Journal of Immunological Methods ( IF 2.2 ) Pub Date : 2020-10-06 , DOI: 10.1016/j.jim.2020.112877 Gábor Szabó , Krisztina Pénzes , Bernadett Torner , Miklós Fagyas , Tünde Tarr , Pál Soltész , Gréta Kis , Miklós Antal , János Kappelmayer
One of the most abundant coagulation proteins is β2-glycoprotein I (β2GPI) that is present in humans at a concentration of around 200 mg/L. Its physiological role is only partially understood, but it adopts several different structural forms the majority of which are the open and closed forms. We isolated native (circular) β2GPI and converted it into an open conformation. The effectiveness of these procedures was assessed by Western blot and negative-staining electron microscopy. We found that in coagulation assays the open form of β2GPI had a significant prolonging effect on fibrin formation in a dilute prothrombin time test (p < 0.001). In the dilute activated partial thromboplastin time test, both conformations had a significant prolonging effect (p < 0.001) but the open conformation was more effective. In a fluorescent thrombin generation assay both conformations slightly delayed thrombin generation with no significant effect on the quantity of formed thrombin. By using surface plasmon resonance assays, the equilibrium dissociation constants of both the open and closed conformations of β2GPI showed a similar and strong affinity to isolated anti-β2GPI autoantibodies (Kd closed β2GPI = 5.17 × 10−8 M, Kd open β2GPI = 5.56 × 10−8 M) and the open form had one order of magnitude stronger affinity to heparin (Kd = 0.30 × 10−6 M) compared to the closed conformation (Kd = 3.50 × 10−6 M). The two different forms of β2GPI have distinct effects in functional tests and in ligand binding, which may considerably affect the intravascular events related to this abundant plasma protein in health and disease.
中文翻译:
β的不同和重叠效果2在配体的相互作用和功能分析糖蛋白I的构象变体
其中最丰富的凝血蛋白的是β 2 -糖蛋白I(β 2 GPI)是在约200毫克/升的浓度存在于人类。它的生理作用只是部分被理解,但是它采用了几种不同的结构形式,其中大多数是开放形式和封闭形式。我们分离的天然(圆形)β 2 GPI并转化成开放构象。这些程序的有效性通过蛋白质印迹和阴性染色电子显微镜进行了评估。我们发现,在凝固分析的β开放形式2在稀释的凝血酶原时间测试中,GPI对纤维蛋白的形成具有显着的延长作用(p <0.001)。在稀活化的部分凝血活酶时间试验中,两种构象都有明显的延长作用(p <0.001),但开放构象更有效。在荧光凝血酶生成测定中,两种构象都会稍微延迟凝血酶生成,而对形成的凝血酶的量没有明显影响。通过使用表面等离振子共振测定法,β的打开和闭合构象二者的平衡解离常数2 GPI显示对分离的抗β类似和强亲和力2 GPI自身抗体(K d关闭β 2 GPI = 5.17×10 -8 中号,K d开β2 GPI = 5.56×10 -8 M), 与封闭构型(K d = 3.50×10 -6 M)相比,开放形式对肝素的亲和力(K d = 0.30×10 -6 M)强一个数量级。。的两种不同形式的β 2 GPI具有在功能测试和在配体结合,这可能显着地影响与在健康和疾病这种丰富的血浆蛋白血管内的事件不同的作用。