별불가사리(Patiria pectinifera) 유래 새로운 cysteine-rich Macin 의 발견과 재조합 생산을 통한 항균 활성 및 특성 연구

Abstract

The ocean is a reservoir of diverse life forms including various marine animals that are constantly exposed to abundant microorganisms, including pathogenic bacteria. Marine invertebrates, such as starfish, lack adaptive immunity and rely heavily on their innate immune systems to defend against these microorganisms. A key factor in the innate immune response is antimicrobial peptides (AMPs). This study is the first to identify Macin in the starfish Patiria pectinifera and to measure its antimicrobial activity. This AMP, which exhibits both antimicrobial and neuroregenerative activities, has not been previously reported in echinoderms before. This study also investigates the effects of different cysteine connectivities and immune challenges on PpMacin’s antimicrobial activity and transcriptional expression levels, respectively. The full nucleotide and amino acid (AA) sequences of PpMacin were determined through cDNA cloning, revealing a total sequence of 1527 bp, including a 5ʹ untranslated region (UTR) of 140 bp, an open reading frame (ORF) of 264 bp, and a 3ʹ UTR of 1123 bp. The ORF encodes a total of 87 AAs, including a signal peptide of 24 AAs and a mature peptide of 63 AAs. The recombinant PpMacin (rPpMacin) was successfully produced through a heterologous expression system with the pET-28a(+)-TrxA vector system and Escherichia coli BL21 (DE3). The antimicrobial activity of the three rPpMacin forms (refolded, reduced, and one missing a disulfide bond) was investigated using the ultrasensitive radial diffusion assay, with results showing that cysteine connectivity significantly affects PpMacin’s antimicrobial capacity. The bacteriolytic activity of rPpMacin was confirmed using a chromogenic plate assay. Furthermore, NPN and ONPG assays demonstrated that rPpMacin weakly interacted with bacterial membranes at the experimental concentration. The transcriptional expression levels of PpMacin in starfish tissues were evaluated using RT-qPCR, revealing high PpMacin expression in coelomocytes and coelomic epithelium. Finally, temporal expression of PpMacin following immune challenge was highest in coelomocytes at 4 h and 24 h.