Procollagen C-proteinase Enhancer Protein Research Articles

C17ORF58 Is Upregulated in Peripheral T-Cell Lymphomas and Encodes a Protein Critical for Survival of PTCL Cells

Peripheral T-cell lymphomas (PTCL) constitute a group of heterogeneous T- and NK-cell malignancies, accounting for about 15% of Non-Hodgkin lymphomas (NHL). Most PTCL subtypes exhibit an aggressive disease presentation, carrying a worse prognosis than B-cell lymphomas. PTCL - Not Otherwise Specified (PTCL-NOS), the largest subgroup, is categorized by exclusion, with a diagnosis given if the patient does not meet the diagnostic criteria of other PTCL subtypes. PTCL-NOS is a heterogeneous group with diverse cells of origin, varied cytogenic, molecular, and morphological phenotypes, and antigen variability. Despite the increase in specific T-cell lymphoma treatment options, such as belinostat, romidepsin, pralatrexate, and brentuximab vedotin, alongside conventional non-targeted chemotherapy like CHOP and R-CHOP, the overall 5 year survival remains only at 20-30%. A deeper understanding of the pathways and genes critical for PTCL-NOS survival is urgently needed. In our quest to better understand the molecular landscape, we performed a gene expression analysis of ten PTCL-NOS lymphomas and five normal samples using RNAseq analysis. Our study identified ~400 genes consistently upregulated in lymphoma samples, indicating potential involvement in tumor maintenance. Following the exclusion of common essential genes, we performed a shRNA-mediated knockdown screen of the remaining candidates in the human PTCL-NOS cell line T8ML-1. This approach unveiled several lethal genes, including MYBL2, TRIP13, KIF18B, C17ORF58, and others. Cell cycle analysis demonstrated that while knockdown of MYBL2, TRIP13, and KIF18B induced G2-M arrest, downregulation of C17ORF58 primarily induced apoptosis. Notably, downregulation of C17ORF58 did not impact the proliferation of human embryonic kidney fibroblasts HEK 293T, suggesting that normal cells might not require this protein for growth. However, its downregulation in additional hematologic cell lines, such as Cutaneous T-cell lymphoma cell lines (HH, Hut78) and T-cell lymphoblastic leukemia (Jurkat), also induced apoptosis, which suggests a key role for C17ORF58 in T-cell malignancies. Analysis of publicly available RNAseq data revealed overexpression of C17ORF58 in the majority of PTCL-NOS, Anaplastic Large Cell, Hepatosplenic T-cell, and malignant NK/T-cell lymphomas, relative to normal T-cells, thus highlighting its possible role in tumor development and maintenance. C17ORF58 encodes a predicted protein of 339 amino acids, with an estimated molecular weight of 37 kDa. This protein features two putative domains: the PRK07764 domain, which is homologous to DNA polymerase III subunits gamma and tau, and the netrin module, a 130 amino acid residue domain found within the C-terminal sections of various proteins, including netrins, complement proteins C3-C5, secreted frizzled-related proteins, and type I procollagen C-proteinase enhancer proteins. The predicted promoter has binding sites for Oct-B1-3, Pax-2, POU2F1-2, 2B, and 2C, suggesting that the C17ORF58-encoded protein may participate in normal development. In conclusion, our data indicate that upregulation of C17ORF58 may contribute to the progression and maintenance of PTCL and could serve as a potential target for treating T-cell malignancies. Investigations into its physiological role in normal T-cells and pathways deregulated upon its inactivation are ongoing.

Up-regulation of PCOLCE by TWIST1 promotes metastasis in Osteosarcoma.

Procollagen C-proteinase enhancer protein (PCOLCE) was originally identified as an enhancer to facilitate the catalysis of procollagens by BMP1. PCOLCE participates in the reconstitution of extracellular and corneal repair. The elevation of PCOLCE in blood indicates that breast cancer has metastasized into the bones. However, direct research on PCOLCE has not been reported.Methods: ECM candidates were identified by RNA-seq analysis from 4 normal and 16 osteosarcoma tissues. The in vitro migration and invasion abilities of osteosarcoma cells were determined by a Transwell assay. A spontaneous metastatic osteosarcoma model was established to assess osteosarcoma metastasis in vivo. The N-linked glycosylated amino acids were identified by PNGase F treatment combined with Western blotting. The mechanism of TWIST1 regulating PCOLCE transcription was elucidated by luciferase, qPCR and ChIP assays.Results: PCOLCE was markedly up-regulated in human osteosarcoma tissues compared to its expression in noncancerous adjacent tissues; high PCOLCE expression in tissues correlated with a poor patient prognosis, and the knockdown of PCOLCE by shRNAs impaired the migration, invasion and lung metastasis of osteosarcoma cells. The overexpression of wild-type PCOLCE, but not its N29Q mutant, promoted migration, invasion and metastasis, indicating that the glycosylation of PCOLCE at Asn29 is necessary for its functions in osteosarcoma. TWIST1, a key transcription factor in metastasis, was also overexpressed in osteosarcoma tissues and positively correlated with either PCOLCE or its potential procollagen substrates, such as COL1A1, COL1A2, COL5A1, COL8A2 and COL10A1.Conclusion: Our findings are the first to provide evidence that PCOLCE plays a critical role in promoting the lung metastasis of osteosarcoma, and this up-regulation of PCOLCE by TWIST1 may lead to a new therapeutic strategy to treat patients with metastatic osteosarcoma.

Label-free relative quantification applied to LC-MALDI acquisition for rapid analysis of chondrocyte secretion modulation.

The present work presents the adaptation of label free relative protein quantification principles to LC-MALDI data to rapidly measure protein fold-changes between samples of relative complexity and its utility to characterize the secreted proteome of human primary chondrocytes. The method was employed to characterize the chondrocyte secretome regulation by TGFβ1 and is proposed as a routine tool to assess the quality of biomaterials designed for cartilage repair and to quantitatively investigate the influence of environmental factors upon it.

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.

Crystal Structure of C5b-6 Suggests Structural Basis for Priming Assembly of the Membrane Attack Complex

The complement membrane attack complex (MAC) forms transmembrane pores in pathogen membranes. The first step in MAC assembly is cleavage of C5 to generate metastable C5b, which forms a stable complex with C6, termed C5b-6. C5b-6 initiates pore formation via the sequential recruitment of homologous proteins: C7, C8, and 12-18 copies of C9, each of which comprises a central MAC-perforin domain flanked by auxiliary domains. We recently proposed a model of pore assembly, in which the auxiliary domains play key roles, both in stabilizing the closed conformation of the protomers and in driving the sequential opening of the MAC-perforin β-sheet of each new recruit to the growing pore. Here, we describe an atomic model of C5b-6 at 4.2 Å resolution. We show that C5b provides four interfaces for the auxiliary domains of C6. The largest interface is created by the insertion of an interdomain linker from C6 into a hydrophobic groove created by a major reorganization of the α-helical domain of C5b. In combination with the rigid body docking of N-terminal elements of both proteins, C5b becomes locked into a stable conformation. Both C6 auxiliary domains flanking the linker pack tightly against C5b. The net effect is to induce the clockwise rigid body rotation of four auxiliary domains, as well as the opening/twisting of the central β-sheet of C6, in the directions predicted by our model to activate or prime C6 for the subsequent steps in MAC assembly. The complex also suggests novel small molecule strategies for modulating pathological MAC assembly.

Processing of Procollagen III by Meprins: New Players in Extracellular Matrix Assembly?

Meprins α and β, a subgroup of zinc metalloproteinases belonging to the astacin family, are known to cleave components of the extracellular matrix, either during physiological remodeling or in pathological situations. In this study we present a new role for meprins in matrix assembly, namely the proteolytic processing of procollagens. Both meprins α and β release the N- and C-propeptides from procollagen III, with such processing events being critical steps in collagen fibril formation. In addition, both meprins cleave procollagen III at exactly the same site as the procollagen C-proteinases, including bone morphogenetic protein-1 (BMP-1) and other members of the tolloid proteinase family. Indeed, cleavage of procollagen III by meprins is more efficient than by BMP-1. In addition, unlike BMP-1, whose activity is stimulated by procollagen C-proteinase enhancer proteins (PCPEs), the activity of meprins on procollagen III is diminished by PCPE-1. Finally, following our earlier observations of meprin expression by human epidermal keratinocytes, meprin α is also shown to be expressed by human dermal fibroblasts. In the dermis of fibrotic skin (keloids), expression of meprin α increases and meprin β begins to be detected. Our study suggests that meprins could be important players in several remodeling processes involving collagen fiber deposition.

Mammalian Tolloid-like 1 Binds Procollagen C-proteinase Enhancer Protein 1 and Differs from Bone Morphogenetic Protein 1 in the Functional Roles of Homologous Protein Domains

Bone morphogenetic protein 1 (BMP1) is the prototype of a subgroup of metalloproteinases with manifold roles in morphogenesis. Four mammalian subgroup members exist, including BMP1 and mammalian Tolloid-like 1 (mTLL1). Subgroup members have a conserved protein domain structure: an NH2-terminal astacin-like protease domain, followed by a fixed order of CUB and epidermal growth factor-like protein-protein interaction motifs. Previous structure/function studies have documented those BMP1 protein domains necessary for secretion, and activity against various substrates. Here we demonstrate that, in contradiction to previous reports, the most NH2-terminal CUB domain (CUB1) is not required for BMP1 secretion nor is the next CUB domain (CUB2) required for enzymatic activity. The same is true for mTLL1. In fact, secreted protease domains of BMP1 and mTLL1, devoid of CUB or epidermal growth factor-like domains, have procollagen C-proteinase (pCP) activity and activity for biosynthetic processing of biglycan, the latter with kinetics superior to those of the full-length proteins. Structure-function analyses herein also suggest differences in the functional roles played by some of the homologous domains in BMP1 and mTLL1. Surprisingly, although BMP1 has long been known to be Ca2+-dependent, a property previously assumed to apply to all members of the subgroup, mTLL1 is demonstrated to be independent of Ca2 levels in its ability to cleave some, but not all, substrates. We also show that pCP activities of only versions of BMP1 and mTLL1 with intact COOH termini are enhanced by the procollagen C-proteinase enhancer 1 (PCOLCE1) and that mTLL1 binds PCOLCE1, thus suggesting reappraisal of the accepted paradigm for how PCOLCE1 enhances pCP activities.

Degradomics: Systems biology of the protease web. Pleiotropic roles of MMPs in cancer

The major role of matrix metalloproteinases (MMPs) is for homeostatic regulation of the extracellular environment, not simply to degrade matrix as their name suggests. We designed and printed a dedicated, focused DNA microarray, the CLIP-CHIP, that enables the analysis of every human and murine protease, protease homologue and inhibitor on a system-wide basis in cancer. We have also developed novel proteomic approaches to identify cleaved substrates of proteases in complex milieu. Isotope coded affinity tag (ICAT) and iTRAQ labeling of conditioned medium proteins secreted by MDA-MB-231 breast carcinoma cells and Mmp2 -/- murine fibroblasts transfected with protease (MT1-MMP or active MMP-2) or their inactive mutant forms enabled quantitative proteomics to be performed. Comparison of the relative abundance ratios of identical peptides from the two samples identified proteins in the conditioned medium that may have been degraded (low ratios) and those that were shed from the cell membrane (high ratios). MS/MS was used to sequence and identify the potential substrates. These analyses have revealed a plethora of new bioactive substrates and biological roles for MMPs. Biochemical confirmation of cleavage of the potential substrates was performed and the cleavage sites identified by MALDI-TOF. In these studies we discovered and confirmed that CTGF, galectin-1, death receptor-6, HSP90alpha, procollagen C-proteinase enhancer protein, the chemokine fractalkine, and cystatin C were novel MT1-MMP or MMP-2 substrates. These sophisticated cellular control functions highlight new intervention points in multiple pathways to treat early stage cancer.

Expression of procollagen C-proteinase enhancer-1 in the remodeling rat heart is stimulated by aldosterone

Excessive collagen deposition is a common complication of myocardial infarction that causes progressive heart disease. Several pro-fibrotic cytokines and hormones, including aldosterone, control this process. Procollagen processing by procollagen C-proteinase(s) is critical for collagen deposition and is potentiated by procollagen C-proteinase enhancer proteins (PCPEs). We have shown previously that, in addition to stimulation of collagen I expression, aldosterone increases PCPE-1 expression in cultured heart fibroblasts. The present study was designed to examine whether aldosterone acts similarly in vivo. Rats underwent coronary artery ligation to induce myocardial infarction. They were then left either untreated (control) or treated with spironolactone (an aldosterone receptor antagonist) for 5 weeks when they were sacrificed and their hearts removed for analysis. In situ hybridization co-localized PCPE-1 and collagen I mRNAs to fibroblasts surrounding the scar region and adjacent blood vessels. The levels of both transcripts in the remodeling myocardium of untreated rats increased twofold as compared to sham-operated controls, an increase greatly reduced by spironolactone. Correspondingly, a 2–5 fold increase in PCPE-1 and collagen I was observed in the hearts of untreated rats as compared to both the spironolactone-treated and sham-operated controls. The results establish aldosterone as a physiological stimulator of PCPE-1 expression in the remodeling myocardium after infarction. Since PCPE-1 itself is a positive regulator of collagen deposition, this finding suggests PCPE-1 as a new potential target for intervention with cardiac fibrosis.

Functional Insights from the Structure of the Multifunctional C345C Domain of C5 of Complement

The complement protein C5 initiates assembly of the membrane attack complex. This remarkable process results in lysis of target cells and is fundamental to mammalian defense against infection. The 150-amino acid residue domain at the C terminus of C5 (C5-C345C) is pivotal to C5 function. It interacts with enzymes that convert C5 to C5b, the first step in the assembly of the membrane attack complex; it also binds to the membrane attack complex components C6 and C7 with high affinity. Here a recombinant version of this C5-C345C domain is shown to adopt the oligosaccharide/oligonucleotide binding fold, with two helices packed against a five-stranded beta-barrel. The structure is compared with those from the netrin-like module family that have a similar fold. Residues critical to the interaction with C5-convertase cluster on a mobile, hydrophobic inter-strand loop that protrudes from the open face of the beta-barrel. The opposite, helix-dominated face of C5-C345C carries a pair of exposed hydrophobic side chains adjacent to a striking negatively charged patch, consistent with affinity for positively charged factor I modules in C6 and C7. Modeling of homologous domains from complement proteins C3 and C4, which do not participate in membrane attack complex assembly, suggests that this provisionally identified C6/C7-interacting face is indeed specific to C5.

Profiling of the secreted proteins during 3T3-L1 adipocyte differentiation leads to the identification of novel adipokines.

Adipose tissue is an endocrine organ capable of secreting a number of adipokines with a role in the regulation of adipose tissue and whole-body metabolism. We used two-dimensional gel electrophoresis combined with mass spectrometry to profile the secreted proteins from (pre)adipocytes. The culture medium of 3T3-L1 cells during adipocyte differentiation was screened, and 41 proteins that responded to blocking of secretion by 20 degrees C treatment and/or brefeldin A treatment were identified. Prohibitin, stress-70 protein, and adhesion-regulating molecule 1 are reported for the first time as secreted proteins. In addition, procollagen C-proteinase enhancer protein, galectin-1, cyclophilin A and C, and SF20/IL-25 are newly identified as adipocyte secreted factors. Secretion profiles indicated a dynamic environment including an actively remodeling extracellular matrix and several factors involved in growth regulation.

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

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.

Stabilization of RNA Strands in Protein Synthesis by Type I Procollagen C-Proteinase Enhancer Protein, a Potential RNA-Binding Protein, in Hepatic Stellate Cells

Type I procollagen C-proteinase enhancer (PCPE) exists in hepatic stellate cells (HSCs) which can produce collagen. The deduced amino acid sequence of PCPE contains motifs specific for RNA-binding proteins. The effect of PCPE on the syntheses of collagen and noncollagenous protein was studied using an HSC clone derived from cirrhotic rat liver. When the cells were cultured in the presence of an antisense oligonucleotide (AS) against PCPE mRNA, the synthesis of noncollagenous protein as well as collagen was reduced compared to the cells cultured with addition of a nonsense oligonucleotide (NS). The extent of the reduction was similar in both syntheses. The total RNA content of the AS-treated cells and NS-treated cells did not differ. In the presence of actinomycin D, however, such total RNA content was decreased more rapidly in the AS-treated cells than in the NS-treated cells. PCPE may be involved in stabilization of RNA strands in noncollagenous protein synthesis as well as collagen synthesis.

Assignment1 of the mouse Pcolce2 gene, which encodes procollagen C-proteinase enhancer protein 2, to chromosome 9 and localization of PCOLCE2 to human chromosome 3q23

Assignment<footref rid="foot01">1</footref> of the mouse Pcolce2 gene, which encodes procollagen C-proteinase enhancer protein 2, to chromosome 9 and localization of PCOLCE2 to human chromosome 3q23

Identification and Expression of a Novel Type I Procollagen C-Proteinase Enhancer Protein Gene from the Glaucoma Candidate Region on 3q21–q24

A novel human Type I procollagen C-proteinase enhancer protein-like gene, PCOLCE2, was identified by sequencing an EST in the primary open-angle glaucoma (POAG) region on 3q21. The total cDNA encoded a 415-amino-acid protein that has 43% identity to the Type I procollagen C-proteinase enhancer protein (PCOLCE1). PCOLCE2 contains two CUB domains, which are thought to be involved in protein–protein interactions, and an NTR module. PCOLCE2 message is expressed in the trabecular meshwork, lungs, heart, brain, liver, skeletal muscle, kidney, pancreas, and placenta as a 2-kb message. PCOLCE2, a 52-kDa protein, is expressed in the trabecular meshwork. A novel gene, PCOLCE2, has been identified and characterized. Based upon its homology with collagen-binding proteins, its expression in the trabecular meshwork, and its chromosome location, PCOLCE2 is a candidate gene for GLC1C. However, no coding sequence mutations were detected in PCOLCE2 in a POAG patient from the GLC1C family.

Mechanical Load Enhances Procollagen Processing in Dermal Fibroblasts by Regulating Levels of Procollagen C-Proteinase

Mechanical forces are emerging as key regulators of cell function. We hypothesize that mechanical load may influence dermal fibroblast activity. We assessed the direct effects of mechanical load on human dermal fibroblast procollagen synthesis and processing in vitro. Cells were loaded in a biaxial loading system (Flexercell 3000). Hydroxyproline levels were measured in the medium and cell layer as an estimate of procollagen synthesis and processing to insoluble collagen. Mechanical load (in the presence of serum or TGF-β) enhanced procollagen synthesis by 45 ± 3% (P < 0.001), and 38 ± 4% (P < 0.001), respectively, over unloaded growth factor controls after 48 h. Insoluble collagen deposition was enhanced in the same cultures by 115 ± 8% (P < 0.01) and 72% ± 9% (P < 0.01), respectively. This effect was inhibited using l-arginine suggesting that procollagen C-proteinase, the enzyme which directly cleaves the C-terminal propeptide of procollagen to form insoluble collagen, is required for the fiber formation observed. Procollagen mRNA levels in loaded samples increased by more than two-fold in both serum and TGF-β-treated cultures at 48 h. Procollagen C-proteinase mRNA levels were also enhanced by a similar magnitude, although the increase was observed at 24 h. Procollagen C-proteinase protein levels were also increased at this time. Protein and mRNA levels of the procollagen C-proteinase enhancer protein, which binds the C-terminal propeptide of procollagen to enhance the rate of peptide cleavage, were unaffected by mechanical load. This study demonstrates that mechanical load promotes procollagen synthesis in dermal fibroblasts by enhancing gene expression and posttranslational processing of procollagen.

The NTR module: domains of netrins, secreted frizzled related proteins, and type I procollagen C-proteinase enhancer protein are homologous with tissue inhibitors of metalloproteases.

Using homology search, structure prediction, and structural characterization methods we show that the C-terminal domains of (1) netrins, (2) complement proteins C3, C4, C5, (3) secreted frizzled-related proteins, and (4) type I procollagen C-proteinase enhancer proteins (PCOLCEs) are homologous with the N-terminal domains of (5) tissue inhibitors of metalloproteinases (TIMPs). The proteins harboring this netrin module (NTR module) fulfill diverse biological roles ranging from axon guidance, regulation of Wnt signaling, to the control of the activity of metalloproteases. With the exception of TIMPs, it is not known at present what role the NTR modules play in these processes. In view of the fact that the NTR modules of TIMPs are involved in the inhibition of matrixin-type metalloproteases and that the NTR module of PCOLCEs is involved in the control of the activity of the astacin-type metalloprotease BMP1, it seems possible that interaction with metzincins could be a shared property of NTR modules and could be critical for the biological roles of the host proteins.

Up-regulation of type I procollagen C-proteinase enhancer protein messenger RNA in rats with CCl4-induced liver fibrosis.

Using a polyclonal antibody raised against a liver stellate cell (LSC) line derived from a rat CCl4-cirrhotic liver, we isolated 14 clones from a complementary DNA library prepared with total RNA extracted from the same cell line, with nucleotide sequences homologous to that of the type I procollagen C-proteinase enhancer protein (PCPE) gene. The longest PCPE insert of 1,530 base pairs contained an open reading frame coding for 468 amino acids. PCPE cDNA recognized by Northern blot a 1.7-kilobase messenger RNA (mRNA) in total RNA extracted from freshly isolated and early passaged LSC, LSC lines derived from normal (NFSC) and cirrhotic (CFSC) rat livers, and various LSC clones derived from CFSC. The expression of PCPE mRNA was increased threefold in CFSC compared with NFSC. PCPE mRNA was not detected in total rat liver, freshly isolated hepatocytes, or endothelial or Kupffer cells. However, the expression of PCPE mRNA was induced in fibrotic livers of rats treated with CCl4. PCPE mRNA expression in LSC was up-regulated by transforming growth factor beta1 (TGF-beta1) and down-regulated by tumor necrosis factor alpha (TNF-alpha), similar to the changes in alpha1 (1) procollagen mRNA induced by these cytokines. PCPE was not detectable in liver biomatrix proteins obtained from normal liver. However, PCPE was increased in liver biomatrix proteins from cirrhotic livers and was proportional to the amount of collagen. These data suggest that PCPE may play an important role in the processing of type I collagen during liver fibrogenesis, and that TGF-beta1 and TNF-alpha regulate its expression.

Up-regulation of type I procollagen C-proteinase enhancer protein messenger RNA in rats with CCl4-induced liver fibrosis

Using a polyclonal antibody raised against a liver stellate cell (LSC) line derived from a rat CCl4-cirrhotic liver, we isolated 14 clones from a complementary DNA library prepared with total RNA extracted from the same cell line, with nucleotide sequences homologous to that of the type I procollagen C-proteinase enhancer protein (PCPE) gene. The longest PCPE insert of 1,530 base pairs contained an open reading frame coding for 468 amino acids. PCPE cDNA recognized by Northern blot a 1.7-kilobase messenger RNA (mRNA) in total RNA extracted from freshly isolated and early passaged LSC, LSC lines derived from normal (NFSC) and cirrhotic (CFSC) rat livers, and various LSC clones derived from CFSC. The expression of PCPE mRNA was increased threefold in CFSC compared with NFSC. PCPE mRNA was not detected in total rat liver, freshly isolated hepatocytes, or endothelial or Kupffer cells. However, the expression of PCPE mRNA was induced in fibrotic livers of rats treated with CCl4. PCPE mRNA expression in LSC was up-regulated by transforming growth factor beta1 (TGF-beta1) and down-regulated by tumor necrosis factor alpha (TNF-alpha), similar to the changes in alpha1 (1) procollagen mRNA induced by these cytokines. PCPE was not detectable in liver biomatrix proteins obtained from normal liver. However, PCPE was increased in liver biomatrix proteins from cirrhotic livers and was proportional to the amount of collagen. These data suggest that PCPE may play an important role in the processing of type I collagen during liver fibrogenesis, and that TGF-beta1 and TNF-alpha regulate its expression. (Hepatology 1997 Sep;26(3):611-7)

Type I procollagen C-proteinase from mouse fibroblasts. Purification and demonstration of a 55-kDa enhancer glycoprotein.

The enzyme procollagen C-proteinase removes the carboxy-terminal propeptide from procollagen. In the present study we describe an improved procedure for the purification of this enzyme. From the medium of cultured mouse fibroblasts, consisting of ammonium sulfate precipitation, gel filtration and affinity chromatography on a lysyl-Sepharose column, followed by chromatography on a column of Sepharose coupled to the carboxy-terminal propeptide of type I procollagen (PP-Sepharose). This procedure yielded a practically homogeneous, 18,500-fold-purified enzyme preparation and the molecular mass of the purified C-proteinase as determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis was 80 kDa. The lysyl-Sepharose step separated the enzyme from the majority of the contaminating proteins, including a 55-kDa protein which was further purified by PP-Sepharose chromatography and identified as an additional form of the 36-kDa and 34-kDa procollagen C-proteinase enhancer proteins described before [Adar et al. (1986) Collagen Relat. Res. 6,267-277]. It enhanced the C-proteinase activity, bound to the carboxyl propeptide of type I procollagen, cross-reacted immunologically with the 36-kDa as well as the 34-kDa enhancer proteins, and in common with the latter proteins, it was glycosylated. In the course of PP-Sepharose chromatography, a large proportion of the 55-kDa protein disappeared with the concomitant appearance of the smaller enhancer proteins. All these findings suggest that the 55-kDa protein is a precursor of the low molecular mass enhancer proteins. Also suggested from this study is that lysyl-Sepharose chromatography is a highly beneficial purification step which may find use in the purification of the C-proteinase from other sources as well.