Identification of unknown components in insect prophenoloxidase activating system.

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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.