William E. Boeglin

The Structure of Human 5-Lipoxygenase

Substitution of a destabilizing sequence has allowed crystallization of a key enzyme of the inflammatory response. The synthesis of both proinflammatory leukotrienes and anti-inflammatory lipoxins requires the enzyme 5-lipoxygenase (5-LOX). 5-LOX activity is short-lived, apparently in part because of an intrinsic instability of the enzyme. We identified a 5-LOX–specific destabilizing sequence that is involved in orienting the carboxyl terminus, which binds the catalytic iron. Here, we report the crystal structure at 2.4 angstrom resolution of human 5-LOX stabilized by replacement of this sequence.

15-Lipoxygenase Metabolism of 2-Arachidonylglycerol GENERATION OF A PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR α AGONIST

The recent demonstrations that cyclooxygenase-2 and leukocyte-type 12-lipoxygenase (LOX) efficiently oxygenate 2-arachidonylglycerol (2-AG) prompted an investigation into related oxygenases capable of metabolizing this endogenous cannabinoid receptor ligand. We evaluated the ability of six LOXs to catalyze the hydroperoxidation of 2-AG. Soybean 15-LOX, rabbit reticulocyte 15-LOX, human 15-LOX-1, and human 15-LOX-2 oxygenate 2-AG, providing 15(S)-hydroperoxyeicosatetraenoic acid glyceryl ester. In contrast, potato and human 5-LOXs do not efficiently metabolize this endocannabinoid. Among a series of structurally related arachidonyl esters, arachidonylglycerols serve as the preferred substrates for 15-LOXs. Steady-state kinetic analysis demonstrates that both 15-LOX-1 and 15-LOX-2 oxygenate 2-AG comparably or preferably to arachidonic acid. Furthermore, 2-AG treatment of COS-7 cells transiently transfected with human 15-LOX expression vectors or normal human epidermal keratinocytes results in the production and extracellular release of 15-hydroxyeicosatetraenoic acid glyceryl ester (15-HETE-G), establishing that lipoxygenase metabolism of 2-AG occurs in an eukaryotic cellular environment. Investigations into the potential biological actions of 15-HETE-G indicate that this lipid, in contrast to its free-acid counterpart, acts as a peroxisome proliferator-activated receptor α agonist. The results demonstrate that 15-LOXs are capable of acting on 2-AG to provide 15-HETE-G and elucidate a potential role for endocannabinoid oxygenation in the generation of peroxisome proliferator-activated receptor α agonists.

15-Lipoxygenase-2 (15-LOX-2) Is Expressed in Benign Prostatic Epithelium and Reduced in Prostate Adenocarcinoma

Human 15S-lipoxygenase-2 (15-LOX-2) is a recently identified lipoxygenase that has approximately 40% sequence identity to the known human 5S-, 12S-, and 15S-lipoxygenases. 15-LOX-2 has a limited tissue distribution, with mRNA detected in prostate, lung, skin, and cornea, but not in numerous other tissues, including peripheral blood leukocytes. In the current study, we have characterized the distribution of 15-LOX-2 in the human prostate by immunohistochemistry, demonstrated the ability of benign prostate tissue to form 15S-hydroxyeicosatetraenoic acid (15S-HETE) from exogenous arachidonic acid (AA), and begun characterizing possible alterations in 15-LOX-2 in prostate adenocarcinoma. Incubation of benign prostate tissue with [14C]AA resulted in formation of [14C]15-HETE, as determined by reverse- and straight-phase high-performance liquid chromatography. 15-HETE was the major AA metabolite formed. By immunohistochemistry, 15-LOX-2 is located in secretory cells of peripheral zone glands and large prostatic ducts and somewhat less uniformly in apical cells of transition and central zone glands. 15-LOX-2 was not detected in the basal cell layer, stroma, ejaculatory ducts, seminal vesicles, or transitional epithelium. Immunostaining of 18 radical prostatectomy specimens showed a loss of 15-LOX-2 in the majority of prostate adenocarcinomas; 14 of 18 cases showed loss of 15-LOX-2 in >25% of the tumor (mean, 74.9% negative for 15-LOX-2; range, 38.9% to 100%). Incubation of paired pure benign and pure malignant prostate tissue from the same radical prostatectomies showed that 15-HETE formation was markedly reduced (>90%) or undetectable in incubations of prostate adenocarcinoma. 15-LOX-2 is a novel human lipoxygenase with a limited tissue distribution that is strongly expressed in benign prostate glandular epithelium and lost to a variable degree in the majority of prostate adenocarcinomas.

Molecular Cloning and Functional Expression of a Phorbol Ester-inducible 8S-Lipoxygenase from Mouse Skin

One of the effects of topical application of phorbol ester to mouse skin is the induction of an 8S-lipoxygenase in association with the inflammatory response. Here we report the molecular cloning and characterization of this enzyme. The cDNA was isolated by polymerase chain reaction from mouse epidermis and subsequently from a mouse epidermal cDNA library. The cDNA encodes a protein of 677 amino acids with a calculated molecular mass of 76 kDa. The amino acid sequence has 78% identity to a 15S-lipoxygenase cloned recently from human skin and approximately 40% identity to other mammalian lipoxygenases. When expressed in vaccinia virus-infected Hela cells, the mouse enzyme converts arachidonic acid exclusively to 8S-hydroperoxyeicosatetraenoic acid while linoleic acid is converted to 9S-hydroperoxy-linoleic acid in lower efficiency. Phorbol ester treatment of mouse skin is associated with strong induction of 8S-lipoxygenase mRNA and protein. By Northern analysis, expression of 8S-lipoxygenase mRNA was also detected in brain. Immunohistochemical analysis of phorbol ester-treated mouse skin showed the strongest reaction to 8S-lipoxygenase in the differentiated epidermal layer, the stratum granulosum. The inducibility may be a characteristic feature of the mouse 8S-lipoxygenase and its human 15S-lipoxygenase homologue.

Lipoxygenases Mediate the Effect of Essential Fatty Acid in Skin Barrier Formation: A PROPOSED ROLE IN RELEASING OMEGA-HYDROXYCERAMIDE FOR CONSTRUCTION OF THE CORNEOCYTE LIPID ENVELOPE*♦

A barrier to water loss is vital to maintaining life on dry land. Formation of the mammalian skin barrier requires both the essential fatty acid linoleate and the two lipoxygenases 12R-lipoxygenase (12R-LOX) and epidermal lipoxygenase-3 (eLOX3), although their roles are poorly understood. Linoleate occurs in O-linoleoyl-ω-hydroxyceramide, which, after hydrolysis of the linoleate moiety, is covalently attached to protein via the free ω-hydroxyl of the ceramide, forming the corneocyte lipid envelope, a scaffold between lipid and protein that helps seal the barrier. Here we show using HPLC-UV, LC-MS, GC-MS, and 1H NMR that O-linoleoyl-ω-hydroxyceramide is oxygenated in a regio- and stereospecific fashion by the consecutive actions of 12R-LOX and eLOX3 and that these products occur naturally in pig and mouse epidermis. 12R-LOX forms 9R-hydroperoxy-linoleoyl-ω-hydroxyceramide, further converted by eLOX3 to specific epoxyalcohol (9R,10R-trans-epoxy-11E-13R-hydroxy) and 9-keto-10E,12Z esters of the ceramide; an epoxy-ketone derivative (9R,10R-trans-epoxy-11E-13-keto) is the most prominent oxidized ceramide in mouse skin. These products are absent in 12R-LOX-deficient mice, which crucially display a near total absence of protein-bound ω-hydroxyceramides and of the corneocyte lipid envelope and die shortly after birth from transepidermal water loss. We conclude that oxygenation of O-linoleoyl-ω-hydroxyceramide is required to facilitate the ester hydrolysis and allow bonding of the ω-hydroxyceramide to protein, providing a coherent explanation for the roles of multiple components in epidermal barrier function. Our study uncovers a hitherto unknown biochemical pathway in which the enzymic oxygenation of ceramides is involved in building a crucial structure of the epidermal barrier.

Identification of amino acid determinants of the positional specificity of mouse 8S-lipoxygenase and human 15S-lipoxygenase-2.

Phorbol ester-inducible mouse 8S-lipoxygenase (8-LOX) and its human homologue, 15S-lipoxygenase-2 (15-LOX-2), share 78% identity in amino acid sequences, yet there is no overlap in their positional specificities. In this study, we investigated the determinants of positional specificity using a random chimeragenesis approach in combination with site-directed mutagenesis. Exchange of the C-terminal one-third of the 8-LOX with the corresponding portion of 15-LOX-2 yielded a chimeric enzyme with exclusively 15S-lipoxygenase activity. The critical region was narrowed down to a cluster of five amino acids by expression of multiple cDNAs obtained by in situ chimeragenesis in Escherichia coli. Finally, a pair of amino acids, Tyr603 and His604, was identified as the positional determinant by site-directed mutagenesis. Mutation of both of these amino acids to the corresponding amino acids in 15-LOX-2 (Asp and Val, respectively) converted the positional specificity from 8S to 90% 15S without yielding any other by-products. Mutation of the corresponding residues in 15-LOX-2 to the 8-LOX sequence changed specificity to 50% oxygenation at C-8 for one amino acid substitution and 70% at C-8 for the double mutant. Based on the crystal structure of the reticulocyte 15-LOX, these two amino acids lie opposite the open coordination position of the catalytic iron in a likely site for substrate binding. The change from 8 to 15 specificity entails a switch in the head to tail binding of substrate. Enzymes that react with substrate “head first” (5-LOX and 8-LOX) have a bulky aromatic amino acid and a histidine in these positions, whereas lipoxygenases that accept substrates “tail first” (12-LOX and 15-LOX) have an aliphatic residue with a glutamine or aspartate. Thus, this positional determinant of the 8-LOX and 15-LOX-2 may have significance for other lipoxygenases.

The 1.85 A structure of an 8R-lipoxygenase suggests a general model for lipoxygenase product specificity.

Lipoxygenases (LOX) play pivotal roles in the biosynthesis of leukotrienes and other biologically active eicosanoids derived from arachidonic acid. A mechanistic understanding of substrate recognition, when lipoxygenases that recognize the same substrate generate different products, can be used to help guide the design of enzyme-specific inhibitors. We report here the 1.85 A resolution structure of an 8R-lipoxygenase from Plexaura homomalla, an enzyme with a sequence approximately 40% identical to that of human 5-LOX. The structure reveals a U-shaped channel, defined by invariant amino acids, that would allow substrate access to the catalytic iron. We demonstrate that mutations within the channel significantly impact enzyme activity and propose a novel model for substrate binding potentially applicable to other members of this enzyme family.

Purification and Molecular Cloning of an 8R-Lipoxygenase from the Coral Plexaura homomalla Reveal the Related Primary Structures of R- and S-Lipoxygenases

Lipoxygenases that form S configuration fatty acid hydroperoxides have been purified or cloned from plant and mammalian sources. Our objectives were to characterize one of the lipoxygenases with R stereospecificity, many of which are described in marine and freshwater invertebrates. Characterization of the primary structure of an R-specific enzyme should help provide a new perspective to consider the enzyme-substrate interactions that are the basis of the specificity of all lipoxygenases. We purified an 8R-lipoxygenase of the prostaglandin-containing coral Plexaura homomalla by cation and anion exchange chromatography. This yielded a colorless enzyme preparation, a band of ∼100 kDa on SDS-polyacrylamide gel electrophoresis, and turnover numbers of 4000 min−1 of 8R-lipoxygenase activity in peak chromatographic fractions. The full-length cDNA was cloned by PCR using peptide sequence from the purified protein and by 5′- and 3′-rapid amplification of cDNA ends. The cDNA encodes a polypeptide of 715 amino acids, including over 70 amino acids identified by peptide microsequencing. A peptide presequence of 52 amino acids is cleaved to give the mature protein of 76 kDa; the difference from the estimated size by SDS-PAGE implies a post-translational modification of the P. homomalla enzyme. All of the iron-binding histidines of S-lipoxygenases are conserved in the 8R-lipoxygenase. However, the C-terminal amino acid is a threonine, as opposed to the isoleucine that provides the carboxylate ligand to the iron in all known S-lipoxygenases. These results establish that the 8R-lipoxygenase is related in primary structure to the S-lipoxygenases. A model of the basis of R and S stereospecificity is described.

Conversion of human 5-lipoxygenase to a 15-lipoxygenase by a point mutation to mimic phosphorylation at Serine-663.

The enzyme 5‐lipoxygenase (5‐LOX) initiates biosynthesis of the proinflammatory leukotriene lipid mediators and, together with 15‐LOX, is also required for synthesis of the anti‐inflammatory lipoxins. The catalytic activity of 5‐LOX is regulated through multiple mechanisms, including Ca2+‐targeted membrane binding and phosphorylation at specific serine residues. To investigate the consequences of phosphorylation at S663, we mutated the residue to the phosphorylation mimic Asp, providing a homogenous preparation suitable for catalytic and structural studies. The S663D enzyme exhibits robust 15‐LOX activity, as determined by spectrophotometric and HPLC analyses, with only traces of 5‐LOX activity remaining; synthesis of the anti‐inflammatory lipoxin A4 from arachidonic acid is also detected. The crystal structure of the S663D mutant in the absence and presence of arachidonic acid (in the context of the previously reported Stable‐5‐LOX) reveals substantial remodeling of helices that define the active site so that the once fully encapsulated catalytic machinery is solvent accessible. Our results suggest that phosphorylation of 5‐LOX at S663 could not only down‐regulate leukotriene synthesis but also stimulate lipoxin production in inflammatory cells that do not express 15‐LOX, thus redirecting lipid mediator biosynthesis to the production of proresolving mediators of inflammation.—Gilbert, N. C., Rui, Z., Neau, D. B., Waight, M. T., Bartlett, S. G., Boeglin, W. E., Brash, A. R., Newcomer, M. E. Conversion of human 5‐lipoxygenase to a 15‐lipoxygenase by a point mutation to mimic phosphorylation at Serine‐663. FASEB J. 26, 3222–3229 (2012). www.fasebj.org

The hepoxilin connection in the epidermis

The recent convergence of genetic and biochemical evidence on the activities of lipoxygenase (LOX) enzymes has implicated the production of hepoxilin derivatives (fatty acid epoxyalcohols) in the pathways leading to formation of the water‐impermeable barrier of the outer epidermis. The enzymes 12R‐LOX and eLOX3 are mutated in a rare form of congenital ichthyosis, and, in vitro, the two enzymes function together to convert arachidonic acid to a specific hepoxilin. Taken together, these lines of evidence suggest an involvement of these enzymes and their products in skin barrier function in all normal subjects. The natural occurrence of the specific hepoxilin products, and their biological role, whether structural or signaling, remain to be defined.