Manduca sexta proprophenoloxidase activating proteinase-3 (PAP3) stimulates melanization by activating proPAP3, proSPHs, and proPOs

Abstract

Melanization participates in various insect physiological processes including antimicrobial immune responses. Phenoloxidase (PO), a critical component of the enzyme system catalyzing melanin formation, is produced as an inactive precursor prophenoloxidase (proPO) and becomes active via specific proteolytic cleavage by proPO activating proteinase (PAP). In Manduca sexta, three PAPs can activate proPOs in the presence of two serine proteinase homologs (SPH1 and SPH2). While the hemolymph proteinases (HPs) that generate the active PAPs are known, it is unclear how the proSPHs (especially proSPH1) are activated. In this study, we isolated from plasma of bar-stage M. sexta larvae an Ile-Glu-Ala-Arg-p-nitroanilide hydrolyzing enzyme that cleaved the proSPHs. This proteinase, PAP3, generated active SPH1 and SPH2, which function as cofactors for PAP3 in proPO activation. Cleavage of the purified recombinant proSPHs by PAP3 yielded 38 kDa bands similar in mobility to the SPHs formed in vivo. Surprisingly, PAP3 also can activate proPAP3 to stimulate melanization in a direct positive feedback loop. The enhanced proPO activation concurred with the cleavage activation of proHP6, proHP8, proPAP1, proPAP3, proSPH1, proSPH2, proPOs, but not proHP14 or proHP21. These results indicate that PAP3, like PAP1, is a key factor of the self-reinforcing mechanism in the proPO activation system, which is linked to other immune responses in M. sexta.

Multifaceted biological insights from a draft genome sequence of the tobacco hornworm moth, Manduca sexta

“…Protease cascades in M. sexta hemolymph have been extensively investigated through reconstitution of pathways using purified natural and recombinant proteins and by ex vivo analysis of proteins added to hemolymph plasma (An et al, 2009; Gorman et al, 2007; Jiang, 2008; Kanost et al, 2004; Wang and Jiang, 2007, 2008; Wang et al, 2014). In the current studies, we found that serine proteases and catalytically inactive pseudoproteases, termed serine protease homologs (SPH), are encoded by 193 genes in M. sexta .…”

“…Pathways of clip domain proteases and SPHs (acting as cofactors with poorly understood molecular function) that activate proPO and the Toll ligand spaetzle have been identified in M. sexta hemolymph, involving 6 clip SPs and 2 SPHs (Fig. 9) (Kanost and Nardi, 2010; Wang et al, 2014). Sequence alignment and phylogenetic analysis revealed evolutionary relationships among the clip-domain proteins (Fig.…”

CLIPB8 is part of the prophenoloxidase activation system in Anopheles gambiae mosquitoes

“…An alternative and more parsimonious explanation is that CLIPB8 is required for the activation of one or several proteolytically inactive CLIPAs that complement the proPO cascade. Published examples for proteolytic activation of CLIPAs by CLIPBs exist from other insect species (Kan et al, 2008; Wang et al, 2014). A candidate for such a proteinase homolog is CLIPA8, which is required for melanization of bacteria, fungi and P. berghei (Schnitger et al, 2007; Volz et al, 2006; Yassine et al, 2012).…”

In search of a function of Manduca sexta hemolymph protease-1 in the innate immune system

“…In the proteomic analysis of larval hemolymph, we identified 36 SPs and SPHs, nine of which are known to be components of the SP-SPH system (Zhang et al, 2014; He et al, 2016). They are: HP14 (HP for hemolymph protease), HP21, HP6, HP8, proPO activating proteases (PAP1–3), SPH1, and SPH2 (Jiang et al, 2010; Wang et al, 2014). We isolated SP and SPH cDNAs (or genes) from hemocytes or fat body for HP1–4, PAP1–3, HP5–24, SPH1, and SPH2 (Jiang et al, 1998, 1999, 2003a, 2003b and 2005; Wang et al, 2006; Yu et al, 2003).…”