A new easter-type serine protease cleaves a masquerade-like protein during prophenoloxidase activation in Holotrichia diomphalia larvae
A new easter-type serine protease cleaves a masquerade-like protein during prophenoloxidase activation in Holotrichia diomphalia larvae
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DOI：
10.1074/jbc.m205508200
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发表时间：
2002-10-18
影响因子：
4.8
通讯作者：
Lee, BL
中科院分区：
文献类型：
--
作者：
Kim, MS;Baek, MJ;Lee, BL
酚氧化酶原（proPO）激活途径，如脊椎动物补体系统，由蛋白酶级联反应组成，在这些动物中作为非自我识别系统发挥作用。确定模式识别分子区分非自我和自我的分子机制以及刺激防御反应的信号是理解先天免疫系统调节方式的关键。然而，proPO系统在分子水平上的定义很差。昆虫Holotrichia diomphalia的proPO激活系统包括几种组分，其中一些组分已经被克隆和表征，例如来自H. diomphalia幼虫和两种酚氧化酶原。PPAF-III基因编码一个由351个氨基酸残基组成的酯型丝氨酸蛋白酶酶原，分子量为40 kDa。纯化的27-kDa PPAF-III特异性地切割55-kDa proPPAF-II以产生45-kDa PPAF-II（有或没有Ca 2+存在）。此外，两个Holotrichia prophenoloxidases（proPO-I和41）的特点，并在激活过程中的结构变化进行了研究，在体外重建实验。当proPO与PPAF-I孵育时，79-kDa proPO转化为76-kDa proPO，其不显示任何酚氧化酶（PO）活性。然而，当proPO与PPAF-I、proPPAF-II和PPAF-III在Ca 2+存在下同时孵育时，除了76-kDa proPO-II蛋白外，还检测到具有PO活性的60-kDa蛋白（PO-1）。这些结果表明，Holotrichia proPOs转化为具有酶活性的酚氧化酶是通过PPAF-I，PAF-II和PPAF-III在Ca 2+存在下通过两步有限蛋白水解完成的。
The prophenoloxidase (proPO) activation pathway, like the vertebrate complement system, consists of a protease cascade and functions as a non-self-recognition system in these animals. Determining the molecular mechanism by which pattern recognition molecules differentiate non-self from self and transduce signals that stimulate defense responses is a key for understanding the ways in which innate immune systems are regulated. However, the proPO system is poorly defined at the molecular level. The proPO-activating system of the insect Holotrichia diomphalia comprises several components, some of which have been cloned and characterized, such as the novel 27-kDa proPO-activating factor-III (PPAF-III) from the plasma of H. diomphalia larvae and two prophenoloxidases. The PPAF-III gene encodes an easter-type serine protease zymogen consisting of 351 amino acid residues with a mass of 40 kDa. The purified 27-kDa PPAF-III specifically cleaved a 55-kDa proPPAF-II to generate a 45-kDa PPAF-II with or without Ca2+ present. Furthermore, two Holotrichia prophenoloxidases (proPO-I and 41) have been characterized, and their structural changes during activation were examined by in vitro reconstitution experiments. When the proPOs were incubated with PPAF-I, the 79-kDa proPOs were converted to 76-kDa proPOs, which did not exhibit any phenoloxidase (PO) activity. However, when the proPOs were incubated simultaneously with PPAF-I, proPPAF-II, and PPAF-III in the presence of Ca2+, a 60-kDa protein (PO-1) with PO activity was detected in addition to the 76-kDa proPO-II protein. These results indicate that the conversion of Holotrichia proPOs to enzymatically active phenoloxidase is accomplished by PPAF-I, PAF-II, and PPAF-III through a two-step limited proteolysis in the presence of Ca2+.