Probiotics and Antimicrobial Proteins

, Volume 9, Issue 4, pp 473–482 | Cite as

Molecular Characterisation of a Novel Isoform of Hepatic Antimicrobial Peptide, Hepcidin (Le-Hepc), from Leiognathus equulus and Analysis of Its Functional Properties In Silico

  • Aishwarya Nair
  • K. S. Sruthy
  • E. R. Chaithanya
  • T. P. Sajeevan
  • I. S. Bright Singh
  • Rosamma Philip


Hepcidin represents a family of cysteine-rich antimicrobial peptides that are mainly expressed in the liver of living organisms. In this study, we have identified and characterised a novel isoform of hepcidin from the common pony fish, Leiognathus equulus (Le-Hepc). A 261-bp fragment cDNA coding for 86 amino acids was obtained. Homologous analysis showed that Le-Hepc belongs to the hepcidin super family and shares sequence identity with other known fish pre-propeptide hepcidin sequences. The ORF encodes for a 24-amino acid (aa) signal peptide coupled to a 36-aa prodomain followed by a 26-aa mature peptide. The mature peptide region has a calculated molecular weight of 2.73 kDa, a net positive charge of +2 and a theoretical pI of 8.23. Phylogenetic analysis of Le-Hepc showed a strong relationship with other fish hepcidin sequences and clustered into HAMP2 group hepcidins. Secondary structural analysis indicated that Le-Hepc mature peptide contains two antiparallel β-sheets strengthened by four disulphide bonds formed by eight conserved cysteine residues. The physicochemical properties of the peptide and its structural parameters are in agreement with characteristic features of an antimicrobial peptide. This is the first report of an antimicrobial peptide from the common pony fish, L. equulus.


Antimicrobial peptide Hepcidin Pony fish Leiognathus equulus HAMP2 



The authors are grateful to the Director, Centre for Marine Living Resources and Ecology (CMLRE) and Ministry of Earth Sciences (MoES), Govt. of India, for the research grant (MoES/10-MLR/01/2012) and scientific support for the work. The authors also thank the Coordinator, National Centre for Aquatic and Animal Health (NCAAH), for scientific support. The first author gratefully acknowledges UGC-BSR (University Grant Commission-Basic Scientific Research) for the award of a fellowship.

Compliance with Ethical Standards


The study was funded by the Ministry of Earth Sciences (MoES), Govt. of India (MoES/10-MLR/01/2012).

Conflict of Interest

Aishwarya Nair, Sruthy K. S., Chaithanya E.R., Sajeevan T.P., I. S. Bright Singh and Rosamma Philip declare that they have no conflict of interest.

This article does not contain any studies with human participants or animals performed by any of the authors.


  1. 1.
    Magnadóttir B (2006) Innate immunity of fish (overview). Fish Shellfish Immunol 20(2):137–151. doi: 10.1016/j.fsi.2004.09.006 CrossRefGoogle Scholar
  2. 2.
    Workenhe ST, Rise ML, Kibenge MJ, Kibenge FS (2010) The fight between the teleost fish immune response and aquatic viruses. Mol Immunol 47(16):2525–2536. doi: 10.1016/j.molimm.2010.06.009 CrossRefGoogle Scholar
  3. 3.
    Ravichandran S, Kumaravel K, Rameshkumar G, AjithKumar TT (2010) Antimicrobial peptides from the marine fishes. Res J Immunol 3(2):146–156. doi: 10.3923/rji.2010.146.156 CrossRefGoogle Scholar
  4. 4.
    Silphaduang U, Noga EJ (2001) Antimicrobials: peptide antibiotics in mast cells of fish. Nature 414(6861):268–269. doi: 10.1038/35104690 CrossRefGoogle Scholar
  5. 5.
    Zou J, Mercier C, Koussounadis A, Secombes C (2007) Discovery of multiple beta-defensin like homologues in teleost fish. Mol Immunol 44:638–647. doi: 10.3410/f.1031751.368863 CrossRefGoogle Scholar
  6. 6.
    Shike H, Lauth X, Westerman ME, Ostland VE, Carlberg JM, Van Olst JC, Shimizu C, Bulet P, Burns JC (2002) Bass hepcidin is a novel antimicrobial peptide induced by bacterial challenge. Eur J Biochem 269:2232–2237. doi: 10.1046/j.1432-1033.2002.02881 CrossRefGoogle Scholar
  7. 7.
    Uzzell T, Stolzenberg ED, Shinnar AE, Zasloff M (2003) Hagfish intestinal antimicrobial peptides are ancient cathelicidins. Peptides 24:1655–1667. doi: 10.1016/j.peptides.2003.08.024 CrossRefGoogle Scholar
  8. 8.
    Birkemo GA, Luders T, Andersen O, Nes IF, Nissen-Meyer J (2003) Hipposin, a histone-derived antimicrobial peptide in Atlantic halibut (Hippoglossus hippoglossus L.) Biochim Biophys Acta 1646:207–215. doi: 10.1016/S1570-9639(03)00018-9 CrossRefGoogle Scholar
  9. 9.
    Rajanbabu V, Chen JY (2011) Applications of antimicrobial peptides from fish and perspectives for the future. Peptides 32(2):415–420. doi: 10.1016/j.peptides.2010.11.005 CrossRefGoogle Scholar
  10. 10.
    Park CH, Valore EV, Waring AJ, Ganz T (2001) Hepcidin, a urinary antimicrobial peptide synthesized in the liver. J Biol Chem 276(11):7806–7810. doi: 10.1074/jbc CrossRefGoogle Scholar
  11. 11.
    Krause A, Neitz S, Mägert HJ, Schulz A, Forssmann WG, Schulz-Knappe P, Adermann K (2000) LEAP-1, a novel highly disulfide-bonded human peptide, exhibits antimicrobial activity. FEBS Lett 480:147–150. doi: 10.1016/S0014-5793(00)01920-7 CrossRefGoogle Scholar
  12. 12.
    Douglas SE, Gallant JW, Liebscher RS, Dacanay A, Tsoi SCM (2003) Identification and expression analysis of hepcidin-like antimicrobial peptides in bony fish. Dev Comp Immunol 27:589–601. doi: 10.1016/S0145-305X(03)00036-3 CrossRefGoogle Scholar
  13. 13.
    Bao B, Peatman E, Li P, He C, Liu Z (2005) Catfish hepcidin gene is expressed in a wide range of tissues and exhibits tissue-specific upregulation after bacterial infection. Dev Comp Immunol 29(11):939–950. doi: 10.1016/j.dci.2005.03.006 CrossRefGoogle Scholar
  14. 14.
    Chen SL, Xu MY, Ji XS, Yu GC, Liu Y (2005) Cloning, characterization, and expression analysis of hepcidin gene from red sea bream (Chrysophrys major). Antimicrob Agents Chemother 49:1608–1612. doi: 10.1128/AAC.49.4.1608-1612.2005 CrossRefGoogle Scholar
  15. 15.
    Masso-Silva JA, Diamond G (2014) Antimicrobial peptides from fish. Pharmaceuticals 7(3):265–310. doi: 10.3390/ph7030265 CrossRefGoogle Scholar
  16. 16.
    Zhou JG, Wei JG, Xu D, Cui HC, Yan Y, Ou-Yang ZL, Huang XH, Huang YH, Qin QW (2011) Molecular cloning and characterization of two novel hepcidins from orange-spotted grouper, Epinephelus coioides. Fish Shellfish Immunol 30:559–568. doi: 10.1016/j.fsi.2010.11.021 CrossRefGoogle Scholar
  17. 17.
    Xu T, Sun Y, Shi G, Wang R (2012) Miiuy croaker hepcidin gene and comparative analyses reveal evidence for positive selection. PLoS One 7(4):e35449. doi: 10.1371/journal.pone.0035449 CrossRefGoogle Scholar
  18. 18.
    Liang T, Ji W, Zhang GR, Wei KJ, Feng K, Wang WM, Zou GW (2013) Molecular cloning and expression analysis of liver-expressed antimicrobial peptide 1 (LEAP-1) and LEAP-2 genes in the blunt snout bream(Megalobrama amblycephala). Fish Shellfish Immunol 35:553–563. doi: 10.1016/j.fsi.2013.05.021 CrossRefGoogle Scholar
  19. 19.
    Li H, Zhang F, Guo H, Zhu Y, Yuan J, Yang G, An L (2013) Molecular characterization of hepcidin gene in common carp (Cyprinus carpio L.) and its expression pattern responding to bacterial challenge. Fish Shellfish Immunol 35:1030–1038. doi: 10.1016/j.fsi.2013.07.001 CrossRefGoogle Scholar
  20. 20.
    Gong LC, Wang H, Deng L (2014) Molecular characterization, phylogeny and expression of a hepcidin gene in the blotched snakehead Channa maculata. Dev Comp Immunol 44:1–11. doi: 10.1016/j.dci.2013.11.007 CrossRefGoogle Scholar
  21. 21.
    Chi JR, Liao LS, Wang RG, Jhu CS, Wu JL, Hu SY (2014) Molecular cloning and functional characterization of the hepcidin gene from the convict cichlid (Amatitlania nigrofasciata) and its expression pattern in response to lipopolysaccharide challenge. Fish Physiol Biochem 41:449–461. doi: 10.1007/s10695-014-9996-6 CrossRefGoogle Scholar
  22. 22.
    Sang C, Lin Y, Jiang K, Zhang F, Song W (2015) Molecular cloning and mRNA expression of a hepcidin gene from the spinyhead croaker, Collichthys lucidus. Genet Mol Res 14(4):16050–16059. doi: 10.4238/2015 CrossRefGoogle Scholar
  23. 23.
    Chaturvedi P, Dhanik M, Pande V, Pande A (2016) Identification and in-silico analysis of hepcidin from Tor putitora (Hamilton). Indian J Comp Microbiol Immunol Infect Dis 37(2):67–76. doi: 10.5958/0974-0147.2016.00013.1 Google Scholar
  24. 24.
    Ren G, Shen WY, Li WF, Zhu YR (2014) Molecular characterization and expression pattern of a liver-expressed antimicrobial peptide 2 (LEAP-2) gene in yellow catfish (Pelteobagrus fulvidraco). J Aquac Res Development 5:229. doi: 10.4172/2155-9546.1000229 Google Scholar
  25. 25.
    Liu QN, Xin ZZ, Zhang DZ, Jiang SH, Chai XY, Wang ZF, Li CF, Zhou CL, Tang BP (2017) cDNA cloning and expression analysis of a hepcidin gene from yellow catfish Pelteobagrus fulvidraco (Siluriformes: Bagridae). Fish Shellfish Immun 60:247–254. doi: 10.1016/j.fsi.2016.10.049 CrossRefGoogle Scholar
  26. 26.
    Shi J, Camus AC (2006) Hepcidins in amphibians and fishes: antimicrobial peptides or iron-regulatory hormones? Dev Comp Immunol 30(9):746–755. doi: 10.1016/j.dci.2005.10.009 CrossRefGoogle Scholar
  27. 27.
    Ramey G, Deschemin JC, Durel B, Canonne-Hergaux F, Nicolas G, Vaulont S (2010) Hepcidin targets ferroportin for degradation in hepatocytes. Haematologica 95(3):501–504. doi: 10.3324/haematol.2009.014399 CrossRefGoogle Scholar
  28. 28.
    Lauth X, Babon JJ, Stannard JA, Singh S, Nizet V, Carlberg JM, Ostland VE, Pennington MW, Norton RS, Westerman ME (2005) Bass hepcidin synthesis, solution structure, antimicrobial activities and synergism, and in vivo hepatic response to bacterial infections. J Biol Chem 280(10):9272–9282. doi: 10.1074/jbc.M411154200 CrossRefGoogle Scholar
  29. 29.
    Chang WT, Pan CY, Rajanbabu V, Cheng CW, Chen JY (2011) Tilapia (Oreochromis mossambicus) antimicrobial peptide, hepcidin 1–5, shows antitumor activity in cancer cells. Peptides 32(2):342–352. doi: 10.1016/j.peptides.2010.11.003 CrossRefGoogle Scholar
  30. 30.
    Shike H, Shimizu C, Lauth X, Burns JC (2004) Organization and expression analysis of the zebrafish hepcidin gene, an antimicrobial peptide gene conserved among vertebrates. Dev Comp Immunol 28(7–8):747–754. doi: 10.1016/j.dci.2003.11.009 CrossRefGoogle Scholar
  31. 31.
    Hilton KB, Lambert LA (2008) Molecular evolution and characterization of hepcidin gene products in vertebrates. Gene 415:40–48. doi: 10.1016/j.gene.2008.02.016 CrossRefGoogle Scholar
  32. 32.
    Chaithanya ER, Philip R, Sathyan N, Anil Kumar PR, Antony SP, Sanjeevan VN, Bright Singh IS (2013) A novel isoform of the hepatic antimicrobial peptide, hepcidin (Hepc-CB1), from a deep-sea fish, the spinyjaw greeneye Chlorophthalmus bicornis (Norman, 1939): molecular characterisation and phylogeny. Probiotics Antimicrob Proteins 5(1):1–7. doi: 10.1007/s12602-012-9120-0 CrossRefGoogle Scholar
  33. 33.
    Xu Q, Cheng CH, Hu P, Ye H, Chen Z, Cao L, Chen L, Shen Y, Chen L (2008) Adaptive evolution of hepcidin genes in Antarctic notothenioid fishes. Mol Biol Evol 25:1099–1112. doi: 10.1093/molbev/msn056 CrossRefGoogle Scholar
  34. 34.
    Yang M, Wang KJ, Chen JH, Qu HD, Li SJ (2007) Genomic organization and tissue specific expression analysis of hepcidin-like genes from black porgy (Acanthopagrus schlegelii B). Fish Shellfish Immunol 23:1060–1071. doi: 10.1016/j.fsi.2007.04.011 CrossRefGoogle Scholar
  35. 35.
    Rodrigues PN, Vazquez-Dorado S, Neves JV, Wilson JM (2006) Dual function of fish hepcidin: response to experimental iron overload and bacterial infection in sea bass (Dicentrarchus labrax). Dev Comp Immunol 30:1156–1167. doi: 10.1016/j.dci.2006.02.005 CrossRefGoogle Scholar
  36. 36.
    Cho YS, Lee SY, Kim KH, Kim SK, Kim DS, Nam YK (2009) Gene structure and differential modulation of multiple rockbream (Oplegnathus fasciatus) hepcidin isoforms resulting from different biological stimulations. Dev Comp Immunol 33:46–58. doi: 10.1016/j.dci.2008.07.009 CrossRefGoogle Scholar
  37. 37.
    Hunter HN, Fulton DB, Ganz T, Vogel HJ (2002) The solution structure of human hepcidin, a peptide hormone with antimicrobial activity that is involved in iron uptake and hereditary hemochromatosis. J Biol Chem 277(40):37597–37603. doi: 10.1074/jbc.M205305200 CrossRefGoogle Scholar
  38. 38.
    Ren H-L, Wang K-J, Zhou H-L, Yang M (2006) Cloning and organisation analysis of a hepcidin-like gene and cDNA from Japan sea bass, Lateolabrax japonicus. Fish Shellfish Immunol 21:221–227. doi: 10.1016/j.fsi.2005.10.011 CrossRefGoogle Scholar
  39. 39.
    Ganz T (2003) Hepcidin, a key regulator of iron metabolism and mediator of anemia of inflammation. Blood 102:783–788. doi: 10.1182/blood-2003-03-0672 CrossRefGoogle Scholar
  40. 40.
    Valore EV, Ganz T (2008) Posttranslational processing of hepcidin in human hepatocytes is mediated by the prohormone convertase furin. Blood Cells Mol Dis 40:132–138. doi: 10.1016/j.bcmd.2007.07.009 CrossRefGoogle Scholar
  41. 41.
    Nakayama K (1997) Furin: a mammalian subtilisin/Kex2p-like endoprotease involved in processing of a wide variety of precursor proteins. Biochem J 327:625–635. doi: 10.1042/bj3270625 CrossRefGoogle Scholar
  42. 42.
    Chaturvedi P, Dhanik M, Pande A (2014) Characterization and structural analysis of hepcidin like antimicrobial peptide from Schizothorax richardsonii (Gray). Protein J 33:1–10CrossRefGoogle Scholar
  43. 43.
    Huang PH, Chen JY, Kuo CM (2007) Three different hepcidins from tilapia, Oreochromis mossambicus: analysis of their expressions and biological functions. Mol Immunol 44:1922–1934. doi: 10.1016/j.molimm.2006.09.031 CrossRefGoogle Scholar
  44. 44.
    Wang KJ, Cai JJ, Cai L, Qu HD, Yang M, Zhang M (2009) Cloning and expression of a hepcidin gene from a marine fish (Pseudosciaena crocea) and the antimicrobial activity of its synthetic peptide. Peptides 30(4):638–646. doi: 10.1016/j.peptides.2008.12.014 CrossRefGoogle Scholar
  45. 45.
    Carugo O, Cemazar M, Zahariev S, Hudaky I, Gaspari Z, Perczel A, Pongor S (2003) Vicinal disulfide turns. Protein Eng 16:637–639. doi: 10.1093/protein/gzg088 CrossRefGoogle Scholar
  46. 46.
    Chekmenev EY, Jones SM, Nikolayeva YN, Vollmar BS, Wagner TJ, Gor'kov PL, Brey WW, Manion MN, Daugherty KC, Cotten M (2006) High-field NMR studies of molecular recognition and structure-function relationships in antimicrobial piscidins at the water-lipid bilayer interface. J Am Chem Soc 128:5308–5309. doi: 10.1021/ja058385e CrossRefGoogle Scholar
  47. 47.
    Lynch M, O’Hely M, Walsh B, Force A (2001) The probability of preservation of a newly arisen gene duplicate. Genetics 159:1789–1804Google Scholar
  48. 48.
    Rastogi S, Liberles DA (2005) Subfunctionalization of duplicated genes as a transition state to neofunctionalization. BMC Evol Biol 5:28. doi: 10.1186/1471-2148-5-28 CrossRefGoogle Scholar
  49. 49.
    He X, Zhang J (2005) Rapid subfunctionalization accompanied by prolonged and substantial neofunctionalization in duplicate gene evolution. Genetics 169:1157–1164. doi: 10.1534/genetics.104.037051 CrossRefGoogle Scholar
  50. 50.
    Nemeth E, Ganz T (2006) Regulation of iron metabolism by hepcidin. Annu Rev Nutr 26:323–342. doi: 10.1146/annurev.nutr.26.061505.111303 CrossRefGoogle Scholar
  51. 51.
    Padhi A, Verghese B (2007) Evidence for positive Darwinian selection on the hepcidin gene of Perciform and Pleuronectiform fishes. Mol Divers 11:119–130. doi: 10.1007/s11030-007-9066-4 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Aishwarya Nair
    • 1
  • K. S. Sruthy
    • 1
  • E. R. Chaithanya
    • 1
  • T. P. Sajeevan
    • 2
  • I. S. Bright Singh
    • 2
  • Rosamma Philip
    • 1
  1. 1.Department of Marine Biology, Microbiology and Biochemistry, School of Marine SciencesCochin University of Science and TechnologyKochiIndia
  2. 2.National Centre for Aquatic Animal HealthCochin University of Science and TechnologyKochiIndia

Personalised recommendations