Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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The Antimicrobial Characteristics of McSSP-31 Purified from the Hemocyte of the Hard-shelled Mussel, Mytilus coruscus

참담치(Mytilus coruscus) 혈구(hemocyte)에서 분리한 McSSP-31의 항균 특성 분석

  • 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) ;
  • 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)
  • 오륜경 (국립수산과학원 생명공학과) ;
  • 이민정 (국립수산과학원 생명공학과) ;
  • 김영옥 (국립수산과학원 생명공학과) ;
  • 남보혜 (국립수산과학원 생명공학과) ;
  • 공희정 (국립수산과학원 생명공학과) ;
  • 김주원 (국립수산과학원 생명공학과) ;
  • 박중연 (국립수산과학원 생명공학과) ;
  • 서정길 (군산대학교 식품생명과학부) ;
  • 김동균 (국립수산과학원 생명공학과)
  • Received : 2017.09.27
  • Accepted : 2017.10.19
  • Published : 2017.11.30
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Abstract

This study isolated and purified the antimicrobial peptide McSSP-31 from an acidified hemocyte extract of a Mytilus coruscus. The antimicrobial peptide was purified by using a $C_{18}$ reversed-phase high-performance liquid chromatography (HPLC). The peptide was determined to be 3330.549 Da by matrix assisted-laser desorption ionization time-of-flight mass spectrophotometer (MALDI-TOF/MS). The N-terminus of a 14 amino-acid sequence was identified as P-S-P-T-R-R-S-T-S-R-S-K-S-R by Edman degradation method. The acquired sequence showed a 93% similarity with the sperm-specific protein Phi-1, which is from M. californianus. The identified open-reading frame (ORF) of peptide was 306 bp encoding 101 amino acids, which was analyzed by rapid amplification of cDNA ends (RACE), cloning and sequencing analysis. We compared the full sequence with other known proteins that reveal the sperm-specific protein Phi-1 (93.5%) of M. californianus. Synthesized antimicrobial peptide (McSSP-31) showed antibacterial activity against gram-positive bacteria including B. subtilis, S. mutans, S. aureus and gram-negative bacteria including E. coli, K. pneumoniae, P. mirabilis, P. aeruginosa and fungi, C. albicans. Also, synthesized peptide showed strong antibacterial activity against antibiotic-resistant strains, including S. aureus. The cytotoxicity of the peptide was determined by using the HUVEC human cell line. The peptide did not exhibit any significant cytotoxic effects on the normal human cell line, and it had very low hemolytic activity with flounder hemoglobin. The results demonstrated that peptide purified from the hemocyte of a M. coruscus exhibits antibacterial activity against various bacteria and has the potential to be an alternative antibiotic agent.

참담치 hemocyte에 존재하는 항균 펩타이드를 역상 HPLC column을 사용하여 분리 및 정제하였다. 정제된 펩타이드는 matrix-assisted laser desorption ionization time-of-flight mass spectrophotometer (MALDI-TOF/MS) 분석을 통해 분자량이 3330.549 Da이며, edman 분해법을 통해 14개의 N-말단 아미노산 서열을 확보하였다. 분석한 N-말단 서열은 M. californianus의 sperm-specific protein Phi-1과 protamine-like PL-III protein과 각각 93%와 87%의 유사도를 나타냈으며, M. edulis의 sperm-specific protein Phi-1과 87% 일치함을 확인하였다. 또한 open-reading frame (ORF)은 306 bp의 길이에 101개의 아미노산을 코딩하고 있음을 밝혔으며, 이는 M. californianus의 sperm-specific protein Phi-1와 93.5% 유사하였다. 분자량과 아미노산 서열에 근거하여 31개 아미노산으로 구성된 펩타이드를 합성하였으며 이는 그람 양성균인 B. subtilis, S. mutans, S. aureus와 그람 음성균인 E. coli, K. pneumoniae, P. mirabilis, P. aeruginosa 그리고 진균류인 C. albicans에 항균 활성을 보였다. 합성한 펩타이드는 항생제 내성균주인 S. aureus CCARM 0203와 S. aureus CCARM 0204에 항균 활성을 보였다. 합성 항균 펩타이드는 넙치 혈장에 대한 용혈현상은 없었고, 세포독성을 확인한 결과 HUVEC cell line에 전혀 독성을 보이지 않았다. 본 연구결과, 참담치의 혈구로부터 분리 및 정제한 sperm-specific protein 유래 항균 펩타이드는 다양한 균주에 항균 활성을 보였고 낮은 세포독성을 가졌으며, 이러한 특성은 본 실험에서 분리한 항균 펩타이드가 항생제 대체재로서 개발 가능성을 제시하고 있다.

Keywords

References

  1. Ausio, J. 1999. Histone H1 and evolution of sperm nuclear basic proteins. J. Biol. Chem. 274, 31115-31118. https://doi.org/10.1074/jbc.274.44.31115
  2. Balhorn, R. 2007. The protamine family of sperm nuclear proteins. Genome Biol. 8, 227 https://doi.org/10.1186/gb-2007-8-9-227
  3. Carlos, S., Jutglar, L., Borrell, I., Hunt, D. F. and Ausio, J. 1993. Sequence and characterization of a sperm-specific histone H1-like protein of Mytilus californianus. J. Biol. Chem. 268, 185-194.
  4. Cha, Y. K., Kim, Y. S. and Chio, Y. S. 2012. Antimicrobial peptides as natural antibiotic materials. Biotechnol. Bioprocess Eng. 27, 9-15.
  5. 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
  6. Eirín-Lopez, J. M., Frehlick, L. J. and Ausio, J. 2006. Protamines, in the footsteps of linker histone evolution. J. Biol. Chem. 281, 1-4. https://doi.org/10.1074/jbc.R500018200
  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. Heath, D. D. and Hilbish, T. J. 1998. Mytilus protamine-like sperm-specific protein genes are multicopy, dispersed, and closely associated with hypervariable RFLP regions. Genome 41, 587-596. https://doi.org/10.1139/g98-057
  10. John, D. and Ausio, J. 2002. Protamine-like proteins: evidence for a novel chromatin structure. Biochem. Cell Biol. 80, 353-361. https://doi.org/10.1139/o02-083
  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. Lehrer, R. I., Rosenman, M., Harwig, S. S. L., Jackson, R. and Eisenhaur, P. 1991. Ultrasensitive assay for endogenous antimicrobial polypeptides. J. Immunol. Methods 137, 167-173. https://doi.org/10.1016/0022-1759(91)90021-7
  13. Lewis, J. D., Saperas, N., Song, Y., Zamora, M. J., Chiva, M. and Ausio, J. 2003. Histone H1 and the origin of protamines. Proc. Natl. Acad. Sci. USA 101, 4148-4152.
  14. Mitta, G., Hubert, F., Dyrynda, E. A., Boundry, 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
  15. Mitta, G., Vandenbulcke, F., Hubert, F., Salzet, M. and Roch, P. 2000. Involvement of mytilins in mussel antimicrobial defense. J. Biol. Chem. 275, 12954-12962. https://doi.org/10.1074/jbc.275.17.12954
  16. Mitta, G., Vandenbulcke, F., Noel, T., Romestand, B., Beauvillain, J. C., Salzet, M. and Roch, P. 2000. Differential distribution and defence involvement of antimicrobial peptides in mussel. J. Cell Sci. 113, 2759-2769.
  17. Mitta, G., Vandenbulcke, F. and Roch, P. 2000. Original involvement of antimicrobial peptides in mussel innate immunity. FEMS Lett. 486, 185-190.
  18. Oh, R., Lee, M. J., Kim, Y. O., Nam, B. H., Kong, H. J., Kim, J. W., An, C. H. and Kim, D. G. 2016. Isolation and purification of antimicrobial peptide from hard-shelled mussel, Mytilus coruscus. J. Life Sci. 26, 1259-1268. https://doi.org/10.5352/JLS.2016.26.11.1259
  19. 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.
  20. Ruiz-Lara, S., Prats, E., Casas, M. T. and Cornudella, L. 1993. Molecular cloning and sequence of a cDNA for the spermspecific protein ${\phi}1$ from the mussel Mytilus edulis. Nucleic Acids Res. 21, 11.
  21. Santel, A., Thomas, W., Nicole, B. and Renate, P. 1997. The Drosophila don juan (dj) gene encodes a novel sperm specific protein component characterized by an unusual domain of a repetitive amino acid motif. Mech. Develop. 64, 19-30. https://doi.org/10.1016/S0925-4773(97)00031-2
  22. Seo, J. K., Kim., D. G., Oh., R., Park, K. S., Lee, I. A., Cho, S. M., Lee, K. Y. and Nam, B. H. 2017. Antimicrobial effect of the 60S ribosomal protein L29(cgRPL29), purified from the gill of pacific oyster, Crassostrea gigas. Fish Shellfish Immunol. 67, 675-683. https://doi.org/10.1016/j.fsi.2017.06.058
  23. Shai, Y. 2002. Mode of action of membrane active antimicrobial peptides. Biopolymers 66, 236-248. https://doi.org/10.1002/bip.10260
  24. 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
  25. 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
  26. 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
  27. 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.
  28. Wade, D., Andreu, D., Mitchell, S. A., Silveria, A. M. V., Boman, A., Boman, H. G. and Merrifield, R. B. 1992. Antibacterial peptides designed as analogs or hybrids of cecropins and melittin. Int. J. Pept. Protein Res. 40, 429-436.
  29. Yechiel, S. 2000. Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by K-helical antimicrobial and cell non-selective membranelytic peptides. Biochim. Biophys. Acta. 1462, 55-79.
  30. Yuan, T., Zhang, X., Hu, Z., Wang, F., and Lei, M. 2012. Molecular dynamics studies of the antimicrobial peptides piscidin 1 and its mutants with a DOPC lipid bilayer. Biopolymers 97, 998-1009 https://doi.org/10.1002/bip.22116
  31. Zasloff, M. 2002. Antimicrobial peptide of multicellular organisms. Nature 415, 389-395. https://doi.org/10.1038/415389a
  32. Zhu, H., Yang, X., Liu, J., Ge, Y., Qin, Q., Lu, J., Zhan, L., Liu, Z., Zhang, H., Chen, X., Zhang, C., Xu, L., Cheng, H. and Sun, X. 2014. Melittin radiosensitizes esophageal squamous cell carcinoma with induction of apoptosis in vitro and in vivo. Tumour Biol. 35, 8699-8705. https://doi.org/10.1007/s13277-014-2146-z