Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh 202002, India.
CSIR, Central Drug Research Institute, Lucknow 226031, India.
Biochim Biophys Acta Proteins Proteom. 2018 Sep;1866(9):989-1000. doi: 10.1016/j.bbapap.2018.06.006. Epub 2018 Jun 28.
Hyperglycaemic conditions facilitate the glycation of serum proteins which may have predisposition to aggregation and thus lead to complications. The current study investigates the glycation induced structural and functional modifications of chickpea cystatin (CPC) as well as biological toxicity of the modified protein forms, using CPC-glucose as a model system. Several structural intermediates were formed during the incubation of CPC with glucose (day 4, 8, 12, & 16) as revealed by circular dichroism (CD), altered intrinsic fluorescence, and high ANS binding. Further incubation of CPC with glucose (day 21) formed abundant β structures as revealed by Fourier transform infrared spectroscopy and CD analysis which may be due to the aggregation of protein. High thioflavin T fluorescence intensity and increased Congo red absorbance together with enhanced turbidity and Rayleigh scattering by this modified form confirmed the aggregation. Electron microscopy finally provided the valid physical authentication about the presence of aggregate structures. Functional inactivation of glucose incubated CPC was also observed with time. Single cell electrophoresis of lymphocytes and plasmid nicking assays in the presence of modified CPC showed the DNA damage which confirmed its biological toxicity. Hence, our study suggests that glycation of CPC not only leads to structural and functional alterations in proteins but also to biotoxic AGEs and aggregates.
高血糖条件促进血清蛋白的糖化，这可能使它们易于聚集，从而导致并发症。本研究以鹰嘴豆半胱氨酸蛋白酶抑制剂（CPC）-葡萄糖为模型系统，研究了糖化诱导的 CPC 结构和功能改变以及修饰蛋白形式的生物学毒性。圆二色性（CD）、内源荧光变化和高 ANS 结合表明，在 CPC 与葡萄糖孵育的第 4、8、12 和 16 天，形成了几种结构中间体。进一步孵育 CPC 与葡萄糖（第 21 天）形成丰富的β结构，傅里叶变换红外光谱和 CD 分析表明这可能是由于蛋白质的聚集。这种修饰形式的硫代黄素 T 荧光强度增加、刚果红吸收增加以及浊度和瑞利散射增强证实了聚集的发生。电子显微镜最终提供了关于存在聚集结构的有效物理验证。用时间扫描法观察到葡萄糖孵育的 CPC 功能失活。在修饰的 CPC 存在下进行淋巴细胞单细胞电泳和质粒切口测定显示 DNA 损伤，证实了其生物毒性。因此，我们的研究表明，CPC 的糖化不仅导致蛋白质的结构和功能改变，还导致生物毒性 AGEs 和聚集物的形成。
