An MBT domain containing anti-lipopolysaccharide factor (PtALF8) from Portunus trituberculatus is involved in immune response to bacterial challenge

Abstract

Anti-lipopolysaccharide factors are effective antimicrobial peptides that can bind and neutralize lipopolysaccharide (LPS). In the present study, a new sequence encoding for ALF (designated as PtALF8) was cloned by suppression subtractive hybridization method using ovary of swimming crab Portunus trituberculatus as material. The full-length cDNA of PtALF8 consisted of 531 bp with an ORF of 348 bp encoding a peptide of 115 amino acids containing a putative signal peptide of 19 amino acids. The mature PtALF8 had a predicted molecular weight (MW) of 11.28 kDa and theoretical isoelectricpoint (pI) of 5.11. The PtALF8 contains an MBT domain which was not found in the other 7 isoforms of ALF reported in P. trituberculatus. Unlike most ALFs expressed in hemocytes, PtALF8 transcript was predominantly detected in hepatopancreas. After challenge with Vibrio alginolyticus, the temporal expression level of PtALF8 transcript in hemocytes reached the highest level at 3 h, then decreased to the lowest level at 24 h, and started to increase at 48 h. The recombinant protein showed antimicrobial and bactericidal activity against several bacteria, such as Gram-positive bacteria, Staphylococcus aureus, Micrococcus luteus and Gram-negative bacteria, V. alginolyticus, indicated that the PtALF8 isoform might play protective function against invading bacteria in P. trituberculatus.