Mammalian arachidonate 15-lipoxygenases: Structure, function, and biological implications

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Abstract

Lipoxygenases (LOXs) constitute a heterogeneous family of lipid peroxidizing enzymes capable of oxygenating polyunsaturated fatty acids to their corresponding hydroperoxy derivatives. In mammals, LOXs are classified with respect to their positional specificity of arachidonic acid oxygenation into 5-, 8-, 12-, and 15-LOXs. Arachidonate 15-LOXs may be sub-classified into a reticulocyte-type (type-1) and an epidermis-type (type-2) enzyme. Since the leukocyte-type 12-LOXs are very similar to the reticulocyte-type 15-LOXs, these enzymes are designated 12/15-LOXs. Several LOX isoforms, in particular the reticulocyte-type 15-LOX and the human 5-LOX, are well characterized with respect to their structural and functional properties On the other hand, the biological role of most LOX-isozymes including the reticulocyte-type 15-LOC is far from clear. This review is intended to summarize the recent developments in 15-LOX research with particular emphasis to molecular enzymology and regulation of gene expression. In addition, the major hypotheses on the physiological and patho-physiological roles of 15-LOXs will be discussed briefly.

Introduction

15-Lipoxygenases (LOXs) are lipid peroxidizing enzymes that catalyze the stereoselective introduction of molecular dioxygen at carbon 15 (C-15) of arachidonic acid [1], [2]. With linoleic acid as substrate, oxygen is introduced at C-13. The primary products of the 15-LOX reaction are n-6 hydroperoxy fatty acids containing a Z,E-conjugated diene system. In cellular systems, the fatty acid hydroperoxides are rapidly reduced to the corresponding hydroxy derivatives. LOXs have been described in plants [3], [4] and in the animal kingdom [1], [2]. They can also be found in lower marine organisms, such as algae, sea urchin, star fish, surf clam, and corals [5], [6], [7], [8], as well as in fungi [9], [10]. Most recently [11], LOX sequences have also been detected in bacteria (P. aeruginosa, accession no. AE004547; S. cellulosum, acession no. AX024393) and it may be of particular interest that the bacterial LOXs, which have not been characterized with respect to their positional specificity, appear to be more closely related to the human enzymes than to plant LOXs [11]. This unusual phylogenetic relatedness rises the question of whether these enzymes are introduced into bacteria by a horizontal transfer event. Despite this heterogeneity, most of our knowledge on the enzymology and structural biology of LOXs originates from studies on the soybean LOX-1 that was discovered some 60 years ago. This enzyme was characterized in detail with respect to its protein chemical and enzymatic properties.

For a long time, it was believed that LOXs in general and 15-LOX in particular might not occur in animal tissues. However, in 1975, a 15-LOX was described in rabbit reticulocytes, which was capable of oxidizing phospholipids and biomembranes [12]. This enzyme was purified to homogeneity from rabbit immature red blood cells and was well characterized [13]. In 1988, a similar enzyme was purified from human eosinophils [14] and comparison of the enzymatic properties led to the conclusion that this enzyme was the human orthologe of the rabbit 15-LOX. The search for reticulocyte-type 15-LOXs in other mammalian species remained unsuccessful until now. However, in mice [15], pigs [16], and rats [17], a leukocyte-type 12-LOX was identified and several lines of experimental evidence suggest that these enzymes may be considered the functional equivalent of the reticulocyte-type 15-LOX in these species. (i) The human and rabbit reticulocyte-type 15-LOXs share a high degree of phylogenetic relatedness with the leukocyte-type 12-LOXs from pig, rat and mouse [2]. In contrast, there is only a low degree of sequence conservation between the leukocyte-type 12-LOXs and other 12-LOX isoforms. (ii) The leukocyte-type 12-LOXs and the reticulocyte-type 15-LOXs have similar enzymatic properties (substrate specificity, reaction kinetic). (iii) Both, the human reticulocyte-type 15-LOX and the murine leukocyte-type 12-LOX share similar mechanisms in cytokine-dependent regulation of gene expression [18], [19], [20]. (iv) Completion of the human genome project indicated that there is no separate gene encoding a leukocyte-type 12-LOX in humans. A search of the publicly available murine genome databases neither revealed any evidence for a reticulocyte-type 15-LOX gene. However, in rabbit separate genes for both, a reticulocyte-type 15-LOX and a leukocyte-type 12-LOX, appear to exist. The high degree (>99%) of sequence conservation between these isoforms suggests that they may originate from gene duplication [21].

In 1997, a second type of 15-LOX was cloned from human hair roots [22]. This enzyme, which was named epidermis-type 15-LOX or 15-LOX-2, only shares a low degree of sequence homology with the reticulocyte-type 15-LOX and strongly differs from the latter isoform with respect to its protein chemical and enzymatic properties. It is expressed in skin, prostate, lung, and cornea [22], but PCR screening of 16 other tissues including peripheral blood leukocytes revealed negative results. The cDNA encodes for 676 amino acids and the predicted molecular weight was 76 kDa. When expressed in HEK 293 cells, the enzyme converts arachidonic acid exclusively to 15S-HPETE. In contrast to the reticulocyte-type 15-LOX, linoleic acid is less well metabolized. Although the biological role of this LOX isoform is far from clear, it may be related to skin functionality and prostate cancer. Interestingly, the orthologe murine enzyme is an arachidonate 8-LOX [23], and a combined strategy of site directed mutagenesis and chimera formation indicated the structural basis for the difference in the positional specificity of the two isoforms [24].

This review is intended to summarize and critically review the recent developments in 15-LOX research. Since the volume contains separate chapters on other LOX isoforms, this paper will concentrate on the reticulocyte-type 15-LOX. The leukocyte-type 12-LOXs and the human epidermis-type 15-LOX will be discussed elsewhere (I-3, I-4). In the plant kingdom, several arachidonate 15-LOXs have also been identified, but because of space limitations it is impossible to include plant enzymes in the discussion.

Section snippets

Enzymatic properties

As other LOXs, the reticulocyte-type 15-LOX is a single polypeptide (MW=75 kDa) folded into a two-domain structure (Fig. 1). It contains one non-heme iron per mole enzyme and four histidines (H361, H366, H541, H545) and the C-terminal isoleucine constitute the protein iron ligands (see Section 2.2).

The reticulocyte-type 15-LOXs are characterized by a rather broad substrate specificity [13], [25]. All major naturally occurring polyenoic fatty acids, such as (5Z,8Z,11Z,14Z

Regulation of cellular LOX activity

The intracellular 15-LOX activity is strongly regulated [51] and elements of transcriptional, translational, and post-translational regulation have been described in various cellular systems (Fig. 4).

Biological role of 12/15-LOXs

According to the conventional view of the arachidonic acid cascade bioactive eicosanoids are formed from free arachidonic acid upon cell stimulation [80]. Because of their enzymatic properties, mammalian 15-LOXs may exhibit biological activities outside the arachidonic acid cascade. Liberation of arachidonic acid from the phospholipid stores is not an absolute precondition for the enzymatic activity of this LOX isoform. The mechanism of the intracellular formation of free hydroxy fatty acids

Perspectives

After the enormous progress in molecular biology and completion of the human and murine genome projects, the sequences of all LOX genes present in these medically most relevant animal species are known. As in other areas of biomedical research, functional genomics will now become a major topic in LOX-research. Knockout mice for the leukocyte-type 12-LOX, the platelet-type 12-LOX and the 5-LOX are commercially available and are expected to be used more intensively to study LOX function.

Acknowledgements

Financial aid of Deutsche Forschungsgemeinschaft (Ku 961/6-1 and Ku 961/7-1) is acknowledged.

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