Identification of unknown components in insect prophenoloxidase activating system.

昆虫血淋巴的酚氧化酶原（proPO）级联对于入侵微生物周围的黑化和外来生物的识别是重要的。这种级联反应是由微生物细胞壁成分引发的，如真菌中的β-1,3-葡聚糖和细菌中的肽聚糖。虽然肽聚糖识别蛋白（PGRP-S1）与肽聚糖的相互作用被认为在肽聚糖介导的级联途径中启动了一系列酶促反应，但该途径的激活机制尚不清楚。本研究明确了以下几点：1。我证明了革兰氏阴性细菌结合蛋白（GNBP）是家蚕proPO级联中肽聚糖介导途径的重要组成部分，该蛋白不直接与肽聚糖结合，而是与肽聚糖和PGRP-S1的复合物结合，形成一个三联合物。这些结果表明，GNBP与PGRP-S1共同参与了昆虫先天免疫对细菌肽聚糖的识别。我从蚕的血浆中提纯了一种新的重要成分，因子H。该组分可与肽聚糖、PGRP-S1和GNBP的三元配合物相互作用，自身激活，随后激活蛋白酶酶原因子s。通过对因子H内部序列的分析，发现该蛋白含有少量的sushi结构域和一个丝氨酸蛋白酶结构域，提示其是一种新的丝氨酸蛋白酶酶原。系统研究了添加多糖和Ca^<2+>-调节蛋白对蛋白酶活性的影响，以稳定因子H。β-硫酸环糊精和钙调素结合蛋白作为非竞争性抑制因子抑制凝血酶活性，提示有可能抑制因子H的自发活化。

The prophenoloxidase (proPO) cascade of insect hemolymph is important for melanization around microbes invading the organisms and for recognition of foreignness. The cascade is triggered by microbial cell wall components such as β-1,3-glucan from fungi and peptidoglycan from bacteria. Although the interaction of peptidoglycan recognition protein (PGRP-S1) and peptidoglycan is thought to initiate a chain of enzymatic reactions in the peptidoglycan-mediated pathway of the cascade, the activating mechanism of the pathway has not been worked out yet. In this research the followings were made clear.1.I demonstrated that Gram-negative bacteria-binding protein (GNBP) is an essential component of the peptidoglycan-mediated pathway in the proPO cascade of the silkworm, Bombyx mori and this protein does not directly bind to the peptidoglycan but bind to a complex of the peptidoglycan and PGRP-S1, forming a ternary complex. These results indicate that GNBP works together with PGRP-S1 for the recognition of bacterial peptidoglycan in insect innate immunity.2.I purified a new essential component, Factor H, of the cascade from the silkworm plasma. The component could interacted with the ternary complex of peptidoglycan, PGRP-S1 and GNBP, and activates by itself, followed by the activation of a protease zymogen, Factor S. From analysis of internal sequences of Factor H, this protein was revealed to contain few sushi domains and a serine protease domain, suggesting that it is a novel zymogen of serine protease.3.The effects of addition of polysaccharides and Ca^<2+>-regulatory protein to protease activity were systematically investigated for stabilization of Factor H. β-Cyclodextrin-sulfate and calmodulin-binding protein inhibited thrombin activity as a noncompetitive inhibition, suggesting that there are a possibility of suppressing spontaneous activation of Fatcor H.