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PubMed1983

Nucleotide sequences of complementary deoxyribonucleic acids for the pro alpha 1 chain of human type I procollagen. Statistical evaluation of structures that are conserved during evolution.

Nucleotide sequences were determined for two cloned cDNAs encoding for over three-fourths of the pro alpha 1 (I) chain of type I procollagen from man. Comparison with previously published data on amino acid sequences of the alpha 1 (I) chain of type I collagen made it possible to examine mutations in the transcribed products of the gene which have occurred during the evolution of man, calf, rat, mouse, and chick. Comparison of the nucleotide sequences with the corresponding sequences of cDNAs from chick [Fuller, F., & Boedtker, H. (1981) Biochemistry 20, 996] and with cDNAs for the pro alpha 2(I) chain from man [Bernard, M.P., Myers, J. C., Chu, M.-L., Ramirez, F., Eikenberry, E. F., & Prockop, D. J. (1983) Biochemistry 22, 1139] demonstrated that selective pressure during evolution for 250 million or more years acted more strongly on the structure of the pro alpha 1 (I) chain than on the pro alpha 2(I) chain. To improve the reliability of the comparison, the nucleotide sequences were examined with a modification of previous procedures for evaluating mutations in replacement sites and silent sites. The corrected divergence for replacement sites between the alpha 1 (I) chains was 6 +/- 0.8% whereas it was 15 +/- 1.9% for the alpha 2(I) chains. The C-propeptide domain of the pro alpha 1 (I) chain was also highly conserved with a corrected divergence at replacement sites of 5 +/- 0.9%, a value that was not distinguishable from the value previously found for the C-propeptide of the pro alpha 2(I) chain. Therefore, a large part of the structure of both C-propeptides appears to be under selective pressure. Inspection of changes in the C-propeptide of the pro alpha 1 (I) chain suggested that there was a highly conserved region around the carbohydrate attachment site similar to the highly conserved region of 37 amino acids previously found in the C-propeptide of the pro alpha 2(I) chain. Two statistical tests, however, were unable to confirm nonrandom distribution of changes in the C-propeptide of the pro alpha 1(I) chain. The same tests established the presence of a nonrandom distribution in nucleotide changes of the C-propeptide of the pro alpha 2(I) chain. The 3'-noncoding region of the cDNA for pro alpha 1(I) of human type I procollagen showed no homology with the same region in the chick.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1984

Neutral protease cleaving the N-terminal propeptide of type III procollagen: partial purification and characterization of the enzyme from smooth muscle cells of bovine aorta.

Procollagen type III amino-terminal protease was detected in cultures of smooth muscle cells of fetal calf aorta, and this protease was purified about 400-fold. Only about half of the enzyme activity was consistently attached to concanavalin A-agarose (Con A-agarose). After affinity chromatography on type III pN-collagen-Sepharose, the Con A bound fraction showed only one major band with a molecular weight of about 72000, this value corresponding well with the elution position of enzyme activity in gel filtration. The enzyme did not cleave procollagens type I or type IV, and denatured type III pN-collagen also remained uncleaved. The Km of the enzyme activity for iodo[14C]acetamide-labeled type III pN-collagen was 0.76 microM. Neutral pH and Ca2+ wer required for maximal enzymic activity. The metal chelators ethylenediaminetetraacetic acid and ethylene glycol bis(beta-amino-ethyl ether)-N,N,N',N'-tetraacetic acid inhibited the activity as well as did dithiothreitol, but there was only little if any inhibition by several other proteinase inhibitors tested.

Animals↗

PubMed1987

Transcriptional and posttranscriptional effects of dexamethasone on albumin and procollagen messenger RNAs in murine schistosomiasis.

We have previously shown that dexamethasone increases albumin mRNA and decreases procollagen steady-state mRNA levels in rat hepatocyte cultures. These studies were extended by evaluating an in vivo model of fibrogenesis (murine schistosomiasis) and by determining a more precise level of gene expression responsible for these changes. Control mice and litter mates infected with Schistosomiasis mansoni were evaluated at 8 weeks postinfection when the livers of the infected mice had become fibrotic and their serum albumin levels significantly decreased. The addition of 4 micrograms/mL dexamethasone to the drinking water of half of the infected mice led to a 75% decrease in the liver collagen content as determined by high-performance liquid chromatography. RNA was extracted from the livers of mice under three conditions: control and infected +/- dexamethasone. This RNA was then hybridized with cDNA probes to determine steady-state levels of specific mRNAs. In the infected mice, albumin mRNA levels were decreased compared to control; however, infected mice treated with dexamethasone increased their albumin mRNA content by 3-fold at 8 weeks. Types I and IV procollagen steady-state mRNA levels in infected mice were increased compared to control while dexamethasone suppressed the mRNA level of collagen in infected mice by 50%. The level of gene expression responsible for these steady-state changes was evaluated by nuclear run-on analysis. While the effect of schistosomiasis on these genes was primarily at a transcriptional level, dexamethasone exerted its effect on different genes in the injured liver by diverse mechanisms, i.e., decreasing collagen synthesis at a transcriptional level and increasing albumin by posttranscriptional mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1981

Regulation or procollagen messenger ribonucleic acid levels in Rous sarcoma virus transformed chick embryo fibroblasts.

Using cloned cDNAs for pro-alpha 1 and pro-alpha 2 collagen messenger ribonucleic acid (mRNA), we have investigated the regulation of collagen mRNA levels in Rous sarcoma virus (RSV) transformed chick embryo fibroblasts (CEF). We find that both pro-alpha 1 and pro-alpha 2 mRNA levels are decreased approximately 10-fold in CEF transformed by either the Bryan high-titer strain or the Schmidt-Ruppin strain of RSV. Using temperature-sensitive mutants in the transforming gene src, we also investigated the rate of change in the levels of the two mRNA species. We employed mutants of both the Bryan high-titre strain (BHTa) and the Schmidt-Ruppin strain (ts68). With both mutants the results were similar. Upon shift from the permissive temperature (35 degrees C) to the non-permissive temperature (41 degrees C), collagen mRNA synthesis, did not increase until more than 5 h had passed, suggesting that action of src on collagen gene expression is indirect. Upon shift from 41 to 35 degrees C, collagen mRNA levels fell with a half-life of 10 h. Whether this fall reflects the half-life of procollagen mRNA or an effect of src on procollagen RNA stability is unclear. Both pro-alpha 1 and pro-alpha 2 mRNA levels were coordinately controlled.

Animals↗

PubMed1978

Translation of chick calvarial procollagen messenger RNA'S by a messenger RNA dependent reticulocyte lysate.

A method was developed for the extraction of RNA from chick embryo calvaria which should be generally applicable to other connective tissues. Total RNA prepared by this method was translated by a mRNA-dependent reticulocyte lysate into discrete pro alpha chains. Several criteria were used to identify these translation products, including (1) preferential labeling with [3H]proline, (2) appropriate migration on sodium dodecyl sulfate-polyacrylamide gels, (3) selective sensitivity to collagenase digestion, and (4) specific precipitability by two different antisera against procollagen. Data from the immunoprecipitation experiments indicated that the majority of the pro alpha chains contained the carboxy-terminal antigenic determinants. These results demonstrate that this translation system can be used as an assay for intact procollagen mRNAs and as a source of in vitro synthesized pro alpha chains for future structural analysis.

Animals↗

PubMedDate not supplied

Subtle structural alterations in the chains of type I procollagen produce osteogenesis imperfecta type II.

Although the perinatal lethal form of osteogenesis imperfecta (OI type II) occasionally results from large rearrangements within the genes encoding type I collagen, most mutations are far more subtle. The complexity of the human collagen genes precludes cloning and sequencing each gene from every patient, and we have therefore developed an approach to localizing mutations at the protein level. We report here that cells cultured from 15 infants with OI type II synthesized both normal type I procollagen and a form that was unstable, poorly secreted and excessively modified. Abnormal procollagen from different strains was overmodified to different extents. The patterns of overmodification we observed are best explained by mutations that disrupt the Gly-X-Y sequence of pro alpha chains, and thus alter the rate of propagation of triple helix from COOH-terminus to NH2-terminus. As a consequence, a given mutation allows overmodification of all three chains in a molecule NH2-terminal to its position in the triple helix.

Collagen↗

PubMed1973

Evidence that chick tendon procollagen must be denatured to serve as substrate for proline hydroxylase.

The ability of chick-embryo proline hydroxylase to hydroxylate [(14)C]proline-labelled procollagen was investigated between 23 degrees and 37 degrees C. The amount of hydroxy[(14)C]proline that could be formed increased sharply between 26 degrees and 30 degrees C. This corresponded to the temperature interval in which the [(14)C]procollagen substrate was thermally denatured, and the results therefore indicate that only denatured molecules can be hydroxylated.

Animals↗

PubMed1979

Amino acid sequence of the N-terminal non-collagenous segment of dermatosparactic sheep procollagen type I.

The non-collagenous N-terminal segment of type I procollagen from dermatosparactic sheep skin was isolated in the form of the peptide Col 1 from a collagenase digest of the protein. The peptide has a blocked N-terminus, which was identified as pyrrolid-2-one-5-carboxylic acid. Appropriate overlapping fragments were prepared from reduced and alkylated peptide Col 1 by cleavage with trypsin at lysine, arginine and S-aminoethyl-cysteine residues and by cleavage with staphylococcal proteinase at glutamate residues. Amino acid sequence analysis of these fragments by Edman degradation and mass spectrometry established the whole sequence of peptide Col 1 except for a peptide junction (7--8) and a single Asx residue (44), and demonstrated that peptide Col 1 consists of 98 amino acid residues. The N-terminal portion of peptide Col 1 (86 residues) shows an irregular distribution of glycine, whereas the C-terminal portion (12 residues) possesses the triplet structure Gly-Xy and is apparently derived from the precursor-specific collagenous domain of procollagen. The central region of the peptide contains ten cysteine residues located between positions 18 and 73 and shows alternating polar and hydrophobic sequence elements. The regions adjacent to the cysteine-rich portion have a hydrophilic nature and are abundant in glutamic acid. The data are consistent with previous physicochemical and immunological evidence that distinct regions at the N- and C-termini of the non-collagenous domain possess a less rigid conformation than does the central portion of the molecule.

Amino Acid Sequence↗

PubMed1980

Protein disulphide-isomerase activity in chick-embryo tissues. Correlation with the biosynthesis of procollagen.

Protein disulphide-isomerase activity was determined in homogenates of chick-embryo tissues. Activities were highest in tissues active in procollagen synthesis and were maximal at the developmental stage of maximal procollagen synthesis. These variations in protein disulphide-isomerase activity correlate closely with those previously observed for protocollagen hydroxylase activities.

Animals↗

PubMed1988

Structure of a full-length cDNA clone for the prepro alpha 2(I) chain of human type I procollagen. Comparison with the chicken gene confirms unusual patterns of gene conservation.

A cDNA clone from a human placental library was found to consist of an essentially full-length cDNA of 4.6 kb for the prepro alpha 2(I) chain of type I procollagen. Nucleotide sequencing of the 5'-end of the cDNA provided a sequence of 1617 nucleotide residues and codons for 539 amino acid residues not previously defined. Comparison of the complete structure of the prepro alpha 2(I) cDNA with previously reported sequences for the chicken pro alpha 2(I) gene indicated that 83% of 1366 total amino acid residues were conserved. In the alpha-chain domain 84% of 1014 amino acid residues were conserved. Also, there was conservation of the previously noted preference for U and C in the third position of codons for glycine, proline and alanine. One major difference between the human and the chicken prepro alpha 2(I) chain was that the human chain contained 21 fewer proline residues, an observation that probably explains why the triple helix of human type I procollagen unfolds at temperatures that are 1-2 degrees C lower. In parallel experiments, sequencing of intron-exon boundaries for nine exons of genomic subclones confirmed and extended previous observations that the pro alpha 2(I) gene, like other genes from fibrillar collagens, has an unusual 54-base pattern of exon sizes that is highly conserved through evolution.

Amino Acid Sequence↗

PubMed1991

Substitution of cysteine for glycine-alpha 1-691 in the pro alpha 1(I) chain of type I procollagen in a proband with lethal osteogenesis imperfecta destabilizes the triple helix at a site C-terminal to the substitution.

Skin fibroblasts from a proband with lethal osteogenesis imperfecta synthesized a type I procollagen containing a cysteine residue in the alpha 1(I) helical domain. Assay of thermal stability of the triple helix by proteinase digestion demonstrated a decreased temperature for thermal unfolding of the protein. Of special importance was the observation that assays of thermal stability by proteinase digestion revealed two bands present in a 2:1 ratio of about 140 and 70 kDa; the 140 kDa band was reducible to a 70 kDa band. Further analysis of the fragments demonstrated that the cysteine mutation produced a local unfolding of the triple helix around residue 700 and apparently exposed the arginine residue at position 704 in both the alpha 1(I) and alpha 2(I) chains. Analysis of cDNAs and genomic DNAs demonstrated a single-base mutation that changed the GGT codon for glycine-691 of the alpha 1(I) chain to a TGT codon for cysteine. The mutation was not found in DNA from either of the proband's parents. Since the proteinase assay of helical stability generated a fragment of 700 residues that retained disulphide-bonded cysteine residues at alpha 1-691, the results provide one of the first indications that glycine substitutions in type I procollagen can alter the conformation of the triple helix at a site that is C-terminal to the site of the substitution.

Alleles↗

PubMed1992

Structural analysis of the regulatory elements of the type-II procollagen gene. Conservation of promoter and first intron sequences between human and mouse.

Transcription of the type-II procollagen gene (COL2A1) is very specifically restricted to a limited number of tissues, particularly cartilages. In order to identify transcription-control motifs we have sequenced the promoter region and the first intron of the human and mouse COL2A1 genes. With the assumption that these motifs should be well conserved during evolution, we have searched for potential elements important for the tissue-specific transcription of the COL2A1 gene by aligning the two sequences with each other and with the available rat type-II procollagen sequence for the promoter. With this approach we could identify specific evolutionarily well-conserved motifs in the promoter area. On the other hand, several suggested regulatory elements in the promoter region did not show evolutionary conservation. In the middle of the first intron we found a cluster of well-conserved transcription-control elements and we conclude that these conserved motifs most probably possess a significant function in the control of the tissue-specific transcription of the COL2A1 gene. We also describe locations of additional, highly conserved nucleotide stretches, which are good candidate regions in the search for binding sites of yet-uncharacterized cartilage-specific transcription regulators of the COL2A1 gene.

Amino Acid Sequence↗

PubMed2002

Preliminary evaluation of soluble IL-2 receptor and type III procollagen N-terminal aminopeptide in pleural fluid for differentiating tuberculous, carcinomatous and parapneumonic pleural effusions.

OBJECTIVE: The aim of the study was to clarify the possibility of using pleural fluid levels of soluble IL-2 receptor (sIL-2R) and type III procollagen N-terminal aminopeptide (PIIIP) for differentiating tuberculous, carcinomatous and parapneumonic pleural effusions. METHODOLOGY: Fifty patients with pleural effusions were investigated retrospectively. The pleural effusions were due to tuberculosis (n = 11), carcinoma (n = 22), pneumonia (n = 9) and heart failure (n = 8). The concentrations of sIL-2R and PIIIP were measured in pleural effusion using commercially available kits. RESULTS: Soluble IL-2 receptor concentrations were highest in the patients with tuberculosis (6,856 +/- 3,212 U/mL), followed by those with carcinoma (4,680 +/- 1,872 U/mL), pneumonia (2,227 +/- 525 U/mL) and heart failure (1,439 +/- 244 U/mL). Significant differences were found between tuberculosis and carcinoma (P = 0.023), carcinoma and pneumonia (P = 0.015), and pneumonia and heart failure (P = 0.002) groups. Type III procollagen N-terminal aminopeptide concentrations were higher in the patients with tuberculosis (262.5 +/- 157.9 U/mL) and pneumonia (257.0 +/- 96.7 U/mL) than in those with carcinoma (48.0 +/- 27.7 U/mL) and heart failure (10.9 +/- 5.6 U/mL). Significant differences were found between tuberculosis and carcinoma (P < 0.001) and pneumonia and carcinoma (P < 0.001). To differentiate effusions, the cutoff points of sIL-2R (2,980 U/mL) and PIIIP (110.0 U/mL) were obtained from the highest concentration in the pneumonia group and in the carcinoma group, respectively. Using these criteria, the sensitivities for differentiating tuberculous, carcinomatous, and parapneumonic effusions were 90.9, 86.4 and 88.9%, respectively, with 100, 95 and 100% specificity, respectively. CONCLUSIONS: The simultaneous determination of sIL-2R and PIIIP concentrations in pleural effusions may be clinically useful in differentiating tuberculous, carcinomatous, and parapneumonic effusions. Further assessments are required to determine the broad clinical application of this assay.

Adult↗

PubMed2001

The serum level of the amino-terminal propeptide of type I procollagen is a sensitive marker for prostate cancer metastasis to bone.

OBJECTIVE: To assess the level of a bone-formation marker, the amino-terminal propeptide of type I procollagen (PINP), for its utility in indicating bone metastasis in patients with prostate cancer. PATIENTS AND METHODS: Several bone formation markers, i.e. PINP, the carboxy-terminal propeptide of type I procollagen (PICP), bone-specific alkaline phosphatase (BALP), and bone Gla protein (BGP), a bone resorption marker (pyridinoline cross-linked carboxy-terminal telopeptide, ICTP), and prostate specific antigen (PSA) were measured in 40 patients without and 25 patients with bone metastasis. No patient had undergone previous treatment, except for six who developed bone metastasis while undergoing hormone therapy. RESULTS: All markers except BGP were significantly higher in patients with bone metastasis than in those without. The levels of PINP correlated best with the extent of disease, although the levels of PSA, BALP and ICTP also correlated well. While PINP had the largest area under the receiver-operator characteristic curve, PSA, BALP and ICTP also produced useful curves. CONCLUSION: The bone formation marker PINP seems to be useful for discriminating patients with and without bone metastasis. PINP may help in the early and accurate diagnosis of bone metastasis in such patients.

Aged↗

PubMed1980

Parathyroid hormone alters collagen synthesis and procollagen mRNA levels in fetal rat calvaria.

Parathyroid hormone decreased the incorporation of [3H]proline into collagenase-digestible protein in cultured 21-day fetal rat calvaria but had little effect on the labeling of noncollagen protein. After 24 hr of culture, there was a 50% reduction in collaghen synthesis and a 40% decrease in the level of functional procollagen mRNA as measured in a reticulocyte lystate translation assay. The effect of parathyroid hormone on both parameters was detectable after 6 hr of treatment. In these cultures, there was also a substantial degradation or release of newly synthesized collagen from the calvaria, but parathyroid hormone had little effect on the release of collagen into the medium. These results suggest that parathyroid hormone inhibits collagen synthesis primarily by decreasing the steady-state level of procollagen mRNA.

Animals↗

PubMed1980

Biosynthesis of A,B procollagen.

Interest in cell-associated collagens led others to isolate A and B collagen chains, also known as type V collagen, from many tissues, but only after pepsin cleavage. Soluble precursors of these chains are synthesized in vitro by crop, a chicken embryo muscle tissue, and are converted by at least two processing steps from procollagens via intermediates to final forms which are large than the pepsin-derived A and B chains. Heterotrimeric, disulfide-bridged procollagen molecules corresponding to B2A exist. Components were separated by ion exchange chromatography, velocity sedimentation, and electrophoresis, and the relationships between them were established by sequential radioactive labeling and comparison of peptides generated by protease cleavage.

Animals↗

PubMed1985

Nonrandom association of a type II procollagen genotype with achondroplasia.

Achondroplasia is an autosomal dominant disorder that involves defective endochondral bone formation. Type II collagen is the predominant collagen of cartilage. We found a HindIII polymorphic site in the normal Caucasian population by using the type II procollagen gene probe pgHCol(II)A. The presence of this site yields a 7.0-kilobase (kb) band; its absence yields a 14.0-kb band. We found a significant deviation in genotype distribution and allele frequencies in a population of unrelated individuals with sporadic achondroplasia, compared with the normal control population. The HindIII genotype frequencies in 32 individuals with achondroplasia are 0.41 for the 7/7 genotype (controls, 0.08), 0.34 for the 7/14 genotype (controls, 0.54), and 0.25 for the 14/14 genotype (controls, 0.37). The apparent equilibrium excess of the "7" allele in individuals with achondroplasia may reflect either a predisposition for the mutation that causes achondroplasia or it could be the result of the achondroplasia-causing mutation. In either case, these findings suggest an association of the type II procollagen gene with achondroplasia.

Achondroplasia↗

PubMed1999

Interaction of recombinant procollagen and properdin modules of thrombospondin-1 with heparin and fibrinogen/fibrin.

Many properties have been assigned to the procollagen and properdin (Type I) modules of thrombospondin-1 (TSP1) based on activities of large proteolytic fragments of TSP1 or peptides containing TSP1-derived sequences. To examine the activities of the modules more exactly, we expressed the first properdin module (P1); the third properdin module (P3); the first and second properdin modules (P12); the first, second, and third properdin modules (P123); and the procollagen module with the first, second, and third properdin modules (CP123) in the GELEX expression vector (GE1) using the baculovirus system. GE1 encodes the pre-pro sequence, the transglutaminase cross-linking site(s), the protease-sensitive site, and the gelatin binding domain from the amino terminus of rat fibronectin. All five recombinant proteins were expressed by insect cells, secreted into the culture medium, and purified by gelatin-agarose affinity chromatography. P123 shared with TSP1 a resistance to trypsin unless reduced and alkylated. P12/GE1, P123/GE1, and CP123/GE1 bound poorly to heparin-agarose except in the absence of sodium chloride, whereas peptides based on P2 are known to bind to heparin in up to 150 mM sodium chloride. In cross-linking experiments employing activated recombinant factor XIII and the transglutaminase cross-linking site in the fibronectin-derived sequence, P12/GE1, P123/GE1, CP123/GE1, and P3/GE1 but not P1/GE1 became incorporated into a fibrin clot more than GE1 alone. Analysis of the complex indicated that cross-linking was to the portion of the fibrin alpha-chain remaining in the D-dimer of plasmin digests. P123 also cross-linked to the Aalpha-chain of unclotted fibrinogen. P123 competed for 125I-TSP1 incorporation into the fibrin clot. P123 did not cross-link to plasminogen, histidine-rich glycoprotein, fibronectin, or plasma globulins other than fibrinogen/fibrin. These results indicate that the properdin modules of TSP1 specifically interact with fibrinogen/fibrin but not with heparin under physiologic conditions.

Animals↗