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PubMed2004

Biosynthetic processing of the Pro-alpha1(V)Pro-alpha2(V)Pro-alpha3(V) procollagen heterotrimer.

Type V collagen is a quantitatively minor fibrillar collagen comprised of different chain compositions in different tissues. The most widely distributed form, an alpha1(V)2alpha2(V) heterotrimer, regulates the physical properties of type I/V heterotypic collagen fibrils via partially processed NH2-terminal globular sequences. A less characterized alpha1(V)alpha2(V)alpha3(V) heterotrimer has a much more limited distribution of expression and unknown function(s). We characterized the biosynthetic processing of pro-alpha1(V)2pro-alpha2(V) procollagen previously and showed it to differ in important ways from biosynthetic processing of the major fibrillar procollagens I-III. Here we have successfully produced recombinant pro-alpha1(V)pro-alpha2(V)pro-alpha3(V) heterotrimers. We use these, and mouse embryo fibroblasts doubly homozygous null for the Bmp1 gene, which encodes the metalloproteinase bone morphogenetic protein-1 (BMP-1), and for a gene encoding the closely related metalloproteinase mammalian Tolloid-like 1, to characterize biosynthetic processing of pro-alpha1(V)pro-alpha2(V)pro-alpha3(V) heterotrimers, thus completing characterization of type V collagen biosynthetic processing. Whereas pro-alpha1(V) and pro-alpha2(V) processing in pro-alpha1(V)pro-alpha2(V)pro-alpha3(V) heterotrimers is similar to that which occurs in pro-alpha1(V)2pro-alpha2(V) heterotrimers, the processing of pro-alpha3(V) by BMP-1 occurs at an unexpected site within NH2-terminal globular sequences. We also demonstrate that, despite similarities in NH2-terminal domain structures, pro-alpha2(V) NH2-terminal globular sequences are not cleaved by ADAMTS-2, the metalloproteinase that cleaves the N-propeptides of the major fibrillar procollagen chains.

ADAM Proteins↗
PubMed2005

Alternative splicing of type II procollagen exon 2 is regulated by the combination of a weak 5' splice site and an adjacent intronic stem-loop cis element.

Alternative splicing of the type II procollagen gene (COL2A1) is developmentally regulated during chondrogenesis. Chondroprogenitor cells produce the type IIA procollagen isoform by splicing (including) exon 2 during pre-mRNA processing, whereas differentiated chondrocytes synthesize the type IIB procollagen isoform by exon 2 skipping (exclusion). Using a COL2A1 mini-gene and chondrocytes at various stages of differentiation, we identified a non-classical consensus splicing sequence in intron 2 adjacent to the 5' splice site, which is essential in regulating exon 2 splicing. RNA mapping confirmed this region contains secondary structure in the form of a stem-loop. Mutational analysis identified three cis elements within the conserved double-stranded stem region that are functional only in the context of the natural weak 5' splice site of exon 2; they are 1) a uridine-rich enhancer element in all cell types tested except differentiated chondrocytes; 2) an adenine-rich silencer element, and 3) an enhancer cis element functional in the context of secondary structure. This is the first report identifying key cis elements in the COL2A1 gene that modulate the cell type-specific alternative splicing switch of exon 2 during cartilage development.

Alternative Splicing↗
PubMed2001

Coordinated regulation of procollagens I and III and their post-translational enzymes by dissipation of mechanical tension in human dermal fibroblasts.

Mechanical tension governs fibroblast proliferation and survival and the homeostasis of the extracellular matrix to adapt its resistance to the mechanical requirements of the organs. To consolidate this view, we analysed the effect of tension release on the expression of molecules involved in the architecture and stabilisation of the collagen fibres, namely the procollagens type I and III, the amino- and carboxy-procollagen peptidases (N-pCP and C-pCP) and lysyl oxidase. Cells were cultured in conditions of high mechanical stress in monolayer on a collagen coat and under reduced tension by disruption of the cytoskeleton upon treatment with cytochalasin D in monolayer on a collagen coat or by integrin-mediated stress relaxation in a freely retracting collagen gel. The mRNAs were measured by quantitative RT-PCR monitored by simultaneous reverse-transcription and amplification of an original internal standard. Tension relaxation resulted in a decreased expression of the procollagens type I and III, of the two expressed forms of C-pCP, of the two forms of N-pCP and of lysyl oxidase. Type III collagen, known to control diameter of the fibres, was less down-regulated than type I collagen. Interestingly, the expression of the two alternatively spliced forms of the N-pCP was dissimilarly regulated. These data suggest that mechanical tension may modulate the stiffness of the extracellular matrix by controlling not only the level of expression of its fibrillar constituents but also that of the enzymes participating in their extracellular processing and mechanical stabilisation.

Adolescent↗
PubMed1998

Differential splicing of type II procollagen mRNA in canine retina.

Canine retina expressed two forms of Type II procollagen mRNA, IIA and IIB, due to alternate splicing of exon 2 at the N-propeptide region. The N-propeptide region of Type II procollagen showed a high degree of homology with human, approximately 96% both at the nucleotide and the amino acid levels. Expression of IIA and IIB forms of Type II procollagen from early stage of development to adult life suggests the important role of both forms for the development as well as maintenance of the ocular structure.

Alternative Splicing↗
PubMed1991

Expression of two nonallelic type II procollagen genes during Xenopus laevis embryogenesis is characterized by stage-specific production of alternatively spliced transcripts.

The pattern of type II collagen expression during Xenopus laevis embryogenesis has been established after isolating specific cDNA and genomic clones. Evidence is presented suggesting that in X. laevis there are two transcriptionally active copies of the type II procollagen gene. Both genes are activated at the beginning of neurula stage and steady-state mRNA levels progressively increase thereafter. Initially, the transcripts are localized to notochord, somites, and the dorsal region of the lateral plate mesoderm. At later stages of development and parallel to increased mRNA accumulation, collagen expression becomes progressively more confined to chondrogenic regions of the tadpole. During the early period of mRNA accumulation, there is also a transient pattern of expression in localized sites that will later not undergo chondrogenesis, such as the floor plate in the ventral neural tube. At later times and coincident with the appearance of chondrogenic tissues in the developing embryo, expression of the procollagen genes is characterized by the production of an additional, alternatively spliced transcript. The alternatively spliced sequences encode the cysteine-rich globular domain in the NH2-propeptide of the type II procollagen chain. Immunohistochemical analyses with a type II collagen monoclonal antibody documented the deposition of the protein in the extracellular matrix of the developing embryo. Type II collagen expression is therefore temporally regulated by tissue-specific transcription and splicing factors directing the synthesis of distinct molecular forms of the precursor protein in the developing Xenopus embryo.

Amino Acid Sequence↗
PubMed1984

Specific binding of fibronectin--antifibronectin immune complexes to procollagen: a new pitfall in immunostaining.

We observed intense intracellular immunofluorescence of rat lung fibroblasts stained with hybridoma culture supernatant containing monoclonal antibodies to human plasma fibronectin, but no pericellular matrix staining. Immunoprecipitation and absorption experiments revealed that this intracellular staining by hybridoma-conditioned medium was due to binding of fibronectin-antifibronectin immune complexes via the fibronectin to intracellular procollagen. The anomalous staining patterns we encountered were not revealed by the usual controls for immunohistochemical specificity, and also occurred in rat tissue sections. This general phenomena--binding of serum antigens present in hybridoma medium to cellular components--could in principle result in artifactual staining with monoclonal antibodies to other serum components, so investigators using monoclonal antibodies should be aware of this new artifact. Our results also demonstrate that fibronectin binds specifically to native procollagen. Monoclonal antibodies may be useful for studying fibronectin-procollagen and other macromolecular interactions.

Animals↗
PubMed1997

The C-propeptide domain of procollagen can be replaced with a transmembrane domain without affecting trimer formation or collagen triple helix folding during biosynthesis.

The folding and assembly of procollagen occurs within the cell through a series of discrete steps leading to the formation of a stable trimer consisting of three distinct domains: the N-propeptide, the C-propeptide and the collagen triple helix flanked at either end by short telopeptides. We have established a semi-permeabilized cell system which allows us to study the initial stages in the folding and assembly of procollagen as they would occur in the intact cell. By studying the folding and assembly of the C-propeptide domain in isolation, and a procollagen molecule which lacks the C-propeptide, we have shown that this domain directs the initial association event and is required to allow triple helix formation. However, the essential function of this domain does not include triple helix nucleation or alignment, since this can occur when the C-propeptide is substituted with a single transmembrane domain. Also the telopeptide region is not involved in triple helix nucleation; however, a minimum of two hydroxyproline-containing Gly-X-Y triplets at the C-terminal end of the triple helix are required for nucleation to occur. Thus, the C-propeptide is required solely to ensure association of the monomeric chains; once these are brought together, the triple helix is able to nucleate and fold to form a correctly aligned triple helix.

Amino Acid Sequence↗
PubMed1982

Construction and partial characterization of two recombinant cDNA clones for procollagen from chicken cartilage.

Type II procollagen mRNA has been partially purified from embryonic chick sternal cartilage by guanidine hydrochloride extraction, sucrose gradient sedimentation and Sepharose 4B chromatography. Double stranded cDNA was synthesized using AMV reverse transcriptase and E. coli DNA polymerase I, tailed using terminal transferase and inserted into the Pst I site of pBR322. Two putative type II procollagen cDNA clones have been characterized. Both plasmids hybridize to 2 sternal RNA species, a major species of 5.3 kb and a minor species of 7 kb. These RNAs are present in total RNA from sterna and differentiated limb bud cultures but are not detected in RNA from stage 24 limb bud which has not yet differentiated to cartilage or in RNA from calvaria. The time of appearance of these RNAs during the differentiation of limb mesenchyme in culture parallels the appearance of translatable type II procollagen mRNA.

Animals↗
PubMed1997

Formation of recombinant human procollagen I heterotrimers in a baculovirus expression system.

The present study describes the production of human procollagen I in a baculovirus expression system. Recombinant baculovirus carrying pro alpha1(I) or pro alpha2(I) cDNA was constructed and infected to Sf9 cells. Full-length pro alpha1(I) or pro alpha2(I) chains were synthesized by the cells infected with either of the recombinant viruses. The pro alpha1(I) chains formed pepsin-resistant homotrimers stabilized by interchain disulfide bonds, a small proportion of which was secreted into the culture medium. The pro alpha2(I) chains were not linked into trimers by disulfide bonds and failed to form stable triple helices, although some chains were suggested to exist as dimers or unstable trimers in which only two chains were linked by disulfide bonds. In spite of their non-helicity, the pro alpha2(I) chains were secreted at a higher rate than the pro alpha1(I) chains. Sf9 cells simultaneously synthesized both pro alpha1(I) and pro alpha2(I) chains when the cells were co-infected with the two recombinant viruses. Pepsin-treatment of the product clearly demonstrated the production of procollagen I heterotrimers composed of two pro alpha1(I) chains and one pro alpha2(I) chain, homotrimers of the pro alpha1(I) chains being negligible. This expression system appears to offer a unique means of studying the mechanism of chain association and secretion during procollagen biosynthesis.

Baculoviridae↗
PubMed1987

The carboxylpropeptide of type I procollagen in skin fibrillogenesis.

Previous studies suggested that the aminopropeptide of type I procollagen may initiate fibril formation. The purpose of this investigation was to study the location of the carboxylpropeptide of type I procollagen during collagen fibrillogenesis. Chick embryonic and posthatching skin specimens were studied by immunofluorescence and immunoelectron microscopy and by immunoblotting with antibodies against the amino and carboxylpropeptide of type I procollagen. The carboxylpropeptide was demonstrated at the surface of collagen fibrils, 20-40 nm in diameter (10-day embryos) and in fibrils, 40-65 nm (21-day embryos). In addition, the carboxylpropeptide was found at the cell surface and free in the ground substance. The aminopropeptide was only seen in fibrils, 20-30 nm in diameter, as previously reported. Ratios of pN-collagen/pC-collagen increased from 16 days embryonic to 3 and 9 days postembryonic skins. This study suggests that both pN-collagen (aminopropeptide plus collagen) and pC-collagen (carboxylpropeptide plus collagen) participate in fibrillogenesis.

Animals↗
PubMed1988

Collagen type IV and procollagen type III during granulation tissue formation: a serological, biochemical, immunohistochemical and morphometrical study on the viscose cellulose sponge rat model.

The serum concentrations of collagen type IV,7S, collagen type IV,nc1, and aminoterminal type III procollagen peptide immunoreactive components were measured by means of specific radioimmunoassays during development of granulation tissue in rats. The results were compared with tissue deposition of basement membranes and interstitial collagens in the granulation as measured morphometrically. A parallel sequential pattern in tissue deposition of collagen types III and IV, and serum increase of collagen types III- and IV-related fragments, was observed. Serum collagen type IV was less sensitive as a marker for development of granulation tissue than the serum procollagen type III N-peptide. This was in accordance with a low collagen type IV/interstitial collagen ratio in the granulation tissue. However, a cross-sectional study showed that serum collagens types IV,7S and IV,nc1 may be useful as early quantitative indicators of granulation tissue formation. Simultaneously, measurement of collagen type IV- and procollagen type III N-peptide-related antigens in serum provides a differentiated reflection of the dynamic matrix processes in developing granulation tissue.

Animals↗
PubMed1977

Intermediates in the conversion of procollagen to collagen. Evidence for stepwise limited proteolysis of the COOH-terminal peptide extensions.

Intermediates in the conversion of procollagen to collagen were isolated from radioactively labeled chick cranial bones by ion-exchange chromatography. Cleavage of these proteins with vertebrate collagenase revealed that each of the several forms of these intermediates lacked NH2-terminal but retained COOH-terminal extensions. The chain composition of each intermediate was resolved by two-dimensional slab gel electrophoresis. The intermediates differed from each other in having sustained cleavages in zero, one or two pcalpha chains. The relative proportions of intermediates with different intact pcalpha chains, observed in conversion of procollagen, have enabled us to construct a detailed model of the stepwise limited proteolysis of procollagen.

Amino Acids↗
PubMed1991

Genomic organization of the human procollagen alpha 1(II) collagen gene.

The nucleotide sequence of the human procollagen alpha 1(II) collagen gene extending from within the first intron through exon 15, and part of the 15th intron has been determined. This sequence analysis (7056 bases) identifies the intron/exon organization of the region of this gene encoding the N-propeptide and part of the triple-helical domain. Structural comparison of this with the genes of other human fibrillar collagens shows considerable diversity in terms of size and number of introns and exons that encodes the N-propeptide domain. Although the genomic structure of the human procollagen alpha 1(II) gene is quite different from the rat procollagen alpha 1(II) gene, the nucleotide coding sequences are 89% identical.

Base Sequence↗
PubMed1997

Molecular characterisation of integrin-procollagen C-propeptide interactions.

The carboxyl-terminal propeptide of type I procollagen (CPP-I) plays a key role in regulation of collagen fibrillogenesis, and may exert feedback control of collagen biosynthesis. We have previously shown that CPP-I is a ligand for the integrin alpha2beta1 [Weston, S. A., Hulmes, D. J. S., Mould, A. P., Watson, R. B. & Humphries, M. J. (1994) Identification of the integrin alpha2beta1 as a cell surface receptor for the C-propeptide of type I procollagen, J. Biol. Chem. 269, 20982-20986] suggesting that some of the phenotypic effects of C-propeptides may be mediated by adhesion receptors. Here we have extended this work to study the molecular basis of this interaction. We have broadened the ligand range by demonstrating that the C-terminal propeptide of type II procollagen supports alpha2beta1-mediated binding of NHS human fibroblasts in cell attachment assays. Also, we have used function-blocking antibodies in cell attachment and solid-phase binding assays with purified integrin to expand the CPP-I receptor family, showing that integrin alpha1beta1 is also a receptor for CPP-I. Integrin alpha-subunit A-domains are known to be major ligand-binding sites and recombinant alpha1 and alpha2 subunit A-domains were able to bind CPP-I. Finally we have shown that peptides corresponding to potential integrin-binding sequences in CPP-I do not mediate integrin-CPP-I adhesion. Taken together, these studies indicate that the interactions between C-propeptides and integrins are more numerous than previously reported, that C-propeptides are a new class of molecule which bind to A-domains, and that the integrin-C-propeptide interaction does not utilise established peptide motifs.

Amino Acid Sequence↗
PubMedDate not supplied

Diagnostic value of serum procollagen peptide measurements in alcoholic liver disease.

Procollagen type I carboxyterminal and type III aminoterminal peptide concentrations were measured in sera of 60 patients with alcoholic and 14 with nonalcoholic liver disease to study whether these assays are useful as clinical tests to differentiate various stages of alcoholic liver injury. Both propeptides were markedly elevated in alcoholic hepatitis and cirrhosis: procollagen type III peptide in 90% and type I peptide in 60-80% of these patients. Moderately increased values were found less frequently in patients with fatty liver. These tests did not differentiate patients with simple fatty liver from those with fatty liver and early fibrosis. There was a significant difference in serum procollagen type III peptide between fatty liver and both alcoholic hepatitis and cirrhosis (p less than 0.001), and in type I peptide between fatty liver and alcoholic hepatitis (p less than 0.005). Although serum peptide values correlated with the degree of liver fibrosis, appreciable overlap of values was found between the various groups. The peptide concentrations also seemed to be related to the degree of hepatic inflammation, and the highest values were observed in a subgroup of patients with alcoholic hepatitis in whom numerous Mallory bodies were found. The data suggest that in alcoholic liver diseases, serum collagen propeptide determination may be useful in diagnosing severe alcoholic hepatitis.

Adult↗
PubMed1992

Cyclosporin-A increases type I procollagen production and mRNA level in human gingival fibroblasts in vitro.

In order to study the pathogenesis of gingival overgrowth induced by the immunosuppressive drug cyclosporine-A (CyA), we investigated its effect on 3H thymidine incorporation and on collagen production and mRNA levels in fibroblast cultures obtained from normal human gingiva. At concentrations of 100, 500 and 1000 ng/ml, CyA did not modify thymidine incorporation after 24 and 72 h of incubation. However, after 24 h it significantly increased the level of 3H proline-containing proteins in the medium. In addition, CyA increased alpha-procollagen chains by up to three times. This CyA-induced change was related to a rise in the level of type I procollagen. The CyA effect on fibroblasts was markedly reduced by cycloheximide, an inhibitor of protein synthesis, and it correlated well with an increase of type I procollagen mRNA. Overall, our data indicate a direct stimulatory action of CyA on collagen synthesis, but not on DNA synthesis, in human gingival fibroblasts.

Collagen↗
PubMed2006

Procollagen C proteinase enhancer 1 genes are important determinants of the mechanical properties and geometry of bone and the ultrastructure of connective tissues.

Procollagen C proteinases (pCPs) cleave type I to III procollagen C propeptides as a necessary step in assembling the major fibrous components of vertebrate extracellular matrix. The protein PCOLCE1 (procollagen C proteinase enhancer 1) is not a proteinase but can enhance the activity of pCPs approximately 10-fold in vitro and has reported roles in inhibiting other proteinases and in growth control. Here we have generated mice with null alleles of the PCOLCE1 gene, Pcolce, to ascertain in vivo roles. Although Pcolce-/- mice are viable and fertile, Pcolce-/- male, but not female, long bones are more massive and have altered geometries that increase resistance to loading, compared to wild type. Mechanical testing indicated inferior material properties of Pcolce-/- male long bone, apparently compensated for by the adaptive changes in bone geometry. Male and female Pcolce-/- vertebrae both appeared to compensate for inferior material properties with thickened and more numerous trabeculae and had a uniquely altered morphology in deposited mineral. Ultrastructurally, Pcolce-/- mice had profoundly abnormal collagen fibrils in both mineralized and nonmineralized tissues. In Pcolce-/- tendon, 100% of collagen fibrils had deranged morphologies, indicating marked functional effects in this tissue. Thus, PCOLCE1 is an important determinant of bone mechanical properties and geometry and of collagen fibril morphology in mammals, and the human PCOLCE1 gene is identified as a candidate for phenotypes with defects in such attributes in humans.

Alleles↗
PubMed1999

Detection of meningeal fibrosis after subarachnoid haemorrhage by assaying procollagen propeptides in cerebrospinal fluid.

OBJECTIVE: To study whether meningeal collagen synthesis under normal conditions is reflected in the CSF and whether a meningeal fibroproliferative reaction or fibrosis after subarachnoid haemorrhage can be detected by measuring markers of collagen synthesis in the CSF. METHODS: Serum samples and CSF were collected from 56 patients with various neurological symptoms and from nine patients with a recent subarachnoid haemorrhage. The concentrations of the carboxyterminal propeptide of type I procollagen (PICP) and the aminoterminal propeptide of type III procollagen (PIIINP) were measured using radioimmunoassays. RESULTS: The mean(SD) concentration of PICP was 75.2 (SD 13.6) microgram/l and that of PIIINP 3.56 (SD 0.91) microgram/l in the CSF of the controls, and the CSF/serum ratios were 0.74 (SD 0.24) for PICP and 1.34 (SD 0.48) for PIIINP. A 1.4-fold increase in both the PICP (p=0.001) and the PIIINP (p=0.001) concentration was found in patients with a neurological disease and with an abnormal CSF leucocyte count or protein concentration. In eight patients with a recent subarachnoid haemorrhage the PICP was 5.9-fold higher (p<0.001) and the PIIINP concentration 7.7-fold higher (p<0.001) than that in the controls, whereas no difference was found in the serum values. Similar high concentrations were also found in a patient from whom the CSF sample was obtained before operation for aneurysm. CONCLUSIONS: The intrathecal compartment is a site for active collagen synthesis under normal conditions. The synthesis rate is markedly increased in patients with a recent subarachnoid haemorrhage, suggesting a fibroproliferative reaction or fibrosis. Assays of procollagen propeptides may be useful in the clinical diagnosis of meningeal fibrosis and their use may enable the identification of diseases and symptoms aetiologically related to meningeal fibrosis.

Adult↗