Abstract
Melanin synthesis is essential for defense and development but must be tightly controlled because systemic hyperactivation of the prophenoloxidase and excessive melanin synthesis are deleterious to the hosts. The melanization cascade of the arthropods can be activated by bacterial lysine-peptidoglycan (PGN), diaminopimelic acid (DAP)-PGN, or fungal beta-1,3-glucan. The molecular mechanism of how DAP- or Lys-PGN induces melanin synthesis and which molecules are involved in distinguishing these PGNs are not known. The identification of PGN derivatives that can work as inhibitors of the melanization cascade and the characterization of PGN recognition molecules will provide important information to clarify how the melanization is regulated and controlled. Here, we report that a novel synthetic Lys-PGN fragment ((GlcNAc-MurNAc-L-Ala-D-isoGln-L-Lys-D-Ala)2, T-4P2) functions as a competitive inhibitor of the natural PGN-induced melanization reaction. By using a T-4P2-coupled column, we purified the Tenebrio molitor PGN recognition protein (Tm-PGRP) without causing activation of the prophenoloxidase. The purified Tm-PGRP recognized both Lys- and DAP-PGN. In vitro reconstitution experiments showed that Tm-PGRP functions as a common recognition molecule of Lys- and DAP-PGN-dependent melanization cascades.
Highlights
When we examined the effect of lipid A on Tenebrio melanin synthesis, lipid A did not induce the activation of the proPO system and melanin synthesis
-1,3-glucan, Lys-PGN, or diaminopimelic acid (DAP)-PGN-dependent proPO fractions generated new protein bands on the gel after 30 min of incubation. When we determined their N-terminal sequences of these three bands, we could confirm that all three N-terminal sequences were perfectly matched with Tenebrio PO (Fig. 7C), which are generated from proPO as we previously reported [42]. These results suggest that these proPO system fractions contain all of the essential components necessary for activation by either -1,3-glucan or Lys-PGN and DAP-PGN, and it will be possible to purify and characterize melanization-regulatory proteins involved in these three different melanization cascades as we have demonstrated with Tenebrio molitor PGN recognition protein (Tm-PGN recognition protein (PGRP)) here
The other is that Tm-PGRP showing high homology with Dm-PGRP-SA functions as a common PGN recognition molecule of DAP- or Lys-type PGN-dependent melanization cascade
Summary
Mechanism of how PGN or -1,3-glucan can activate proPO system and what proteins are involved in PGN or -1,3-glucan specific proPO cascade are not fully understood at the molecular level One reason for this is that it is difficult to obtain a fraction showing specific PO activity induced by either PGN or -1,3-glucan. An important approach to understanding how natural PGN or -1,3-glucan induce the activation of proPO system is to obtain a fraction from the crude hemolymph showing either a PGN or a -1,3glucan specific PO activities and purify proteins involved in each pathway. Recent studies have demonstrated that several PGRPs have been shown to bind directly to PGN, each with distinct preferences for binding Lys- or DAP-PGN (24 –27) This binding discrimination to Lys-PGN or DAP-PGN leads to the activation of specific immune signaling pathways (Toll and Imd, respectively, in Drosophila). We demonstrate that Tenebrio PGRP purified by using the T-4P2-coupled column works as a common PGN recognition molecule on both Lys- and DAP-PGN-dependent melanization responses
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