Mark Linda, Lee Wen H, Spiller O Brad, Proctor David, Blackbourn David J, Villoutreix Bruno O, Blom Anna M
Department of Clinical Chemistry, Lund University, University Hospital Malmö, S-20502 Malmö, Sweden.
J Biol Chem. 2004 Oct 22;279(43):45093-101. doi: 10.1074/jbc.M407558200. Epub 2004 Aug 10.
Kaposi's sarcoma-associated human herpesvirus (KSHV) is thought to cause Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. Previously, we reported that the KSHV complement control protein (KCP) encoded within the viral genome is a potent regulator of the complement system; it acts both as a cofactor for factor I and accelerates decay of the C3 convertases (Spiller, O. B., Blackbourn, D. J., Mark, L., Proctor, D. G., and Blom, A. M. (2003) J. Biol. Chem. 278, 9283-9289). KCP is a homologue to human complement regulators, being comprised of four complement control protein (CCP) domains. In this, the first study to identify the functional sites of a viral homologue at the amino acid level, we created a three-dimensional homology-based model followed by site-directed mutagenesis to locate complement regulatory sites. Classical pathway regulation, both through decay acceleration and factor I cleavage of C4b, required a cluster of positively charged amino acids in CCP1 stretching into CCP2 (Arg-20, Arg-33, Arg-35, Lys-64, Lys-65, and Lys-88) as well as positively (Lys-131, Lys-133, and His-135) and negatively (Glu-99, Glu-152, and Asp-155) charged areas at opposing faces of the border region between CCPs 2 and 3. The regulation of the alternative pathway (via factor I-mediated C3b cleavage) was found to both overlap with classical pathway regulatory sites (Lys-64, Lys-65, Lys-88 and Lys-131, Lys-133, His-135) as well as require unique, more C-terminal residues in CCPs 3 and 4 (His-158, His-171, and His-213) and CCP 4 (Phe-195, Phe-207, and Leu-209). We show here that KCP has evolved to maintain the spatial structure of its functional sites, especially the positively charged patches, compared with host complement regulators.
卡波西肉瘤相关人类疱疹病毒（KSHV）被认为可引发卡波西肉瘤、原发性渗出性淋巴瘤和多中心性Castleman病。此前，我们报道过病毒基因组中编码的KSHV补体控制蛋白（KCP）是补体系统的强效调节剂；它既作为I因子的辅因子发挥作用，又能加速C3转化酶的衰变（Spiller, O. B., Blackbourn, D. J., Mark, L., Proctor, D. G., and Blom, A. M. (2003) J. Biol. Chem. 278, 9283 - 9289）。KCP是人类补体调节蛋白的同源物，由四个补体控制蛋白（CCP）结构域组成。在这项首次在氨基酸水平鉴定病毒同源物功能位点的研究中，我们构建了基于三维同源性的模型，随后进行定点诱变以定位补体调节位点。经典途径的调节，无论是通过衰变加速还是I因子对C4b的裂解，都需要CCP1中延伸至CCP2的一簇带正电荷的氨基酸（Arg - 20、Arg - 33、Arg - 35、Lys - 64、Lys - 65和Lys - 88），以及CCP2和CCP3之间边界区域相对面上的带正电区域（Lys - 131、Lys - 133和His - 135）和带负电区域（Glu - 99、Glu - 152和Asp - 155）。发现替代途径的调节（通过I因子介导的C3b裂解）既与经典途径调节位点重叠（Lys - 64、Lys - 65、Lys - 88和Lys - 131、Lys - 133、His - 135），也需要CCP3和CCP4中独特的、更靠近C端的残基（His - 158、His - 171和His - 213）以及CCP4中的残基（Phe - 195、Phe - 207和Leu - 209）。我们在此表明，与宿主补体调节蛋白相比，KCP已进化以维持其功能位点的空间结构，尤其是带正电荷的区域。
