A clip domain serine protease regulates the expression of proPO and hemolymph clotting in mud crab, Scylla paramamosain
Abstract The clip domain serine proteinases (clip-SPs) play vital roles in embryonic development and in various innate immune functions in invertebrates such as antimicrobial activity, cell adhesion, hemolymph clotting, pattern recognition and regulation of the prophenoloxidase system. However, little is known about the role of the clip domain serine proteinase in Scylla paramamosain (designated Sp cSP) immunity. In the present study, we cloned a clip-SP from S. paramamosain hemocytes using rapid amplification of cDNA end (RACE) approach. The full-length cDNA of Sp cSP was 1823 bp, containing a 5′ untranslated region (UTR) of 334 bp, an open reading frame of 1122 bp, and a 3′ UTR of 367 bp. The open reading frame encoded a polypeptide of 373 amino acids with a calculated molecular weight of 39.7 kDa and an isoelectric point of 6.64. Structurally, Sp cSP has a predicted 21-residue signal peptide and possessed the characteristic features of the clip domain family of serine proteases, namely one clip domain in the amino-terminal with six highly conserved cysteine residues and one enzyme active serine proteinase domain in the carboxyl-terminal with a highly conserved catalytic triad (His 156 , Asp 226 , Ser 321 ). Phylogenetic analysis showed that Sp cSP was clustered together with Pt cSP (clip domain serine proteinase from Portunus trituberculatus ). Quantitative real-time PCR (qPCR) analysis showed that the mRNA of Sp cSP was constitutively expressed at different levels in all tested tissues in untreated S. paramamosain , with hemocytes and skin expressing the most. The transcriptional level of Sp cSP in hemocytes was significantly up-regulated upon challenge with V. parahaemolyticus and LPS, indicating its involvement in antibacterial immune response. Indirect immunofluorescence analysis showed that Sp cSP was expressed in the cytoplasm of all three hemocyte cell types (hyaline, semigranular and granular cells). Further, recombinant Sp cSP protein exhibited strong binding ability and has antimicrobial activity against both Gram-positive and Gram-negative bacteria as well as fungi. Moreover, knockdown of Sp cSP resulted in increased hemolymph clotting time and decreased the mRNA expression of Sp proPO mRNA in hemocytes. These findings therefore suggest that Sp cSP plays an important role in the antimicrobial defense mechanism of S. paramamosain by regulating the expression of Sp proPO and hemolymph clotting in S. paramamosain. Highlights Full-length of Sp cSP with 1823 bp was isolated from mud crab. Recombinant Sp cSP protein exhibited strong binding ability and has antimicrobial activity ability to specific pathogen. SpcSP might function in hemolymph clotting and proPO in hemocytes in mud crab.
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