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

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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