Department of Chemistry and Biochemistry, University of California Santa Cruz, Santa Cruz, CA 95064.
Department of Pharmaceutical Chemistry, School of Pharmacy, University of California San Francisco, San Francisco, CA 94143.
J Lipid Res. 2020 Jul;61(7):1087-1103. doi: 10.1194/jlr.RA120000777. Epub 2020 May 13.
The two oxylipins 7S,14S-dihydroxydocosahexaenoic acid (diHDHA) and 7S,17S-diHDHA [resolvin D5 (RvD5)] have been found in macrophages and infectious inflammatory exudates and are believed to function as specialized pro-resolving mediators (SPMs). Their biosynthesis is thought to proceed through sequential oxidations of DHA by lipoxygenase (LOX) enzymes, specifically, by human 5-LOX (h5-LOX) first to 7(S)-hydroxy-4Z,8E,10Z,13Z,16Z,19Z-DHA (7S-HDHA), followed by human platelet 12-LOX (h12-LOX) to form 7(S),14(S)-dihydroxy-4Z,8E,10Z,12E,16Z,19Z-DHA (7S,14S-diHDHA) or human reticulocyte 15-LOX-1 (h15-LOX-1) to form RvD5. In this work, we determined that oxidation of 7(S)-hydroperoxy-4Z,8E,10Z,13Z,16Z,19Z-DHA to 7S,14S-diHDHA is performed with similar kinetics by either h12-LOX or h15-LOX-1. The oxidation at C14 of DHA by h12-LOX was expected, but the noncanonical reaction of h15-LOX-1 to make over 80% 7S,14S-diHDHA was larger than expected. Results of computer modeling suggested that the alcohol on C7 of 7S-HDHA hydrogen bonds with the backbone carbonyl of Ile399, forcing the hydrogen abstraction from C12 to oxygenate on C14 but not C17. This result raised questions regarding the synthesis of RvD5. Strikingly, we found that h15-LOX-2 oxygenates 7S-HDHA almost exclusively at C17, forming RvD5 with faster kinetics than does h15-LOX-1. The presence of h15-LOX-2 in neutrophils and macrophages suggests that it may have a greater role in biosynthesizing SPMs than previously thought. We also determined that the reactions of h5-LOX with 14(S)-hydroperoxy-4Z,7Z,10Z,12E,16Z,19Z-DHA and 17(S)-hydroperoxy-4Z,7Z,10Z,13Z,15E,19Z-DHA are kinetically slow compared with DHA, suggesting that these reactions may be minor biosynthetic routes in vivo. Additionally, we show that 7S,14S-diHDHA and RvD5 have anti-aggregation properties with platelets at low micromolar potencies, which could directly regulate clot resolution.
两种氧代二十碳六烯酸 7S,14S-二羟基二十二碳六烯酸（diHDHA）和 7S,17S-二 HDHA [分辨率 D5（RvD5）] 在巨噬细胞和感染性炎症渗出物中被发现，被认为是具有特殊抗炎作用的介质（SPMs）。它们的生物合成被认为是通过脂氧合酶（LOX）酶对 DHA 的顺序氧化进行的，具体来说，首先是人类 5-LOX（h5-LOX）将 7S-羟基-4Z,8E,10Z,13Z,16Z,19Z-DHA（7S-HDHA）氧化为 7（S）-羟基-4Z,8E,10Z,12E,16Z,19Z-DHA（7S-HDHA），然后是人类血小板 12-LOX（h12-LOX）形成 7S,14S-二羟基-4Z,8E,10Z,12E,16Z,19Z-DHA（7S,14S-二 HDHA），或者人类网织红细胞 15-LOX-1（h15-LOX-1）形成 RvD5。在这项工作中，我们确定了 7（S）-过氧-4Z,8E,10Z,13Z,16Z,19Z-DHA 到 7S,14S-二 HDHA 的氧化由 h12-LOX 或 h15-LOX-1 以相似的动力学进行。h12-LOX 对 DHA 进行 C14 氧化是预期的，但 h15-LOX-1 的非典型反应生成超过 80%的 7S,14S-二 HDHA 比预期的要大。计算机建模的结果表明，7S-HDHA 上 C7 的醇与 Ile399 的骨架羰基形成氢键，迫使 C12 上的氢从 C17 转移到 C14 上，但不转移到 C17 上。这一结果引发了关于 RvD5 合成的问题。引人注目的是，我们发现 h15-LOX-2 几乎完全在 C17 上氧化 7S-HDHA，形成 RvD5 的动力学比 h15-LOX-1 更快。h15-LOX-2 在中性粒细胞和巨噬细胞中的存在表明，它在合成 SPMs 方面可能比以前认为的更重要。我们还确定了 h5-LOX 与 14（S）-过氧-4Z,7Z,10Z,12E,16Z,19Z-DHA 和 17（S）-过氧-4Z,7Z,10Z,13Z,15E,19Z-DHA 的反应与 DHA 相比动力学缓慢，这表明这些反应可能是体内次要的生物合成途径。此外，我们还表明，7S,14S-二 HDHA 和 RvD5 以低微摩尔效力与血小板具有抗聚集特性，这可能直接调节血栓溶解。
