Manduca sexta Serpin-7, a Putative Regulator of Hemolymph Prophenoloxidase Activation.
Department of Biochemistry, Kansas State University, Manhattan, KS 66506, USA.Insect biochemistry and molecular biology (Impact Factor: 3.45). 04/2013; 43(7). DOI: 10.1016/j.ibmb.2013.03.015
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.
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- "to the serpin class have been identified in many insect species and 69 their functions in the regulation of the proPO system have been clearly demonstrated (Ahmad 70 et al., 2009; An and Kanost, 2010; An et al., 2011, 2012; De Gregorio et al., 2002; Gupta et 71 al., 2005; Jiang et al., 2003; Ligoxygakis et al., 2002; Liu et al., 2015a; Scherfer et al., 2008; 72 Suwanchaichinda et al., 2013; Tang et al., 2008; Tong et al., 2005; Wang and Jiang, 2004, 73 2006; Zhu et al., 2003; Zou and Jiang, 2005; Zou et al., 2010). Although, a MIP homolog 74 (PmMIP) was identified in the shrimp Penaeus monodon, only the nucleotide sequence and 75 transcript expression profiles have been reported (Angthong et al., 2010). "
ABSTRACT: Pacifastin is a recently classified family of serine proteinase inhibitors that play essential roles in various biological processes, including in the regulation of the melanization cascade. Here, a novel pacifastin-related gene, termed PmPacifastin-like, was identified from a reverse suppression subtractive hybridization (SSH) cDNA library created from hemocytes of the prophenoloxidase PmproPO1/2 co-silenced black tiger shrimp Penaeus monodon. The full-length sequences of PmPacifastin-like and its homologue LvPacifastin-like from the Pacific white shrimp Litopenaeus vannamei were determined. Sequence analysis revealed that both sequences contained thirteen conserved pacifastin light chain domains (PLDs), followed by two putative kunitz domains. Expression analysis demonstrated that the PmPacifastin-like transcript was expressed in all tested shrimp tissues and larval developmental stages, and its expression responded to Vibrio harveyi challenge. To gain insight into the functional roles of PmPacifastin-like protein, the in vivo RNA interference experiment was employed; the results showed that PmPacifastin-like depletion strongly increased PO activity. Interestingly, suppression of PmPacifastin-like also down-regulated the expression of the proPO-activating enzyme PmPPAE2 transcript; the PmPacifastin-like transcript was down-regulated after the PmproPO1/2 transcripts were silenced. Taken together, these results suggest that PmPacifastin-like is important in the shrimp proPO system and may play an essential role in shrimp immune defense against bacterial infection. These results also expand the knowledge of how pacifastin-related protein participates in the negative regulation of the proPO system in shrimp. Copyright © 2015. Published by Elsevier Ltd.
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- "Serpin-1, -2, -3, -4, -5, -6, -7 have previously been identified and characterized from the tobacco hornworm Manduca sexta. All of them could inhibit and regulate proteases that lead to activation of the pro-PO system and the cytokine spatzle function in cascades , –. "
ABSTRACT: Destruxin A is a mycotoxin that is secreted by entomopathogenic fungi which has a broad-spectrum insecticidal effect. Previous transcript and protein profiling analysis showed that destruxin A has significant effects on the expression of serine protease inhibitor genes (serpin-2, 4, 5) in the larvae of Plutella xylostella. In the current study, we aimed to understand the role of serpins under application of destruxin A. We obtained two full-length cDNA sequences of P. xylostella serpins, named serpin-4 and serpin-5, and cloned the serpin-2 gene whose full-length has already been published. Phylogenetic analysis indicated that these two serpin genes were highly clustered with other serpins associated with the immune response in other insects. The temporal and spatial expression of serpin-2, serpin-4 and serpin-5 were determined to be the highest in the fat body and hemolymph of 4th larval stage using qRT-PCR and western blot detection techniques. RNA interference (RNAi) mediated knockdown of P. xylostella serpin genes was carried out by microinjection of double-stranded RNA (dsRNA). The expression levels of serpins decreased significantly after RNAi. Results showed that the depletion of serpins induced cecropins expression, increased phenoloxidase (PO) activity, body melanization and mortality in the larvae of P. xylostella under the same lethal concentration of destruxin A. The superimposed effects of serpins RNAi were similar with the destruxin A treatment upon mortality of P. xylostella larvae. We discovered for the first time that serpins play indispensable role in P. xylostella when challenged by destruxin A and deduced the possible function mechanism of destruxin A. Our findings are conducive to fully understanding the potential insecticidal mechanism of destruxin A and constitute a well-defined potential molecular target for novel insecticides.
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- "We now know that HP1, HP6, HP8, HP14, HP17, HP21, PAPs, SPH1, and SPH2 are probably involved in proPO and pro-spätzle activation. Serpin-1 variants and serpins 3 through 7 form covalent complexes with HPs (Ragan et al., 2010; Zhu et al., 2003; Christen et al., 2012; Tong et al., 2005; Zou et al., 2005; Suwanchaichinda et al., 2013). "
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.
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