recognized molecular causes of vEDS.

Fibrillar Collagens.

Fibrillar collagens (types I, II, III, V, XI, XXIV and XXVII) constitute a sub-group within the collagen family (of which there are 28 types in humans) whose functions are to provide three-dimensional frameworks for tissues and organs. These networks confer mechanical strength as well as signalling and organizing functions through binding to cellular receptors and other components of the extracellular matrix (ECM). Here we describe the structure and assembly of fibrillar collagens, and their procollagen precursors, from the molecular to the tissue level. We show how the structure of the collagen triple-helix is influenced by the amino acid sequence, hydrogen bonding and post-translational modifications, such as prolyl 4-hydroxylation. The numerous steps in the biosynthesis of the fibrillar collagens are reviewed with particular attention to the role of prolyl 3-hydroxylation, collagen chaperones, trimerization of procollagen chains and proteolytic maturation. The multiple steps controlling fibril assembly are then discussed with a focus on the cellular control of this process in vivo. Our current understanding of the molecular packing in collagen fibrils, from different tissues, is then summarized on the basis of data from X-ray diffraction and electron microscopy. These results provide structural insights into how collagen fibrils interact with cell receptors, other fibrillar and non-fibrillar collagens and other ECM components, as well as enzymes involved in cross-linking and degradation.

Determination of the substrate repertoire of ADAMTS2, 3, and 14 significantly broadens their functions and identifies extracellular matrix organization and TGF-ß signaling as primary targets.

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-ß binding protein 1, TGF-ß 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-ß 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-ß signaling as primary targets.

Proteolytic control of TGF-ß co-receptor activity by BMP-1/tolloid-like proteases revealed by quantitative iTRAQ proteomics.

The metalloproteinase BMP-1 (bone morphogenetic protein-1) plays a major role in the control of extracellular matrix (ECM) assembly and growth factor activation. Most of the growth factors activated by BMP-1 are members of the TGF-ß superfamily known to regulate multiple biological processes including embryonic development, wound healing, inflammation and tumor progression. In this study, we used an iTRAQ (isobaric tags for relative and absolute quantification)-based quantitative proteomic approach to reveal the release of proteolytic fragments from the cell surface or the ECM by BMP-1. Thirty-eight extracellular proteins were found in significantly higher or lower amounts in the conditioned medium of HT1080 cells overexpressing BMP-1 and thus, could be considered as candidate substrates. Strikingly, three of these new candidates (betaglycan, CD109 and neuropilin-1) were TGF-ß co-receptors, also acting as antagonists when released from the cell surface, and were chosen for further substrate validation. Betaglycan and CD109 proved to be directly cleaved by BMP-1 and the corresponding cleavage sites were extensively characterized using a new mass spectrometry approach. Furthermore, we could show that the ability of betaglycan and CD109 to interact with TGF-ß was altered after cleavage by BMP-1, leading to increased and prolonged SMAD2 phosphorylation in BMP-1-overexpressing cells. Betaglycan processing was also observed in primary corneal keratocytes, indicating a general and novel mechanism by which BMP-1 directly affects signaling by controlling TGF-ß co-receptor activity. The proteomic data have been submitted to ProteomeXchange with the identifier PXD000786 and doi: 10.6019/PXD000786 .

Procollagen C-proteinase enhancer grasps the stalk of the C-propeptide trimer to boost collagen precursor maturation.

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.

Metalloproteases meprin α and meprin ß are C- and N-procollagen proteinases important for collagen assembly and tensile strength.

Type I fibrillar collagen is the most abundant protein in the human body, crucial for the formation and strength of bones, skin, and tendon. Proteolytic enzymes are essential for initiation of the assembly of collagen fibrils by cleaving off the propeptides. We report that Mep1a(-/-) and Mep1b(-/-) mice revealed lower amounts of mature collagen I compared with WT mice and exhibited significantly reduced collagen deposition in skin, along with markedly decreased tissue tensile strength. While exploring the mechanism of this phenotype, we found that cleavage of full-length human procollagen I heterotrimers by either meprin α or meprin ß led to the generation of mature collagen molecules that spontaneously assembled into collagen fibrils. Thus, meprin α and meprin ß are unique in their ability to process and release both C- and N-propeptides from type I procollagen in vitro and in vivo and contribute to the integrity of connective tissue in skin, with consequent implications for inherited connective tissue disorders.

Clinical, structural, biochemical and X-ray crystallographic correlates of pathogenicity for variants in the C-propeptide region of the COL3A1 gene.

Vascular Ehlers-Danlos syndrome (vEDS) is a heritable disorder of connective tissue caused by pathological variants in the COL3A1 gene, which encodes the α1 chain of type III collagen. Type III collagen is a major component of skin, arterial walls, and the gastrointestinal tract. Collagen III protein deficiency manifests as an increased risk of rupture, perforation, and dissection of these structures. The most disruptive gene variants affect the collagen helix via glycine substitutions or splice donor site mutations. The C-propeptide region of COL3A1 includes exons 49-52 and has a crucial role in initiating the C-terminal assembly of procollagen monomers in the early stages of collagen biosynthesis. Nineteen COL3A1 variants have previously been reported in these exons, of which four were associated with a severe vEDS phenotype. We identified two novel C-propeptide missense variants; p.Pro1440Leu, p.Arg1432Leu, and a non-stop mutation, c.4400A > T, p. (*1467Leuext*45). These variants produce variable phenotypes ranging from obvious acrogeria to classical or hypermobile EDS. A previously reported variant p.Lys1313Arg is of unknown clinical significance but likely benign, based on this study. Assigning disease pathogenicity remains complex, clinical phenotyping and crystal structure evidence being crucial. We briefly compare reported phenotypes for patients with missense variants in the C-propeptide domain for other human collagen disorders including COL1A1 and COL1A2 (osteogenesis imperfecta).

Type I procollagen C-propeptide defects: study of genotype-phenotype correlation and predictive role of crystal structure.

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.

Sizzled is unique among secreted frizzled-related proteins for its ability to specifically inhibit bone morphogenetic protein-1 (BMP-1)/tolloid-like proteinases.

BMP-1/tolloid-like proteinases (BTPs) are major enzymes involved in extracellular matrix assembly and activation of bioactive molecules, both growth factors and anti-angiogenic molecules. Although the control of BTP activity by several enhancing molecules is well established, the possibility that regulation also occurs through endogenous inhibitors is still debated. Secreted frizzled-related proteins (sFRPs) have been studied as possible candidates, with highly contradictory results, after the demonstration that sizzled, a sFRP found in Xenopus and zebrafish, was a potent inhibitor of Xenopus and zebrafish tolloid-like proteases. In this study, we demonstrate that mammalian sFRP-1, -2, and -4 do not modify human BMP-1 activity on several of its known substrates including procollagen I, procollagen III, pN-collagen V, and prolysyl oxidase. In contrast, Xenopus sizzled appears as a tight binding inhibitor of human BMP-1, with a K(i) of 1.5 ± 0.5 nM, and is shown to strongly inhibit other human tolloid isoforms mTLD and mTLL-1. Because sizzled is the most potent inhibitor of human tolloid-like proteinases known to date, we have studied its mechanism of action in detail and shown that the frizzled domain of sizzled is both necessary and sufficient for inhibitory activity and that it acts directly on the catalytic domain of BMP-1. Residues in sizzled required for inhibition include Asp-92, which is shared by sFRP-1 and -2, and also Phe-94, Ser-43, and Glu-44, which are specific to sizzled, thereby providing a rational basis for the absence of inhibitory activity of human sFRPs.

Procollagen C-proteinase enhancer stimulates procollagen processing by binding to the C-propeptide region only.

Bone morphogenetic protein-1 (BMP-1) and the tolloid-like metalloproteinases control several aspects of embryonic development and tissue repair. Unlike other proteinases whose activities are regulated mainly by endogenous inhibitors, regulation of BMP-1/tolloid-like proteinases relies mostly on proteins that stimulate activity. Among these, procollagen C-proteinase enhancers (PCPEs) markedly increase BMP-1/tolloid-like proteinase activity on fibrillar procollagens, in a substrate-specific manner. Here, we performed a detailed quantitative study of the binding of PCPE-1 and of its minimal active fragment (CUB1-CUB2) to three regions of the procollagen III molecule: the triple helix, the C-telopeptide, and the C-propeptide. Contrary to results described elsewhere, we found the PCPE-1-binding sites to be located exclusively in the C-propeptide region. In addition, binding and enhancing activities were found to be independent of the glycosylation state of the C-propeptide. These data exclude previously proposed mechanisms for the action of PCPEs and also suggest new mechanisms to explain how these proteins can stimulate BMP-1/tolloid-like proteinases by up to 20-fold.

Development of ex vivo organ culture models to mimic human corneal scarring.

PURPOSE: To develop ex vivo organ culture models of human corneal scarring suitable for pharmacological testing and the study of the molecular mechanisms leading to corneal haze after laser surgery or wounding. METHODS: Corneas from human donors were cultured ex vivo for 30 days, either at the air-liquid interface (AL) or immersed (IM) in the culture medium. Histological features and immunofluorescence for fibronectin, tenascin C, thrombospondin-1, and α-smooth muscle actin were graded from 0 to 3 for control corneas and for corneas wounded with an excimer laser. The effects of adding 10 ng/ml transforming growth factor-ß1 (TGF-ß1) to the culture medium and of prior complete removal of the epithelium and limbus, thus preventing reepithelialization, were also analyzed on wounded corneas. Collagen III expression was detected with real-time PCR. RESULTS: Wounding alone was sufficient to induce keratocyte activation and stromal disorganization, but it was only in the presence of added TGF-ß1 that intense staining for fibronectin and tenascin C was found in the AL and IM models (as well as thrombospondin-1 in the AL model) and that α-smooth muscle actin became detectable. The scar-like appearance of the corneas was exacerbated when TGF-ß1 was added and reepithelialization was prevented, resulting in the majority of corneas becoming opaque and marked upregulation of collagen III. CONCLUSIONS: THE MAIN FEATURES OF CORNEAL SCARRING WERE REPRODUCED IN THESE TWO COMPLEMENTARY MODELS: the AL model preserved differentiation of the epithelium and permits the topical application of active molecules, while the IM model ensures better perfusion by soluble compounds.

Dynamics of the secreted frizzled related protein Sizzled and potential implications for binding to bone morphogenetic protein-1 (BMP-1).

Sizzled (Szl) is both a secreted frizzled related protein (sFRP) and a naturally occurring inhibitor of the zinc metalloproteinase bone morphogenetic protein-1 (BMP-1), a key regulator of extracellular matrix assembly and growth factor activation. Here we present a new crystal structure for Szl which differs from that previously reported by a large scale (90°) hinge rotation between its cysteine-rich and netrin-like domains. We also present results of a molecular docking analysis showing interactions likely to be involved in the inhibition of BMP-1 activity by Szl. When compared with known structures of BMP-1 in complex with small molecule inhibitors, this reveals features that may be helpful in the design of new inhibitors to prevent the excessive accumulation of extracellular matrix that is the hallmark of fibrotic diseases.

Role of the netrin-like domain of procollagen C-proteinase enhancer-1 in the control of metalloproteinase activity.

The netrin-like (NTR) domain is a feature of several extracellular proteins, most notably the N-terminal domain of tissue inhibitors of metalloproteinases (TIMPs), where it functions as a strong inhibitor of matrix metalloproteinases and some other members of the metzincin superfamily. The presence of a C-terminal NTR domain in procollagen C-proteinase enhancers (PCPEs), proteins that stimulate the activity of astacin-like tolloid proteinases, raises the possibility that this might also have inhibitory activity. Here we show that both long and short forms of the PCPE-1 NTR domain, the latter beginning at the N-terminal cysteine known to be critical for TIMP activity, show no inhibition, at micromolar concentrations, of several members of the metzincin superfamily, including matrix metalloproteinase-2, bone morphogenetic protein-1 (a tolloid proteinase), and different ADAMTS (a disintegrin and a metalloproteinase with thrombospondin motifs) proteinases from the adamalysin family. In contrast, we report that the NTR domain within PCPE-1 leads to superstimulation of bone morphogenetic protein-1 activity in the presence of heparin and heparan sulfate. These observations point to a new mechanism whereby binding to cell surface-associated or extracellular heparin-like sulfated glycosaminoglycans might provide a means to accelerate procollagen processing in specific cellular and extracellular microenvironments.

Strong cooperativity and loose geometry between CUB domains are the basis for procollagen c-proteinase enhancer activity.

Procollagen C-proteinase enhancers (PCPE-1 and -2) specifically activate bone morphogenetic protein-1 (BMP-1) and other members of the tolloid proteinase family during C-terminal processing of fibrillar collagen precursors. PCPEs consist of two CUB domains (CUB1 and CUB2) and one NTR domain separated by one short and one long linker. It was previously shown that PCPEs can strongly interact with procollagen molecules, but the exact mechanism by which they enhance BMP-1 activity remains largely unknown. Here, we used a series of deletion mutants of PCPE-1 and two chimeric constructs with repetitions of the same CUB domain to study the role of each domain and linker. Out of all the forms tested, only those containing both CUB1 and CUB2 were capable of enhancing BMP-1 activity and binding to a mini-procollagen substrate with nanomolar affinity. Both these properties were lost by individual CUB domains, which had dissociation constants at least three orders of magnitude higher. In addition, none of the constructs tested could inhibit PCPE activity, although CUB2CUB2NTR was found to modulate BMP-1 activity through direct complex formation with the enzyme, resulting in a decreased rate of substrate processing. Finally, increasing the length of the short linker between CUB1 and CUB2 was without detrimental effect on both activity and substrate binding. These data support the conclusion that CUB1 and CUB2 bind to the procollagen substrate in a cooperative manner, involving the short linker that provides a flexible tether linking the two binding regions.

Extracellular and cell surface proteases in wound healing: new players are still emerging.

Tissue remodelling results from the concerted action of numerous extracellular and cell surface proteases. These act to synchronize the synthesis and degradation of the extracellular matrix with the control of cytokine activity and cell signalling in order to create appropriate environments for cell proliferation, migration and differentiation. Wound healing is a complex example of tissue remodelling that includes several steps occurring either concomitantly or successively during the process of repair: haemostasis, inflammation, angiogenesis, re-epithelialisation, granulation tissue formation, wound contraction and matrix remodelling. The main extracellular and cell surface proteases involved in wound healing are serine proteases, especially plasmin, and metalloproteases of the metzincin family (MMPs, ADAM(TS)s, tolloids, meprins, pappalysins) with cysteine proteases playing less prominent roles. Several regulatory proteins and hundreds of substrates have been identified for these proteases, either in vitro or in vivo. The aim of this review is not to present an exhaustive list of proteases and related molecules but to give an overview of the proteolytic events that are potentially relevant during tissue repair. New developments aimed at approaching a more integrative view of all the molecular events involved in tissue remodelling are also discussed.

Trimerization of collagen IX alpha-chains does not require the presence of the COL1 and NC1 domains.

Collagen IX is a heterotrimer of three alpha-chains, which consists of three COL domains (collagenous domains) (COL1-COL3) and four NC domains (non-collagenous domains) (NC1-NC4), numbered from the C-terminus. Although collagen IX chains have been shown to associate via their C-terminal NC1 domains and form a triple helix starting from the COL1 domain, it is not known whether chain association can occur at other sites and whether other collagenous and non-collagenous regions are involved. To address this question, we prepared five constructs, two long variants (beginning at the NC4 domain) and three short variants (beginning at the COL2 domain), all ending at the NC2 domain (or NC2 replaced by NC1), to study association and selection of collagen IX alpha-chains. Both long variants were able to associate with NC1 or NC2 at the C-terminus and form various disulfide-bonded trimers, but the specificity of chain selection was diminished compared with full-length chains. Trimers of the long variant ending at NC2 were shown to be triple helical by CD. Short variants were not able to assemble into disulfide-bonded trimers even in the presence of both conserved cysteine residues from the COL1-NC1 junction. Our results demonstrate that collagen IX alpha-chains can associate in the absence of COL1 and NC1 domains to form a triple helix, but the COL2-NC2 region alone is not sufficient for trimerization. The results suggest that folding of collagen IX is a co-operative process involving multiple COL and NC domains and that the COL1-NC1 region is important for chain specificity.

Roles of the procollagen C-propeptides in health and disease.

The procollagen C-propeptides of the fibrillar collagens play key roles in the intracellular assembly of procollagen molecules from their constituent polypeptides chains, and in the extracellular assembly of collagen molecules into fibrils. Here we review recent advances in understanding the molecular mechanisms controlling C-propeptide trimerization which have revealed the importance of inter-chain disulphide bonding and a small number of charged amino acids in the stability and specificity of different types of chain association. We also show how the crystal structure of the complex between the C-propeptide trimer of procollagen III and the active fragment of procollagen C-proteinase enhancer-1 leads to a detailed model for accelerating release of the C-propeptides from procollagen by bone morphogenetic protein-1 and related proteinases. We then discuss the effects of disease-related missense mutations in the C-propeptides in relation to the sites of these mutations in the three-dimensional structure. While in general there is a good correlation between disease severity and structure-based predictions, there are notable exceptions, suggesting new interactions involving the C-propeptides yet to be characterized. Mutations affecting proteolytic release of the C-propeptides from procollagen are discussed in detail. Finally, the roles of recently discovered interaction partners for the C-propeptides are considered during fibril assembly and cross-linking.

COL1A1 C-propeptide mutations cause ER mislocalization of procollagen and impair C-terminal procollagen processing.

Mutations in the type I procollagen C-propeptide occur in ~6.5% of Osteogenesis Imperfecta (OI) patients. They are of special interest because this region of procollagen is involved in α chain selection and folding, but is processed prior to fibril assembly and is absent in mature collagen fibrils in tissue. We investigated the consequences of seven COL1A1 C-propeptide mutations for collagen biochemistry in comparison to three probands with classical glycine substitutions in the collagen helix near the C-propeptide and a normal control. Procollagens with C-propeptide defects showed the expected delayed chain incorporation, slow folding and overmodification. Immunofluorescence microscopy indicated that procollagen with C-propeptide defects was mislocalized to the ER lumen, in contrast to the ER membrane localization of normal procollagen and procollagen with helical substitutions. Notably, pericellular processing of procollagen with C-propeptide mutations was defective, with accumulation of pC-collagen and/or reduced production of mature collagen. In vitro cleavage assays with BMP-1 ±â€¯PCPE-1 confirmed impaired C-propeptide processing of procollagens containing mutant proα1(I) chains. Overmodified collagens were incorporated into the matrix in culture. Dermal fibrils showed alterations in average diameter and diameter variability and bone fibrils were disorganized. Altered ER-localization and reduced pericellular processing of defective C-propeptides are expected to contribute to abnormal osteoblast differentiation and matrix function, respectively.

Procollagen C-proteinase enhancer-1 (PCPE-1) interacts with beta2-microglobulin (beta2-m) and may help initiate beta2-m amyloid fibril formation in connective tissues.

Dialysis related amyloidosis (DRA) is a progressive and serious complication in patients under long-term hemodialysis and mainly leads to osteo-articular diseases. Although beta(2)-microglobulin (beta2-m) is the major structural component of beta2-m amyloid fibrils, the initiation of amyloid formation is not clearly understood. Here, we have identified procollagen C-proteinase enhancer-1 (PCPE-1) as a new interacting protein with beta2-m by screening a human synovium cDNA library. The interaction of beta2-m with full-length PCPE-1 was confirmed by immunoprecipitation, solid-phase binding and pull-down assays. By yeast two-hybrid analysis and pull-down assay, beta2-m appeared to interact with PCPE-1 via the NTR (netrin-like) domain and not via the CUB (C1r/C1s, Uegf and BMP-1) domain region. In synovial tissues derived from hemodialysis patients with DRA, beta2-m co-localized and formed a complex with PCPE-1. beta2-m did not alter the basal activity of bone morphogenetic protein-1/procollagen C-proteinase (BMP-1/PCP) nor BMP-1/PCP activity enhanced by PCPE-1. PCPE-1 did not stimulate beta2-m amyloid fibril formation from monomeric beta2-m in vitro under acidic and neutral conditions as revealed by thioflavin T fluorescence spectroscopy and electron microscopy. Since PCPE-1 is abundantly expressed in connective tissues rich in type I collagen, it may be involved in the initial accumulation of beta2-m in selected tissues such as tendon, synovium and bone. Furthermore, since such preferential deposition of beta2-m may be linked to subsequent beta2-m amyloid fibril formation, the disruption of the interaction between beta2-m and PCPE-1 may prevent beta2-m amyloid fibril formation and therefore PCPE-1 could be a new target for the treatment of DRA.