Abstract
BackgroundSNAP25 is an essential SNARE protein for regulated exocytosis in neuronal cells. Differential splicing of the SNAP25 gene results in the expression of two transcripts, SNAP25a and SNAP25b. These splice variants differ by only 9 amino acids, and studies of their expression to date have been limited to analysis of the corresponding mRNAs. Although these studies have been highly informative, it is possible that factors such as differential turnover of the SNAP25 proteins could complicate interpretations based entirely on mRNA expression profiles.ResultsWe report the generation and characterization of antibodies that distinguish between SNAP25a and SNAP25b isoforms, and their use to investigate the expression profile of these proteins in rat and human brain. In rat brain, SNAP25b protein expression increased dramatically during post-natal development, whereas the increase in SNAP25a expression was more modest and variable. The extent of this up-regulation in SNAP25b expression was similar across cortex, cerebellum and hippocampus. The SNAP25 isoforms also displayed distinct regional expression patterns, with SNAP25a very weakly expressed in both rat and human cerebellum. Quantitative analysis revealed that SNAP25b was the dominant isoform in all adult human brain regions examined.ConclusionsSNAP25a and SNAP25b display distinct developmental and regional expression profiles in rat and human brain. These differences might reflect distinct functions of these highly conserved isoforms in membrane fusion pathways in the brain. The antibodies generated and characterized in this study represent important tools for future analyses of these essential SNARE protein isoforms.
Highlights
synaptosomal-associated protein of 25 kDa (SNAP25) is an essential SNARE protein for regulated exocytosis in neuronal cells
The exocytosis of synaptic vesicles is driven by interactions between the plasma membrane SNARE proteins syntaxin 1 and SNAP25 and the vesicle SNARE VAMP2 [1,2,3]
Generation and characterization of antibodies that selectively recognize either SNAP25a or SNAP25b To study the expression profile of SNAP25a and SNAP25b proteins, rabbits were immunized with peptides that correspond to non-conserved regions of the isoforms present within the differentially spliced exon 5 (Figure 1A)
Summary
SNAP25 is an essential SNARE protein for regulated exocytosis in neuronal cells. Differential splicing of the SNAP25 gene results in the expression of two transcripts, SNAP25a and SNAP25b. Exocytosis, the fusion of intracellular secretory vesicles with the plasma membrane, is essential for protein targeting and for secretion of soluble vesicle components to the extracellular milieu This pathway occurs constitutively in all cell types but can be a highly regulated process, such as synaptic vesicle exocytosis in neurons. The exocytosis of synaptic vesicles is driven by interactions between the plasma membrane SNARE proteins syntaxin 1 and SNAP25 and the vesicle SNARE VAMP2 [1,2,3] These neuronal SNARE proteins are specific targets of the potent botulinum and tetanus neurotoxins, emphasizing their essential functions in synaptic vesicle fusion events [4,5,6,7,8,9]. These differences in the cysteine-rich domains of SNAP25a and SNAP25b may affect their interaction with palmitoyl transferases [14], and the precise intracellular targeting of the proteins [15,16]
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