Serpin-1 splicing isoform J inhibits the proSpätzle-activating proteinase HP8 to regulate expression of antimicrobial hemolymph proteins in Manduca sexta |
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Authors: | Chunju AnEmily J Ragan Michael R Kanost |
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Institution: | Department of Biochemistry, Kansas State University, Manhattan, KS 66506, USA |
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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 × 104 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. |
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Keywords: | BSA bovine serum albumin HP6 hemolymph proteinase 6 HP8 hemolymph proteinase 8 IEARpNa acetyl-Ile-Glu-Ala-Arg-p-nitroanilide SDS-PAGE SDS-polyacrylamide gel electrophoresis |
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