A single whey acidic protein domain (SWD)-containing peptide from fleshy prawn with antimicrobial and proteinase inhibitory activities

Abstract

With their inherent immune system, shrimps survive in marine environments despite the presence of different stressors, such as bacteria, fungi, and virus. In order to fight against and subsist even in the presence of such pathogens, the shrimps' ability to produce and release antimicrobial peptides are indeed important. In view of this, a new type of genetic material, an antimicrobial peptide containing a single whey acidic protein domain (SWD), was cloned from the haemocytes of fleshy prawns ( Fenneropenaeus chinensis). Hence, this peptide is named Fc-SWD. The full-length cDNA of Fc-SWD was 399 bp, and it contained a single open reading frame (ORF) of 273 bp encoding an Fc-SWD of 90 amino acids, including a putative 24-amino acid signal peptide and a mature 66 residue peptide. Sequence comparison and phylogenetic analysis of SWDs and other whey acidic protein (WAP) domains containing proteins in crustaceans suggested that the crustacean WAP containing proteins can be divided into four classes: crustin I, crustin II, SWD, and carcinin and carcinin-like proteins. Apparently, the Fc-SWD is a new type in the SWD subgroup. Northern blot analysis demonstrated that Fc-SWD transcripts were constitutively expressed in the haemocytes of non-infected shrimps and increased in the haemocytes of infected shrimps. Meanwhile, Fc-SWD mRNA was not detected in the hearts, hepatopancreas, stomachs, gills, intestines, or ovaries of the non-infected shrimps nor the infected ones. Reverse transcription polymerase chain reaction (RT-PCR) experiments showed that the Fc-SWD signal was detected in the haemocytes, hearts, and gills of both the unchallenged and the challenged shrimps, and in the stomach of the challenged shrimps. Western blot analysis indicated that the Fc-SWD protein appeared to be synthesized in the haemocytes with a signal peptide and then secreted to the haemolymph as a mature peptide when the F. chinensis was infected by pathogens. Thus, the Fc-SWD (for the full-length peptide including the signal peptide), the mFc-SWD (for the mature peptide), and the Fc-WAPD (for the single WAP domain) were manifested in the Escherichia coli, respectively . Further findings indicated that the purified recombinant Fc-SWD and the mFc-SWD have antimicrobial activities against Gram-positive (G +) and Gram-negative (G −) bacteria and fungi, as well as a strong inhibitory activity against subtilisin A and protein K. The Fc-WAPD has anti-protease activities and low antimicrobial activity. In view of the foregoing findings, it can be concluded that Fc-SWD is one of the factors that affect shrimps' immunity against various pathogens.