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At least 19 recordsLinked to original sources
PubMed1977

[Biochemistry of collagen and the locomoter apparatus (Hereditary connective tissue diseases and rheumatic diseases. Part I].

The elementary collagen molecule consists of three chains rolled into a spiral and ending in nonhelical telopeptides. In cutaneous collagen, there are two types of chains, in cartilaginous collagen there is only one. In the synthesis, several enzymes play successive parts: proline hydroxylase, lysine hydroxylase, then pro-collagen peptidase and finally, lysine oxidase and hydroxylysine oxidase; their coordinated actions ultimately allow the chains to establish the transverse intra and intermolecular links which give the collagen fiber its cohesion. The type and number of these transverse links vary from one tissue to another. Specific collagenases make collagen degradation possible.

Animals↗
PubMed1977

[Hereditary deficiency in the enzymes of the biosynthesis of collagen. The Ehlers-Danlos syndromes (author's transl)].

The mechanism of the biosynthetic pathway of collagen is briefly summarised. The hereditary enzyme deficiencies of this pathway concern some of the Ehlers-Danlos syndromes. Seven clinically well defined varieties of these syndromes have been recognized, all presenting, as common feature, an hyperextensivitry of joints and hyperelastic, excessively fragile skin. In three of these seven varieties, the enzyme defect has been recently discovered: the type V (associated with chromosome X) is characterized by the deficiency in the lysyl-oxidase, the type VI (ocular) by the deficiency in lysyl-hydroylase; in the type VII (arthrolaxis multiplex congenita) the activity of tropocollagen-peptidase is practically absent. These enzyme deficiencies provide a molecular basis for the interpretation of the pathogenesis of these varieties of the Ehlers-Danlos syndrome.

Amino Acid Oxidoreductases↗
PubMed1987

Cloning of a complementary DNA for rabbit proactivator. A metalloproteinase that activates synovial cell collagenase, shares homology with stromelysin and transin, and is coordinately regulated with collagenase.

Rabbit proactivator is a neutral metalloproteinase that activates another metalloproteinase, procollagenase, and degrades noncollagenous matrix. We describe the construction of an activator complementary DNA (cDNA) clone, which is 1.9 kb, that selects a 2.1-kb messenger RNA (mRNA) in Northern blot hybridizations. Nucleic acid sequence studies of the activator cDNA indicate 1) that it encodes protein Mr 53,881, 2) that this protein exhibits approximately 80% homology with rat transin, an oncogene-induced protein with a previously unknown function, and 3) that, in the first 172 residues, it is virtually identical to the rabbit metalloproteinase, stromelysin. Homology between rabbit activator and human skin collagenase is approximately 50%. Activator and collagenase mRNA are coordinately regulated; untreated cultures of rabbit synovial fibroblasts produce low levels of each protein, but addition of phorbol myristate acetate (10(-8)M) results in an increase in mRNA for both proteins by 2.5-5 hours. Adding all-trans-retinoic acid (10(-6)M) or dexamethasone (10(-7)M) to phorbol-stimulated cells coordinately suppresses both activator and collagenase mRNA. Our data suggest the existence of coordinately regulated metalloproteinases that are important in the modulation of connective tissue metabolism.

Amino Acid Sequence↗
PubMed1985

Enzymes converting procollagens to collagens.

Conversion from procollagen to collagen is a specific process that is a requirement for proper alignment of collagen molecules to form functional fibers. This process is catalyzed by at least three structurally and functionally distinct enzymes cleaving collagen types I-III. The cleavage processes possibly taking place in the more recently discovered collagen types are not known to any extent at this time. Two amino-terminal proteinases, one cleaving type I and type II procollagens and the other cleaving type III procollagen, have been purified close to homogeneity, and the more unspecific activity of carboxy-terminal proteinase has been isolated from several tissues. In our experimental model, however, cleavage of the carboxy-terminal propeptides of types I and III procollagen is differently affected by lysine. This suggests the presence of at least two distinct enzymes for the removal of carboxyl-terminal propeptides. The regulation of the reaction process from procollagen to collagen is not well known at present. The importance of the phenomenon in terms of fibril formation, however, is demonstrated by several elegant studies in vitro; and certain genetic disorders in which this process is defective demonstrate the significance in vivo. Moreover, the factors shown to effect the cleavage process may be potentially beneficial in the treatment of the pathological processes with abnormal collagen accumulation such as fibrosis. In this paper we briefly review the current knowledge of the converting enzymes, including some very recent findings of our laboratory as well as the evidence presented for the biological significance of the conversion process.

Animals↗
PubMed1976

Connective tissue metabolism in culture fibroblasts of a patient with Ehlers-Danlos syndrome type I.

Study on connective tissue metabolism was conducted with a patient with Ehlers-Danlos syndrome (E-D) of Type I who visited our institute. The conversion of procollagen into tropocollagen in the medium of cultured fibroblasts was assayed by the chase technique using 3H-proline. The conversion was inhibited in the cultured fibroblasts of the patient. The components of glycosaminoglycans in cultured medium and fibroblasts from E-D were within normal ranges, however, the ratio of glycoprotein to glycosaminoglycans of intra- and extra-cellular fractions of E-D fibroblasts was higher than the normal one. These findings suggest that the insufficient maturation of collagen fiber may be considered fundamental disorders of E-D.

Adolescent↗
PubMed1979

Activity of procollagen-prolyl-hydroxylase and N-acetyl-beta-glucosaminidase in liver biopsies from patients with chronic liver diseases.

In 5 mg of liver tissue obtained by needle biopsy from patients with chronic liver diseases, activity of both Procollagen-prolyl-hydroxylase and N-Acetyl-beta-glucosaminidase was measured. The activity of both enzymes increased due to the activity of fibrosis, but prolylhydroxylase increased more. The relation of these two enzyme activities in the development of liver fibrosis is discussed.

Acetylglucosaminidase↗
PubMed1985

A sensitive and rapid method for assaying the activity of type III procollagen amino-terminal proteinase.

A rapid assay procedure was developed for measuring the rate of cleavage of the amino-terminal propeptide of type III procollagen. The method was based on the sequential precipitation of type III collagen and uncleaved pN-collagen by 30% ammonium sulfate, while the free amino-terminal propeptide remained in solution and could be further precipitated by 60% ammonium sulfate. Consistently better results were obtained than with the earlier method in which absolute ethanol was used as the precipitant, and selective precipitation was confirmed by polyacrylamide gel electrophoresis of the pellets. The high sensitivity of this method facilitates relatively rapid assays even from small amounts of cultured cells.

Amino Acids↗
PubMed1990

C1q, a collagen-like complement subcomponent, in dermatosparactic cattle: its extracellular modification is not affected by lack of procollagen N-terminal proteinase (pN-proteinase).

C1q, a collagen-like complement protein, was purified from the serum of a dermatosparactic calf which lacks procollagen N-terminal proteinase (pN-proteinase). The specific hemolytic activity of the serum C1q from the dermatosparactic animal was identical to that of C1q from a normal calf. Gel-filtration of serum from the dermatosparactic calf, on Sepharose 6B, showed the presence of C1q-antigenic material at only one position which was identical to the elution position of normal bovine C1q. No difference, under dissociating conditions, could be seen in the size of the chains of C1q in specific immunoprecipitates isolated from the sera of dermatosparactic and normal animals, as judged by polyacrylamidegel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS). The C1q from the dermatosparactic animal showed the same N-terminal amino acid and tryptic-digest peptide pattern on HPLC as C1q from the normal calf. These results strongly suggest that pN-proteinase is not involved in the extracellular processing of C1q.

Animals↗
PubMed2026

Hypoxia reprograms VEGF signaling to differentially control ADAMTS2 and ADAMTS3 expression in endothelial cells.

ADAMTS2/-3, key metalloproteinases involved in collagen processing and extracellular matrix dynamics, remain insufficiently characterized in terms of their transcriptional regulation under hypoxic and pro-angiogenic conditions. In this study, we demonstrate that VEGF₁₆₅ robustly enhances ADAMTS2/-3 expression in endothelial cells, with hypoxia providing a striking amplification of this response. Bioinformatic analyses revealed that hypoxia and VEGF induced HIF-mediated and time-varying expression responses in ADAMTS2/-3. Using HUVECs exposed to CoCl₂-induced hypoxia, VEGF stimulation led to substantial increases in ADAMTS2 (approximately 19-fold at 3 h) and ADAMTS3 (approximately 46-fold at 3 h) mRNA levels, accompanied by concordant protein upregulation. Promoter-reporter assays revealed strong VEGF responsiveness in defined ADAMTS2 (-658/+112) and ADAMTS3 (-131/+40; -1340/+40) promoter fragments, particularly under hypoxic conditions. Pharmacological inhibition showed that JNK, MAPK/ERK, p38, and PI3K pathways each contributed partially to VEGF-mediated transcription, indicating multi-pathway convergence rather than single-pathway dependency. This finding is consistent with RNA-seq analyses showing that VEGF-related signaling is extensively re-regulated under hypoxic conditions. Extension of these analyses to MG-63 and SAOS-2 cell lines revealed modest but consistent VEGF-induced upregulation, supporting a tissue-independent regulatory axis. Collectively, these findings position ADAMTS2/-3 as potent hypoxia- and VEGF-responsive genes, uncovering their integration into HIF-1α-dependent transcriptional networks and VEGF-activated signaling cascades. This work highlights the relevance of ADAMTS2/-3 in angiogenesis-associated extracellular matrix remodeling and identifies them as promising biomarkers and potential therapeutic targets in hypoxia-driven vascular pathology.

Humans↗
PubMed1985

Sequential cleavage of type I procollagen by procollagen N-proteinase. An intermediate containing an uncleaved pro alpha 1(I) chain.

The conversion of type I procollagen to type I collagen was studied by cleaving the protein with partically purified type I procollagen N-proteinase from chick embryos. Examination of the reaction products after incubation for varying times at 30 degrees C indicated that, during the initial stages of the reaction, pro alpha 1(I) and pro alpha 2(I) chains were cleaved at about the same rate. As a result, all the pro alpha 2(I) chains were converted to pC alpha 2(I) chains well before all the pro alpha 1 chains were cleaved. When the reaction products were examined by gel electrophoresis without reduction of interchain disulfide bonds, a distinct band of an intermediate was detected. The same intermediate was seen when the reaction was carried out at 35, 37, and 40 degrees C. The data established that over two-thirds of the type I procollagen was converted to the intermediate and that this intermediate was then slowly converted to the final product of pCcollagen. The kinetics for the reaction, however, did not fit a simple model for precursor-product relationship among substrate, intermediate, and product. Examination of the reaction products with a two-step gel procedure demonstrated that the intermediate consisted of three polypeptide chains in which the N propeptide was cleaved from one pro alpha 1 chain and one pro alpha 2(I) chain but the N propeptide was still present on one of the pro alpha 1(I) chains. In further experiments it was demonstrated that a similar intermediate was seen when a homotrimer of pro alpha 1(I) chains was partially cleaved by the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1986

Iron-containing metallocenes as active site directed inhibitors of the proteinase that cleaves the NH2-terminal propeptides from type I procollagen.

Derivatives of ferrocene (dicyclopentadienyliron) (Fc) were examined as active site directed inhibitors of type I procollagen N-proteinase, the enzyme that cleaves the NH2-terminal propeptides from type I procollagen. The compounds were shown here to be reversible, competitive inhibitors of the enzyme. The effectiveness of the Fc inhibitors varied with modification of the cyclopentadienyl (cp) rings. The monocarboxylic acid (I) and the 1,1'-dicarboxylic acid (II) derivatives of Fc inhibited 50% of the enzymic activity (I50) at concentrations of 1.0 and 0.5 mM, respectively. The Ki values were 0.3 mM for both I and II. Derivatization of the carbonyl alpha to the cp ring of compound I (FcCOCH2CH2COOH, III) increased the inhibitory activity (I50 = 0.100 mM; Ki = 0.065 mM). Removal of the carbonyl alpha to the cp ring of III did not improve inhibitory activity: FcCH2CH2COOH, I50 = 2 mM; FcCH = CHCOOH, I50 = 1.5 mM. The active inhibitory species apparently contained iron in the 3+ valence state since two ferrocenium derivatives were very effective inhibitors: ferrocenium tetrachloroferrate, IV (I50 = 0.030 mM; Ki = 0.004 mM), and carboxyferrocenium hexafluorophosphate, V (I50 less than 0.1 mM; Ki less than 0.05 mM). In addition, reduction of III with ascorbic acid abolished its inhibitory activity. Compounds I and III stabilized the enzyme to heat denaturation in the absence of exogenous calcium; compound IV did not stabilize the enzyme. Further observations indicated that Fc derivatives were specific inhibitors of procollagen N-proteinase.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Mutations that alter the primary structure of type I procollagen have long-range effects on its cleavage by procollagen N-proteinase.

Type I/II procollagen N-proteinase was partially purified from chick embryos and used to examine the rate of cleavage of a series of purified type I procollagens synthesized by fibroblasts from probands with heritable disorders of connective tissue. The rate of cleavage was normal with procollagen from a proband with osteogenesis imperfecta that was overmodified by posttranslational enzymes. Therefore, posttranslational overmodification of the protein does not in itself alter the rate of cleavage under the conditions of the assay employed. Cleavage of the procollagen, however, was altered in several procollagens with known mutations in primary structure. Two of the procollagens had in-frame deletions of 18 amino acids encoded by exons 11 and 33 of the pro alpha 2(I) gene. In both procollagens, both the pro alpha 1(I) and the pro alpha 2(I) chains were totally resistant to cleavage. With a procollagen in which glycine-907 of the alpha 2(I) chain domain was substituted with aspartate, both pro alpha chains were cleaved but at a markedly decreased rate. The results, therefore, establish that mutations that alter the primary structure of the pro alpha chains of procollagen at sites far removed from the N-proteinase cleavage site can make the protein resistant to cleavage by the enzyme. The long-range effects of in-frame deletions or other changes in amino acid sequence are probably explained by their disruption of the hairpin structure that is formed by each of the three pro alpha chains in the region containing the cleavage site and that is essential for cleavage of the procollagen molecule by N-proteinase.

Animals↗
PubMed1992

Evidence for a relationship between Ehlers-Danlos type VII C in humans and bovine dermatosparaxis.

Ehlers-Danlos (ED) syndrome type VII is characterized by the accumulation of collagen precursors in connective tissues. ED VII A and B are caused by mutations in the genes of alpha 1 and alpha 2 collagen I which result in the disruption of the cleavage site of procollagen I N-proteinase. The existence of ED VII C in humans has been hypothesized on the basis of a disorder in cattle and sheep related to the absence of the enzyme. We now present evidence for the existence of this disease in humans, characterized by skin fragility, altered polymers seen as hieroglyphic pictures with electron microscopy, accumulation of p-N-alpha 1 and p-N-alpha 2 collagen type I in the dermis and absence of processing of the p-N-I polypeptides in fibroblast cultures.

Animals↗
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↗