重组金黄色葡萄球菌次黄嘌呤单核苷酸脱氢酶与脂蛋白(a)的相互作用

The interaction between Lipoprotein(a) and recombinant inosine 5'-monophosphate dehydrogenase derived from Staphylococcus aureus

摘要:

次黄嘌呤单核苷酸脱氢酶(IMPDH)是金黄色葡萄球菌(S. aureus)表面的纤溶酶原(Plg)受体之一, 它可以通过赖氨酸结合位点(LBS)与Plg相结合。脂蛋白(a)[Lp(a)]中的载脂蛋白(a)[Apo(a)]与Plg有很高的同源性, 即两者的Kringle结构域都含有LBS, 其中Apo(a)的KIV10含有强的LBS。因此本实验提出了Lp(a)应该能够与S. aureus表面的Plg受体相结合, 进而可能竞争性抑制S. aureus与Plg结合的假说。本研究克隆了S. aureus 的IMPDH基因, 酶切后将其连接到表达载体pASK-IBA37中, 并在大肠杆菌BL21中表达了该重组蛋白(rIMPDH)。通过酶联免疫吸附试验(ELISA)、亲和色谱层析及Western blot对rIMPDH与Lp(a)的相互作用机制进行了研究。结果表明rIMPDH可以通过LBS与Lp(a)和rKIV10发生特异性结合, 而且一定浓度的赖氨酸类似物6-氨基己酸(EACA)可以抑制这种结合, 然而本研究并未发现Lp(a)和rKIV10对rIMPDH与Plg的相互作用有明显的抑制。

Abstract:

Inosine 5'-monophosphate dehydrogenase (IMPDH) is one of plasminogen (Plg) receptors on the surface of Staphylococcus aureu (S. aureus). Plg through its lysine binding sites (LBS) binds to IMPDH. Apolipoprotein(a) [Apo(a)], which is one component of Lipoprotein(a) [Lp(a)], has a high homology with Plg and both of them contain LBS in their Kringle (K) domains, and a strong LBS is identified in KIV10 of Apo(a). Therefore, we previously reported that Lp(a) might bind to Plg receptor on the surface of S. aureus, subsequently competitively inhibiting the interaction of S. aureus with Plg. To further test our hypothesis, the IMPDH gene of S. aureus was cloned into pASK-IBA37 and the re-combinant IMPDH(rIMPDH) was expressed in E. coli BL21. The interaction between rIMPDH and Lp(a) was investigated by enzyme-linked immunosorbent assay (ELISA), and affinity chromatogra-phy-binding assay followed by Western blotting. The results indicated that rIMPDH could specifically bind to purified Lp(a) and rKIV10, and the lysine analog EACA inhibited the binding. Unexpectedly, Lp(a) and rKIV10 did not significantly inhibit the interaction between rIMPDH and Plg.