Synaptotagmin在免疫细胞胞吐中的作用

Synaptotagmin(Syt)是一大类膜转运蛋白,现已发现13个成员,被认为是神经、内分泌、免疫以及其它细胞胞吐过程中的主要钙传感器(calcium sensor).本文综述了Syt的结构、基因、功能以及其在几种免疫细胞中的表达和作用.     

Synaptotagmin Ⅱ在RBL-2H3细胞系表达及在胞吐中的作用研究

研究SynaptotagminII(简称Syt2)在大鼠嗜碱性白血病细胞(RBL-2H3,简称RBL)中的表达、分布及其在胞吐中的作用。通过Westernblot检测Syt2在RBL中的表达情况。用高保真酶扩增Syt2,构建pEGFP-N1-Syt2表达质粒,电穿孔转染RBL,G418筛选获得稳定表达Syt2-EGFP的细胞。通过钙离子载体和抗原刺激,应用Westernblot检测稳定转染细胞和对照细胞分泌的组织蛋白酶D,分析Syt2对胞吐的影响。结果显示,在RBL中检测到Syt2表达。构建了pEGFP-N1-Syt2质粒,插入片段测序结果与GenBank登录号NM012665(ratSyt2)序列完全一致。经转染和G418筛选,获得了稳定表达细胞RBL-Syt2-S,经刺激后,其分泌的组织蛋白酶D较对照明显减少。证实Syt2主要分布在RBL的质膜,在胞浆中表达较少,Syt2对于RBL溶酶体胞吐起负调控作用。     

Synaptotagmin在免疫细胞胞吐中的作用

Synaptotagmin(Syt)是一大类膜转运蛋白，现已发现13个成员，被认为是神经、内分泌、免疫以及其它细胞胞吐过程中的主要钙传感器(calcium sensor)。本文综述了Syt的结构、基因、功能以及其在几种免疫细胞中的表达和作用。     

钙网蛋白与心血管疾病

钙网蛋白是内质网中主要的Ca2 结合分子伴侣,具有调控细胞钙稳态、蛋白质合成与修饰等作用,在生物体内钙网蛋白具有调节细胞凋亡、应激、心血管炎症反应等多种生理和病理生理过程的功能。钙网蛋白在心脏发育、心肌肥大、缺血再灌注和血管生成等过程中的作用是心血管领域的研究热点问题之一。     

反义RNA抑制synaptotagminⅡ在RBL-2H3的表达及对胞吐的作用

目的：研究SynaptotagminⅡ（简称syt2）在RBL-2H3（简称RBL）中的表达及其在胞吐中的作用。方法：通过Westernblot检测Syt2在RBL中的表达情况。用高保真酶扩增Syt2，与pEGFP-N1构建全长反义基因表达载体，电穿孔转染RBL，G418筛选获得稳定转染细胞。通过钙离子载体和抗原刺激，应用Westernblot检测稳定转染细胞和对照细胞分泌的组织蛋白酶D，分析Syl2对胞吐的影响。结果：在RBL中检测到Syt2表达。构建了pEGFP-N1-Syt2-AS质粒，插入片段测序结果与GenBank登录号NM012665（ratsyt2）序列完全一致。经转染和G418筛选，获得了稳定转染细胞RBL-Syt2-AS，Westernblot结果显示，两株RBL-Syt2-AS表达的syt2均明显减少，分别只有对照的8％和10％。经刺激后，RBL-Syt2．AS分泌的组织蛋白酶D较对照明显增加。结论：Syt在RBL中表达，其对RBL溶酶体胞吐起负调控作用。     

突触结合蛋白-1与学习记忆的研究进展

<正>突触结合蛋白-1(synaptotagmin-1,Syt-1)是脑内小突触囊泡和大致密核心囊泡特有的膜内在蛋白质,是在膜融合机制中起重要作用的一个蛋白,可以作为Ca2+感受器调节刺激偶联的快速化学突触传递,对胞吐及递质释放过程皆有影响。本文旨在讨论Syt-1通过调节神经递质释放进而影响到     

25kD蛋白在人骨骼肌中的定位及在人其他组织中的表达

25 k D蛋白是一种新的与重症肌无力相关的蛋白质。为了检测该蛋白在人体不同组织中的表达并明确其在人骨骼肌中的亚细胞定位。本研究提取和纯化 2 5 k D蛋白 ,免疫小鼠制备了 2 5 k D蛋白的抗体 ;用免疫印迹方法验证了抗体的特异性并检测2 5 k D蛋白在成年人及胎儿 8种不同组织中的表达 ;用免疫组化和免疫电镜方法明确了 2 5 k D蛋白在骨骼肌中的细胞及亚细胞定位。结果显示 ,2 5 k D蛋白抗体只与肌肉匀浆样品中 2 5 k D蛋白特异性反应。在成年人及胎儿 8种不同组织中 ,2 5 k D蛋白只见于骨骼肌。免疫组化可见在肌膜下和横纹上有不均匀的免疫染色 ;免疫电镜显示在肌膜下有电子致密物沉积 ,肌丝中未见有电子致密物沉积。这些结果表明 :2 5 k D蛋白抗体具有很高的特异性 ,2 5 k D蛋白只表达于人骨骼肌中 ,其亚细胞定位在骨骼肌的肌膜下。     

人鼻咽癌细胞系CNE2分泌蛋白质谱的初步建立

目的：建立鼻咽癌（nasopharyngeal carcinoma,NPC）细胞系CNE2细胞的分泌蛋白质谱。方法：采用无血清培养法培养CNE2细胞,超滤法脱盐并浓缩细胞培养上清制备分泌蛋白质,采用双向凝胶电泳技术（two-dimensional electrophoresis,2-DE)分离蛋白质,应用基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption/ionization time of flight mass spectrometry, MALDI-TOF-MS)鉴定蛋白质,利用生物信息学资源对所鉴定的蛋白质的功能和亚细胞定位进行分析。结果：建立了CNE2细胞分泌蛋白质的2-DE图谱,鉴定了52种非冗余的CNE2细胞分泌蛋白质,并对52种蛋白质按功能和亚细胞分布进行了初步分类。结论：初步建立CNE2细胞的分泌蛋白质谱,为研究鼻咽癌分泌蛋白质在鼻咽癌发生发展中的作用提供了有意义的资料。     

SM蛋白家族在分泌过程中的作用机制研究进展

Secl/Munc-18(SM)蛋白是一类与分泌相关的亲水性蛋白质,能以多种结合方式与可溶性N-乙基马莱酰胺敏感因子(NSF)附着蛋白受体(SNARE)的Syntaxin蛋白家族结合,进而参与细胞的分泌调控。虽然SM蛋白对于细胞分泌至关重要,但是SM蛋白调节细胞分泌的分子机制尚不清楚。SM蛋白可能主要在囊泡的锚定(docking)过程中起作用,但是也有文献表明SM蛋白可能也参与锚定以后的启动(priming)、融合(fusion)等分泌步骤中的调控。     

Copines蛋白家族的研究进展

Copines蛋白家族是新近发现的一类分布广泛、进化保守的Ca2 依赖性磷脂结合蛋白,它们的结构特征为:N端有2个C2结构域,与突触结合蛋白(synaptotagmin)和蛋白激酶C的C2结构域不但高度同源,而且同样具有Ca2 依赖性的磷脂结合功能;C端有1个A结构域,与整合蛋白A结构域部分同源,是蛋白与蛋白相互作用的结构域。近年来研究表明,Copines家族成员与细胞的膜转运、胞内信号转导、肿瘤发生等重大生物过程都密切相关。     

Synaptotagmin function in dense core vesicle exocytosis studied in cracked PC12 cells

Ca(2+)-triggered dense-core vesicle exocytosis in PC12 cells does not require vesicular synaptotagmins 1 and 2, but may use plasma membrane synaptotagmins 3 and 7 as Ca(2+) sensors. In support of this hypothesis, C(2) domains from the plasma membrane but not vesicular synaptotagmins inhibit PC12 cell exocytosis. Ca(2+) induces binding of both plasma membrane and vesicular synaptotagmins to phospholipids and SNAREs (soluble N-ethylmaleimide-sensitive attachment protein receptors), although with distinct apparent Ca(2+) affinities. Here we used gain-of-function C(2)-domain mutants of synaptotagmin 1 and loss-of-function C(2)-domain mutants of synaptotagmin 7 to examine how synaptotagmins function in dense-core vesicle exocytosis. Our data indicate that phospholipid- but not SNARE-binding by plasma membrane synaptotagmins is the primary determinant of Ca(2+)-triggered dense-core vesicle exocytosis. These results support a general lipid-based mechanism of action of synaptotagmins in exocytosis, with the specificity of various synaptotagmins for different types of fusion governed by their differential localizations and Ca(2+) affinities.     

Functional involvement of synaptotagmin I/II C2A domain in neurite outgrowth of chick dorsal root ganglion neuron

Synaptotagmin I or II (Syt I/II) is involved in Ca²⁺-regulated exocytosis of secretory vesicles, probably serving as a Ca²⁺-sensor via its C2A domain. Synaptotagmin is also known to be expressed in neuronal growth cone vesicles, but its functional involvement in neurite outgrowth remains largely unknown. In this study, we examined the function of Syt I/II in neurite outgrowth in cultured chick dorsal root ganglion neurons using an anti-synaptotagmin I and II C2A domain (anti-STI/II-C2A) antibody that inhibits Ca²⁺-regulated exocytosis. Immunoblots confirmed the high specificity of the anti-STI/II-C2A antibody and showed the expression of synaptotagmin I or II in chick dorsal root ganglion neurons. Immunocytochemistry revealed that synaptotagmin I or II is enriched at the growth cone region of chick dorsal root ganglion neurons, in both lamellipodia and filopodia. Whole or Fab-fragment of the anti-STI/II-C2A antibody loaded into dorsal root ganglion neurons by trituration significantly inhibited neurite outgrowth, whereas preimmune IgG had no effect. These results showed that the C2A domain of synaptotagmin I or II plays a crucial role in neurite outgrowth.     

Recessive congenital myasthenic syndrome caused by a homozygous mutation in SYT2 altering a highly conserved C‐terminal amino acid sequence

Defects in the gene encoding synaptotagmin 2 (SYT2) have been linked to a presynaptic congenital myasthenic syndrome (CMS) and motor neuropathies. However, to date only dominant forms of the disease have been described. We report here a consanguineous patient with a severe recessive form of presynaptic CMS and denervation atrophy caused by the homozygous mutation c.1191delG, p.Arg397Serfs*37 in SYT2. The affected 2‐year‐old girl had profound weakness and areflexia with moderate bulbar deficit. Repetitive nerve stimulation revealed an extreme reduction of compound muscle action potential amplitudes at rest, with a striking facilitation followed by a progressive decline at fast stimulation rates. These findings were reminiscent, but not identical to those seen in the Lambert–Eaton myasthenic syndrome. 3,4 diaminopyridine and pyridostigmine were effective to ameliorate muscle fatigue, but albuterol was ineffective. Modeling of the mutation using the rat Syt1 C2B x‐ray structure revealed that Arg397Serfs*37 disrupts a highly conserved amino acid sequence at the bottom face of the C2B domain not directly involved in calcium binding, but crucial for synaptotagmin‐SNARE interaction and exocytosis. Thus, this report describes a recessive form of synaptotagmin 2‐CMS and highlights the importance of the synaptotagmin C‐terminal on synaptic vesicle fusion and exocytosis.     

G protein betagamma directly regulates SNARE protein fusion machinery for secretory granule exocytosis

The activation of G protein-coupled receptors (GPCRs) can result in an inhibition of Ca(2+)-dependent hormone and neurotransmitter secretion. This has been attributed in part to G protein inhibition of Ca(2+) influx. However, a frequently dominant inhibitory effect, of unknown mechanism, also occurs distal to Ca(2+) entry. Here we characterize direct inhibitory actions of G protein betagamma (Gbetagamma) on Ca(2+)-triggered vesicle exocytosis in permeable PC12 cells. Gbetagamma inhibition was rapid (<1 s) and was attenuated by cleavage of synaptosome-associated protein of 25 kD (SNAP25). Gbetagamma bound soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes, and binding was reduced to SNARE complexes containing cleaved SNAP25 or by Ca(2+)-dependent synaptotagmin binding. Here we show inhibitory coupling between GPCRs and vesicle exocytosis mediated directly by Gbetagamma interactions with the Ca(2+)-dependent fusion machinery.     

Synaptotagmin-7 is a principal Ca2+ sensor for Ca2+-induced glucagon exocytosis in pancreas

Hormones such as glucagon are secreted by Ca²⁺-induced exocytosis of large dense-core vesicles, but the mechanisms involved have only been partially elucidated. Studies of pancreatic β-cells secreting insulin revealed that synaptotagmin-7 alone is not sufficient to mediate Ca²⁺-dependent insulin granule exocytosis, and studies of chromaffin cells secreting neuropeptides and catecholamines showed that synaptotagmin-1 and -7 collaborate as Ca²⁺ sensors for exocytosis, and that both are equally involved. As no other peptide secretion was analysed, it remains unclear whether synaptotagmins generally act as Ca²⁺ sensors in large dense-core vesicle exocytosis in endocrine cells, and if so, whether synaptotagmin-7 always functions with a partner in that role. In particular, far less is known about the mechanisms underlying Ca²⁺-triggered glucagon release from α-cells than insulin secretion from β-cells, even though insulin and glucagon together regulate blood glucose levels. To address these issues, we analysed the role of synaptotagmins in Ca²⁺-triggered glucagon exocytosis. Surprisingly, we find that deletion of a single synaptotagmin isoform, synaptotagmin-7, nearly abolished Ca²⁺-triggered glucagon secretion. Moreover, single-cell capacitance measurements confirmed that pancreatic α-cells lacking synaptotagmin-7 exhibited little Ca²⁺-induced exocytosis, whereas all other physiological and morphological parameters of the α-cells were normal. Our data thus identify synaptotagmin-7 as a principal Ca²⁺ sensor for glucagon secretion, and support the notion that synaptotagmins perform a universal but selective function as individually acting Ca²⁺ sensors in neurotransmitter, neuropeptide, and hormone secretion.     

The Distribution of Synaptotagmin Ⅱ in RBL-2H3 and Its Regulation on Exocytosis of Lysosomes in RBL-2H3

Synaptotagmin （Syt） constitutes a family of membrane-trafficking proteins, so far nearly 20 Syts have been discovered. Extensive work showed that synatotagmins were a potential Ca^2＋ sensor for regulated exocytosis. This study was to investigate the expression and location of synaptotagmin II （Syt2） in RBL-2H3 （RBL） and its role in regulating exocytosis of RBL. The expression of Syt2 in RBL was confirmed by Western blot. The recombinant expression vector pEGFP-N1-Syt2 was constructed and transfected into RBL by electroporation, the stable transfectant RBL-Syt2-S expressing fusion protein Syt2-EGFP were obtained and Syt2 was highly concentrated at plasma membrane with little detected in cytoplasm. To analyze the role of Syt2 during exocytosis of RBL, the release of cathepsin D was assayed by immunoblotting. Compared with control, the release of cathepsin D by RBL-Syt2-S was markedly decreased. The results indicated that Syt2 played a negative regulation in exocytosis of lysosomes in RBL. Cellular ＆ Molecular Immunology. 2005;2（3）：205-209.     

Expression of synaptotagmin 1 in the taste buds of rat gustatory papillae

Synapses between taste receptor cells and primary sensory afferent fibers transmit the output signal from taste buds to the central nervous system. The synaptic vesicle cycle at the synapses involves vesicle docking, priming, fusion, endocytosis, and recycling. Many kinds of synaptic vesicle proteins participate in synaptic vesicle cycles. One of these, synaptotagmin 1, binds Ca(2+) phospholipids with high affinity and plays a role in Ca(2+) regulated neurotransmitter release in the central and peripheral nervous systems. However, the expression patterns of synaptotagmin 1 in rat taste tissues have not been determined. We therefore examined the expression patterns of synaptotagmin 1 and several cell specific markers of type II and III cells in rat taste buds. RT-PCR assay showed that synaptotagmin 1 mRNA was expressed in circumvallate papillae. In fungiform, foliate, and circumvallate papillae, the antibody against synaptotagmin 1 yielded the labeling of a subset of taste bud cells and intra- and subgemmal nerve processes. Double labeled experiments showed that synaptotagmin 1 positive cells co-expressed type III cell markers, PGP 9.5, and NCAM. Intragemmal nerve processes positive for synaptotagmin 1 co-expressed PGP 9.5. Conversely, all synaptotagmin 1 expressing cells did not co-expressed type II cell markers, PLCbeta2, or gustducin. These results show that synaptotagmin 1 may play some regulatory roles in vesicle membrane fusion events with the plasma membrane at the synapses of type III cells in rat taste buds.     

Properties of exocytotic response in vertebrate photoreceptors

Synaptic transmission at the photoreceptor synapse is characterized by continuous release of glutamate in darkness. Release is regulated by the intracellular calcium concentration ([Ca2+]i). We here examined the physiological properties of exocytosis in tiger salamander (Ambystoma tigrinum) retinal rods and cones. Patch-clamp capacitance measurements were used to monitor exocytosis elicited by a rapid and uniform increase in [Ca2+]i by photolysis of the caged Ca2+ compound NP-EGTA. The amplitude of flash-induced increases in membrane capacitance (Cm) varied monotonically with [Ca2+]i beyond approximately 15 microM. The following two types of kinetic responses in Cm were recorded in both rods and cones: 1) a single exponential rise (39% of cells) or 2) a double-exponential rise (61%). Average rate constants of rapid and slow exocytotic responses were 420 +/- 168 and 7.85 +/- 5.02 s-1, respectively. The rate constant for the single exponential exocytotic response was 17.5 +/- 12.4 s-1, not significantly different from that of the slow exocytotic response. Beyond the threshold [Ca2+]i of approximately 15 microM, the average amplitude of rapid, slow, and single Cm response were 0.84 +/- 0.35, 0.82 +/- 0.20, and 0.70 +/- 0.23 pF, respectively. Antibodies against synaptotagmin I, a vesicle protein associated with fast exocytosis, strongly stained the synaptic terminal of isolated photoreceptors, suggesting the presence of fusion-competent vesicles. Our results confirm that photoreceptors possess a large rapidly releasable pool activated by a low-affinity Ca2+ sensor whose kinetic and calcium-dependent properties are similar to those reported in retinal bipolar cells and cochlear hair cells.     

Formation, stabilisation and fusion of the readily releasable pool of secretory vesicles

Calcium-triggered exocytosis of neurotransmitter or hormone-filled vesicles has developed as the main mechanism for cell-to-cell communication in animals. Consequently, in the course of evolution this form of exocytosis has been optimized for speed. Since many of the maturation processes of vesicles are intrinsically slow, the solution has been to develop a pool of vesicles that are fully matured and can be fused very rapidly upon stimulation. Vesicles in this readily releasable pool are characterized by very low release rate constants at the resting cytosolic [Ca²⁺] ([Ca²⁺]_i) and very high release rate constants at stimulated [Ca²⁺]_i. Here I review the kinetic and molecular requirements for the existence of such a pool of vesicles, focusing on chromaffin cells of the adrenal medulla. I discuss how the use of assay methods with different time resolution may lead to fundamentally different conclusions about the role of proteins in exocytosis. Finally, I review recent evidence that the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, formed between proteins residing in the vesicle and the plasma membrane, is involved in formation and stabilization of the readily releasable vesicle pool, whereas synaptotagmin, a Ca²⁺- and phospholipid-binding vesicular protein, is involved in setting the Ca²⁺ dependence of the fusion process itself. Future studies are likely to focus on the interaction between these two classes of proteins.     

Sodium-dependent potassium channels of a Slack-like subtype contribute to the slow afterhyperpolarization in lamprey spinal neurons

Synaptic vesicles aggregate at the presynaptic terminal during synapse formation via mechanisms that are poorly understood. Here we have investigated the role of the putative calcium sensor synaptotagmin I in vesicle aggregation during the formation of soma–soma synapses between identified partner cells using a simple in vitro synapse model in the mollusc Lymnaea stagnalis . Immunocytochemistry, optical imaging and electrophysiological recording techniques were used to monitor synapse formation and vesicle localization. Within 6 h, contact between appropriate synaptic partner cells up-regulated global synaptotagmin I expression, and induced a localized aggregation of synaptotagmin I at the contact site. Cell contacts between non-synaptic partner cells did not affect synaptotagmin I expression. Application of an human immunodeficiency virus type-1 transactivator (HIV-1 TAT)-tagged peptide corresponding to loop 3 of the synaptotagmin I C2A domain prevented synaptic vesicle aggregation and synapse formation. By contrast, a TAT-tagged peptide containing the calcium-binding motif of the C2B domain did not affect synaptic vesicle aggregation or synapse formation. Calcium imaging with Fura-2 demonstrated that TAT–C2 peptides did not alter either basal or evoked intracellular calcium levels. These results demonstrate that contact with an appropriate target cell is necessary to initiate synaptic vesicle aggregation during nascent synapse formation and that the initial aggregation of synaptic vesicles is dependent on loop 3 of the C2A domain of synaptotagmin I.