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

我们先前在大鼠嗜铬细胞瘤细胞系PC12中发现分泌型磷脂酶A2(SPLA2)和溶血磷脂酰胆碱(LPC)具有神经营养素样神经发生活性。此外，SPLA2和LPC可使小脑颗粒神经元免于去极化刺激所致的细胞死亡。在本研究中，我们进一步分析了SPLA2具有轴突诱导活性的机制。外源添加哺乳动物X组SPLA2(SPLA2-X)，而IB组和IIA组SPLA2不能诱导神经再生，这与SPLA2-X将LPC释放到培养上清液中的能力有关。相应地，补充牛血清白蛋白或磷脂酶B阻断LPC的作用，可减弱SPLA2或LPC的神经再生。参与LPC信号转导的G蛋白偶联受体G2a的过度表达或抑制可导致SPLA2诱导的神经再生增强或减少。这些结果表明，Neu…与哺乳动物的SPLA2不同，微生物SPLA2的生理功能知之甚少。我们从丝状真菌米曲霉中克隆并鉴定了编码SPLA2的SPAA和SPAB。PLA2活性测定表明，重组SPAA和SPAB具有不同的酶学性质。免疫印迹和免疫荧光分析表明，虽然SPAA主要分泌到培养液中，但SPAB与内质网和/或脂体的细胞内结构有关。SPAA在碳饥饿、氧化胁迫和分生孢子的诱导下表达，而SPAB在很低的稳定水平上结构性表达，在冷胁迫条件下表达上调。SPAA和SPAB高表达菌株表现为弱分生孢子表型，后者表现明显。虽然在SPAA、SPAB和SPAA/SPAB干扰物中没有发现形态异常，但我们发现SPAA干扰物的分生孢子和SPAB干扰物的菌丝对氧化胁迫变得敏感。这些结果表明，真菌SPLA2在氧化应激防御系统中发挥作用。较少

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