Biochemical interactions of the neuronal pentraxins. Neuronal pentraxin (NP) receptor binds to taipoxin and taipoxin-associated calcium-binding protein 49 via NP1 and NP2.

Neuronal pentraxin 1 (NP1), neuronal pentraxin 2 (NP2), and neuronal pentraxin receptor (NPR) are members of a new family of proteins identified through interaction with a presynaptic snake venom toxin taipoxin. We have proposed that these three neuronal pentraxins represent a novel neuronal uptake pathway that may function during synapse formation and remodeling. We have investigated the mutual interactions of these proteins by characterizing their enrichment on taipoxin affinity columns; by expressing NP1, NP2, and NPR singly and together in Chinese hamster ovary cells; and by generating mice that fail to express NP1. NP1 and NP2 are secreted, exist as higher order multimers (probably pentamers), and interact with taipoxin and taipoxin-associated calcium-binding protein 49 (TCBP49). NPR is expressed on the cell membrane and does not bind taipoxin or TCBP49 by itself, but it can form heteropentamers with NP1 and NP2 that can be released from cell membranes. This is the first demonstration of heteromultimerization of pentraxins and release of a pentraxin complex by proteolysis. These processes are likely to directly effect the localization and function of neuronal pentraxins in neuronal uptake or synapse formation and remodeling.

Neuronal pentraxin receptor, a novel putative integral membrane pentraxin that interacts with neuronal pentraxin 1 and 2 and taipoxin-associated calcium-binding protein 49.

We have identified the first putative integral membrane pentraxin and named it neuronal pentraxin receptor (NPR). NPR is enriched by affinity chromatography on columns of a snake venom toxin, taipoxin, and columns of the taipoxin-binding proteins neuronal pentraxin 1 (NP1), neuronal pentraxin 2 (NP2), and taipoxin-associated calcium-binding protein 49 (TCBP49). The predominant form of NPR contains an putative NH2-terminal transmembrane domain and all forms of NPR are glycosylated. NPR has 49 and 48% amino acid identity to NP1 and NP2, respectively, and NPR message is expressed in neuronal regions that express NP1 and NP2. We suggest that NPR, NP1, NP2, and TCBP49 are involved in a pathway responsible for the transport of taipoxin into synapses and that this may represent a novel neuronal uptake pathway involved in the clearance of synaptic debris.

Mirror image motifs mediate the interaction of the COOH terminus of multiple synaptotagmins with the neurexins and calmodulin.

I have previously reported that the COOH-terminal 34 amino acids of synaptotagmin 1 are capable of interacting with the presynaptic proteins, the neurexins. Multiple synaptotagmins and a synaptotagmin-like protein, rabphilin 3A, are conserved in this domain, raising the possibility that many different synaptotagmins may interact with neurexins. Here 1 report that the COOH termini of synaptotagmins 1, 2, 4, 5, 6, 7, and 9 and rabphilin 3A are capable of interacting with neurexins. The COOH terminus of rabphilin 3A is still capable or substantial enrichment of neurexins from solubilized brain membranes even though only 11 of 33 residues are identical with the COOH terminus of synaptotagmin 1. Like the purification of neurexins on the COOH terminus of synaptotagmin 1, purification by the COOH terminus of rabphilin 3A is calcium-independent. The conservation between carboxyl termini of these proteins suggests symmetrical motifs are necessary for neurexin binding. These include the sequence Leu-X-His-Trp, followed by 13 amino acids, and the sequence Trp-His-X-Lcu. Deletion of the first motif or substitution of residues in the second of these motifs greatly reduces neurexin enrichment. Interestingly, these same COOH termini yield substantial calcium-dependent enrichment of calmodulin mediated by the first of these sequence motifs. This correlates with the binding of 125I-labeled calmodulin by recombinant pieces of synaptotagmn 1 containing the carboxyl terminus. These data suggest that multiple synaptotagmins may interact with neurexins to mediate docking or regulation of neurotransmitter release and that synaptotagmins may be calcium-regulated via interaction with calmodulin.

Mouse and human neuronal pentraxin 1 (NPTX1): conservation, genomic structure, and chromosomal localization.

We have previously identified novel members of the pentraxin family (neuronal pentraxin 1 and 2) that are expressed in the nervous system. Neuronal pentraxin 1 (NP1) was identified as a rat protein that may mediate the uptake of synaptic material and the presynaptic snake venom toxin, taipoxin. NP2 was identified as a separate gene discovered by screening for a human homolog for NP1. Here, we report human cDNA and mouse genomic DNA sequences for NP1 (gene symbol NPTX1). Human NP1 and mouse NP1 show 95 and 99% amino acid identity, respectively, with rat NP1 and conserve all potential glycosylation sites. Like rat NP1, human NP1 message is large (6.5 kb) and is exclusively localized to the nervous system. The mouse NP1 gene is 13 kb in length and contains four introns that break the coding sequence of NP1 in the same positions as the introns of the human NP2 gene. The human and mouse NP1 genes are localized to chromosome 17q25.1-q25.2 and chromosome 11e2-e1.3, respectively. These data demonstrate the existence of a separate family of pentraxin proteins that are expressed in the human brain and other tissues and that may play important roles in the uptake of extracellular material.

Human neuronal pentraxin II (NPTX2): conservation, genomic structure, and chromosomal localization.

We have previously identified a novel rat neuronal member of the pentraxin family (neuronal pentraxin) that may mediate the uptake of synaptic material and the presynaptic snake venom toxin, taipoxin. Here we report human cDNA and genomic sequences of a second neuronal pentraxin. This pentraxin, which we propose to name neuronal pentraxin II (NPII; gene symbol NPTX2), shows 54% amino acid identity to rat neuronal pentraxin (NPI) with 69% identity over the carboxyl-terminal half of NPI and is 88% identical to a newly identified sperm acrosomal pentraxin p50/apexin. Northern blot analysis reveals that NPII message is present in brain, testis, pancreas, liver, heart, and skeletal muscle, so, unlike NPI, NPII is not exclusively localized to neurons. Like NPI, NPII has potential N-linked glycosylation sites. The human NPII gene is 11 kb in length, contains four introns, and is localized to chromosome 7q21.3-q22.1. These data demonstrate the existence of a family of pentraxin proteins that are expressed in the brain and other tissues and that may play important roles in the uptake of extracellular material.

Novel reticular calcium binding protein is purified on taipoxin columns.

We identified, by affinity chromatography, two putative binding proteins for the presynaptic snake venom toxin taipoxin. We have previously characterized one of these proteins [neuronal pentraxin (NP)] as a neuronally secreted protein with homology to acute-phase proteins. Here we report the identification of the second protein as a 49-kDa lumenal calcium binding protein that we have named taipoxin-associated calcium binding protein 49 (TCBP-49). This protein contains six EF-hand putative calcium binding domains and the carboxyl-terminal sequence His-Asp-Glu-Leu (HDEL), identical to the yeast endoplasmic reticulum retention signal. Message for this protein is present in brain, liver, muscle, heart, kidney, and testis. Antibodies to this protein label reticular organelles of neurons and glia. This localization and the specific enrichment of native and recombinant TCBP-49 on columns of immobilized taipoxin raise the possibility that this protein interacts with internalized taipoxin, perhaps mediating its activation. The availability of pure TCBP-49 will allow direct tests of whether TCBP-49 alters the integrity of the oligomeric structure, phospholipase activity, or toxicity of taipoxin.

Neuronal pentraxin, a secreted protein with homology to acute phase proteins of the immune system.

We have identified, by affinity chromatography, a binding protein for the snake venom toxin taipoxin. The sequence of this 47 kDa protein is unique, is characteristic of a secreted protein, and has homology to the acute phase proteins serum amyloid P protein and C-reactive protein of the pentraxin family. We have named this protein neuronal pentraxin (NP), as Northern analysis and in situ hybridization demonstrate high message levels in neurons of cerebellum, hippocampus, and cerebral cortex. Because NP may be released synaptically and has homology to immune proteins potentially involved in uptake of lipidic, toxic, or other antigenic material, we suggest that NP may be involved in a general uptake of synaptic macromolecules.

Genetic and electrophysiological studies of Drosophila syntaxin-1A demonstrate its role in nonneuronal secretion and neurotransmission.

Cloning and characterization of the Drosophila syntaxin-1A gene, syx-1A, reveal that it is present in several tissues but is predominantly expressed in the nervous system, where it is localized to axons and synapses. We have generated an allelic series of loss-of-function mutations that result in embryonic lethality with associated morphological and secretory defects dependent on the severity of the mutant allele. Electrophysiological recordings from partial loss-of-function mutants indicate absence of endogenous synaptic transmission at the neuromuscular junction and an 80% reduction of evoked transmission. Complete absence of syx-1A causes subtle morphological defects in the peripheral and central nervous systems, affects nonneural secretory events, and entirely abolishes neurotransmitter release. These data demonstrate that syntaxin plays a key role in nonneuronal secretion and is absolutely required for evoked neurotransmission.

The COOH terminus of synaptotagmin mediates interaction with the neurexins.

The interaction of the synaptic vesicle protein, synaptotagmin, and the presynaptic alpha-latrotoxin receptor, a neurexin, has been proposed to be involved in docking of synaptic vesicles at active sites or modulation of neurotransmitter release. Here I report the investigation of the domain of synaptotagmin responsible for this interaction. Pieces of synaptotagmin containing the carboxyl terminus are capable of purifying neurexins from solubilized brain homogenates. Pieces as small as a synthesized peptide corresponding to the COOH-terminal 34 amino acids are capable of enriching neurexins 100-fold. The binding of neurexins to synaptotagmin is calcium-independent and of moderate affinity. This COOH-terminal segment of synaptotagmin is conserved in all species characterized to date. Reflective of this, a synthetic peptide corresponding to the carboxyl terminus of Drosophila synaptotagmin is capable of purification of rat neurexins, suggesting the possibility that this interaction may also exist in Drosophila. I propose that the carboxyl terminus of synaptotagmin binds to the carboxyl terminus of the neurexins and that this interaction may mediate docking of synaptic vesicles or modulation of neurotransmitter release.

Expression of synaptotagmin in Drosophila reveals transport and localization of synaptic vesicles to the synapse.

Synaptotagmin is a synaptic vesicle-specific integral membrane protein that has been suggested to play a key role in synaptic vesicle docking and fusion. By monitoring Synaptotagmin's cellular and subcellular distribution during development, it is possible to study synaptic vesicle localization and transport, and synapse formation. We have initiated the study of Synaptotagmin's expression during Drosophila neurogenesis in order to follow synaptic vesicle movement prior to and during synapse formation, as well as to localize synaptic sites in Drosophila. In situ hybridizations to whole-mount embryos show that synaptotagmin (syt) message is present in the cell bodies of all peripheral nervous system neurons and many, if not all, central nervous system neurons during neurite outgrowth and synapse formation, and in mature neurons. Immunocytochemical staining with antisera specific to Synaptotagmin indicates that the protein is present at all stages of the Drosophila life cycle following germ band retraction. In embryos, Synaptotagmin is only transiently localized to the cell body of neurons and is transported rapidly along axons during axonogenesis. After synapse formation, Synaptotagmin accumulates in a punctate pattern at all identifiable synaptic contact sites, suggesting a general role for Synaptotagmin in synapse function. In embryos and larvae, the most intense staining is found along two broad longitudinal tracts on the dorsal side of the ventral nerve cord and the brain, and at neuromuscular junctions in the periphery. In the adult head, Synaptotagmin localizes the discrete regions of the neurophil where synapses are predicted to occur. These data indicate that synaptic vesicles are present in axons before synapse formation, and become restricted to synaptic contact sites after synapses are formed. Since a similar expression pattern of Synaptotagmin has been reported in mammals, we propose that the function of Synaptotagmin and the mechanisms governing localization of the synaptic vesicle before and after synapse formation are conserved in invertebrate and vertebrate species. The ability to mark synapses in Drosophila should facilitate the study of synapse formation and function, providing a new tool to dissect the molecular mechanisms underlying these processes.

Exo-endocytotic recycling of synaptic vesicles in developing processes of cultured hippocampal neurons.

In mature neurons synaptic vesicles (SVs) undergo cycles of exo-endocytosis at synapses. It is currently unknown whether SV exocytosis and recycling occurs also in developing axons prior to synapse formation. To address this question, we have developed an immunocytochemical assay to reveal SV exo-endocytosis in hippocampal neurons developing in culture. In this assay antibodies directed against the lumenal domain of synaptotagmin I (Syt I), an intrinsic membrane protein of SVs, are used to reveal exposure of SV membranes at the cell surface. Addition of antibodies to the culture medium of living neurons for 1 hr at 37 degrees C resulted in their rapid and specific internalization by all neuronal processes and, particularly, by axons. Double immunofluorescence and electron microscopy immunocytochemistry indicated that the antibodies were retained within SVs in cell processes and underwent cycles of exo-endocytosis in parallel with SV membranes. In contrast, another endocytotic marker, wheat germ agglutinin, was rapidly cleared from the processes and transported to the cell body. Antibody-labeled SVs were still present in axons several days after antibody loading and became clustered at presynaptic sites in parallel with synaptogenesis. These results demonstrate that SVs undergo multiple cycles of exo-endocytosis in developing neuronal processes irrespective of the presence of synaptic contacts.

Binding of synaptotagmin to the alpha-latrotoxin receptor implicates both in synaptic vesicle exocytosis.

A vertebrate neurotoxin, alpha-latrotoxin, from black widow spider venom causes synaptic vesicle exocytosis and neurotransmitter release from presynaptic nerve terminals. Although the mechanism of action of alpha-latrotoxin is not known, it does require binding of alpha-latrotoxin to a high-affinity receptor on the presynaptic plasma membrane. The alpha-latrotoxin receptor seems to be exclusively at the presynaptic plasmamembrane. Here we report that the alpha-latrotoxin receptor specifically binds to a synaptic vesicle protein, synaptotagmin, and modulates its phosphorylation. Synaptotagmin is a synaptic vesicle-specific membrane protein that binds negatively charged phospholipids and contains two copies of a putative Ca(2+)-binding domain from protein kinase C (the C2-domain), suggesting a regulatory role in synaptic vesicle fusion. Our findings suggest that a physiological role of the alpha-latrotoxin receptor may be the docking of synaptic vesicles at the active zone. The direct interaction of the alpha-latrotoxin receptor with a synaptic vesicle protein also suggests a mechanism of action for this toxin in causing neurotransmitter release.

Structure of the 116-kDa polypeptide of the clathrin-coated vesicle/synaptic vesicle proton pump.

A 116-kDa polypeptide has recently been found to be a common component of vacuolar proton pumps isolated from a variety of sources. The 116-kDa subunit of the proton pump was purified from clathrin-coated vesicles of bovine brain, and internal sequences were obtained from proteolytic peptides. Oligonucleotide probes designed from these peptide sequences were utilized in polymerase chain reactions to isolate partial bovine cDNA clones for the protein. Sequences from these were then utilized to isolate rat brain cDNA clones containing the full-length coding region. RNA blots indicate the presence of an abundant 3.9-kilobase message for the 116-kDa subunit in brain, and primer extension analysis demonstrates that the cloned sequence is full-length. The rat cDNA sequences predict synthesis of a protein of 96,267 Da. Analysis of the deduced amino acid sequence of the 116-kDa subunit suggests that it consists of two fundamental domains: a hydrophilic amino-terminal half that is composed of greater than 30% charged residues, and a hydrophobic carboxyl-terminal half that contains at least six transmembrane regions. The structural properties of the 116-kDa proton pump polypeptide agree well with its proposed function in coupling ATP hydrolysis by the cytoplasmic subunits to proton translocation by the intramembranous components of the pump.

Domain structure of synaptotagmin (p65)

Synaptotagmin (p65) is an abundant and evolutionarily conserved protein of synaptic vesicles that contains two copies of an internal repeat homologous to the regulatory region of protein kinase C. In the current study, we have investigated the biochemical properties of synaptotagmin, demonstrating that it contains five protein domains: an intravesicular amino-terminal domain that is glycosylated but lacks a cleavable signal sequence; a single transmembrane region; a sequence separating the transmembrane region from the two repeats homologous to protein kinase C; the two protein kinase C-homologous repeats; and a conserved carboxyl-terminal sequence following the two repeats homologous to protein kinase C. Sucrose density gradient centrifugations and gel electrophoresis indicate that synaptotagmin monomers associate into dimers and are part of a larger molecular weight complex. A sequence predicted to form an amphipathic alpha-helix that may cause the stable dimerization of synaptotagmin is found in its third domain between the transmembrane region and the protein kinase C-homologous repeats. Synaptotagmin contains a single hypersensitive proteolytic site that is located immediately amino-terminal to the amphipathic alpha-helix, suggesting that synaptotagmin contains a particularly exposed region as the peptide backbone emerges from the dimer. Finally, subcellular fractionation and antibody bead purification demonstrate that synaptotagmin co-purifies with synaptophysin and other synaptic vesicle markers in brain. However, in the adrenal medulla, synaptotagmin was found in both synaptophysin-containing microvesicles and in chromaffin granules that are devoid of synaptophysin, suggesting a shared role for synaptotagmin in the exocytosis of small synaptic vesicles and large dense core catecholaminergic vesicles.

Structural and functional conservation of synaptotagmin (p65) in Drosophila and humans.

Synaptotagmin (p65) is an abundant synaptic vesicle protein that contains two copies of a sequence that is homologous to the regulatory region of protein kinase C. Full length cDNAs encoding human and Drosophila synaptotagmins were characterized to study its structural and functional conservation in evolution. The deduced amino acid sequences for human and rat synaptotagmins show 97% identity, whereas Drosophila and rat synaptotagmins are only 57% identical but exhibit a selective conservation of the two internal repeats that are homologous to the regulatory region of protein kinase C (78% invariant residues in all three species). The two internal repeats of synaptotagmin are only slightly more homologous to each other than to protein kinase C, and the differences between the repeats are conserved in evolution, suggesting that they might not be functionally equivalent. The cytoplasmic domains of human and Drosophila synaptotagmins produced as recombinant proteins in Escherichia coli specifically bound phosphatidylserine similar to rat synaptotagmin. They also hemagglutinated trypsinized erythrocytes at nanomolar concentrations. Hemagglutination was inhibited both by negatively charged phospholipids and by a recombinant fragment from rat synaptotagmin that contained only a single copy of the two internal repeats. Together these results demonstrate that synaptotagmin is highly conserved in evolution compatible with a function in the trafficking of synaptic vesicles at the active zone. The similarity of the phospholipid binding properties of the cytoplasmic domains of rat, human, and Drosophila synaptotagmins and the selective conservation of the sequences that are homologous to protein kinase C suggest that these are instrumental in phospholipid binding. The human gene for synaptotagmin was mapped by Southern blot analysis of DNA from somatic cell hybrids to chromosome 12 region cen-q21, and the Drosophila gene by in situ hybridization to 23B.

Two novel annexins from Drosophila melanogaster. Cloning, characterization, and differential expression in development.

The annexins are a family of homologous Ca2(+)- and phospholipid-binding proteins that until now have only been found in vertebrates. cDNA clones encoding two novel annexins from Drosophila melanogaster were isolated and characterized. RNA blots indicate that the messages for the two Drosophila proteins are differentially expressed in development, with one message being expressed throughout development, while the other is only found in early embryos and adult flies. In situ hybridizations localize the two Drosophila genes to 93B and 19A-4,7. A similarly high degree of homology relates Drosophila annexins to different vertebrate annexins, indicating that the Drosophila annexins are not the invertebrate homologues of particular mammalian annexins but that they constitute novel members of the annexin gene family. In continuation with a recently established terminology, the Drosophila annexins will be named annexins IX and X. The biochemical properties of Drosophila annexin X were investigated using recombinant protein. Similar to vertebrate annexins, annexin X bound to liver membranes and liposomes containing phosphatidylserine in a calcium-dependent manner but not to liposomes containing phosphatidylcholine. In addition, annexin X partitioned into the detergent phase of Triton X-114 as a function of calcium. The conservation of the annexin family of Ca2(+)-binding proteins in invertebrates suggests that they have a basic function in cells which is not peculiar to vertebrate biology, and the availability of the Drosophila sequences will open avenues for mutational studies of these functions.

Phospholipid binding by a synaptic vesicle protein homologous to the regulatory region of protein kinase C.

Neurotransmitters are released at synapses by the Ca2(+)-regulated exocytosis of synaptic vesicles, which are specialized secretory organelles that store high concentrations of neurotransmitters. The rapid Ca2(+)-triggered fusion of synaptic vesicles is presumably mediated by specific proteins that must interact with Ca2+ and the phospholipid bilayer. We now report that the cytoplasmic domain of p65, a synaptic vesicle-specific protein that binds calmodulin contains an internally repeated sequence that is homologous to the regulatory C2-region of protein kinase C (PKC). The cytoplasmic domain of recombinant p65 binds acidic phospholipids with a specificity indicating an interaction of p65 with the hydrophobic core as well as the headgroups of the phospholipids. The binding specificity resembles PKC, except that p65 also binds calmodulin, placing the C2-regions in a context of potential Ca2(+)-regulation that is different from PKC. This is a novel homology between a cellular protein and the regulatory domain of protein kinase C. The structure and properties of p65 suggest that it may have a role in mediating membrane interactions during synaptic vesicle exocytosis.

rab3 is a small GTP-binding protein exclusively localized to synaptic vesicles.

rab3, a low molecular weight GTP-binding protein, is primarily expressed in brain, where it is present in soluble and membrane-bound forms. Membrane-bound rab3 in brain is exclusively localized on synaptic vesicles, the secretory organelles of the synapse that store and release neurotransmitters. rab3 is also expressed in endocrine tissues such as the adrenal medulla, where it is found together with other synaptic vesicle proteins on microvesicles distinct from chromaffin granules. The tight binding of rab3 to membranes correlates with hydrophobic modifications that are different in the membrane-bound and soluble forms of rab3. The results demonstrate the exclusive targeting of a small GTP-binding protein to secretory vesicles of a subset of the regulated pathway of secretion.

Synapsins: mosaics of shared and individual domains in a family of synaptic vesicle phosphoproteins.

Synapsins are neuronal phosphoproteins that coat synaptic vesicles, bind to the cytoskeleton, and are believed to function in the regulation of neurotransmitter release. Molecular cloning reveals that the synapsins comprise a family of four homologous proteins whose messenger RNA's are generated by differential splicing of transcripts from two genes. Each synapsin is a mosaic composed of homologous amino-terminal domains common to all synapsins and different combinations of distinct carboxyl-terminal domains. Immunocytochemical studies demonstrate that all four synapsins are widely distributed in nerve terminals, but that their relative amounts vary among different kinds of synapses. The structural diversity and differential distribution of the four synapsins suggest common and different roles of each in the integration of distinct signal transduction pathways that modulate neurotransmitter release in various types of neurons.

Interactions of liposomes with planar bilayer membranes.

The adhesion to horizontal, planar lipid membranes of lipid vesicles containing calcein in the aqueous compartment or fluorescent phospholipids in the membranes has been examined by phase contrast, differential interference contrast and fluorescence microscopy. With water-immersion lenses, it was possible to study the interactions of vesicles with planar bilayers at magnifications up to the useful limit of light microscopy. In the presence of 15 mM calcium chloride, vesicles composed of phosphatidylserine and either phosphatidylethanolamine or soybean lipids adhere to the torus, bilayer and lenses of planar bilayers of the same composition. Lenses of solvent appear at the site where vesicles attach to decane-based bilayers and lipid fluorophores move from the vesicles to the lenses. Because the calcein contained in such vesicles is not released, we interpret this as indicating fusion of only the outer monolayer (hemifusion) of the vesicles with the decane lenses. In the case of squalene-based black lipid membranes (BLMs), in contrast, vesicles do not nucleate lenses but they apparently do fuse with the torus at the bilayer boundary. Interactions leading to hemifusions between vesicles and planar membranes thus occur predominantly in regions where hydrocarbon solvent is present. Osmotic water flow, induced by addition of urea to the compartment containing vesicles, causes coalescence of lenses in decane-based BLMs as well as coalescence of the aqueous spaces of the vesicles that have undergone hemifusion with the lenses. We did not observe transfer of the aqueous phase of vesicles to the trans side of either decane- or squalene-based planar membranes; however, we cannot rule out the possibility particularly in the latter case, that rupture of the planar membrane may have been an immediate result of vesicle fusion and thus precluded its detection.