We herein demonstrate that mast cells express all known members of the group II subfamily of secretory phospholipase A2 (sPLA2) isozymes, and those having heparin affinity markedly enhance the exocytotic response. Rat mastocytoma RBL-2H3 cells transfected with heparin-binding (sPLA2-IIA, -V, and -IID), but not heparin-nonbinding (sPLA2-IIC), enzymes released more granule-associated markers (beta-hexosaminidase and histamine) than mock- or cytosolic PLA2alpha (cPLA2alpha)-transfected cells after stimulation with IgE and Ag. Site-directed mutagenesis of sPLA2-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 sPLA2-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 sPLA2s 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 sPLA2-IIA, -IID, and -V in the regulation of degranulation, only sPLA2-V had the ability to markedly augment IgE/Ag-stimulated immediate PGD2 production, which reached a level comparable to that elicited by cPLA2alpha. The latter observation reveals an unexplored functional segregation among the three related isozymes expressed in the same cell population.

译文

:我们在本文中证明肥大细胞表达分泌性磷脂酶A2(sPLA2)同工酶II组的所有已知成员,而具有肝素亲和力的细胞显着增强胞吐反应。用肝素结合(sPLA2-IIA,-V和-IID)转染大鼠肥大细胞瘤RBL-2H3细胞,但未结合肝素不结合(sPLA2-IIC),酶释放的颗粒相关标志物(β-己糖胺酶和组胺)多于用IgE和Ag刺激后,模拟或胞质PLA2alpha(cPLA2alpha)转染的细胞。 sPLA2-IIA和-V的定点诱变表明,催化结构域和肝素结合结构域对于该功能都是必不可少的。共聚焦激光和电子显微镜分析表明,存储在未刺激细胞分泌颗粒中的sPLA2-IIA积累在膜质部位,在质膜中质膜和颗粒膜之间发生融合,并在活化细胞中发生。这些结果表明,与肝素结合的sPLA2s通过其肝素结合结构域与外周膜结合,并且通过其酶促作用原位产生的溶血磷脂可以促进正在进行的膜融合。与sPLA2-IIA,-IID和-V在脱粒调节中的冗余作用相反,只有sPLA2-V具有显着增加IgE / Ag刺激的PGD2立即产生的能力,该水平达到了与诱发的水平相当的水平。由cPLA2alpha。后者的观察结果揭示了在同一细胞群体中表达的三种相关同工酶之间尚未探索的功能分离。

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