Functional Characterization, Antimicrobial Effects, and Potential Antibacterial Mechanisms of HM4, a Derived Peptide of Hemocyanin.

Hemocyanins present in the hemolymph of invertebrates are multifunctional proteins that are responsible for oxygen transport and play crucial roles in the immune system. They have also been identified as a source of antimicrobial peptides during infection in mollusks. Hemocyanin has also been identified in the cephalopod ancestor Nautilus, but antimicrobial peptides derived from the hemocyanin of have not been reported. Here, the bactericidal activity of six predicted peptides from hemocyanin and seven mutant peptides was analyzed. Among those peptides, a mutant peptide with 15 amino acids (1RVFAGFLRHGIKRSR15), HM4, showed relatively high antibacterial activity. HM4 was determined to have typical antimicrobial peptide characteristics, including a positive charge (+5.25) and a high hydrophobic residue ratio (40%), and it was predicted to form an alpha-helical structure. In addition, HM4 exhibited significant antibacterial activity against Gram-negative bacteria (MBC = 30 μM for ), with no cytotoxicity to mammalian cells even at a high concentration of 180 µM. Upon contact with cells, we confirmed that the bactericidal activity of HM4 was coupled with membrane permeabilization, which was further confirmed via ultrastructural images using a scanning electron microscope. Therefore, our study provides a rationalization for the development and optimization of antimicrobial peptide from the cephalopod ancestor Nautilus, paving the way for future novel AMP development with broad applications.