Dissection of the molecular mechanism of the novel neurotrophin-like effects of secretory phospholipase A_2

我们以前证明，分泌型磷脂酶A_2（sPLA_2）和溶血磷脂酰胆碱（LPC）在大鼠嗜铬细胞瘤细胞系PC 12中表现出神经营养因子样的神经轴突发生活性。此外，sPLA_2和LPC还可使小脑颗粒神经元免于去极化刺激剥夺引起的细胞凋亡。在本研究中，我们进一步分析了sPLA_2显示神经突起诱导活性的机制。外源性sPLA_2-X（sPLA_2-X）能诱导神经突的发生，而IB和IIA型sPLA_2不能诱导神经突的发生。因此，通过补充牛血清白蛋白或磷脂酶B阻断LPC的作用，可减弱sPLA_2或LPC的神经突起形成。G2 A是一种参与LPC信号转导的G蛋白偶联受体，其过量产生或抑制可导致sPLA_2诱导的神经突形成的增强或减弱。这些结果表明， ...更多信息 sPLA_2的致敏作用是通过产生LPC和随后激活G2 A来介导的，与哺乳动物sPLA_2不同，微生物sPLA_2的生理功能知之甚少。我们克隆并鉴定了丝状真菌Aspergillus spaA和spaB编码的sPLA_2。PLA_2活性测定表明，重组SpaA和SpaB具有不同的酶学特性。免疫印迹和免疫荧光分析表明，虽然SpaA主要分泌到培养基中，SpaB与内质网和/或脂质体的细胞内结构联想到。spaA的表达在碳饥饿、氧化胁迫和分生孢子形成过程中被诱导，而spaB的组成型表达在非常低的稳定水平，在冷胁迫条件下表达上调。spaA和spaB过表达菌株的产孢表型较差，其中spaB过表达菌株的产孢表型较差。虽然没有发现形态异常的spaA，spaB，和spaA/spaB干扰，我们发现，spaA干扰的分生孢子和spaB干扰的菌丝变得敏感的氧化胁迫。这些结果表明真菌sPLA_2在抗氧化系统中起重要作用。少

We previously demonstrated that secretory phospholipase A_2 (sPLA_2) and lysophosphatidylcholine (LPC) exhibit neurotrophin-like neuritogenic activity in the rat pheochromocytoma cell line PC12. Furthermore, sPLA_2 and LPC rescued cerebellar granule neurons from apoptotic cell death induced by deprivation of depolarizing stimulus. In the current study, we further analyzed the mechanism whereby sPLA_2 displays neurite-inducing activity. Exogenously-added mammalian group X sPLA_2 (sPLA_2-X), but not group IB and IIA sPLA_2s, induced neuritogenesis, which correlated with the ability of sPLA_2-X to liberate LPC into the culture media. In accordance, blocking the effect of LPC by supplementation of bovine serum albumin or phospholipase B attenuated neuritogenesis by sPLA_2 or LPC. Overproduction or suppression of G2A, a G protein-coupled receptor involved in LPC signaling, resulted in the enhancement or reduction of neuritogenesis induced by sPLA_2 treatment. These results indicate that neu … More ritogenic effect of sPLA_2 is mediated by generation of LPC and subsequent activation of G2A.In contrast to mammalian sPLA_2s, much less is known for the physiological function of microbial sPLA_2. We cloned and characterized spaA and spaB encoding putative sPLA_2s from a filamentous fungus Aspergillus oryzae. PLA_2 activity assays showed that recombinant SpaA and SpaB had different enzymatic characteristics. Western blot and immunofluorescence analyzes demonstrated that, while SpaA was mainly secreted to culture medium, SpaB was associated with the intracellular structures reminiscent of the endoplasmic reticulum and/or lipid body. The expression of spaA was induced in response to carbon starvation, oxidative stress and conidiation, while spaB was constitutively expressed at very low steady level and upregulated in the cold-stressed condition. spaA- and spaB-overexpressing strains showed poor-conidiation phenotype which was remarkable in the latter. Although no morphological abnormalities were found in spaA, spaB, and spaA/spaB disruptants, we found that conidia of spaA disruptant and hyphae of spaB disruptant became sensitive to oxidative stress. These results suggest that fungal sPLA_2s play roles in defense system to oxidative stress. Less