Yousefi Reza, Khazaei Sima, Moosavi-Movahedi Ali-Akbar
Protein Chemistry Laboratory (PCL), Department of Biology, College of Sciences, Shiraz University, Shiraz, Iran.
Protein Pept Lett. 2013 Aug;20(8):932-41. doi: 10.2174/0929866511320080011.
Various chemical modifications can reduce chaperone activity of α-crystallin (α-Cry) and the loss of which has been implicated in the development of cataract diseases. The side chains of lysine residues are the target of both glycation and homocysteinylation, and lysine modification by the two reactions may similarly affect the structure and function of α- Cry. In this study, α-Cry was incubated with homocysteine thiolactone (HCTL), resulting in significant protein homocysteinylation, as determined with Ellman's assay. Homocysteinylation of α-Cry resulted in the reduction in surface hydrophobicity and alpha-helix to beta-sheet transition, as observed respectively with fluorescence and circular dichroism (CD) spectroscopy. The structural alteration of homocysteinylated α-Cry was accompanied by protein aggregation, including the formation of amyloid fibrils as detected by thioflavin T (ThT) fluorescence and Congo red (CR) absorption spectroscopy. The mobility shifts of homocysteinylated α-Cry on reducing and non-reducing SDS-PAGEs suggest that disulfide cross-linking in addition to lysine modification, also plays a role in aggregation of this protein. The chaperone activities of α-Cry, namely to prevent aggregation, to assist refolding and to restore activity of thermally stressed α-glucosidase (α-Gls) were reduced after homocysteinylation. Overall, this study suggests that similar to non-enzymatic glycation, homocysteinylation of α-Cry is a risk factor for the development of cataract disorders, for instance during hyperhomocysteinemia which is linked to the various ocular pathological disorders.
多种化学修饰可降低α-晶状体蛋白（α-Cry）的伴侣活性，其活性丧失与白内障疾病的发生有关。赖氨酸残基的侧链是糖基化和同型半胱氨酸化的靶点，这两种反应对赖氨酸的修饰可能同样会影响α-Cry的结构和功能。在本研究中，α-Cry与同型半胱氨酸硫内酯（HCTL）孵育，通过埃尔曼测定法确定，导致蛋白质显著同型半胱氨酸化。α-Cry的同型半胱氨酸化导致表面疏水性降低以及α-螺旋向β-折叠转变，分别通过荧光和圆二色性（CD）光谱观察到。同型半胱氨酸化的α-Cry的结构改变伴随着蛋白质聚集，包括通过硫黄素T（ThT）荧光和刚果红（CR）吸收光谱检测到的淀粉样原纤维的形成。同型半胱氨酸化的α-Cry在还原和非还原SDS-PAGE上的迁移率变化表明，除赖氨酸修饰外，二硫键交联也在该蛋白质的聚集中起作用。同型半胱氨酸化后，α-Cry的伴侣活性，即防止聚集、协助重折叠和恢复热应激α-葡萄糖苷酶（α-Gls）活性的能力降低。总体而言，本研究表明，与非酶糖基化类似，α-Cry的同型半胱氨酸化是白内障疾病发生的一个风险因素，例如在与各种眼部病理疾病相关的高同型半胱氨酸血症期间。
