Manduca sexta serpin-7, a putative regulator of hemolymph prophenoloxidase activation

Abstract

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.

Changes in the Plasma Proteome of Manduca sexta Larvae in Relation to the Transcriptome Variations after an Immune Challenge: Evidence for High Molecular Weight Immune Complex Formation

“…Fortunately, proteolytic activation of the precursors of SPs (including HPs, PAPs), SPHs, POs, PSP, and spä tzle-1 has been well documented in M. sexta (2,16,32,43). It is also known that serpins and other protease inhibitors are involved in the control of immune SPs (19,(33)(34)(35)44). While the current analysis lacks the resolution needed to reveal components of the serpin-protease complexes, it did provide consistent results and new leads (e.g.…”

Ostrinia furnacalis serpin-3 regulates melanization cascade by inhibiting a prophenoloxidase-activating protease

“…2A). This immune-responsive expression profile has been observed in M. sexta serpin-4 through -7 (Suwanchaichinda et al, 2013;Zou and Jiang, 2005) and B. mori serpin-3 (Zou et al, 2009). It is likely that O. furnacalis serpin-3 controls a specific protease reaction in the hemolymph.…”

“…We infer there is other serpin(s) in the hemolymph, which work together with serpin-3 to regulate the proPO activation in O. furnacalis. For example, at least five serpins (serpin-3 through -7) were present in M. sexta hemolymph to cooperatively regulate the proPO activation cascade Zou and Jiang, 2005;Suwanchaichinda et al, 2013). We currently identified several transcripts other than serpin-3 from O. furnacalis transcriptome, which also encode potential serpins for the regulation of proPO activation.…”

“…Serpins have been reported in three different of insect orders (flies, beetle, and moths) to participate in the regulation of melanization reactions. They include SRPN1 and SRPN2 in Aedes agypti (Zou et al, 2010), SRPN2 in Anopheles gambiae (An et al, 2011a;Michel et al, 2005), anti-trypsin and anti-chymotrypsin in Bombyx mori (Sasaki, 1991;Sasaki and Kobayashi, 1984), Spn27A, Spn28D and Spn77Ba in Drosophila melanogaster (De Gregorio et al, 2002;Ligoxygakis et al, 2002;Scherfer et al, 2008;, serpin-1, -3 through À5, and À7 in Manduca sexta Jiang et al, 2003;Suwanchaichinda et al, 2013;Zhu et al, 2003;Zou and Jiang, 2005), and SPN40, SPN55 and SPN48 in Tenebrio molitor . In most of these cases, the involvement of serpins in regulating the melanization reaction is determined, but the protease the serpin inhibits has not been clearly demonstrated.…”

“…In M. sexta, serpin-1 splicing isoform J (serpin-1J), serpin-3, serpin-6, and serpin-7 regulate proPO activation in plasma by inhibiting PAPs Suwanchaichinda et al, 2013;Zhu et al, 2003;Zou and Jiang, 2005). Serpin-1I, serpin-4 and serpin-5 block proPO activation by inhibiting the proteases upstream of PAPs Wang and Jiang, 2006).…”