Folding and activity of recombinant human procollagen C‐proteinase enhancer

Moschcovich, Laura; Bernocco, Simonetta; Font, B.; Rivkin, Hadassah; Eichenberger, D.; Chejanovsky, Nor; Hulmes, David J.S.; Kessler, Efrat

doi:10.1046/j.1432-1327.2001.02189.x

Cited by 39 publications

(46 citation statements)

References 38 publications

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“…As expected (30,44), maximum enhancement with wild-type PCPE-1 was obtained at an enhancer:substrate ratio of 1:1. In contrast, for the mutants F90A and D109A, increasing the enhancer:substrate ratio up to 5:1 led only to a gradual increase in enhancing activity, with no sign of a plateau, and enhancing activity remained considerably less than that of the control (Fig.…”

Section: Identification Of Targetsupporting

confidence: 80%

Insights into How CUB Domains Can Exert Specific Functions while Sharing a Common Fold

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Eichenberger

et al. 2007

Journal of Biological Chemistry

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Procollagen C-proteinase enhancers (PCPE-1 and -2) are extracellular glycoproteins that can stimulate the C-terminal processing of fibrillar procollagens by tolloid proteinases such as bone morphogenetic protein-1. They consist of two CUB domains (CUB1 and -2) that alone account for PCPE-enhancing activity and one C-terminal NTR domain. CUB domains are found in several extracellular and plasma membrane-associated proteins, many of which are proteases. We have modeled the structure of the CUB1 domain of PCPE-1 based on known threedimensional structures of CUB-containing proteins. Sequence alignment shows conserved amino acids, notably two acidic residues (Asp-68 and Asp-109) involved in a putative surface-located calcium binding site, as well as a conserved tyrosine residue (Tyr-67). In addition, three residues (Glu-26, Thr-89, and Phe-90) are found only in PCPE CUB1 domains, in putative surface-exposed loops. Among the conserved residues, it was found that mutations of Asp-68 and Asp-109 to alanine almost completely abolished PCPE-1 stimulating activity, whereas mutation of Tyr-67 led to a smaller reduction of activity. Among residues specific to PCPEs, mutation of Glu-26 and Thr-89 had little effect, whereas mutation of Phe-90 dramatically decreased the activity. Changes in activity were paralleled by changes in binding of different PCPE-1 mutants to a mini-procollagen III substrate, as shown by surface plasmon resonance. We conclude that PCPE-stimulating activity requires a calcium binding motif in the CUB1 domain that is highly conserved among CUB-containing proteins but also that PCPEs contain specific sites that could become targets for the development of novel anti-fibrotic therapies. CUB3 domains are widely occurring structural motifs, found almost exclusively in extracellular and plasma membrane-associated proteins. These proteins are involved in a wide range of biological functions, including complement activation (1, 2), developmental patterning (3, 4), tissue repair (5), axon guidance and angiogenesis (6, 7), cell signaling (8), fertilization (9), hemostasis (10), inflammation (11), neurotransmission (12), receptor-mediated endocytosis (13,14), and tumor suppression (15, 16). Many CUB domain-containing proteins are proteases (1,2,5,10,(17)(18)(19). Although the roles of the CUB domains are largely unexplored, a number of them have been shown to be involved in oligomerization and/or recognition of substrates and binding partners.The protein families from which the CUB domain derives its name are the complement serine proteases C1r, C1s, MASP-1, MASP-2, and MASP-3 and the bone morphogenetic protein-1/ tolloid metalloproteases BMP-1, mTLD, mTLL-1, and mTLL-2 (or their counterparts xolloids, tolloids, and SpAN/BP10 in Xenopus, Drosophila, and sea urchin, respectively). Each consists of a catalytic domain (either N-terminal in tolloids or C-terminal in complement proteases) together with several CUB domains interspersed by calcium-binding EGF domains. In the case of the complement proteases, the CUB domain...

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Section: Identification Of Targetsupporting

confidence: 80%

Insights into How CUB Domains Can Exert Specific Functions while Sharing a Common Fold

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Eichenberger

et al. 2007

Journal of Biological Chemistry

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“…Proteins and Antibodies-Recombinant human PCPE as well as the C-terminal propeptide trimer from procollagen III (CPIII) were expressed using baculovirus systems (19,37,38). Procollagen I and its C-propeptide trimer domain (CPI) were purified from the culture media of chick embryo tendon fibroblasts (39) or chick embryo tendons (40), respectively.…”

Section: Methodsmentioning

confidence: 99%

“…41). Reduced and alkylated CPIII was prepared as described (19). The binding epitopes of the monoclonal antibodies 48D34, 48B14, and 48D19 mapped to sites within residues 1-30, 31-207, and 208 -245, respectively, of the CPIII polypeptide chain as described (42).…”

Section: Methodsmentioning

confidence: 99%

Interaction Properties of the Procollagen C-proteinase Enhancer Protein Shed Light on the Mechanism of Stimulation of BMP-1

Ricard‐Blum

Bernocco

Font

et al. 2002

Journal of Biological Chemistry

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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 Cpropeptide 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.Bone morphogenetic protein-1 (BMP-1) 1 and other tolloidrelated metalloproteinases (1, 2), also known as procollagen C-proteinases (PCPs), have recently been shown to be involved in the control of a variety of morphogenetic events during development and tissue repair. These include: (i) the deposition of collagen fibrils in the extracellular matrix following the processing of procollagen propeptides (3-6); (ii) dorsoventral patterning (7-10) through the cleavage of the growth factor inhibitors chordin and SOG; (iii) collagen and elastin crosslinking by the processing of the inactive precursors of lysyl oxidases (11, 12); and (iv) adhesion/migration of epithelial cells by the cleavage of laminin 5 chains (13-15). The activities of PCPs on procollagen substrates may be stimulated up to 20-fold by another glycoprotein of the extracellular matrix, procollagen C-proteinase enhancer (PCPE) (16 -19), which lacks intrinsic proteinase activity. Similarly, in the case of chordin and SOG, the protein TSG or its homologues stimulates cleavage by tolloid proteinases (20, 21), thus raising the possibility that PCP processing of different substrates might be specifically regulated by distinct enhancer proteins.Both tolloid proteinases and PCPE are multidomain glycoproteins containing multiple copies of the so-called CUB domain (22), a protein module common to several extracellular and plasma membrane-associated...

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“…BMP6 and BMP7 were detected immunohistochemically as described previously. 54,55 rhBMP1-3 and synthetic peptides from specific protein domains were used to immunize mice and produce hybridomas for the production of monoclonal BMP1-3 antibodies (Genera Reasearch Lab).…”

Section: Antibodiesmentioning

confidence: 99%

Circulating Bone Morphogenetic Protein 1–3 Isoform Increases Renal Fibrosis

Grgurević¹,

Maček²,

Healy³

et al. 2011

Journal of the American Society of Nephrology

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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 TGF␤1 and connective tissue growth factor. In HEK293 cells and remnant kidneys, BMP1-3 increased the transcription of collagen type I, TGF␤1, ␤-catenin, and BMP7 via a BMP-and Wnt-independent mechanism that involved signaling through an integrin ␤1 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.

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Folding and activity of recombinant human procollagen C‐proteinase enhancer

Cited by 39 publications

References 38 publications

Insights into How CUB Domains Can Exert Specific Functions while Sharing a Common Fold

Insights into How CUB Domains Can Exert Specific Functions while Sharing a Common Fold

Interaction Properties of the Procollagen C-proteinase Enhancer Protein Shed Light on the Mechanism of Stimulation of BMP-1

Circulating Bone Morphogenetic Protein 1–3 Isoform Increases Renal Fibrosis

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