Defensin-neurotoxin dyad in a basally branching metazoan sea anemone.

Recent studies suggest that vertebrate and invertebrate defensins have evolved from two independent ancestors, and that both defensins could share origins with animal toxins. Here, we purified novel sea anemone neurotoxin (BDS)-like antimicrobial peptides (AMPs)-Crassicorin-I and its putative homolog (Crassicorin-II)-from the pharynx extract of an anthozoan sea anemone (Urticina crassicornis). Based on structural analyses and cDNA cloning, mature Crassicorin-I represents a cationic AMP likely generated from a precursor and comprising 40 amino acid residues, including six cysteines forming three intramolecular disulfide bonds. Recombinant Crassicorin-I produced in a heterologous bacterial-expression system displayed antimicrobial activity against both a gram-positive bacterium (Bacillus subtilis) and gram-negative bacteria (Escherichia coli and Salmonella enterica). The Crassicorin-I transcript was upregulated by immune challenge, suggesting its involvement in defense mechanisms against infectious pathogens in sea anemone. Sequence alignment and three-dimensional molecular modeling revealed that Crassicorin-I exhibits high degrees of structural similarity to sea anemone neurotoxins that share β-defensin fold which is found in vertebrate defensins and invertebrate big-defensins. Consistent with its structural similarity to neurotoxins, Crassicorin-I exhibited paralytic activity toward a crustacean. These findings motivated our investigation and subsequent discovery of antimicrobial activity from other known sea anemone neurotoxins, such as APETx1 and ShK. Collectively, our work signified that Crassicorin-I is the first AMP identified from a sea anemone and provided evidence of a functional linkage between AMPs and neurotoxins in a basally branching metazoan.