title: Serpin-1 splicing isoform J inhibits the proSpätzle-activating proteinase HP8 to regulate expression of antimicrobial hemolymph proteins in Manduca sexta.
abstract: The innate immune system of insects include the Toll pathway, which is mediated by an extracellular serine proteinase cascade. In the tobacco hornworm, Manduca sexta, hemolymph proteinase 8 (HP8) promotes the synthesis of antimicrobial proteins by cleaving proSpätzle, the putative ligand of M. sexta Toll. HP8 has been observed to form a complex in hemolymph with M. sexta serpin-1, which has multiple alternative splicing isoforms. To investigate the regulation of HP8 and its processing of proSpätzle, we characterized the interaction of recombinant HP8 with serpin-1 isoform J (serpin-1J). Recombinant serpin-1J formed an SDS-stable complex with HP8 in vitro. The association rate constant of serpin-1J and HP8 was 1.3×10(4)M(-1)s(-1), with a stoichiometry of inhibition of 5.4. Serpin-1J inhibited the cleavage of proSpätzle by HP8. Injection of serpin-1J into M. sexta larvae resulted in decreased bacteria-induced antimicrobial activity in hemolymph and reduced expression of cecropin, attacin and hemolin mRNA in fat body. Altogether, these results suggest that serpin-1J functions to inhibit HP8 and thereby modulates the concentration of active Spätzle to regulate the Toll pathway response in M. sexta.
authors.affiliation: Department of Biochemistry, Kansas State University, Manhattan, KS 66506, USA.
authors.forename: Chunju
authors.initials: C
authors.lastname: An
authors.forename: Emily J
authors.initials: EJ
authors.lastname: Ragan
authors.forename: Michael R
authors.initials: MR
authors.lastname: Kanost
mesh_terms: D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000276:immunology*; D005786:Gene Expression Regulation; D006458:Hemolymph / Q000276:immunology*; D007113:Immunity, Innate; D019476:Insect Proteins / Q000276:immunology*; D018402:Manduca / Q000201:enzymology* / Q000276:immunology*; D020033:Protein Isoforms; D015398:Signal Transduction / Q000187:drug effects; D000515:alpha 1-Antitrypsin / Q000276:immunology*
chemical_list: D023181:Antimicrobial Cationic Peptides; D019476:Insect Proteins; D020033:Protein Isoforms; D000515:alpha 1-Antitrypsin