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PubMedDate not supplied

[Procollagen type 111 peptide levels in patients with bronchial asthma and COPD].

The aim of these study was to evaluate if the inflammatory injury of parenchymal cells can lead to proliferation of fibroblasts and deposition of increased amounts of collagen, measured by the increased concentration of procollagen in blood, in patients with asthma and COPD. We evaluated 22 patients with asthma that has lasted more than 5 years, 18 patients with COPD and 20 healthy subjects. RIA-gnost method (Behring) was used to measure the procollagen peptide concentrations in blood. Our results showed that the concentration of procollagen peptide in blood samples from patients with asthma was 5.8 +/- 2.4, 4.9 +/- 1.8 in patients with COPD and 11.1 +/- 3.6 in healthy subjects. There was no significant difference between patients with asthma, COPD and healthy subjects (p less than 0.01). It can be concluded that there is no increased deposition of collagen in patients with long lasting asthma and COPD. Further studies of active collagen deposition in the early acute forms of these diseases are in progress.

Adult↗
PubMedDate not supplied

Procollagen type III peptide in the assessment of liver fibrosis in children with congenital alpha-1-antitrypsin deficiency.

In 31 children with congenital alpha 1-antitrypsin deficiency the concentration of procollagen type III peptide was determined in a trial establishing liver fibrosis degree and monitoring of fibrosis progression. No correlation was found between the degree liver fibrosis determined by histological examination and the serum concentration of procollagen type III peptide. The concentration of procollagen type III peptide was higher with coexistent cholestasis and in case of inflammatory processes outside the liver (pneumonia). In progressing cirrhosis with inflammatory reaction the concentration was higher than in advanced cirrhosis and non-inflammatory liver fibrosis.

Adolescent↗
PubMed1991

A mutation in the pro alpha 2(I) gene (COL1A2) for type I procollagen in Ehlers-Danlos syndrome type VII: evidence suggesting that skipping of exon 6 in RNA splicing may be a common cause of the phenotype.

Fibroblasts from a proband with Ehlers-Danlos syndrome type VII synthesized approximately equal amounts of normal and shortened pro alpha 2(I) chains of type I procollagen. Nuclease S1 probe protection experiments with mRNA demonstrated that the pro alpha 2(I) chains were shortened because of a deletion of most or all of the 54 nucleotides in exon 6, the exon that contains codons for the cleavage site for procollagen N-proteinase. Sequencing of genomic clones revealed a single-base mutation that converted the first nucleotide of intron 6 from G to A. Therefore, the mutation was a change, in the -GT-consensus splice site, that produced efficient exon skipping. Allele-specific oligonucleotide hybridizations demonstrated that the proband's mother, father, and brother did not have the mutation. Therefore, the mutation was a sporadic one. Analysis of potential 5' splice sites in the 5' end of intron 6 indicated that none had favorable values by the two commonly employed techniques for evaluating such sites. The proband is the fourth reported proband with Ehlers-Danlos syndrome VII with a single-base mutation that causes skipping of exon 6 in the splicing of RNA from either the COL1A1 gene or COL1A2 gene. No other mutations in the two type I procollagen genes have been found in the syndrome. Therefore, such mutations may be a common cause of the phenotype. The primers developed should be useful in screening for the same or similar mutations causing the disease.

Alleles↗
PubMed1991

A single base mutation in type I procollagen (COL1A1) that converts glycine alpha 1-541 to aspartate in a lethal variant of osteogenesis imperfecta: detection of the mutation with a carbodiimide reaction of DNA heteroduplexes and direct sequencing of products of the PCR.

Skin fibroblasts from a proband with a lethal variant of osteogenesis imperfecta synthesized both apparently normal type I procollagen and a type I procollagen that had slow electrophoretic mobility because of posttranslational overmodifications. The thermal unfolding of the collagen molecules as assayed by protease digestion was about 2 degrees C lower than normal. It is surprising, however, that collagenase A and B fragments showed an essentially normal melting profile. Assay of cDNA heteroduplexes with a new technique involving carbodiimide modification indicated a mutation at about the codon for amino acid 550 of the alpha 1(I) chain. Subsequent amplification of the cDNA by the PCR and nucleotide sequencing revealed a single-base mutation that substituted an aspartate codon for glycine at position alpha 1-541 in the COL1A1 gene. The results here confirm previous indications that the effects of glycine substitutions in type I procollagen are highly position specific. They also demonstrate that a recently described technique for detecting single-base differences by carbodiimide modification of DNA heteroduplexes can be effectively employed to locate mutations in large genes.

Adult↗
PubMed1990

Procollagen type-III aminoterminal peptide in serum and synovial fluid of dogs with hip dysplasia and coxarthrosis.

Hip dysplasia is an affection of the coxofemoral joint that progresses until stabilization is caused by fibrosis and osteoarthritic changes. This stabilization process can be examined by clinical and radiographic methods. The capability of evaluating the procollagen concentrations in liquids, such as serum and synovial fluid, has further offered the basis for an objective biochemical evaluation of the stabilization process. Our study was performed to evaluate whether determination of procollagen concentrations was suitable for the use in practice. The procollagen type-III aminoterminal peptide (P-III-NP) concentration was measured in serum and in synovial fluid from coxofemoral joints in 20 dogs. Dogs were grouped on the basis of evidence of dysplasia and osteoarthritic changes of the hip: (1) a control group of 6 dogs without clinical or radiographic signs of hip dysplasia, and (2) dysplastic group of 14 dogs, which was further grouped with respect to the coxofemoral joint laxity, as determined by the Ortolani test. Synovial fluid concentration of P-III-NP was significantly (P less than 0.05) higher in fluid from dysplastic joints than in fluid from normal joints. Serum concentrations of P-III-NP were significantly (P less than 0.05) higher in dogs in which results of the Ortolani test were positive.

Animals↗
PubMed1990

Inheritance of an RNA splicing mutation (G+ 1 IVS20) in the type III procollagen gene (COL3A1) in a family having aortic aneurysms and easy bruisability: phenotypic overlap between familial arterial aneurysms and Ehlers-Danlos syndrome type IV.

Inheritance of a single base mutation in the type III procollagen gene (COL3A1) was studied in a family with aortic aneurysms and easy bruisability. The mutation was a substitution of A for G+ 1 of intron 20 of the gene and caused aberrant splicing of RNA transcribed from the mutated allele. The phenotype in the family included aortic aneurysms that ruptured and produced death. It also included easy bruisability, but it did not include other characteristic features of Ehlers-Danlos syndrome type IV, such as ecchymoses, abnormal scarring, or prominent subcutaneous blood vessels. The data from the family, together with a review of other probands with mutations in the type III procollagen gene, indicated that there is phenotypic overlap between Ehlers-Danlos syndrome type IV and familial arterial aneurysms not associated with any overlap between Ehlers-Danlos syndrome type IV and familial arterial aneurysms not associated with any of the striking changes in skin originally cited as a characteristic feature of Ehlers-Danlos syndrome type IV. In addition, the results suggested that DNA tests for mutations in the type III procollagen gene may be useful to identify individuals predisposed to developing arterial aneurysms.

Adult↗
PubMed1990

Differential expression of a cysteine-rich domain in the amino-terminal propeptide of type II (cartilage) procollagen by alternative splicing of mRNA.

Type II collagen, like other fibrillar collagens, is synthesized as a procollagen containing amino (NH2)-and carboxyl (COOH)-terminal extension peptides. Based on cDNA cloning of human (Baldwin, C. T., Reginato, A. M., Smith, C., Jimenez, S. A., and Prockop, D. J. (1989) Biochem. J. 262, 521-528) and rat (Kohno, K., Martin, G. R., and Yamada, Y. (1984) J. Biol. Chem. 259, 13668-13673) type II procollagen, it was concluded that much of the NH2-terminal propeptide seen in pro-alpha 1(I) was missing. Analysis of human genomic clones for type II collagen revealed an additional exon encoding a 69-amino acid cysteine-rich domain in the NH2-terminal propeptide. This exon (exon 2) is expressed in the mRNA population of chondrocytes isolated from human fetal skeleton and notochord, juvenile costal cartilage, and bovine articular cartilage. Oligonucleotide probes spanning specific exon boundaries were used to detect two populations of procollagen mRNA by Northern blot analysis. Amplification of cDNA templates using polymerase chain reaction provided direct evidence for two distinct pro-alpha 1(II) collagen mRNAs. DNA sequence analysis showed that the two mRNAs resulted from the alternative splicing of exon 2. The protein domain encoded by exon 2 is conserved between the fibrillar collagens and two other extracellular matrix proteins, thrombospondin and von Willebrand factor. In fibrillar collagens, this protein domain may play a regulatory role in fibrillogenesis and feedback inhibition of collagen biosynthesis. Consequently, the differential expression of this protein domain could alter the biosynthesis or fibril formation of type II collagen. In addition, the expression of exon 2 may be a marker for a distinct population of chondrocytes.

Amino Acid Sequence↗
PubMed1987

Type I and type III procollagen peptides during hepatic fibrogenesis. An immunohistochemical and ELISA serum study in the CCl4 rat model.

Serum assays of procollagen peptides have been suggested as useful clinical indicators of hepatic fibrogenesis. To evaluate these assays in an experimental situation, the carbon tetrachloride model of hepatic fibrosis was produced in the rat. With affinity-purified antibodies, ELISA assays were developed and used for detecting nanogram quantities of the aminopropeptides of Types I and III procollagen (pro-I and pro-III) in rat serum. Serum SGPT levels were also determined. Routine histologic staining, as well as immunofluorescent staining for localization of Types I, III, and IV collagen and the aminopropeptides of Types I and III procollagen were performed on corresponding livers. It was found that serum pro-III levels were elevated after 45 and 70 days of treatment (28 +/- 15.2 ng/ml and 21 +/- 3.4 ng/ml, respectively) but returned to normal levels after 90 days of treatment (less than 3 ng/ml). Serum pro-I levels were elevated after 45 days of treatment (1028 +/- 504 ng/ml) but were normal at 70 and 90 days of treatment. Serum SGPT values were elevated at 45 and 70 days of treatment but were normal at 90 days of treatment. The decline in serum pro-III levels appeared to parallel the noted decline in SGPT values. Linear regression analysis revealed a good correlation between pro-III levels and histologic inflammation (r = 0.886) but a poor correlation between pro-III levels and histologic fibrosis (r = 0.116). Immunohistochemistry revealed that the pro-III antigen persists extracellularly for at least 45 days. Therefore, serum assays of pro-III might reflect extracellular collagen degradation as well as active collagen secretion. This would limit the clinical utility of the pro-III assay as an unequivocal marker of active hepatic collagen deposition.

Animals↗
PubMed1987

Anchoring fibrils contain the carboxyl-terminal globular domain of type VII procollagen, but lack the amino-terminal globular domain.

Type VII procollagen has been characterized as a product of epithelial cell lines. As secreted, it contains a large triple-helical domain terminated by a multi-globular-domained carboxyl terminus (NC-1), and a smaller amino-terminal globule (NC-2). The triple helix and the NC-1 domain have previously been identified in anchoring fibril-containing tissues by biochemical and immunochemical means, leading to the conclusion that type VII collagen is a major component of anchoring fibrils. In order to better characterize the tissue form of type VII collagen, we have produced a panel of monoclonal antibodies which recognize the NC-1 domain. Peptide mapping of these epitopes indicate that they are independent and span approximately 125,000 kDa of the total 150,000 kDa of each alpha chain contained in NC-1. All these antibodies elicit immunofluorescent staining of the basement membrane zone in tissues. Type VII collagen has been extracted from tissues. As previously reported, it is smaller than type VII procollagen, (Woodley, D. T., Burgeson, R. E., Lunstrum, G. P., Bruckner-Tuderman, L., and Briggaman, R. A., submitted for publication), and we now find that it predominantly occurs as a dimer. Following clostridial collagenase digestion, intact NC-1 has been recognized, indicating that the difference in apparent Mr between the tissue form of the molecule and type VII procollagen results from modification of the amino terminus. The size of the amino-terminal globule has been determined to be between approximately 96 and 102 kDa. Rotary shadowing analyses of extracted molecules indicate that dimeric molecules contain the NC-1 domain, but are missing intact NC-2. We propose that the tissue form monomer, Mr = 960,000, be referred to as "type VII collagen." These studies strongly suggest that anchoring fibrils contain dimeric molecules with intact NC-1 domains. The data also support the previous suggestion that the NC-2 domain is involved in the formation of disulfide bond-stabilized type VII collagen dimers, and is subsequently removed by physiological proteolytic processing.

Amnion↗
PubMed1989

Localization of type III procollagen mRNA in areas of liver fibrosis by in situ hybridization.

Localization of type III procollagen mRNA in human liver was studied by in situ hybridization using human alpha 1(III) procollagen cDNA. Frozen and paraformaldehyde-fixed sections of biopsied human liver from patients with chronic hepatitis and cirrhosis were examined using the digoxigenin-labeled cDNA probe. Localization of the type III procollagen mRNA was demonstrated not only in the cytoplasm of mesenchymal cells but also in a large number of hepatocytes, in proportion to the extent of fibrosis. These results suggest that hepatocytes play an important role in fibrogenesis in the liver.

DNA Probes↗
PubMed1986

Large complex globular domains of type VII procollagen contribute to the structure of anchoring fibrils.

Type VII collagen, in the form of an antiparallel dimer, is a major protein component of anchoring fibrils. The ultrastructural appearance of these fibrils suggests that they may serve to anchor the basement membrane zone to the underlying connective tissue matrix. We report here the identification and initial characterization of Type VII procollagen, recovered from the media of epidermoid carcinoma cell cultures. Immunoblotting using monospecific antibodies to Type VII procollagen identifies a single, homogeneous band of at least Mr 320,000 following disulfide bond reduction. This chain contains 170 kDa of collagen triple helix and 150 kDa of non-helical domain at the carboxyl terminus. Pepsin digestion of this material yields Type VII collagen identical to that isolated from whole tissue and a series of quasi-stable peptides derived from the carboxyl-terminal region. Cell extracts contain procollagen chains identical in size to those secreted into the media. There is no evidence for processing of this material in cell culture. Partial purification by velocity sedimentation and transmission electron microscopic observation following rotary shadowing reveals both monomers (426 nm) and dimers (785 nm). Dimers are antiparallel and interact through 60-nm overlap, with amino-terminal globular domains present at the ends of the overlap. The multi-domain carboxyl-terminal region appears as three similar arms originating from a centralized globular region adjacent to the collagen helix. The carboxyl globular domain is present in whole tissue and may participate in the unique fibril form of this collagen. The amino-terminal globule may function in the antiparallel assembly of dimers.

Antibodies↗
PubMed1986

Deletion of 24 amino acids from the pro-alpha 1(I) chain of type I procollagen in a patient with the Ehlers-Danlos syndrome type VII.

A child with the type VII form of the Ehlers-Danlos syndrome was shown to have a structural defect in the amino terminus of the pro-alpha 1(I) chain of type I procollagen. Normal and mutant amino-terminal cyanogen bromide peptides (pN-alpha 1(I) CB0,1 peptides) were purified from the medium of the patient's cultured fibroblasts. Amino acid sequencing of tryptic peptides derived from the mutant pN-alpha 1(I) CB0,1 peptide showed that an expected sequence of 24 amino acids (positions 136-159 of the normal pN-alpha 1(I) CB0,1 peptide) was deleted. The segment deleted from the mutant pro-alpha 1(I) chain contains the small globular region of the NH2-propeptide, the procollagen N-proteinase cleavage site, the NH2-telopeptide, and first triplet of the helix of the alpha I(I) collagen chain (Chu, M.-L., de Wet, W., Bernard, M., Ding, J.F., Morabito, M., Myers, J., Williams, C., and Ramirez, F. (1984) Nature 310, 337-340). Loss of the procollagen N-proteinase cleavage site from the mutant pro-alpha 1(I) chain accounted for the persistence of its NH2-propeptide despite normal production of the N-proteinase by cultured mutant fibroblasts. Collagen production by mutant fibroblasts was doubled possibly due to reduced feedback inhibition by the NH2-propeptides. The child appeared to be heterozygous for the peptide deletion and, as the parents did not show any evidence of the deletion, it is likely that the child had a new mutation of one allele of the pro-alpha 1(I) gene. The deleted peptide corresponds precisely to the sequence coded by exon 46 of the normal pro-alpha 1(I) gene (Chu, M.-L., de Wet, W., Bernard, M., Ding, J.F., Morabito, M., Myers, J., Williams, C., and Ramirez, F. (1984) Nature 310, 337-340).

Amino Acids↗
PubMed1986

Suppression of synthesis of pro-alpha 1(I) and production of altered pro-alpha 2(I) procollagen subunits in 4-nitroquinoline-1-oxide-transformed fibroblasts.

The collagen phenotype of a 4-nitroquinoline-1-oxide-transformed line of Syrian hamster embryo fibroblasts, NQT-SHE, was markedly altered from that of normal Syrian hamster embryo cells, which synthesized mainly type I procollagen [pro-alpha 1(I)]2 pro-alpha 2(I). Total collagen synthesis in the transformant was reduced to about 30% of the control level primarily because synthesis of the pro-alpha 1(I) subunit was completely suppressed. The major collagenous products synthesized consisted of two polypeptides, designated as N-33 and N-50, which could be completely separated by precipitation with ammonium sulfate at 33 and 50% saturation, respectively. N-33 migrated similarly to pro-alpha 2(I) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and N-50 migrated slightly more slowly. The collagenous regions of these chains were more sensitive to protease than the analogous region of procollagen I, but alpha-chains could be obtained by digestion for 2 h at 4 degrees C with high ratios of protein:pepsin. Staphylococcus V8 protease and cyanogen bromide peptide maps of N-33 alpha and N-50 alpha chains indicated that the chains were homologous with, but different than, alpha 2(I) chains and that they differed from each other. Considering their similarity to pro-alpha 2(I), it was surprising to find that the N-collagens were secreted to the same extent as was type I procollagen from Syrian hamster embryo cells and that there were no disulfide bonds between N-collagen chains. Intrachain disulfides were present. One possible explanation for the unusual collagen phenotype of NQT-SHE cells is that transformation induced one or more mutations in the pro-alpha 2(I) structural gene while suppression of synthesis of the pro-alpha 1(I) subunit may be due to a mutation in the regulatory region of its gene or in a general regulatory gene.

4-Nitroquinoline-1-oxide↗
PubMed1988

Regulation of mRNAs for type-1 plasminogen activator inhibitor, fibronectin, and type I procollagen by transforming growth factor-beta. Divergent responses in lung fibroblasts and carcinoma cells.

Transforming growth factor-beta (TGF beta) is a growth modulator which stimulates the growth of fibroblasts but acts as a strong growth inhibitor for cells of epithelial origin. TGF beta also influences the production of extracellular matrix proteins and of proteases and protease inhibitors by cultured cells. One important protease inhibitor whose production is affected rapidly by TGF beta is the type-1 plasminogen activator inhibitor (PAI-1). To investigate the relationships between PAI-1 and the extracellular matrix, we analyzed the regulation by TGF beta of PAI-1, fibronectin, and type I procollagen in malignant human lung carcinoma cells (A549) and in human lung fibroblasts (WI-38). The expression of the respective genes was examined by polypeptide analyses and by measurements of the steady-state levels of the corresponding mRNAs by Northern hybridization. The mRNA levels for PAI-1 were elevated rapidly by TGF beta in both cell lines. This induction occurred in the presence of cycloheximide and thus was not dependent on protein synthesis. In fact, the effects of TGF beta and cycloheximide on PAI-1 mRNA were additive. In contrast, the induction of fibronectin, beta-actin, and type I procollagen (synthesized only in WI-38 cells) was abrogated by cycloheximide. In general, the effects of TGF beta on the steady-state levels of mRNAs for fibronectin, actin, and type I procollagen mRNA were similar in the two cell types. However, the production of PAI-1 mRNA in response to TGF beta differed in the two cell types, being transient (peak within 5 h) in the carcinoma cells and more persistent in fibroblasts. Thus, the major difference in TGF beta regulation of PAI-1 mRNA between lung fibroblasts and carcinoma cells was the duration of the effect. These results, together with previous data, suggest that PAI-1 and plasminogen activators may be regulated oppositely by TGF beta. We therefore propose a model for TGF beta in the regulation of extracellular proteolytic activity.

Actins↗
PubMed1988

Uncoupled expression of mRNAs for alpha 1(I) and alpha 2(I) procollagen chains in chemically transformed Syrian hamster fibroblasts.

Syrian hamster embryo fibroblasts transformed by 4-nitroquinoline-1-oxide (NQT-SHE cells) failed to synthesize the pro-alpha 1(I) subunit of type I procollagen but continued to synthesize altered forms of the other subunit, pro-alpha 2(I) (Peterkofsky, B., and Prather, W. (1986) J. Biol. Chem. 261, 16818-16826). This was unusual, since synthesis of the two subunits generally is coordinately regulated. Present experiments using cell-free translation and hybridization of RNA from normal and transformed Syrian hamster fibroblasts with labeled pro-alpha 1(I) DNA probes show that mRNA for pro-alpha 1(I) is absent from the transformant. In contrast, dot-blot and Southern blot hybridizations of cellular DNAs with pro-alpha 1(I) DNA probes demonstrated that the transformed cells contained pro-alpha 1(I) gene sequences and that the gross structure of the gene was unchanged by transformation. mRNA for the other type I procollagen subunit, pro-alpha 2(I), was present in transformed cells and the major collagenous polypeptide translated from this RNA migrated like the normal pro-alpha 2 subunit during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The translated procollagen chain was cleaved to an alpha 2(I)-sized collagen chain by pepsin at 4 degrees C. These studies provide a molecular basis for the observed collagen phenotype of NQT-SHE cells.

4-Nitroquinoline-1-oxide↗
PubMed1977

Proteolysis of procollagen I.

1. Digestion of procollagen I which trypsin, pepsin or pronase performed at 20 degrees C causes the release of acidic non-collagenous fragments and hydroxyproline-rich fraction. Enzymatic proteolysis performed at 41 degrees C (above the temperature of denaturation) results in degradation of procollagen I to low-molecular peptides. 2. The hydroxyproline-rich fraction obtained by limited proteolysis of procollagen I with pepsin (at 20 degrees C) contains a material corresponding to alpha and beta subunits of tropocollagen. Reduction of the hydroxyproline-rich fraction released by trypsin or pronase (at 20 degrees C) causes the appearance of polypeptides similar to pro-alpha subunits.

Molecular Weight↗
PubMed1986

Ultrastructural localization of type III procollagen in baboon liver.

The localization of the extension aminopropeptides of Type III procollagen was studied in baboon liver by the immunoperoxidase method. The aminopropeptides were localized along the collagen fibrils around terminal hepatic venules, in the space of Disse, and in portal tracts of control baboons as well as of alcohol-fed animals. The labeling showed a characteristic periodicity of 60-70 nm, corresponding to the D (67 nm) stagger of collagen fibrils. The data indicate that some extension aminopropeptides of Type III procollagen are not cleaved from procollagen prior to its incorporation into collagen fibrils but that they exist as constituents of mature fibrils.

Animals↗
PubMed1987

[Various characteristics of the structure and synthesis of procollagens produced by cultured skin fibroblasts from patients with Danlos-Ehlers syndrome type I].

The electrophoretic mobilities of the collagen and procollagen type I and III chains synthesized by the fibroblasts isolated from patients with type I Ehlers-Danlos syndrome as well as a set of peptides obtained by splitting of pro alpha 1(I) and pro alpha 2(I) type I procollagens by cyanbromide are not different from the normal ones. The fact demonstrates the absence of long insertions or deletions, or the sufficient defects in intracellular chain modifications. The changes were also nor registered for the ratio of type I and III collagens from the digested by pepsin preparations of protein accumulating in the culture media of the cultured skin fibroblasts from patients. The studied strains of cultured fibroblasts from patients suffering the Ehlers-Danlos syndrome have the trend to increased accumulation of partially processed chains of proc alpha 1(I) and proc alpha 2(I) type I procollagen and to the increased ratio of pro alpha 1(I) to pro alpha 2(I).

Amino Acid Sequence↗