Altered positional specificity of 15-Lipoxygenase-1 creates new pathways for the resolution of inflammation
Abstract
The oxylipins 7S,14S-diHDHA and 7S,17S-diHDHA (RvD5) have been found in macrophages exudates and are believed to function as specialized pro-resolving mediators of inflammation. Their biosynthesis has been proposed to proceed through sequential oxidations of docosahexaenoic acid (DHA) by lipoxygenase enzymes, specifically by h5-LOX performing the first oxidation to 7S-HDHA followed by h12-LOX oxidation to form 7S,14S-diHDHA or h15-LOX-1 oxidation to form RvD5. In this work, we determine that the oxidation of 7S-HpDHA to 7S,14S-diHDHA can be carried out by either h12-LOX or h15-LOX-1, with similar kinetics. The oxidation at C14 of DHA by h12-LOX was expected, but the non-canonical reaction of h15-LOX-1 to make 7S,14S-diHDHA was unexpected. This result raised questions regarding biosynthesis of RvD5. Strikingly, we find that h15-LOX-2 oxygenates 7S-HDHA almost exclusively at C17 to form RvD5, with faster kinetics than that of h15-LOX-1. We also determine that the reactions of h5-LOX with 14S-HpDHA and 17S-HpDHA are kinetically slow, suggesting these may be minor biosynthetic routes in vivo.
We determine that altered positional specificity of h15-LOX-1 extends to 5S-HETE and show that 15-LOX-2 plays a greater role in generating 5S,15S-diHETE, while h15-LOX-1 primarily generates the non-canonical product, 5S,12S-diHETE.
Previous work has indicated numerous active-site amino acids that influence regiospecificity of h15-LOX-1 with AA. We extend this work to oxylipins and show that the depth of the active site as determined by I417 and F352 strongly affects the regiospecificity with 7S-HDHA and 5S-HETE, with F352W almost entirely reversing the altered positional specificity seen in wildtype 15-LOX-1.
Our results may extend beyond the resolution of inflammation. Specifically, we show that both 7S,14S-diHDHA and RvD5 have anti-aggregation properties with platelets at low micro-molar concentrations, which could directly regulate clot resolution. 15-(S)hydroxyeicosatrienoic acid (15-HETrE), generated by reaction of the omega-6 polyunsaturated fatty acid dihomo-γ-linolenic acid (DGLA) with h15-LOX-1, is known to inhibit platelet activation through an unknown mechanism. To investigate the basis of this inhibition, human platelets were treated with 15-HETrE and platelet aggregation was assayed by various methods. 15-HETrE was shown to inhibit platelet activation through activation of PPARβ and inhibition of 12-lipoxygenase (12-LOX).