Serpins regulate various physiological reactions in humans and insects, including certain immune responses, primarily through inhibition of serine proteases. Six serpins have previously been identified and characterized in the tobacco hornworm Manduca sexta. In this study, we obtained a full-length cDNA sequence of another Manduca serpin, named serpin-7. The open reading frame of serpin-7 encodes a polypeptide of 400 amino acid residues with a predicted signal peptide of the first 15 residues. Multiple protein sequence alignment of the reactive center loop region of the M. sexta serpins indicated that serpin-7 contains Arg–Ile at the position of the predicted scissile bond cleaved by protease in the serpin inhibition mechanism. The same residues occur in the scissile bond of the reactive center loop in M. sexta serpin-4 and serpin-5, which are protease inhibitors that can block prophenoloxidase activation in plasma. Serpin-7 transcript was detected in hemocytes and fat body, and its expression increased in fat body after injection of larvae with Micrococcus luteus. Recombinant serpin-7 added to larval plasma inhibited spontaneous melanization and decreased prophenoloxidase activation stimulated by bacteria. Serpin-7 inhibited prophenoloxidase-activating protease-3 (PAP3), forming a stable serpin-protease complex. Considering that serpin-3 and serpin-6 are also efficient inhibitors of PAP3, it appears that multiple serpins present in plasma may have redundant or overlapping functions. We conclude that serpin-7 has serine protease inhibitory activity and is likely involved in regulation of proPO activation or other protease-mediated aspects of innate immunity in M. sexta.
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)
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