Degradation of fibrin-β amyloid co-aggregate: A novel function attributed to ubiquitin

Human placental extract contains numerous bioactive components that are effective wound healing, antimicrobial and anti-inflammatory agents. During our investigation on the therapeutic potency of human placental extract, we have purified ubiquitin-like molecules that showed strong fibrino(geno)lytic activity. Further investigation confirmed similar potency of ubiquitin purified from adult human erythrocyte. Additionally, ubiquitin efficiently degraded disordered amyloid β 42 peptide (Aβ42) aggregate and fibrin-Aβ42 co-aggregate in vitro and reduced co-aggregate induced cytotoxicity in SH-SY5Y human neuroblastoma cells as compared to plasmin. Ubiquitin also degraded abnormal co-aggregates of fibrin with other plasma proteins such as fibronectin, albumin, lysozyme, tranthyretin and α-synuclein. To elucidate the mechanism of degradation, synthetic peptides (ADG, GKT, DQQ, QRL, LIF, AGK, HLVL) derived from ubiquitin template as well as synthetic ubiquitin (8565.32?Da) were employed. Synthetic ubiquitin completely degraded preformed Aβ 42 aggregate and fibrin-Aβ42 co-aggregate, whereas, the smaller synthetic peptides showed varying degrees of degradation. These observations attribute a novel function of ubiquitin that may be used for degrading abnormal fibrin clots in human body. Thorough investigation might unfold a novel molecular mechanism of ubiquitin in protein homeostasis.     

A ubiquitin-like peptide from the mushroom Pleurotus sajor-caju exhibits relatively potent translation-inhibitory and ribonuclease activities

The fruiting bodies of the edible mushroom Pleurotus sajor-caju were extracted with an aqueous buffer and then subjected to affinity chromatography on Affi-gel Blue gel, ion exchange chromatography on DEAE-cellulose and gel filtration on Superdex 75. From the fraction of the extract adsorbed on Affi-gel Blue gel and unadsorbed on DEAE-cellulose, a 9.5 kDa peptide with an N-terminal sequence similar to ubiquitin was isolated with a yield of 0.25 mg/kg mushroom. The peptide inhibited cell-free translation with an IC(50) of 30 nM. It exhibited a ribonuclease activity of 450 U/mg toward yeast transfer RNA. The activities were substantially more potent than those of previously isolated mushroom ubiquitin-like protein and peptide.     

Cloning and sequence analysis of a novel metalloprotease gene from Vibrio parahaemolyticus 04

The metalloprotease gene (vppC) from Vibrio parahaemolyticus 04 has been cloned and sequenced. The vppC gene contains an open reading frame of 2442 nucleotides encoding a polypeptide of 814 amino acids with a calculated molecular mass of 89,833 Da. The predicted amino acid sequence of VppC containing a zinc metalloprotease HEXXH consensus motif displays extensive homology to the collagenase from Vibrio alginolyticus. The activity of the recombinant protease produced in Escherichia coli was examined by gelatin zymography and proteolytic activity assays. The substrate specificity study showed that the type I collagen and synthetic collagenase substrate carbobenzoxy-glycyl-L-prolyl-glycyl-glycyl-L-prolyl-L-alanine were the best substrates, indicating that the cloned metalloprotease is indeed a collagenase. Multiple alignment analysis of the amino acid sequences and the enzymatic properties such as molecular mass and substrate specificity revealed three distinct classes of Vibrio metalloproteases. The identification of a new metalloprotease gene expands the role of Vibrio metalloproteases as a virulence factor for host infection.     

Identification of a metalloproteinase co-purifying with rat tumour collagenase and the characteristics of fragments of both enzymes

A metalloproteinase similar or identical to stromelysin was shown to co-purify with interstitial collagenase from the rat mammary carcinoma cell line, BC1. The mixture of BC1 metalloproteinase and collagenase degraded casein, gelatin, fibronectin, fibrinogen, laminin, proteoglycan and type IV collagen, in addition to types I and II collagen. Using SDS-PAGE and zymography, the Mr of both enzymes was 51.10(3). During storage, the 51.10(3) protein converted to fragments of Mr 34.10(3) and 24.10(3), and isoelectric points of 4.6-5.3 and 5.7-6.0, respectively. The fragments were separated from the intact (Mr 51.10(3) enzymes by DEAE-Sepharose chromatography, but intact metalloproteinase and collagenase activities resisted separation by a range of chromatographic methods. The Mr 34.10(3) fragment retained the proteinolytic activities of the intact enzymes, excepting collagenase cleavage of collagen types I and II. The Mr 24.10(3) fragment had no proteinolytic activity, showed an increase in Mr of 6.10(3) upon reduction, in common with the intact enzymes, and also had similar chromatographic properties to the intact enzymes. The data presented are consistent with a pattern of breakdown which is common to both collagenase and the metalloproteinase, and suggest that both enzymes are comprised of two protein domains.     

Effects of RU486 on the interstitial collagenase in the process of cervical ripening in the pregnant rat.

Changes in interstitial collagenase activity in the rat uterine cervix during ripening were clarified in a time-dependent manner. Premature delivery was induced by an antiprogesterone agent, RU486, for rats in late pregnancy. The presence of interstitial collagenase in the extract from the rat cervical tissue was demonstrated, by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis using the natural and unaffected collagen as a substrate. The collagenase activity was determined as the release of digested peptides from the radio-labeled collagen. Our experiments with RU486 were performed in rats on the 18th day of pregnancy. A single administration of RU486 (15 mg/kg) resulted in the premature delivery of all treated rats within 30 h after the injection (average time was 23.9 h). The marked increase in cervical wet weight was observed up to the time to premature delivery along with a significant acceleration from 18 h after the administration of RU486. In this state, the cervical collagenase activity was enhanced, the highest levels being recorded at 21 h after the administration. The interstitial collagenase in the uterine cervix appears to play a significant role in the regulation mechanisms of cervical ripening in late pregnant rats.     

Discovery and development of a type II collagen neoepitope (TIINE) biomarker for matrix metalloproteinase activity: from in vitro to in vivo

Destruction of cartilage by matrix metalloproteinases (MMPs) plays a significant role in the pathology of osteoarthritis (OA). A translatable biomarker of MMP activity would enable development of MMP inhibitors for the treatment of OA and potentially the improved diagnosis of OA. A directed approach to identifying specific MMP cleavage products as potential biomarkers has been undertaken. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify peptides generated by MMP-driven degradation of human articular cartilage (HAC) in vivo. It was shown that a 45-mer peptide fragment of collagen type II with five hydroxyprolines (OH) can be selectively produced by the activity of collagenase, an enzyme purported to be involved in the pathology of OA. This 45-mer is the most abundant neoepitope peptide found in biological fluids such as urine and synovial fluid. An immunoaffinity LC-MS/MS assay has been developed to quantify collagen type II neoepitope peptides as biomarkers of collagenase modulation. The lower limit of quantification for this assay was established to be 0.035 nM. The assay was used to measure the levels of collagen type II peptides in the urine of both clinical (healthy human subjects) and preclinical species. The urinary levels of the most abundant peptides are reported for rat, rabbit, guinea pig, dog, and healthy human adult subjects. The utility of this peptide to monitor collagenase activity in vivo has been demonstrated through its detailed characterization in HAC explants as well as in the urine of human and other preclinical species.     

Purification and properties of individual collagenases fromStreptomyces sp. strain 3B

Streptomyces strain 3B constitutively secreted collagenolytic enzymes during the post-exponential growth phase. Purification is described here leading to two collagenases (I and II) with specific activity of 3350 and 3600 U/mg, respectively, the highest activity obtained as yet for any streptomycete collagenase. Analysis of the purified enzymes by the method of zymography revealed that both I and II were homogeneous, with molar mass 116 and 97 kDa, respectively. Both collagenases were identical in their pH (7.5) and temperature optimum (37 degrees C). The inhibition profile of the enzymes by EDTA and 1,10-phenanthroline confirmed these enzymes to be metalloproteinases. By testing the activity with insoluble collagen, acid soluble collagen, gelatin, casein, elastin and Pz-PLGPR it was established that I and II are very specific for insoluble collagen and gelatin, showing a high activity toward acid soluble collagen and Pz-PLGPR. However, collagenases I and II failed to hydrolyze casein and elastin; they belong to true collagenases and resemble the clostridial enzymes.     

A vertebrate interstitial collagenase inhibitor from bovine scapular cartilage: purification and characterization

A collagenase inhibitor was purified from bovine cartilage by a combination of gel filtration, ion exchange, concanavalin A-Sepharose affinity chromatography, and elution from preparative sodium dodecyl sulfate-polyacrylamide gels. The inhibitor was purified 370-fold and migrated as a single polypeptide with an Mr of 19,000 on SDS-polyacrylamide gels. It stained positively for carbohydrate with periodic acid-Schiff's reagent and bound to lectins, indicating that it is a glycoprotein. The inhibitory activity was stable to heating up to 60 degrees C and between pH 4 and 10. The inhibition of collagenase by the cartilage inhibitor could not be reversed by trypsin or mersalyl. The inhibitory activity did not require the presence of free sulfhydryl groups, and it could be removed from the cartilage extract by incubating with native collagen, suggesting that the inhibitor binds to collagen. The cartilage inhibitor was effective against human and mouse interstitial collagenases, but it did not inhibit trypsin or bacterial collagenase.     

Pichia anomala DBVPG 3003 Secretes a Ubiquitin-Like Protein That Has Antimicrobial Activity

The yeast strain Pichia anomala DBVPG 3003 secretes a killer toxin (Pikt) that has antifungal activity against Brettanomyces/Dekkera sp. yeasts. Pikt interacts with β-1,6-glucan, consistent with binding to the cell wall of sensitive targets. In contrast to that of toxin K1, secreted by Saccharomyces cerevisiae, Pikt killer activity is not mediated by an increase in membrane permeability. Purification of the toxin yielded a homogeneous protein of about 8 kDa, which showed a marked similarity to ubiquitin in terms of molecular mass and N-terminal sequences. Pikt is also specifically recognized by anti-bovine ubiquitin antibodies and, similar to ubiquitin-like peptides, is not absorbed by DEAE-cellulose. However, Pikt differs from ubiquitin in its sensitivity to proteolytic enzymes. Therefore, Pikt appears to be a novel ubiquitin-like peptide that has killer activity.     

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.     

Production and properties of two collagenases from bacteria and their application for collagen extraction

Two collagenolytic protease (collagenase) producing bacteria, a Gram positive Bacillus cereus CNA1 and a Gram negative Klebsiella pneumoniae CNL3, were isolated under alkaline and acidic conditions, respectively. The production of collagenase by these two bacteria was optimized. Glycerol was the suitable carbon source for collagenase production by both strains. The optimal initial pH values for collagenase production by CNA1 and CNL3 were 7.5 and 6.0, respectively, and the optimal temperature was 37°C for both strains. The maximum activity of the partially purified collagenase from CNA1 was at pH 7.0 and 45°C. Its pH and thermal stability were in the range of 6-8 and below 40°C, respectively. The maximum activity of the partially purified collagenase from CNL3 was at pH 6.0 and 40°C. Its pH and thermal stability were in the range of 5-7 and below 37°C, respectively. The collagenase from CNL3 was more stable at a low pH compared with that from CNA1. Collagenases from both strains were used to extract collagen from salmon fish skin. The use of collagenases from CNA1 and CNL3 combined with acid treatment yielded a high collagen extraction of 54.6% and 53.0%, of the fish skin dry weight, respectively.     

A simple vertebrate collagenase assay using soluble radioactive collagen substrate

A highly sensitive assay for vertebrate collagenase has been developed using [14C]proline- or [3H]proline-labeled collagen as soluble substrate. The substrate was easy to prepare, gave high specific activity (1.4 X 10(6) cpm/mg collagen), and was stable at -20 degrees C for a long period. The digestion reaction for the assay was done at 21 degrees C to minimize the cleavage of collagen by proteases other than collagenase and to protect the 3/4 and 1/4 cleavage fragments of collagen from being further attacked by proteases. The cleaved products were denatured and then separated from undigested native collagen by precipitation with 1 M NaCl at pH 3.5. The conditions selected for denaturation and separation gave better discrimination between the cleaved products and uncleaved substrate than did conditions used in some other assays. The digestion products can be examined further by gel electrophoresis at the end of the assay to confirm the activity of vertebrate collagenase. This assay can also be adapted to assess telopeptidase activity independently of collagenase activity.     

Identification and partial characterization of an inhibitor of collagenase from rabbit bone

Bone explants from foetal and newborn rabbits synthesize and release a collagenase inhibitor into culture media. Inhibitor production in the early days of culture is followed first by latent collagenase and subsequently active collagenase in the culture media. A reciprocal relationship exists between the amounts of free inhibitor and latent collagenase in culture media, suggesting strongly that the inhibitor is a component of the latent form of the enzyme. Over 90% of the inhibitory activity of culture media is associated with a fraction of apparent mol.wt. 30000 when determined by gel filtration on Ultrogel AcA 44. The inhibitor blocks the action of rabbit collagenase on both reconstituted collagen fibrils and collagen in solution. It inhibits the action of either active collagenase or latent collagenase activated by 4-aminophenylmercuric acetate. Latent collagenase activated by trypsin is usually much less susceptible to inhibition. The activity of the inhibitor is destroyed by heat, by incubation with either trypsin or chymotrypsin and by 4-aminophenylmercuric acetate. Collagenase activity can be recovered from complexes of enzyme (activated with 4-aminophenylmercuric acetate) with free inhibitor by incubation with either trypsin or 4-aminophenylmercuric acetate, at concentrations similar to those that activate latent collagenase from culture media. The rabbit bone inhibitor does not affect the activity of bacterial collagenase, but blocks the action of collagenases not only from a variety of rabbit tissues but also from other mammalian species.     

Characterization and serum inhibition of neutral collagenase from cultured dog gingival tissue.

1. Explants of dog gingiva, maintained in culture for 9 days in the absence of serum, released a collagenase (EC 3.4.24.3) into the medium. The yield of active enzyme reached a maximum after 5-8 days with concomitant release of collagen degradation products from the explants. 2. The enzyme attacked undenatured collagen in solution at 25 degrees C resulting in a 58% loss of specific viscosity and producing the two characteristic products TCA(3/4) and TCB(1/4). Electron microscopy of segment-long-spacing crystallites of these reaction products showed the cleavage locus of the collagen molecule at interband 40. 3. Optimal enzyme activity was observed over the pH range 7.5-8.5 and a molecular weight of approximately 35,000 was derived from gel filtration studies. EDTA, 1,10-phenanthroline, cysteine and dithiothreitol all inhibited collagenase activity. Proteoglycan derived from porcine and human cartilage did not inhibit the enzyme. 4. The enzyme was inhibited by the dog serum proteins alpha2-macroglobulin and a smaller component of molecular weight approximately 40,000. This small component was purified by column chromatography utilising Sephadex G-200, DEAE A-50, and G-100 (superfine grade). Agarose electrophoresis of the purified component showed it to represent a beta-serum protein. alpha1-Antitrypsin did not inhibit the enzyme. 5. The physiological importance of the natural serum inhibitors and gingival collagenase are discussed in relation to latent enzyme and periodontal disease.     

Purification and physico-chemical properties of collagenase synthesized by a bacterium of the type Acinetobacter sp.]

The Acinetobacter spec collagenase has been almost completely purified. This enzyme is a true collagenase the activity of which is high on collagen. The enzyme is active on insoluble collagen, gelatin and the synthetic Pz-peptide, but has no proteolytic activity on casein or bovine serum-albumin. The collagenase was obtained on a simple medium with gelatin and yeast extract. The enzyme was purified by (NH4)2SO4 precipitation. DEAE cellulose column chromatography, Sephadex G 200 gel-filtration. The molecular weight of the enzyme was found to be 102 000 daltons, and its isoelectric point was found to be 7,7 +/- 0,2. The optimum pH and temperature for insoluble collagen hydrolysis were 7.6 and 37 degrees C, respectively; so, this collagenase corresponds to true collagenase. Hydrolysis of Pz-peptide is activated by Ca2+ and inhibited by metal ions (Cu2+, Fe3+, Zn2+, Pb2+, Hg2+). EDTA and o-phenanthroline induced a very significant reduction in enzyme activity. Iodoacetate and p-CMB induced a slight reduction in enzyme activity only at high concentrations (10-2M). The collagenase is most stable for temperatures less than or equal to 50 degrees C.     

Induction of collagenase production in Vibrio B-30.

The inducible nature of an extracellular collagenase produced by a marine Vibrio (Vibrio B-30, ATCC 21250) was demonstrated by observing the increase in extracellular collagenase activity after the addition of collagen to cell cultures in the latter part of the exponential growth phase. When collagenase-hydrolyzed collagen was added, the lag time required before collagenase production was detected decreased significantly compared with cultures receiving collagen. Cells preinduced to synthesize collagenase did not produce the enzyme when collagen was removed from the culture medium. Incorporation of penicillin G had no effect on final collagenase activity levels in suspensions of Vibrio B-30 in complete medium supplemented with collagen. However, chloramphenicol and tetracycline inhibited collagenase production, indicating that de novo protein synthesis was necessary for the appearance of activity. Attempts to isolate the inducing substance(s) involved filtering hydrolyzed collagen through a series of ultrafiltration membranes. The lowest-molecular-weight fraction of collagen hydrolysate with inducing ability was between 1,000 and 10,000. Gel filtration of this fraction on Sephadex G-50 resulted in the appearance of three protein peaks, two of which were capable of inducing collagenase production. Results from amino acid composition and N-terminal amino acid analysis suggest that the inducing substance originates from the polar helical portion of the collagen molecule.     

A modified collagenase assay method based on the use of p-dioxane.

Radioactive collagen synthesized by human skin fibroblasts in monolayer culture was used as a substrate for collagenase. The high specific activity of this substrate (75,000 cpm/μg) and the use of p-dioxane as a precipitant of the undigested collagen permit this enzyme to be assayed with collagen in solution at 35°C and pH 7.5. The dilutions used are sufficient to prevent the collagen molecules from aggregating, thus precluding the use of inhibitors of gel formation which tend to decrease the activity of the enzyme. Using a 1-h incubation, the procedure is reproducible (SD ± 2.3%) and linear over the range from 10 to 100 ng of bacterial collagenase. Vertebrate collagenase activity is also easily measured with this method.