Prophenoloxidases of Anopheles gambiae

描述(由申请人提供)：酚氧化酶参与昆虫的关键生理过程，包括色素沉着、角质层晒黑、伤口愈合和防御反应。一些蚊子酚氧化酶产生活性化学物质和真黑素来杀死和隔离疟疾寄生虫。虽然反向遗传分析揭示了这种酶系统的成分，但由于缺乏生化分析，重要的知识差距仍然存在。在对水葫芦原酚氧化酶(PPO)激活系统的研究中，我们积累了丰富的关于PPO、PPO激活酶及其调节因子的知识。我们克隆了所有9个冈比亚按蚊PPO的cDNA，在大肠杆菌中以功能蛋白的形式表达，结晶了3个，并解决了1个PPO结构。初步动力学分析表明，底物选择性和催化效率存在显著差异。我们验证了半定量检测蚊子中PPO和其他蛋白质的蛋白质组学方法，并使用多克隆抗体检测组织中的PPO。通过将编码PPO保守区的dsRNA注射到雌性成虫体内，我们显著降低了它们的mRNA和蛋白质水平。在此基础上，我们建议对冈比亚按蚊PPO1-9的结构、功能和表达进行研究。所获得的知识和试剂将有助于探索寄生虫如何逃避或抑制宿主的免疫机制。

DESCRIPTION (provided by applicant): Phenoloxidases take part in key insect physiological processes including pigmentation, cuticle tanning, wound healing, and defense responses. Some mosquito phenoloxidases generate active chemicals and eumelanin to kill and sequester malaria parasites. While reverse genetic analyses have revealed components of this enzyme system, important knowledge gaps still exist due to lack of biochemical analysis. In studies of the Manduca sexta prophenoloxidase (PPO) activation system, we accumulated wealthy knowledge on PPOs, PPO activating proteases, and their regulators. We cloned cDNAs of all nine Anopheles gambiae PPOs, expressed them as functional proteins in E. coli, crystallized three, and solved one PPO structure. Initial kinetic analysis revealed striking differences in substrate preference and catalytic efficiency. We validated the proteomic approach to semi-quantify PPOs and other proteins in the mosquito and detected PPOs in tissues using polyclonal antibodies. By injecting into female adults dsRNA encoding a conserved region in PPOs, we substantially reduced their mRNA and protein levels. Based on these advances, we propose to study the structures, functions, and expression of A. gambiae PPO1-9. Acquired knowledge and reagents will be useful for exploring how parasites evade or suppress the host immune mechanism in the future.