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

• DOI: 10.1046/j.1432-1327.2000.01695.x
• Publication Date: 2003-03-11T17:56:57Z
• Authors: Tae Hyuk Kwon; Moon Suk Kim; Hye Won Choi; Chang Hun Joo; Mi Young Cho; Bok Luel Lee
• Keywords: Enzyme Precursors; 0303 health sciences; Hemocytes; Sequence Homology, Amino Acid; Monophenol Monooxygenase; Molecular Sequence Data; Serine Endopeptidases; Recombinant Proteins; Coleoptera; 03 medical and health sciences; Catalytic Domain; Hemolymph; Larva; Animals; Drosophila Proteins; Insect Proteins; Amino Acid Sequence; Cloning, Molecular; Sequence Alignment; Catechol Oxidase; Peptide Hydrolases

Previously, we reported the molecular cloning of cDNA for prophenoloxidase activating factor‐I (PPAF‐I) that encoded a member serine proteinase group with disulfide‐knotted motif at N‐terminus and trypsin‐like catalytic domain C‐terminus [Lee, S.Y., Cho, M.Y., Hyun, J.H., Lee, K.M., Homma, K.I., Natori, S., Kawabata, S.I., Iwanaga, S. & B.L. (1998) Eur. J. Biochem . 257 , 615–621]. PPAF‐I is directly involved in activation pro‐phenoloxidase (pro‐PO) by limited proteolysis overall structure highly similar to Drosophila easter protease, an essential protease zymogen pattern formation normal embryonic development. Here, report purification another 45‐kDa novel PPAF from hemocyte lysate Holotrichia diomphalia larvae. The gene encodes homologue consisting 415 amino‐acid residues mass 45 256 Da. protein masquerade, expressed during embryogenesis, larval, pupal development melanogaster contained C‐terminus, except substitution Ser active site triad Gly had N‐terminus. A was also cloned larval library coleopteran, Tenebrio molitor By vitro reconstitution experiments, found purified homologue, pro‐PO were necessary expressing phenoloxidase activity system. However, incubation either or protein, no observed. Interestingly, when incubated absence, but not presence Ca 2+ cleaved 35‐kDa protein. RNA blot hybridization revealed expression increased hemolymph after Escherichia coli challenge.