The plasma and cytoplasmic forms of human gelsolin differ in disulfide structure
The plasma and cytoplasmic forms of human gelsolin differ in disulfide structure
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DOI：
10.1021/bi960920n
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发表时间：
1996-07-30
期刊：
影响因子：
2.9
通讯作者：
Pepinsky, RB
中科院分区：
文献类型：
--
作者：
Wen, DY;Corina, K;Pepinsky, RB
凝溶胶蛋白是一种广泛分布的肌动蛋白结合蛋白，调节肌动蛋白丝的长度。它以细胞内和细胞外形式存在，通过选择性剪接衍生自单个基因。这两种形式都含有负责毛皮功能的六个同源结构域。关于这些形式之间的差异知之甚少。我们已经使用了半胱氨酸特异性修饰与3-乙烯基吡啶，HPLC肽图谱方法，和质谱分析的二硫键结构的人血浆和细胞质凝溶胶蛋白的组合。在人凝溶胶蛋白序列中的五个Cys残基中，所有Cys残基都以游离巯基形式存在于人细胞质凝溶胶蛋白中，而其中只有三个Cys残基是人血浆形式中的巯基。血浆凝溶胶蛋白结构域2中的Cys残基188和201是二硫键连接的。还研究了已在大肠杆菌细胞内表达的重组人血浆凝溶胶蛋白和作为来自Cos绿色猴细胞的分泌蛋白。急诊大肠杆菌产物缺乏二硫键，但可以通过温和氧化转化为血浆样结构，而哺乳动物产物在分离前形成正确的二硫键。通过纤溶酶的有限蛋白水解也检测到结构差异。蛋白水解敏感性的差异也是由于结构域2的扰动。这些研究表明，细胞内和细胞外凝溶胶蛋白在结构上是不同的，并表明，至少有一些被用于研究结构/功能的重组凝溶胶蛋白的制剂可能是不正确的折叠。实验还提供了一种确定蛋白质二硫键位置的通用方法。
Gelsolin is a widely distributed actin binding protein that regulates actin filament length. It exists in both an intracellular and an extracellular form that is derived from a single gene by alternative splicing. Both forms contain the six homologous domains that are responsible fur function. Little is known regarding differences between the forms. We have used a combination of cysteine-specific modification with 3-vinylpyridine, HPLC peptide mapping methods, and mass spectrometry to analyze the disulfide structures of human plasma and cytoplasmic gelsolin. Of the five Cys residues in the human gelsolin sequence, all were present in the free thiol form in human cytoplasmic gelsolin while only three of them were foe thiols in the human plasma form. Cys residues 188 and 201 in domain 2 of plasma gelsolin were disulfide linked. Recombinant human plasma gelsolin that had been expressed intracellularly In Escherichia call and as a secreted protein from Cos green monkey cells was also investigated. The E. coli product lacked the disulfide but could be converted to the plasma-like structure with mild oxidation while the mammalian product formed the correct disulfide prior to isolation. Structural differences were also detected by limited proteolysis with plasmin. The differences in proteolytic susceptibility were also due to perturbations in domain 2. These studies demonstrate that the intracellular and extracellular gelsolins are structurally distinct and suggest that at least some of the preparations of recombinant gelsolin that are being used to study structure/function may be improperly folded. The experiments also demonstrate a general method fur the location of disulfide bonds in proteins.