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

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.

참담치(Mytilus coruscus)의 혈구 유래의 항균 펩타이드를 역상 column들을 사용한 reversed-phase high-performance liquid chromatography (RP-HPLC)로 분리 및 정제하였다. 정제된 펩타이드는 matrix-assisted laser desorption ionization time-of-flight mass spectrophotometer (MALDI-TOF/MS)를 통해 분자량이 4041.866 Da으로 밝혀졌으며 Edman degradation법을 통해 25개의 N-말단 서열을 확보하였다. 이는 참담치의 mytilin B precursor와 100%, mytilin 8 precursor, mytilin 4 precursor와 96% 일치하였다. 또한 103개의 아미노산 서열을 코딩하고 있는 312 bp의 open-reading frame (ORF)을 밝혔으며 이는 참담치의 mytilin B precursor와 100% 일치하였다. 밝혀진 분자량과 아미노산 서열을 바탕으로 C-말단 alanine 잔기의 유무에 따라 2개의 펩타이드를 합성하였으며 이는 mytilin B1과 B2라고 명명하였다. 이들은 그람 양성 균주 Bacillus cereus, Streptococcus parauberis [minimal effective concentrations, MECs $41.6-89.7{\mu}g/ml$], 그람 음성 균주 Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Providencia stuartii, Pseudomonas aeruginosa, Vibrio ichthyoenteri [MECs $7.4-39.5{\mu}g/ml$] 그리고 진균류인 Candida albicans [MECs $26.0-31.8{\mu}g/ml$]에 항균활성을 나타냈다. 본 연구 결과, 참담치 혈구 유래 mytilin B1과 mytilin B2는 넓은 항균 스펙트럼을 가지고 열과 염분에 대한 안정성이 높으며 용혈현상과 세포독성은 나타나지 않았다. 이러한 특성은 기능성 사료첨가제 및 항생제 대체제로써 충분히 안정적인 역할을 할 뿐만 아니라 추후 mytilin의 구조적 중요성과 참담치의 면역학적 측면에서 다양한 자료를 제시할 것으로 사료된다.

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.

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.

Fig. 3. The molecular weight of the purified peptide was determined using an ultraflleXtreme^TM 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.

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.

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.

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).

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.

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

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

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

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

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