title: Recombinant Drosophila prophenoloxidase 1 is sequentially cleaved by α-chymotrypsin during in vitro activation.
abstract: Insect prophenoloxidase (PPO) is an essential innate immunity protein to induce pathogen into melanization. In Bombyx mori, pro-phenoloxidase-activating enzyme (PPAE) can directly cleave and activate PPO. However, PPO in Manduca sexta cannot be cleaved into active phenoloxidase (PO) by serine proteases unless cofactors are involved, which indicates that PPO activation is complicated. Here we use recombinant Drosophila melanogaster prophenoloxidase 1 (rPPO1) to study the mechanism of PPO activation induced by a typical serine protease, α-chymotrypsin. Small amounts of α-chymotrypsin cleave rPPO1 at the N- and C-terminus to produce a large fragment rPPO1(N1/C1) that needs further cleavage by α-chymotrypsin to produce a smaller fragment rPO1(60-kD) with PO activity. rPO1(60-kD) oxidizes dopamine without being affected by high temperature, or by having salt and Ethylene diamine tetraacetic acid (EDTA) in the solution. After incubation with dopamine, rPO1(60-kD) cannot be detected using reducing SDS-PAGE due to formation of a large complex. Trypsin, another typical serine protease, cleaves rPPO1 at the N- and C-terminus to produce a small fragment rPPO1(N'/C') without PO activity. Several rPPO1 mutants were created through over-expressing active fragments that have direct PO activity. They are easily cleaved by low amounts of α-chymotrypsin without increasing PO activity. Therefore, rPPO1 can be sequentially cleaved in at least three places by α-chymotrypsin to produce activated rPO1(60-kD).
authors.affiliation: Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, People's Republic of China.
authors.forename: Anrui
authors.initials: A
authors.lastname: Lu
authors.affiliation: Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, People's Republic of China.
authors.forename: Xuquan
authors.initials: X
authors.lastname: Li
authors.affiliation: Department of Biological Sciences and Institute for Global Health, Vanderbilt University, VU Station B 35-1634, Nashville, TN 37235, USA.
authors.forename: Julián F
authors.initials: JF
authors.lastname: Hillyer
authors.affiliation: Department of Veterinary Pathobiology, University of Missouri, Columbia, MO 65211, USA.
authors.forename: Brenda T
authors.initials: BT
authors.lastname: Beerntsen
authors.affiliation: Department of Comparative Physiology, Uppsala University, Norbyvägen 18 A, 752 18 Uppsala, Sweden.
authors.forename: Kenneth
authors.initials: K
authors.lastname: Söderhäll
authors.affiliation: Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, People's Republic of China. Electronic address: erjunling@sippe.ac.cn.
authors.forename: Erjun
authors.initials: E
authors.lastname: Ling
mesh_terms: D000818:Animals; D004156:Catechol Oxidase / Q000737:chemistry / Q000378:metabolism*; D002918:Chymotrypsin / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000201:enzymology; D004789:Enzyme Activation / Q000235:genetics*; D004792:Enzyme Precursors / Q000737:chemistry / Q000378:metabolism*; D007113:Immunity, Innate / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D004792:Enzyme Precursors; D011994:Recombinant Proteins; C038451:pro-phenoloxidase; D004156:Catechol Oxidase; D002918:Chymotrypsin; C117608:alpha-chymotrypsin
keywords: Immunity; Prophenoloxidase; Structure; α-chymotrypsin