Mass spectrometrical characterisation of mouse and rat synapsin isoforms 2a and 2b.

Synapsin 2 proteins are key elements of the synaptic machinery and still hold the centre stage in neuroscience research. Although fully sequenced at the nucleic acid level in mouse and rat, structural information on amino acid sequences and post-translational modifications (PTMs) is limited. Knowledge on protein sequences and PTMs, however, is mandatory for several purposes including conformational studies and the generation of antibodies. Hippocampal proteins from rat and mouse were extracted, run on two-dimensional gel electrophoresis and multi-enzyme digestion was carried out to generate peptides for mass spectrometrical analysis [nano-LC-ESI-(CID/ETD)-MS/MS]. As much as 12 synapsin 2 proteins (6 alpha and 6 beta isoforms) in the mouse and 13 synapsin 2 proteins (6 alpha and 7 beta isoforms) were observed in the rat. Protein sequences were highly identical to nucleic acid sequences, and only few amino acid exchanges probably representing polymorphisms or sequence conflicts were detected. Mouse and rat synapsins 2a differed in three amino acids, while rat and mouse synapsins 2b differed in two amino acids. As much as 13 phosphorylation sites were determined by MS/MS data in rat and mouse hippocampus and 5 were verified by phosphatase treatment. These findings are important for interpretation of previous results and design of future studies on synapsins.

Interactions of synapsin I with phospholipids: possible role in synaptic vesicle clustering and in the maintenance of bilayer structures.

Synapsin I is a synaptic vesicle-specific phosphoprotein composed of a globular and hydrophobic head and of a proline-rich, elongated and basic tail. Synapsin I binds with high affinity to phospholipid and protein components of synaptic vesicles. The head region of the protein has a very high surface activity, strongly interacts with acidic phospholipids and penetrates the hydrophobic core of the vesicle membrane. In the present paper, we have investigated the possible functional effects of the interaction between synapsin I and vesicle phospholipids. Synapsin I enhances both the rate and the extent of Ca(2+)-dependent membrane fusion, although it has no detectable fusogenic activity per se. This effect, which appears to be independent of synapsin I phosphorylation and localized to the head region of the protein, is attributable to aggregation of adjacent vesicles. The facilitation of Ca(2+)-induced liposome fusion is maximal at 50-80% of vesicle saturation and then decreases steeply, whereas vesicle aggregation does not show this biphasic behavior. Association of synapsin I with phospholipid bilayers does not induce membrane destabilization. Rather, 31P-nuclear magnetic resonance spectroscopy demonstrated that synapsin I inhibits the transition of membrane phospholipids from the bilayer (L alpha) to the inverted hexagonal (HII) phase induced either by increases in temperature or by Ca2+. These properties might contribute to the remarkable selectivity of the fusion of synaptic vesicles with the presynaptic plasma membrane during exocytosis.

Matrix-assisted laser desorption mass spectrometric peptide mapping of proteins separated by two-dimensional gel electrophoresis: determination of phosphorylation in synapsin I.

A technique is described for the rapid, sensitive analysis of posttranslational modifications of proteins that have been separated by 2-dimensional electrophoresis and blotted onto a membrane with a cationic surface. The isolated protein spots visualized by reverse staining of the blotting membrane are excised, washed, and subjected to chemical (cyanogen bromide) and/or enzymatic (endoproteinase Lys-C) degradation directly on the membrane. The resulting mixture of peptide fragments is extracted from the membrane into a solution that is compatible with matrix-assisted laser desorption mass spectrometric analysis and analyzed without fractionation. Relatively accurate (+/- 1 Da) mass determination of these peptide fragments provides a facile and sensitive means for detecting the presence of modifications and for correlating such modifications with the differential mobility of different isoforms of a given protein during 2-dimensional electrophoresis. The technique is applied to the determination of sites of phosphorylation in synapsins Ia and Ib, neuronal phosphoproteins that are believed to function in the regulation of neurotransmitter release and are substrates for cAMP and Ca2+/calmodulin-dependent protein kinases, which appear to control their biological activity.

Neuronal compartments and axonal transport of synapsin I.

Studies on the transport kinetics and the posttranslational modification of synapsin I in mouse retinal ganglion cells were performed to obtain an insight into the possible factors involved in forming the structural and functional differences between the axon and its terminals. Synapsin I, a neuronal phosphoprotein associated with small synaptic vesicles and cytoskeletal elements at the presynaptic terminals, is thought to be involved in modulating neurotransmitter release. The state of phosphorylation of synapsin I in vitro regulates its interaction with both synaptic vesicles and cytoskeletal components, including microtubules and microfilaments. Here we present the first evidence that in the mouse retinal ganglion cells most synapsin I is transported down the axon, together with the cytomatrix proteins, at the same rate as the slow component b of axonal transport, and is phosphorylated at both the head and tail regions. In addition, our data suggest that, after synapsin I has reached the nerve endings, the relative proportions of variously phosphorylated synapsin I molecules change, and that these changes lead to a decrease in the overall content of phosphorus. These results are consistent with the hypothesis that, in vivo, the phosphorylation of synapsin I along the axon prevents the formation of a dense network that could impair organelle movement. On the other hand, the dephosphorylation of synapsin I at the nerve endings may regulate the clustering of small synaptic vesicles and modulate neurotransmitter release by controlling the availability of small synaptic vesicles for exocytosis.

Cloning and sequencing analysis of a human synapsin IIb-encoding brain cDNA.

Cloning and sequencing of the gene encoding human synapsin IIb, an abundant peripheral membrane protein of synaptic vesicles, is described. The human and rat coding sequences showed 89% nucleotide and 94% amino acid sequence homology.

Determination of synapsin I and synaptophysin in body fluids by two-site enzyme-linked immunosorbent assays.

Two-site enzyme-linked immunosorbent assays (ELISA) have been established for the specific and sensitive determination of two membrane proteins of the small synaptic vesicles (SSV), namely: peripheral synapsin I and integral synaptophysin. The ELISA used highly specific capture monoclonal antibodies (mAB) and polyclonal antibodies (pAB) as detectors. For synapsin I, the mAB were newly generated, whereas for synaptophysin, the commercially available mAB SY38 was applied. In order to calibrate the ELISA and to raise pAB, both proteins were purified in the mg-range. Synapsin I was purified by conventional means from human and porcine brain and synaptophysin was purified by immunoaffinity chromatography from porcine brain. Using the ELISA, neither synapsin I nor synaptophysin could be determined in serum or cerebrospinal fluid (CSF) from healthy donors or patients suffering various neurological disorders or pheochromocytomas. For this reason, the degradation of both proteins in serum and CSF was investigated. With the exception of synaptophysin measured in serum, both proteins exhibited fast rates of degradation. Despite the negative results in human body fluids, the two ELISA are appropriate for the quantification of these membrane proteins in neuronal or neuroendocrine cell extracts or preparations of SSV.

Synapsin I identified as a novel brain-specific autoantigen.

BACKGROUND: We report the identification and characterization of a novel 74-kd brain-specific autoantigen that is reactive with serum from a patient with discoid lupus erythematosus and chronic lymphocytic leukemia. METHODS: We determined the molecular weight, tissue distribution and subcellular distribution of the autoantigen and obtained limited amino acid sequence after purification by ion-exchange chromatography and trypsin digestion. RESULTS: We identified the 74-kd autoantigen as synapsin I on the basis of the following observations. First, the autoantigen has properties consistent with synapsin I: molecular weight of approximately equals 74 kd, brain-specific distribution, presence in cytosol and on synaptosomes, and association with taxol-stabilized microtubules. Second, limited amino acid sequence determination after trypsin digestion of the autoantigen shows identity with synapsin I. Third, the autoimmune serum immunoblots fusion proteins that incorporate rat synapsin Ia. The autoantibodies reactive to synapsin Ia are of immunoglobulin (Ig) G and IgM class. CONCLUSIONS: This is the first report of autoantibodies that are reactive to synapsin Ia. Autoantibodies that are reactive to synapsin Ia are not restricted to discoid lupus erythematosus patients, because we found identical reactivity in two of 18 sera from dsDNA-positive systemic lupus erythematosus patients and in two of 14 rheumatoid factor-positive sera. Whether autoantibodies to synapsin I are associated with neuropsychiatric manifestations is currently unknown.

Development of a high-throughput AlphaScreen assay for modulators of synapsin I phosphorylation in primary neurons.

Alterations in synaptic transmission have been implicated in a number of psychiatric and neurological disorders. The discovery of small-molecule modulators of proteins that regulate neurotransmission represents a novel therapeutic strategy for these diseases. However, high-throughput screening (HTS) approaches in primary neurons have been limited by challenges in preparing and applying primary neuronal cultures under conditions required for generating sufficiently robust and sensitive HTS assays. Synapsin I is an abundant presynaptic protein that plays a critical role in neurotransmission through tethering synaptic vesicles to the actin cytoskeleton. It has several phosphorylation sites that regulate its modulation of synaptic vesicle trafficking and, therefore, the efficacy of synaptic transmission. Here, we describe the development of a rapid, sensitive, and homogeneous assay to detect phospho-synapsin I (pSYN1) in primary cortical neurons in 384-well plates using AlphaScreen technology. From results of a pilot screening campaign, we show that the assay can identify compounds that modulate synapsin I phosphorylation via multiple signaling pathways. The implementation of the AlphaScreen pSYN1 assay and future development of additional primary neuronal HTS assays provides an attractive approach for discovery of novel classes of therapeutic candidates for a variety of CNS disorders.

Synapsin I is a major endogenous substrate for protein L-isoaspartyl methyltransferase in mammalian brain.

The accumulation of potentially deleterious L-isoaspartyl linkages in proteins is prevented by the action of protein L-isoaspartyl O-methyltransferase, a widely distributed enzyme that is particularly active in mammalian brain. Methyltransferase-deficient (knock-out) mice exhibit greatly increased levels of isoaspartate and typically succumb to fatal epileptic seizures at 4-10 weeks of age. The link between isoaspartate accumulation and the neurological abnormalities of these mice is poorly understood. Here, we demonstrate that synapsin I from knock-out mice contains 0.9 +/- 0.3 mol of isoaspartate/mol of synapsin, whereas the levels in wild-type and heterozygous mice are undetectable. Transgenic mice that selectively express methyltransferase only in neurons show reduced levels of synapsin damage, and the degree of reduction correlates with the phenotype of these mice. Isoaspartate levels in synapsin from the knock-out mice are five to seven times greater than those in the average protein from brain cytosol or from a synaptic vesicle-enriched fraction. The isoaspartyl sites in synapsin from knock-out mice are efficiently repaired in vitro by incubation with purified methyltransferase and S-adenosyl-L-methionine. These findings demonstrate that synapsin I is a major substrate for the isoaspartyl methyltransferase in neurons and suggest that isoaspartate-related alterations in the function of presynaptic proteins may contribute to the neurological abnormalities of mice deficient in this enzyme.

Simultaneous purification of the neuroproteins synapsin I and synaptophysin.

A procedure for the simultaneous purification of synapsin I and synaptophysin from calf brain was developed. Demyelinated membranes were extracted with 2% Triton X-100 and 2 M KCl. The extracted proteins were separated by weak cation-exchange chromatography on carboxymethyl-Sepharose Fast Flow. Synaptophysin was finally purified by preparative sodium dodecyl sulphate-polyacrylamine gel electrophoresis and synapsin I by affinity chromatography using a calmodulin-Sepharose column. The recovery obtained was 40 micrograms/g in brain for synaptophysin and 25 micrograms/g in brain for synapsin I.

Electrotransfer of fixed phosphoproteins from pieces of dried polyacrylamide gel to small disks of nitrocellulose, nylon or polyvinylidene difluoride.

A simple method for the transfer of 32P-labeled proteins from dried polyacrylamide gels to small disks of nitrocellulose, nylon or polyvinylidene difluoride (PVDF) is described. Gel pieces containing the desired phosphoprotein are rehydrated in buffer containing sodium dodecyl sulfate (SDS) and sealed in agarose in a glass tube over a supporting gel of polyacrylamide. Protein is transferred upwards through a discontinuous density gradient of SDS-buffer and methanol to a disk of membrane sealed to the mouth of the tube with dialysis membrane. The method allows the concentration of a phosphoprotein present in several gel pieces to a single disk of immobilized membrane. Recovery of phosphoprotein was at least as good as obtained with conventional electroblotting. Application of the method to the analysis of the phosphoamino acid content of the astrocyte marker, glial fibrillary acidic protein, is described.

Synapsin IIa bundles actin filaments.

Synapsins are neuron-specific phosphoproteins associated with small synaptic vesicles in the presynaptic nerve terminal. Synapsin I, which has been demonstrated to bundle F-actin in vitro, has been postulated to regulate neurotransmitter release by cross-linking synaptic vesicles to the actin cytoskeleton. To investigate the possible interaction of synapsin II with actin filaments, we expressed synapsin II in Spodoptera frugiperda and High Five insect cells using a recombinant baculovirus. Purified recombinant synapsin IIa was incubated with F-actin, and bundle formation was evaluated by light scattering and electron microscopy. Synapsin IIa was found to bundle actin filaments. Dose-response curves indicated that synapsin IIa was more potent than synapsin I in bundling actin filaments. These data suggest that synapsin IIa may cross-link synaptic vesicles and actin filaments in the nerve terminal.

Neuronal nitric-oxide synthase localization mediated by a ternary complex with synapsin and CAPON.

The specificity of the reactions of nitric oxide (NO) with its neuronal targets is determined in part by the precise localizations of neuronal NO synthase (nNOS) within the cell. The targeting of nNOS is mediated by adapter proteins that interact with its PDZ domain. Here, we show that the nNOS adapter protein, CAPON, interacts with synapsins I, II, and III through an N-terminal phosphotyrosine-binding domain interaction, which leads to a ternary complex comprising nNOS, CAPON, and synapsin I. The significance of this ternary complex is demonstrated by changes in subcellular localization of nNOS in mice harboring genomic deletions of both synapsin I and synapsin II. These results suggest a mechanism for specific actions of NO at presynaptic sites.

Synapsin IIa: expression in insect cells, purification, and characterization.

Synapsin IIa belongs to a family of neuron-specific phosphoproteins called synapsins, which are associated with synaptic vesicles in presynaptic nerve terminals. In order to examine the biochemical properties of synapsin IIa, and ultimately its physiological function, purified protein is required. Since attempts to purify significant quantities of synapsin IIa, an isoform of the synapsins, from mammalian brain have proven difficult, we undertook the production of recombinant synapsin IIa by utilizing the baculovirus expression system. Rat synapsin IIa cDNA was introduced into the baculovirus genome via homologous recombination, and the recombinant baculovirus was purified. Spodoptera frugiperda (Sf9) cells infected with this virus expressed synapsin IIa as 5% of the total cellular protein. The recombinant protein was extracted from the particulate fraction of the infected Sf9 cells with salt and a nonionic detergent and purified by immunoaffinity chromatography. The purified synapsin IIa was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase to a stoichiometry of 0.8 mol of phosphate/mol of protein. Metabolic labeling with [32P]Pi demonstrated synapsin IIa phosphorylation in infected Sf9 cells. Using a homogenate of uninfected Sf9 cells, a cAMP-dependent protein kinase activity which can phosphorylate synapsin IIa was detected. Limited proteolysis of recombinant synapsin IIa phosphorylated in vitro and in vivo resulted in identical phosphopeptide maps. Further, synapsin IIa, like synapsin I, binds with high affinity in a saturable manner to synaptic vesicles purified from rat cortex.

Cyclic inhibition-potentiation of the crosslinking of synapsin I with brain microtubules by protein kinase FA (an activator of ATP.Mg-dependent protein phosphatase).

The ATP.Mg-dependent type-1 protein phosphatase activating factor (FA) was identified as a protein kinase that could phosphorylate synapsin I, a neuronal protein that coats synaptic vesicles, binds to cytoskeleton and is believed to be involved in the modulation of neurotransmission. More importantly, more than 90% of the phosphates in 32P-synapsin I phosphorylated by FA could be removed by the activated ATP.Mg-dependent type-1 protein phosphatase and the synapsin I phosphatase activity was found to be strictly FA-dependent. Functional study further revealed that as a synapsin I kinase, factor FA could phosphorylate synapsin I and thereby inhibits crosslinking of synapsin I with tubulin, while as a synapsin I phosphatase activator, FA could promote the crosslinking copolymerization of synapsin I with tubulin. Taken together, the results provide initial evidence that a cyclic modulation of the crosslinking copolymerization of synapsin I with brain microtubules can be controlled by factor FA, representing an efficient cyclic cascade control mechanism for the regulation of axonal transport process during neurotransmission.

Development of synapsin I and synapsin II in intraocular hippocampal transplants.

Previous studies have indicated that the appearance of synaptic vesicle-associated proteins known as the synapsins is one indicator of synapse formation. In this study, the levels and morphological distribution of synapsin I and synapsin IIa and IIb were studied in intraocular hippocampal transplants and in situ in the intact hippocampus. No detectable levels of either synapsin I or synapsin II were found in the fetal brain. The in situ levels of the synapsins exhibited parallel increases rapidly after birth, reaching peak levels at 8 weeks, after which a slight decline was noted in synapsin I and synapsin IIb. In hippocampal transplants, a comparable increase in the synapsins was seen during the first 8 weeks in oculo. It is likely that the synapse formation in the hippocampal transplants represents synapses from neurons within the transplant, as well as from various peripheral ganglia that send collaterals into the graft. Peripheral and central synapses express different synapsin I: synapsin IIa and IIb ratios. When the ratios of the synapsin proteins in hippocampal transplants were examined ratios essentially identical to those seen in the normal hippocampus were found, despite the numerous peripheral neurites innervating the grafts. Immunohistochemical studies supported the immunoblot data, showing no detectable immunofluorescence with synapsin antibodies in fetal or newborn hippocampal formation. The density of immunoreactive profiles increased substantially both in transplants and in the hippocampal formation in situ during the first 2 postnatal months. In conclusion, the present data demonstrate that hippocampal transplants in oculo can develop significant levels of the synapsins and that there is no time lag in development in these levels compared to the hippocampal formation in situ.

Synapsin is a novel Rab3 effector protein on small synaptic vesicles. I. Identification and characterization of the synapsin I-Rab3 interactions in vitro and in intact nerve terminals.

Synapsins, a family of neuron-specific phosphoproteins, have been demonstrated to regulate the availability of synaptic vesicles for exocytosis by binding to both synaptic vesicles and the actin cytoskeleton in a phosphorylation-dependent manner. Although the above-mentioned observations strongly support a pre-docking role of the synapsins in the assembly and maintenance of a reserve pool of synaptic vesicles, recent results suggest that the synapsins may also be involved in some later step of exocytosis. In order to investigate additional interactions of the synapsins with nerve terminal proteins, we have employed phage display library technology to select peptide sequences binding with high affinity to synapsin I. Antibodies raised against the peptide YQYIETSMQ (syn21) specifically recognized Rab3A, a synaptic vesicle-specific small G protein implicated in multiple steps of exocytosis. The interaction between synapsin I and Rab3A was confirmed by photoaffinity labeling experiments on purified synaptic vesicles and by the formation of a chemically cross-linked complex between synapsin I and Rab3A in intact nerve terminals. Synapsin I could be effectively co-precipitated from synaptosomal extracts by immobilized recombinant Rab3A in a GTP-dependent fashion. In vitro binding assays using purified proteins confirmed the binding preference of synapsin I for Rab3A-GTP and revealed that the COOH-terminal regions of synapsin I and the Rab3A effector domain are required for the interaction with Rab3A to occur. The data indicate that synapsin I is a novel Rab3 interactor on synaptic vesicles and suggest that the synapsin-Rab3 interaction may participate in the regulation of synaptic vesicle trafficking within the nerve terminals.

Src interacts with dynamin and synapsin in neuronal cells.

The nonreceptor tyrosine kinase Src is expressed at a high level in cells that are specialized for regulated secretion, such as the neuron, and is concentrated on secretory vesicles or at the site of exocytosis. To investigate the possibility that Src may play a role in regulating membrane traffic, we searched for neuronal proteins that will interact with Src. The SH3 domain of Src, but not that of the splice variant N-Src, bound to three proteins from mouse synaptosomes or PC12 cells: dynamin, synapsin Ia, and synapsin Ib. Dynamin and the synapsins coprecipitated with Src from PC12 cell extracts, and they colocalized with a subset of Src in the PC12 cell by immunofluorescence. Neither dynamin nor the synapsins were phosphorylated by Src, suggesting that the interaction of these proteins serves to direct the kinase activity of Src toward other proteins in the vesicle population. In immunoprecipitates containing Src and dynamin, the clathrin adaptor protein alpha-adaptin was also found. The association of Src and synapsin suggests a role for Src in the life cycle of the synaptic vesicle. The identification of a complex containing Src, dynamin, and alpha-adaptin indicates that Src may play a more general role in membrane traffic as well.

Synapsins as major neuronal Ca2+/S100A1-interacting proteins.

The mammalian S100A1 protein can activate the invertebrate myosin-associated giant protein kinase twitchin in a Ca(2+)-dependent manner by more than 1000-fold in vitro; however, no mammalian S100-dependent protein kinases are known. In an attempt to identify novel mammalian Ca(2+)/S100A1-regulated protein kinases, brain extracts were subjected to combined Ca(2+)-dependent affinity chromatography with S100A1 and an ATP analogue. This resulted in the purification to near-homogeneity of the four major synapsin isoforms Ia, Ib, IIa and IIb. All four synapsins were specifically affinity-labelled with the ATP analogue 5'-p-fluorosulphonylbenzoyladenosine. S100A1 bound to immobilized synapsin IIa in BIAcore experiments in a Ca(2+)-dependent and Zn(2+)-enhanced manner with submicromolar affinity; this interaction could be competed for with synthetic peptides of the proposed S100A1-binding sites of synapsin. Double-labelling confocal immunofluorescence microscopy demonstrated that synapsins and S100A1 are both present in the soma and neurites of PC12 cells, indicating their potential to interact in neurons in vivo.

Acidic calmodulin binding protein, ACAMP-81, is MARCKS protein interacting with synapsin I.

ACAMP-81 is an acidic calmodulin binding protein with molecular mass of 81 kDa. We report partial amino acid analysis of ACAMP-81 and its interaction with synapsin I. 123 amino acids of ACAMP-81 were determined and the sequence was completely identical with that of MARCKS protein which was thought to be a substrate for calcium/phospholipid dependent protein kinase (PKC). We found ACAMP-81 bound to synapsin I with 125I-labeled ACAMP-81 overlay method. ACAMP-81 bound to the cysteine specific cleaved 51 kDa fragment derived from middle/tail region of synapsin I.