Expression and characterization of a serine proteinase homologue from black tiger shrimp Penaeus monodon

Abstract

The full-length cDNAs of the serine proteinase homologue from Penaeus monodon (PmMasSPH) and Litopenaeus vannamei (LvMasSPH) were identified by rapid amplification cDNA end (RACE) method. The complete cDNA sequence of PmMasSPH (1,958 bp) contained an open reading frame (ORF) of 1,572 bp encoding a 523 amino acid protein. A full-length cDNA of LvMasSPH (1,955 bp) consists of an ORF of 1,536 bp encoding for a polypeptide of 511 amino acids. Both of the shrimp SPHs contained a glycine-rich repeated region, a clip domain at the N-terminus and a SP-like domain at the C terminus. Sequence comparison showed that the deduced amino acid of PmMasSPH had a sequence identity of 58%, 55% and 53% to those of Callinectes sapidus PPAF, Apis mellifera PPAF and Bombyx mori masquerade-like, respectively and the deduced amino acid of LvMasSPH showed 93% identity to that of PmMasSPH. A phylogenetic tree clearly revealed that PmMasSPH and LvMasSPH were more closely related to non-catalytic SPHs than to the active SP. In situ hybridization analysis showed that PmMasSPH expressed in hemocytes and was up-regulated within 6 h after Vibrio harveyi injection. Moreover, Real-time RT-PCR analyses showed that the transcript of LvMasSPH was induced in response to V. harveyi infection. To further characterize the functions of PmMasSPH protein, the C-terminal SP-like domain was expressed in Escherichia coli Rosetta (DE3) and purified by a Ni NTA column. The refolded recombinant SP-like domain protein was then assayed for various biological functions. The refolded recombinant SP-like domain protein lacks proteolytic activity but mediates hemocyte adhesion, displays binding activity to Gram-negative and Gram-positive bacteria and cell wall components, suggesting that PmMasSPH may act as a pattern recognition molecule. These results suggested that PmMasSPH plays an important role in shrimp defense.