分泌型磷脂酶 A2非酶活性依赖的神经元保护作用

【目的】鉴定中华眼镜蛇(Najanajaatra)蛇毒中的神经保护因子,研究其抗神经元凋亡作用机制。【方法】连续柱层析法分离、纯化蛇毒神经保护因子。用EdmanN-末端测序法取得蛋白质一级序列,BLAST软件进行蛋白质鉴定。使用低钾诱导体外培养小脑颗粒神经元凋亡模型,研究其神经元保护作用。【结果】从中华眼镜蛇蛇毒中分离出一种,可浓度依赖性地抑制低钾诱导的小脑颗粒神经元凋亡。神经保护因子(Cobravenomneu-ronalprotectivefactor,CVNPF)此神经保护因子N-末端20个氨基酸序列为NLYQFKNMIQCTVPSRSWWD-,BLAST序列同源比对确定该保护因子为中华眼镜蛇分泌型磷脂酶A2(secretedphospholipaseA2,sPLA2)。进一步发现来源于蜂毒、莫桑比克眼镜蛇(Njajanajamossambica)蛇毒、西部菱斑响尾蛇(Crotalusatroxalso)蛇毒的sPLA2也对小脑颗粒神经元具有保护作用;其作用不为磷脂酶A2酶活性抑制剂7,7-Dimethyleicosadienoicacid(DEDA)和Manoalide阻断。【结论】CVNPF属于sPLA2。多种sPLA2可以抗神经元凋亡,其保护机制与磷脂酶A2的酶活性无关。

Secreted Phospholipase A2s Protect Neurons from Apoptosis Independent of Enzymatic Activity

To isolate and identify a neuronal protective factor from Naja naja atra venom, and then explore its protective mechanism against neuronal apoptosis. The neuronal protective component was isolated and purified from Naja naja atra crude venom by continuous chromatography. The protein sequencing was carried out using Edman N terminal sequencing method, and sequence alignment was performed by BLAST (basic local sequence alignment searching tool). Neuronal protection was confirmed by the neuronal apoptosis model ( cerebellar granule neuronal apoptosis model induced by low potassium)A neuronal protective factor (Cobra venom neuronal protective factor,CVNPF) from Naja naja atra venom was isolated, which could protect cerebellar granule neurons from apoptosis. The first 20 amino acid sequence of CVNPF from N terminal as NLYQFKNMIQCTVPSRSWWD was obtained, and CNPF was identified as a member of secreted phospholipase A2s(sPLA2s) by BLAST sequence alignment. Inhibitors of sPLA2s (7,7 Dimethyleico sadienoic acid and Manoalide) can not block its neuronal protection.Furthermore,some other sPLA2s (from bee venom, Naja naja mossambica venom, Crotalus atroxalso venom respectively) were found to have neuronal protective function. [Conclusion]CVNPF belongs to sPLA2 family, CVNPF and some other members of sPLA2s family can protect cerebellar granule neurons from apoptosis induced by low potassium. The neuronal protection of CVNPF is independent of sPLA2s enzymatic activity.