Increased association of synaptosome-associated protein of 25 kDa with syntaxin and vesicle-associated membrane protein following acrosomal exocytosis of sea urchin sperm

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a palmitoylated integral membrane protein expressed almost exclusively in neuronal and neuroendocrine tissues. This protein forms a ternary complex with vesicle-associated membrane protein (VAMP) and syntaxin, which is thought to regulate the fusion of plasma and vesicle membranes during exocytosis. We report the identification of SNAP-25 expressed in sea urchin sperm. Sea urchin SNAP-25 shares greater identity with mammalian SNAP-25 than with mammalian SNAP-23, a ubiquitously expressed homologue believed to regulate membrane fusion in non-neuronal tissues. Sea urchin sperm contain a single exocytotic vesicle, the acrosomal vesicle, whose contents are exposed during the acrosome reaction. Fusion of the plasma membrane with the acrosomal vesicle membrane at multiple points (vesiculation) results in the release of SNAP-25 with the shed acrosome reaction vesicles. A complex containing SNAP-25, syntaxin, and VAMP is present in sperm, as detected by affinity chromatography and immunoprecipitation. Although this complex is present prior to the acrosome reaction, the amount of complex increases over 4-fold following acrosomal exocytosis. These findings support the involvement of SNAP-25 in the invertebrate sperm acrosome reaction, possibly through increased association with VAMP and syntaxin driving the fusion of plasma and acrosomal membranes.     

Differential expression of SNAP-25 isoforms and SNAP-23 in the adrenal gland

In the rat adrenal gland, we previously observed that SNAP-25 is not restricted to the plasmalemma in noradrenergic cells as it is in adrenergic cells, and hypothesized that SNAP-25 isoform expression is different in the two phenotypes. Expression of SNAP-25 isoforms and SNAP-23 was examined by immunoblotting, immunofluorescence, and RT-PCR. Amplifications of SNAP-25 mRNAs were combined with Southern hybridization, restriction enzyme analysis, and sequencing of cloned PCR products to compare SNAP-25 isoform expression in rat and bovine adrenal glands. SNAP-25 and SNAP-23 mRNA and protein are expressed in the glands; SNAP-23 is enriched in the adrenal cortex, whereas SNAP-25 is restricted to the adrenal medulla. Furthermore, high levels of SNAP-25 and low levels of SNAP-23 are observed in the PC12 cells, whereas both SNAP-25 and SNAP-23 are expressed in adrenal medullary cultures. In all extracts, the SNAP-23 mRNA corresponded to SNAP-23a. SNAP-25a is the major form expressed in rat adrenal glands (75%), as it is in PC12 cells (80%), but both SNAP-25a and SNAP-25b (40% vs. 60%) are expressed in bovine adrenal medulla in situ and in culture. In addition, an enriched population of adrenergic cells (93%) expressed a higher level of SNAP-25b (70%), suggesting that this isoform may not be restricted to fast neurotransmission.     

Association of syntaxin with SNAP-25 and VAMP (synaptobrevin) during axonal transport

Cluster headache is characterized by regular periodicity, high frequency during a cluster period, relative brevity, and extreme intensity. Lancinations, as in trigeminal neuralgia, are rare. An important behavioral difference between migraine and cluster headache is that the patient is usually hyperactive during a cluster headache, whereas the migraineur retreats to a dark, quiet room. Cluster headache is more common in middle-aged men; migraine is more common in young women. Intermediate or overlap syndromes occur. Many of the same prophylactic and abortive treatments are effective in both, although in cluster headache there is a premium on rapid action.     

Three novel proteins of the syntaxin/SNAP-25 family

Intracellular membrane traffic is thought to be regulated in part by soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (SNAREs) through the formation of complexes between these proteins present on vesicle and target membranes. All known SNARE-mediated fusion events involve members of the syntaxin and vesicle-associated membrane protein families. The diversity of mammalian membrane compartments predicts the existence of a large number of different syntaxin and vesicle-associated membrane protein genes. To further investigate the spectrum of SNAREs and their roles in membrane trafficking we characterized three novel members of the syntaxin and SNAP-25 (synaptosome-associated protein of 25 kDa) subfamilies. The proteins are broadly expressed, suggesting a general role in vesicle trafficking, and localize to distinct membrane compartments. Syntaxin 8 co-localizes with markers of the endoplasmic reticulum. Syntaxin 17, a divergent member of the syntaxin family, partially overlaps with endoplasmic reticulum markers, and SNAP-29 is broadly localized on multiple membranes. SNAP-29 does not contain a predicted membrane anchor characteristic of other SNAREs. In vitro studies established that SNAP-29 is capable of binding to a broad range of syntaxins.     

Protease resistance of syntaxin.SNAP-25.VAMP complexes. Implications for assembly and structure

A stable ternary complex formed with vesicle-associated membrane protein 2 (VAMP2) and plasma membrane proteins syntaxin 1A and synaptosome-associated protein of 25 kDa (SNAP-25) is proposed to function in synaptic vesicle exocytosis. To analyze the structural characteristics of this synaptic protein complex, recombinant binary (syntaxin 1A.SNAP-25), recombinant ternary, and native ternary complexes were subjected to limited trypsin proteolysis. The protected fragments, defined by amino-terminal sequencing and mass spectrometry, included a carboxyl-terminal region of syntaxin 1A, the cytoplasmic domain of VAMP2, and amino- and carboxyl-terminal regions of SNAP-25. Furthermore, separate amino- and carboxyl-terminal fragments of SNAP-25, when combined with VAMP2 and syntaxin 1A, were sufficient for stable complex assembly. Analysis of ternary complexes formed with full-length proteins revealed that the carboxyl-terminal transmembrane anchors of both syntaxin 1A and VAMP2 were protected from trypsin digestion. Moreover, the stability of ternary complexes was increased by inclusion of these transmembrane domains. These results suggest that the transmembrane domains of VAMP2 and syntaxin 1A contribute to complex assembly and stability and that amino- and carboxyl-terminal regions of SNAP-25 may function as independent domains.     

Vam7p, a SNAP-25-like molecule, and Vam3p, a syntaxin homolog, function together in yeast vacuolar protein trafficking

A genetic screen to isolate gene products required for vacuolar morphogenesis in the yeast Saccharomyces cerevisiae identified VAM7, a gene which encodes a protein containing a predicted coiled-coil domain homologous to the coiled-coil domain of the neuronal t-SNARE, SNAP-25 (Y. Wada and Y. Anraku, J. Biol. Chem. 267:18671-18675, 1992; T. Weimbs, S. H. Low, S. J. Chapin, K. E. Mostov, P. Bucher, and K. Hofmann, Proc. Natl. Acad. Sci. USA 94:3046-3051, 1997). Analysis of a temperature-sensitive-for-function (tsf) allele of VAM7 (vam7(tsf)) demonstrated that the VAM7 gene product directly functions in vacuolar protein transport. vam7(tsf) mutant cells incubated at the nonpermissive temperature displayed rapid defects in the delivery of multiple proteins that traffic to the vacuole via distinct biosynthetic pathways. Examination of vam7(tsf) cells at the nonpermissive temperature by electron microscopy revealed the accumulation of aberrant membranous compartments that may represent unfused transport intermediates. A fraction of Vam7p was localized to vacuolar membranes. Furthermore, VAM7 displayed genetic interactions with the vacuolar syntaxin homolog, VAM3. Consistent with the genetic results, Vam7p physically associated in a complex containing Vam3p, and this interaction was enhanced by inactivation of the yeast NSF (N-ethyl maleimide-sensitive factor) homolog, Sec18p. In addition to the coiled-coil domain, Vam7p also contains a putative NADPH oxidase p40(phox) (PX) domain. Changes in two conserved amino acids within this domain resulted in synthetic phenotypes when combined with the vam3(tsf) mutation, suggesting that the PX domain is required for Vam7p function. This study provides evidence for the functional and physical interaction between Vam7p and Vam3p at the vacuolar membrane, where they function as part of a t-SNARE complex required for the docking and/or fusion of multiple transport intermediates destined for the vacuole.     

Phosphorylation of 25-kDa synaptosome-associated protein. Possible involvement in protein kinase C-mediated regulation of neurotransmitter release

Protein kinase C-mediated phosphorylation of a 25-kDa synaptosome-associated protein (SNAP-25) was examined in living PC12 cells. Phorbol 12-myristate 13-acetate treatment enhanced high potassium-induced [3H]-norepinephrine release, and a 28-kDa protein recognized by an anti-SNAP-25 antibody was phosphorylated on Ser residues. The molecular size of the phosphorylated band decreased slightly following treatment with Clostridium botulinum type A neurotoxin, whereas the band disappeared after treatment with botulinum type E neurotoxin, indicating that the 28-kDa protein was SNAP-25. A phosphorylation is likely to occur at Ser187, as this is the only Ser residue located between the cleavage sites of botulinum type A and E neurotoxins. SNAP-25 of PC12 cells was phosphorylated by purified protein kinase C in vitro, and the amount of syntaxin co-immunoprecipitated with SNAP-25 was decreased by phosphorylation. These results suggest that the phosphorylation of SNAP-25 may be involved in protein kinase C-mediated regulation of catecholamine release from PC12 cells.     

Multiple loci for synapse protein SNAP-25 in the tetraploid goldfish

The common goldfish Carassius auratus is tetraploid and has 100 chromosomes. We describe here goldfish cDNA clones for SNAP-25, a 200-amino-acid synaptosome-associated protein that has remained highly conserved during evolution. SNAP-25 occurs as a single-copy gene in mouse, chicken, and Drosophila melanogaster. Sequences of six distinct goldfish cDNA clones and Southern hybridizations show that the goldfish has three, or possibly four, SNAP-25 loci rather than two as expected. A gene duplication early in actinopterygian fish evolution gave rise to the loci SnapA and SnapB. The proteins SNAP-A and SNAP-B are 94% and 91% identical to the mouse protein but are only 91% identical to each other. SNAP-B has a larger number of unique amino acid replacements than SNAP-A and also has more dramatic replacements. The tetraploidization resulted in two SnapB loci whose divergence from each other is consistent with a tetraploidization event 15-20 million years ago. The presence of duplicate SnapA loci has not yet been possible to confirm, possibly because they are still very similar to each other. Two of the SnapA cDNA clones and one SnapB cDNA clone have frameshift mutations. As these aberrant alleles otherwise display high sequence identity to the functional alleles, they probably became nonfunctional recently. The findings of allelic variability and aberrant alleles emphasize the importance of characterizing multiple DNA clones in tetraploid species.     

Assembly of a ternary complex by the predicted minimal coiled-coil-forming domains of syntaxin, SNAP-25, and synaptobrevin. A circular dichroism study

The assembly of target (t-SNARE) and vesicle-associated SNAP receptor (v-SNARE) proteins is a critical step for the docking of synaptic vesicles to the plasma membrane. Syntaxin-1A, SNAP-25, and synaptobrevin-2 (also known as vesicle-associated membrane protein, or VAMP-2) bind to each other with high affinity, and their binding regions are predicted to form a trimeric coiled-coil. Here, we have designed three peptides, which correspond to sequences located in the syntaxin-1A H3 domain, the C-terminal domain of SNAP-25, and a conserved central domain of synaptobrevin-2, that exhibit a high propensity to form a minimal trimeric coiled-coil. The peptides were synthesized by solid phase methods, and their interactions were studied by CD spectroscopy. In aqueous solution, the peptides were unstructured and showed no interactions with each other. In contrast, upon the addition of moderate amounts of trifluoroethanol (30%), the peptides adopted an alpha-helical structure and displayed both homomeric and heteromeric interactions. The interactions observed in ternary mixtures induce a stabilization of peptide structure that is greater than that predicted from individual binary interactions, suggesting the formation of a higher order structure compatible with the assembly of a trimeric coiled-coil.     

Heat shock protein 27 kDa expression and phosphorylation regulates endothelial cell migration

The effects of enhanced HSP27 expression or expression of a nonphosphorylatable form of HSP27 on the migration of bovine arterial endothelial cells was assessed. Expression of the wild-type protein enhanced migration by twofold compared to control transfectants, whereas expression of the mutant protein retarded migration by 40%. Since homologs of the small heat shock protein inhibit F-actin polymerization in vitro and may alter basolateral F-actin content in vivo, it was postulated that the 27 kDa heat shock protein affects microfilament extension essential for cell motility. Expression of the wild-type protein promoted the generation of long cellular extensions, whereas expression of the dominant negative mutant protein resulted in a marked reduction of lamellipodia and generated aberrant microfilament morphology at the wound edge. Immunofluorescence combined with phalloidin staining demonstrated the colocalization of the HSP27 gene products with lamellipodial microfilament structures. These data suggest that the 27 kDa heat shock protein regulates migration by affecting the generation lamellipodia microfilaments.     

Distinct exocytotic responses of intact and permeabilised chromaffin cells after cleavage of the 25-kDa synaptosomal-associated protein (SNAP-25) or synaptobrevin by botulinum toxin A or B

Botulinum neurotoxin (BoNT) types A and B are Zn2+-requiring endoproteases which potently block neurotransmitter release by cleavage of a 25-kDa synaptosomal-associated protein (SNAP-25) and synaptobrevin, respectively. Synaptobrevin is important for the exocystosis of catecholamines from dense-core granules and evidence is presented here for the involvement of SNAP-25 in this process in neuroendocrine cells. The effects of BoNT/A and BoNT/B on regulated secretion were compared in intact bovine chromaffin cells to investigate the consequences of cleavage of the different targets. Catecholamine secretion elicited by Ba2+, by elevated K+ concentrations or by nicotine was prevented by each toxin. A very good correlation was observed between the extents of SNAP-25 cleavage or synaptobrevin cleavage and inhibition of secretion by BoNT/A or BoNT/B, respectively, which indicates the importance of SNAP-25 and synaptobrevin in regulated exocytosis. Despite truncation of almost the entire SNAP-25 pool by exposure of the cells to BoNT/A, a residual fraction of secretion persisted that was induced by 20microM Ca2+ (and to a lesser extent by 1 mM Ba2+) following permeabilisation. Addition of more BoNT/A failed to reduce this level of secretion. Inclusion of Mg.ATP, which greatly enhanced secretion from permeabilised cells, was required for Ca2+-stimulated or Ba2+-stimulated BoNT/A-resistant secretion. Furthermore, synaptobrevin is essential for this response because the response was not observed in BoNT/B treated cells. In view of the ability of BoNT/E to abolish secretion from permeabilised cells and to delete 26 amino acids from the C-terminus of SNAP-25, it can be deduced that cleavage of only nine residues by BoNT/A does not prevent the resultant truncated form exhibiting attenuated activity under the conditions created by permeabilisation. This identification of a novel component of secretion from permeabilised cells should facilitate investigation of the functional interaction of SNAP-25 with other proteins involved in regulated exocytosis.     

Regulation of CFTR chloride channels by syntaxin and Munc18 isoforms

The cystic fibrosis gene encodes a cyclic AMP-gated chloride channel (CFTR) that mediates electrolyte transport across the luminal surfaces of a variety of epithelial cells. The molecular mechanisms that modulate CFTR activity in epithelial tissues are poorly understood. Here we show that CFTR is regulated by an epithelially expressed syntaxin (syntaxin 1A), a membrane protein that also modulates neurosecretion and calcium-channel gating in brain. Syntaxin 1A physically interacts with CFTR chloride channels and regulates CFTR-mediated currents both in Xenopus oocytes and in epithelial cells that normally express these proteins. The physical and functional interactions between syntaxin 1A and CFTR are blocked by a syntaxin-binding protein of the Munc18 protein family (also called n-Secl). Our results indicate that CFTR function in epithelial cells is regulated by an interplay between syntaxin and Munc18 isoforms.     

Characterization of the palmitoylation domain of SNAP-25

SNAP-25 (synaptosomal associated protein of 25 kDa) is a neural specific protein that has been implicated in the synaptic vesicle docking and fusion process. It is tightly associated with membranes, and it is one of the major palmitoylated proteins found in neurons. The functional role of palmitoylation for SNAP-25 is unclear. In this report, we show that the palmitate of SNAP-25 is rapidly turned over in PC12 cells, with a half-life of approximately 3 h, and the half-life for the protein is 8 h. Mutation of Cys to Ser at positions 85, 88, 90, and 92 reduced the palmitoylation to 9, 21, 42, and 35% of the wild-type protein, respectively. Additional mutations of either Cys(85,88) or Cys(90,92) nearly abolished palmitoylation of the protein. A similar effect on membrane binding for the mutant SNAP-25 was observed, which correlated with the degree of palmitoylation. These results suggest that all four Cys residues are involved in palmitoylation and that membrane association of SNAP-25 may be regulated through dynamic palmitoylation.     

Proteolysis of SNAP-25 by types E and A botulinal neurotoxins

Clostridial neurotoxins, tetanus toxin (TeTx) and the seven related but serologically distinct botulinal neurotoxins (BoNT/A to BoNT/G), are potent inhibitors of synaptic vesicle exocytosis in nerve endings. Recently it was reported that the light chains of clostridial neurotoxins act as zinc-dependent metalloproteases which specifically cleave synaptic target proteins such as synaptobrevin/VAMPs, HPC-1/syntaxin (BoNT/C1), and SNAP-25 (BoNT/A). We show here that BoNT/E, like BoNT/A, cleaves SNAP-25, as generated by in vitro translation or by expression in Escherichia coli. BoNT/E cleaves the Arg180-Ile181 bond. This site is different from that of BoNT/A, which cleaves SNAP-25 between the amino acid residues Gln197 and Arg198. These findings further support the view that clostridial neurotoxins have evolved from an ancestral protease recognizing the exocytotic fusion machinery of synaptic vesicles whereby individual toxins target different members of the membrane fusion complex.     

Immunological analysis of potato leafroll luteovirus (PLRV) P1 expression identifies a 25 kDa RNA-binding protein derived via P1 processing

Mono- and polyclonal antibodies directed against different domains of the potato leafroll luteovirus (PLRV) P1 (ORF1) protein were applied to the analysis of P1 expression during PLRV replication in planta. Western analyses detected P1 and a protein of approximately 25 kDa (P1-C25) that accumulated to readily detectable amounts in PLRV-infected plants, but was not detected by in vitro cell-free translation of P1. P1-C25 represents the C-terminus of P1 and is a proteolytic cleavage product produced during P1 processing. On the basis of its molecular weight, the N-terminus of P1-C25 is either identical to or located adjacent to the previously identified PLRV genome-linked protein, VPg. P1-C25 is not associated with virus particles, and subcellular localization experiments detected P1-C25, but not P1, in the membrane and cytoplasmic fractions of PLRV-infected cells. In addition, P1-C25 exhibits nucleic acid-binding properties. On the basis of its biosynthesis, localization and biochemical properties, P1-C25 may facilitate the formation of P1/PLRV RNA complexes in which the spatial proximity allows for covalent bond formation between PLRV RNA and VPg.     

SNAP-23 is not cleaved by botulinum neurotoxin E and can replace SNAP-25 in the process of insulin secretion

The synaptosomal-associated protein of 25 kDa (SNAP-25) is expressed in neurons and endocrine cells. It has been shown to play an important role in the release mechanism of neurotransmitters and peptide hormones, including insulin. Thus, when insulin-secreting cells are permeabilized and treated with botulinum neurotoxin E (BoNT/E), SNAP-25 is hydrolyzed, and insulin secretion is inhibited. Recently SNAP-23, a more generally expressed isoform of SNAP-25, has been described. The functional role of SNAP-23 has not been investigated to date. It is now shown that SNAP-23 is resistant to cleavage by BoNT/E. It was therefore possible to test whether transfection of HIT (transformed pancreatic B-) cells with SNAP-23 reconstitutes insulin release from BoNT/E treated cells, in which SNAP-25 is inactivated by the toxin. The results show that SNAP-23 is able to replace SNAP-25 when it is overexpressed. While these results demonstrate that SNAP-23 is a functional homologue of SNAP-25, able to function in regulated exocytosis, they indicate that SNAP-23 may be inefficient in this process. This suggests that both isoforms may have their own specific binding partners and discrete, albeit mechanistically similar, functional roles within the cell.     

Accumulation of SNAP-25 immunoreactive material in axons of Alzheimer's disease

Neurofibrillary tangles and neuropil threads, both made of hyperphosphorylated tau proteins, point to an alteration of microtubules in Alzheimer's disease. The aim of this study was to test the consequences of these lesions on axoplasmic flow, which is dependent on intact microtubule assembly. We assessed the transport of synaptic proteins from the neuronal cell body to axonal terminals, using SNAP-25 (synaptosomal-associated protein of 25 kD) immunohistochemistry as a marker of impaired axonal transport. A sample from the supra-marginalis gyrus was obtained from 29 individuals over 75 years of age whose cognitive function had been prospectively assessed. Accumulation of immunoreactive material in swollen axons was observed in the white matter of severely demented individuals, and their number was correlated with the density of neurofibrillary tangles (r = 0.53, p = 0.005) and of focal Abeta deposits (r = 0.61, p = 0.001). This supports the hypothesis of a dysfunction of the cytoskeleton in Alzheimer's disease. An unexpected finding was the lack of correlation between SNAP-25 immunohistochemistry in the grey matter and the intellectual status or the density of neurofibrillary tangles, focal Abeta deposits and neuronal profiles. These results which question the role of synaptic markers as correlates of dementia, should be extended to other brain areas.     

Proteolysis of SNAP-25 isoforms by botulinum neurotoxin types A, C, and E: domains and amino acid residues controlling the formation of enzyme-substrate complexes and cleavage

Tetanus toxin and the seven serologically distinct botulinal neurotoxins (BoNT/A to BoNT/G) abrogate synaptic transmission at nerve endings through the action of their light chains (L chains), which proteolytically cleave VAMP (vesicle-associated membrane protein)/synaptobrevin, SNAP-25 (synaptosome-associated protein of 25 kDa), or syntaxin. BoNT/C was reported to proteolyze both syntaxin and SNAP-25. Here, we demonstrate that cleavage of SNAP-25 occurs between Arg198 and Ala199, depends on the presence of regions Asn93 to Glu145 and Ile156 to Met202, and requires about 1,000-fold higher L chain concentrations in comparison with BoNT/A and BoNT/E. Analyses of the BoNT/A and BoNT/E cleavage sites revealed that changes in the carboxyl-terminal residues, in contrast with changes in the amino-terminal residues, drastically impair proteolysis. A proteolytically inactive BoNT/A L chain mutant failed to bind to VAMP/synaptobrevin and syntaxin, but formed a stable complex (KD = 1.9 x 10(-7) M) with SNAP-25. The minimal essential domain of SNAP-25 required for cleavage by BoNT/A involves the segment Met146-Gln197, and binding was optimal only with full-length SNAP-25. Proteolysis by BoNT/E required the presence of the domain Ile156-Asp186. Murine SNAP-23 was cleaved by BoNT/E and, to a reduced extent, by BoNT/A, whereas human SNAP-23 was resistant to all clostridial L chains. Lys185Asp or Pro182Arg mutations of human SNAP-23 induced susceptibility toward BoNT/E or toward both BoNT/A and BoNT/E, respectively.