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Mytilin B, an Antimicrobial Peptide from the Hemocyte of the Hard-shelled Mussel, Mytilus coruscus : Isolation, Purification, and Characterization

참담치(Mytilus coruscus) 혈구(hemocyte) 유래 항균 펩타이드 mytilin B의 정제 및 특성 분석

  • Lee, Min Jeong (Biotechnology Research Division, National Institute of Fisheries Science) ;
  • Oh, Ryunkyoung (Biotechnology Research Division, National Institute of Fisheries Science) ;
  • Kim, Young-Ok (Biotechnology Research Division, National Institute of Fisheries Science) ;
  • Nam, Bo-Hye (Biotechnology Research Division, National Institute of Fisheries Science) ;
  • Kong, Hee Jeong (Biotechnology Research Division, National Institute of Fisheries Science) ;
  • Kim, Joo-Won (Biotechnology Research Division, National Institute of Fisheries Science) ;
  • Park, Jung Youn (Biotechnology Research Division, National Institute of Fisheries Science) ;
  • Seo, Jung-Kil (Department of Food Science and Biotechnology, Kunsan National University) ;
  • Kim, Dong-Gyun (Biotechnology Research Division, National Institute of Fisheries Science)
  • 이민정 (국립수산과학원 생명공학과) ;
  • 오륜경 (국립수산과학원 생명공학과) ;
  • 김영옥 (국립수산과학원 생명공학과) ;
  • 남보혜 (국립수산과학원 생명공학과) ;
  • 공희정 (국립수산과학원 생명공학과) ;
  • 김주원 (국립수산과학원 생명공학과) ;
  • 박중연 (국립수산과학원 생명공학과) ;
  • 서정길 (군산대학교 식품생명과학부) ;
  • 김동균 (국립수산과학원 생명공학과)
  • Received : 2018.10.08
  • Accepted : 2018.11.13
  • Published : 2018.11.30

Abstract

We purified an antimicrobial peptide from the acidified hemocyte extract of Mytilus coruscus by $C_{18}$ reversed-phase high-performance liquid chromatography (RP-HPLC). The peptide was 4041.866 Da based on matrix-assisted laser desorption ionization time-of-flight mass spectrophotometer (MALDI-TOF/MS) and the 25 amino acids of the N-terminus sequence were identified. Comparison of this sequence of the purified peptide with the N-terminus sequences of other antimicrobial peptides revealed 100% identity with the mytilin B precursor of Mytilus coruscus. We also identified a 312 bp open-reading frame (ORF) encoding 103 amino acids based on the obtained amino acid residues. The nucleotide sequence of this ORF and the amino acid sequence also revealed 100% identity with the mytilin B precursor of Mytilus coruscus. We synthesized two antimicrobial peptides with an alanine residue in the C-terminus, and designated them mytilin B1 and B2. These two antimicrobial peptides showed antimicrobial activity against gram-positive bacteria, including Bacillus cereus and Streptococcus parauberis (minimal effective concentration, MECs $41.6-89.7{\mu}g/ml$), gram-negative bacteria, including Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Providencia stuartii, Pseudomonas aeruginosa, and Vibrio ichthyoenteri (MECs $7.4-39.5{\mu}g/ml$), and the fungus Candida albicans (MECs $26.0-31.8{\mu}g/ml$). This antimicrobial activity was stable under heat and salt conditions. Furthermore, the peptides did not exhibit significant hemolytic activity or cytotoxic effects. These results suggest that mytilin B could be applied as alternative antibiotic agent, and they add to the understanding of the innate immunity of hard-shelled mussels.

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Fig. 1. Purification and antimicrobial activity of hemocyte extract of M. coruscus. The extract was fractionated by the CapCell-Pak C18 reversed-phase column. Elution was performed with a linear gradient of 5-65% CH3CN in 0.1% TFA for 60 min at a flow rate of 1 ml/min. The eluate was monitored at 220 nm. Fraction of the absorbance peak (indicated by the arrow) showed antimicrobial activity against B. subtilis (inset). The elution point of the active peak was at 29% CH3CN. Scale bar indicates 5 mm.

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Fig. 2. Final purification of pooled active fraction and antimicrobial activity. The active fraction was applied to a PEPTIDE XB-C18 reversed-phase column. Elution was performed with a linear gradient of 20-40% CH3CN in 0.1% TFA for 30 min at a flow rate of 0.5 ml/min. The eluate was monitored at 220 nm. The elution point of the active peak was at 33% CH3CN (indicated by the arrow). Antimicrobial activity of the purified peak (before) and proteinase K treated purified peak (after) against B. subtlis. Scale bar indicates 5 mm.

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Fig. 3. The molecular weight of the purified peptide was determined using an ultraflleXtremeTM MALDI TOF/MS spectrometer equipped with a pulsed smart beam II in linear mode. The molecular weight of the purified peptide is 4041.866 Da.

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Fig. 4. Multiple alignment of nucleotide sequences of mytilin B precursor and mytilin B isoforms. Signal peptide is indicated by grey box. The mature peptide and polyadenylation signal is indicated by black outlined. Conserved residues are indicated by dot.

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Fig. 5. Amino acid sequence alignment of mytilin B precursor with 4 isoform of mytilin B. Signal peptide and mature peptide regions were outlined by black boxes. Conserved residues of amino acids are indicated by dot.

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Fig. 6. Hemolytic activity of mytilin B1, mytilin B2 and piscidin 1 against erythrocytes of flounder (Paralichthys olivaceus). (A) The concentration of each sample was 100 μg/ml. (B) Effect of hemolytic activity depend on the concentrations of each samples (100, 50, 25, 12.5, 6.5 μg/ml).

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Fig. 7. Morphological differences of HUVEC after 24 hr growth with each specimens. (A) HUVEC control (B) HUVEC treated with mytilin B1 (C) HUVEC treated with mytilin B2 (D) HUVEC treated with melittin.

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Fig. 8. Cell viability of HUVEC treated with mytilin B after 24 hr growth. Error bars represent the mean ± SD of three technical replicates.

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Fig. 9. Quantitative analysis of the mytilin B gene expression level from the various tissues. HEP; hepatopancreas, SIP; siphon, GIL; gill, ADD; adductor muscle, FOO; foot, HEM; hemocyte, MAN: mantle. Error bars represent the mean ± SD of three technical replicates.

Table 1. Pathogenic bacteria strains used in this study

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Table 2. Antimicrobial activity of hemocyte extract of M. coruscus against pathogenic bacteria

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Table 3. Antimicrobial activity and stability of mytilin B1 and mytilin B2 against pathogenic bacteria under heat & salt conditions

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Table 4. Minimal effective concentrations (MECs, μg/ml) of mytilin B antimicrobial peptides

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Acknowledgement

Grant : 수산생명자원유래 항미생물 천연소재 개발

Supported by : 국립수산과학원

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