The LO has so far only been found in humans

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 Magnolol sale 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.
Materials and methods
Results
Discussion We have in this study used semi-nested degenerative PCR to identify the lipoxygenases expressed in the lung tissue from M. fascicularis and report on the identification of 5- and 12/15-LO transcripts from this tissue. Although we did not find transcripts encoding other lipoxygenases, we cannot rule out the possibility that such still are present, albeit as very low-copy number transcripts. To define the ratio between different transcripts a more quantitative method such as quantitative-PCR should be used. Nevertheless, our results indicate that the 5- and 12/15-LOs were the most abundantly expressed lipoxygenases in the investigated sample. The partial sequence obtained for 12/15-LO did not provide insight into the positional specificity of the lipoxygenase. Thus, we cloned the full-length coding sequence of the 12/15-LO enzyme from rhesus monkey. Some amino acid residues are known to play a key role in the positional specificity in 12/15-LO enzymes. In the human enzyme these are Phe 352, Ile 417, Met 418 and Ile592. All the known primate 12/15-lipoxygenases has a conserved phenylalanine at position 352 and an isoleucine at 592. Most likely, the amino acid residues at position 417 and 418 will thus govern the positional specificity of the primate 12/15-lipoxygenases. This is exemplified by the activity of the rhesus and the orang-utan enzymes characterized in this paper. Interestingly, the primate 12/15-LOs show a high degree of identity with the human enzyme. Despite of this, they can easily be distinguished as enzymes with mainly 12- or 15-lipoxygenase activity. In the rat and mouse 12/15-LO, the amino acid residue corresponding to the human residue 417 are Ala and Val, respectively, and the activities of these enzymes are predominately 12-lipoxygenase activities. Both Ala and Val are smaller amino acids than Ile found in the human enzyme which is thought to facilitate the entrance of arachidonic acid into the active site, leading to hydrogen abstraction at C-10 and consequently oxygenation at C-12. With this in mind and having both the rhesus and the orang-utan sequence, we assumed that the rhesus 12/15-LO enzyme had mostly 12-LO activity whereas the orang-utan enzyme should display mostly 15-LO activity. This assumption was confirmed and thus further lending support to the theory by Kuhn et al. [24] suggesting that the amino acid at position 352 is a primary sequence determinant. If this residue is rather bulky and space filling, positions 417 and 418 will be important for the positional specificity. Furthermore, our results suggest that the concentration of the substrate can affect the ratio between 12- and 15-HETE formed by the rhesus enzyme. This is most likely due to the fact that the fatty acid is presented to the enzyme in different biophysical forms, i.e. as monomers at low concentrations, as polymers or microaggregates at intermediate concentrations or as larger secondary structures such as aggregates, micelles or lipid bilayers at higher concentrations. Indeed, the length of the fatty acid chain and the degree, type and position of unsaturation has been shown to affect the pKa of lipids by the formation of secondary structures due to van der Waals forces and interactions between the polar carboxylate groups [33], [34]. Presumably, when the substrate is in a more rigid physical state than monomers, it may not align deep enough in the active site to allow for 12-lipoxygenation. Thus, caution should be taken when interpreting positional specificity for 12/15-lipoxygenases since it can be affected by the substrate concentration.