A New Easter-type Serine Protease Cleaves a Masquerade-like Protein during Prophenoloxidase Activation in Holotrichia diomphalia Larvae

Abstract

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 Ca 2؉ present. Furthermore, two Holotrichia prophenoloxidases (proPO-I and -II) 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 Ca 2؉ , 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 twostep limited proteolysis in the presence of Ca 2؉ .The prophenoloxidase (proPO) 1 -activating system in invertebrates plays an important role in defense against pathogens and parasites and during cuticular sclerotization. The activation of the proPO system is triggered by elicitors derived from microbial cell walls, such as lipopolysaccharide (LPS), peptidoglycan, and ␤-1,3-glucan (1-3). Several pattern recognition molecules involved in the proPO system, such as peptidoglycan-binding proteins (4), proteins that bind both LPS and ␤-1,3-glucan (5, 6), and ␤-1,3-glucan-binding proteins (7, 8), have been found in various invertebrates. However, the key question is how these pattern recognition molecules can induce activation of the proPO system in response to microbial infection. One hypothesis is that the pattern recognition molecules make a complex with the proPO-activating enzyme(s) and microbial cell wall components, and then activated proPO activating enzyme(s) will convert proPO to active phenoloxidase (PO) by limited proteolysis (1-3).Recently, we characterized two new proPO-activating factors (PPAF-I and PPAF-II) from the coleopteran Holotrichia diomphalia larvae (9, 10). The overall structure of the 37-kDa proPPAF-I is highly similar to that of Drosophila easter, a serine protease that is ...

Reconstitution of a branch of the Manduca sexta prophenoloxidase activation cascade in vitro: Snake-like hemolymph proteinase 21 (HP21) cleaved by HP14 activates prophenoloxidase-activating proteinase-2 precursor

“…We expressed both pro-SPHs in a baculovirus-insect cell system and found them inactive as "cofactors" (data not shown). Probably, as demonstrated in a beetle (Kim et al, 2002), pro-SPHs also need proteolytic activation -cleaved SPHs are catalytically inactive but active in assisting PAP to produce active PO. In that case, what is M. sexta pro-SPH activating proteinase and how does it become active in response to pathogen infection?…”

“…At least in some insects, proPO activating proteinases (PAPs) require one or two non-catalytic serine proteinase homologs (SPHs) as a "cofactor" to generate active PO (Jiang et al, 1998;Lee et al, 1998;Kwon et al, 2000;Yu et al, 2003). Like PAPs, SPHs need cleavage activation by a serine proteinase in the cascade (Kim et al, 2002).…”

Peptidoglycan Recognition Proteins Involved in 1,3-β-D-Glucan-dependent Prophenoloxidase Activation System of Insect

“…We were able to reconstitute the downstream components of the proPO cascade using biochemically purified proteins from a large beetle, Holotrichia diomphalia (19). Using this in vitro system, we were able to show that the downstream part of the proPO system is regulated by two easter-type serine proteases and a masquerade-type serine protease homologue and involves a two-step proteolysis of proPOs before they exhibit any PO activity.…”

“…The soluble proteins of plasma, hemocyte lysate, and fat body were precipitated with trichloroacetic acid and subjected to SDS-PAGE and then immunoblotting with affinity-purified Hd-PGRP-1 and Hd-PGRP-2 antibodies, respectively. Previously it was reported that insect proPO system induced the activation of serine protease zymogen to active serine protease during 1,3-␤-D-glucan-dependent proPO activation (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)30). To explore the existence of serine protease activity during 1,3-␤-D-glucan-specific proPO activation, we examined the amidase activity in the presence of 1,3-␤-D-glucan and Ca 2ϩ by using commercially available trypsin substrate, Boc-PheSer-Arg-MCA.…”

“…This enzyme produces toxic compounds to microorganisms by oxidizing phenols to melanin, and it also participates in the sclerotization of the cuticle, which is vital for the survival of insects (18). Many reports have been published about the proPO of the invertebrate and its activation mechanism (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24). A key question regarding the proPO activation system is how pattern recognition molecules can induce activation of the system in response to microbial infection.…”