Determination of the active site of the complement regulatory protein CD59

CD59通过与新生膜攻击复合体中的C8和C9结合以及在C5b-8和C9聚合中抑制C9与C8的结合来抑制人补体的膜攻击复合体(MAC)的形成。考虑到CD59的5个二硫键，我们将分子分成两部分，合成了两个多肽。一个代表氨基末端的一半，P1-41，由残基1-41组成，而另一个代表羧基末端的一半，P42-77，由残基42-77组成。P1-41对MAC形成的抑制作用比P42-77强得多，说明氨基末端半部分含有活性部位。我们进一步合成了由4到18个残基组成的P4-18和由19到41个残基组成的P19-41。P4-18活性低于P19-41。令人惊讶的是，P19-41的活性高于P1-41，与尿CD59相当。残基19至41进一步分为两部分：由残基20至25组成的P20-25和由残基27至38组成的P27-38。虽然它们的活性显著低于P19-41的活性，但P27-38的活性高于P20-25。残基27至38进一步分为三个部分：P27-32由残基27至32组成，P30-34由残基30至34组成，P33-38由残基33至38组成。当对这些多肽进行活性分析时，它们都显示出显著的活性，尽管它们所需的浓度是P19-41的10倍。这些数据表明，残基27到38是构成C8和C9结合部位的活性部位。

CD59 inhibits the formation of membrane attack complex (MAC) of human complement by binding to C8 and C9 in the nascent membrane attack complex and inhibiting C9 binding to C8 in C5b-8 and C9 polymerization. Considering 5 disulfide bridges of CD59, we divided the molecule into two portions and synthesized the two peptides. One represented an amino-terminal half, P1-41, consisting of residues 1 to 41 while another represented a carboxyl-terminal half, P42-77, consisting of residues 42 to 77. P1-41 inhibited the MAC formation much more strongly than P42-77, indicating that the amino-terminal half contained the active site. We further synthesized P4-18 that consisted of residues 4 to 18 and P19-41 that consisted of residues 19 to 41. The activity of P4-18 was less than that of P19-41. Surprisingly P19-41 showed higher activity than P1-41 and was comparable to urine CD59. The residues 19 to 41 were further divided into two portions : P20-25 which consisted of residues 20 to 25 and P27-38 which consisted of residues 27 to 38. Although their activities were significantly less than the activity of P19-41, P27-38 showed higher activity than P20-25. The residues 27 to 38 were further divided into three portions ; P27-32 which consisted of residues 27 to 32, P30-34 which consisted of residues 30 to 34 and P33-38 which consisted of residues 33 to 38. When these peptides were assayd for the activities, all of them showed significant activities, even though they needed 10-fold more concentrations than P19-41. These data suggest that the residues 27 to 38 is the active site constituting the binding site to C8 and C9.