Data comparing the kinetics of procollagen type I processing by bone morphogenetic protein 1 (BMP-1) with and without procollagen C-proteinase enhancer 1 (PCPE-1)

This article provides kinetic constants for C-terminal processing of procollagen type I by bone morphogenetic protein 1 (BMP-1; the major procollagen C-proteinase), a reaction stimulated by the connective tissue glycoprotein procollagen C-proteinase enhancer 1 (PCPE-1). Reported are Km, Vmax, Kcat and Kcat/Km (catalytic coefficient) values for BMP-1 alone, BMP-1 with intact PCPE-1, BMP-1 with the CUB (Complement C1r/C1s, Uegf, BMP-1) domains fragment of PCPE-1 as well as its NTR (netrin-like) domain

Folding and activity of recombinant human procollagen C-proteinase enhancer.

International audienceRecombinant human procollagen C-proteinase enhancer (rPCPE) was expressed using a baculovirus system and purified to homogeneity using a three-step procedure including heparin affinity chromatography. Heparin binding was dependent on the C-terminal netrin-like domain. The recombinant protein was found to be active, increasing the activity of procollagen C-proteinase/bone morphogenetic protein-1 on type I procollagen in a manner comparable to the native protein. Enhancing activity was dependent on intact disulfide bonding within the protein. By circular dichroism, the observed secondary structure of rPCPE was consistent with the known three-dimensional structures of proteins containing homologous domains.Recombinant human procollagen C-proteinase enhancer (rPCPE) was expressed using a baculovirus system and purified to homogeneity using a three-step procedure including heparin affinity chromatography. Heparin binding was dependent on the C-terminal netrin-like domain. The recombinant protein was found to be active, increasing the activity of procollagen C-proteinase/bone morphogenetic protein-1 on type I procollagen in a manner comparable to the native protein. Enhancing activity was dependent on intact disulfide bonding within the protein. By circular dichroism, the observed secondary structure of rPCPE was consistent with the known three-dimensional structures of proteins containing homologous domains

Loss of fibulin-4 disrupts collagen synthesis and maturation: implications for pathology resulting from EFEMP2

Homozygous recessive mutations in either EFEMP2 (encoding fibulin-4) or FBLN5 (encoding fibulin-5), critical genes for elastogenesis, lead to autosomal recessive cutis laxa types 1B and 1A, respectively. Previously, fibulin-4 was shown to bind lysyl oxidase (LOX), an elastin/collagen cross-linking enzyme, in vitro. Consistently, reported defects in humans with EFEMP2 mutations are more severe and broad in range than those due to FBLN5 mutations and encompass both elastin-rich and collagen-rich tissues. However, the underlying disease mechanism in EFEMP2 mutations has not been fully addressed. Here, we show that fibulin-4 is important for the integrity of aortic collagen in addition to elastin. Smooth muscle-specific Efemp2 loss in mouse (termed SMKO) resulted in altered fibrillar collagen localization with larger, poorly organized fibrils. LOX activity was decreased in Efemp2-null cells, and collagen cross-linking was diminished in SMKO aortas; however, elastin cross-linking was unaffected and the level of mature LOX was maintained to that of wild-type aortas. Proteomic screening identified multiple proteins involved in procollagen processing and maturation as potential fibulin-4-binding partners. We showed that fibulin-4 binds procollagen C-endopeptidase enhancer 1 (Pcolce), which enhances proteolytic cleavage of the procollagen C-terminal propeptide during procollagen processing. Interestingly, however, procollagen cleavage was not affected by the presence or absence of fibulin-4 in vitro. Thus, our data indicate that fibulin-4 serves as a potential scaffolding protein during collagen maturation in the extracellular space. Analysis of collagen in other tissues affected by fibulin-4 loss should further increase our understanding of underlying pathologic mechanisms in patients with EFEMP2 mutations

Circulating Bone Morphogenetic Protein 1–3 Isoform Increases Renal Fibrosis

Bone morphogenetic proteins (BMPs) participate in organ regeneration through autocrine and paracrine actions, but the existence and effects of these proteins in the systemic circulation is unknown. Using liquid chromatography–mass spectrometry, we identified BMP6, GDF15, and the BMP1–3 isoform of the Bmp1 gene in plasma samples from healthy volunteers and patients with CKD. We isolated the endogenous BMP1–3 protein and demonstrated that it circulates as an active enzyme, evidenced by its ability to cleave dentin matrix protein-1 in vitro. In rats with CKD, administration of recombinant BMP1–3 increased renal fibrosis and reduced survival. In contrast, administration of a BMP1–3-neutralizing antibody reduced renal fibrosis, preserved renal function, and increased survival. In addition, treating with the neutralizing antibody was associated with low plasma levels of TGFβ1 and connective tissue growth factor. In HEK293 cells and remnant kidneys, BMP1–3 increased the transcription of collagen type I, TGFβ1, β-catenin, and BMP7 via a BMP- and Wnt-independent mechanism that involved signaling through an integrin β1 subunit. The profibrotic effect of BMP1–3 may, in part, be a result of the accompanied decrease in decorin (DCN) expression. Taken together, inhibition of circulating BMP1–3 reduces renal fibrosis, suggesting that this pathway may be a therapeutic target for CKD

Enzymatic cleavage specificity of the proα1(V) chain processing analysed by site-directed mutagenesis

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 proα1(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 proα1(V) chain, referred to as Nα1, was recombinantly expressed to be used for enzymatic assays and for antibody production. Structural analysis showed that Nα1 is a monomer composed of a compact globule and an extended tail, which correspond respectively to the non-collagenous Nα1 subdomains, TSPN-1 (thrombospondin-1 N-terminal domain-like) and the variable region. Nα1 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