Prophenoloxidase-activating Enzyme of the Silkworm, Bombyx mori

Abstract

Prophenoloxidase-activating enzyme (PPAE) was purified to homogeneity as judged by SDS-polyacrylamide gel electrophoresis from larval cuticles of the silkworm, Bombyx mori. The purified PPAE preparation was shown to be a mixture of the isozymes of PPAE (PPAE-I and PPAE-II), which were eluted at different retention times in reversed-phase high performance liquid chromatography. PPAE-I and PPAE-II seemed to be post translationally modified isozymes and/or allelic variants. Both PPAE isozymes were proteins composed of two polypeptides (heavy and light chains) that are linked by disulfide linkage(s) and glycosylated serine proteases. The results of cDNA cloning, peptide mapping, and amino acid sequencing of PPAE revealed that PPAE is synthesized as prepro-PPAE with 441 amino acid residues and is activated from pro-PPAE by cleavage of a peptide bond between Lys 152 and Ile 153 . The homology search showed 36.9% identity of PPAE to easter, which is a serine protease involved in dorsoventral pattern formation in the Drosophila embryo, and indicated the presence of two consecutive clip-like domains in the light chain. A single copy of the PPAE gene was suggested to be present in the silkworm genome. In the fifth instar larvae, PPAE transcripts were detected in the integument, hemocytes, and salivary glands but not in the fat body or mid gut. A polypeptide cross-reactive to mono-specific anti-PPAE/IgG was transiently detected in the extract of eggs between 1 and 3 h after they were laid.

Cuticular Pro-phenoloxidase of the Silkworm, Bombyx mori

“…ProPO-activating Enzyme (PPAE)-Crude PPAE preparation referred to as PPAE (AS-salt) and purified PPAE preparation were obtained from larval cuticles of silkworms as previously reported (14).…”

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

“…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.…”

Characterization and Properties of a 1,3-β-d-Glucan Pattern Recognition Protein of Tenebrio molitor Larvae That Is Specifically Degraded by Serine Protease during Prophenoloxidase Activation

“…Microbial carbohydrates such as LPS, PGN, or 1,3-D-␤-glucan will first react with pattern recognition proteins, which then will induce activation of several serine proteases within the proPO system (7)(8)(9)(10)(11)(12). It is known that one of the serine proteases in the cascade, which is named proPO-activating enzyme or factor, will cleave proPO to generate the active enzyme, phenoloxidase (PO) (13)(14)(15)(16). This enzyme can produce 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 (17).…”