The t-SNARE protein SNAP-25 (synaptosome-associated protein of 25 kDa) plays an essential role in regulating fusion between the vesicle and plasma membranes during exocytosis. To clone and characterize SNAP-25 gene, the first step in the functional study of SNARE proteins in marine teleostean, was to obtain the cDNA of sea perch SNAP-25 (SPsn25) by RT-PCR and RACE-PCR amplification of a Japanese sea perch. The full-length cDNA of 831bp contains a CDS of 615 bp, coding 204 amino acid residues, and a 5′UTR of 219bp. Bioinformatic analysis revealed that SPsn25 corresponds with SNAP-25a isoform and shares 91.1% identity with SNAP-25a of a goldfish and a zebrafish. The SPsn25 expression in both mRNA and protein levels in the Japanese sea perch had been identified through semi-quantitative RT-PCR and Western Blot assay. Together, these data again confirmed the nerve tissue specificity of the fish SNAP-25 gene expression.
The whole length SPV2 gene of 715 bp,encoding VAMP-2 protein of 110 amino acids from Japanese sea perch,Lateolabrax japonicus,was obtained by using both RT-PCR and anchored PCR strategies while we initiated the structural and functional study on SNARE proteins in marine teleostean. Analysis of the deduced amino acid sequence indicated that SPV2 has its core arginine residue,a potential N-linked glycosylation site near its N-terminal,and one transmembrane domain in its C-terminal. Advanced structural analysis of bioinformatics approach predicts a coiled-coil α-helix backbone as the characteristic of SPV2 main conformational structure,identical to the structure of rat VAMP-2 obtained by crystallography. Semi-quantitative RT-PCR revealed that SPV2 was generally expressed in 10 neural and non-neural tissues,with the highest concentration in brain and the least in muscle.
