Serpin-1 splicing isoform J inhibits the proSpätzle-activating proteinase HP8 to regulate expression of antimicrobial hemolymph proteins in Manduca sexta.
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.
An
Chunju
C
Department of Biochemistry, Kansas State University, Manhattan, KS 66506, USA.
Ragan
Emily J
EJ
Kanost
Michael R
MR
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*
D023181:Antimicrobial Cationic Peptides; D019476:Insect Proteins; D020033:Protein Isoforms; D000515:alpha 1-Antitrypsin