Distinct self-oligomerization activities of synaptotagmin family. Unique calcium-dependent oligomerization properties of synaptotagmin VII

Synaptotagmins constitute a large protein family, characterized by one transmembrane region and two C2 domains, and can be classified into several subclasses based on phylogenetic relationships and biochemical activities (Fukuda, M., Kanno, E., and Mikoshiba, K. (1999) J. Biol. Chem. 274, 31421-31427). Synaptotagmin I (Syt I), a possible Ca(2+) sensor for neurotransmitter release, showed both Ca(2+)-dependent (via the C2 domain) and -independent (via the NH(2)-terminal domain) self-oligomerization, which are thought to be important for synaptic vesicle exocytosis. However, little is known about the relationship between these two interactions and the Ca(2+)-dependent oligomerization properties of other synaptotagmin isoforms. In this study, we first examined the Ca(2+)-dependent self-oligomerization properties of synaptotagmin family by co-expression of T7- and FLAG-tagged Syts (full-length or cytoplasmic domain) in COS-7 cells. We found that Syt VII is a unique class of synaptotagmins that only showed robust Ca(2+)-dependent self-oligomerization at the cytoplasmic domain with EC(50) values of about 150 micrometer Ca(2+). In addition, Syt VII preferentially interacted with the previously described subclass of Syts (V, VI, and X) in a Ca(2+)-dependent manner. Co-expression of full-length and cytoplasmic portion of Syts VII (or II) indicate that Syt VII cytoplasmic domain oligomerizes in a Ca(2+)-dependent manner without being tethered at the NH(2)-terminal domain, whereas Ca(2+)-dependent self-oligomerization at the cytoplasmic domain of other isoforms (e.g. Syt II) occurs only when the two molecules are tethered at the NH(2)-terminal domain.     

Expression and regulation of SNAP-25 and synaptotagmin VII in developing mouse ovarian follicles via the FSH receptor

Soluble-NSF attachment protein receptor (SNARE) proteins play a role in vesicle fusion, exocytosis, and intracellular trafficking in neuronal cells as well as in fertilization and embryogenesis. We investigated the expression patterns of two SNARE proteins, SNAP-25 and synaptotagmin VII (SytVII), and their regulation by pregnant mare serum gonadotropin (PMSG) during mouse ovarian follicular development. Ovaries were obtained at 0, 12, 24, 36, and 48 h post-PMSG injection of immature mice. SNAP-25 and SytVII mRNA expression levels increased gradually in a time-dependant manner. However, protein levels revealed different patterns of expression, suggesting different translational regulation following PMSG stimulation. SNAP-25 and SytVII expression was closely associated with thickening of the granulosa cell (GC) layer and follicle morphological changes from a flattened to a cuboidal shape. To explore follicle stimulating hormone receptor (FSHR)-mediated regulation of their expression, GCs from preantral follicles were cultured to examine the effects of FSHR siRNA knockdown. FSHR siRNA abolished upregulation of the SNAREs in both PMSG and FSH-stimulated GCs. This abolished gene expression was rescued by adding dibutyryl cyclic AMP to the cultures. These results suggest that SNAP-25 and SytVII expression is regulated via the FSHR-cAMP pathway during follicular development.     

C-terminal ECFP fusion impairs synaptotagmin 1 function: crowding out synaptotagmin 1

To allow the monitoring of synaptotagmin 1 trafficking in vivo, we generated transgenic mice expressing a synaptotagmin 1-enhanced cyan fluorescent protein (ECFP) fusion protein under control of the Thy1 promoter. Transgenic synaptotagmin 1-ECFP is expressed throughout the brain where it localizes to synapses and marks synapses in vivo. However, when we crossed transgenic synaptotagmin 1-ECFP mice with synaptotagmin 1 knock-out mice, we detected no rescue of survival or function. Furthermore, viral overexpression of synaptotagmin 1-ECFP in synaptotagmin 1-deficient neurons failed to restore normal Ca2+-triggered release, whereas overexpression of wild type synaptotagmin 1 did so efficiently. To determine whether synaptotagmin 1-ECFP is non-functional because the ECFP-fusion interferes with its biochemical activities, we measured Ca2+-independent binding of synaptotagmin 1-ECFP to SNARE complexes, and Ca2+-dependent binding of synaptotagmin 1-ECFP to phospholipids and to itself. Although the apparent Ca2+ affinity of synaptotagmin 1-ECFP was decreased compared with wild type synaptotagmin 1, we observed no major changes in Ca2+-dependent or -independent activities, indicating that the non-functionality of the synaptotagmin 1-ECFP fusion protein was not because of inactivation of its biochemical properties. These data suggest that synaptotagmin 1-ECFP is suitable for monitoring synaptic vesicle traffic in vivo because the synaptotagmin 1-ECFP marks synaptic vesicles without participating in exocytosis. In addition, the data demonstrate that synaptotagmin 1 function requires a free C terminus, possibly because of spatial constraints at the release sites.     

Regulation of insulin secretion and GLUT4 trafficking by the calcium sensor synaptotagmin VII

Insulin regulates blood glucose by promoting uptake by fat and muscle, and inhibiting production by liver. Insulin-stimulated glucose uptake is mediated by GLUT4, which translocates from an intracellular compartment to the plasma membrane. GLUT4 traffic and insulin secretion both rely on calcium-dependent, regulated exocytosis. Deletion of the voltage-gated potassium channel Kv1.3 results in constitutive expression of GLUT4 at the plasma membrane. Inhibition of channel activity stimulated GLUT4 translocation through a calcium dependent mechanism. The synaptotagmins (Syt) are calcium sensors for vesicular traffic, and Syt VII mediates lysosomal and secretory granule exocytosis. We asked if Syt VII regulates insulin secretion by pancreatic beta cells, and GLUT4 translocation in insulin-sensitive tissues mouse model. Syt VII deletion (Syt VII -/-) results in glucose intolerance and a marked decrease in glucose-stimulated insulin secretion in vivo. Pancreatic islet cells isolated from Syt VII -/- cells secreted significantly less insulin than islets of littermate controls. Syt VII deletion disrupted GLUT4 traffic as evidenced by constitutive expression of GLUT4 present at the plasma membrane of fat and skeletal muscle cells and unresponsiveness to insulin. These data document a key role for Syt VII in peripheral glucose homeostasis through its action on both insulin secretion and GLUT4 traffic.     

There's more to life than neurotransmission: the regulation of exocytosis by synaptotagmin VII

Among the 16 known vertebrate synaptotagmins, only Syt I, IV and VII are also present in C. elegans and Drosophila, suggesting that these isoforms play especially important roles in vivo. Extensive evidence indicates that Syt I is a synaptic vesicle Ca(2+) sensor essential for rapid neurotransmitter release. It has been suggested that the ubiquitously expressed Syt VII also regulates synaptic vesicle exocytosis, despite its presence in several tissues in addition to the brain. Here, we discuss recent genetic and biochemical evidence that does not support this view. Syt VII null mutants do not have a neurological phenotype, and the protein is found on the membrane of lysosomes and some non-synaptic secretory granules, where it regulates Ca(2+)-triggered exocytosis and plasma membrane repair.     

Mechanism of the calcium-dependent multimerization of synaptotagmin VII mediated by its first and second C2 domains

The Ca(2+)-dependent oligomerization activity of the second C2 (C2B) domain of synaptotagmin I (Syt I) has been hypothesized to regulate neurotransmitter release. We previously showed that the cytoplasmic domains of several other Syt isoforms also show Ca(2+)-dependent oligomerization activity (Fukuda, M., and Mikoshiba, K. (2000) J. Biol. Chem. 275, 28180-28185), but little is known about the involvement of their C2 domains in Ca(2+)-dependent oligomerization. In this study, we analyzed the Ca(2+)-dependent oligomerization properties of the first (C2A) and the second C2 (C2B) domains of Syt VII. Unlike Syt I, both C2 domains of Syt VII contribute to Ca(2+)-dependent homo- and hetero-oligomerization with other isoforms. For instance, the Syt VII C2A domain Ca(2+)-dependently binds itself and the C2A domain of Syt VI but not its C2B domain, whereas the Syt VII C2B domain Ca(2+)-dependently binds itself and the C2B domain of Syt II but not its C2A domain. In addition, we showed by gel filtration that a single Syt VII C2 domain is sufficient to form a Ca(2+)-dependent multimer of very high molecular weight. Because of this "two handed" structure, the Syt VII cytoplasmic domain has been found to show the strongest Ca(2+)-dependent multimerization activity in the Syt family. We also identified Asn-328 in the C2B domain as a crucial residue for the efficient Ca(2+)-dependent switch for multimerization by site-directed mutagenesis. Our results suggest that Syt VII is a specific isoform that can cluster different Syt isoforms with two hands in response to Ca(2+).     

Palmitoylation-dependent association with CD63 targets the Ca2+ sensor synaptotagmin VII to lysosomes

Syt VII is a Ca(2+) sensor that regulates lysosome exocytosis and plasma membrane repair. Because it lacks motifs that mediate lysosomal targeting, it is unclear how Syt VII traffics to these organelles. In this paper, we show that mutations or inhibitors that abolish palmitoylation disrupt Syt VII targeting to lysosomes, causing its retention in the Golgi complex. In macrophages, Syt VII is translocated simultaneously with the lysosomal tetraspanin CD63 from tubular lysosomes to nascent phagosomes in a Ca(2+)-dependent process that facilitates particle uptake. Mutations in Syt VII palmitoylation sites block trafficking of Syt VII, but not CD63, to lysosomes and phagosomes, whereas tyrosine replacement in the lysosomal targeting motif of CD63 causes both proteins to accumulate on the plasma membrane. Complexes of CD63 and Syt VII are detected only when Syt VII palmitoylation sites are intact. These findings identify palmitoylation-dependent association with the tetraspanin CD63 as the mechanism by which Syt VII is targeted to lysosomes.     

The calcium-binding loops of the tandem C2 domains of synaptotagmin VII cooperatively mediate calcium-dependent oligomerization

Synaptotagmin VII (Syt VII), a proposed regulator for Ca2+-dependent exocytosis, showed a robust Ca2+-dependent oligomerization property via its two C2 domains (Fukuda, M., and Mikoshiba, K. (2001) J. Biol. Chem. 276, 27670-27676), but little is known about its structure or the critical residues directly involved in the oligomerization interface. In this study, site-directed mutagenesis and chimeric analysis between Syt I and Syt VII showed that three Asp residues in Ca2+-binding loop 1 or 3 (Asp-172, Asp-303, and Asp-357) are crucial to robust Ca(2+)-dependent oligomerization. Unlike Syt I, however, the polybasic sequence in the beta4 strands of the C2 structures (so-called "C2 effector domain") is not involved in the Ca2+-dependent oligomerization of Syt VII. The results also showed that the Ca2+-binding loops of the two C2 domains cooperatively mediate Syt VII oligomerization (i.e. the presence of redundant Ca2+-binding site(s)) as well as the importance of Ca2+-dependent oligomerization of Syt VII in Ca2+-regulated secretion. Expression of wild-type tandem C2 domains of Syt VII in PC12 cells inhibited Ca2+-dependent neuropeptide Y release, whereas mutant fragments lacking Ca2+-dependent oligomerization activity had no effect. Finally, rotary-shadowing electron microscopy showed that the Ca2+-dependent oligomer of Syt VII is "a large linear structure," not an irregular aggregate. By contrast, in the absence of Ca2+ Syt VII molecules were observed to form a globular structure. Based on these results, we suggest that the linear Ca2+-dependent oligomer may be aligned at the fusion site between vesicles and plasma membrane and modulate Ca2+-regulated exocytosis by opening or dilating fusion pores.     

Autonomous function of synaptotagmin 1 in triggering synchronous release independent of asynchronous release

Ca(2+) triggers neurotransmitter release in at least two principal modes, synchronous and asynchronous release. Synaptotagmin 1 functions as a Ca(2+) sensor for synchronous release, but its role in asynchronous release remains unclear. We now show that in cultured cortical neurons stimulated at low frequency (or Hz), deletion of synaptotagmin 1 also alters only synchronous, not asynchronous, release during the stimulus train, but dramatically enhances "delayed asynchronous release" following the stimulus train. Thus synaptotagmin 1 functions as an autonomous Ca(2+) sensor independent of asynchronous release during isolated action potentials and action potential trains, but restricts asynchronous release induced by residual Ca(2+) after action potential trains. We propose that synaptotagmin 1 occupies release "slots" at the active zone, possibly in a Ca(2+)-independent complex with SNARE proteins that are freed when action potential-induced Ca(2+) influx activates synaptotagmin 1.     

凝血因子VII基因表达载体的构建及其在BHK细胞中的表达

凝血因子Ⅶ是凝血块形成的起始关键分子之一,它对外源性凝血途径的启动具有重要的作用。为表达具有促凝活性的重组人凝血因子Ⅶ,通过PCR的方法从质粒pUC-FⅦ中扩增人凝血因子ⅦcDNA,并将其定向克隆入真核表达载体pIRESneo中,构建重组表达载体pIRES-FⅦ,测序正确后用脂质体介导的方法转染BHK-21细胞,经G418加压筛选、细胞有限稀释等方法获得克隆细胞株,收集无皿清培养上清,进行SDS-PAGE、Western blot和活性鉴定。结果成功构建了重组表达载体pIRES-FⅦ,实现了其在BHK-21细胞中的表达,且表达产物具有促凝活性。重组人凝血因子Ⅶ在哺乳动物细胞中的成功表达,为整体止血剂的研究奠定了基础。     

Impaired cytolytic activity in calreticulin-deficient CTLs

Calreticulin is an endoplasmic reticulum-resident chaperone that is stored in the cytotoxic granules of CTLs and NK cells and is released with granzymes and perforin upon recognition of target cells. To investigate the role of calreticulin in CTL-mediated killing, we generated CTL lines from crt(+/+) and crt(-/-) mice expressing a constitutively active form of calcineurin in the heart. Crt(-/-) CTLs showed reduced cytotoxic activity toward allogeneic target cells despite normal production, intracellular localization, and activity of granzymes and despite perforin overexpression. Comparable or higher amounts of granzymes were degranulated by crt(-/-) cells in response to immobilized anti-CD3 Abs, indicating that calreticulin is dispensable for the signal transduction that leads to granule exocytosis. The ability to form conjugates with target cells was affected in the crt(-/-) CTLs, explaining the observed reduction in cytotoxicity. Conjugate formation and cytotoxicity were completely restored by treatments that facilitate recognition and contact with target cells, a prerequisite for degranulation and killing. Therefore, we conclude that calreticulin is dispensable for the cytolytic activity of granzymes and perforin, but it is required for efficient CTL-target cell interaction and for the formation of the death synapse.     

Examining synaptotagmin 1 function in dense core vesicle exocytosis under direct control of Ca2+

We tested the long-standing hypothesis that synaptotagmin 1 is the Ca2+ sensor for fast neurosecretion by analyzing the intracellular Ca2+ dependence of large dense-core vesicle exocytosis in a mouse strain carrying a mutated synaptotagmin C2A domain. The mutation (R233Q) causes a twofold increase in the KD of Ca2+-dependent phospholipid binding to the double C2A-C2B domain of synaptotagmin. Using photolysis of caged calcium and capacitance measurements we found that secretion from mutant cells had lower secretory rates, longer secretory delays, and a higher intracellular Ca2+-threshold for secretion due to a twofold increase in the apparent KD of the Ca2+ sensor for fast exocytosis. Single amperometric fusion events were unchanged. We conclude that Ca2+-dependent phospholipid binding to synaptotagmin 1 mirrors the intracellular Ca2+ dependence of exocytosis.     

Calcium-dependent and -independent hetero-oligomerization in the synaptotagmin family

Synaptotagmins constitute a family of membrane proteins that are characterized by one transmembrane region and two C2 domains. Recent genetic and biochemical studies have indicated that oligomerization of synaptotagmin (Syt) I is important for expression of function during exocytosis of synaptic vesicles. However, little is known about hetero-oligomerization in the synaptotagmin family. In this study, we showed that the synaptotagmin family is a type I membrane protein (N(lumen)/C(cytoplasm)) by introducing an artificial N-glycosylation site at the N-terminal domain, and systematically examined all the possible combinations of hetero-oligomerization among synaptotagmin family proteins (Syts I-XI). We classified the synaptotagmin family into four distinct groups based on differences in Ca(2+)-dependent and -independent oligomerization activity. Group A Syts (III, V, VI, and X) form strong homo- and hetero-oligomers by disulfide bonds at an N-terminal cysteine motif irrespective of the presence of Ca(2+) [Fukuda, M., Kanno, E., and Mikoshiba, K. (1999) J. Biol. Chem. 274, 31421-31427]. Group B Syts (I, II, VIII, and XI) show moderate homo-oligomerization irrespective of the presence of Ca(2+). Group C synaptotagmins are characterized by weak Ca(2+)-dependent (Syts IX) or no homo-oligomerization activity (Syt IV). Syt VII (Group D) has unique Ca(2+)-dependent homo-oligomerization properties with EC(50) values of about 150 microM Ca(2+) [Fukuda, M., and Mikoshiba, K. (2000) J. Biol. Chem. 275, 28180-28185]. Syts IV, VIII, and XI did not show any apparent hetero-oligomerization activity, but some sets of synaptotagmin isoforms can hetero-oligomerize in a Ca(2+)-dependent and/or -independent manner. Our data suggest that Ca(2+)-dependent and -independent hetero-oligomerization of synaptotagmins may create a variety of Ca(2+)-sensors.     

Synaptotagmin在神经递质释放过程中的作用

神经突触间递质的释放是神经系统完成其生理功能最重要的生物现象之一。在贮存递质的突触囊泡上存在一些神经细胞所特有的囊泡蛋白,如突触素（synapsin）、synaptobrevin和synaptotagmin等。其中synaptotagmins是膜转运蛋白中的一个家族,它们的特征是含有两个钙结合区：C2A和C2B。到目前为止,在哺乳动物中已经发现了15种synaptotagmin同形物（isoforms）。神经递质释放是由Ca^2＋内流以诱导突触囊泡发生胞吐而引起的,Ca^2＋需与细胞内部的Ca^2＋感受器相结合来协同控制囊泡胞吐释放,SynaptotagminⅠ可能作为快速同步释放的Ca^2＋感受器而发挥作用。现在已知synaptotagminⅠ在胞吐和胞吞两个过程中都扮演重要角色。     

SNAP-25 and synaptotagmin 1 function in Ca2+-dependent reversible docking of granules to the plasma membrane

In neuroendocrine cells, Ca²⁺ triggers fusion of granules with the plasma membrane and functions at earlier steps by increasing the size of the readily releasable pool of vesicles. The effect of Ca²⁺ at early steps of secretion may be due to the recruitment at the plasma membrane of granules localized in the cytoplasm. To study the mechanism of granule docking, a new in vitro assay is designed using membrane fractions from mouse pituitary AtT-20 cells. By using this assay, it is found that granule docking to the plasma membrane is controlled by Ca²⁺ concentrations in the micromolar range, is reversible and requires intact SNAP-25, but not VAMP-2. In the docking assay, addition of Ca²⁺ induces the formation of a SNAP-25-Synaptotagmin 1 complex. The cytosolic domain C2AB of Synaptotagmin 1 and anti-Synaptotagmin 1 antibodies block granule docking. These results show that Ca²⁺ modulates dynamic docking of granules to the plasma membrane and that this process is due to a Ca²⁺-dependent interaction between SNAP-25 and Synaptotagmin 1 .     

Protein kinase C-mediated phosphorylation of the two polybasic regions of synaptotagmin VI regulates their function in acrosomal exocytosis

We have previously reported that synaptotagmin VI is present in human sperm cells and that a recombinant protein containing the C2A and C2B domains abrogates acrosomal exocytosis in permeabilized spermatozoa, an effect that was regulated by phosphorylation. In this report, we show that each individual C2 domain blocks acrosomal exocytosis. The inhibitory effect was completely abrogated by phosphorylation of the domains with purified PKCbetaII. We found by site-directed mutagenesis that Thr418 and/or Thr419 in the polybasic region (KKKTTIK) of the C2B domain--a key region for the function of synaptotagmins--are the PKC target that regulates its inhibitory effect on acrosomal exocytosis. Similarly, we showed that Thr284 in the polybasic region of C2A (KCKLQTR) is the target for PKC-mediated phosphorylation in this domain. An antibody that specifically binds to the phosphorylated polybasic region of the C2B domain recognized endogenous phosphorylated synaptotagmin in the sperm acrosomal region. The antibody was inhibitory only at early stages of exocytosis in sperm acrosome reaction assays, and the immunolabeling decreased upon sperm stimulation, indicating that the protein is dephosphorylated during acrosomal exocytosis. Our results indicate that acrosomal exocytosis is regulated through the PKC-mediated phosphorylation of conserved threonines in the polybasic regions of synaptotagmin VI.     

Expression and function of periostin-isoforms in bone

Periostin was originally identified in MC3T3-E1 osteoblast-like cells. We have identified an isoform of periostin referred to as periostin-like-factor (PLF). It is homologous to other proteins such as fasciclin I (fas I), MPB70, betaIG-H3, and Algal-CAMs. All of these proteins are implicated in regulating cell adhesion. PLF and the other isoforms of periostin differ in their C-terminal sequences. PLF and periostin differ in two specific regions, between 673 and 699 amino acids (aa) and 785-812 aa. Periostin isoforms are expressed in vivo and in vitro during the stages of osteoblast differentiation and maturation. Their mRNAs are present in pre-osteoblast cells as detected by in situ hybridization, and the proteins are between 86 and 93 kD in size as determined by Western blot analysis. Antisense oligonucleotides and antibodies directed against the isoforms of periostin were used to block the activity of these proteins. In both cases, the levels of osteoblast-specific-differentiation markers were markedly reduced suggesting a role for these proteins in osteoblast differentiation.