Biosynthesis and functions of eicosanoids generated by the coelomocytes of the starfish, Asterias rubens

Eicosanoids are a group of oxygenated fatty acid derivatives formed from C20 polyunsaturated fatty acids, including arachidonic and eicosapentaenoic acids. The potential of the coelomocytes of the starfish, Asterias rubens, to generate eicosanoids through the cyclooxygenase (COX) and lipoxygenase (LOX) pathways was investigated using reverse-phase high performance liquid chromatography, enzyme immunoassay and gas chromatography–mass spectrometry. The principal LOX product was identified as 8-hydroxyeicosatetraenoic acid (8-HETE) with 8-hydroxyeicosapentaenoic acid (8-HEPE) synthesised at significantly lower levels. No classical prostaglandins (PG), such as PGE₂ or PGD₂, were found to be generated by ionophore-challenged coelomocytes. Incubation of coelomocytes with lipopolysaccharides from either Escherichia coli or Salmonella abortus failed to induce an increase in generation of LOX products and the presence of 8-HETE (0–25 μM) had no significant effect on the in vitro phagocytic activity of Asterias coelomocytes. Neither indomethacin (a COX inhibitor) or esculetin (a LOX inhibitor) had any effect on the clearance of the bacterium, Vibrio splendidus, from the coelomic cavity of starfish suggesting that products of these enzymes are not involved in such coelomocyte responses to foreign particles.     

Citrus abscission and Arabidopsis plant decline in response to 5-chloro-3-methyl-4-nitro-1H-pyrazole are mediated by lipid signalling

The compound 5-chloro-3-methyl-4-nitro-1H-pyrazole (CMNP) is a pyrazole-derivative that induces abscission selectively in mature citrus (Citrus sinensis) fruit when applied to the canopy and has herbicidal activity on plants when applied to roots. Despite the favourable efficacy of this compound, the mode of action remains unknown. To gain information about the mode of action of CMNP, the effect of application to mature citrus fruit and Arabidopsis thaliana roots was explored. Peel contact was essential for mature fruit abscission in citrus, whereas root drenching was essential for symptom development and plant decline in Arabidopsis. CMNP was identified as an uncoupler in isolated soybean (Glycine max) mitochondria and pea (Pisum sativum) chloroplasts and an inhibitor of alcohol dehydrogenase in citrus peel, but not an inhibitor of protoporphyrinogen IX oxidase. CMNP treatment reduced ATP content in citrus peel and Arabidopsis leaves. Phospholipase A₂ (PLA₂) and lipoxygenase (LOX) activities, and lipid hydroperoxide (LPO) levels increased in flavedo of citrus fruit peel and leaves of Arabidopsis plants treated with CMNP. An inhibitor of PLA₂ activity, aristolochic acid (AT), reduced CMNP-induced increases in PLA₂ and LOX activities and LPO levels in citrus flavedo and Arabidopsis leaves and greatly reduced abscission in citrus and delayed symptoms of plant decline in Arabidopsis. However, AT treatment failed to halt the reduction in ATP content. Reduction in ATP content preceded the increase in PLA₂ and LOX activities, LPO content and the biological response. The results indicate a link between lipid signalling, abscission in citrus and herbicidal damage in Arabidopsis.     

Purification of enzymatically active human lysyl oxidase and lysyl oxidase-like protein from Escherichia coli inclusion bodies

Lysyl oxidase (LOX) is an extracellular copper dependent enzyme catalyzing lysine-derived cross-links in extracellular matrix proteins. Recent molecular cloning has revealed the existence of a LOX family consisting of LOX and four lysyl oxidase-like proteins (LOXLs; LOXL, LOXL2, LOXL3, and LOXL4). Each member of the LOX family contains a copper-binding domain, residues for lysyl-tyrosyl quinone, and a cytokine receptor-like domain. Very recently, novel functions, such as tumor suppression, cellular senescence, and chemotaxis, have been attributed to this family of amine oxidases, but functional differences among the family members have yet to be determined. For efficient expression and purification, we cloned the cDNAs corresponding to proteolytically processed forms of LOX (LOX-p) and LOXL (LOXL-p1 and LOXL-p2) into a bacterial expression vector pET21a with six continuous histidine codons attached to the 3^′ of the gene. The recombinant proteins were purified with nickel-chelating affinity chromatography and converted into enzymatically active forms by stepwise dialysis in the presence of N-lauroylsarcosinate and Cu²⁺. The purified LOX-p, LOXL-p1, and LOXL-p2 proteins showed specific amine oxidase activity of 0.097, 0.054, and 0.150 U/mg, respectively, which was inhibited by β-aminopropionitrile (BAPN), a specific inhibitor of LOX. Availability of these pure and active forms of LOX and LOXLs will be significantly helpful in functional studies related to substrate specificity and crystal structures of this family of amine oxidases.     

The jasmonate pathway is involved differentially in the regulation of different defence responses in tobacco cells

Jasmonic acid, a product of the lipoxygenase (LOX) pathway, has been proposed to be a signal transducer of defence reactions in plants. We have reported previously that methyl jasmonate (MJ) induced accumulation of proteinase inhibitors in tobacco cell suspensions (Rickauer et al., 1992, Plant Physiol Biochem 30: 579–584). The role of this compound in the induction of this and of other defence reactions is further studied in this paper. Treatment of tobacco cell suspensions with an elicitor from Phytophthora parasitica var. nicotianae induced a rapid and transient increase in jasmonic acid levels, which was abolished when cells were preincubated with eicosatetraynoic acid (ETYA), an inhibitor of LOX. Pretreatment with ETYA also inhibited the induction of proteinase inhibitors by fungal elicitor, but not by MJ. Linolenic acid, a precursor of jasmonate biosynthesis, induced this defence response, whereas linoleic acid had no effect. Expression of defence-related genes encoding proteinase inhibitor II, hydroxyproline-rich or glycine-rich glycoproteins, glucanase and chitinase, was induced in a basically similar manner by fungal elicitor or MJ. However, ETYA did not inhibit, or only partially inhibited, the elicitation of these defence genes. Expression of the sesquiterpene cyclase (5-epi-aristolochene synthase) gene was not induced by MJ, but only by fungal elicitor, and ETYA pretreatment had no effect on this induction. The obtained results indicate that synthesis of jasmonate via the LOX pathway seems to be only part of a complex regulatory mechanism for the onset of many, but not all, defence reactions. Received: 4 July 1996 / Accepted: 23 November 1996     

Jasmonic Acid is Induced in a Biphasic Manner in Response of Pea Seedlings to Wounding

The role of jasmonic acid (JA) in plant wounding response has been demonstrated. However, the source of JA in wound signaling remains unclear. In the present study, pea seedlings were used as material to investigate the systemic induction of JA and the activation of lipoxygenase (LOX)-dependent octadecanoid pathway upon wounding. The results showed that endogenous JA could induce two peaks in the wounded leaves and the stalks, while only one peak in the systemic leaves.LOX activity and its protein amount were also induced and the stimulation mainly occurred in the late phase, while one peak of induction was present after pretreatment with JA. Applied nordihydroguaiaretic acid (NDGA), an inhibitor of LOX activity, only inhibited the induction of JA in the late phase, and the resistance of pea was impaired. Furthermore, 13(S)-hydroperoxy-9(Z), 11 (E)-octadecadienoic acid (13(S)-H(P)ODE) was confirmed to be the main product of LOX throughout the experimental time. In addition, immunocytochemical analysis also revealed the occurrence of JA biosynthesis and transport upon wounding. These results demonstrated that wound-induced JA in wounded leaves resulted from Its biosynthesis and conversion from its conjugates, while in systemic leaves resulted from its transport and biosynthesis; and proved that the LOX pathway was vital to the wound-induced defense response involved in JA biosynthesis.     

The human lysyl oxidase-like 2 protein functions as an amine oxidase toward collagen and elastin

The lysyl oxidase-like 2 (LOXL2) protein is a human paralogue of lysyl oxidase (LOX) that functions as an amine oxidase for formation of lysine-derived cross-links found in collagen and elastin. In addition to the C-terminal domains characteristic to the LOX family members, LOXL2 contains four scavenger receptor cysteine-rich (SRCR) domains in the N-terminus. In order to assess the amine oxidase activity of LOXL2, we expressed a series of recombinant LOXL2 proteins with deletions in the SRCR domains, using an Escherichia coli expression system. All of the purified recombinant LOXL2 proteins, with or without the SRCR domains in the N-terminus, showed significant amine oxidase activity toward several different types of collagen and elastin in in vitro amine oxidase assays, indicating deletion of the SRCR domains does not interfere with amine oxidase activity of LOXL2. Further, amine oxidase activity of LOXL2 was not susceptible to inhibition by β-aminopropionitrile, an irreversible inhibitor of LOX, suggesting a different enzymatic mechanism between these two paralogues.     

Induction of a new Lipoxygenase Form in Cucumber Leaves by Salicylic Acid or 2,6-Dichloroisonicotinic Acid*

Changes in lipoxygenase (LOX) protein pattern and/or activity were investigated in relation to acquired resistance of cucumber (Cucumis sativus L.) leaves against two powdery mildews, Sphaerotheca fuliginea (Schlecht) Salmon and Erysiphe cichoracearum DC et Merat. Acquired resistance was established by spraying leaves with salicylic acid (SA) or 2,6-dichloroisonicotinic acid (INA) and estimated in whole plants by infested leaf area compared to control plants. SA was more effective than INA. According to Western blots, untreated cucumber leaves contained a 97 kDa LOX form, which remained unchanged for up to 48 h after pathogen inoculation. Upon treatment with SA alone for 24 h or with INA plus pathogen, an additional 95 kDa LOX form appeared which had an isoelectric point in the alkaline range. For the induction of this form, a threshold concentration of 1 mM SA was required, higher SA concentrations did not change LOX-95 expression which remained similar between 24 h and 96 h but further increased upon mildew inoculation. Phloem exudates contained only the LOX-97 form, in intercellular washing fluid no LOX was detected. dichloroisonicotinic localization revealed LOX protein in the cytosol of the mesophyll cells without differences between the forms.     

Antisense suppression of LOX3 gene expression in rice endosperm enhances seed longevity

Lipid peroxidation plays a major role in seed longevity and viability. In rice grains, lipid peroxidation is catalyzed by the enzyme lipoxygenase 3 (LOX3). Previous reports showed that grain from the rice variety DawDam in which the LOX3 gene was deleted had less stale flavour after grain storage than normal rice. The molecular mechanism by which LOX3 expression is regulated during endosperm development remains unclear. In this study, we expressed a LOX3 antisense construct in transgenic rice (Oryza sativa L.) plants to down‐regulate LOX3 expression in rice endosperm. The transgenic plants exhibited a marked decrease in LOX mRNA levels, normal phenotypes and a normal life cycle. We showed that LOX3 activity and its ability to produce 9‐hydroperoxyoctadecadienoic acid (9‐HPOD) from linoleic acid were significantly lower in transgenic seeds than in wild‐type seeds by measuring the ultraviolet absorption of 9‐HPOD at 234 nm and by high‐performance liquid chromatography. The suppression of LOX3 expression in rice endosperm increased grain storability. The germination rate of TS‐91 (antisense LOX3 transgenic line) was much higher than the WT (29% higher after artificial ageing for 21 days, and 40% higher after natural ageing for 12 months). To our knowledge, this is the first report to demonstrate that decreased LOX3 expression can preserve rice grain quality during storage with no impact on grain yield, suggesting potential applications in agricultural production.     

Role of Lysyl Oxidase Propeptide in Secretion and Enzyme Activity

Lysyl oxidase (LOX) is secreted as a proenzyme (proLOX) that is proteolytically processed in the extracellular milieu to release the propeptide and mature, active LOX. LOX oxidizes lysyl residues of a number of protein substrates in the extracellular matrix and on the cell surface, which impacts several physiological and disease states. Although the LOX propeptide (LOX‐PP) is glycosylated, little is known about the role of this modification in LOX secretion and activity. To gain insight into this issue, cells were transfected with native, full‐length LOX cDNA (pre‐pro‐LOX), the N‐glycosylation null pre‐[N/Q]pro‐LOX cDNA and the deletion mutant pre‐LOX cDNA, referred to as secretory LOX, in which mature LOX is targeted to the secretory pathway without its N‐terminal propeptide sequence. The results show that glycosylation of the LOX‐PP is not required for secretion and extracellular processing of pro‐LOX but it is required for optimal enzyme activity of the resulting mature LOX. Complete deletion of the propeptide sequence prevents mature LOX from exiting the endoplasmic reticulum (ER). Taken together, our study points out the requirement of the LOX‐PP for pro‐LOX exit from the ER and is the first to highlight the influence of LOX‐PP glycosylation on LOX enzyme activity. J. Cell. Biochem. 111: 1231–1243, 2010. © 2010 Wiley‐Liss, Inc.     

Cunxi Wang · Kevan P.C. Croft · David F. Hildebrand

 Auxin [α-naphthaleneacetic acid (NAA) or indole-3-acetic acid] can induce the expression of lipoxygenases (LOXs) in cultured immature zygotic embryo cotyledons of soybean [Glycine max. (L.) Merr]. These auxin-induced LOXs are different from those normally expressed in seeds but have the same isoelectric points (pI) as those found in seedlings. The pIs of the two seedling LOXs were determined to be 5.09 and 5.23. One of the auxin-induced LOXs has the same pI (5.09) and molecular mass (94 kDa) as seedling LOX4. The partial amino acid sequences from the purified NAA-induced pI-5.09 LOX are identical to those of LOX4. RNA protection assays showed that NAA induces the expression of LOX4 and LOX5 mRNAs in cultured embryo cotyledons where they are not normally expressed. Soybean genotypes with a polymorphic variant of LOX4 in hypocotyls showed the same variation as NAA-induced LOXs in the embryo cotyledons. These results demonstrate that the NAA-induced pI-5.09 LOX is seedling LOX4 and also suggest that auxin might be directly or indirectly involved in seedling LOX expression during seed germination. Received: 10 January 2000 / Revision received: 16 June 2000 / Accepted: 29 June 2000     

Designing of enzyme inhibitors based on active site specificity: lessons from methyl gallate and its lipoxygenase inhibitory profile

Methyl gallate was purified, by lipoxygenase (LOX) inhibitory activity-guided method since its alleged anti-inflammatory property, from Bergenia ligulata (Wall), a plant used in the traditional, Ayurvedic system of medicine extensively. The LOX inhibitory property of methyl gallate was studied by enzyme kinetics, isothermal titration calorimetry and molecular docking followed by molecular simulation studies. The wet-laboratory experiments and in silico studies showed complete agreement, and promise of methyl gallate as a drug-lead molecular scaffold for anti-inflammatory therapy, based on LOX inhibition. The expressed work shows the need of nonactive site binding parameters to be considered while designing of inhibitors based on the specificities toward active sites of enzymes.     

Lipoxygenase is an abundant protein in cucumber exudates

The presence of lipoxygenase (LOX) has been reported in many plant organs. High LOX activity (1–2 katal/mg protein) was detected in exudates from cut cucumber (Cucumis sativus L.) stems and petioles. Exudate LOX had a pH optimum of 5.0, an estimated molecular weight of 95 kDa and cross-reacted on sodium-dodecyl-sulfate gels with anti-LOX antibodies raised against soybean leaf LOX isoenzymes. Lipoxygenase activity was detected on native gels stained with o-dianisidine using linoleic acid as a substrate. Enzyme activity was similar with linoleic and linolenic acid and 2 times greater with arachidonic acid as substrate. At pH 6.8, LOX metabolized linoleic acid into 13- and 9-hydroperoxides at a ratio of 12. Linolenic acid was preferentially oxidized at carbon 13. Lipoxygenase activity was inhibited by n-propyl gallate (IC₅₀ 300 nM) and nordihydroguaiaretic acid (IC₅₀ 25 nM), but not by nonsteroidal anti-inflammatory drugs. LOX activity was enhanced 4.5-fold by 300 mM Ca²⁺. Spermine at 1 mM, and putrescine and spermidine at 2 mM completely inhibited LOX activity, but at low concentrations spermine (100 mM) and spermidine (100–500 mM) significantly stimulated LOX activity: 8- and 4.5-fold, respectively. Tissue printing of stem, petiole and hypocotyl sections with subsequent incubation with the antiserum raised against soybean leaf LOX revealed the presence of LOX in the internal and external phloem and in the sieve tubes.Abbreviations DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycol-bis(-aminoethyl ether) N,N,N,N-tetraacetic acid - 9(S)-HpOD 9-(S)-hydroperoxy-(E,Z)10,12-octadecadienoic acid - 13(S)-HpOD 13-(S)-hydroperoxy(Z,E)-9,11-octadecadienoic acid - IC inhibition constant - IEF isoelectrofocusing - LOX lipoxygenase - NDGA nordihydroguaiaretic acid - PBS phosphate buffered saline - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate We would like to thank Ulla Jarlfors for exellent technical assistance with the histological analysis. The research reported in this paper was supported in part by grants to J.K. from the R.J. Reynolds Tobacco Company and Cooperative Agreement 43YK-5-0030 of the USDA-ARS. Journal paper 93-11-12 of the Kentucky Agricultural Experiment Station, Lexington.     

Lipoxygenase Pathway and Membrane Permeability and Composition during Storage of Potato Tubers (Solanum tuberosum L. cv Bintje and Désirée) in Different Conditions

Abstract: Potato tubers ( Solanum tuberosum L. cv Bintje and Désirée) were stored for 12 months under three different storage conditions: 4 °C, 20 °C with sprout inhibitor and 20 °C without sprout inhibitor. Independent of the storage conditions, our results show that the increase of membrane permeability, as revealed by electrolyte leakage, is not correlated with the lipid saturation status. Moreover, there is no simple correlation between cold sweetening and membrane permeability or lipid saturation status. During storage at 20 °C without sprout inhibitor, the increase in membrane permeability is inversely correlated to sucrose accumulation, but this is not the case when tubers were stored with sprout inhibitors. Lipoxygenase (LOX) is often proposed as responsible for peroxidative damage to membrane lipids. The gradual peroxidation resulting in double bond index decrease is regarded as a cause of senescence sweetening. Our results revealed that the role of LOX in aging and senescence of potato tubers is far from clear. LOX activity and gene expression are not correlated with the fatty acids composition of the membrane. Moreover, LOX activity and fatty acid hydroperoxide content are low in older tubers, whatever the storage conditions or the varieties. On the basis of our results, the correlation between sugar accumulation (low temperature and senescence sweetening) and peroxidative damage occurring during storage of potato tubers is discussed.     

Expression of cucumber lipid-body lipoxygenase in transgenic tobacco: lipid-body lipoxygenase is correctly targeted to seed lipid bodies

 A particular isoform of lipoxygenase (LOX, EC 1.13.11.12) localized on lipid bodies has been shown by earlier investigations to play a role during seed germination in initiating the mobilization of triacylglycerols. On lipid bodies of germinating cucumber (Cucumis sativus L.) seedlings, the modification of linoleoyl moieties by this LOX precedes the hydrolysis of the ester bonds. We analyzed the expression and intracellular location of this particular LOX form in leaves and seeds of tobacco (Nicotiana tabacum L.) transformed with one construct coding for cucumber lipid-body LOX and one construct coding for cucumber LOX fused with a hemagglutinin epitope. In both tissues, the amount of lipid-body LOX was clearly detectable. Biochemical analysis revealed that in mature seeds the foreign LOX was targeted to lipid bodies, and the preferred location of the LOX on lipid bodies was verified by immunofluorescence microscopy. Cells of the endosperm and of the embryo exhibited fluorescence based on the immunodecoration of LOX protein whereas very weak fluorescent label was visible in seeds of untransformed control plants. Further cytochemical analysis of transformed plants showed that the LOX protein accumulated in the cytoplasm when green leaves lacking lipid bodies were analyzed. Increased LOX activity was shown in young leaves of transformed plants by an increase in the amounts of endogenous (2E)-hexenal and jasmonic acid. Received: 9 August 1999 / Accepted: 28 September 1999