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Biochemical Journal logoLink to Biochemical Journal
. 2002 Dec 1;368(Pt 2):611–620. doi: 10.1042/BJ20020980

Anti-microbial properties of histone H2A from skin secretions of rainbow trout, Oncorhynchus mykiss.

Jorge M O Fernandes 1, Graham D Kemp 1, M Gerard Molle 1, Valerie J Smith 1
PMCID: PMC1222992  PMID: 12164782

Abstract

Skin exudates of rainbow trout contain a potent 13.6 kDa anti-microbial protein which, from partial internal amino acid sequencing, peptide mass fingerprinting, matrix-associated laser desorption/ionization MS and amino acid analysis, seems to be histone H2A, acetylated at the N-terminus. The protein, purified to homogeneity by ion-exchange and reversed-phase chromatography, exhibits powerful anti-bacterial activity against Gram-positive bacteria, with minimal inhibitory concentrations in the submicromolar range. Kinetic analysis revealed that at a concentration of 0.3 microM all test bacteria lose viability after 30 min incubation. Weaker activity is also displayed against the yeast Saccharomyces cerevisiae. The protein is salt-sensitive and has no haemolytic activity towards trout erythrocytes at concentrations below 0.3 microM. Reconstitution of the protein in a planar lipid bilayer strongly disturbs the membrane but does not form stable ion channels, indicating that its anti-bacterial activity is probably not due to pore-forming properties. This is the first report to show that, in addition to its classical function in the cell, histone H2A has extremely strong anti-microbial properties and could therefore help contribute to protection against bacterial invasion.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. Bevins C. L. Antimicrobial peptides as agents of mucosal immunity. Ciba Found Symp. 1994;186:250–269. doi: 10.1002/9780470514658.ch15. [DOI] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  4. Buchmann K. Lectins in fish skin: do they play a role in host-monogenean interactions? J Helminthol. 2001 Sep;75(3):227–231. [PubMed] [Google Scholar]
  5. Cho Ju Hyun, Park In Yup, Kim Hun Sik, Lee Won Taek, Kim Mi Sun, Kim Sun Chang. Cathepsin D produces antimicrobial peptide parasin I from histone H2A in the skin mucosa of fish. FASEB J. 2002 Jan 30;16(3):429–431. doi: 10.1096/fj.01-0736fje. [DOI] [PubMed] [Google Scholar]
  6. Cole A. M., Weis P., Diamond G. Isolation and characterization of pleurocidin, an antimicrobial peptide in the skin secretions of winter flounder. J Biol Chem. 1997 May 2;272(18):12008–12013. doi: 10.1074/jbc.272.18.12008. [DOI] [PubMed] [Google Scholar]
  7. Connor W., States J. C., Mezquita J., Dixon G. H. Organization and nucleotide sequence of rainbow trout histone H2A and H3 genes. J Mol Evol. 1984;20(3-4):236–250. doi: 10.1007/BF02104730. [DOI] [PubMed] [Google Scholar]
  8. Deutschlander M. E., Greaves D. K., Haimberger T. J., Hawryshyn C. W. Functional mapping of ultraviolet photosensitivity during metamorphic transitions in a salmonid fish, Oncorhynchus mykiss. J Exp Biol. 2001 Jul;204(Pt 14):2401–2413. doi: 10.1242/jeb.204.14.2401. [DOI] [PubMed] [Google Scholar]
  9. Dong F., van Holde K. E. Nucleosome positioning is determined by the (H3-H4)2 tetramer. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10596–10600. doi: 10.1073/pnas.88.23.10596. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ebran N., Julien S., Orange N., Auperin B., Molle G. Isolation and characterization of novel glycoproteins from fish epidermal mucus: correlation between their pore-forming properties and their antibacterial activities. Biochim Biophys Acta. 2000 Aug 25;1467(2):271–280. doi: 10.1016/s0005-2736(00)00225-x. [DOI] [PubMed] [Google Scholar]
  11. Ebran N., Julien S., Orange N., Saglio P., Lemaître C., Molle G. Pore-forming properties and antibacterial activity of proteins extracted from epidermal mucus of fish. Comp Biochem Physiol A Mol Integr Physiol. 1999 Feb;122(2):181–189. doi: 10.1016/s1095-6433(98)10165-4. [DOI] [PubMed] [Google Scholar]
  12. Friedrich C., Scott M. G., Karunaratne N., Yan H., Hancock R. E. Salt-resistant alpha-helical cationic antimicrobial peptides. Antimicrob Agents Chemother. 1999 Jul;43(7):1542–1548. doi: 10.1128/aac.43.7.1542. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Frohm M., Gunne H., Bergman A. C., Agerberth B., Bergman T., Boman A., Lidén S., Jörnvall H., Boman H. G. Biochemical and antibacterial analysis of human wound and blister fluid. Eur J Biochem. 1996 Apr 1;237(1):86–92. doi: 10.1111/j.1432-1033.1996.0086n.x. [DOI] [PubMed] [Google Scholar]
  14. HIRSCH J. G. Bactericidal action of histone. J Exp Med. 1958 Dec 1;108(6):925–944. doi: 10.1084/jem.108.6.925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hikima J., Hirono I., Aoki T. Characterization and expression of c-type lysozyme cDNA from Japanese flounder (Paralichthys olivaceus). Mol Mar Biol Biotechnol. 1997 Dec;6(4):339–344. [PubMed] [Google Scholar]
  16. Huang H. W. Action of antimicrobial peptides: two-state model. Biochemistry. 2000 Jul 25;39(29):8347–8352. doi: 10.1021/bi000946l. [DOI] [PubMed] [Google Scholar]
  17. Kashima M. H1 histones contribute to candidacidal activities of human epidermal extract. J Dermatol. 1991 Dec;18(12):695–706. doi: 10.1111/j.1346-8138.1991.tb03160.x. [DOI] [PubMed] [Google Scholar]
  18. Kragol G., Lovas S., Varadi G., Condie B. A., Hoffmann R., Otvos L., Jr The antibacterial peptide pyrrhocoricin inhibits the ATPase actions of DnaK and prevents chaperone-assisted protein folding. Biochemistry. 2001 Mar 13;40(10):3016–3026. doi: 10.1021/bi002656a. [DOI] [PubMed] [Google Scholar]
  19. Lehrer R. I., Rosenman M., Harwig S. S., Jackson R., Eisenhauer P. Ultrasensitive assays for endogenous antimicrobial polypeptides. J Immunol Methods. 1991 Mar 21;137(2):167–173. doi: 10.1016/0022-1759(91)90021-7. [DOI] [PubMed] [Google Scholar]
  20. Ludtke S. J., He K., Heller W. T., Harroun T. A., Yang L., Huang H. W. Membrane pores induced by magainin. Biochemistry. 1996 Oct 29;35(43):13723–13728. doi: 10.1021/bi9620621. [DOI] [PubMed] [Google Scholar]
  21. Matsuzaki K., Yoneyama S., Fujii N., Miyajima K., Yamada K., Kirino Y., Anzai K. Membrane permeabilization mechanisms of a cyclic antimicrobial peptide, tachyplesin I, and its linear analog. Biochemistry. 1997 Aug 12;36(32):9799–9806. doi: 10.1021/bi970588v. [DOI] [PubMed] [Google Scholar]
  22. Montal M., Mueller P. Formation of bimolecular membranes from lipid monolayers and a study of their electrical properties. Proc Natl Acad Sci U S A. 1972 Dec;69(12):3561–3566. doi: 10.1073/pnas.69.12.3561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Murphy C. J., Foster B. A., Mannis M. J., Selsted M. E., Reid T. W. Defensins are mitogenic for epithelial cells and fibroblasts. J Cell Physiol. 1993 May;155(2):408–413. doi: 10.1002/jcp.1041550223. [DOI] [PubMed] [Google Scholar]
  24. Oren Z., Shai Y. A class of highly potent antibacterial peptides derived from pardaxin, a pore-forming peptide isolated from Moses sole fish Pardachirus marmoratus. Eur J Biochem. 1996 Apr 1;237(1):303–310. doi: 10.1111/j.1432-1033.1996.0303n.x. [DOI] [PubMed] [Google Scholar]
  25. Park C. B., Kim H. S., Kim S. C. Mechanism of action of the antimicrobial peptide buforin II: buforin II kills microorganisms by penetrating the cell membrane and inhibiting cellular functions. Biochem Biophys Res Commun. 1998 Mar 6;244(1):253–257. doi: 10.1006/bbrc.1998.8159. [DOI] [PubMed] [Google Scholar]
  26. Park C. B., Kim M. S., Kim S. C. A novel antimicrobial peptide from Bufo bufo gargarizans. Biochem Biophys Res Commun. 1996 Jan 5;218(1):408–413. doi: 10.1006/bbrc.1996.0071. [DOI] [PubMed] [Google Scholar]
  27. Park I. Y., Park C. B., Kim M. S., Kim S. C. Parasin I, an antimicrobial peptide derived from histone H2A in the catfish, Parasilurus asotus. FEBS Lett. 1998 Oct 23;437(3):258–262. doi: 10.1016/s0014-5793(98)01238-1. [DOI] [PubMed] [Google Scholar]
  28. Patrzykat A., Zhang L., Mendoza V., Iwama G. K., Hancock R. E. Synergy of histone-derived peptides of coho salmon with lysozyme and flounder pleurocidin. Antimicrob Agents Chemother. 2001 May;45(5):1337–1342. doi: 10.1128/AAC.45.5.1337-1342.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Perkins D. N., Pappin D. J., Creasy D. M., Cottrell J. S. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis. 1999 Dec;20(18):3551–3567. doi: 10.1002/(SICI)1522-2683(19991201)20:18<3551::AID-ELPS3551>3.0.CO;2-2. [DOI] [PubMed] [Google Scholar]
  30. Relf J. M., Chisholm J. R., Kemp G. D., Smith V. J. Purification and characterization of a cysteine-rich 11.5-kDa antibacterial protein from the granular haemocytes of the shore crab, Carcinus maenas. Eur J Biochem. 1999 Sep;264(2):350–357. doi: 10.1046/j.1432-1327.1999.00607.x. [DOI] [PubMed] [Google Scholar]
  31. Richards R. C., O'Neil D. B., Thibault P., Ewart K. V. Histone H1: an antimicrobial protein of Atlantic salmon (Salmo salar). Biochem Biophys Res Commun. 2001 Jun 15;284(3):549–555. doi: 10.1006/bbrc.2001.5020. [DOI] [PubMed] [Google Scholar]
  32. Robinette D., Wada S., Arroll T., Levy M. G., Miller W. L., Noga E. J. Antimicrobial activity in the skin of the channel catfish Ictalurus punctatus: characterization of broad-spectrum histone-like antimicrobial proteins. Cell Mol Life Sci. 1998 May;54(5):467–475. doi: 10.1007/s000180050175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  34. Shai Y. Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by alpha-helical antimicrobial and cell non-selective membrane-lytic peptides. Biochim Biophys Acta. 1999 Dec 15;1462(1-2):55–70. doi: 10.1016/s0005-2736(99)00200-x. [DOI] [PubMed] [Google Scholar]
  35. Simmaco M., Mangoni M. L., Boman A., Barra D., Boman H. G. Experimental infections of Rana esculenta with Aeromonas hydrophila: a molecular mechanism for the control of the normal flora. Scand J Immunol. 1998 Oct;48(4):357–363. doi: 10.1046/j.1365-3083.1998.00407.x. [DOI] [PubMed] [Google Scholar]
  36. Smith V. J., Fernandes J. M., Jones S. J., Kemp G. D., Tatner M. F. Antibacterial proteins in rainbow trout, Oncorhynchus mykiss. Fish Shellfish Immunol. 2000 Apr;10(3):243–260. doi: 10.1006/fsim.1999.0254. [DOI] [PubMed] [Google Scholar]
  37. Thompson J. D., Higgins D. G., Gibson T. J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994 Nov 11;22(22):4673–4680. doi: 10.1093/nar/22.22.4673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Wachinger M., Kleinschmidt A., Winder D., von Pechmann N., Ludvigsen A., Neumann M., Holle R., Salmons B., Erfle V., Brack-Werner R. Antimicrobial peptides melittin and cecropin inhibit replication of human immunodeficiency virus 1 by suppressing viral gene expression. J Gen Virol. 1998 Apr;79(Pt 4):731–740. doi: 10.1099/0022-1317-79-4-731. [DOI] [PubMed] [Google Scholar]
  39. Winder D., Günzburg W. H., Erfle V., Salmons B. Expression of antimicrobial peptides has an antitumour effect in human cells. Biochem Biophys Res Commun. 1998 Jan 26;242(3):608–612. doi: 10.1006/bbrc.1997.8014. [DOI] [PubMed] [Google Scholar]
  40. Zasloff Michael. Antimicrobial peptides of multicellular organisms. Nature. 2002 Jan 24;415(6870):389–395. doi: 10.1038/415389a. [DOI] [PubMed] [Google Scholar]
  41. Zhong R., Roeder R. G., Heintz N. The primary structure and expression of four cloned human histone genes. Nucleic Acids Res. 1983 Nov 11;11(21):7409–7425. doi: 10.1093/nar/11.21.7409. [DOI] [PMC free article] [PubMed] [Google Scholar]

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