Serine Protease Mauritanicain from Euphorbia mauritanica and Phorbol-12-myristate-13-acetate Modulate the IL-8 Release in Fibroblasts and HaCaT Keratinocytes.

In recent years, skin reactions such as phytophotodermatitis, contact dermatitis, and other inflammatory responses after contact with chemicals from various plants, e.g., Heracleum mantegazzianum or Hippomane mancinella, are one of the hot topics in phytobiology. Occupational skin inflammation after contact with latices of plants from Euphorbiaceae are common among people who work with plants of this family. Activation of protein kinase C by G protein-coupled receptors such as protease-activated receptors is associated with skin inflammation. In this study, we focused on the inflammatory modulation potential of proteases combined with diterpenes on human skin. Because of its role as a proinflammatory cytokine, we concentrated on the release of IL-8 by fibroblasts and keratinocytes. Therefore, primary human dermal fibroblasts and the HaCaT keratinocytes cell line were used as a model. The results indicated that the combination of the protease mauritanicain from Euphorbia mauritanica and phorbol-12-myristate-13-acetate induced a significantly increased IL-8 release in HaCaT keratinocytes compared to single treatments. The obtained results also suggest that mauritanicain has an anti-inflammatory effect on primary human dermal fibroblasts.

Probing substrate-product relationships by natural abundance deuterium 2D NMR spectroscopy in liquid-crystalline solvents: epoxidation of linoleate to vernoleate by two different plant enzymes.

Natural abundance deuterium 2D NMR spectroscopy in weakly ordering, polypeptide chiral liquid crystals is a powerful technique that enables determination of enantiotopic isotopic ratios ((2)H/(1)H)( i ) at the methylene groups of long-chain fatty acids. This technique has been used to study the bioconversion of linoleic acid to vernoleic acid with the objective of establishing the in-vivo site-specific fractionation of (2)H associated with this process. The fractionation pattern was investigated in Euphorbia lagascae and Vernonia galamensis, plants that use different enzyme systems to perform the Δ(12)-epoxidation: a cytochrome P450 monooxygenase in the former and a di-iron dioxygenase in the latter. The specific interest in this study was to ascertain whether different ((2)H/(1)H)( i ) isotopic ratios in substrate and product might reflect distinct features of the nature of the reaction centre. However, both the linoleate (substrate) samples and both vernoleate (product) samples isolated from the seed oils of the two plants had remarkably similar (2)H isotope profiles, with selection against (2)H in the positions around the Δ(12)-epoxidation site. This is interpreted as indicating that, despite differences in the form in which the activated Fe is presented and in the architecture of the active site, the ((2)H/(1)H)( i ) isotopic pattern is determined by features common to the reaction. It is suggested that the effects acting as the overall determinants of the final ((2)H/(1)H)( i ) distribution in the product are the encumbrance of the active site pocket and constraints to conformational readjustment during the linoleate to vernoleate transformation.

Purification and physicochemical characterization of a serine protease with fibrinolytic activity from latex of a medicinal herb Euphorbia hirta.

A 34 kDa serine protease, designated as hirtin, with fibrinolytic activity was purified to homogeneity from the latex of Euphorbia hirta by the combination of ion exchange and gel filtration chromatography. The N-terminal sequence of hirtin was found to be YAVYIGLILETAA/NNE. Hirtin exhibited esterase and amidase activities along with azocaseinolytic, gelatinolytic, fibrinogenolytic and fibrinolytic activities. It preferentially hydrolyzed Aα and α-chains, followed by Bß and ß, and γ and γ-γ chains of fibrinogen and fibrin clot respectively. The optimum pH and temperature for enzyme activity was found to be pH 7.2 and 50 °C respectively. Enzymatic activity of hirtin was significantly inhibited by PMSF and AEBSF. It showed higher specificity for synthetic substrate p-tos-GPRNA for thrombin. The CD spectra of hirtin showed a high content of ß-sheets as compared to α-helix. The results indicate that hirtin is a thrombin-like serine protease and may have potential industrial and therapeutic applications.

Molecular characterization and expression analysis of a gene encoding for farnesyl diphosphate synthase from Euphorbia pekinensis Rupr.

The root of Euphorbia pekinensis as a traditional herbal medicine has been recorded in Chinese pharmacopoeias for the treatment of oedema, gonorrhea, migraine and wart cures. In this work, we reported on the cDNA cloning and characterization of a novel farnesyl diphosphate synthase (FPS) from E. pekinensis. The full-length cDNA named EpFPS (Genbank Accession Number FJ755465) contained 1431 bp with an open reading frame of 1029 bp encoding a polypeptie of 342 amino acids. The deduced amino acid sequence of the EpFPS named EpFPS exhibited a high homology with other plant FPSs, and contained five conserved domains. Phylogenetic analysis showed that EpFPS belonged to the plant FPS group. Southern blot analysis revealed that there exists a small FPS gene family in E. pekinensis. Expression pattern analysis revealed that EpFPS expressed strongly in root, weak in leaf and stem. In callus, expression of EpFPS gene and biosynthesis of triterpenoids were strongly induced by Methyl jasmonate and slightly induced by Salicylic acid. Functional complementation of EpFPS in an ergosterol auxotrophic yeast strain indicated that the cloned cDNA encoded a functional farnesyl diphosphate synthase.

Biochemical and spectroscopic characterization of a novel metalloprotease, cotinifolin from an antiviral plant shrub: Euphorbia cotinifolia.

A high molecular mass novel metalloprotease, cotinifolin is purified from the latex of Euphorbia cotinifolia by a combination of anion exchange and hydrophobic interaction chromatography. The nonglycosylated enzyme has a molecular mass of 79.76 kDa (ESI-MS) and the isoelectric point of the enzyme is pH 7.7. Cotinifolin hydrolyzes denatured natural substrates such as casein, azoalbumin, and hemoglobin with high specific activity. The K(m) value of the enzyme was found to be 20 µM with azocasein. The enzyme is not prone to autolysis even at very low concentrations. Polyclonal antibodies specific to enzyme was raised and immunodiffusion reveals that the enzyme has unique antigenic determinants. Maximum caseinolytic activity of cotinifolin is observed in the range of pH 7.0-8.0 and temperature of 50 °C. Using 0.2 mL of 1 mM solution of each metal ion, the purified protease was inhibited slightly by Ba²âº and Mn²âº, moderately by Mg²âº, Ca²âº and Cs²âº and significantly by Zn²âº, Cu²âº and Co²âº. On the other hand, substantial activation in caseinolytic activity was achieved by Ni²âº. The enzyme activity was also inhibited by EDTA and o-phenanthroline but not by any other protease inhibitors. Perturbation studies by temperature, pH, and chaotrophs of the enzyme also reveal its high stability as seen by CD, fluorescence and proteolytic activity. Spectroscopic studies reveal that cotinifolin has secondary structural features with α/ß type with approximately 9% of α-helicity. Easy availability and simple purification procedure makes the enzyme a good system for biophysical study, biotechnological and industrial applications.

DGAT1, DGAT2 and PDAT expression in seeds and other tissues of epoxy and hydroxy fatty acid accumulating plants.

Triacylglycerol (TAG) is the main storage lipid in plants. Acyl-CoA: diacylglycerol acyltransferase (DGAT1 and DGAT2) and phospholipid: diacylglycerol acyltransferase (PDAT) can catalyze TAG synthesis. It is unclear how these three independent genes are regulated in developing seeds, and particularly if they have specific functions in the high accumulation of unusual fatty acids in seed oil. The expression patterns of DGAT1, DGAT2 and a PDAT in relation to the accumulation of oil and epoxy and hydroxy fatty acids in developing seeds of the plant species Vernonia galamensis, Euphorbia lagascae, Stokesia laevis and castor that accumulate high levels of these fatty acids in comparison with soybean and Arabidopsis were investigated. The expression patterns of DGAT1, DGAT2 and the PDAT are consistent with all three enzymes playing a role in the high epoxy or hydroxy fatty acid accumulation in developing seeds of these plants. PDAT and DGAT2 transcript levels are present at much higher levels in developing seeds of epoxy and hydroxy fatty acid accumulating plants than in soybeans or Arabidopsis. Moreover, PDAT, DGAT1 and DGAT2 are found to be expressed in many different plant tissues, suggesting that these enzymes may have other roles in addition to seed oil accumulation. DGAT1 appears to be a major enzyme for seed oil accumulation at least in Arabidopsis and soybeans. For the epoxy and hydroxy fatty acid accumulating plants, DGAT2 and PDAT also show expression patterns consistent with a role in the selective accumulation of these unusual fatty acids in seed oil.

Molecular cloning, characterization and function analysis of the gene encoding HMG-CoA reductase from Euphorbia Pekinensis Rupr.

A new full-length cDNA encoding 3-hydroxy-3-methylglutoryl-Coenzyme A reductase (HMGR; EC1.1.1.34), which catalyzes the first committed step of isoprenoids biosynthesis in MVA pathway, was isolated from young leaves of Euphorbia Pekinensis Rupr. by rapid amplification of cDNA ends (RACE) for the first time. The full-length cDNA of HMGR (designated as EpHMGR, GenBank Accession NO. EF062569) was 2,200 bp containing a 1,752 bp ORF encoding 583 amino acids. Bioinformatic analyzes revealed that the deduced EpHMGR had extensive homology with other plant HMGRs and contained two transmembrane domains and a catalytic domain. The predicted 3-D model of EpHMGR had a typical spatial structure of HMGRs. Southern blot analysis indicated that at most two copies of EpHMGR gene existed in E. Pekinensis genome. Tissue expression analysis revealed that EpHMGR expressed strongly in roots, weakly in stems and leaves. The functional colour complementation assay indicated that EpHMGR could accelerate the biosynthesis of carotenoids in the Escherichia coli transformant, demonstrating that EpHMGR plays an influential role in isoprenoid biosynthesis.

Catalase and antiquitin from Euphorbia characias: two proteins involved in plant defense?

Here we report the cDNA nucleotide sequences of a calmodulin-binding catalase and an antiquitin from the latex of the Mediterranean shrub Euphorbia characias. Present findings suggest that catalase and antiquitin might represent additional nodes in the Euphorbia defense systems, and a multi-enzymatic interaction contributing to plant's protection against biotic and abiotic stresses is proposed to occur in E. characias laticifers.

Highly stable glycosylated serine protease from the medicinal plant Euphorbia milii.

A serine protease, named as "Milin" was purified to homogeneity from the latex of Euphorbia milii, a medicinal plant of Euphorbiaceae family. The molecular mass (SDS-PAGE), optimum pH and temperature of the enzyme were 51kDa, pH 8.0 and 60 degrees C, respectively. Milin retains full proteolytic activity over a wide range of pH (5.5-12) and temperature (up to 65 degrees C) with casein and azoalbumin as substrates. The activity of milin is inhibited by serine proteases inhibitors like PMSF, APMSF and DFP, but not by any other protease inhibitors such as E-64 and PCMB. Like the other serine proteases from the genus Euphorbia, the activity of milin was not inhibited by the proteinaceous inhibitor soyabean trypsin inhibitor (SBTI) even at very high concentrations that is naturally present in plants. The specific extinction coefficient (epsilon(280 nm)(1%)), molar extinction coefficient (a(m)) and isoelectric point of the enzyme were found to be 29, 152,500 M(-1) cm(-1) and pH 7.2, respectively. The enzyme is a glycoprotein with detectable carbohydrate moiety (7-8%) in its constitution, which is essential for the activity. The numbers of tryptophan, tyrosine and cysteine residues in the sequence of milin were estimated chemically and are 23, 14 and 14, respectively. Of the 14-cysteine residues, 12 constituted 6-disulfide linkages while two are free cysteines. The N-terminal sequence (first 12 amino acid residues) was determined and does not match with any sequence of known plant serine proteases. Perturbation studies by temperature, pH and chaotropes of the enzyme also reveal its high stability as seen by CD, fluorescence and proteolytic activity. Thus, this serine protease may have potential applications in food industry.

Transgenic production of epoxy fatty acids by expression of a cytochrome P450 enzyme from Euphorbia lagascae seed.

Seed oils of a number of Asteraceae and Euphorbiaceae species are enriched in 12-epoxyoctadeca-cis-9-enoic acid (vernolic acid), an unusual 18-carbon Delta(12)-epoxy fatty acid with potential industrial value. It has been previously demonstrated that the epoxy group of vernolic acid is synthesized by the activity of a Delta(12)-oleic acid desaturase-like enzyme in seeds of the Asteraceae Crepis palaestina and Vernonia galamensis. In contrast, results from metabolic studies have suggested the involvement of a cytochrome P450 enzyme in vernolic acid synthesis in seeds of the Euphorbiaceae species Euphorbia lagascae. To clarify the biosynthetic origin of vernolic acid in E. lagascae seed, an expressed sequence tag analysis was conducted. Among 1,006 randomly sequenced cDNAs from developing E. lagascae seeds, two identical expressed sequence tags were identified that encode a cytochrome P450 enzyme classified as CYP726A1. Consistent with the seed-specific occurrence of vernolic acid in E. lagascae, mRNA corresponding to the CYP726A1 gene was abundant in developing seeds, but was not detected in leaves. In addition, expression of the E. lagascae CYP726A1 cDNA in Saccharomyces cerevisiae was accompanied by production of vernolic acid in cultures supplied with linoleic acid and an epoxy fatty acid tentatively identified as 12-epoxyoctadeca-9,15-dienoic acid (12-epoxy-18:2Delta(9,15)) in cultures supplied with alpha-linolenic acid. Consistent with this, expression of CYP726A1 in transgenic tobacco (Nicotiana tabacum) callus or somatic soybean (Glycine max) embryos resulted in the accumulation of vernolic acid and 12-epoxy-18:2Delta(9,15). Overall, these results conclusively demonstrate that Asteraceae species and the Euphorbiaceae E. lagascae have evolved structurally unrelated enzymes to generate the Delta(12)-epoxy group of vernolic acid.

Structure and nucleotide sequence of Euphorbia characias copper/TPQ-containing amine oxidase gene.

A cDNA encoding for a copper containing amine oxidase has been isolated and sequenced from young leaves of Euphorbia characias, a perennial mediterranean shrub. A single long open reading frame of 2068 pb encodes a protein composed of 653 amino acids with a molecular mass of about 74 kDa. A putative 24-aminoacid signal peptide precedes the sequence of the mature protein, with characteristics of a secretion signal peptide. Alignments of Euphorbia amine oxidase cDNA nucleotide sequence with that of amine oxidase from the seedlings of the pulses lentil, pea, and chickpea reveal several conserved regions, especially in the C-terminus, with a homology 90%-97%. The near 5' region shows several insertions, deletions, and different nucleotide sequence with ca. 60% homology. The enzyme contains 1%-2% carbohydrate deduced by deglycosylation experiments. Five cysteine residues are present in the deduced aminoacid sequence with a single disulfide bridge as judged by titration with cysteine reagents.