Purification and characterization of a novel antimicrobial peptide from visceral tissue of sea urchin, Heliocidaris crassispina.

Antimicrobial peptides (AMPs) are crucial components of the innate immune system and are increasingly recognized as potential alternatives to traditional antibiotics. In this study, we report the isolation and characterization of Heliocidin, a novel AMP purified from the visceral tissue of the sea urchin Heliocidaris crassispina. Heliocidin was purified using a series of HPLC steps, and its molecular mass was determined by LC-MS. Edman degradation identified the N-terminal 17 amino acid residues, and cDNA cloning revealed the full nucleotide sequence of 838 bp encoding a precursor protein of 107 amino acids. This precursor protein comprised a signal peptide with 30 AAs and a mature peptide with 17 AAs. Heliocidin shared no significant sequence homology with other known peptides. The C-terminal amidation of Heliocidin was confirmed through HPLC retention time analysis, which was further supported by the cloning results showing a glycine residue juxtaposed to Heliocidin's C-terminus. Synthetic Heliocidin with amidated C-terminus showed antimicrobial activity against Gram-positive and Gram-negative bacteria and a fungus. This antimicrobial activity is attributed to the presence of C-terminal amidation, as the OH-type Heliocidin exhibited no detectable antimicrobial effect. Moreover, Heliocidin's antimicrobial mode of action is likely through interactions with the bacterial outer membrane consistent with inner and outer membrane permeability assay results. Transcriptional expression analysis demonstrated that Heliocidin's mRNA was predominantly expressed in coelomocytes, immune cells crucial to the sea urchin's defense system. Heliocidin transcript was upregulated following immune challenge, indicating its role in the innate immune response of H. crassispina. This study represents the first report of an antimicrobial peptide in H. crassispina, expanding our understanding of echinoderm immune defense and providing a foundation for the discovery of novel AMPs in marine invertebrates.