Tanihiro Yoshimoto

Polyphenols of Cocoa: Inhibition of Mammalian 15-Lipoxygenase

Abstract Some cocoas and chocolates are rich in ()epicatechin and its related oligomers, the procyanidins. Fractions of these compounds, isolated from the seeds of Theobroma cacao, caused dosedependent inhibition of isolated rabbit 15-lipoxygenase-1 with the larger oligomers being more active; the decamer fraction revealed an IC 50 of 0.8 M. Among the monomeric flavanols, epigallocatechin gallate (IC 50 = 4 M) and epicatechin gallate (5 M) were more potent than ()epicatechin (IC50 = 60 M). ()Epicatechin and procyanidin nonamer also inhibited the formation of 15-hydroxyeicosatetraenoic acid from arachidonic acid in rabbit smooth muscle cells transfected with human 15-lipoxygenase-1. In contrast, inhibition of the lipoxygenase pathway in J774A.1 cells transfected with porcine leukocytetype 12- lipoxygenase (another representative of the 12/15- lipoxygenase family) was only observed upon sonication of the cells, suggesting a membrane barrier for flavanols in these cells. Moreover, epicatechin (IC50 approx. 15 M) and the procyanidin decamer inhibited recombinant human platelet 12-lipoxygenase. These observations suggest general lipoxygenase inhibitory potency of flavanols and procyanidins that may contribute to their putative beneficial effects on the cardiovascular system in man. Thus, they may provide a plausible explanation for recent literature reports indicating that procyanidins decrease the leukotriene/prostacyclin ratio in humans and human aortic endothelial cells.

Arachidonate 12-lipoxygenases

Arachidonate 12-lipoxygenase introduces a molecular oxygen at carbon 12 of arachidonic acid to generate a 12-hydroperoxy derivative. The enzymes generate 12-hydroperoxy derivatives with either S- or R-configurations. There are three isoforms of 12S-lipoxygenases named after the cells where they were first identified; platelet, leukocyte and epidermis. The leukocyte-type enzyme is widely distributed among cells, but the tissue distribution varies substantially from species to species. The platelet and epidermal enzymes are present in only a relatively limited number of cell types. Although the structures and enzymatic properties of the three isoforms of 12S-lipoxygenases have been elucidated, the physiological roles of the 12S-lipoxygenases are not yet fully understood. There are important roles for the enzymes and their products in several biological systems including those involved in atherosclerosis and neurotransmission.

Panaxynol, a polyacetylene compound isolated from oriental medicines, inhibits mammalian lipoxygenases.

Panaxynol is a polyacetylene compound isolated from commonly used oriental medicines, and its effects on various cyclooxygenases and lipoxygenases were investigated. The compound had only a marginal effect on cyclooxygenase activities (IC50 values >> 100 microM), but inhibited lipoxygenases; 5-lipoxygenase (IC50, 2 microM), two isoforms of 12-lipoxygenase (leukocyte-type, 1 microM; platelet-type, 67 microM) and 15-lipoxygenase (4 microM). Thus, panaxynol inhibited leukocyte-type 12-lipoxygenase much more effectively than platelet-type 12-lipoxygenase. Falcarindiol, an analogue of panaxynol, inhibited these lipoxygenases with higher IC50 values than panaxynol. These compounds could provide a clue to develop a selective inhibitor of one isoform of 12-lipoxygenase.

Site-directed mutagenesis studies on the iron-binding domain and the determinant for the substrate oxygenation site of porcine leukocyte arachidonate 12-lipoxygenase.

cDNA for arachidonate 12-lipoxygenase of porcine leukocytes was expressed in Escherichia coli. The recombinant 12-lipoxygenase was purified by immunoaffinity chromatography to near homogeneity with a specific activity of about 1.5 mumol/min per mg protein. Each of eight histidine residues, which were well-conserved among various mammalian lipoxygenases and presumed as ligands for non-heme iron, was substituted with leucine by site-directed mutagenesis. Each mutant enzyme was immunoaffinity-purified to near homogeneity. Mutations of His-361, -366 and -541 caused a total loss of enzyme activity, and the iron content was much lower (0.10, 0.06 and 0.06 g atom/mol protein) than that of the wild-type enzyme (0.53). Mutations of His-128 and -356 gave 159% and 162% specific activity of the wild-type enzyme, and the iron contents were 0.55 and 0.52 g atom/mol protein. Substitution of His-426 decreased the activity to 5%, but the iron content was 0.4 g atom/mol protein. The expression level of mutants at His-384 and -393 was too low to precisely determine the iron content. Taken together, His-361, -366 and -541 may play important roles for iron-binding in catalytically active 12-lipoxygenase. Since a high homology of amino acid sequence was known between porcine leukocyte 12-lipoxygenase and mammalian 15-lipoxygenases, we attempted to convert the 12-lipoxygenase to a 15-lipoxygenase. A double mutation of Val-418 and -419 to Ile and Met increased the ratio of 15- and 12-lipoxygenase activities from 0.1 to 5.7.

Molecular cloning and expression of human arachidonate 12-lipoxygenase

The cDNA for a 12-lipoxygenase was isolated from cDNA library of human erythroleukemia cells. The cDNA had an open reading frame encoding 663 amino acids with a calculated molecular weight of 75,513. The deduced amino acid sequence of human 12-lipoxygenase exhibited 41.5%, 65.3% and 65.4% identity with human 5-lipoxygenase, human 15-lipoxygenase and porcine 12-lipoxygenase, respectively. Blot hybridization analysis of RNA from human erythroleukemia cells demonstrated a single species (3.1 kb) of mRNA with the cDNA probe for 12-lipoxygenase of these cells, but not with the cDNA for porcine leukocyte enzyme. The cytosol of Escherichia coli transformed with a recombinant pUC19 plasmid oxygenated the position 12 of arachidonic acid.

Partial purification and characterization of arachidonate 12-lipoxygenase from rat lung

Incubation of rat lung cytosol with arachidonic acid produced 12-hydroxy-5,8,10,14-eicosatetraenoic acid as a major product, which was identified by gas chromatography-mass spectrometry. By ammonium sulfate fractionation and DEAE-cellulose chromatography the arachidonate 12-lipoxygenase was purified about 30-fold from the rat lung cytosol. The partially purified enzyme was mostly free of the glutathione peroxidase activity and transformed arachidonic acid to its 12-hydroperoxide. 5,8,11,14,17-Eicosapentaenoic acid was also an active substrate, and the oxygenation at C-12 was confirmed by mass spectrometry. A significant amount of 12-lipoxygenase activity was also found in the microsomes and other particulate fractions.

The transcriptional regulation of human arachidonate 12-lipoxygenase gene by NFκB/Rel

As examined by the luciferase assay, a negative regulatory region including the NFκB motif was found in the 5′‐flanking region of the 12‐lipoxygenase gene in human erythroleukemia cells. The negative control was abolished by a site‐specific mutation of the NFκB motif. Probes including the NFκB region gave positive bands upon a gel‐shift assay. The bands were supershifted by antibodies for NFκB p50, NFκB p65 and c‐Rel, and were lost by a NFκB competitor DNA. Furthermore, the NFκB sequence was protected in DNase I footprinting. Thus, two kinds of heterodimer (p50 and p65; p50 and c‐Rel) seemed to control the over‐expression of the human 12‐lipoxygenase gene.

Immunoaffinity purification and cDNA cloning of human platelet prostaglandin endoperoxide synthase (cyclooxygenase).

The cDNA for prostaglandin endoperoxide synthase (cyclooxygenase) was cloned from human platelets by the polymerase chain reaction amplification method, and the primary structure of the enzyme was deduced from the nucleotide sequence. The enzyme was composed of 599 amino acids including 23-amino acid signal sequence, and the calculated molecular weight of the mature protein was 65,995. The enzyme was immunoaffinity-purified from human platelets. The N-terminal amino acid sequence determined by Edman degradation was Ala-Asp-Pro-Gly-Ala-Pro-Thr-Pro-, and the result confirmed the primary structure of the enzyme, which was deduced from the cDNA sequence.

Arachidonate 12-lipoxygenase of rat pineal glands : catalytic properties and primary structure deduced from its cDNA

When a crude extract of rat pineal glands (the 1000 x g supernatant of a homogenate) was incubated with arachidonic acid, 12-hydroxy-5,8,10,14-eicosatetraenoic acid was found as a major product. The 12-lipoxygenase of rat pineal gland also reacted with linoleic and alpha-linolenic acids at 35% and 101% the rate of arachidonate 12-oxygenation, respectively. Upon Western blot analysis using polyclonal antibody against porcine leukocyte 12-lipoxygenase, the cytosol fraction of rat pineal gland showed a positive band with a molecular weight of approx. 74 kDa. A full-length cDNA for this enzyme was cloned from a cDNA library of rat pineal gland and the identity of the 12-lipoxygenase cDNA was confirmed by its expression in E. coli. The amino acid sequence of the enzyme was deduced from the nucleotide sequence of the cDNA, encoding 663 amino acids with a calculated molecular weight of 75,305. The enzyme showed 72% identity of amino acid sequence with porcine leukocyte 12-lipoxygenase and 73% with bovine tracheal 12-lipoxygenase, but only 59% with human platelet 12-lipoxygenase. Taken together, the high reactivity with C-18 fatty acids, the immunoreactivity and the amino acid homology data indicate that the rat pineal 12-lipoxygenase is more closely related to leukocyte 12-lipoxygenase than to platelet 12-lipoxygenase. Upon RNA blot analysis, by far the highest content of 12-lipoxygenase mRNA was observed in the pineal gland and negligible amounts of mRNA were detected in other parts of the brain. The predominant presence of 12-lipoxygenase mRNA in pineal gland was confirmed by in situ hybridization of rat brain. Significant amounts of 12-lipoxygenase mRNA were also detected in rat spleen, aorta, lung and leukocytes.

Arachidonate 5-lipoxygenase and its new inhibitors

The 5-lipoxygenases of guinea pig peritoneal polymorphonuclear leukocytes and of rat basophilic leukemia cells have been solubilized, purified partially by affinity chromatography, and shown to convert arachidonic acid principally to 5-hydroperoxy-6,8,11,14- eicosatetraenoic acid. The activity of both 5-lipoxygenases is calcium dependent and enhanced by adenosine triphosphate and other nucleotides in the presence of calcium ion. Both 5-lipoxygenase activity and leukotriene generation by sensitized guinea pig lung tissue challenged with antigen were suppressed substantially by specific benzoquinone and flavonoid inhibitors. The in vivo significance of the findings will be explored with potent and selective lipoxygenase inhibitors, which are delineated in the 5-lipoxygenase model systems.