A new anti-lipopolysaccharide factor isoform (PtALF4) from the swimming crab Portunus trituberculatus exhibited structural and functional diversity of ALFs

Anti-lipopolysaccharide factors (ALFs) are the potent antimicrobial peptides that can bind and neutralize lipopolysaccharide (LPS). In this study, a new isoform of the ALF homologs (PtALF4) was cloned from eyestalk cDNA library of swimming crab Portunus trituberculatus. PtALF4 shared lower amino acid similarity with other ALFs, yet it contained the conserved LPS-binding domain and was clearly member of the ALF family. The genomic sequence of PtALF4 consisted of three exons interrupted by two introns. Several tandem repeats were found in both introns. Unlike most ALFs expressed in hemocytes, PtALF4 transcript was predominantly detected in eyestalk. After challenge with Vibrio alginolyticus, the temporal expression level of PtALF4 transcript in hemocytes showed a clear time-dependent response expression pattern with two significant peaks. The recombinant proteins of PtALF1, PtALF3 and PtALF4 revealed different antimicrobial activities against bacteria or fungus. These results together suggest that PtALF isoforms might be potent immune effectors to provide multiple protective functions against invading bacteria or fungus in P. trituberculatus.