Localization of the phosphatidylserine-binding site of glyceraldehyde-3-phosphate dehydrogenase responsible for membrane fusion.
Localization of the phosphatidylserine-binding site of glyceraldehyde-3-phosphate dehydrogenase responsible for membrane fusion.
复制标题
负责膜融合的甘油醛-3-磷酸脱氢酶的磷脂酰丝氨酸结合位点的定位。
DOI：
10.1093/oxfordjournals.jbchem.a021886
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发表时间：
1997
影响因子：
2.7
通讯作者：
M. Umeda
中科院分区：
文献类型：
--
作者：
M. Kaneda;K. Takeuchi;K. Inoue;M. Umeda
在这项研究中，我们证明了 3-磷酸甘油醛脱氢酶 (GAPDH) 是一种磷脂酰丝氨酸 (PS) 结合蛋白，并定位了参与该酶诱导的膜融合的假定 PS 结合位点。为了鉴定 PS 结合蛋白，我们制备了针对 15 个氨基酸的合成肽（磷脂酰丝氨酸脱羧酶的氨基酸残基 390-403）的多克隆抗体，该抗体被证明与 PS 特异性结合。一种多克隆抗体，命名为aPSD-2，与GAPDH发生交叉反应，其与GAPDH的结合被PS抑制，但不被其他磷脂如磷脂酰乙醇胺和磷脂酰肌醇抑制。通过测量表面等离子体共振对GAPDH与磷脂膜结合的动力学分析表明，GAPDH与含有PS的磷脂膜结合迅速(k[on] =2.8 X 10(4) M(-1) X s[-1])并且解离极其缓慢(k[off]=5.9 X 10(-5) s[-1])，解离常数低 (KD=2.6nM). GAPDH 与没有 PS 的膜的结合效率较低，解离常数为 0.2 microM。 GAPDH 诱导的囊泡融合也被 aPSD-2 抑制，表明该抗体识别 GAPDH 上参与酶诱导的膜融合的推定 PS 结合位点。用溴化氰对 GAPDH 进行化学断裂，然后对反应肽进行分离和序列分析，结果鉴定出具有 GAPDH 氨基酸残基 45-103 序列的单个反应肽。对aPSD-2与衍生自相应区域的合成肽的结合分析将抗体结合位点定位于GAPDH的氨基酸残基70-94。 25 个氨基酸的合成肽（GAPDH 的氨基酸残基 70-94）和针对该肽产生的多克隆抗体均抑制 GAPDH 诱导的膜融合，表明这些氨基酸残基在该膜融合过程中发挥着至关重要的作用。
In this study, we demonstrated that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a phosphatidylserine (PS)-binding protein and localized the putative PS-binding site involved in the membrane fusion induced by this enzyme. In an attempt to identify the PS-binding proteins, we raised polyclonal antibodies against a 15-amino-acid synthetic peptide (amino acid residues 390-403 of phosphatidylserine decarboxylase), which was shown to bind specifically to PS. One polyclonal antibody, designated aPSD-2, crossreacted with GAPDH, and its binding to GAPDH was inhibited by PS but not by other phospholipids such as phosphatidylethanolamine and phosphatidylinositol. Kinetic analysis of GAPDH binding to phospholipid membranes by measuring surface plasmon resonance showed that GAPDH associated with the phospholipid membrane containing PS rapidly (k[on] =2.8 X 10(4) M(-1) X s[-1]) and dissociated extremely slowly (k[off]=5.9 X 10(-5) s[-1]), giving a low dissociation constant (KD=2.6nM). GAPDH bound less effectively to membranes without PS with a dissociation constants of 0.2 microM. GAPDH-induced vesicle fusion was also inhibited by aPSD-2, suggesting that this antibody recognizes the putative PS-binding site on GAPDH involved in the enzyme-induced membrane fusion. Chemical fragmentation of GAPDH with cyanogen bromide followed by separation and sequence analysis of the reactive peptide resulted in the identification of a single reactive peptide with the sequence of amino acid residues 45-103 of GAPDH. Analysis of aPSD-2 binding to synthetic peptides derived from the corresponding region localized the antibody-binding site to amino acid residues 70-94 of GAPDH. Both the 25-amino-acid synthetic peptide (amino acid residues 70-94 of GAPDH) and polyclonal antibody raised against this peptide inhibited GAPDH-induced membrane fusion, suggesting that these amino acid residues play a crucial role in this membrane fusion process.