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Molecular Cloning of Hemocyanin cDNA from Fenneropenaeus chinensis and Antimicrobial Analysis of Two C-terminal Fragments

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Abstract

Peptides derived from shrimp hemocyanin have antimicrobial properties. This is the first report of hemocyanin cDNA (FCHc) cloned from Fenneropenaeus chinensis and recombinant expression of two C-terminal fragments. Based on sequence analysis of Fenneropenaeus chinensis hemocyanin FCHc, we subcloned two FCHc fragments by designing special primers. Two antimicrobial peptides (AMPs) were derived from FCHc (FCHc-C1 and FCHc-C2). The recombinant sequence of FCHc-C1 consisted of 207 bp encoding 69 amino acids and the recombinant sequence of FCHc-C2 consisted of 120 bp encoding 40 amino acids. The results of Tricine–sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting indicated that recombinant FCHc-C1 and FCHc-C2 peptides (rFCHc-C1 and rFCHc-C2) were expressed successfully. An inhibition assay showed that FCHc-C1 and FCHc-C2 were anionic AMPs with antifungal and antibacterial activities.

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References

  • Bhanot V, Balamurugan V, Bhanuprakash V, Venkatesan G, Sen A, Yadav V, Yogisharadhya R, Singh RK (2009) Expression of P32 protein of goatpox virus in Pichia pastoris and its potential use as a diagnostic antigen in ELISA. J Virol Methods 162(1–2):251–257

    Article  CAS  PubMed  Google Scholar 

  • Brake AJ, Merryweather JP, Coit DG, Heberlein UA, Masiarz FR, Mullenbach GT, Urdea MS, Valenzuela P, Barr PJ (1984) Alpha-factor-directed synthesis and secretion of mature foreign proteins in Saccharomyces cerevisiae. PNAS 81(15):4642–4646

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Bulet P, Dimarcq J, Hetru C, Lagueux M, Charlet M, Hegy G, Van Dorsselaer A, Hoffmann J (1993) A novel inducible antibacterial peptide of Drosophila carries an O-glycosylated substitution. J Biol Chem 268(20):14893–14897

    CAS  PubMed  Google Scholar 

  • Cereghino GPL, Cregg JM (1999) Applications of yeast in biotechnology: protein production and genetic analysis. Curr Opin Biotechnol 10(5):422–427

    Article  CAS  PubMed  Google Scholar 

  • Cerenius L, Jiravanichpaisal P, Liu H, Soderhall I (2011) Crustacean immunity. Invertebrate Immunity 708:239–259

    Article  Google Scholar 

  • Cuthbertson BJ, Deterding LJ, Williams JG, Tomer KB, Etienne K, Blackshear PJ, Büllesbach EE, Gross PS (2008) Diversity in penaeidin antimicrobial peptide form and function. DCI 32(3):167–181

    CAS  Google Scholar 

  • Cytryńska M, Mak P, Zdybicka-Barabas A, Suder P, Jakubowicz T (2007) Purification and characterization of eight peptides from Galleria mellonella immune hemolymph. Peptides 28(3):533–546

    Article  PubMed  Google Scholar 

  • Decker H, Jaenicke E (2004) Recent findings on phenoloxidase activity and antimicrobial activity of hemocyanins. Dev Comp Immunol 28:673–687

    Article  CAS  PubMed  Google Scholar 

  • Dennison SR, Howe J, Morton LHG, Brandenburg K, Harris F, Phoenix DA (2006a) Interactions of an anionic antimicrobial peptide with Staphylococcus aureus membranes. Biochem Bioph Res Co 347(4):1006–1010

    Article  CAS  Google Scholar 

  • Dennison SR, Morton LHG, Brandenburg K, Harris F, Phoenix DA (2006b) Investigations into the ability of an oblique α-helical template to provide the basis for design of an antimicrobial anionic amphiphilic peptide. FEBS J 273(16):3792–3803

    Article  CAS  PubMed  Google Scholar 

  • Destoumieux-Garzón D, Saulnier D, Garnier J, Jouffrey C, Bulet P, Bachère E (2001) Crustacean immunity. J Biol Chem 276(50):47070–47077

    Article  PubMed  Google Scholar 

  • Diego-García E, Batista CVF, García-Gómez BI, Lucas S, Candido DM, Gómez-Lagunas F, Possani LD (2005) The Brazilian scorpion Tityus costatus Karsch: genes, peptides and function. Toxicon 45(3):273–283

    Article  PubMed  Google Scholar 

  • Ellison RT, Boose D, LaForce FM (1985) Isolation of an antibacterial peptide from human lung lavage fluid. J Infect Dis 151(6):1123

    Article  CAS  PubMed  Google Scholar 

  • Flegel TW, Lightner DV, Lo C, Owens L (2008) Shrimp disease control: past, present and future. Diseases in Asian Aquaculture VI Fish Health Section, Asian Fisheries Society Manila, Philippines: pp. 355–378

  • Harris F, Dennison SR, Phoenix DA (2009) Anionic antimicrobial peptides from eukaryotic organisms. Curr Protein Pept Sc 10(6):585–606

    Article  CAS  Google Scholar 

  • Heidari M, Hamir A, Cutlip RC, Brogden KA (2002) Antimicrobial anionic peptide binds in vivo to Mannheimia (Pasteurella) haemolytica attached to ovine alveolar epithelium. Int J Antimicrob Ag 20(1):69–72

    Article  CAS  Google Scholar 

  • Hetru C, Bulet P (1997) Strategies for the isolation and characterization of antimicrobial peptides of invertebrates. MIMB 78:35–50

    CAS  Google Scholar 

  • Huang WS, Wang KJ, Yang M, Cai JJ, Li SJ, Wang GZ (2006) Purification and part characterization of a novel antibacterial protein Scygonadin, isolated from the seminal plasma of mud crab, Scylla serrata (Forskål, 1775). J Exp Mar Biol Ecol 339(1):37–42

    Article  CAS  Google Scholar 

  • Jaenicke E, Decker H (2004) Functional changes in the family of type 3 copper proteins during evolution. ChemBioChem 5(2):163–169

    Article  CAS  PubMed  Google Scholar 

  • Jaenicke E, Föll R, Decker H (1999) Spider hemocyanin binds ecdysone and 20-OH-ecdysone. J Biol Chem 274:34267–34271

    Article  CAS  PubMed  Google Scholar 

  • Jin F, Xu X, Wang L, Zhang W, Gu D (2006) Expression of recombinant hybrid peptide cecropinA(1–8)-magainin2(1–12) in Pichia pastoris: purification and characterization. Protein Expres Purif 50(2):147–156

    Article  CAS  Google Scholar 

  • LaForce FM, Boose DS (1984) Effect of zinc and phosphate on an antibacterial peptide isolated from lung lavage. Infect Immun 45(3):692–696

    PubMed Central  CAS  PubMed  Google Scholar 

  • Lai R, Liu H, Hui Lee W, Zhang Y (2002) An anionic antimicrobial peptide from toad Bombina maxima. Biochem Bioph Res Co 295(4):796–799

    Article  CAS  Google Scholar 

  • Lai R, Lomas LO, Jonczy J, Turner PC, Rees HH (2004) Two novel non-cationic defensin-like antimicrobial peptides from haemolymph of the female tick, Amblyomma hebraeum. Biochem J 379:681–5

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Lai Y, Gallo RL (2009) AMPed up immunity: how antimicrobial peptides have multiple roles in immune defense. Trends Immunol 30(3):131–141

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Lee SY, Lee BL, Söderhäll K (2003) Processing of an antibacterial peptide from hemocyanin of the freshwater crayfish Pacifastacus leniusculus. J Biol Chem 278(10):7927–7933

    Article  CAS  PubMed  Google Scholar 

  • Li L, Wang J-X, Zhao X-F, Kang C-J, Liu N, Xiang J-H, Li F-H, Sueda S, Kondo H (2005) High level expression, purification, and characterization of the shrimp antimicrobial peptide, Ch-penaeidin, in Pichia pastoris. Protein Expres Purif 39(2):144–151

    Article  CAS  Google Scholar 

  • Lightner DV, Redman RM (1998) Shrimp diseases and current diagnostic methods. Aquaculture 164(1–4):201–220

    Article  Google Scholar 

  • Littauer UZ, Sela M (1962) An ultracentrifugal study of the efficiency of some macromolecular inhibitors of ribonuclease. BBA-Specialized Section on Nucleic Acids and Related Subjects 61(4):609–611

    CAS  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, New York

    Google Scholar 

  • Markl J (1986) Evolution and function of structurally diverse subunits in the respiratory protein hemocyanin from arthropods. Biol Bull-US 171:90–115

    Article  CAS  Google Scholar 

  • Markl J, Decker H (1992) Molecular structure of the arthropod hemocyanins in blood and tissue oxygen carriers. Springer, Berlin

    Google Scholar 

  • Miller KI, Eldred NW, Arisaka F, Van Holde KE (1977) Structure and function of hemocyanin from thalassinid shrimp. J Comp Physiol 115:171–184

    Article  CAS  Google Scholar 

  • Nakamura T, Furunaka H, Miyata T, Tokunaga F, Muta T, Iwanaga S, Niwa M, Takao T, Shimonishi Y (1988) Tachyplesin, a class of antimicrobial peptide from the hemocytes of the horseshoe crab (Tachypleus tridentatus). Isolation and chemical structure. J Biol Chem 263(32):16709–16713

    CAS  PubMed  Google Scholar 

  • Paul R, Bergner B, Pfeffer-Seidl A, Decker H, Efinger R, Storz H (1994) Gas transport in the haemolymph of arachnids-oxygen transport and the physiological role of haemocyanin. J Exp Biol 188:25–46

    CAS  PubMed  Google Scholar 

  • Paul RJ, Pirow R (1998) The physiological significance of respiratory proteins in invertebrates. Zoology 100:319–327

    Google Scholar 

  • Schägger H, Von Jagow G (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166(2):368–379

    Article  PubMed  Google Scholar 

  • Schlievert P, Johnson W, Galask R (1976) Bacterial growth inhibition by amniotic fluid. VI. Evidence for a zinc-peptide antibacterial system. AJOG 125(7):906

    CAS  Google Scholar 

  • Sellos D, Lemoine S, Van Wormhoudt A (1997) Molecular cloning of hemocyanin cDNA from Penaeus vannamei (Crustacea, Decapoda): structure, evolution and physiological aspects. FEBS Lett 407(2):153–158

    Article  CAS  PubMed  Google Scholar 

  • Smith VJ, Desbois AP, Dyrynda EA (2010) Conventional and unconventional antimicrobials from fish, marine invertebrates and micro-algae. Mar Drugs 8(4):1213–1262

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Smith VJ, Fernandes JMO, Kemp GD, Hauton C (2008) Crustins: enigmatic WAP domain-containing antibacterial proteins from crustaceans. DCI 32(7):758–772

    CAS  Google Scholar 

  • Söderhäll K, Cerenius L (1998) Role of the prophenoloxidase-activating system in invertebrate immunity. Curr Opin Immunol 10:23–28

    Article  PubMed  Google Scholar 

  • Sritunyalucksana K, Cerenius L, Söderhäll K (1999) Molecular cloning and characterization of prophenoloxidase in the black tiger shrimp, Penaeus monodon. DCI 23(3):179–186

    CAS  Google Scholar 

  • Steiner H, Hultmark D, Engström Å, Bennich H, Boman H (1981) Sequence and specificity of two antibacterial proteins involved in insect immunity. Nature 292:246–248

    Article  CAS  PubMed  Google Scholar 

  • Vandendriessche L (1956) Inhibitors of ribonuclease activity. Arch Biochem Biophys 65(1):347–353

    Article  CAS  PubMed  Google Scholar 

  • Volbeda A, Hol W (1989) Crystal structure of hexameric haemocyanin from Panulirus interruptus refined at 3.2 Å resolution. J Mol Biol 209:249–279

    Article  CAS  PubMed  Google Scholar 

  • Yu X, Li Z, Xia X, Fang H, Zhou C, Chen H (2007) Expression and purification of ancrod, an anticoagulant drug, in Pichia pastoris. Protein Expr Purif 55(2):257–261

    Article  CAS  PubMed  Google Scholar 

  • Zhang X, Huang C, Qin Q (2004) Antiviral properties of hemocyanin isolated from shrimp Penaeus monodon. Antivir Res 61(2):93–99

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by the National Key Technology Support Program (2012BAD17B03) and the National High Technology Research and Development Program of China (863 Program) (2006AA100311).

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Authors and Affiliations

  1. R&D Center of Marine Biotechnology, Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao, 266071, People’s Republic of China

    Chuwen Qiu, Mei Liu, Baojie Wang, Keyong Jiang, Shujuan Sun, Xiaolin Meng, Zuoyong Luo & Lei Wang

  2. University of the Chinese Academy of Sciences, Beijing, 100039, People’s Republic of China

    Chuwen Qiu, Xiaolin Meng & Zuoyong Luo

  3. Shandong Peanut Research Institute, 126 Fushan Road, Qingdao, 266100, People’s Republic of China

    Jie Sun

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  1. Chuwen Qiu
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Correspondence to Mei Liu or Lei Wang.

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Qiu, C., Sun, J., Liu, M. et al. Molecular Cloning of Hemocyanin cDNA from Fenneropenaeus chinensis and Antimicrobial Analysis of Two C-terminal Fragments. Mar Biotechnol 16, 46–53 (2014). https://doi.org/10.1007/s10126-013-9519-y

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  • DOI: https://doi.org/10.1007/s10126-013-9519-y

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