Mammalian ALOX15 orthologs exhibit pronounced dual positional specificity with docosahexaenoic acid

Mammalian lipoxygenases (LOX) have been implicated in cell differentiation and in the pathogenesis of inflammatory, hyperproliferative and neurological diseases. Although the reaction specificity of mammalian LOX with n − 6 fatty acids (linoleic acid, arachidonic acid) has been explored in detail little information is currently available on the product patterns formed from n − 3 polyenoic fatty acids, which are of particular nutritional importance and serve as substrate for the biosynthesis of pro-resolving inflammatory mediators such as resolvins and maresins. Here we expressed the ALOX15 orthologs of eight different mammalian species as well as human ALOX12 and ALOX15B as recombinant his-tag fusion proteins and characterized their reaction specificity with the most abundantly occurring polyunsaturated fatty acids (PUFAs) including 5,8,11,14,17-eicosapentaenoic acid (EPA) and 4,7,10,13,16,19-docosahexaenoic acid (DHA). We found that the LOX isoforms tested accept these fatty acids as suitable substrates and oxygenate them with variable positional specificity to the corresponding n − 6 and n − 9 hydroperoxy derivatives. Surprisingly, human ALOX15 as well as the corresponding orthologs of chimpanzee and orangutan, which oxygenates arachidonic acid mainly to 15S-H(p)ETE, exhibit a pronounced dual reaction specificity with DHA forming similar amounts of 14- and 17-H(p)DHA. Moreover, ALOX15 orthologs prefer DHA and EPA over AA when equimolar concentrations of n − 3 and n − 6 PUFA were supplied simultaneously. Taken together, these data indicate that the reaction specificity of mammalian LOX isoforms is variable and strongly depends on the chemistry of fatty acid substrates. Most mammalian ALOX15 orthologs exhibit dual positional specificity with highly unsaturated n − 3 polyunsaturated fatty acids.     

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.     

Growth inhibition and chromosomal instability of cultured marsupial (opossum) cells after treatment with DNA polymerase α inhibitor

The DNA replication mechanism has been well established for eutherian mammals (placental mammals such as humans, mice, and cattle), but not, to date, for metatherian mammals (marsupials such as kangaroos, koalas, and opossums). In this study, we found that dehydroaltenusin, a selective inhibitor of mammalian (eutherian) DNA polymerase α, clearly suppressed the growth of metatherian (opossum and rat kangaroo) cultured cells. In cultured opossum (OK) cells, dehydroaltenusin also suppressed the progression of DNA replication. These results suggest that dehydroaltenusin inhibits metatherian as well as eutherian DNA replication. Dehydroaltenusin treatment of OK cells engendered fluctuations in the numbers of chromosomes in the OK cells as well as inhibition of cell growth and DNA replication. This suggests that partial inhibition of DNA replication by dehydroaltenusin causes chromosomal instability in cultured cells.     

Phosphorylation mimicking mutations of ALOX5 orthologs of different vertebrates do not alter reaction specificities of the enzymes

5-Lipoxygenase (ALOX5) plays a key role in the biosynthesis of pro-inflammatory leukotrienes whereas 15-lipoxygenases (ALOX15) have been implicated in the formation of pro-resolving eicosanoids (lipoxins, resolvins). Recently, it has been suggested that a phosphorylation mimicking mutant (Ser663Asp) of a stabilized variant of human ALOX5 exhibits dominant arachidonic acid 15-lipoxygenase activity (> 95%). To test whether similar alterations in the reaction specificity can also be observed for ALOX5 orthologs of other species we expressed wildtype and phosphorylation mimicking mutants (Ser271Asp, Ser523Asp, Ser663Asp, Ser663Glu) of human, mouse and zebrafish ALOX5 in pro- and eukaryotic overexpression systems and characterized their reaction specificities. We found that neither of the phosphorylation mimicking mutants produced significant amounts of 15-hydroperoxyeicosatetraenoic acid and the 5-lipoxygenation/15-lipoxygenation ratio for all wildtype and mutant enzyme species was lower than 100:2. Taken together, this data suggest that phosphorylation of native ALOX5 orthologs of different vertebrates may not induce major alterations in the reaction specificity and thus may not inverse their biological activity.     

A role of Gln596 in fine-tuning mammalian ALOX15 specificity,protein stability and allosteric properties

His596 of human ALOX12 has been suggested to interact with the COO⁻-group of arachidonic acid during ALOX catalysis. In mammalian ALOX15 orthologs Gln596 occupies this position and this amino acid exchange might contribute to the functional differences between the two ALOX-isoforms. To explore the role of Gln596 for ALOX15 functionality we mutated this amino acid to different residues in rabbit and human ALOX15 and investigated the impact of these mutations on structural, catalytic and allosteric enzyme properties. To shed light on the molecular basis of the observed functional alterations we performed in silico substrate docking studies and molecular dynamics simulations and also explored the impact of Gln596 exchange on the protein structure. The combined theoretical and experimental data suggest that Gln596 may not directly interact with the COO⁻-group of arachidonic acid. In contrast, mutations at Gln596 destabilize the secondary and tertiary structure of ALOX15 orthologs, which may be related to a disturbance of the electrostatic interaction network with other amino acids in the immediate surrounding. Moreover, our MD-simulations suggest that the geometry of the dimer interface depends on the structure of substrate bound inside the substrate-binding pocket and that Gln596Ala exchange impairs the allosteric properties of the enzyme. Taken together, these data indicate the structural and functional importance of Gln596 for ALOX15 catalysis.     

Molecular basis for the catalytic inactivity of a naturally occurring near-null variant of human ALOX15

Mammalian lipoxygenases belong to a family of lipid-peroxidizing enzymes, which have been implicated in cardiovascular, hyperproliferative and neurodegenerative diseases. Here we report that a naturally occurring mutation in the hALOX15 gene leads to expression of a catalytically near-null enzyme variant (hGly422Glu). The inactivity may be related to severe misfolding of the enzyme protein, which was concluded from CD-spectra as well as from thermal and chemical stability assays. In silico mutagenesis experiments suggest that most mutations at hGly422 have the potential to induce sterical clash, which might be considered a reason for protein misfolding. hGly422 is conserved among ALOX5, ALOX12 and ALOX15 isoforms and corresponding hALOX12 and hALOX5 mutants also exhibited a reduced catalytic activity. Interestingly, in the hALOX5 Gly429Glu mutants the reaction specificity of arachidonic acid oxygenation was shifted from 5S- to 8S- and 12R-H(p)ETE formation. Taken together, our data indicate that the conserved glycine is of functional importance for these enzyme variants and most mutants at this position lose catalytic activity.     

Applicability of the Triad Concept for the Positional Specificity of Mammalian Lipoxygenases

The nomenclature of lipoxygenases (LOXs) is partly based on the positional specificity of arachidonic acid oxygenation, but there is no unifying concept explaining the mechanistic basis of this enzyme property. According to the triad model, Phe-353, Ile-418, and Ile-593 of the rabbit 12/15-LOX form the bottom of the substrate-binding pocket, and introduction of less space-filling residues at either of these positions favors arachidonic acid 12-lipoxygenation. The present study was aimed at exploring the validity of the triad concept for two novel primate 12/15-LOX (Macaca mulatta and Pongo pygmaeus) and for five known members of the mammalian LOX family (human 12/15-LOX, mouse 12/15-LOX, human 15-LOX2, human platelet type 12-LOX, and mouse (12R)-LOX). The enzymes were expressed as N-terminal His tag fusion proteins in E. coli, the potential sequence determinants were mutated, and the specificity of arachidonic acid oxygenation was quantified. Taken together, our data indicate that the triad concept explains the positional specificity of all 12/15-LOXs tested (rabbit, human, M. mulatta, P. pygmaeus, and mouse). For the new enzymes of M. mulatta and P. pygmaeus, the concept had predictive value because the positional specificity predicted on the basis of the amino acid sequence was confirmed experimentally. The specificity of the platelet 12-LOX was partly explained by the triad hypothesis, but the concept was not applicable for 15-LOX2 and (12R)-LOX.     

A gene cluster encoding human epidermis-type lipoxygenases at chromosome 17p13.1: cloning, physical mapping, and expression

Epidermis-type lipoxygenases, a distinct subclass within the multigene family of mammalian lipoxygenases (LOX), comprise recently discovered novel isoenzymes isolated from human and mouse skin including human 15-LOX-2, human and mouse 12R-LOX, mouse 8S-LOX, and mouse e-LOX-3. We have isolated the human homologue of mouse e-LOX-3. The cDNA of 3362 bp encodes a 711-amino-acid protein displaying 89% sequence identity with the mouse protein and exhibiting the same unusual structural feature, i.e., an extra segment of 41 amino acids, which can be located beyond the N-terminal beta-barrel domain at the surface of the C-terminal catalytic domain. The gene encoding e-LOX-3, ALOXE3, was found to be part of a gene cluster of approximately 100 kb on human chromosome 17p13.1 containing in addition the 12R-LOX gene, ALOX12B, the 15-LOX-2 gene, ALOX15B, and a novel 15-LOX pseudogene, ALOX15P. ALOXE3 and ALOX12B are arranged in a head-to-tail fashion separated by 8.5 kb. The genes are split into 15 exons and 14 introns spanning 22 and 15 kb, respectively. ALOX15P was found on the opposite DNA strand directly adjacent to the 3'-untranslated region of ALOX12B. ALOX15B is located in the same orientation 25 kb downstream of ALOX12B, and is composed of 14 exons and 13 introns spanning a total of 9.7 kb of genomic sequence. RT-PCR analysis demonstrated a predominant expression of ALOXE3, ALOX12B, and ALOX15B in skin.     

Alterations in leukotriene synthase activity of the human 5-lipoxygenase by site-directed mutagenesis affecting its positional specificity

The positional specificity of arachidonic acid oxygenation is currently the decisive parameter for classification of lipoxygenases. Although the mechanistic basis of lipoxygenase specificity is not completely understood, sequence determinants for the positional specificity have been identified for various isoenzymes. In this study we altered the positional specificity of the human 5-lipoxygenase by multiple site-directed mutagenesis and assayed the leukotriene A(4) synthase activity of the mutant enzyme species with (5S,6E,8Z,11Z,14Z)-5-hydroperoxy-6,8,11,14-eicos atetraenoic acid (5S-HpETE) as substrate. The wild-type 5-lipoxygenase converts 5S-HpETE almost exclusively to leukotriene A(4) as indicated by the dominant formation of leukotriene A(4) hydrolysis products. Since leukotriene synthesis involves a hydrogen abstraction from C(10), it was anticipated that the 15-lipoxygenating quadruple mutant F359W + A424I + N425M + A603I might not exhibit a major leukotriene A(4) synthase activity. Surprisingly, we found that this quadruple mutant exhibited a similar leukotriene synthase activity as the wild-type enzyme in addition to its double oxygenation activity. The leukotriene synthase activity of the 8-lipoxygenating double mutant F359W + A424I was almost twice as high, and similar amounts of leukotriene A(4) hydrolysis products and double oxygenation derivatives were detected with this enzyme species. These data indicate that site-directed mutagenesis of the human 5-lipoxygenase that leads to alterations in the positional specificity favoring arachidonic acid 15-lipoxygenation does not suppress the leukotriene synthase activity of the enzyme. The residual 8-lipoxygease activity of the mutant enzyme and its augmented rate of 5-HpETE conversion may be discussed as major reasons for this unexpected result.     

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.     

Structural basis for lipoxygenase specificity. Conversion of the human leukocyte 5-lipoxygenase to a 15-lipoxygenating enzyme species by site-directed mutagenesis

Mammalian lipoxygenases constitute a heterogeneous family of lipid-peroxidizing enzymes, and the various isoforms are categorized with respect to their positional specificity of arachidonic acid oxygenation into 5-, 8-, 12-, and 15-lipoxygenases. Structural modeling suggested that the substrate binding pocket of the human 5-lipoxygenase is 20% bigger than that of the reticulocyte-type 15-lipoxygenase; thus, reduction of the active-site volume was suggested to convert a 5-lipoxygenase to a 15-lipoxygenating enzyme species. To test this "space-based" hypothesis of the positional specificity, the volume of the 5-lipoxygenase substrate binding pocket was reduced by introducing space-filling amino acids at critical positions, which have previously been identified as sequence determinants for the positional specificity of other lipoxygenase isoforms. We found that single point mutants of the recombinant human 5-lipoxygenase exhibited a similar specificity as the wild-type enzyme but double, triple, and quadruple mutations led to a gradual alteration of the positional specificity from 5S- via 8S- toward 15S-lipoxygenation. The quadruple mutant F359W/A424I/N425M/A603I exhibited a major 15S-lipoxygenase activity (85-95%), with (8S,5Z,9E,11Z,14Z)-8-hydroperoxyeicosa-5,9 ,11, 14-tetraenoic acid being a minor side product. These data indicate the principle possibility of interconverting 5- and 15-lipoxygenases by site-directed mutagenesis and appear to support the space-based hypothesis of positional specificity.     

Gene mapping in marsupials: Detection of an ancient autosomal gene cluster

The genes HRAS, HBB, and CAT, which are located together on the short arm of human chromosome 11, appear to be part of a conserved synteny group found in many eutherian mammals. These genes were mapped to the chromosomes of two marsupial (metatherian) species by in situ hybridization. All three genes were located together on chromosome 3 in Macropus eugenii. Only HRAS and CAT were used to probe Dasykaluta rosamondae metaphases and these genes both mapped to chromosome 4. This suggests that the HRAS-HBB-CAT gene cluster has been conserved at least since the metatherians and eutherians diverged some 130 million years ago. These findings support the concept of a mammalian genome that has remained highly conserved throughout evolution.     

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.     

Cloning,purification and characterization of non-human primate 12/15-lipoxygenases

The enzyme 15-lipoxygenase-1 (15-LO-1) possesses mainly 15-LO activity and has so far only been described in human cells and rabbit reticulocytes. The animal ortholog, except rabbit reticulocytes, is an enzyme with predominantly a 12-lipoxygenase activity, commonly referred to as 12/15-LO. We describe herein the characterization of the 12/15-LOs in Macaca mulatta (rhesus monkey) and in Pongo pygmaeus (orang-utan). The rhesus and the orang-utan enzymes have mainly 12-lipoxygenase and 15-lipoxygenase activity, respectively, and they display 94% and 98% identity to the human 15-LO-1 protein. The rhesus enzyme was functionally different from the human enzyme with respect to substrate utilization in that anandamide was used differently and that the rhesus enzymes positional specificity could be affected by the substrate concentration. Furthermore, genomic data indicate that chimpanzees express an enzyme with mainly 15-lipoxygenase activity whereas marmosets express an enzyme with mainly 12-LO activity. Taken together, the switch during evolution from a 12-lipoxygenating enzyme in lower primates to a 15-lipoxygenating enzyme in higher primates and man might be of importance for the biological function of this enzyme.     

Sex chromosome homology and incomplete, tissue-specific X-inactivation suggest that monotremes represent an intermediate stage of mammalian sex chromosome evolution

Female mammals have two X chromosomes and males have a single X and a smaller, male-determining Y chromosome. The dosage of X-linked gene products is equalized between the sexes by the genetic inactivation of one X chromosome in females. The characteristics of the mechanism of X-chromosome inactivation differ in eutherian and metatherian mammals, and it has been suggested that the metatherian system represents a more primitive stage. The present study of monotreme sex chromosomes and X-chromosome inactivation suggests that the prototherian mammals may represent an even more primitive stage. There is extensive G-band homology between the monotreme X and Y chromosomes, and differences in the patterns of replication of the two X chromosomes in females suggest that X inactivation is tissue specific and confined to the unpaired segment of the X. On the basis of these results, we propose a model for the differentiation of mammalian sex chromosomes and the evolution of the mechanism of X-chromosome inactivation. This model involves a gradual reduction of the Y chromosome and an accompanying gradual recruitment of (newly unpaired) X-linked loci under the control of a single inactivation center.     

Stereocontrol of arachidonic acid oxygenation by vertebrate lipoxygenases: newly cloned zebrafish lipoxygenase 1 does not follow the Ala-versus-Gly concept

Animal lipoxygenases (LOXs) are classified according to their specificity of arachidonic acid oxygenation, and previous sequence alignments suggested that S-LOXs contain a conserved Ala at a critical position at the active site but R-LOXs carry a Gly instead. Here we cloned, expressed, and characterized a novel LOX isoform from the model vertebrate Danio rerio (zebrafish) that carries a Gly at this critical position, classifying this enzyme as putative arachidonic acid R-LOX. Surprisingly, the almost exclusive arachidonic acid oxygenation product was 12S-H(p)ETE (hydro(pero)xyeicosatetraenoic acid), and extensive mutation around Gly-410 failed to induce R-lipoxygenation. This finding prompted us to explore the importance of the corresponding amino acids in other vertebrate S-LOXs. We found that Ala-to-Gly exchange in human 15-LOX2 and human platelet 12-LOX induced major alterations in the reaction specificity with an increase of specific R-oxygenation products. For mouse 5-LOX and 12/15-LOX from rabbits, men, rhesus monkeys, orangutans, and mice, only minor alterations in the reaction specificity were observed. For these enzymes, S-HETE (hydroxyeicosatetraenoic acid) isomers remained the major oxygenation products, whereas chiral R-HETEs contributed only 10-30% to the total product mixture. Taken together these data indicate that the Ala-versus-Gly concept may not always predict the reaction specificity of vertebrate LOX isoforms.     

Biting through constraints: cranial morphology, disparity and convergence across living and fossil carnivorous mammals

Carnivory has evolved independently several times in eutherian (including placental) and metatherian (including marsupial) mammals. We used geometric morphometrics to assess convergences associated with the evolution of carnivory across a broad suite of mammals, including the eutherian clades Carnivora and Creodonta and the metatherian clades Thylacoleonidae, Dasyuromorphia, Didelphidae and Borhyaenoidea. We further quantified cranial disparity across eutherians and metatherians to test the hypothesis that the marsupial mode of reproduction has constrained their morphological evolution. This study, to our knowledge the first to extensively sample pre-Pleistocene taxa, analysed 30 three-dimensional landmarks, focused mainly on the facial region, which were digitized on 130 specimens, including 36 fossil taxa. Data were analysed with principal components (PC) analysis, and three measures of disparity were compared between eutherians and metatherians. PC1 showed a shift from short to long faces and seemed to represent diet and ecology. PC2 was dominated by the unique features of sabre-toothed forms: dramatic expansion of the maxilla at the expense of the frontal bones. PC3, in combination with PC1, distinguished metatherians and eutherians. Metatherians, despite common comparisons with felids, were more similar to caniforms, which was unexpected for taxa such as the sabre-toothed marsupial Thylacosmilus. Contrary to previous studies, metatherian carnivores consistently exhibited disparity which exceeded that of the much more speciose eutherian carnivore radiations, refuting the hypothesis that developmental constraints have limited the morphological evolution of the marsupial cranium.     

Gene mapping in marsupials and monotremes, V. Synteny between hypoxanthine phosphoribosyltransferase and phosphoglycerate kinase in the platypus

In order to extend comparative mapping studies to the monotreme mammals (subclass Prototheria), somatic-cell hybrids were obtained between Chinese-hamster cells deficient in hypoxanthine phosphoribosyltransferase (HPRT) and platypus fibroblasts. The characteristics of these hybrids closely resemble those of metatherian x eutherian hybrids, in that they are recovered at low frequency and they rapidly segregate and fragment platypus chromosomes. Biochemical and cytological studies of the hybrids, their subclones and HPRT-deficient revertants indicate that phosphoglycerate kinase is syntenic with HPRT in the platypus (as it is in other mammals); however, the studies do not permit chromosomal assignment of the syntenic group. The implications of the chromosomal location of this ancient synteny group for the evolution of the mammalian X chromosome are discussed.