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  • br Materials and methods br Results br

    2023-01-28


    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 Xylazine HCl 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. The phylogenetic relationship demonstrates that both bovine and porcine 12/15-LO is more related to the human enzyme than the rabbit enzyme, despite the fact that the rabbit reticulocyte enzyme is a 15-lipoxygenase. The rabbit is one single species that expresses a 12/15 LO with 15-LO activity while in its most related species the corresponding enzyme has 12-LO activity. In fact, not even all primates express an enzyme with 15-LO activity. This seems to be restricted to the hominids which are the members of the biological family Hominidae including humans, chimpanzees, gorillas and orang-utans. If the restricted expression of a 15-LO-1 enzyme in hominids can be coupled to formation of eoxins or certain airway inflammatory diseases, such as asthma, in these species remains to be investigated. The expression of the second form of 15-LO, i.e. 15-LO-2, in rhesus and orang-utan has in contrast to human cells [35], [36], so far not been described. The relevance for the difference between man and macaca in using anandamide as a substrate is presently unclear but it might indicate differences in metabolism of endocannabinoids in these two species.