Evolutionary aspects of lipoxygenases and genetic diversity of human leukotriene signaling

Leukotrienes are pro-inflammatory lipid mediators, which are biosynthesized via the lipoxygenase pathway of the arachidonic acid cascade. Lipoxygenases form a family of lipid peroxidizing enzymes and human lipoxygenase isoforms have been implicated in the pathogenesis of inflammatory, hyperproliferative (cancer) and neurodegenerative diseases. Lipoxygenases are not restricted to humans but also occur in a large number of pro- and eucaryotic organisms. Lipoxygenase-like sequences have been identified in the three domains of life (bacteria, archaea, eucarya) but because of lacking functional data the occurrence of catalytically active lipoxygenases in archaea still remains an open question. Although the physiological and/or pathophysiological functions of various lipoxygenase isoforms have been studied throughout the last three decades there is no unifying concept for the biological importance of these enzymes. In this review we are summarizing the current knowledge on the distribution of lipoxygenases in living single and multicellular organisms with particular emphasis to higher vertebrates and will also focus on the genetic diversity of enzymes and receptors involved in human leukotriene signaling.     

Structural basis for pH-dependent alterations of reaction specificity of vertebrate lipoxygenase isoforms

Lipoxygenases have been classified according to their specificity of fatty acid oxygenation and for several plant enzymes pH-dependent alterations in the product patterns have been reported. Assuming that the biological role of mammalian lipoxygenases is based on the formation of specific reaction products, pH-dependent alterations would impact enzymes' functionality. In this study we systematically investigated the pH-dependence of vertebrate lipoxygenases and observed a remarkable stability of the product pattern in the near physiological range for the wild-type enzyme species. Site-directed mutagenesis of selected amino acids and alterations in the substrate concentrations induced a more pronounced pH-dependence of the reaction specificity. For instance, for the V603H mutant of the human 15-lipoxygenase-2 8-lipoxygenation was dominant at acidic pH (65%) whereas 15-H(p)ETE was the major oxygenation product at pH 8. Similarly, the product pattern of the wild-type mouse 8-lipoxygenase was hardly altered in the near physiological pH range but H604F exchange induced strong pH-dependent alterations in the positional specificity. Taken together, our data suggest that the reaction specificities of wild-type vertebrate lipoxygenase isoforms are largely resistant towards pH alterations. However, we found that changes in the assay conditions (low substrate concentration) and introduction/removal of a critical histidine at the active site impact the pH-dependence of reaction specificity for some lipoxygenase isoforms.     

The N-terminal β-barrel domain of mammalian lipoxygenases including mouse 5-lipoxygenase is not essential for catalytic activity and membrane binding but exhibits regulatory functions

Mammalian lipoxygenases (LOXs) have been implicated in cell differentiation and in the pathogenesis of inflammatory and hyperproliferative diseases. The available structural information indicated that lipoxygenases constitute single polypeptide chain enzymes consisting of a small N-terminal β-barrel domain and a larger C-terminal subunit that harbors the catalytic non-heme iron. Because of its structural similarity to C2-domains of lipases the N-terminal β-barrel domain of lipoxygenases, which comprises about 110 amino acids, has been implicated in membrane binding and activity regulation. To explore the functional relevance of the C2-domain in more detail and to develop a more comprehensive hypothesis on the biological role of this structural subunit we performed gene technical truncation on various mammalian LOX isoforms (12/15-LOXs of various species, human 15-LOX2, mouse 5-LOX) and quantified catalytic activity and membrane binding properties of the truncated recombinant enzyme species. We found that the C2-domain is not essential for catalytic activity and does hardly impact reaction specificity. Truncated enzyme species exhibit impaired membrane binding properties and altered reaction kinetics. Taken together, our data suggests a regulatory importance of the N-terminal β-barrel domain for mammalian lipoxygenase isoforms.     

The cDNA cloning of a pea (Pisum sativum) seed lipoxygenase. Sequence comparisons of the two major pea seed lipoxygenase isoforms.

Cloning and sequencing of two cDNAs from mRNA of maturing pea (Pisum sativum) seeds allowed the deduction of the complete amino acid sequence of a lipoxygenase polypeptide which is most similar to that of soya-bean lipoxygenase 2. The predicted Mr of this polypeptide is 97134, and its sequence permits comparisons between the lox2-type and the lox3-type lipoxygenase isoforms from pea and soya bean (Glycine max).     

Metabolic regulation of glyoxylate-cycle enzyme synthesis in detached cucumber cotyledons and protoplasts

Cucumber (Cucumis sativus L.) cotyledons, a plant model system for studying changes in metabolic compartmentation, contain at least six forms of lipoxygenase. The intracellular location and organellar topology of lipoxygenase forms in lipid bodies, microsomes, and cytosol were investigated. A protocol was worked out to solubilize and prepare lipid-body lipoxygenase in an enzymatically active form. The methodology required for the solubilization of the lipid-body form differed from the procedure applicable for solubilization of two lipoxygenase forms from the microsomal membranes. Three cytosolic lipoxygenases were purified and found to be distinguishable from each other in size and charge. Further characterization and differentiation of all cellular lipoxygenase isoforms was achieved by comparison of the enzymatic properties. Marked differences in pH optima of the particle-bound lipoxygenases were found: optimal pH of 8.5 for lipid-body lipoxygenase and pH 5.5 for the microsomal lipoxygenases. In addition, analysis of the products formed showed that the catalytic properties of lipidbody lipoxygenase and microsomal lipoxygenase are clearly distinguishable from each other and from the soluble forms.Abbreviations Brij-99 eikosaethyleneglycol monooleyl ether - LOX lipoxygenase The investigations were supported by the Deutsche Forschungsgemeinschaft. I.F. was supported by a stipend from the Friedrich-Ebert-Stiftung.     

Membrane-Associated and Soluble Lipoxygenase Isoforms in Tomato Pericarp (Characterization and Involvement in Membrane Alterations)

Membrane-associated and soluble lipoxygenases from green tomato (Lycopersicon esculentum Mill. cv Ailsa Craig) fruit have been identified. Microsomal lipoxygenase was localized partly in the plasma membrane and tonoplast fractions. The possibilities of glycosyl-phosphatidylinositol or transmembrane polypeptide anchors in the membrane were ruled out by differential solubilization and temperature-induced phase separation in Triton X-114. High performance liquid chromatography of reaction products combined with polarography showed that tomato lipoxygenase is capable of specific oxygenation of fatty acids esterified in phospholipids. This possibility of direct action on membrane phospholipids strengthened the hypothesis of a role for lipoxygenase in plant senescence and membrane turnover. Membrane-associated lipoxygenase is polymorphic, with two forms differing by their isoelectric points (pls) (around 4.2 and 5.1). The pl of the soluble lipoxygenase corresponds to the minor microsomal enzyme, with a pl of 5.1. The charge-differing isoforms were separated and analyzed by western blotting using anti-soybean lipoxygenase antibodies. A single polypeptide with an apparent molecular weight of 92,000 was identified in each case for the soluble and microsomal enzymes. It is suggested that a charge modification of the soluble lipoxygenase allows its association with the membrane.     

Isolation of lipoxygenase isoforms from Glycine max embryo axes based on apparent cross-reactivity with anti-myosin antibodies

Three lipoxygenase isoforms were isolated from Glycine max embryo axes. A number of proteins around 97 kDa cross-reacted with several anti-actin and anti-myosin antibodies and these were used to follow their purification through gel filtration, hydroxyapatite and anion exchange columns. The 97-kDa cross-reactive material eluted in the unbound fractions of the last anion exchange column, and displayed two components of pI's 6.2 and 6.3. Further phase partition of this fraction in TX-114 yielded a hydrophobic 97 kDa protein. Additionally, a 95-kDa protein was retained and eluted from this last column. Partial peptide sequences indicated that the 95 kDa protein was soybean lipoxygenase-1, the first 97 kDa protein was lypoxygenase-3, and the hydrophobic 97 kDa protein was lipoxygenase-2. Several possible reasons for the cross-reactivity with the antibodies are discussed. To our knowledge, this is the first example of individual lipoxygenase isoforms isolated from soybean embryo axes.     

Structural biology of mammalian lipoxygenases: enzymatic consequences of targeted alterations of the protein structure

Lipoxygenases form a heterogeneous family of lipid peroxidizing enzymes, which have been implicated in the pathogenesis of diseases with major health political relevance (bronchial asthma, atherosclerosis, cancer, and osteoporosis). The crystal structures of one mammalian lipoxygenase and of two plant isoenzymes have been solved and the structural bases of important enzyme properties (reaction specificity, membrane binding, and suicidal inactivation) have been investigated in the past. This review will briefly summarize our current understanding on the structural biology of the most important mammalian lipoxygenase isoforms and will also address selected mechanistic features of the lipoxygenase reaction.     

The N-terminal beta-barrel structure of lipid body lipoxygenase mediates its binding to liposomes and lipid bodies.

Phospholipase A2 and a particular isoform of lipoxygenase are synthesized and transferred to lipid bodies during the stage of triacylglycerol mobilization in germinating cucumber seedlings. Lipid body lipoxygenase (LBLOX) is post-translationally transported to lipid bodies without proteolytic modification. Fractionation of homogenates from cucumber cotyledons or transgenic tobacco leaves expressing LBLOX showed that a small but significant amount was detectable in the microsomal fraction. A beta-barrel-forming N-terminal domain in the structure of LBLOX, as deduced from sequence data, was shown to be crucial for selective intracellular transport from the cytosol to lipid bodies. Although a specific signal sequence for targeting protein domains to the lipid bodies could not be established, it was evident that the beta-barrel represents a membrane-binding domain that is functionally comparable with the C2 domains of mammalian phospholipases. The intact beta-barrel of LBLOX was demonstrated to be sufficient to target in vitro a fusion protein of LBLOX beta-barrel with glutathione S-transferase (GST) to lipid bodies. In addition, binding experiments on liposomes using lipoxygenase isoforms, LBLOX deletions and the GST-fusion protein confirmed the role of the beta-barrel as the membrane-targeting domain. In this respect, the cucumber LBLOX differs from cytosolic isoforms in cucumber and from the soybean LOX-1. When the beta-barrel of LBLOX was destroyed by insertion of an additional peptide sequence, its ability to target proteins to membranes was abolished.     

Identification and characterization of lipoxygenase isoforms in senescing carnation petals

A membrane-associated lipoxygenase and a soluble lipoxygenase have been identified in carnation (Dianthus caryophyllus L. cv Rêve) petals. Treatments of microsomal membranes by nonionic or zwitterionic detergents indicated that lipoxygenase is tightly bound to the membranes. By phase separation in Triton X-114, microsomal lipoxygenase can be identified in part as an integral membrane protein. Soluble lipoxygenase had an optimum pH range of 4.9 to 5.8, whereas microsomal lipoxygenase exhibited maximum activity at pH 6.1. Both soluble and membrane-associated lipoxygenases produced carbonyl compounds and hydroperoxides simultaneously, in the presence of oxygen. The membranous enzyme was fully inhibited by 0.1 millimolar n-propyl gallate, nordihydroguaiaretic acid, or salicylhydroxamic acid, but the effect of the three inhibitors on the soluble enzyme was much lower. The soluble lipoxygenase is polymorphic and three isoforms greatly differing by their isoelectric points were identified. Lipoxygenase activity in flowers was maximal at the beginning of withering, both in the microsomal and the soluble fractions. Substantial variations in the ratio of the two forms of lipoxygenase were noted at different sampling dates. Our results allowed us to formulate the hypothesis of a strong association of one soluble form with defined membrane constituents.     

Differential Expression of Lipoxygenase Isoenzymes in Embryos of Germinating Barley

Expression of lipoxygenase was studied in barley (Hordeum distichum L.) embryos during germination. Total lipoxygenase activity was high in quiescent grains, dropped during the 1st d of germination, and subsequently increased to a level similar to that in quiescent grains. The contribution of two isoenzymes, lipoxygenases 1 (LOX-1) and 2 (LOX-2), was studied at the activity, protein, and mRNA levels. Activity ratios of the two isoforms were determined via the ratio of 9- and 13-hydroperoxides, which are formed from linoleic acid. Isoenzyme protein levels were determined using specific monoclonal antibodies. mRNA levels were studied using the specific cDNA probes LoxA and LoxC, which correspond to LOX-1 and LOX-2, respectively. The major difference in temporal expression of LOX-1 and LOX-2 was observed in quiescent grains. At this stage, LOX-1 contributed almost exclusively to total lipoxygenase activity. LOX-2 activity rapidly increased until d 2 of germination. From this time point onward, LOX-1 and LOX-2 showed similar patterns at both activity and protein levels. The tissue distribution of the two isoenzymes in the germinating embryo was closely similar, with the highest expression levels in leaves and roots. The levels of LOX-1 and LOX-2 may be regulated mainly pretranslationally, as suggested by the similarity of the protein and mRNA patterns corresponding to the two isoforms.     

Specific lipoxygenase isoforms accumulate in distinct regions of soybean pod walls and mark a unique cell layer

Developing seeds constitute a strong sink for the plant and rely on the turnover and mobilization of carbon and nitrogen assimilates to supply the nutrients needed for their maturation. In large part these nutrients emanate from the vegetative organs including leaves and pod walls. Vegetative lipoxygenases (VLXs) accumulate in the paraveinal mesophyll cell layer of soybean (Glycine max L.) leaves where individual isoforms are proposed to play a role(s) as active enzymes or as transient storage proteins. VLXs also are prominent proteins in soybean pod walls, representing approximately 12% of the total soluble protein. Examining the temporal, tissue, and subcellular patterns of individual VLX isoform accumulation and of lipoxygenase activity through pod wall development indicates that VLXD is the principal VLX isoform playing a role in storage in this organ. The major accumulation of VLXD occurs just prior to seed fill within the endocarp middle zone, and protein extracted from this region shows relatively low levels of lipoxygenase activity, suggesting the middle zone may act as a storage tissue. Three other VLX isoforms, VLXA, VLXB, and VLXC colocalize to the cytoplasm of a single discrete cell layer in the mesocarp. Thus, the patterns of VLX cellular and subcellular localization in pod walls suggest independent functions for these different isoforms while also serving as specific markers for a novel cell layer in the pod wall.     

Lowering of Endogenous Lipoxygenase Activity in Pisum sativum Foliage by Cytokinin as Related to Senescence

Cytokinin (CK), when applied to intact pea plants, considerably lowered endogenous lipoxygenase levels. Furthermore it was demonstrated that the triggering of senescence induced by leaf detachment was invariably accompanied by a significant increase of lipoxygenase activity and the CK application considerably decelerates lipoxygenase increment. It is suggested that the lipoxygenase repression induced by CK is a contributing mechanism to the overall antisenescence action of the hormone, and that this may have biological implications.     

Lipoxygenase pathway in olive callus cultures (Olea europaea)

Stimulation of the lipoxygenase pathway in olive fruit initiates a cascade of reactions that begins with the regio- and stereospecific di-oxygenation of polyunsaturated fatty acids containing a cis, cis-1,4 pentadiene moiety. Later products of the pathway include volatiles that influence the organoleptic properties of harvested olive oil. In this study, we have investigated lipoxygenase activity in olive callus cultures, and found that there is evidence of several isoforms of the enzyme with different pH optima and substrate specificities. Endogenous lipoxygenase activity was detected throughout the growth cycle of olive callus, particularly during the log phase of growth, suggesting that olive lipoxygenases are intimately involved in growth. The most prominent lipoxygenase activity in tissue cultures was found to be soluble but significant activities were detected in the plastid fraction. In addition, hydroperoxide lyase (HPL) activity was measured in the calli; both 13- and 9-HPL activities were found which were particulate.     

Characterisation of lipoxygenase isoforms from olive callus cultures

Two lipoxygenase isoforms from olive callus cultures were separated from each other. Acetone powders were made to stabilise activity and remove lipids. Separation was then achieved by salt precipitation and ion-exchange chromatography. Both isoforms had comparable activity with linoleic and α-linolenic acid substrates, a basic pH optimum and had molecular masses of around 95 kDa. The callus extracts preferentially formed the 13-hydroperoxy products, in keeping with the pattern of volatile derivatives found in olive tissues and oils derived therefrom.     

The production and characterisation of products of the lipoxygenase enzyme system released by rat peritoneal macrophages

Rat peritoneal monocytes and macrophages when exposed to the ionophore A23187 release products of the lipoxygenase pathway of arachidonic acid metabolism which cause the aggregation and chemokinesis of polymorphonuclear leucocytes suspensions. The major biologically active compound released was leukotriene B which accounted for greater than 80% of the activity. The remaining biological activity was due to the release of a more polar as yet unidentified compound. In addition rat macrophages release 5, 12 and 15-HETE but these mono-HETEs do not significantly contribute to the biological activity.     

The production and characterisation of products of the lipoxygenase enzyme system released by rat peritoneal macrophages

Rat peritoneal monocytes and macrophages when exposed to the ionophore A23187 release products of the lipoxygenase pathway of arachidonic acid metabolism which cause the aggregation and chemokinesis of polymorphonuclear leucocytes suspensions. The major biologically active compound released was leukotriene B which accounted for >80% of the activity. The remaining biological activity was due to the release of a more polar as yet unidentified compound. In addition rat macrophages release 5, 12 and 15-HETE but these mono-HETEs do not significantly contribute to the biological activity.     

Crystal structures of vegetative soybean lipoxygenase VLX-B and VLX-D, and comparisons with seed isoforms LOX-1 and LOX-3

The lipoxygenase family of lipid-peroxidizing, nonheme iron dioxygenases form products that are precursors for diverse physiological processes in both plants and animals. In soybean (Glycine max), five vegetative isoforms, VLX-A, VLX-B, VLX-C, VLX-D, VLX-E, and four seed isoforms LOX-1, LOX-2, LOX-3a, LOX-3b have been identified. In this study, we determined the crystal structures of the substrate-free forms of two major vegetative isoforms, with distinct enzymatic characteristics, VLX-B and VLX-D. Their structures are similar to the two seed isoforms, LOX-1 and LOX-3, having two domains with similar secondary structural elements: a beta-barrel N-terminal domain containing highly flexible loops and an alpha-helix-rich C-terminal catalytic domain. Detailed comparison of the structures of these two vegetative isoforms with the structures of LOX-1 and LOX-3 reveals important differences that help explain distinct aspects of the activity and positional specificity of these enzymes. In particular, the shape of the three branches of the internal subcavity, corresponding to substrate-binding and O(2) access, differs among the isoforms in a manner that reflects the differences in positional specificities.     

The lipoxygenase pathway of Tupaia belangeri representing Scandentia. Genomic multiplicity and functional characterization of the ALOX15 orthologs in the tree shrew

The tree shrew (Tupaia belangeri) is a rat-sized mammal, which is more closely related to humans than mice and rats. However, the use of tree shrew to explore the patho-mechanisms of human inflammatory disorders has been limited since nothing is known about eicosanoid metabolism in this mammalian species. Eicosanoids are important lipid mediators exhibiting pro- and anti-inflammatory activities, which are biosynthesized via lipoxygenase and cyclooxygenase pathways. When we searched the tree shrew genome for the presence of cyclooxygenase and lipoxygenase isoforms we found copies of functional COX1, COX2 and LOX genes. Interestingly, we identified four copies of ALOX15 genes, which encode for four structurally distinct ALOX15 orthologs (tupALOX15a-d). To explore the catalytic properties of these enzymes we expressed tupALOX15a and tupALOX15c as catalytically active proteins and characterized their enzymatic properties. As predicted by the Evolutionary Hypothesis of ALOX15 specificity we found that the two enzymes converted arachidonic acid predominantly to 12S-HETE and they also exhibited membrane oxygenase activities. However, their reaction kinetic properties (K_M for arachidonic acid and oxygen, T- and pH-dependence) and their substrate specificities were remarkably different. In contrast to mice and humans, tree shrew ALOX15 isoforms are highly expressed in the brain suggesting a role of these enzymes in cerebral function. The genomic multiplicity and the tissue expression patterns of tree shrew ALOX15 isoforms need to be considered when the results of in vivo inflammation studies obtained in this animal are translated into the human situation.     

Carotenoids as cellular antioxidants.

Consumption of carotenoids is associated with an enhanced immune response and protection against neoplasia and atherosclerosis. Because these effects have been achieved using carotenoids with no pro-vitamin A activity, they are assumed to be due to the antioxidant properties of carotenoids. Carotenoids protect against photosensitized oxidation by quenching singlet oxygen. In addition, beta-carotene reacts chemically with peroxyl radicals to produce epoxide and apocarotenal products. To investigate the potential significance of these reactions to biological systems, we have used soybean lipoxygenase to generate peroxyl radical enzymatically. beta-Carotene inhibits the oxidation of linoleic acid by soybean lipoxygenase as well as the formation of the hydroperoxide product. In addition, the absorption of beta-carotene is diminished (bleached) by soybean lipoxygenase. The potential significance of these antioxidant reactions of carotenoids to biological function is discussed.