Diversities and functions of phospholipase A_2s in mast cells

在本研究中，我们发现肥大细胞表达分泌型磷脂酶A_2（sPLA_2）Ⅱ亚家族的所有已知成员，并且那些具有肝素亲和力的肥大细胞显著增强胞吐反应。大鼠肥大细胞瘤RBL-2 H3细胞经肝素结合型（sPLA_2-IIA、-V和-IID）转染，而非肝素非结合型（sPLA_2-IIC）转染，在IgE和抗原刺激后，比模拟或胞浆型PLA_2α（cPLA_2α）转染的细胞释放更多的颗粒相关标志物（β-氨基己糖苷酶和组胺）。sPLA_2-IIA和sPLA_2-V的定点突变表明，催化结构域和肝素结合结构域都是这一功能所必需的。激光共聚焦显微镜和电子显微镜分析表明，未受刺激时sPLA_2-IIA储存在分泌颗粒中，而在活化细胞中，sPLA_2-IIA聚集在质膜与颗粒膜融合的膜上。这些结果表明，肝素结合sPLA_2通过其肝素结合域与颗粒周围膜结合，并且通过其酶促作用原位产生的溶血磷脂可能促进正在进行的膜融合。与sPLA_2-IIA、-IID和-V在脱颗粒调节中的冗余作用相反，只有sPLA_2-V能显著增加IgE/抗原刺激的脂质介质如前列腺素D_2、白三烯C_4和血小板活化因子（PAF）的立即产生，其水平与cPLA_2α相当。利用sPLA_2突变体的分析以及免疫细胞化学研究表明，sPLA_2-V和sPLA_2-X（如果人工过量表达）产生脂质介质的能力归因于它们相对于其它sPLA_2同工酶对富含磷脂酰胆碱的质膜表面的高结合能力。

In this study, we found that mast cells express all known members of the group II subfamily of secretory phospholipase A_2 (sPLA_2) isozymes and those having heparin affinity markedly enhance the exocytotic response. Rat mastocytoma RBL-2H3 cells transfected with heparin-binding (sPLA_2-IIA,-V and-IID), but not heparin-non-binding (sPLA_2-IIC), enzymes released more granule-associated markers (β-hexosaminidase and histamine) than mock-or cytosolic PLA_2α (cPLA_2α)-transfected cells after stimulation with IgE and antigen. Site-directed mutagenesis of sPLA_2-IIA and-V revealed that both the catalytic and heparin-binding domains are essential for this function. Confocal laser and electron microscopic analyses revealed that sPLA_2-IIA, which was stored in secretory granules in unstimulated cells, accumulated on the membranous sites where fusion between the plasma membrane and granule membranes occurred in activated cells. These results suggest that the heparin-binding sPLA_2s bind to the perigranular membranes through their heparin-binding domain, and lysophospholipids produced in situ by their enzymatic action may facilitate the ongoing membrane fusion. In contrast to the redundant role of sPLA_2-IIA,-IID and-V in the regulation of degranulation, only sPLA_2-V had the ability to markedly augment IgE/antigen-stimulated immediate production of lipid mediators such as prostaglandin D_2, leukotriene C_4 and platelet-activating factor (PAF), which reached a level comparable to that elicited by cPLA_2α. Analyses using sPLA_2 mutants as well as immunocytochemical studies demonstrated that the lipid mediator-producing ability of sPLA_2-V, and sPLA_2-X if artificially overexpressed, was attributed to their high binding ability, relative to other sPLA_2 isozymes, to phosphatidylcholine-rich plasma membrane surfaces.