Regulation of Manduca sexta hemolymph proteinase 8 by serpin‐1 isoforms

Abstract

Some insect immune responses, including prophenoloxidase activation and Toll pathway initiation, are mediated by serine proteinase cascades and regulated by serpins. Hemolymph proteinase 8 (HP8), a trypsin‐like serine proteinase, is involved in a Toll‐like pathway by cleaving pro‐sp ?tzle, the putative ligand of M. sexta Toll. The M. sexta serpin‐1 gene is expressed as 12 isoforms, which vary in their reactive center loop due to alternative splicing of exon 9. Serpin‐1 isoforms A, E, and J can all inhibit a fungal trypsin‐like protease, PR2, in vitro. To identify the roles of serpins in the Toll pathway in M. sexta, we purified recombinant serpin‐1A, ‐1E, ‐1J and a mutant HP8 (proHP8Xa) in which the activation site was changed from NNDH to IEGR, allowing activation by bovine Factor Xa. Recombinant serpin‐1A, ‐1E, and ‐1J formed SDS‐stable complexes with HP8Xa. Serpin‐1J was the most effective inhibitor of HP8 among the tested serpin‐1 isoforms. The association rate constant of serpin‐1J and HP8 was 1.3×104 M‐1s‐1, indicating that serpin‐1J may contribute to the inhibitory regulation of HP8 in the hemolymph. Injection of serpin‐1J into M. sexta larvae resulted in a significant decrease in bacteria‐induced mRNA levels for the antibacterial peptides attacin and cecropin. These results suggest that serpin‐1J regulates the activity of HP8 to modulate the Toll pathway response in M. sexta. Supported by NIH grant GM41247.