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PubMed1978

The simultaneous release by bone explants in culture and the parallel activation of procollagenase and of a latent neutral proteinase that degrades cartilage proteoglycans and denatured collagen.

1. A latent neutral proteinase was found in culture media of mouse bone explants. Its accumulation during the cultures is closely parallel to that of procollagenase; both require the presence of heparin in the media. 2. Latent neutral proteinase was activated by several treatments of the media known to activate procollagenase, such as limited proteolysis by trypsin, chymotrypsin, plasmin or kallikrein, dialysis against 3 M-NaSCN at 4 degrees C and prolonged preincubation at 25 degrees C. Its activation often followed that of the procollagenase present in the same media. 3. Activation of neutral proteinase (as does that of procollagenase) by trypsin or plasmin involved two successive steps: the activation of a latent endogenous activator present in the media followed by the activation of neutral proteinase itself by that activator. 4. The proteinase degrades cartilage proteoglycans, denatured collagen (Azocoll) and casein at neutral pH; it is inhibited by EDTA, cysteine or serum. Collagenase is not inhibited by casein or Azocoll and is less resistant to heat or to trypsin than is the proteinase. Partial separation of the two enzymes was achieved by gel filtration of the media but not by fractional (NH4)2SO4 precipitation, by ion exchange or by affinity chromatography on Sepharose-collagen. These fractionations did not activate latent enzymes. 5. Trypsin activation decreases the molecular weight of both latent enzymes (60 000-70 000) by 20 000-30 000, as determined by gel filtration of media after removal of heparin. 6. The latency of both enzymes could be due either to a zymogen or to an enzyme-inhibitor complex. A thermostable inhibitor of both enzymes was found in some media. However, combinations of either enzyme with that inhibitor were not reactivated by trypsin, indicating that this inhibitor is unlikely to be the cause of the latency.

Animals↗
PubMed1986

Proactivator-dependent activation of procollagenase induced by treatment with EGTA.

A new mechanism for activation of the proactivator of procollagenase [Vater, Nagase & Harris (1983) J. Biol. Chem. 258, 9374-9382] has been found. Collagenolytic and other proteolytic enzyme activities in the medium of cultured rabbit synovial fibroblasts were found to be activated by a new mechanism: short-term incubation at 37 degrees C performed in the presence of EGTA followed by replacement of Ca2+ during enzyme assay. The crucial event in procollagenase activation is the production of a functional activator enzyme. Activation of procollagenase in the culture medium did not occur when proactivator was removed by immunoprecipitation. Proteolytic activity of proactivator was fully activated, whereas procollagenase alone could not be activated by the same sequence. EGTA treatment of the culture medium at 0 degrees C did not result in enzyme activation if Ca2+ was replaced before incubation at 37 degrees C. Certain other bivalent metal ions (e.g. Sn2+, Cd2+, Zn2+ and Mn2+) could substitute for Ca2+ to stabilize the proactivator as a zymogen and therefore prevent the appearance of proteolytic activity in culture medium. Isolation of proactivator and procollagenase from EGTA-treated radiolabelled culture medium by immunoprecipitation and subsequent analyses by fluorography revealed that a time-dependent proteolysis of both molecules occurred after replacement of Ca2+ and incubation at 37 degrees C. However, comparison of enzyme activity with fluorographic analyses showed that the maximal activation of both enzymes was achieved before any detectable decrease in Mr. The results suggest that the activation of proactivator and the subsequent activation of procollagenase may be initiated by conformational changes in structure of the proactivator molecule produced by removal of stabilizing bivalent metal ions.

Animals↗
PubMed1982

Abnormalities in collagenase expression as in vitro markers for recessive dystrophic epidermolysis bullosa.

In order to correlate a characteristic clinical phenotype with biochemical abnormalities in recessive dystrophic epidermolysis bullosa, fibroblast cultures were established from 4 typical patients with the severe form of the disease. Collagenase, the enzyme implicated in the pathogenesis of blistering, was present in vitro in 2- to 4-fold greater concentrations than in control fibroblast cultures. Partially purified preparations of this enzyme displayed marked thermal lability and diminished affinity for Ca2+, a metal cofactor, suggesting the existence of a mutant enzyme. The data suggest that these 3 biochemical abnormalities, increased synthesis, decreased thermal stability and diminished affinity for Ca2+, should serve as reliable in vitro markers for genetic discrimination of recessive dystrophic epidermolysis bullosa.

Cells, Cultured↗
PubMed1988

New thiol inhibitors of Clostridium histolyticum collagenase. Importance of the P3' position.

An extensive series of synthetic mercaptotripeptides (HS-CH2-CH2-CO-Pro-Yaa) was prepared, and the inhibitory constants were determined on the Clostridium histolyticum collagenase. Among the factors which control the optimal binding of these inhibitors, we found that the presence of a free C-terminal carboxylate group in the position P3' of the compounds is of primary importance. In general, the esterification of this carboxylate group decreased the potency of the inhibitors by two orders of magnitude. We observed also that the enzyme favored the inhibitors having a long linear apolar or basic side-chain at the position P3'. The present data suggest a large S3' subsite of the C. histolyticum collagenase. The compound which contains a homoarginine residue at the P3' position with a Ki of 0.2 microM proved to be the most potent synthetic inhibitor known to date for the C. histolyticum collagenase.

Binding Sites↗
PubMed1982

Human skin collagenase in recessive dystrophic epidermolysis bullosa. Purification of a mutant enzyme from fibroblast cultures.

Recessive dystrophic epidermolysis bullosa, a genodermatosis characterized by dermolytic blister formation in response to minor trauma, is characterized by an incresaed collagenase synthesis by skin fibroblasts in culture. Since preliminary studies of partially purified recessive dystrophic epidermolysis bullosa collagenase suggested that the protein itself was aberrant, efforts were made to purify this enzyme to homogeneity, so that detailed biochemical and immunologic comparisons could be made with normal human skin fibroblast collagenase. Recessive dystrophic epidermolysis bullosa skin fibroblasts obtained from a patient documented to have increased synthesis of the enzyme were grown in large scale tissue culture and both serum-free and serum-containing medium collected as a source of collagenase. The recessive dystrophic epidermolysis bullosa collagenase was purified to electrophoretic homogeneity using a combination of salt precipitation, ion-exchange, and gel-filtration chromatography. In contrast to the normal enzyme, the recessive dystrophic epidermolysis bullosa collagenase bound to carboxymethyl-cellulose at Ca(2+) concentrations at least 10 times higher than those used with the normal enzyme. Additionally, this enzyme was significantly more labile to chromatographic manipulations, particularly when serum-free medium was used. However, rapid purification from serum-containing medium yielded a preparation enzymatically equivalent to normal human skin collagenase. Like the normal enzyme, the recessive dystrophic epidermolysis bullosa collagenase was secreted as a set of two closely related zymogens of approximately 60,000 and approximately 55,000 daltons that could be activated by trypsin to form enzymically active species of approximately 50,000 and approximately 45,000 daltons, respectively. Amino acid analysis suggested slight variations between the normal and recessive dystrophic epidermolysis bullosa collagenases. Cyanogen bromide digests demonstrated peptides unique to the enzyme from each source. The recessive dystrophic epidermolysis bullosa proenzyme was significantly more thermolabile at 60 degrees C than the normal, a finding that correlated with an approximate fourfold decrease in the affinity of the mutant enzyme for Ca(2+), a known activator and stabilizer of human skin collagenase. Aside from the altered affinity for this metal cofactor, kinetic analysis of the structurally altered recessive dystrophic epidermolysis bullosa collagenase revealed that its reaction rates and substrate specificity for human collagen types I-V were identical to those for the normal enzyme. Likewise, enzymes from both sources displayed identical energies of activation and deuterium isotope effects. Antisera were raised to the normal and putatively mutant procollagenases respectively, and, although they displayed a reaction of identity in double diffusion analysis, immunologic differences were present in enzyme inhibition and quantitative precipitation studies. These studies indicate that recessive dystrophic epidermolysis bullosa is characterized by the increased synthesis of an enzymically normal, but structurally aberrant, collagenase.

Amino Acids↗
PubMed1992

Polymerization of pNcollagen I and copolymerization of pNcollagen I with collagen I. A kinetic, thermodynamic, and morphologic study.

Previous observations established that pNcollagen III copolymerized with collagen I and decreased the diameter of the fibrils formed (Romanic, A.M., Adachi, E., Kadler, K.E., Hojima, Y., and Prockop, D.J. (1991) J. Biol. Chem. 266, 12703-12709). Here, procollagen I alone or mixtures of procollagen I and pCcollagen I were incubated with procollagen C-proteinase to generate pNcollagen I or mixtures of pNcollagen I and collagen I. The results confirmed previous reports that pNcollagen I assembles into sheet-like structures. They also demonstrated that polymerization of pNcollagen I exhibits a lag period and propagation phase similar to those seen with other protein self-assembly systems. In addition, the results demonstrated that pNcollagen I formed true copolymers with collagen I in that the presence of pNcollagen I increased the lag time, decreased the propagation rate, and increased the concentration of collagen I in solution at equilibrium. Copolymerization of pNcollagen I with collagen I, however, differed in two features from copolymerization of pNcollagen III with collagen I. One was that, in confirmation of previous work, copolymerization of pNcollagen I with collagen I markedly altered the circularity of the fibrils formed. The second difference was that the copolymerization increased the concentration in solution at equilibrium of pNcollagen I whereas copolymerization with collagen I was previously shown to decrease the concentration in solution of pNcollagen III. The increase in concentration in solution of pNcollagen I was explicable either by the assembly of soluble oligomers of pNcollagen I and collagen I, or by subtle changes in the activities of pNcollagen I and collagen I in the solid-phase. Comparison with previous data with pNcollagen III indicated that although pNcollagen I and pNcollagen III copolymerize with collagen I, there are marked differences in the two kinds of copolymers.

Collagen↗
PubMed1990

Relaxin modulates synthesis and secretion of procollagenase and collagen by human dermal fibroblasts.

Relaxin is believed to play a role in connective tissue remodeling during pregnancy (Bell, R.J., Eddie, L. W., Lester, A. R., Wood, E. C., Johnston, P.D., and Niall, H. D. (1987) Obstet. Gynecol. 69, 585-589; MacLennan, A. H. (1983) Clin. Reprod. Fertil. 2, 77-95). In the present study, normal human fibroblasts exposed to concentrations of a synthetic bioactive relaxin peptide from 0.1 to 10 ng/ml synthesized and secreted the metalloproteinase procollagenase, which was immunoprecipitable as a doublet of 52 and 57 kDa by a monoclonal antibody to human collagenase. The stimulation in procollagenase protein expression was reflected in an elevation in procollagenase mRNA levels. Media conditioned for 48 h by relaxin-treated fibroblasts (0.1 ng/ml) contained 1.7 units/ml activatable collagenase compared with 0.2 units/ml by untreated fibroblasts. In addition, relaxin caused a modest decrease in the levels of tissue inhibitor of metalloproteinases, as detected by reverse zymography and Northern analysis. Relaxin was also a potent modulator of the collagen secretory phenotype of these fibroblasts. Relaxin at 100 ng/ml down-regulated collagen secretion by 40%. When fibroblasts were treated simultaneously with cytokines such as transforming growth factor beta or interleukin 1 beta, which stimulated collagen synthesis to at least 9-fold of basal levels, relaxin at 100 ng/ml was able to down-regulate collagen expression by up to 88%. This decrease was reflected by changes at the mRNA level. These results indicate that relaxin can cause significant collagen turnover both by stimulating collagenase expression and by down-modulating collagen synthesis and secretion.

Cells, Cultured↗
PubMed1990

Rat collagenase. Cloning, amino acid sequence comparison, and parathyroid hormone regulation in osteoblastic cells.

We have isolated clones for rat collagenase from a rat osteoblastic cell cDNA library. These clones have been sequenced and the amino acids deduced. The calculated molecular weight is 51,352 for the proenzyme and 42,229 for the active enzyme. The deduced amino acid sequence was compared to those previously reported for: 1) human collagenase, 2) rat transin 1 (stromelysin), 3) human stromelysin, and 4) rabbit collagenase. The number of amino acids conserved was 47, 47, 50, and 47%, respectively. We also compared the collagenase mRNA and protein in different rat cells (osteoblast, uterine smooth muscle, synovial fibroblast) and determined that in rat uterine cells the message is slightly larger, although collagenase protein in all three cell types was identical in size. Parathyroid hormone dramatically induces the 2.9-kilobase collagenase mRNA in the rat osteoblastic cells, UMR 106-01. Nuclear run-on studies in UMR 106-01 cells demonstrated a 4-8-fold induction in the rate of synthesis of collagenase mRNA at 2 and 4 after parathyroid hormone treatment, with steady state levels of mRNA increased 100-fold at 4 h. Thus, parathyroid hormone regulation of the collagenase gene in UMR 106-01 cells is in part transcriptional.

Amino Acid Sequence↗
PubMed1987

Monocyte procollagenase and tissue inhibitor of metalloproteinases. Identification, characterization, and regulation of secretion.

Alveolar macrophages have been shown to secrete a procollagenase and the tissue inhibitor of metalloproteinases (TIMP), which are similar or identical to the corresponding proteins of human skin fibroblasts. Little is known, however, about the collagenolytic activity of normal human monocytes. We have applied immunologic, biochemical, and molecular biologic tools to examine the collagenolytic profile of freshly isolated peripheral blood monocytes. Our studies indicate that: 1) monocytes are capable of producing both procollagenase and TIMP that are identical to the corresponding products of skin fibroblasts, alveolar macrophages, and U-937 cells; 2) unstimulated monocytes in vitro secrete high levels of TIMP, but little or no procollagenase; 3) an as yet unidentified component(s) of serum are required for in vitro production of TIMP (but not procollagenase) by monocytes; 4) even when stimulated, monocytes secrete much smaller quantities of procollagenase in comparison with macrophages; and 5) regulation of the secretion of procollagenase and TIMP by monocytes exhibits a high degree of individual variability, but is nevertheless subject to clearly different control mechanisms than our previous findings would indicate for alveolar macrophages. Monocytes thus express a macrophage-like, rather than a neutrophil-like, profile of proteins capable of mediating collagen turnover, the regulation of which is distinct from that of more differentiated alveolar macrophages. Further study of monocyte and macrophage collagenolytic activities may provide insights into both the cell biology of mononuclear phagocyte maturation and the mechanisms by which such cells mediate the turnover of interstitial collagens.

Animals↗
PubMed1986

Human fibroblast collagenase. Complete primary structure and homology to an oncogene transformation-induced rat protein.

We have determined the complete sequence of the cDNA clone representing the full size human skin collagenase mRNA. Collagenase is synthesized in preproenzyme form, Mr 54,092, with a 19 amino acid long signal peptide. The primary secretion products of the enzyme consist of a minor glycosylated form, Mr 57,000, and a major unmodified polypeptide of predicted Mr 51,929. Proteolytic activation of human skin procollagenase results in removal of 81 amino acid residues from the amino-terminal portion of the proenzyme. Both potential N-glycosylation sites are contained within the proteolytically activated form of the enzyme. The primary structure of the coding region of the presented clone is homologous to an oncogene-induced rat protein whose function is still unknown, although preliminary observations suggest that it is not rat skin collagenase.

Amino Acid Sequence↗