SpTGase plays an important role in the hemolymph clotting in mud crab (Scylla paramamosain).

Affiliation

Guangdong Provincial Key Laboratory of Marine Biology, Shantou University, Shantou, 515063, China; Marine Biology Institute, Shantou University, Shantou, 515063, China; STU-UMT Joint Shellfish Research Laboratory, Shantou University, Shantou, 515063, China. Electronic address: [Email]

Abstract

Transglutaminase (TGase) is important in blood coagulation, a conserved immunological defense mechanism among invertebrates. This study is the first report of the TGase in mud crab (Scylla paramamosain) (SpTGase) with a 2304 bp ORF encoding 767 amino acids (molecular weight 85.88 kDa). SpTGase is acidic, hydrophilic, stable and thermostable, containing three transglutaminase domains, one TGase/protease-like homolog domain (TGc), one integrin-binding motif (Arg270, Gly271, Asp272) and three catalytic sites (Cys333, His401, Asp424) within the TGc. Neither a signal peptide nor a transmembrane domain was found, and the random coil is dominant in the secondary structure of SpTGase. Phylogenetic analysis revealed a close relation between SpTGase to its homolog EsTGase 1 from Chinese mitten crab (Eriocheir sinensis). Expression of SpTGase was investigated using qRT-PCR (1) in eight tissues from healthy mud crabs, with the highest expression in hemocytes, and (2) in response to various immune challenges (Vibrio parahaemolyticus, lipopolysaccharide (LPS) or Poly I:C infection), revealing a major up-regulation in hemocytes, skin, and hepatopancreas during the 96-h post injection. The recombinant SpTGase showed a capacity of agglutination activities on both Gram-negative bacteria and yeast. SpTGase was found to directly interact with another important blood coagulation component clip domain serine protease (SpcSP). Moreover, knockdown of SpTGase resulted in a decreased expression of both clotting protein precursor (SppreCP) and SpcSP and an increase of duration time in the blood coagulation. Taken together, the findings of this study suggest SpTGase play an important role in the hemolymph clotting in mud crab S. paramamosain.

Keywords

Hemolymph clotting,Scylla paramamosain,Transglutaminase,