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(SPLA2)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了胞吐反应。大鼠肥大细胞瘤RBL-2H3细胞转染肝素结合素(SPLA2-IIA、-V和-IID)，但未转非肝素结合(SPLA2-IIC)的细胞，经β和抗原刺激后，酶释放的颗粒相关标志物(α-己糖氨酸酶和组胺)明显多于对照组或胞浆α(CPLA_2)。SPLA2-IIA和-V的定点突变表明，催化区和肝素结合区都是这一功能所必需的。激光共聚焦和电子显微镜分析表明，未受刺激的细胞分泌颗粒中的SPLA2-IIA积聚在活化细胞质膜和颗粒膜融合的膜部。这些结果表明，肝素结合的SPLA2S通过其肝素结合区与肾周膜结合，其酶作用原位产生的溶血磷脂可能促进了膜的融合。与SPLA2-IIA、-IID和-V在调节脱颗粒中的多余作用相反，只有SPLA2-V能显著增加Ig E/抗原刺激的脂质介质前列腺素D_2、白三烯C_4和血小板活化因子的即刻产生，其水平可与CplA_2α相当。利用SPLA2突变体的分析和免疫细胞化学研究表明，如果人工过表达，SPLA2-V和SPLA2-X产生脂质介质的能力是由于它们相对于其他SPLA2同工酶与富含磷脂酰胆碱的质膜表面具有较高的结合能力。

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.