Cloning and characterization of PmSERPIN6, a serine proteinase inhibitor, from the black tiger shrimp Penaeus monodon

Abstract

Serine proteinase inhibitors (SERPINs or serpins) have been found as a regulator of various biological processes in a diverse range of organisms. In Penaeid shrimp, a few serpins have been identified and their functions have not been clearly characterized. Herein, eight serpin genes, namely PmSERPIN1 - 8, were identified from the Penaeus monodon EST database (http://pmonodon.biotec.or.th/home.jsp). Protein sequence alignment revealed a high similarity of PmSERPIN6 to the serpin-6 from Manduca sexta which are an inhibitor in prophenoloxidase (proPO) system, an important defense mechanism in arthropod immunity. Among those, PmSERPIN6 was selected for further characterization. Tissue distribution analysis revealed that PmSERPIN6 transcripts mainly expressed in the lymphoid organ, hemocyte, heart and gill, but not in the hepatopancreas. To investigate the participation of PmSERPIN6 in response to pathogen challenge, semi-quantitative RT-PCR analysis of PmSERPIN6 at 0 - 48 h after pathogen challenge was performed. The PmSERPIN6 transcript expression levels in hemocytes was slightly decreased after systemic white spot syndrome virus (WSSV) injection but remained unchanged upon Vibrio harveyi injection. Interestingly, immunocytochemistry showed that the number of PmSERPIN6 producing hemocyte was increased at 72 h after both V. harveyi- and WSSV- infection indicating the response of PmSERPIN6 in the late phase of pathogen challenge. Furthermore, the recombinant protein of PmSERPIN6 (rPmSERPIN6) was produced in order to assay for its proteinase inhibitory activity. After incubation of various commercial protinases with rPmSERPIN6 at the mole ratio of about 1:400, the remaining activity of trypsin, subtilisin A, chymotrypsin, and elastase decreased to 13, 13, 39, and 66%, respectively, implying that rPmSERPIN6 could inhibit the activity of all tested proteinases at different strength. In vitro assay for inhibition of prophenoloxidase activation revealed that PmSERPIN6 might not involve in regulation of the proPO system.