Radboud Institute for Molecular Life Sciences, Department of Pediatric Nephrology, Amalia Children's Hospital, Radboud University Medical Center, Nijmegen, the Netherlands;
Department of Laboratory Medicine, Radboud University Medical Center, Nijmegen, the Netherlands.
J Immunol. 2021 Nov 15;207(10):2465-2472. doi: 10.4049/jimmunol.2100238. Epub 2021 Oct 11.
The complement system is an important part of innate immunity. Complement activation leads to formation of convertase enzymes, switch of their specificity from C3 to C5 cleavage, and generation of lytic membrane attack complexes (C5b-9) on surfaces of pathogens. Most C5 cleavage occurs via the complement alternative pathway (AP). The regulator properdin promotes generation and stabilization of AP convertases. However, its role in C5 activation is not yet understood. In this work, we showed that serum properdin is essential for LPS- and zymosan-induced C5b-9 generation and C5b-9-mediated lysis of rabbit erythrocytes. Furthermore, we demonstrated its essential role in C5 cleavage by AP convertases. To this end, we developed a hemolytic assay in which AP convertases were generated on rabbit erythrocytes by using properdin-depleted serum in the presence of C5 inhibitor (step 1), followed by washing and addition of purified C5-C9 components to allow C5b-9 formation (step 2). In this assay, addition of purified properdin to properdin-depleted serum during convertase formation (step 1) was required to restore C5 cleavage and C5b-9-mediated hemolysis. Importantly, C5 convertase activity was also fully restored when properdin was added together with C5b-9 components (step 2), thus after convertase formation. Moreover, with C3-depleted serum, not capable of forming new convertases but containing properdin, in step 2 of the assay, again full C5b-9 formation was observed and blocked by addition of properdin inhibitor Salp20. Thus, properdin is essential for the convertase specificity switch toward C5, and this function is independent of properdin's role in new convertase formation.
补体系统是先天免疫系统的重要组成部分。补体激活导致转化酶酶的形成，其特异性从 C3 向 C5 切割的转换，以及在病原体表面生成溶细胞攻击复合物（C5b-9）。大多数 C5 切割通过补体替代途径（AP）发生。调节剂properdin 促进 AP 转化酶的生成和稳定。然而，其在 C5 激活中的作用尚不清楚。在这项工作中，我们表明血清 properdin 对于 LPS 和酵母聚糖诱导的 C5b-9 生成和 C5b-9 介导的兔红细胞裂解是必不可少的。此外，我们证明了它在 AP 转化酶介导的 C5 切割中的重要作用。为此，我们开发了一种溶血测定法，其中在存在 C5 抑制剂的情况下，使用 properdin 耗尽的血清在兔红细胞上生成 AP 转化酶（步骤 1），然后洗涤并添加纯化的 C5-C9 成分以允许 C5b-9 形成（步骤 2）。在该测定中，在转化酶形成期间（步骤 1）将纯化的 properdin 添加到 properdin 耗尽的血清中是恢复 C5 切割和 C5b-9 介导的溶血所必需的。重要的是，当与 C5b-9 成分一起添加 properdin 时，C5 转化酶活性也完全恢复（步骤 2），因此在转化酶形成之后。此外，在步骤 2 的测定中，用 C3 耗尽的血清，不能形成新的转化酶，但含有 properdin，再次观察到完全的 C5b-9 形成，并通过添加 properdin 抑制剂 Salp20 阻止。因此，properdin 对于转化酶向 C5 的特异性转换是必不可少的，并且该功能独立于 properdin 在新转化酶形成中的作用。
