Abstract
Melanization reaction, resulting from the activation of prophenoloxidase, is a vital immune response in insects for encapsulating and killing the invasive organisms. Prophenoloxidase needs to be proteolytically activated by its upstream prophenoloxidase-activating protease (PAP) in melanization. Identification and characterization of PAPs facilitates the understanding of the molecular mechanisms involved in insect immunity. We here cloned a full-length cDNA for a serine protease, named as SP105, from Asian corn borer, Ostrinia furnacalis (Guenée). The open reading frame of SP105 encodes 424-amino acid residue protein with a 19-residue signal peptide. Sequence comparison indicates that SP105 is most similar to Manduca sexta PAP3, a defined prophenoloxidase-activating protease. qRT-PCR analysis showed that SP105 mRNA levels increased significantly after a bacterial injection. Recombinant SP105 directly cleaved and activated Asian corn borer prophenoloxidase and therefore acted as the prophenoloxidase-activating protease. Additionally, SP105 formed SDS-stable complexes with a serine protease inhibitor, serpin-3, and its activity in activating prophenoloxidase was efficiently inhibited by serpin-3. Our work thus illustrated a prophenoloxidase-activating protease and revealed its regulation by serpin-3. The results would allow further advances in the understanding of the melanization in Asian corn borer and other insects.
Highlights
Require the specific proteolytic cleavage at the activation site for conducting functions[3,27]
The cDNA sequences of serine protease 8 (SP8) and SP105 were successfully submitted to NCBI, with GenBank accession number as KT751522 and KT751521, respectively
The conceptual protein deduced from nucleotide sequence of SP8 and SP105 consists of 424 amino acid residues, including a predicted 19-residue secretion signal peptide
Summary
Require the specific proteolytic cleavage at the activation site for conducting functions[3,27]. This activation is often regulated by members of the serine protease inhibitor (serpin) superfamily[29,30,31]. We have characterized the transcriptome of Asian corn borer larvae[43], and indicated that two serine proteases, SP1 and SP13 mediated the melanization response[44]. We cloned a full-length cDNA for a serine protease, named as SP105 (GenBank: KT751521) from Asian corn borer. Recombinant SP105 directly cleaved and activated Asian corn borer prophenoloxidase. The activity of SP105 in cleaving prophenoloxidase was regulated by a defined serine protease inhibitor, serpin-3
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