Center for Free Radical and Antioxidant Health, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Biochemistry. 2012 Dec 4;51(48):9736-50. doi: 10.1021/bi301024e. Epub 2012 Nov 19.
Ca(2+)-independent lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) is a member of the phospholipase A(2) superfamily with a distinguishing characteristic of high specificity for oxidatively modified sn-2 fatty acid residues in phospholipids that has been especially well characterized for peroxidized species of phosphatidylcholines (PC). The ability of Lp-PLA(2) to hydrolyze peroxidized species of phosphatidylserine (PS), acting as a recognition signal for clearance of apoptotic cells by professional phagocytes, as well as the products of the reaction has not been investigated. We performed liquid chromatography-electrospray ionization mass spectrometry-based structural characterization of oxygenated, hydrolyzed molecular species of PS-containing linoleic acid in either the sn-2 position (C(18:0)/C(18:2)) or in both sn-1 and sn-2 positions (C(18:2)/C(18:2)), formed in the cytochrome c- and H(2)O(2)-driven enzymatic oxidation reaction. Cytochrome c has been chosen as a catalyst of peroxidation reactions because of its likely involvement in PS oxidation in apoptotic cells. We found that Lp-PLA(2) catalyzed the hydrolysis of both nontruncated and truncated (oxidatively fragmented) species of oxidized PS species, albeit with different efficiencies, and performed detailed characterization of the major reaction products: oxygenated derivatives of linoleic acid as well as nonoxygenated and oxygenated species of lyso-PS. Among linoleic acid products, derivatives oxygenated at the C(9) position, including 9-hydroxyoctadecadienoic acid (9-HODE), a potent ligand of G protein-coupled receptor G2A, were the most abundant. Computer modeling of interactions of Lp-PLA(2) with different PS-oxidized species indicated that they are able to bind in the proximity (<5 Å) of Ser273 and His351 of the catalytic triad. For 9-hydroxy and 9-hydroperoxy derivatives of oxidized PS, the sn-2 ester bond was positioned very close (<3 Å) to the Ser273 residue, a nucleophile directly attacking the sn-2 bond, thus favoring the hydrolysis reaction. We suggest that oxidatively modified free fatty acids and lyso-PS species generated by Lp-PLA(2) may represent important signals facilitating and regulating the execution of apoptotic and phagocytosis programs essential for the control of inflammation.
钙（Ca(2+)）非依赖型脂蛋白相关磷脂酶 A2（Lp-PLA2）是磷脂酶 A2 超家族的成员，其特征为对氧化修饰的 sn-2 脂肪酸残基具有高度特异性，这种特异性在过氧化磷脂酰胆碱（PC）中得到了很好的描述。Lp-PLA2 能够水解过氧化的磷脂酰丝氨酸（PS），作为专业吞噬细胞清除凋亡细胞的识别信号，以及反应产物尚未被研究。我们使用液相色谱-电喷雾电离质谱法对 sn-2 位（C(18:0)/C(18:2)）或 sn-1 和 sn-2 位（C(18:2)/C(18:2)）含有亚油酸的含氧化合、水解的 PS 分子种类进行了结构特征描述，这些分子是在细胞色素 c 和 H2O2 驱动的酶促氧化反应中形成的。细胞色素 c 被选为过氧化物反应的催化剂，因为它可能参与凋亡细胞中 PS 的氧化。我们发现，Lp-PLA2 催化非截断和截断（氧化片段化）的 PS 氧化产物的水解，尽管效率不同，并对主要反应产物进行了详细的特征描述：亚油酸的含氧衍生物以及非氧合和含氧合的溶菌 PS 衍生物。在亚油酸产物中，C(9)位氧化的衍生物，包括 9-羟基十八碳二烯酸（9-HODE），一种 G 蛋白偶联受体 G2A 的有效配体，是最丰富的。Lp-PLA2 与不同 PS 氧化产物相互作用的计算机建模表明，它们能够在靠近（<5 Å）催化三联体的 Ser273 和 His351 附近结合。对于氧化 PS 的 9-羟基和 9-过氧基衍生物，sn-2 酯键与 Ser273 残基非常接近（<3 Å），亲核试剂直接攻击 sn-2 键，从而有利于水解反应。我们认为，Lp-PLA2 产生的氧化修饰的游离脂肪酸和溶菌 PS 衍生物可能代表重要的信号，促进和调节凋亡和吞噬程序的执行，这对于炎症的控制至关重要。
