铜绿假单胞菌二氢硫辛酸酰胺脱氢酶的重组表达及其与脂蛋白(a)的相互作用

The interaction between lipoprotein(a) and recombinant dihydrolipoamide dehydrogenase derived from Pseudomonas aeruginosa

摘要:

【目的】二氢硫辛酸酰胺脱氢酶(Dihydrolipoamide dehydrogenase，Lpd)是铜绿假单胞菌(Pseudomonas aeruginosa)表面的一种纤溶酶原(Plasminogen，Plg)受体，旨在研究Lpd与脂蛋白(a)[Lipoprotein(a)，Lp(a)]以及Plg之间的相互作用。【方法】用大肠杆菌表达rLpd及其突变分子(rLpdK476A、rLpdK477A、rLpdΔKKR)，用酶联免疫吸附实验(ELISA)、亲和色谱层析及Western blot等技术检测rLpd及其突变分子与Lp(a)、Plg的相互作用。【结果】ELISA及亲和色谱层析实验结果表明，rLpd可以与Lp(a)结合但不与LDL结合，Lp(a)与rLpdΔKKR的结合能力显著低于其与rLpd的结合能力。1 mmol/L的赖氨酸类似物6-氨基己酸(EACA)对rLpd与Lp(a)的结合有显著的抑制作用。1 000 μg/L的Lp(a)对rLpd与Plg的结合起到显著的抑制作用。【结论】Lpd能够与Lp(a)特异性结合，其476和477两个相邻的赖氨酸残基是与Lp(a)结合的主要位点，Lp(a)可以竞争性地抑制rLpd与Plg的结合。

关键词:铜绿假单胞菌，二氢硫辛酸酰胺脱氢酶，脂蛋白(a)，纤溶酶原

Abstract:

[Objective] To investigate the interaction between plasminogen (Plg), lipoprotein(a) [Lp(a)] and dihydrolipoamide dehydrogenase (Lpd) on the surface of P. aeruginosa. [Methods] Recombinant Lpd and its mutants (rLpdK476A, rLpdK477A, rLpdΔKKR) were used to explore the interaction with enzyme-linked immunosorbent assay (ELISA) and affinity chromatography-binding assay followed by Western blot analysis. [Results] The recombinant rLpd and its mutants were expressed and purified from Escherichia coli. The results indicated that rLpd could specifically bind to Lp(a) but not to LDL. The binding capacity of rLpdΔKKR to Lp(a) is significantly lower than that of rLpd. The binding of rLpd to Lp(a) could be significantly inhibited by 1 mmol/L of EACA. 1 000 μg/L of Lp(a) could inhibit the interaction between rLpd and Plg. [Conclusion] Lpd could bind to Lp(a) and the 476th and 477th lysine residues of Lpd were main binding sites. Lp(a) could significantly inhibit the binding of Plg to Lpd.

Key words:Pseudomonas aeruginosa, Dihydrolipoamide dehydrogenase, Lipoprotein(a), Plasminogen