title: A newly identified anti-lipopolysaccharide factor from the swimming crab Portunus trituberculatus with broad spectrum antimicrobial activity.
abstract: Anti-lipopolysaccharide factors (ALFs), exhibiting binding and neutralizing activities to lipopolysaccharide (LPS), are the potent antimicrobial peptides of innate immunity in crustaceans. In this study, a unique isoform of ALF (PtALF6) was identified from eyestalk cDNA library of the swimming crab Portunus trituberculatus. The full-length cDNA of PtALF6 was 669 bp encoding 115 amino acids, relatively short to other known ALFs. The deduced peptide of PtALF6 was conserved; it contained the signal peptide and LPS-binding domain, especially the two conserved cysteine residues at both ends of the domain. Predicted tertiary structures of PtALF6 containing four β-strands and three α-helices were similar to that described in Limulus polyphemus. The genomic fragment of PtALF6 contained three exons separated by two introns. Unlike most ALFs expressed in hemocytes, PtALF6 transcript was predominantly detected in gill with 14.05-fold higher than that in hemocytes. After challenge with Vibrio alginolyticus, the temporal expression level of PtALF6 transcript in hemocytes showed a clear time-dependent response expression pattern with two significant peaks at 12 h and 32 h post-injection. The recombinant PtALF6 protein revealed antimicrobial activity against the test Gram-negative and Gram-positive bacteria, but did not inhibit the growth of fungus Pichia pastoris. These results together indicate that PtALF6 is a potential antimicrobial protein against Gram-negative and Gram-positive bacteria infection, and may play an important role in innate immune response of P. trituberculatus.
authors.affiliation: Key Laboratory of Experimental Marine Biology, EMBL, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Liu
authors.forename: Zhaoxia
authors.initials: Z
authors.lastname: Cui
authors.forename: Xihong
authors.initials: X
authors.lastname: Li
authors.forename: Chengwen
authors.initials: C
authors.lastname: Song
authors.forename: Guohui
authors.initials: G
authors.lastname: Shi
mesh_terms: D000595:Amino Acid Sequence; D000704:Analysis of Variance; D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D003386:Brachyura / Q000235:genetics* / Q000276:immunology* / Q000382:microbiology; D017931:DNA Primers / Q000235:genetics; D040461:Gene Components; D005786:Gene Expression Regulation / Q000276:immunology*; D015723:Gene Library; D005880:Gills / Q000378:metabolism; D007113:Immunity, Innate / Q000276:immunology*; D008070:Lipopolysaccharides / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D020033:Protein Isoforms / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013997:Time Factors; D044149:Vibrio alginolyticus / Q000276:immunology
chemical_list: D023181:Antimicrobial Cationic Peptides; D017931:DNA Primers; D008070:Lipopolysaccharides; D020033:Protein Isoforms