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Regional and developmental brain expression patterns of SNAP25 splice variants.

Prescott GR, Chamberlain LH - BMC Neurosci (2011)

Bottom Line: Differential splicing of the SNAP25 gene results in the expression of two transcripts, SNAP25a and SNAP25b.The extent of this up-regulation in SNAP25b expression was similar across cortex, cerebellum and hippocampus.The antibodies generated and characterized in this study represent important tools for future analyses of these essential SNARE protein isoforms.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Integrative Physiology, School of Biomedical Sciences, Hugh Robson Building, University of Edinburgh, Edinburgh EH8 9XD, UK.

ABSTRACT

Background: 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. 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.

Results: We 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.

Conclusions: SNAP25a 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.

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Related in: MedlinePlus

Characterization of 3067 and 3068 antibodies. A) The top schematic (in blue) shows the domain structure of SNAP25; SN1 and SN2 denote the respective SNARE domains. An alignment of the peptide sequences from rat SNAP25a and SNAP25b that were used for antibody production is also shown. The sequence of rat SNAP23 in the corresponding region is provided for comparison. Numbers indicate the position of the peptides in relation to the full-length proteins. The shading highlights conserved residues in SNAP25a and SNAP25b. The shading for SNAP23 highlights amino acids that are identical to those in the corresponding region of SNAP25b. B) HEK293T cells were transfected with plasmids encoding EGFP-tagged SNAP25a, SNAP25b and SNAP23. Equal volumes of lysates from the transfected cells were probed with antibodies against SNAP25a (3067), SNAP25b (3068), SNAP23 (Synaptic Systems), and GFP (Roche). C) Cerebellum lysates from wild-type or SNAP25a/a mice were resolved by SDS-PAGE and transferred to nitrocellulose for immunoblotting analysis using 3067, 3068, pan-SNAP25 and SNAP23 antibodies. The position of molecular weight markers are shown on the left side of panels B and C.
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Figure 1: Characterization of 3067 and 3068 antibodies. A) The top schematic (in blue) shows the domain structure of SNAP25; SN1 and SN2 denote the respective SNARE domains. An alignment of the peptide sequences from rat SNAP25a and SNAP25b that were used for antibody production is also shown. The sequence of rat SNAP23 in the corresponding region is provided for comparison. Numbers indicate the position of the peptides in relation to the full-length proteins. The shading highlights conserved residues in SNAP25a and SNAP25b. The shading for SNAP23 highlights amino acids that are identical to those in the corresponding region of SNAP25b. B) HEK293T cells were transfected with plasmids encoding EGFP-tagged SNAP25a, SNAP25b and SNAP23. Equal volumes of lysates from the transfected cells were probed with antibodies against SNAP25a (3067), SNAP25b (3068), SNAP23 (Synaptic Systems), and GFP (Roche). C) Cerebellum lysates from wild-type or SNAP25a/a mice were resolved by SDS-PAGE and transferred to nitrocellulose for immunoblotting analysis using 3067, 3068, pan-SNAP25 and SNAP23 antibodies. The position of molecular weight markers are shown on the left side of panels B and C.

Mentions: 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). These different peptides did not contain more than four identical contiguous amino acids and only 10 out of 15 residues were identical (Figure 1A). IgG fractions were purified from the collected antisera: IgG #3067 was from rabbits immunized with the SNAP25a peptide and IgG #3068 was from rabbits immunized with the SNAP25b peptide.


Regional and developmental brain expression patterns of SNAP25 splice variants.

Prescott GR, Chamberlain LH - BMC Neurosci (2011)

Characterization of 3067 and 3068 antibodies. A) The top schematic (in blue) shows the domain structure of SNAP25; SN1 and SN2 denote the respective SNARE domains. An alignment of the peptide sequences from rat SNAP25a and SNAP25b that were used for antibody production is also shown. The sequence of rat SNAP23 in the corresponding region is provided for comparison. Numbers indicate the position of the peptides in relation to the full-length proteins. The shading highlights conserved residues in SNAP25a and SNAP25b. The shading for SNAP23 highlights amino acids that are identical to those in the corresponding region of SNAP25b. B) HEK293T cells were transfected with plasmids encoding EGFP-tagged SNAP25a, SNAP25b and SNAP23. Equal volumes of lysates from the transfected cells were probed with antibodies against SNAP25a (3067), SNAP25b (3068), SNAP23 (Synaptic Systems), and GFP (Roche). C) Cerebellum lysates from wild-type or SNAP25a/a mice were resolved by SDS-PAGE and transferred to nitrocellulose for immunoblotting analysis using 3067, 3068, pan-SNAP25 and SNAP23 antibodies. The position of molecular weight markers are shown on the left side of panels B and C.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3104942&req=5

Figure 1: Characterization of 3067 and 3068 antibodies. A) The top schematic (in blue) shows the domain structure of SNAP25; SN1 and SN2 denote the respective SNARE domains. An alignment of the peptide sequences from rat SNAP25a and SNAP25b that were used for antibody production is also shown. The sequence of rat SNAP23 in the corresponding region is provided for comparison. Numbers indicate the position of the peptides in relation to the full-length proteins. The shading highlights conserved residues in SNAP25a and SNAP25b. The shading for SNAP23 highlights amino acids that are identical to those in the corresponding region of SNAP25b. B) HEK293T cells were transfected with plasmids encoding EGFP-tagged SNAP25a, SNAP25b and SNAP23. Equal volumes of lysates from the transfected cells were probed with antibodies against SNAP25a (3067), SNAP25b (3068), SNAP23 (Synaptic Systems), and GFP (Roche). C) Cerebellum lysates from wild-type or SNAP25a/a mice were resolved by SDS-PAGE and transferred to nitrocellulose for immunoblotting analysis using 3067, 3068, pan-SNAP25 and SNAP23 antibodies. The position of molecular weight markers are shown on the left side of panels B and C.
Mentions: 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). These different peptides did not contain more than four identical contiguous amino acids and only 10 out of 15 residues were identical (Figure 1A). IgG fractions were purified from the collected antisera: IgG #3067 was from rabbits immunized with the SNAP25a peptide and IgG #3068 was from rabbits immunized with the SNAP25b peptide.

Bottom Line: Differential splicing of the SNAP25 gene results in the expression of two transcripts, SNAP25a and SNAP25b.The extent of this up-regulation in SNAP25b expression was similar across cortex, cerebellum and hippocampus.The antibodies generated and characterized in this study represent important tools for future analyses of these essential SNARE protein isoforms.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre for Integrative Physiology, School of Biomedical Sciences, Hugh Robson Building, University of Edinburgh, Edinburgh EH8 9XD, UK.

ABSTRACT

Background: 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. 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.

Results: We 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.

Conclusions: SNAP25a 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.

Show MeSH
Related in: MedlinePlus