Molecular cloning of cDNA for pro-phenol-oxidase-activating factor I, a serine protease is induced by lipopolysaccharide or 1,3-beta-glucan in coleopteran insect, Holotrichia diomphalia larvae.
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 40193 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 28009 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-beta-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.
Lee
S Y
SY
College of Pharmacy, Pusan National University, Kumjeong Ku, Korea.
Cho
M Y
MY
Hyun
J H
JH
Lee
K M
KM
Homma
K I
KI
Natori
S
S
Kawabata
S I
SI
Iwanaga
S
S
Lee
B L
BL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D001517:Coleoptera / Q000196:embryology / Q000201:enzymology*; D018076:DNA, Complementary; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D005936:Glucans / Q000494:pharmacology*; D007814:Larva / Q000201:enzymology; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000235:genetics* / Q000378:metabolism; D047071:beta-Glucans
D018076:DNA, Complementary; D004792:Enzyme Precursors; D005936:Glucans; D008070:Lipopolysaccharides; D047071:beta-Glucans; C033363:beta-1,3-glucan; D012697:Serine Endopeptidases; C115269:pro-phenol oxidase activating proteinase