br Conflict of interest statement

Conflict of interest statement
Acknowledgement
Introduction Lipoxygenases (LOs) are enzymes catalyzing the positional as well as stereo-specific introduction of molecular oxygen into 1,4-pentadiene structures found in unsaturated fatty acids such as arachidonic L-a-Hydroxyglutaric acid disodium salt or linoleic acid [1], [2], [3]. The positional specificity of the introduction of molecular oxygen in arachidonic acid is commonly used to classify lipoxygenases. Thus, 5-LO introduces oxygen at carbon 5 of arachidonic acid and 12-LO at carbon 12. Lipoxygenases are distributed throughout the animal, plant and fungi kingdom as well as in several prokaryotes [4], [5], [6]. The mammalian 15-LO (EC 1.13.11.33) was first purified and characterized 1975 [7]. Later on it was demonstrated that the enzyme showed a fairly restricted expression pattern with the airway epithelium [8], eosinophils [9], reticulocytes [7], macrophages [10], dendritic cells [11], human mast cells [12] and Hodgkin lymphoma [13] as being the most prominent sites of expression. It has also been shown that the expression can be regulated by inflammatory cytokines such as interleukin-4 (IL-4) and IL-13 [8], [10], [11], [13]. The regulation of 15-LO-1 by pro-inflammatory cytokines is in line with the role of 15-LO-1 in the formation of pro-inflammatory mediators such as the eoxins [14]. These potent mediators have been described to be formed in cells involved in airway inflammation such as eosinophils and mast cells [14]. Several studies have demonstrated increased expression and activity of 15-LO-1 in the bronchial mucosa of patients with asthma compared with control subjects [15], [16]. Furthermore, mice deficient of 12/15-LO had an attenuated allergic airway inflammation compared to wild-type controls [17], [18]. On the other hand, the enzyme can also under certain conditions be involved in the formation of lipoxins which possess anti-inflammatory actions [19]. A second 15-LO was discovered in 1997 and it is hence named 15-LO-2 whereas the first reported 15-lipoxygenase is referred to as 15-LO-1. The 15-LO-1 has so far only been found in humans and rabbit reticulocytes [3], [20] and the rabbit 15-LO-1 protein demonstrate 81% identity to the human 15-LO-1. However, rabbit monocytes express an enzyme with mainly 12-LO activity [20], [21]. The high degree of sequence conservation between these two rabbit enzymes (more than 99%) indicates that these genes might originate from gene duplication. The lipoxygenase activity of 12/15-LO in other species is thought to be an enzyme with primarily 12-activity, earlier named leukocyte type 12-lipoxygenase. The crystal structure of rabbit 15-LO-1 enzyme was solved and it was demonstrated that the enzyme is composed of an N-terminal β-barrel, similar to a domain in mammalian lipases [22]. The substrate binding site was suggested to be a hydrophobic pocket in which the fatty acid is docked with the methyl end extending down into the pocket. The carboxyl group of the fatty acid functions to tether and position the fatty acid by binding to Arg402. The hydrophobic pocket is defined at its base by the side chains of Phe353, Met419, Ile418 and Ile593. Mutagenesis studies have demonstrated that variation of the size of the hydrophobic pocket affects the positional specificity of the enzyme [23], [24], [25]. Thus, enlargement of the pocket of 15-LO-1 increased the amount of 12-lipoxygenase products. Only a few reports describe the formation of lipoxygenase products in cells or tissues from monkeys. Liminga et al. [26] demonstrated the formation of both 12-HETE and 15-HETE in monkey corneal epithelium and Kulkarni et al. [27] demonstrated the formation of 5-HETE and 12-HETE in both cynomolgus and rhesus monkey . Smith et al. [28] examined the expression of LOs in myometrium, cervix, deciduas and chorion of pregnant baboons. Some reports describe the effects of LO-inhibitors in different monkey species, indicating the existence of a 5-LO [29], [30]. None of these reports clarify the nature of the lipoxygenase involved in the formation of 12-hydroxyeicosa-5E,8Z,11Z,14Z-tetraenoic acid (12-HETE) or 15-hydroxyeicosa-5Z,8Z,11Z,13E-tetraenoic acid (15-HETE). Since the ortholog to human 15-LO-1 in animals, except rabbit reticulocytes, possesses mainly 12-LO activity we raised the question when the enzyme during evolution switched from primarily 12-LO activity to primarily 15-LO activity. Therefore, we set out to identify and characterize the 12/15-LO expressed in two non-human primate species.