DOI QR코드

DOI QR Code

Isolation and Purification of Antimicrobial Peptide from Hard-shelled Mussel, Mytilus coruscus

참담치(Mytilus coruscus) 유래 항균 펩타이드 분리 및 정제

  • Oh, Ryunkyoung (Biotechnology Research Division, National Institute of Fisheries Science) ;
  • Lee, Min Jeong (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) ;
  • An, Cheul Min (Biotechnology Research Division, National Institute of Fisheries Science) ;
  • Kim, Dong-Gyun (Biotechnology Research Division, National Institute of Fisheries Science)
  • 오륜경 (국립수산과학원 전략양식부 생명공학과) ;
  • 이민정 (국립수산과학원 전략양식부 생명공학과) ;
  • 김영옥 (국립수산과학원 전략양식부 생명공학과) ;
  • 남보혜 (국립수산과학원 전략양식부 생명공학과) ;
  • 공희정 (국립수산과학원 전략양식부 생명공학과) ;
  • 김주원 (국립수산과학원 전략양식부 생명공학과) ;
  • 안철민 (국립수산과학원 전략양식부 생명공학과) ;
  • 김동균 (국립수산과학원 전략양식부 생명공학과)
  • Received : 2016.09.23
  • Accepted : 2016.10.19
  • Published : 2016.11.30

Abstract

In this study, we investigated antimicrobial peptide from the acidified muscle extract of Mytilus coruscus, which mostly inhabits China, Japan, and Korea, to develop a natural product-derived antibiotics substitution in terms of its abuse and restriction. Antimicrobial peptide was purified by $C_{18}$ reversed-phase high-performance liquid chromatography and was detected as having a molecular mass of 6,701 Da by MALDI-TOF/MS. The N-terminal amino acid sequence of the purified peak was obtained from edman degradation, and 20 identified residues shown 100% identity with the N-terminus region of sperm-specific protein and protamine-like PL-II/PL-IV precursor of Mytilus californianus. We also identified 60 open-reading frame (ORF) encoding amino acids with 183 bp of purified peptide based on the obtained amino acid residues. The amino acid sequence of ORF showed 100% and the nucleotide sequence revealed 97.2% identity with the protamine-like PL-II/PL-IV precursor of Mytilus californianus. Synthesized antimicrobial peptide showed antimicrobial activity against gram-positive bacteria, including Bacillus cereus (minimal effective concentration [MEC], $20.8{\mu}g/ml$), Bacillus subtilis (MEC, $0.2{\mu}g/ml$), Streptococcus mutans (MEC, $0.2{\mu}g/ml$), gram-negative bacteria including Pseudomonas aeruginosa (MEC, $5.7{\mu}g/ml$), Escherichia coli (MEC, $2.6{\mu}g/ml$) and fungi, Candida albicans (MEC, $56.3{\mu}g/ml$). In addition, synthesized peptide showed stable activities under heat and salt conditions against gram-positive and gram-negative bacteria, but was inhibited by salt against only C. albicans. With these results, isolated peptide from M. coruscus could be an alternative agent to antibiotics for defending against pathogenic microorganisms, and helpful information to understand the innate immune system of marine invertebrates.

항생제 남용의 문제점 및 사용제한에 따른 천연물 유래 항생제 대체제 개발을 위하여 우리나라와 중국, 일본에서만 서식하는 고유종인 참담치의 족부 근육 추출물로부터 항균 펩타이드를 분리 및 정제하였다. $C_{18}$ 역상 컬럼을 사용하여 항균 펩타이드를 정제하였으며, MALDI-TOF/MS로 분석 결과 분자량은 6,701 Da에 해당하였다. edman degradation 방법으로 N-말단 아미노산 서열 분석을 통하여 20개의 아미노산 서열을 밝혔으며 이는 캘리포니아 홍합(Mytilus californianus)의 sperm-specific protein 또는 protamine-like PL-II/PL-IV precursor와 100% 상동성을 지님을 밝혀냈다. 이러한 정보를 바탕으로 ORF를 분석한 결과 60개의 아미노산을 코딩하는 183 bp로 캘리포니아 홍합의 protamine-like PL-II/PL-IV precursor와 아미노산 서열이 100% 일치하였고 유전자 염기서열은 97.2%의 상동성을 나타냈다. 합성된 항균 펩타이드는 그람 양성 균주 Bacillus cereus (MEC, $20.8{\mu}g/ml$), Bacillus subtilis (MEC, $0.2{\mu}g/ml$), Streptococcus mutans (MEC, $0.2{\mu}g/ml$), 그람 음성 균주 Pseudomonas aeruginosa (MEC, $5.7{\mu}g/ml$), Escherichia coli (MEC, $2.6{\mu}g/ml$) 그리고 진균류 Candida albicans (MEC, $56.3{\mu}g/ml$)의 항균활성을 나타냈다. 또한 높은 열안정성을 지녔으며, C. albicans를 제외하고 염안정성을 지닌 항균 펩타이드로 확인되었다. 이를 통해 참담치 유래의 항균 펩타이드는 항생제 대체제의 후보소재로 높은 가능성을 지녔다고 할 수 있다. 따라서 본 연구에서 분리한 참담치 족부 근육 유래 항균 펩타이드는 향후 추가적인 연구를 통하여 항생제를 대체할 수 있는 물질의 개발이 가능할 것으로 사료된다. 그리고 해양무척추동물의 선천성 면역 기작에 대한 정보를 제공할 것으로 기대한다.

Keywords

References

  1. Aditya, K., Heinrich, K., Lategan, M. J. and Gibson, L. 2008. Probiotics in aquaculture: The need, principles and mechanisms of action and screening processes. Aquaculture 274, 1-14. https://doi.org/10.1016/j.aquaculture.2007.11.019
  2. Ausio, J. 1999. Histone H1 and evolution of sperm nuclear basic proteins. J. Biol. Chem. 44, 31115-31118.
  3. Ausio J., Eirin-Lopez J M. and Frehlick, L. J. 2006. Protamines, in the footsteps of linker histone evolution. J. Biol. Chem. 281, 1-4. https://doi.org/10.1074/jbc.R500018200
  4. Ausio, J., Lewis, J. D., Saperas, N., Zamora, M. J. and Chiva., M. 2004. Histone H1 and the origin of protamines. Proc. Natil. Acad. Sci. USA 101, 4148-4152. https://doi.org/10.1073/pnas.0308721101
  5. Cha, Y. K., Kim, Y. S. and Chio, Y. S. 2012. Antimicrobial peptdies as natural antibiotic materials. Biotechnol. Bioprocess Eng. 27, 9-15.
  6. Charlet, M., Chernysh, S., Philippe, H., Hetru, C., Hoffmann, J. A. and Bulet, P. 1996. Innate immunity. Isolation of several cysteine-rich antimicrobial peptides from the blood of a mollusc, Mytilus edulis. J. Biol. Chem. 271, 21808-21813. https://doi.org/10.1074/jbc.271.36.21808
  7. Gregory, B. and Gail, S. 1996. Immunity and the Invertebrates. Dev. Immunol. 275, 60-63.
  8. Hancock, R. and Diamond, G. 2000. The role of cationic antimicrobial peptides in innate host defences. Trends Microbiol. 8, 402-410. https://doi.org/10.1016/S0966-842X(00)01823-0
  9. Hubert, F., Noel, T. and Roch, P. 1996. A member of the arthropod defensin family from edible Mediterranean mussels (Mytilus galloprovincialis). Eur. J. Biochem. 240, 302-306. https://doi.org/10.1111/j.1432-1033.1996.0302h.x
  10. James, F. G., Nouth, C., Manker, P. G., Ian, S. W., Jennifer, A.C. and Daniel, M. R. 2003. Phosphorylation of soybean nodulin26 on serine 262 enhances water permeability and is regulated developmentally and by osmotic signals. Plant Cell. 15, 981-991. https://doi.org/10.1105/tpc.009787
  11. Kesarcodi-Watson, A., Kaspar, H., Lategan, M. J. and Gibson, L. 2008. Probiotics in aquaculture: the need, principles and mechanisms of action and screening processes. Aquaculture 274, 1-14. https://doi.org/10.1016/j.aquaculture.2007.11.019
  12. Kim, I. H., Kim, J. W. and Lee, J. H. 2006. Purification of antimicrobial peptide from the marine mussel, Mytilus coruscus. Environ. Mutagens Carcinogens. 26, 25-29.
  13. Kim, W. J., Cho, M. Y., Jee, B., Park, M. A. and Kim, N. Y. 2014. Administration and use of aquaculture drugs in Korea. Kor. J. Fish. Pathol. 27, 67-75. https://doi.org/10.7847/jfp.2014.27.1.067
  14. Kim, Y. H., Kim, S. M. and Lee, S. Y. 2014. Antimicrobial activity of protamine against oral microorganisms. Biocontrol. Sci. 20, 275-280.
  15. Lehrer, R. I., Rosenman, M., Harwig, S. S., Jackson, R. and Eisenhauer, P. 1991. Ultrasensitive assays for endogenous antimicrobial polypeptides. J. Immunol. Methods 137, 167-173. https://doi.org/10.1016/0022-1759(91)90021-7
  16. Liao, Z., Wang, X. C., Liu, H. H., Fan, M. H., Sun, J. J. and Shen, W. 2013. Molecular characterization of a novel antimicrobial peptide from Mytilus coruscus. Fish Shellfish Immunol. 34, 610-616. https://doi.org/10.1016/j.fsi.2012.11.030
  17. Liu, R., Mu, L., Liu, H., Wei, L., Yan, T., Chen, M., Zhang, K., Li, J., You, D. and Lai, R. 2011. Two antimicrobial and nematicidal peptides derived from sequences encoded Picea sitchensis. J. Pept. Sci. 17, 627-631 https://doi.org/10.1002/psc.1380
  18. Mitta, G., Hubert, F., Dyrynda, E. A., Boudry, P. and Roch, P. 2000. Mytilin B and MGD2, two antimicrobial peptides of marine mussels: gene structure and expression analysis. Dev. Comp. Immunol. 24, 381-393. https://doi.org/10.1016/S0145-305X(99)00084-1
  19. Mitta, G., Hubert, F., Noel, T. and Roch, P. 1999. Myticin, a novel cysteine-rich antimicrobial peptide isolated from haemocytes and plasma of the mussel Mytilus galloprovincialis. Eur. J. Biochem. 265, 71-78. https://doi.org/10.1046/j.1432-1327.1999.00654.x
  20. Miyata, T., Tokunaga, F., Yoneya, T., Yoshikawa, K., Iwanaga, S., Niwa, M., Takao, T. and Shimonishi, Y. 1989. Antimicrobial peptides, isolated from horseshoe crab hemocytes, tachyplesin II, and polyphemusins I and II: chemical structures and biological activity. J. Biochem. 106, 663-668. https://doi.org/10.1093/oxfordjournals.jbchem.a122913
  21. Moon, H. S., Kim, Y. K., Lee, M. H., Yoon, N. Y., Lee, D. S., Yoon, H. D. and Seo, J. K. 2011. Isolation and Purification of an Antimicrobial Material from the Jellyfish Nemopilema nomurai. Fish Aquatic Sci. 44, 478-483.
  22. Nam, B. H., Seo, J. K., Lee, M. J., Kim, Y. O., Kim, D. G., An, C. M. and Park, N. G. 2015. Functional analysis of Pacific oyster (Crassostrea gigas) ${\beta}$-thymosin: Focus on antimicrobial activity. Fish Shellfish Immunol. 45, 167-174. https://doi.org/10.1016/j.fsi.2015.03.035
  23. Park, I. S., Oh, R., Lee, M. J., Moon, J, Y., Kim, Y. O., Nam, B. H., Kong, H. J., Kim, W. J., An, C. M. and Kim, D. G., 2015. Antibacterial activity of bacteria isolated from rocks on the seashore. Kor. J. Fish. Aquat. Sci. 48, 904-912.
  24. Parisia, M. G., Lia, H., Toubianaa, M., Parrinellob, N., Cammaratab, M. and Rocha, P. 2009. Polymorphism of my tilin B mRNA is not translated into mature peptide. Mol. Immunol. 46, 384-392. https://doi.org/10.1016/j.molimm.2008.10.009
  25. Qin, C. L., Huang, W., Zhou, S. Q., Wang, X. C., Liu, H. H., Fan, M. H., Wang, R. X., Gao, P. and Liao, Z. 2014. Characterization of a novel antimicrobial peptide with chiting- biding domain from Mytilus coruscus. Fish Shellfish Immunol. 41, 362-370. https://doi.org/10.1016/j.fsi.2014.09.019
  26. Richards, R. C. 2001. Histone H1: an antimicrobial protein of Altantic salmon (Salmo salar). Biochem. Biophys. Res. Commun. 294, 549-555.
  27. Rocha, P., Yangb, Y., Toubianaa, M. and Aumelasba, A. 2008. NMR structure of mussel mytilin, and antiviral antibacterial activities of derived synthetic peptides. Dev. Comp. Immunol. 32, 227-238. https://doi.org/10.1016/j.dci.2007.05.006
  28. Seo, J. K. 2016. Screening and purification of an antimicrobial peptide from the gill of the Manila Clam Ruditapes philippinarum. Kor. J. Fish. Aquat. Sci. 49, 137-145.
  29. Seo, J. K., Crawford, J. M., Stone, K. L. and Noga, E. J. 2005. Purification of a novel arthropod defensin from the American oyster, Crassostrea virginica. Biochem Biophys. Res. Commun. 338, 1998-2004. https://doi.org/10.1016/j.bbrc.2005.11.013
  30. Seo, J. K., Lee, M. J., Go, H. J., Kim, G. D., Jeong, H. D., Nam, B. H. and Park, N. G. 2013. Purification and antimicrobial function of ubiquitin isolated from the gill of Pacific oyster, Crassostrea gigas. Mol. Immunol. 53, 88-98. https://doi.org/10.1016/j.molimm.2012.07.003
  31. Seo, J. K., Lee, M. J., Nam, B. H. and Park, N. G. 2013. cgMolluscidin, a novel dibasic residue repeat rich antimicrobial peptide, purified from the gill of the Pacific oyster, Crassostrea gigas. Fish Shellfish Immunol. 35, 480-488. https://doi.org/10.1016/j.fsi.2013.05.010
  32. Shai, Y. 2002. Mode of action of membrane active antimicrobial peptides. Biopolymers 66, 236-248. https://doi.org/10.1002/bip.10260
  33. Sonthi, M., Cantet, F., Toubiana, M., Trapani, M. R., Parisi, M. G., Cammarata, M. and Roch, P. 2012. Gene expression specificity of the mussel antifungal mytimycin (MytM). Fish Shellfish Immunol. 32, 45-50. https://doi.org/10.1016/j.fsi.2011.10.017
  34. Thomas, B., Norimasa, I. and Isabell, H. 2012. Evolution of the immune system in the lower vertebrates. Annu. Rev. Genomics Hum. Genet. 13, 127-149. https://doi.org/10.1146/annurev-genom-090711-163747
  35. Tincu, J. A. and Taylor, S. W. 2004. Antimicrobial peptides from marine invertebrates. Antimicrob. Agents Chemother. 48, 3645-3654. https://doi.org/10.1128/AAC.48.10.3645-3654.2004
  36. Tom, D., Patrick, S. and Peter, B. 2011. Alternatives to antibiotics for the control of bacterial disease in aquaculture. Curr. Opin. Microbiol. 14, 251-258. https://doi.org/10.1016/j.mib.2011.03.004
  37. Yoo, S. K. 1986. Coastal culture. Gudeok publisher. 141-158.
  38. Van't, H. W., Veerman, E. C, Helmerhorst, E. J. and Amerongen, A. V. 2001. Antimicrobial peptides: properties and applicability. Biol. Chem. 382, 597-619.
  39. Yechiel, S. 2000. Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by Khelical antimicrobial and cell non-selective membrane-lytic peptides. Biochim. Biophys. Acta. 1462, 55-79.
  40. Zasloff, M. 2002. Antimicrobial peptide of multicellular organisms. Nature 415, 389-395. https://doi.org/10.1038/415389a