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

국문 초록 (Abstract)

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 precursor 와 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 μg/ml], 그람 음성 균주 Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Providencia stuartii, Pseudomonas aeruginosa, Vibrio ichthyoenteri [MECs 7.4-39.5 μg/ml] 그리고 진균류인 Candida albicans [MECs 26.0-31.8 μg/ml]에 항균활성을 나타냈다. 본 연구 결과, 참담치 혈구유래 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.

다국어 초록 (Multilingual Abstract)

We purified an antimicrobial peptide from the acidified hemocyte extract of Mytilus coruscus by C18 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 (MALDITOF/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 μg/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 μg/ml), and the fungus Candida albicans (MECs 26.0-31.8 μg/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.

목차 (Table of Contents)

• 서론
• 재료 및 방법
• 결과 및 고찰
• References
• 초록

참고문헌 (Reference)

1 김동균, "패류(Scapharca broughtonii) 유래의 용혈활성 미생물 다양성 분석" 한국생명과학회 22 (22): 642-649, 2012

2 오륜경, "참담치(Mytilus coruscus) 혈구(hemocyte)에서 분리한 McSSP-31의 항균 특성 분석" 한국생명과학회 27 (27): 1276-1289, 2017

3 오륜경, "참담치(Mytilus coruscus) 유래 항균 펩타이드 분리 및 정제" 한국생명과학회 26 (26): 1259-1268, 2016

4 박은희, "북방전복 (Haliotis discus hannai) 의 대식세포이동저해인자 (MIF, Macrophage migration inhibitory factor) 유전자 동정 및 발현분석" 한국패류학회 32 (32): 241-247, 2016

5 Seo, J. K., "cgMolluscidin, a novel dibasic residue repeat rich antimicrobial peptide, purified from the gill of the Pacific oyster, Crassostrea gigas" 35 : 480-488, 2013

6 Wright, R. K., "Urochordates" Academic Press 565-626, 1981

7 Hsieh, I. N., "The role of antimicrobial peptides in influenza virus infection and their potential as antiviral and immunomodulatory therapy" 9 : 53-, 2016

8 Seo, J. K., "Purification and antimicrobial function of ubiquitin isolated from the gill of Pacific oyster, Crassostrea gigas" 53 : 88-98, 2013

9 Boman, H. G., "Peptide antibiotics and their role in innate immunity" 13 : 61-92, 1995

10 Mitta, G., "Original involvement of antimicrobial peptides in mussel innate immunity" 486 : 185-190, 2000

11 Taylor, S. W., "New perspectives in the chemistry and biochemistry of the tunichromes and related compounds" 97 : 333-346, 1997

12 Söderhäll, K., "New direction in invertebrate immunology" SOS Publications 494-, 1996

13 Rosh, P., "NMR structure of mussel mytilin, and antiviral-antibacterial activities of derived synthetic peptides" 32 : 227-238, 2008

14 Mitta, G., "Mytilin B and MGD2, two antimicrobial peptides of marine mussels: gene structure and expression analysis" 24 : 381-393, 2000

15 Mitta, G., "Myticin, a novel cysteine-rich antimicrobial peptide isolated from haemocytes and plasma of the mussel Mytilus galloprovincialis" 265 : 71-78, 1999

16 Leoni, G., "Myticalins: a novel multigenic family of linear, cationic antimicrobial peptides from marine mussels (Mytilus spp.)" 15 : 261-, 2017

17 Mitta, G., "Mussel defensins are synthesized and processed in granulocytes then released into the plasma after bacterial challenge" 112 : 4233-4242, 1999

18 Yuan, T., "Molecular dynamics studies of the antimicrobial peptides piscidin 1 and its mutants with a DOPC lipid bilayer" 97 : 998-1009, 2012

19 Liao, Z., "Molecular characterization of a novel antimicrobial peptide from Mytilus coruscus" 34 : 610-616, 2013

20 Tam, J. P., "Marked increase in membranolytic selectivity of novel cyclic tachyplesins contrained with an antiparallel two-beta strand cysteine knot framework" 267 : 783-790, 2000

21 Jung, S., "Macin family of antimicrobial proteins combines antimicrobial and nerve repair activities" 287 : 14246-14258, 2012

22 Malagili, D., "Lectins and cytokines in celomatic invertebrates: two tales with the same end" 7 : 1-10, 2010

23 Mitta, G., "Involvement of mytilins in mussel antimicrobial defense" 275 : 12954-12962, 2000

24 Charlet, M., "Innate immunity. Isolation of several cysteine-rich antimicrobial peptides from the blood of a mollusc, Mytilus edulis" 271 : 21808-21813, 1996

25 Gerdol, M., "Identification and characterization of a novel family of cysteine-rich peptides (MgCRP-I) from Mytilus galloprovincialis" 7 : 2203-2219, 2015

26 Fred, J. G., "Factors influencing in vitro killing of bacteria by hemocytes of the eastern oyster (Crassostrea virginica)" 65 : 3015-3020, 1999

27 Boehm, T., "Evolution of the immune system in the lower vertebrates" 13 : 127-149, 2012

28 Epand, R. M., "Diversity of antimicrobial peptides and their mechanisms of action" 1462 : 11-28, 1999

29 Mitta, G., "Differential distribution and defence involvement of antimicrobial peptides in mussel" 113 : 2759-2769, 2000

30 Pipe, R. K., "Differential binding of lectins to haemocytes of the mussel Mytilus edulis" 261 : 261-268, 1990

31 Dimarcq, J. L., "Cystein-rich antimicrobial peptides in invertebrates" 47 : 465-477, 1998

32 Bartlett, T. C., "Crustins, homologues of an 11.5-kDa antibacterial peptide, from two species of penaeid shrimp, Litopenaeus vannamei and Litopenaeus setiferus" 4 : 278-293, 2002

33 Yu, G., "Combination effects of antimicrobial peptides" 60 : 1717-1724, 2016

34 Yoo, S. K., "Coastal culture" Gudeok Publisher 141-158, 1986

35 Qin, C. L., "Characterization of a novel antimicrobial peptide with chitin-binding domain from Mytilus coruscus" 41 : 362-370, 2014

36 Shike, H., "Bass hepcidin is a novel antimicrobial peptide induced by bacterial challenge" 269 : 2232-2237, 2002

37 Miyata,T., "Antimicrobial peptides, isolated from horseshoe crab hemocytes, tachyplesin II, and polyphemusins I and II: chemical structures and biological activity" 106 : 663-668, 1989

38 Tincu, J. A., "Antimicrobial peptides from marine invertebrates" 48 : 3645-3654, 2004

39 Seo, J. K., "Antimicrobial function of SHβAP, a novel hemoglobin β chain-related antimicrobial peptide, isolated from the liver of skipjack tuna, Katsuwonus pelamis" 37 : 173-183, 2014

40 Seo, J. K., "Antimicrobial effect of the 60S ribosomal protein L29 (cgRPL29), purified from the gill of pacific oyster, Crassostrea gigas" 67 : 675-683, 2017

41 Anderson, R., "Antibacterial activities of oyster (Crassostrea virginica) and mussel (Mytilus edulis and Geukensia demissa) plasma" 14 : 343-349, 2001

42 Cerne, K., "Analysis of cytotoxicity of melittin on adherent culture of human endothelial cells reveals advantage of fluorescence microscopy overflow cytometry and haemocytometer assay" 250 : 1131-1137, 2013

43 Gerdol, M., "An updated molecular basis for mussel immunity" 46 : 17-38, 2015