Characterization of two isoforms of antiliopolysacchride factors (Sp-ALFs) from the mud crab Scylla paramamosain

Apr 28, 2012Fish & shellfish immunology

Identification of two versions of immune proteins that fight bacteria in the mud crab Scylla paramamosain

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Abstract

Two isoforms of the antilipopolysaccharide factor (Sp-ALF1 and Sp-ALF2) in the mud crab contain 1070 bp and 731 bp of cDNA, respectively.

Sp-ALF1 and Sp-ALF2 have 123 deduced amino acid residues and show significant similarity (57.7%) to other known antilipopolysaccharide factors from crustaceans.
Phylogenetic analysis indicates a close relationship between Sp-ALFs from the Scylla genus and those from the Portunus genus.
Both Sp-ALF1 and Sp-ALF2 are primarily expressed in hemocytes, with mRNA transcripts also found in various developmental stages of the crab, but rarely in the megalopa stage.
Recombinant and synthetic Sp-ALFs demonstrate high effectiveness against tested Gram-positive and Gram-negative bacteria.
Sp-ALFs show strong antiviral activity against the white spot syndrome virus, suggesting a potential role in immune defense against infections.

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In the previous study of the mud crab (Scylla paramamosain) hemocyte proteins, which interacted with a bacterium, Vibrio parahaemolyticus, a protein known as antilipopolysaccharide factor (Sp-ALF) was isolated in addition to a serine proteinase homolog (Sp-SPH) protein. In the present study, we further reported the characterization of two isoforms of the mud crab ALF - Sp-ALFs genes (designated as Sp-ALF1 and Sp-ALF2, respectively) based on our previous result. The Sp-ALF1 and Sp-ALF2 cDNA contained 1070 bp and 731 bp, respectively, with 123 deduced amino acid residues. Alignment of deduced amino acid sequences showed that Sp-ALFs possessed high identity with other known ALFs from crustaceans and exhibited an overall similarity of 57.7% to those of ALFs compared. Phylogenetic tree analysis revealed a clear group of each species and also suggested that ALFs from Scylla genus and those from Portunus genus were closely related. Tissue distribution analysis in adult crab implied that both Sp-ALF1 and Sp-ALF2 were mainly expressed in hemocytes. The mRNA transcripts were also found in embryo (I, II, III and V), zoea-I and juvenile crab, but were rarely observed in the megalopa stage. To further identify the biological activity of Sp-ALFs, recombinant proteins (rSp-ALFs: designated as rSp-ALF1 and rSp-ALF2, respectively) were obtained by expression in Pichia pastris, and the synthetic peptide fragments (sSp-ALFs: designated as sSp-ALF1 and sSp-ALF2, respectively) including the putative LPS binding loop were also prepared for antimicrobial test. The results indicated that both rSp-ALFs and sSp-ALFs were highly effective against most of the Gram-positive bacteria and Gram-negative bacteria tested. In contrast to cecropin P1, a membrane integrity assay revealed that Sp-ALFs did not affect the Escherichia coli by disruption of membrane integrity. Additionally, the recombinant Sp-ALFs proteins exhibited strong antiviral activity against an important aquaculture pathogen, white spot syndrome virus, in crustaceans. Taken together, these data suggested that Sp-ALFs might play a key role in immune defense against microbial infection in the mud crab S. paramamosain.

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Characterization of two isoforms of antiliopolysacchride factors (Sp-ALFs) from the mud crab Scylla paramamosain

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