生物学杂志

生物学杂志 ›› 2024, Vol. 41 ›› Issue (4): 11-.doi: 10.3969/j.issn.2095-1736.2024.04.011

• 研究报告 • 上一篇    下一篇

Gly33的缺失和突变对hTrx-1抗氧化活性的影响

郭席君1,2， 杨吉康1,2， 黄君维1,2， 王　彤1,2， 刘馨乔1,2 ， 齐兴柱1,2   

  1. 1. 海南大学 热带生物资源教育部重点实验室， 海口 570228； 2. 海南大学 药学院， 海口 570228
  • 出版日期:2024-08-18 发布日期:2024-08-14
  • 通讯作者: 齐兴柱，博士，正高级实验师，研究方向为蛋白质分子设计与改造，E-mail:qixingzhu2013@163.com
  • 作者简介:郭席君，硕士研究生，研究方向为抗氧化生物学，E-mail:guoxu@hainanu.edu.cn
  • 基金资助:
    海南省自然科学基金高层次人才项目(821RC534); 国家自然科学基金项目(31560491)

Effect of the deletion and mutation of Gly33 on the antioxidant activity of hTrx-1

GUO Xijun1,2， YANG Jikang1,2， HUANG Junwei1,2， WANG Tong1,2，LIU Xinqiao1,2， QI Xingzhu1,2   

  1. 1. Key Laboratory of Tropical Biological Resources of Ministry of Education, Hainan University,
    Haikou 570228, China; 2. School of Pharmaceutical Science, Hainan University, Haikou 570228, China
  • Online:2024-08-18 Published:2024-08-14

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摘要： 为探讨人硫氧还蛋白hTrx-1活性位点保守序列中Gly33残基在还原氧化性蛋白过程中是否必不可少，通过合成突变基因对hTrx-1的Gly33残基进行缺失和替换（G→A）。合成的突变基因随后在原核细胞中表达并进一步纯化及检测抗氧化活性，结果表明，无论是Gly33残基的缺失还是突变为Ala，突变的hTrx-1均能在原核细胞中高效表达，暗示2种突变型hTrx-1均能形成“Trx折叠”结构。抗氧化活性检测表明Gly33残基缺失的情况下，其抗氧化能力有所下降，当Gly33突变为Ala时，其抗氧化能力大幅下降到接近失去抗氧化能力。推测Trx-1活性中心保守氨基酸残基的改变影响了活性中心区域的几何构象，从而导致底物蛋白难以从空间上靠近。参与形成二硫键巯基的含量检测结果表明，突变型hTrx-1中形成二硫键巯基的含量明显低于野生型hTrx-1中形成二硫键巯基的含量，推测突变型hTrx-1的Cys32与Cys35形成二硫键的能力受到影响。

关键词: 硫氧还蛋白1, Trx折叠, Gly33, 抗氧化, 二硫键

Abstract: In order to investigate whether Gly33 residue in the conserved sequence of the active site of human thioredoxin hTrx-1 is essential in the process of reducing oxidative protein, the Gly33 residue of hTrx-1 was deleted or replaced (G→A) by two synthetic mutant genes. The synthetic mutant genes were then expressed in prokaryotic cells, further purified and tested for antioxidant activity. The results showed that both the deletion of Gly33 residue and the mutation to Ala, the mutated hTrx-1 could be efficiently expressed in prokaryotic cells, indicating that both mutant hTrx-1 could form a “Trx fold” structure. Antioxidant activity testing showed that in the absence of Gly33 residues, its antioxidant capacity decreased, when Gly33 mutated to Ala, its antioxidant capacity significantly decreased to near loss. Therefore, this article speculated that the change of conserved amino acid residues in the Trx-1 active center affects the geometric configuration of the active center region, resulting in difficulty in approaching the substrate protein in space. The detection results of the content of disulfide bond sulfhydryl groups involved in the formation of disulfide bonds showed that the content of disulfide bond sulfhydryl groups formed in mutant hTrx-1 was significantly lower than that in wild-type hTrx-1. It was speculated that the ability of Cys32 and Cys35 to form disulfide bonds in mutant hTrx-1 was affected.

Key words: thioredoxin 1, Trx fold, Gly33, antioxidant, disulfide bond

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