Snap-23 Protein Function, In The Bass Macrophage Interleukin-1�� Secretion

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-αtrafficking 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-1βin 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-αand 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-1βgene. 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-1βsecretion in sea bass macrophage. Also, it was found that the newly synthesized IL-1βwas released out probably via the secretion pathway (Endoplasmic Reticulum-Cytoplasm- Pseudopodia-extracellular) like TNFαmolecular.