title: Purification and characterization of Manduca sexta serpin-6: a serine proteinase inhibitor that selectively inhibits prophenoloxidase-activating proteinase-3.
abstract: The proteolytic activation of prophenoloxidase (proPO) is a critical defense mechanism in insects and crustaceans. We have isolated three prophenoloxidase-activating proteinases (PAPs) from cuticular extracts or hemolymph of Manduca sexta pharate pupae, which are negatively regulated by serpin-1J and serpin-3. To test if other serpins may also inhibit the PAPs, we fractionated the induced hemolymph by ammonium sulfate precipitation, gel filtration, and lectin affinity chromatography. A 47 kDa protein, designated M. sexta serpin-6, was identified in concanavalin A-bound fractions, which formed an SDS-stable complex with PAP-3. This inhibitor, not recognized by the serpin-1 or serpin-3 antibodies, was further purified on HPLC anion exchange and hydroxylapatite columns. The molecular mass and isoelectric point of serpin-6 were found to be 46,710 +/- 10 Da and 5.4. While its amino terminus was blocked, we obtained five internal peptide sequences, one of which is highly similar to M. sexta serpins-1, -2, and -3. Serpin-6 strongly inhibited PAP-3 but not PAP-1 or PAP-2, suggesting that the proPO activation by PAPs is differentially regulated by multiple serpins. When included in the reaction mixture containing proPO, PAP-3, and its cofactor, serpin-6 efficiently blocked the cleavage activation of proPO.
authors.affiliation: Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK 74078, USA.
authors.forename: Yang
authors.initials: Y
authors.lastname: Wang
authors.forename: Haobo
authors.initials: H
authors.lastname: Jiang
mesh_terms: D000581:Amidohydrolases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D004156:Catechol Oxidase / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods; D004789:Enzyme Activation / Q000187:drug effects; D004792:Enzyme Precursors / Q000378:metabolism; D006458:Hemolymph / Q000737:chemistry; D019476:Insect Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007814:Larva / Q000737:chemistry; D018402:Manduca / Q000737:chemistry*; D000073718:Pancreatitis-Associated Proteins; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology*; D015843:Serpins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
chemical_list: D002352:Carrier Proteins; D004792:Enzyme Precursors; D019476:Insect Proteins; D007527:Isoenzymes; D000073718:Pancreatitis-Associated Proteins; C000612141:REG3A protein, human; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D015843:Serpins; C118897:peptidoglycan recognition protein; C038451:pro-phenoloxidase; D004156:Catechol Oxidase; D012697:Serine Endopeptidases; C022435:prephenoloxidase-activating enzyme; D000581:Amidohydrolases