Molecular cloning of cDNA for pro‐phenol‐oxidase‐activating factor I, a serine protease is induced by lipopolysaccharide or 1,3‐β‐glucan in coleopteran insect, Holotrichia diomphalia larvae

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Abstract
Previously, we identified two pro‐phenol oxidase‐activating factors, named PPAF‐I and PPAF‐II, directly involved in the activation of the purified pro‐phenol oxidase (pro‐PO) from the hemolymph of the coleopteran, Holotrichia diomphalia larvae [Lee, S. Y., Kwon, T. H., Hyun, J. H., Choi, J. S., Kawabata, S. I., Iwanga, S, & Lee, B. L. (1998) Eur. J. Biochem. 254, 90−97]. Here, we report molecular cloning of cDNA for PPAF‐I. Based on the sequence of the cloned cDNA, the PPAF‐I gene appears to encode a member of serine protease zymogen consisting of 365 amino acid residues with a molecular mass of 40 193 Da. The 109 amino acid residues preceding the amino‐terminus Ile residue of the mature protein seem to constitute a prepro‐sequence. The mature protein is a serine protease composed of 256 amino acids with a calculated molecular mass of 28 009 Da. The overall structure is highly similar to that of Drosophila easter serine protease (42.9 % identity), an essential serine protease zymogen for pattern formation in normal embryonic development. The locations of disulfide linkages in the pro‐segment of PPAF‐I were similar to those of Tachypleus proclotting enzyme and the mammalian neutrophil‐derived defensin. Furthermore, [3H]diisopropylphosphate (iPr2P)‐labeled PPAF‐I was specifically produced from the crude preparation of PPAF‐I zymogen by incubation with lipopolysaccharide or 1,3‐β‐glucan, whereas [3H]iPr2P‐labeled PPAF‐I was not produced under the same conditions in the absence of these microbial polysaccharides. These results indicate that the pro‐PO‐activation system in H. diomphalia larvae may proceed with the activation of PPAF‐I zymogen by microbial polysaccharides.

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