Nagai Yumiko, Tadokoro Satoshi, Sakiyama Hiroki, Hirashima Naohide
Nagoya City University, Nagoya, Japan.
Biochim Biophys Acta. 2011 Oct;1808(10):2435-9. doi: 10.1016/j.bbamem.2011.07.003. Epub 2011 Jul 20.
Mast cells play a pivotal role in allergic responses. Antigen stimulation causes elevation of the intracellular Ca(2+) concentration, which triggers the exocytotic release of inflammatory mediators such as histamine. Recent research, including our own, has revealed that SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins such as syntaxin-3, -4, SNAP-23, and VAMP-8 are involved in exocytosis. Although exocytosis in mast cells is Ca(2+) dependent, the target molecule that interacts with Ca(2+) is not clear. Synaptotagmin is a Ca(2+) sensor and regulates exocytosis in neuronal cells. However, the role of synaptotagmin 2, a member of the synaptotagmin family, in exocytosis in mast cells remains controversial. In this study, we investigated the role of synaptotagmin 2 by a liposome-based fusion assay. SNARE proteins (SNAP-23, syntaxin-3, VAMP-8) and synaptotagmin 2 were expressed in Escherichia coli and purified as GST-tagged or His-tagged fusion proteins. These SNARE proteins were incorporated by a detergent dialysis method. Membrane fusion between liposomes was monitored by fluorescence resonance energy transfer between fluorescent-labeled phospholipids. In the presence of Ca(2+), low synaptotagmin 2 concentration inhibited membrane fusion between SNARE-containing liposomes, while high synaptotagmin 2 concentration enhanced membrane fusion. This enhancement required phosphatidylserine as a membrane component. These results suggest that synaptotagmin 2 regulates membrane fusion of SNARE-containing liposomes involved in exocytosis in mast cells, and that this regulation is dependent on synaptotagmin 2 concentration, Ca(2+), and phosphatidylserine.
肥大细胞在过敏反应中起关键作用。抗原刺激会导致细胞内钙离子浓度升高，进而触发组胺等炎症介质的胞吐释放。包括我们自己的研究在内，最近的研究表明，诸如 syntaxin-3、-4、SNAP-23 和 VAMP-8 等 SNARE（可溶性 N-乙基马来酰亚胺敏感因子附着蛋白受体）蛋白参与胞吐作用。尽管肥大细胞中的胞吐作用依赖钙离子，但与钙离子相互作用的靶分子尚不清楚。突触结合蛋白是一种钙离子传感器，可调节神经元细胞中的胞吐作用。然而，突触结合蛋白家族成员突触结合蛋白 2 在肥大细胞胞吐作用中的作用仍存在争议。在本研究中，我们通过基于脂质体的融合试验研究了突触结合蛋白 2 的作用。SNARE 蛋白（SNAP-23、syntaxin-3、VAMP-8）和突触结合蛋白 2 在大肠杆菌中表达，并作为 GST 标签或 His 标签融合蛋白进行纯化。这些 SNARE 蛋白通过去污剂透析法掺入。通过荧光标记磷脂之间的荧光共振能量转移监测脂质体之间的膜融合。在钙离子存在的情况下，低浓度的突触结合蛋白 2 抑制含 SNARE 的脂质体之间的膜融合，而高浓度的突触结合蛋白 2 则增强膜融合。这种增强需要磷脂酰丝氨酸作为膜成分。这些结果表明，突触结合蛋白 2 调节肥大细胞中参与胞吐作用的含 SNARE 的脂质体的膜融合，并且这种调节依赖于突触结合蛋白 2 的浓度、钙离子和磷脂酰丝氨酸。
