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; anchored PCR strategies while we initiated the structural; 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,; 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; non-neural tissues, with the highest concentration in brain; the least in muscle.
