Functional characterization of a masquerade-like serine proteinase homologue from the black tiger shrimp Penaeus monodon

Abstract

A cDNA encoding a masquerade-like serine proteinase homologue (PmMasSPH) from the black tiger shrimp, Penaeus monodon, has been cloned and characterized. The transcript of PmMasSPH is induced in response to Vibrio harveyi infection. To further characterize the function(s) of the protein, (i) the N-terminal region comprising the glycine-rich repeats and the clip domain, and (ii) the C-terminal SP-like domain of the PmMasSPH were separately cloned into the pET-28b(+) expression vector and transformed into Escherichia coli Rosetta (DE3). The two recombinant proteins were then assayed for various biological functions; proteinase activity, hemocyte adhesion, bacterial binding, bacterial clearance and antimicrobial activity. The C-terminal SP-like domain lacks proteolytic activity but mediates hemocyte adhesion and displays binding activity to the shrimp pathogenic bacterium, V. harveyi and specific binding to the bacterial cell wall component, lipopolysaccharide (LPS). The N-terminal region exhibited in vitro antimicrobial activity against Gram-positive bacteria. In addition, the in vivo study revealed the opsonic activity of the PmMasSPH protein as shown by a higher bacterial clearance rate of V. harveyi coated with the recombinant proteins as compared with V. harveyi only. The results suggest that the PmMasSPH protein is a multifunctional immune molecule in shrimp defense. © 2009 Elsevier Inc. All rights reserved.