Identification of a novel clip domain serine proteinase (Sp-cSP) and its roles in innate immune system of mud crab Scylla paramamosain

• Full-length of Sp-cSP with 1362 bp was isolated from mud crab.
• Sp-cSP transcripts were significantly up-regulated after immune challenges.
• rSp-cSP showed strong antimicrobial activities against pathogens.

Clip domain serine proteinases and their homologs are involved in the innate immunity of invertebrates. To identify the frontline defense molecules against pathogenic infection, we isolated a novel clip domain serine proteinase (Sp-cSP) from the hemocytes of mud crab Scylla paramamosain. The full-length 1362 bp Sp-cSP contains a 1155 bp open reading frame (ORF) encoding 384 amino acids. Multiple alignment analysis showed that the putative amino acid sequence of Sp-cSP has about 52% and 51% identity with Pt-cSP2 (AFA42360) and Pt-cSP3 (AFA42361) from Portunus trituberculatus, respectively, while the similarity with other cSP sequences was lower than 30%. However, all cSP sequences possess a conserved clip domain at the N-terminal and a Tryp-SPc domain at the C-terminal. The genomic organization of Sp-cSP consists of nine exons and eight introns, with some introns containing one or more tandem repeats. RT-PCR results indicated that Sp-cSP transcripts were predominantly expressed in the subcuticular epidermis, muscle and mid-intestine, but barely detectable in the brain and heart. Further, Sp-cSP transcripts were significantly up-regulated after challenge with lipopolysaccharides (LPS), Vibrio parahaemolyticus, polyinosinic polycytidylic acid (PolyI:C) or white spot syndrome virus (WSSV). Moreover, in vitro, the recombinant Sp-cSP revealed a strong antimicrobial activity against a Gram-positive (Staphylococcus aureus) and four Gram-negative (V. parahaemolyticus, Vibrio alginolyticus, Escherichia coli, Aeromonas hydrophila) bacteria in a dose-dependent manner. Taken together, the acute-phase response to immune challenges and the antimicrobial activity assay indicate that Sp-cSP is a potent immune protector and plays an important role in host defense against pathogen invasion in S. paramamosain.