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Purification and characterization of Manduca sexta serpin-6: a serine proteinase inhibitor that selectively inhibits prophenoloxidase-activating proteinase-3.
Insect Biochem Mol Biol. 2004 Apr; 34(4):387-95.IB

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+Show Affiliations

Wang Y
Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK 74078, USA.
Jiang H
No affiliation info available

MeSH

AmidohydrolasesAmino Acid SequenceAnimalsCarrier ProteinsCatechol OxidaseChromatography, LiquidEnzyme ActivationEnzyme PrecursorsHemolymphInsect ProteinsIsoenzymesLarvaManducaPancreatitis-Associated ProteinsRecombinant ProteinsSerine EndopeptidasesSerine Proteinase InhibitorsSerpinsSpectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

15041022

Citation

Wang, Yang, and Haobo Jiang. "Purification and Characterization of Manduca Sexta Serpin-6: a Serine Proteinase Inhibitor That Selectively Inhibits Prophenoloxidase-activating Proteinase-3." Insect Biochemistry and Molecular Biology, vol. 34, no. 4, 2004, pp. 387-95.
Wang Y, Jiang H. Purification and characterization of Manduca sexta serpin-6: a serine proteinase inhibitor that selectively inhibits prophenoloxidase-activating proteinase-3. Insect Biochem Mol Biol. 2004;34(4):387-95.
Wang, Y., & Jiang, H. (2004). Purification and characterization of Manduca sexta serpin-6: a serine proteinase inhibitor that selectively inhibits prophenoloxidase-activating proteinase-3. Insect Biochemistry and Molecular Biology, 34(4), 387-95.
Wang Y, Jiang H. Purification and Characterization of Manduca Sexta Serpin-6: a Serine Proteinase Inhibitor That Selectively Inhibits Prophenoloxidase-activating Proteinase-3. Insect Biochem Mol Biol. 2004;34(4):387-95. PubMed PMID: 15041022.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Purification and characterization of Manduca sexta serpin-6: a serine proteinase inhibitor that selectively inhibits prophenoloxidase-activating proteinase-3. AU - Wang,Yang, AU - Jiang,Haobo, PY - 2003/11/04/received PY - 2003/12/22/accepted PY - 2004/3/26/pubmed PY - 2004/6/16/medline PY - 2004/3/26/entrez SP - 387 EP - 95 JF - Insect biochemistry and molecular biology JO - Insect Biochem Mol Biol VL - 34 IS - 4 N2 - 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. SN - 0965-1748 UR - https://www.unboundmedicine.com/medline/citation/15041022/Purification_and_characterization_of_Manduca_sexta_serpin_6:_a_serine_proteinase_inhibitor_that_selectively_inhibits_prophenoloxidase_activating_proteinase_3_ DB - PRIME DP - Unbound Medicine ER -