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Peptides Mimicking the Reactive Center Loop (RCL) as Potential Inhibitors of Serpin‐3 in Manduca sexta

Insects employ innate immune responses to eliminate or kill invading pathogens and parasites. Manduca sexta, the tobacco hornworm, is widely used as a model insect for investigating innate immunity, including defenses mediated by serine protease cascade pathways. These pathways are regulated by serpins (serine protease inhibitors). Through forming inactive irreversible complexes with targeted proteases, serpins efficiently block activity of serine proteases in M. sexta plasma after the cascades are triggered by exposure to microorganisms. The hydrolysis of the scissile P1‐P1’ bond in a serpin reactive center loop (RCL) by a protease leads to a large conformational rearrangement, in which the RCL inserts into β‐sheet A in the serpin, resulting in the formation of an inactive complex between the serpin and protease. Previous studies on human antithrombin, α1‐protease inhibitor, and plasminogen activator inhibitor‐1 showed that short peptides mimicking N‐terminal residues of the RCL can insert into β‐sheet A and block the formation of covalent complexes, resulting in loss of serpin inhibitory activity. We are examining the use of serpin RCL peptides in M. sexta to block the function of hemolymph serpins, as a strategy for investigating biological roles of individual serpins. As a model for these studies, we are using M. sexta serpin‐3, which has been well studied and confirmed to effectively inhibit prophenoloxidase‐activating proteinase 3 (PAP3). We designed four peptides, varying from five to eight amino acids in length, whose sequences correspond to the N‐terminal region of M. sexta serpin‐3 RCL (P14 to P7), and synthesized them via solid phase peptide synthesis. The size and purity of these peptides was tested by MALDI and HPLC. Recombinant proPAP3 was expressed in Sf9 cells using a baculovirus system, followed by purification via several chromatography steps. Purified proPAP3 was activated by incubation with its activating protease, HP21. Recombinant serpin‐3 expressed in E. coli was purified by nickel‐affinity chromatography. After incubating serpin‐3 with the RCL peptides, inhibition of PAP3 by serpin‐3 was reduced, with more than 90% loss of inhibitory activity after treatment with the P14‐P9, P14‐P8, or P14‐P7 peptides. This result suggests that the RCL peptides are acting in an insect serpin as demonstrated previously with human serpins to disrupt serpin inhibitory function. Future experiments will focus on optimizing the use of RCL peptides for investigating the functions of serpin‐3 and other hemolymph serpins as regulators of proteases in immune cascades of M. sexta.