A masquerade‐like serine proteinase homologue is necessary for phenoloxidase activity in the coleopteran insect,Holotrichia diomphalialarvae

Abstract

Previously, we reported the molecular cloning of cDNA for the prophenoloxidase activating factor‐I (PPAF‐I) that encoded a member of the serine proteinase group with a disulfide‐knotted motif at the N‐terminus and a trypsin‐like catalytic domain at the C‐terminus [Lee, S.Y., Cho, M.Y., Hyun, J.H., Lee, K.M., Homma, K.I., Natori, S., Kawabata, S.I., Iwanaga, S. & Lee, B.L. (1998) Eur. J. Biochem. 257, 615–621]. PPAF‐I is directly involved in the activation of pro‐phenoloxidase (pro‐PO) by limited proteolysis and the overall structure is highly similar to that of Drosophila easter serine protease, an essential serine protease zymogen for pattern formation in normal embryonic development. Here, we report purification and molecular cloning of cDNA for another 45‐kDa novel PPAF from the hemocyte lysate of Holotrichia diomphalia larvae. The gene encodes a serine proteinase homologue consisting of 415 amino‐acid residues with a molecular mass of 45 256 Da. The overall structure of the 45‐kDa protein is similar to that of masquerade, a serine proteinase homologue expressed during embryogenesis, larval, and pupal development in Drosophila melanogaster. The 45‐kDa protein contained a trypsin‐like serine proteinase domain at the C‐terminus, except for the substitution of Ser of the active site triad to Gly and had a disulfide‐knotted domain at the N‐terminus. A highly similar 45‐kDa serine proteinase homologue was also cloned from the larval cDNA library of another coleopteran, Tenebrio molitor. By in vitro reconstitution experiments, we found that the purified 45‐kDa serine proteinase homologue, the purified active PPAF‐I and the purified pro‐PO were necessary for expressing phenoloxidase activity in the Holotrichia pro‐PO system. However, incubation of pro‐PO with either PPAF‐I or 45‐kDa protein, no phenoloxidase activity was observed. Interestingly, when the 45‐kDa protein was incubated with PPAF‐I and pro‐PO in the absence, but not in the presence of Ca2+, the 45‐kDa protein was cleaved to a 35‐kDa protein. RNA blot hybridization revealed that expression of the 45‐kDa protein was increased in the Holotrichia hemolymph after Escherichia coli challenge.

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

“…However, the partial amino acid sequence of band 5 showed an identity with that of Holotrichia proPO-activating factor II that was previously shown to be involved in Holotrichia proPO activation (Ref. 25, data not shown). Interestingly, the amino-terminal sequences of bands 7 and 8 showed high identities with those of Drosophila peptidoglycan recognition protein-SA (Fig.…”

“…Animals and Collection of Hemolymph-Methods for rearing the insects and collection of hemolymph were as previously described (25). Hemocytes were collected from the hemolymph by centrifugation at 200 ϫ g for 10 min at 4°C, washed with anti-coagulation buffer (30 mM trisodium citrate, 26 mM citric acid, 20 mM EDTA, and 15 mM sodium chloride, pH 4.6, buffer A), and stored at Ϫ80°C.…”

“…The supernatant was taken as plasma, adjusted to pH 4.6 with 1 M citric acid solution, and stored at Ϫ80°C until use. The hemocyte lysate was prepared according to our previously published method (25). Briefly, 5 ml of packed cells were suspended in 10 ml of 50 mM Tris-HCl, 1 mM EDTA, pH 6.5, and then subjected to ultrasonification five times for 3 s at 4°C.…”

Hemolymph Melanization in the Silkmoth Bombyx mori Involves Formation of a High Molecular Mass Complex That Metabolizes Tyrosine

“…This has led to the identification of PRRs in B. mori and several other species that activate the PO cascade when they bind microbial cell wall components (33)(34)(35). Studies in this area have also shed important light on the function of SPHs as PAP cofactors (47,48) and as proteins that bind to PO and microbial cell wall components to form complexes that localize melanin deposition to the surface of bacteria (27,28,38,40).…”

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

“…The enzyme, through sequentially activated serine proteinases, eventually yields a specific proteinase to cleave proPO. 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).…”