Glutathionylation of lens proteins through the formation of thioether bond
Glutathionylation of lens proteins through the formation of thioether bond
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DOI：
10.1007/s11010-005-6908-1
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发表时间：
2005-04-01
影响因子：
4.3
通讯作者：
LeGrand, RD
中科院分区：
文献类型：
--
作者：
Linetsky, M;LeGrand, RD
羊毛硫氨酸（一种脱氢丙氨酸交联）的形成与人类晶状体的老化和白内障的发生有关。在这项研究中，我们研究了谷胱甘肽对晶状体蛋白的修饰是否可以通过替代途径进行：即通过在蛋白质和谷胱甘肽之间形成不可还原的硫醚键。对来自人白内障、老化和牛晶状体的还原的水溶性和水不溶性晶状体蛋白进行直接 ELISA，结果显示仅对人不可还原的谷胱甘肽晶状体蛋白具有浓度依赖性免疫反应性。还原的水不溶性白内障晶状体蛋白显示出最高的免疫反应性，而牛晶状体蛋白则没有表现出任何反应。这些数据通过斑点印迹分析得到证实。在来自老化和白内障晶状体的水不溶性蛋白质中，这种修饰的水平范围为 0.7 至 1.6 nmol/mg 蛋白质。通过用丹磺酰氯对这些制剂进行衍生化，然后进行彻底透析、酸水解和通过 RP-HPLC 进行丹磺酰化氨基酸的荧光检测，对还原和烷基化的晶状体蛋白中的 N 末端氨基酸进行测定，结果表明，N 末端谷氨酸的浓度约为 0.2 nmol/mg 晶状体蛋白。该证据指出，还原的人晶状体蛋白中的不可还原的谷胱甘肽的至少一些N-末端氨基不参与共价键的形成。由于在还原和烷基化的人晶状体蛋白中未检测到二硫化物，因此 GSH 最有可能通过硫醚键连接到晶状体蛋白上。这些结果首次证明人类晶状体蛋白的谷胱甘肽化可以通过形成不可还原的硫醚键来发生。
Formation of lanthionine, a dehydroalanine crosslink, is associated with aging of the human lens and cataractogenesis. In this study we investigated whether modification of lens proteins by glutathione could proceed through an alternative pathway: that is, by the formation of a nonreducible thioether bond between protein and glutathione. Direct ELISA of the reduced water-soluble and water-insoluble lens proteins from human cataractous, aged and bovine lenses showed a concentration-dependent immunoreactivity toward human nonreducible glutathionyl-lens proteins only. The reduced water-insoluble cataractous lens proteins showed the highest immunoreactivity, while bovine lens protein exhibited no reaction. These data were confirmed by dot-blot analysis. The level of this modification ranged from 0.7 to 1.6 nmol/mg protein in water-insoluble proteins from aged and cataractous lenses. N-terminal amino acid determination in the reduced and alkylated lens proteins, performed by derivatization of these preparations with dansyl chloride followed by an exhaustive dialysis, acid hydrolysis and fluorescence detection of dansylated amino acids by RP-HPLC, showed that N-terminal glutamic acid was present in concentration of approximately 0.2 nmol/mg of lens protein. This evidence points out that at least some of the N-terminal amino groups of nonreducible glutathione in the reduced human lens proteins are not involved in a covalent bond formation. Since disulfides were not detected in the reduced and alkylated human lens proteins, GSH is most likely attached to lens proteins through thioether bonds. These results provide, for the first time, evidence that glutathiolation of human lens proteins can occur through the formation of nonreducible thioether bonds.