Chiou Yi-Ling, Lin Shinne-Ren, Hu Wan-Ping, Chang Long-Sen
Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung 804, Taiwan.
Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung 807, Taiwan.
Toxicon. 2014 Dec 15;92:113-22. doi: 10.1016/j.toxicon.2014.10.013. Epub 2014 Oct 17.
Phosphatidylserine (PS) externalization is a hallmark for apoptotic death of cells. Previous studies showed that Naja naja atra phospholipase A2 (NnaPLA2) and Notechis scutatus scutatus notexin induced apoptosis of human cancer cells. However, NnaPLA2 and notexin did not markedly disrupt the integrity of cellular membrane as evidenced by membrane permeability of propidium iodide. These findings reflected that the ability of NnaPLA2 and notexin to hydrolyze membrane phospholipids may be affected by PS externalization. To address that question, this study investigated the membrane-interacted mode and catalytic activity of NnaPLA2 and notexin toward outer leaflet (phosphatidylcholine/sphingomyelin/cholesterol, PC/SM/Chol) and inner leaflet (phosphatidylserine/phosphatidylethanolamine/cholesterol, PS/PE/Chol) of plasma membrane-mimicking vesicles. PS incorporation promoted enzymatic activity of NnaPLA2 and notexin on PC and PC/SM vesicles, but suppressed NnaPLA2 and notexin activity on PC/SM/Chol and PE/Chol vesicles. PS incorporation increased the membrane fluidity of PC vesicles but reduced membrane fluidity of PC/SM, PC/SM/Chol and PE/Chol vesicles. PS increased the phospholipid order of all the tested vesicles. Moreover, PS incorporation did not greatly alter the binding affinity of notexin and NnaPLA2 with phospholipid vesicles. Acrylamide quenching studies and trinitrophenylation of Lys residues revealed that membrane-bound mode of notexin and NnaPLA2 varied with the targeted membrane compositions. The fine structure of catalytic site in NnaPLA2 and notexin in all the tested vesicles showed different changes. Collectively, the present data suggest that membrane-inserted PS modulates PLA2 interfacial activity via its effects on membrane structure and membrane-bound mode of NnaPLA2 and notexin, and membrane compositions determine the effect of PS on PLA2 activity.
磷脂酰丝氨酸（PS）外化是细胞凋亡死亡的一个标志。先前的研究表明，眼镜蛇磷脂酶A2（NnaPLA2）和虎蛇毒神经毒素可诱导人癌细胞凋亡。然而，如碘化丙啶的膜通透性所示，NnaPLA2和虎蛇毒神经毒素并未显著破坏细胞膜的完整性。这些发现反映出NnaPLA2和虎蛇毒神经毒素水解膜磷脂的能力可能受PS外化的影响。为解决该问题，本研究调查了NnaPLA2和虎蛇毒神经毒素对模拟质膜囊泡外小叶（磷脂酰胆碱/鞘磷脂/胆固醇，PC/SM/Chol）和内小叶（磷脂酰丝氨酸/磷脂酰乙醇胺/胆固醇，PS/PE/Chol）的膜相互作用模式及催化活性。PS掺入促进了NnaPLA2和虎蛇毒神经毒素对PC和PC/SM囊泡的酶活性，但抑制了NnaPLA2和虎蛇毒神经毒素对PC/SM/Chol和PE/Chol囊泡的活性。PS掺入增加了PC囊泡的膜流动性，但降低了PC/SM、PC/SM/Chol和PE/Chol囊泡的膜流动性。PS增加了所有测试囊泡的磷脂有序性。此外，PS掺入并未极大改变虎蛇毒神经毒素和NnaPLA2与磷脂囊泡的结合亲和力。丙烯酰胺猝灭研究及赖氨酸残基的三硝基苯化显示，虎蛇毒神经毒素和NnaPLA2的膜结合模式随靶向膜组成而变化。在所有测试囊泡中，NnaPLA2和虎蛇毒神经毒素催化位点的精细结构显示出不同变化。总体而言，目前的数据表明，膜插入的PS通过其对膜结构以及NnaPLA2和虎蛇毒神经毒素膜结合模式的影响来调节PLA2界面活性，且膜组成决定PS对PLA2活性的影响。
