Characterization of a novel anti-lipopolysaccharide factor isoform (SpALF5) in mud crab, Scylla paramamosain

Jan 2, 2015Molecular immunology

Identification of a new infection-fighting protein in mud crab

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Abstract

A novel isoform of anti-lipopolysaccharide factor, SpALF5, was isolated from the mud crab Scylla paramamosain, with a full-length sequence of 975 base pairs encoding 125 amino acids.

SpALF5 contains a conserved amino acid sequence with a signal peptide and a lipopolysaccharide-binding domain.
Genomic analysis shows SpALF5 consists of four exons and three introns, with introns containing tandem repeats.
Unlike most anti-lipopolysaccharide factors found in crab hemocytes, SpALF5 is predominantly expressed in the brain, muscle, and skin.
SpALF5 transcript levels significantly increase following exposure to lipopolysaccharides, certain viruses, and other immunogenic substances.
The recombinant SpALF5 protein exhibits strong antimicrobial activity against several Gram-negative bacteria and fungi, but limited activity against some Gram-positive bacteria.

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Anti-lipopolysaccharide factors (ALFs), the potential antimicrobial peptides that bind and neutralize lipopolysaccharide (LPS), are common effectors of innate immunity in crustaceans. In this study, a novel isoform of ALFs (SpALF5) was isolated from the hemocytes of mud crab Scylla paramamosain. The full-length 975bp SpALF5 contains a 375bp open reading frame (ORF) encoding 125 amino acids. Although SpALF5 exhibits a low degree of nucleotide homology with other reported ALFs, it contains the conserved amino acid sequence with a signal peptide and a LPS-binding domain including two conservative cysteine residues. The genomic organization of SpALF5 consists of four exons and three introns, with each intron containing one or more tandem repeats. Unlike most of ALFs mainly distributed in crab hemocytes, SpALF5 transcript was predominantly observed in the brain, muscle and skin, while barely detected in the hemocytes in our study. In situ hybridization assay also showed that SpALF5 mRNA was localized in brain, muscle and skin tissues of mud crab. Further, SpALF5 transcript was significantly up-regulated after challenge with LPS, polyinosinic polycytidylic acid (PolyI:C) (with the except of that in brain), Vibrio parahemolyticus or white spot syndrome virus (WSSV). The recombinant SpALF5 protein showed a varying degree of binding activity towards bacteria and fungus. Moreover, in vitro, the recombinant SpALF5 revealed a strong antimicrobial activity against Gram-negative bacteria (V. parahemolyticus, Vibrio alginolyticus, Escherichia coli, Aeromonas hydrophila) and fungus (Sacchromyces cerevisiae), but could only inhibited the growth of some Gram-positive bacteria like Staphylococcus aureus. The results suggest that SpALF5 is a potent immune protector and plays an important role in immune defense against invading pathogens in S. paramamosain.

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Characterization of a novel anti-lipopolysaccharide factor isoform (SpALF5) in mud crab, Scylla paramamosain

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