Takata Y, Kinoshita T, Kozono H, Takeda J, Tanaka E, Hong K, Inoue K
J Exp Med. 1987 Jun 1;165(6):1494-507. doi: 10.1084/jem.165.6.1494.
The C5 convertase of the classical complement pathway is a complex enzyme consisting of three complement fragments, C4b, C2a, and C3b. Previous studies have elucidated functional roles of each subunit (4, 6, 7), but little is known about how the subunits associate with each other. In this investigation, we studied the nature of the classical C5 convertase that was assembled on sheep erythrocytes. We found that one of the nascent C3b molecules that had been generated by the C3 convertase directly bound covalently to C4b. C3b bound to the alpha' chain of C4b through an ester bond, which could be cleaved by treatment with hydroxylamine. The ester bond was rather unstable, with a half-life of 7.9 h at pH 7.4 and 37 degrees C. Formation of the C4b-C3b dimer is quite efficient; e.g., 54% of the cell-bound C3b was associated with C4b when 25,000 molecules of C4b and 12,000 molecules of C3b were present per cell. Kinetic analysis also showed the efficient formation of the C4b-C3b dimer; the rate of dimer formation was similar to or even faster than that of cell-bound monomeric C3b molecules. These results indicate that C4b is a highly reactive acceptor molecule for nascent C3b. High-affinity C5-binding sites with an association constant of 2.1 X 10(8) L/M were demonstrated on C4b-C3b dimer-bearing sheep erythrocytes, EAC43 cells. The number of high-affinity C5-binding sites coincided with the number of C4b-C3b dimers, but not with the total number of cell-bound C3b molecules. Anti-C4 antibodies caused 80% inhibition of the binding of C5 to EAC43 cells. These results suggest that only C4b-associated C3b serves as a high-affinity C5 binding site. EAC14 cells had a small amount of high-affinity C5 binding sites with an association constant of 8.1 X 10(7) L/M, 100 molecules of bound C4b being necessary for 1 binding site. In accordance with the hypothesis that C4b-associated C4b might also serve as a high-affinity C5-binding site, a small amount of C4b-C4b dimer was detected on EAC14 cells by SDS-PAGE analysis. Taken together, these observations indicate that the high-affinity binding of C5 is probably divalent, in that C5 recognizes both protomers in the dimers. The high-affinity binding may allow selective binding of C5 to the convertase in spite of surrounding monomeric C3b molecules.
经典补体途径的C5转化酶是一种复合酶，由三个补体片段C4b、C2a和C3b组成。以往的研究阐明了每个亚基的功能作用（4, 6, 7），但对于这些亚基如何相互结合却知之甚少。在本研究中，我们研究了在绵羊红细胞上组装的经典C5转化酶的性质。我们发现，由C3转化酶产生的新生C3b分子之一直接与C4b共价结合。C3b通过酯键与C4b的α'链结合，该酯键可通过羟胺处理裂解。该酯键相当不稳定，在pH 7.4和37℃下的半衰期为7.9小时。C4b - C3b二聚体的形成非常高效；例如，当每个细胞存在25,000个C4b分子和12,000个C3b分子时，54%的细胞结合C3b与C4b相关联。动力学分析也显示了C4b - C3b二聚体的高效形成；二聚体形成的速率与细胞结合的单体C3b分子的速率相似甚至更快。这些结果表明，C4b是新生C3b的高反应性受体分子。在带有C4b - C3b二聚体的绵羊红细胞（EAC43细胞）上证明了具有2.1×10⁸ L/M结合常数的高亲和力C5结合位点。高亲和力C5结合位点的数量与C4b - C3b二聚体的数量一致，但与细胞结合的C3b分子总数不一致。抗C4抗体导致C5与EAC43细胞结合的抑制率达80%。这些结果表明，只有与C4b相关的C3b作为高亲和力C5结合位点。EAC14细胞具有少量结合常数为8.1×10⁷ L/M的高亲和力C5结合位点，1个结合位点需要100个结合的C4b分子。根据与C4b相关的C4b也可能作为高亲和力C5结合位点的假设，通过SDS - PAGE分析在EAC14细胞上检测到少量C4b - C4b二聚体。综上所述，这些观察结果表明，C5的高亲和力结合可能是二价的，因为C5识别二聚体中的两个原体。高亲和力结合可能允许C5选择性地与转化酶结合，尽管周围存在单体C3b分子。
