Molecular Cloning and Biochemical Characterization of Manduca sexta Serpin‐7, a Regulator of Prophenoloxidase Activation

Abstract

Serine protease inhibitors (serpins) are involved in various physiological reactions in humans and insects. Six serpins, SPN1‐SPN6, have been identified and characterized in the tobacco hornworm Manduca sexta. In this study, we obtained a complete DNA sequence of another Manduca serpin gene, SPN7. The putative mature SPN7 protein was 385 amino acid residues long (MW 42.90 kDa, pI 5.32) with a predicted 15 residue signal sequence. Multiple sequence alignment of the reactive center loop region of the M. sexta serpins indicated that SPN7 contained Arg and Ile residues at P1′‐P1 positions, similar to SPN4 and 5. RT‐PCR results demonstrated an up‐regulation of SPN7 expression in the fat body at 24 h after injection of larvae with Micrococcus. The recombinant SPN7 protein was expressed in E. coli and purified using nickel‐affinity chromatography. Addition of recombinant SPN7 significantly inhibited both 1) spontaneous melanization of the 5th instar larval plasma, and 2) phenoloxidase activity of the plasma challenged by Micrococcus bacteria. SPN7 inhibited the enzymatic activity of the active prophenoloxidase‐activating protease‐3 by forming a stable serpin‐protease complex as indicated by SDS‐PAGE under reducing conditions. SPN7 inhibited bovine trypsin but not chymotrypsin. We conclude that SPN7 is inhibitory and likely involved in regulation of innate immunity in the insect. (supported by NIH grant GM41247)