N-Terminal Acetylation of the Neuronal Protein SNAP-25 Is Revealed by the SMI81 Monoclonal Antibody

Abstract

The monoclonal antibody SMI81 binds SNAP-25, a major player in neurotransmitter release, with high affinity and has previously been used to follow changes in the levels of this protein in neuropsychiatric disorders. We report here that the SMI81 epitope is present at the extreme N-terminus of SNAP-25 and, unusually, cannot be recognized when present as an internal sequence. Although it is known that SNAP-25 can be palmitoylated and phosphorylated in brain, we now reveal the existence of a third modification, acetylation of the N-terminus. This acetylation event greatly increases the efficiency of SMI81 antibody binding. We show that this highly specific antibody can be used for studying brain function in many vertebrate organisms.The synaptosomal-associated protein of 25 kDa 1 is a highly conserved synaptic protein and a key component of the synaptic vesicle fusion machinery (1, 2). It was initially identified as the most abundant methionine-rich protein to be rapidly transported along axons, accumulating at synaptic termini (3, 4). Levels of SNAP-25 increase during development, and SNAP-25 expression coincides with the onset of synaptogenesis and neuronal maturation (5, 6). SNAP-25 is a hydrophilic protein but is tightly associated with synaptic membranes because of the attachment of hydrophobic palmitate lipids to four clustered cysteines in the center of the molecule; indeed, SNAP-25 is the major palmitoylation target in brain (3,4,7,8).Key to our understanding of SNAP-25 protein function was the finding that it forms a stoichiometric complex with syntaxin and synaptobrevin in brain (9, 10). The central role of these three soluble NSF-attachment protein receptor (SNARE) proteins, including SNAP-25, in synaptic vesicle release was confirmed upon their identification as the targets of clostridial neurotoxin action (11). Botulinum neurotoxins A and E cleave SNAP-25 at specific points in the amino acid sequence of the C-terminus, rendering the cleaved protein unable to form the SNARE complex which in turn leads to neuromuscular paralysis and death (12, 13).In light of the importance of SNAP-25 for neurotransmission, several antibodies have been developed for recognition of this protein. One such commonly used antibody is mouse monoclonal SMI81, initially generated by Sternberger Monoclonals among a wide panel of antibodies raised against human brain extract (hence the abbreviation SMI81 for Sternberger Monoclonals Inc., clone 81). The SMI81 antibody was later shown to specifically recognize a single 25 kDa protein band by Western immunoblotting in wild-type neurons, but not in neurons from a SNAP-25 null mutant background (14). It recognizes botulinum toxin-truncated protein (15-17) and both alternatively spliced SNAP-25A and -B isoforms, which differ by only nine amino acids. It has been used to detect SNAP-25 in hippocampal slices from mice at various stages of embryonic and postnatal development (18). SMI81 immunoblotting of brain regions of mice suffering from hyperkinesis showed changes in the SNA...

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“…S1, supplemental Table S3). We also found SNAP-25 to be modified by methionine excision and acetylation, which is in agreement with a previous study (24). Syntaxin-1 also exists in two isoforms (1A and 1B) (14,29).…”

SNAP25 expression in mammalian retinal horizontal cells

“…5C, SMI 81 mAb). This pattern indicates that the N-terminus of SNAP25, the epitope recognized by SMI 81, particularly when N-terminal acetylated (Connell et al, 2009), was quite accessible in fixed tissue. Here, both conventional and ribbon synapses showed SNAP25 expression, as would be expected if vesicular release occurs at both types of synapses.…”

“…SMI 81 (Sternberger Monoclonals and ARP; Brandstätter et al, 1996; Morgans and Brandstätter, 2000): The monoclonal antibody was generated against the whole SNAP25 protein in human brain extracts (manufacturer’s datasheet; Connell et al, 2009). Specific labeling in vertical sections of rat retina was abolished when the antibody was preincubated overnight with purified SNAP25 protein on nitrocellulose (Brandstätter et al, 1996).…”

“…Specific labeling in vertical sections of rat retina was abolished when the antibody was preincubated overnight with purified SNAP25 protein on nitrocellulose (Brandstätter et al, 1996). In addition, SMI 81 recognized a 25-kDa band on Western blots of rat retina membrane proteins (Brandstätter et al, 1996), mouse neocortex (Mehta et al, 1996; Grabs et al, 1996), mouse hippocampal neurons (Tafoya et al, 2006), and rat brain (Garbelli et al, 2008; Connell et al, 2009) and hippocampus (Frassoni et al, 2005). The SNAP25 band is eliminated in immunoblots prepared from the SNAP25 null mice (Washbourne et al, 2002; Tafoya et al, 2006).…”

“…The SNAP25 band is eliminated in immunoblots prepared from the SNAP25 null mice (Washbourne et al, 2002; Tafoya et al, 2006). Epitope mapping indicated that SMI 81 recognizes the extreme N-terminus of SNAP25, particularly when it is N-terminal acetylated (Connell et al, 2009). …”