非典型溶血性尿毒综合征(aHUS)是一种罕见的血栓性微血管病变。在约60%的患者中发现补体蛋白的遗传变异。其中,约15%携带补体因子I中的突变。因子I(FI)是一种多结构域丝氨酸蛋白酶,其在辅因子蛋白存在下切割并由此失活C3b和C4b。晶体结构表明FI具有两个钙结合域,LDLRA1和LDLRA2。然而,钙在FI中的作用尚不清楚。我们确定在aHUS中鉴定出9种遗传变异(N151S,G162D,G188A,V230E,A240G,G243R,C247G,A258T,和Q260D)聚集在LDLRA1的钙结合位点周围。使用定点诱变,我们确定除A258T外的所有合成都受到损害,暗示有缺陷的蛋白质折叠,也许是由于钙结合的损失。为了进一步探索这种可能性,我们产生了12个与LDLRA1和LDLRA2中的钙协调的丙氨酸突变体(K239A,D242A,I244A,D246A,D252A,E253A,Y276A,N279A,E281A,D283A,D289A,和D290A),预计会干扰钙结合。除K239A和Y276A外,没有一个突变体被分泌。这些观察结果表明,钙离子在FI中起着关键的结构和功能作用。
Atypical hemolytic uremic syndrome (aHUS) is a rare thrombotic microangiopathy. Genetic variants in complement proteins are found in ≈60% of patients. Of these patients, ≈15% carry mutations in complement factor I (CFI). Factor I (FI) is a multidomain serine protease that cleaves and thereby inactivates C3b and C4b in the presence of cofactor proteins. Crystal structures have shown that FI possesses 2 calcium-binding domains, low-density lipoprotein receptor class A (LDLRA) 1 and LDLRA2. Yet, the role of calcium in FI is unknown. We determined that 9 genetic variants identified in aHUS (N151S, G162D, G188A, V230E, A240G, G243R, C247G, A258T, and Q260D) cluster around the calcium-binding site of LDLRA1. Using site-directed mutagenesis, we established that the synthesis of all, except A258T, was impaired, implying defective protein folding, perhaps due to loss of calcium binding. To further explore this possibility, we generated 12 alanine mutants that coordinate with the calcium in LDLRA1 and LDLRA2 (K239A, D242A, I244A, D246A, D252A, E253A, Y276A, N279A, E281A, D283A, D289A, and D290A) and are expected to perturb calcium binding. Except for K239A and Y276A, none of the mutants was secreted. These observations suggest that calcium ions play key structural and functional roles in FI.