Human phospholipase A₂'s (PLA₂) constitute a superfamily of enzymes that hydrolyze the sn-2 acyl-chain of glycerophospholipids, producing lysophospholipids and free fatty acids. Each PLA₂ enzyme type contributes to specific biological functions based on its expression, subcellular localization, and substrate specificity. Among the PLA₂ superfamily, the cytosolic cPLA₂ enzymes, calcium-independent iPLA₂ enzymes, and secreted sPLA₂ enzymes are implicated in many diseases, but a central issue is the preference for double-bond positions in polyunsaturated fatty acids (PUFAs) occupying the sn-2 position of membrane phospholipids. We demonstrate that each PLA₂ has a unique preference between the specific omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and the omega-6 arachidonic acid (AA), which are the precursors of most pro-inflammatory and anti-inflammatory or resolving eicosanoids and related oxylipins. Surprisingly, we discovered that human cPLA₂ selectively prefers AA, whereas iPLA₂ prefers EPA, and sPLA₂ prefers DHA as substrate. We determined the optimal binding of each phospholipid substrate in the active site of each PLA₂ to explain these specificities. To investigate this, we utilized recently-developed lipidomics-based LC-MS/MS and GC/MS assays to determine the sn-2 acyl chain specificity in mixtures of phospholipids. We performed μs timescale molecular dynamics (MD) simulations to reveal unique active site properties, especially how the precise hydrophobic cavity accommodation of the sn-2 acyl chain contributes to the stability of substrate binding and the specificity of each PLA₂ for AA, EPA, or DHA. This study provides the first comprehensive picture of the unique substrate selectivity of each PLA₂ for omega-3 and omega-6 fatty acids.

中文翻译：

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Omega-3 与 Omega-6 脂肪酸的可用性由磷脂酶 A2 的疏水位点几何形状控制。

人磷脂酶A ₂ (PLA ₂ ) 构成了一个酶超家族，可水解甘油磷脂的sn-2 酰基链，产生溶血磷脂和游离脂肪酸。每种 PLA ₂酶类型都根据其表达、亚细胞定位和底物特异性发挥特定的生物学功能。在 PLA ₂超家族中，胞质 cPLA ₂酶、钙非依赖性 iPLA ₂酶和分泌型 sPLA ₂酶与许多疾病有关，但一个核心问题是多不饱和脂肪酸 (PUFA) 中双键位置的偏好占据膜磷脂的 sn-2 位置。我们证明，每个 PLA ₂在特定的 omega-3 脂肪酸二十碳五烯酸 (EPA) 和二十二碳六烯酸 (DHA) 以及 omega-6 花生四烯酸 (AA) 之间具有独特的偏好，它们是大多数促炎和炎症的前体。抗炎或缓解类二十烷酸和相关的氧脂素。令人惊讶的是，我们发现人cPLA ₂选择性地偏好AA，而iPLA ₂偏好EPA，而sPLA ₂偏好DHA 作为底物。_{我们确定了每个 PLA 2}活性位点中每种磷脂底物的最佳结合，以解释这些特异性。为了研究这一点，我们利用最近开发的基于脂质组学的 LC-MS/MS 和 GC/MS 测定法来确定磷脂混合物中的 sn-2 酰基链特异性。我们进行了 μs 时标分子动力学 (MD) 模拟，以揭示独特的活性位点特性，特别是 sn-2 酰基链的精确疏水腔调节如何有助于底物结合的稳定性以及每个 PLA ₂对 AA、EPA、或DHA。这项研究首次全面展示了每种 PLA ₂对 omega-3 和 omega-6 脂肪酸的独特底物选择性。