TY - JOUR
T1 - Identification of plasma proteinase complexes with serpin-3 in Manduca sexta
AU - Christen, Jayne M.
AU - Hiromasa, Yasuaki
AU - An, Chunju
AU - Kanost, Michael R.
N1 - Funding Information:
We thank Rebekah Woolsey from the Nevada Proteomics Center for sample digestion and MALDI-TOF/TOF analysis. The Nevada Proteomics Center is supported by National Institutes of Health Grant Number P20 RR-016464 from the INBRE Program of the National Center for Research Resources. ESI-MS/MS was performed at the Biotechnology Core/Proteomics Facility at Kansas State University. We also thank Dr. Haobo Jiang for antiserum to HP8 and access to M. sexta EST data and Dr. Neal Dittmer for helpful comments on the manuscript. This is contribution no. 13-015-J from the Kansas Agricultural Experiment Station. This work was supported by National Institutes of Health Grant GM41247 .
PY - 2012/12
Y1 - 2012/12
N2 - Extracellular serine proteinase cascades stimulate prophenoloxidase (proPO) activation and antimicrobial peptide production in insect innate immune responses. Serpins in plasma regulate such cascades by selective inhibition of proteinases, in reactions which result in the formation of covalent serpin-proteinase complexes. We carried out experiments to identify plasma proteinases that are inhibited by Manduca sexta serpin-3, an immune-inducible serpin known to regulate proPO activation. Immunoaffinity chromatography, using antiserum to serpin-3, yielded serpin-3 complexes with proteinases identified by immunoblot analysis as prophenoloxidase-activating proteinase (PAP)-1, PAP-2, PAP-3, and hemolymph proteinase 8 (HP8). HP8 can cleave and activate the Toll ligand, Spätzle, leading to synthesis of antimicrobial peptides. Analysis by mass spectrometry of tryptic peptides derived from the serpin-3 complexes confirmed the presence of PAP-1, PAP-3, and HP8. Purified recombinant serpin-3 and active HP8 formed an SDS-stable complex in vitro. Identification of serpin-3-proteinase complexes in plasma provides insight into proteinase targets of serpin-3 and extends the understanding of serpin/proteinase function in the immune response of M. sexta.
AB - Extracellular serine proteinase cascades stimulate prophenoloxidase (proPO) activation and antimicrobial peptide production in insect innate immune responses. Serpins in plasma regulate such cascades by selective inhibition of proteinases, in reactions which result in the formation of covalent serpin-proteinase complexes. We carried out experiments to identify plasma proteinases that are inhibited by Manduca sexta serpin-3, an immune-inducible serpin known to regulate proPO activation. Immunoaffinity chromatography, using antiserum to serpin-3, yielded serpin-3 complexes with proteinases identified by immunoblot analysis as prophenoloxidase-activating proteinase (PAP)-1, PAP-2, PAP-3, and hemolymph proteinase 8 (HP8). HP8 can cleave and activate the Toll ligand, Spätzle, leading to synthesis of antimicrobial peptides. Analysis by mass spectrometry of tryptic peptides derived from the serpin-3 complexes confirmed the presence of PAP-1, PAP-3, and HP8. Purified recombinant serpin-3 and active HP8 formed an SDS-stable complex in vitro. Identification of serpin-3-proteinase complexes in plasma provides insight into proteinase targets of serpin-3 and extends the understanding of serpin/proteinase function in the immune response of M. sexta.
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U2 - 10.1016/j.ibmb.2012.09.008
DO - 10.1016/j.ibmb.2012.09.008
M3 - Article
C2 - 23063421
AN - SCOPUS:84868507801
SN - 0965-1748
VL - 42
SP - 946
EP - 955
JO - Insect Biochemistry and Molecular Biology
JF - Insect Biochemistry and Molecular Biology
IS - 12
ER -