Abstract
Antilipopolysaccharide factors (ALFs) have been described as highly cationic polypeptides with a broad spectrum of potent antimicrobial activities. In addition, ALFs have been shown to recognize LPS, a major component of the Gram-negative bacteria cell wall, through conserved amino acid residues exposed in the four-stranded β-sheet of their three dimensional structure. In penaeid shrimp, ALFs form a diverse family of antimicrobial peptides composed by three main variants, classified as ALF Groups A to C. Here, we identified a novel group of ALFs in shrimp (Group D ALFs), which corresponds to anionic polypeptides in which many residues of the LPS binding site are lacking. Both Group B (cationic) and Group D (anionic) shrimp ALFs were produced in a heterologous expression system. Group D ALFs were found to have impaired LPS-binding activities and only limited antimicrobial activity compared to Group B ALFs. Interestingly, all four ALF groups were shown to be simultaneously expressed in an individual shrimp and to follow different patterns of gene expression in response to a microbial infection. Group B was by far the more expressed of the ALF genes. From our results, nucleotide sequence variations in shrimp ALFs result in functional divergence, with significant differences in LPS-binding and antimicrobial activities. To our knowledge, this is the first functional characterization of the sequence diversity found in the ALF family.
Highlights
Anti-lipopolysaccharide factors (ALFs) are antimicrobial peptides (AMPs) only found in marine chelicerates and crustaceans, which exhibit a potent antimicrobial activity against a broad range of microorganisms
We provide here the first evidence of functional divergence in shrimp ALFs
Full-length coding sequences (CDS) were kept for subsequent analyses. These data were completed by PCR amplification and sequencing of a novel ALF sequence obtained from the blue shrimp L. stylirostris (GenBank: KC346373)
Summary
Anti-lipopolysaccharide factors (ALFs) are antimicrobial peptides (AMPs) only found in marine chelicerates (horseshoe crabs) and crustaceans, which exhibit a potent antimicrobial activity against a broad range of microorganisms. The spectrum of the antimicrobial activity of ALFs covers a large number of Grampositive and Gram-negative bacteria, filamentous fungi as well as enveloped viruses [1,2,3,4,5]. ALFs are known as highly cationic polypeptides of about 100 residues with a hydrophobic N-terminal region. The horseshoe crab ALF-L and the shrimp ALFPm3, from Penaeus monodon, share a similar threedimensional structure, consisting in three a-helices packed against a four-stranded b-sheet [10,11]. Two conserved cysteine residues are involved in an intramolecular disulfide bridge which delimits the central b-hairpin
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