A clip domain serine protease regulates the expression of proPO and hemolymph clotting in mud crab, Scylla paramamosain

May 11, 2018Fish & shellfish immunology

A specific enzyme controls immune protein production and blood clotting in mud crabs

AI simplified

Circadian Biology on OpenScience ↗PubMed ↗DOI ↗

Abstract

The full-length cDNA of the clip domain serine proteinase from Scylla paramamosain (SpcSP) is 1823 bp and encodes a 373 amino acid polypeptide.

SpcSP possesses characteristic features of the clip domain family of serine proteases, including a signal peptide and conserved domains.
mRNA expression of SpcSP is higher in hemocytes and skin compared to other tissues in untreated S. paramamosain.
Transcription levels of SpcSP significantly increase in hemocytes following exposure to V. parahaemolyticus and lipopolysaccharides (LPS), suggesting a role in antibacterial immune response.
SpcSP is localized in the cytoplasm of all three hemocyte types (hyaline, semigranular, and granular).
Recombinant SpcSP protein shows strong binding and antimicrobial activity against both Gram-positive and Gram-negative bacteria, as well as fungi.
Knockdown of SpcSP leads to prolonged hemolymph clotting time and reduced expression of prophenoloxidase mRNA in hemocytes.

AI simplified

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 SpcSP) 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 SpcSP 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, SpcSP 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, Asp, Ser). Phylogenetic analysis showed that SpcSP was clustered together with PtcSP (clip domain serine proteinase from Portunus trituberculatus). Quantitative real-time PCR (qPCR) analysis showed that the mRNA of SpcSP 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 SpcSP 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 SpcSP was expressed in the cytoplasm of all three hemocyte cell types (hyaline, semigranular and granular cells). Further, recombinant SpcSP protein exhibited strong binding ability and has antimicrobial activity against both Gram-positive and Gram-negative bacteria as well as fungi. Moreover, knockdown of SpcSP resulted in increased hemolymph clotting time and decreased the mRNA expression of SpproPO mRNA in hemocytes. These findings therefore suggest that SpcSP plays an important role in the antimicrobial defense mechanism of S. paramamosain by regulating the expression of SpproPO and hemolymph clotting in S. paramamosain. 156 226 321

Full Text

Full text is available at the source.

View full text (XML) ↗View full text ↗

A clip domain serine protease regulates the expression of proPO and hemolymph clotting in mud crab, Scylla paramamosain

Abstract

Full Text

You found one interesting study. We’ll send the next 7.

what lands in your inbox each week:

Recent issues from the circadian biology brief

Abstract

Full Text

Related papers

You found one interesting study. We’ll send the next 7.

what lands in your inbox each week:

Recent issues from the circadian biology brief