SNAP-23蛋白在鲈鱼巨噬细胞白细胞介素1β分泌过程中的功能

SNARE蛋白家族是所有真核细胞胞吐及分泌作用中的关键因子，由其介导的运输囊泡膜与靶膜的锚靠、融合为胞内蛋白的运出提供了一条重要途径。体外试验表明，Syntaxin6-Syntaxin7-Vti1b，SNAP-23-Syntaxin4等SNARE核心蛋白之间精确的相互作用是哺乳动物巨噬细胞肿瘤坏死因子α (TNF-α)运输和分泌的必备条件，在机体非特异性免疫应答反应过程中起重要作用。 本研究受上述启示，旨在揭示SNARE蛋白在海洋鱼类免疫细胞内重要细胞因子白细胞介素1β (IL-1β)的分泌过程中的作用。参照Percoll密度梯度离心技术，从鲈鱼头肾组织分离纯化巨噬细胞进行稳定培养；利用RT-PCR方法克隆出鲈鱼t-SNARE蛋白SNAP-23和Syntaxin3的部分cDNA序列，再结合先前克隆的VAMP2和已知的鲈鱼IL-1β，TNF-α和IL-8的基因序列，设计特异性引物。利用Real-time PCR技术在mRNA水平上精确测定鲈鱼巨噬细胞中上述6种基因在革兰氏阴性菌脂多糖(LPS)分子刺激下的表达变化，发现SNAP-23基因与三种细胞因子基因的表达正相关；通过免疫印迹检测SNAP-23蛋白表达变化，利用酶联免疫吸附试验(ELISA)检测IL-1β的分泌水平，在蛋白水平上验证了SNAP-23表达与IL-1β分泌的正相关性；利用5`RACE和3`RACE技术克隆出鲈鱼SNAP-23全长基因，结合定点突变策略和靶向PCR克隆手段，构建鲈鱼SNAP-23野生型融合质粒pEGFP-SNAP-23wt，Cys缺失突变融合质粒pEGFP-SNAP-23ΔCys和模拟E型肉毒神经毒素(BoNT/E)切割突变融合质粒pEGFP-SNAP-23ΔBoNT/E，以及鲈鱼IL-1β野生型融合表达质粒IL-1β-pEGFP和IL-1β-pEYFP。所有融合蛋白均在鲈鱼巨噬细胞内成功表达，结合ELISA实验结果发现，SNAP-23野生型的表达对IL-1β的分泌有促进作用，而Cys缺失突变体的表达则抑制IL-1β向胞外分泌。首次证实了鱼类巨噬细胞内SNAP-23蛋白在IL-1β分泌过程中的重要作用。此外通过与GFP共表达，定位了IL-1β分子在巨噬细胞内的分布，发现新合成的IL-1β分子很可能像TNFα一样经“内质网－胞质－伪足－胞外” 的分泌路径运出胞外。

SNARE proteins are key factors during exocytosis and endocytosis in all kinds of eukaryotic cells. The SNARE-mediated docking and fusion between vesicular carriers and target membrane provide an important pathway for protein trafficking and deployment. In vitro studies using cultured cells suggest that the precise interaction between core SNARE membranes Syntaxin6, Syntaxin7 and Vti1b, or between SNAP-23 and Syntaxin4, are indispensable requirements of TNF-α trafficking and secretion pathway in mammalian macrophages. Based on these facts, this study was undertaken to explore the function of SNARE proteins during the secretion of key cytokine Interleukin-1β in marine fish initiated immune cell. Macrophages were cultured after being isolated from sea bass pronephros tissue by using Percoll density gradient centrifugation method. According to partially sequenced results of sea bass t-SANRE SNAP-23 and Syntaxin3 cDNA obtained by RT-PCR amplification and published sequences of VAMP2, IL-1β, TNF-α and IL-8, gene specific primers were designed and used in Real-time PCR analysis to accurately quantify the expression profiles of these genes under LPS stimulation on mRNA level, which revealed that the expression of SNAP-23 gene was up-related to IL-1β gene. Immunoblot analysis on SNAP-23 and ELISA analysis on IL-1β, confirmed the same expression relativity between them on protein level. Full lengthen SNAP-23 gene was obtained by using 5`RACE and 3`RACE methods and wild type recombinant pEGFP-SNAP-23wt and IL-1β-pEYFP was constructed. Also, site point mutant and target-PCR approaches were introduced to obtain the cys-lack recombinant mutant pEGFP-SNAP-23ΔCys and mimic BoNT/E cleaved recombinant mutant pEGFP-SNAP-23ΔBoNT/E. All SNAP-23-GFP and IL-1β-pEYFP recombinants were separately expressed, and the fluorescence signals of transient transfection were detected by confocal imaging, which revealed that expression of wild type SNAP-23 promoted the secretion of IL-1β, while expression of SNAP-23ΔCys mutant suppressed the secretion of IL-1β. These, at first time, suggested that SNAP-23 protein played an important role in IL-1β secretion in sea bass macrophage. Also, it was found that the newly synthesized IL-1β was released out probably via the secretion pathway (Endoplasmic Reticulum-Cytoplasm- Pseudopodia-extracellular) like TNFα molecular.