Cys146-Cys146分子间二硫键在人瘦素二聚化过程中起主导作用

在前期研究中，已发现人瘦素（leptin）在体外再折叠过程中会形成稳定的二聚体，但其二聚化机制尚不清楚. 本研究旨在分析瘦素二聚体的结构特性，并重点研究体外再折叠过程中瘦素二聚化的机制. 相较与瘦素单体，瘦素二聚体保留了约75％免疫活性及15％受体结合活性，同时显示出明显慢的天然电泳迁移率. 圆二色性分析显示,二聚体基本保留了单体α螺旋索结构特征. 还原性及非还原性凝胶电泳分析和自由巯基测定结果表明,瘦素二聚体是由一对分子间二硫键连接2个单体而成的.为了确定瘦素二聚化过程中起主导作用的分子间二硫键，利用PCR定点突变技术构建了C96S和C146S两个突变体瘦素. 通过分析C96S及C146S突变体瘦素的体外再折叠特性及过程,并与野生型瘦素相比较，揭示C96S瘦素的二聚体显示出与野生型瘦素二聚体相似的特性，而C146S瘦素不能形成结构稳定的二聚体. 以上研究结果表明，Cys146-Cys146分子间二硫键在人瘦素二聚化过程中起主导作用.

Human Leptin Dimerization during Refolding is Predominantly Mediated by Cys146-Cys146 Intermolecular Disulfide Bond

Although human leptin has been shown to dimerize during refolding procedure, the exact dimerization mechanism still remains unillustrated. The current paper was aimed to reveal how human leptin dimer is formed during in vitro refolding. The dimer retained about 75％ of immune activity and 15% of the receptor binding activity of the monomer, and exhibited a slower mobility than the monomer on native PAGE gel. Circular Dichroism (CD) analysis indicated that the dimer maintained the key α-helix bundle structure of leptin monomer. Electrophoreticassay and free thiol group determination revealed that the dimer was linked by one intermolecular disulfide bond. To identify which cysteine is involved in dimerization, C96S and C146S mutant leptins were generated by PCR-mediated positional mutation. Refolding analysis indicated that C96S leptin and wild type leptin gave a similar dimer, while very little stable dimer of C146S leptin was observed on native-PAGE gel. In summary, these results suggested that human leptin dimerization during in vitro refolding is predominantly mediated by Cys146-Cys146 intermolecular disulfide bond.