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PubMed1985

Purification and characterization of the N-terminal propeptide of human type III procollagen.

The N-terminal propeptide of type III procollagen was purified from human ascitic fluid by using (NH4)2SO4 precipitation, DEAE-Sephacel chromatography at pH 8.6, Sephacryl S-300 chromatography and another DEAE-Sephacel chromatography at pH 4.5. The Mr of the human peptide was about 42 000, which corresponds in size to the propeptide released by the specific N-proteinase during the extracellular processing of collagen. Bacterial-collagenase digestion of the human peptide produced three fragments, which could be separated on a Bio-Gel P-10 column. The human propeptide and its collagenase-derived fragments, an N-terminal non-collagenous domain Col 1, a C-terminal non-helical domain Col 2 and a collagenous domain Col 3, resembled those derived from the N-terminal segment of bovine type III procollagen in their amino acid composition. The human peptide was found to contain sulphate, which may explain its extremely low isoelectric point (3.1). Antibodies against the human N-terminal propeptide reacted similarly with both the purified human peptide and a corresponding segment of bovine type III procollagen. The human propeptide could be used in developing radioimmunoassays for monitoring fibrotic processes.

Amino Acids↗
PubMed1986

Purification of human procollagen type III N-proteinase from placenta and preparation of antiserum.

Procollagen type III N-proteinase, of Mr about 70,000, was detected in human placental tissue and purified from this source more than 5800-fold. It was found to be a glycoprotein, which was bound to both concanavalin A-Ultrogel and heparin-Sepharose affinity columns. Binding to a type III pN-collagen-Sepharose affinity column was used as the final step in purification. The purified enzyme accepted only native type III procollagen or [14C]carboxymethylated type III pN-collagen as its substrate; type I, type II and type IV procollagen and heat-denatured type III pN-collagen were not cleaved by the enzyme. Antibodies against this purified enzyme protein raised in rabbits demonstrated a high inhibitory effect on the enzyme activity. Immunoblotting of the denatured protein and immunoelectrophoresis of the native enzyme showed only one major antigenic component, again with an Mr of about 70,000. The antibodies cross-reacted with the enzyme preparation from foetal-calf aorta smooth-muscle cells.

Calcium Chloride↗
PubMed1990

Regulation of procollagen synthesis and processing during ascorbate-induced extracellular matrix accumulation in vitro.

Procollagen biosynthesis and matrix deposition were studied in long-term human skin fibroblast cultures exposed to ascorbic acid. Ascorbic acid specifically stimulated types I and III collagen synthesis, reaching a maximum at day 2 and maintaining a specific high rate of production until day 10 of ascorbate exposure, after which collagen production declined. The increased level of collagen synthesis after different exposure times could also be achieved by only brief treatment (10 h) of parallel scorbutic (ascorbic-acid-deficient) cultures with ascorbic acid. This brief exposure did not result in increased collagen mRNA, thus demonstrating that the ascorbate-induced increase in collagen synthesis at all stages of ascorbic acid exposure was due to post-transcriptional mechanisms, most likely a rapid increase in type 1 collagen mRNA translational efficiency. This mechanism, rather than the transcriptional activation, was the primary response and is adequate to explain the ascorbate-induced increase in collagen synthesis. These data also demonstrate that the presence of a collagenous extracellular matrix was not involved in this collagen biosynthetic regulation. During long-term exposure (18 days) to ascorbic acid, a substantial cross-linked collagenous matrix formed, following an approximately sigmoidal time course. The most rapid matrix deposition occurred during the later days of exposure when the rate of collagen synthesis was decreasing, suggesting that the presence of a pre-existing matrix is important for further collagen accumulation. Procollagen was also efficiently processed to collagen during this phase, demonstrating that efficient procollagen processing is an important regulatory event in collagen matrix deposition.

Ascorbic Acid↗
PubMed1993

Two cysteine substitutions in procollagen I: a glycine replacement near the N-terminus of alpha 1(I) chain causes lethal osteogenesis imperfecta and a glycine replacement in the alpha 2(I) chain markedly destabilizes the triple helix.

Cultured skin fibroblasts were examined from two probands with type II (lethal) osteogenesis imperfecta. One proband had a single base mutation which converted the glycine codon at position alpha 1-244 in the alpha 1(I) chain of procollagen I into a cysteine codon whereas the other had a similar mutation that converted the glycine codon at position alpha 2-787 of the alpha 2(I) chain into a cysteine codon. Both mutations produced post-translational overmodification of procollagen I. The Cys alpha 1-244 mutation, however, had a minimal effect on the thermal stability or secretion of the protein whereas the Cys alpha 2-787 mutation markedly decreased the thermal stability and, apparently as a result, essentially none of the mutated protein was secreted. The results provide clear exceptions to two previous generalizations about the position-specificity of glycine substitutions in procollagen I.

Base Sequence↗
PubMed1997

Regulation of the human alpha 2(1) procollagen gene by sequences adjacent to the CCAAT box.

The human, rat, mouse and chicken alpha 2(1) procollagen promoters analysed to date all contain an inverted CCAAT box at -80. In this study we have examined the binding of nuclear proteins to the proximal promotor of the human alpha 2(1) procollagen gene, where an inverted CCAAT box is flanked by a downstream GGAGG sequence and its inverted counterpart (CCTCC) on the upstream end. Each of the GGAGG sequences is separated from the inverted CCAAT box by a single pyrimidine nucleotide (5'-CCTCCCATTGGTGGAGGCCCTTTT-3'). Electrophoretic mobility-shift assays (EMSAs) revealed that two distinct DNA-protein complexes formed on this DNA sequence. Methylation interference analysis and in vitro mutagenesis studies revealed that the integrity of the sequence 5'-CCTCCCATTGG-3' (the GGAGG/CCAAT-binding element or G/CBE) was important for the binding of the CCAAT-binding factor (CBF) (complex I). Competition studies showed that complex formation on the human G/CBE could be competed by mouse CBE and nuclear factor-Y (NF-Y) oligonucleotides, suggesting that mouse CBE and human G/CBE-binding proteins belong to the same family of CCAAT box binding proteins. Furthermore, antibodies to mouse CBF specifically supershifted the G/CBE complex (complex I) in EMSAs. The downstream GGAGG and 3'-flanking sequences (5'-GGAGGCCCTTTT-3') or collagen modulating element (CME), however, were important for the formation of a novel DNA protein complex (complex III). The formation of this complex was not competed out by CBE or NF-Y oligonucleotides, nor was DNA-protein complex formation affected by the anti-CBF antibody. Functional analysis of G/CBE and CME elements subjected to mutagenesis, using promoter-chloroamphenicol acetyl transferase constructs in transient transfection assays, showed that both these elements were essential for activity of the human promoter. These experiments identified a novel regulatory element in the human alpha 2(1) procollagen gene which is not present in the rodent gene.

Cell Line, Transformed↗
PubMed2001

Defects of type I procollagen metabolism correlated with decrease of prolidase activity in a case of lethal osteogenesis imperfecta.

We have studied the structure and metabolism of type I procollagen in a case of perinatal lethal osteogenesis imperfecta (OI) type II. Cultured skin fibroblasts from the proband synthesized both normal and abnormal forms of type I procollagen. Some abnormal, overmodified molecules were secreted by OI cells, although less efficiently than normal molecules from control cells. The OI fibroblasts accumulated large amounts of abnormal proalpha1(I) and proalpha2(I) chains intracellularly. The extracellular collagenolytic activity was decreased compared to control cells. Furthermore, OI cells produced less type I procollagen and demonstrated lower capacity to synthesize DNA than control cells. We have found that in contrast to prolinase activity, the activity of prolidase (an enzyme essential for collagen synthesis and cell growth) is also significantly reduced in OI cells. No differences were found in the amount of the enzyme protein recovered from both the OI and control cells. However, we found that expressions of beta1 integrin and insulin-like growth factor-I receptor (receptors known to play an important role in up regulation of prolidase activity) were decreased in OI cells compared to control cells. The decrease in prolidase activity may provide an important mechanism of altered cell growth and collagen metabolism involved in producing the perinatal lethal form of the OI phenotype.

Adult↗
PubMed2000

Inhibitory effects of antisense oligonucleotides on the expression of procollagen type III gene in mouse hepatic stellate cells transformed by simian virus 40.

The effects of phosphorothioate antisense oligonucleotides (ASO), complementary to the AUG start region, the junctional region of the intron and exon, and to exon of the procollagen type III gene, were investigated in a mouse hepatic stellate cell (HSC) line transformed by the simian virus 40 gene, SV68c-IS cells. ASO were transfected by lipofection. Immunohistochemistry, western and northern blotting showed inhibitory effects on procollagen type III gene expression by ASO that were complementary to the AUG start region and the junctional region of the intron and exon 2. However, ASO complementary to the exon 2 and 3, junctional region of the intron and exon 3, and sense oligonucleotides complementary to each ASO did not show any inhibitory effects. The effects of ASO complementary to the AUG start region were greater than those of ASO complementary to the junctional region. The effects of ASO were transient and a large amount of ASO was required to induce inhibitory effects without lipofection. ASO were effective in inhibiting the expression of the procollagen type III gene in the HSC which is well known to play a critical role in liver fibrosis.

Actins↗
PubMed1976

Segment-long-spacing aggregates and isolation of COOH-terminal peptides from type I procollagen.

Type I procollagen secreted by matrix-free cells from chick embryo tendons was purified by DEAE-cellulose chromatography. Electron microscopy of segment-long-spacing aggregates of the procollagen demonstrated the presence of both NH2-terminal and COOH-terminal extensions not found in collagen. The procollagen was digested with bacterial collagenase and the COOH-terminal fragments were isolated by gel filtration and polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Analysis of tryptic peptides demonstrated that the COOH-terminal extensions on the pro alpha 1 and pro alpha 2 chains had different primary structures.

Amino Acid Sequence↗
PubMed1977

Hexamethylene bisacetamide induces morphologic changes and increased synthesis of procollagen in cell line from glioblastoma multiforme.

Addition to hexamethylene bisacetamide (diacetyldiaminohexane) to cultures of a malignant mesenchymal cell line derived from a human glioblastoma multiforme induces morphological changes and stimulates the synthesis of procollagen. The morphological changes include cell elongation, an increase of extracellular material with staining properties of collagen by light microscopy, and an increase in extracellular 220-A fibrils by electron microscopy. The rate of procollagen synthesis increased as much as 20-fold, and the ratio of type I:type III procollagen changed, with type I becoming the predominant form. The change in type I:type III ratio is similar to that seen in the maturation of normal fetal to adult connective tissue.

Acetamides↗
PubMed1978

Construction and characterization of a 2.5-kilobase procollagen clone.

Recombinant bacterial plasmids have been constructed by inserting double-stranded chicken procollagen cDNA sequences linked to chemically synthesized decanucleotides containing HindIII sites into the HindIII site of pBR322. After transformation of Escherichia coli chi1776, colonies were selected by ampicillin resistance and recombinants containing procollagen sequences were identified by colony hybridization to 32P-labeled procollagen cDNA. The inserts from three recombinant plasmids, pCg10, pCg13, and pCg45, were 1200, 2200, and 2550 base pairs long respectively. Their sequence homology has been established by restriction mapping and crosshybridization of nick-translated plasmids to Southern blots of Hpa II fragments of the inserts, pCg45 has been positively identified as containing the pro alpha2 collagen sequence by partial determination of the DNA sequence of its ends: it has a short thymine-rich sequence at one end and a sequence coding for residues 478--499 in the chicken alpha2 chain at the other end.

Animals↗
PubMed1979

Procollagen segment-long-spacing crystallites: their role in collagen fibrillogenesis.

Naturally occurring segment-long-spacing crystallites of procollagen collagen have been found in the culture medium of fibroblasts from chick embryo tendon, human skin, and dermatosparaxic calf skin; in whole-mount preparations of cultured human skin fibroblasts; in homogenates of lathyritic chick embryo tendon, cartilage, and cornea; and in a partially purified preparation of procollagen. Bundles of similar aggregates occurred within secretory vacuoles of collagen-synthesizing fibroblasts and chondrocytes. These observations suggest that fibroblasts and chondrocytes secrete procollagen assemblies that are stable in the extracellular environment. We propose that subsequent enzymatic processing is accompanied by direct incorporation of such structures into the assembling fibril, which then may be considered as an n X 67 nm staggered array of segment-long-spacing crystallites.

Animals↗
PubMed1982

Type I osteogenesis imperfecta: a nonfunctional allele for pro alpha 1 (I) chains of type I procollagen.

Type I osteogenesis imperfecta (OI) is a dominantly inherited disease characterized clinically by bone fractures during childhood, blue sclerae, and frequent hearing loss accompanied by a decreased content of type I collagen in bone and skin. Cultured skin fibroblasts from three individuals affected with the disease produce half-normal levels of type I procollagen, a disulfide-bonded trimer that contains two pro alpha 1(I) chains and one pro alpha 2(I) chain. In normal cells, pro alpha 1(I) and pro alpha 2(I) are synthesized in a 2:1 ratio and only assembled molecules are secreted. In contrast, the OI cells contain equimolar amounts of pro alpha 1(I) and pro alpha 2(I), which suggests that trimer assembly and secretion are limited by the level of pro alpha 1(I) synthesis. The "extra" pro alpha 2(I) in the OI cells is in a nondisulfide-bonded configuration and is not secreted but apparently contributes to an increased level of intracellular degradation. Thus, decreased production of type I procollagen in these patients is the result of decreased synthesis of pro alpha 1(I). These results suggest that the stoichiometry of pro alpha chains in type I procollagen is determined by the conformation of the chains rather than the ratio in which they are synthesized, that molecules containing more than a single pro alpha 2(I) chain are not assembled, and that the production of this heteropolymeric molecule may be effectively regulated by controlling the synthesis of only one of the subunits.

Adult↗
PubMed1985

Supramolecular assemblies of mRNA direct the coordinated synthesis of type I procollagen chains.

Registration of the three procollagen alpha chains and assembly of the triple-helical procollagen molecules takes place in the rough endoplasmic reticulum, but the exact location and timing of assembly is not known. As part of a study of the mechanism of molecular assembly, intact collagen-producing polyribosomes from embryonic chicken tendon fibroblasts have been examined by the techniques of rotary shadowing and electron microscopy. Intact mRNA strands corresponding in length to approximately 4500 bases and complete procollagen alpha (I) chains have been observed. The mRNA strands are comprised of two mRNA chains. The ribosomes are present in pairs separated along the duplex strand by about 100 nm. The intact polysome is asymmetric; two duplex strands join, and large ribosome aggregates appear. These aggregates are dispersed by collagenase digestion, leaving separate duplex strands with ribosome pairs intact. Ribonuclease digestion yields mixtures of monosomes and ribosome aggregates. Sequential ribonuclease and collagenase digestions yield only monosomes. We propose that each ribosome reads one mRNA chain, so that each pair is thus translating two chains in synchrony. Thus, the complex morphology of the collagen-producing polyribosomes suggests that the organization of a single molecule begins by the organization of the mRNA chains themselves.

Animals↗
PubMed1996

The C-proteinase that processes procollagens to fibrillar collagens is identical to the protein previously identified as bone morphogenic protein-1.

Bone morphogenic protein-1 (BMP-1) was originally identified as one of several BMPs that induced new bone formation when implanted into ectopic sites in rodents. BMP-1, however, differed from other BMPs in that it its structure was not similar to transforming growth factor beta. Instead, it had a large domain homologous to a metalloendopeptidase isolated from crayfish, an epidermal growth-factor-like domain, and three regions of internal sequence homology referred to as CUB domains. Therefore, BMP-1 was a member of the "astacin families" of zinc-requiring endopeptidases. Many astacins have been shown to play critical roles in embryonic hatching, dorsal/ventral patterning, and early developmental decisions. Here, we have obtained amino acid sequences and isolated cDNA clones for procollagen C-proteinase (EC 3.4.24.19), an enzyme that is essential for the processing of procollagens to fibrillar collagens. The results demonstrate that procollagen C-proteinase is identical to BMP-1.

Amino Acid Sequence↗
PubMed1995

Increased alpha 1(I) procollagen gene expression in tight skin (TSK) mice myocardial fibroblasts is due to a reduced interaction of a negative regulatory sequence with AP-1 transcription factor.

The TSK mouse, a model of fibrosis, displays exaggerated connective tissue accumulation in skin and visceral organs including the heart. To study the mechanisms of myocardial fibrosis in TSK mice, we established several strains of TSK mice myocardial fibroblasts in culture and examined the regulation of collagen gene expression in these cells. These strains displayed increased collagen gene expression in comparison with myocardial fibroblasts established from normal mice. On an average, the TSK myocardial fibroblast cultures showed a 4-fold increase in collagen synthesis and 4.4- and 3.6-fold increases, respectively, in alpha 1(I) and alpha 1(III) collagen mRNA steady state levels. The increased alpha 1(I) and alpha 1(III) collagen mRNA levels were mainly due to increased transcription rates (3.4- and 3.8-fold higher, respectively) of the respective genes. Furthermore, we showed that the up-regulation of alpha 1(I) procollagen gene transcription in TSK mice myocardial fibroblasts was due to the lack of the strong inhibitory influence of a regulatory sequence contained in the promoter region encompassing nucleotides -675 to -804. Nuclear extracts from TSK mice myocardial fibroblasts showed lower DNA binding activity to oligonucleotides spanning the mapped regulatory sequence as well as to a consensus AP-1 sequence, but not to a consensus SP-1 sequence, and supershift experiments with an AP-1 antibody confirmed the interaction of these oligonucleotides with AP-1 protein. These observations indicate that a strong negative regulatory sequence contained within -0.675 to -0.804 kilobase of the alpha 1(I) procollagen promoter binds AP-1 transcription factor and mediates inhibition of gene transcription in normal murine myocardial fibroblasts. The TSK mice myocardial fibroblasts lack this inhibitory control, due to lower available amounts and/or decreased binding activity to this inhibitory sequence, and hence display increased alpha 1(I) procollagen gene expression.

Animals↗
PubMed1996

Assembly in vitro of thin and thick fibrils of collagen II from recombinant procollagen II. The monomers in the tips of thick fibrils have the opposite orientation from monomers in the growing tips of collagen I fibrils.

Human type II procollagen was prepared in a recombinant system and cleaved to pC-collagen II by procollagen N-proteinase. The pC-collagen II was then used as a substrate to generate collagen II fibrils by cleavage with procollagen C-proteinase at 37 degrees C. Electron microscopy of the fibrils demonstrated that, at the early stages of fibril assembly, very thin fibrils were formed. As the system approached equilibrium over 7-12 h, however, the thin fibrils were largely but not completely replaced by thick fibrils that had diameters of about 240 nm and a distinct D-period banding pattern. One typical fibril was photographed and analyzed in its entirety. The fibril was 776 D-periods (52 microM) long. It had a central shaft with a uniform diameter that was about 516 D-periods long and two tips of about 100 D-periods each. Most of the central shaft had a symmetrical banding pattern flanked by two transition regions of about 30 D-periods each. Measurements by scanning transmission electron microscopy demonstrated that the mass per unit length from the tips to the shafts increased linearly over approximately 100 D-periods from the fibril end. The linear increase in mass per unit length was consistent with previous observations for collagen I fibrils and established that the tips of collagen II also had a near paraboloidal shape. However, the orientation of monomers in the tips differed from the tips of collagen I fibrils in that the C termini instead of the N termini were directed toward the tips. The thin fibrils that were present at early stages of assembly and at equilibrium were comparable to the collagen II fibrils seen in embryonic tissues and probably represented intermediates on the pathway of thick fibrils formation. The results indicated that the molecular events in the self-assembly of collagen II fibrils are apparently similar to those in self-assembly of collagen I fibrils, but that there are also important differences in the structural information contained in collagen I and collagen II monomers.

Cell Line↗
PubMed1998

Protein disulfide isomerase acts as a molecular chaperone during the assembly of procollagen.

Protein-disulfide isomerase (PDI) has been shown to be a multifunctional enzyme catalyzing the formation of disulfide bonds, as well as being a component of the enzymes prolyl 4-hydroxylase (P4-H) and microsomal triglyceride transfer protein. It has also been proposed to function as a molecular chaperone during the refolding of denatured proteins in vitro. To investigate the role of this multifunctional protein within a cellular context, we have established a semi-permeabilized cell system that reconstitutes the synthesis, folding, modification, and assembly of procollagen as they would occur in the cell. We demonstrate here that P4-H associates transiently with the triple helical domain during the assembly of procollagen. The release of P4-H from the triple helical domain coincides with assembly into a thermally stable triple helix. However, if triple helix formation is prevented, P4-H remains associated, suggesting a role for this enzyme in preventing aggregation of this domain. We also show that PDI associates independently with the C-propeptide of monomeric procollagen chains prior to trimer formation, indicating a role for this protein in coordinating the assembly of heterotrimeric molecules. This demonstrates that PDI has multiple functions in the folding of the same protein, that is, as a catalyst for disulfide bond formation, as a subunit of P4-H during proline hydroxylation, and independently as a molecular chaperone during chain assembly.

Animals↗
PubMed2000

Physical characterization of the procollagen module of human thrombospondin 1 expressed in insect cells.

Thrombospondin 1 (TSP1) is a homotrimeric glycoprotein composed of 150-kDa subunits connected by disulfide bridges. The procollagen module of thrombospondin 1 has been implicated in antiangiogenic activity. Procollagen modules are found in a number of extracellular proteins and are identifiable by 10 cysteines with characteristic spacing. We expressed and studied the procollagen module (C) of human TSP1, both by itself and in the context of the adjoining oligomerization sequence (o) and N-terminal module (N). The coding sequences were introduced into baculoviruses along with an N-terminal signal sequence and C-terminal polyhistidine tag. Proteins were purified from conditioned medium of infected insect cells by nickel-chelate chromatography. NoC is a disulfide bonded trimer and cleaves readily at a site of preferential proteolysis to yield monomeric N and trimeric oC. These are known properties of full-length TSP1. Mass spectroscopy indicated that C is N-glycosylated, and all 10 cysteine residues of C are in disulfides. By equilibrium ultracentrifugation, C is a monomer in physiological salt solution. Circular dichroism, intrinsic fluorescence, and differential scanning calorimetry experiments suggest that the stability of C is determined by the disulfides. The two tryptophans of C are in a polar, exposed environment as assessed by iodide fluorescence quenching and solvent perturbation. The oC far UV circular dichroism spectrum could be modeled as the sum of C and a coiled-coil oligomerization domain. The results indicate that the recombinant C folds autonomously into its native structure, and trimerization of the modules in TSP1 does not perturb their structures.

Amino Acid Sequence↗