Cation metals specific hemocyanin exhibits differential antibacterial property in mud crab, Scylla serrata
Abstract
Serum of mud crab, Scylla serrata had antibacterial activity against certain host specific bacteria as well as other known crustacean pathogenic bacteria. The respective antibacterial molecule was isolated by ion-exchange chromatographic method using diethylaminoethyl-cellulose matrix and eluted with gradient increase of sodium chloride (0-0.5 M). There were three major peaks on the elution profile and fractions from second major peak exhibit differential antibacterial response against Bacillus flexus N3, Escherichia coli, Vibrio harveyi and Vibrio vulnificus. The electrophoretic analyses of this fraction resulted in single protein band and the native molecular weight of this protein was found to be approximately 305 kDa. The bathocuproine sulfonic acid staining (a specific stain for copper), copper/protein ratio (0.129%) reveals this isolated antibacterial protein to be hemocyanin. This antibacterial hemocyanin is also capable of binding to cations, namely Ca2+, K+, Mg2+, Mn2+ and Zn2+ apart from carrying Fe2+ ions. This native antibacterial hemocyanin was found to contain three or four subunits with possible molecular size of 70-98 kDa under reducing conditions. These findings indicate that the respiratory protein hemocyanin in the presence of cations functions as humoral immune molecule and the detailed investigation would reveal additional immune functional characteristics of this respiratory protein as well as subunits-specific functions of this antibacterial hemocyanin.
Acknowledgements
This work was carried out with funding from University Grants Commission (UGC-SAP-BSR programme) and Department of Biotechnology (DBT), New Delhi, awarded to
M.A. V.M. acknowledges UGC, New Delhi, India, for the award of Project Fellow.
References
Adachi K., Hirata T., Nagai K. & Sakaguchi M. 2008. Hemocyanin a most likely inducer of black spots in kuruma prawn Penaeus japonicus during storage. J. Food Sci. 66: 1130–1136.10.1111/j.1365-2621.2001.tb16093.xSuche in Google Scholar
Allison J. & Cole W. 1940. The nitrogen, copper and hemocyanin content of the sera of several arthropods. J. Biol. Chem. 135: 259–265.10.1016/S0021-9258(18)73182-7Suche in Google Scholar
Ansorge W. 1985. Fast and sensitive detection of protein and DNA bands by treatment with potassium permanganate. J. Biochem. Biophys. Methods 11: 13–20.10.1016/0165-022X(85)90037-5Suche in Google Scholar
Arumugam M. & Ravindranath M.H. 1986. Significance of the variation in haemolymph copper-free-and-bound proteins during ageing and time of day in the crab Scylla serrata (Forskal). Arch. Int. Physiol. Biochem. 94: 11–17.10.3109/13813458609069101Suche in Google Scholar
Bachére E., Destoumieux D. & Bulet P. 2000. Penaeidins, antimicrobial peptides of shrimp: a comparison with other effectors of innate immunity. Aquaculture 191: 71–88.10.1016/S0044-8486(00)00419-1Suche in Google Scholar
Battison A.L., Summerfield R. & Patrzykat A. 2008. Isolation and characterisation of two antimicrobial peptides from haemocytes of the American lobster Homarus americanus. Fish Shellfish Immunol. 25: 181–187.10.1016/j.fsi.2008.04.005Suche in Google Scholar
Brenowitz M., Bonaventura C., Bonaventura J. & Gianazza E. 1981. Subunit composition of a high molecular weight oligomer: Limulus polyphemus hemocyanin. Arch. Biochem. Biophys. 210: 748–761.10.1016/0003-9861(81)90242-3Suche in Google Scholar
Bruyninckx W.J., Gutteridge S. & Mason H.S. 1978. Detection of copper on polyacrylamide gels. Anal. Biochem. 89: 174–7.10.1016/0003-2697(78)90738-8Suche in Google Scholar
Chattopadhyay I. & Chatterjee B.P. 1993. A low-molecular weight lectin from the edible crab Scylla serrata hemolymph: purification and partial characterization. Biochem. Arch. 9: 65–72.Suche in Google Scholar
Cornick J.W. & Stewart J.E. 1968. Interaction of the pathogen Gaffkya homari with natural defense mechanisms of Homarus americanus. J. Fish. Res. Board Canada 25: 695–709.10.1139/f68-064Suche in Google Scholar
Decker H. & Jaenicke E. 2004. Recent findings on phenoloxidase activity and antimicrobial activity of hemocyanins. Dev. Comp. Immunol. 28: 673–87.10.1016/j.dci.2003.11.007Suche in Google Scholar PubMed
Destoumieux D., Bulet P., Loew D., Van Dorsselaer A., Rodriguez J. & Bachère E. 1997. Penaeidins, a new family of antimicrobial peptides isolated from the shrimp Penaeus vannamei (Decapoda). J. Biol. Chem. 272: 28398–28406.10.1074/jbc.272.45.28398Suche in Google Scholar
Dolashka P., Dolashki A., Voelter W., Van Beeumen J. & Stevanovic S. 2015. Antimicrobial activity of peptides from the hemolymph of Helix lucorum snails. Int. J. Curr. Microbiol. Appl. Sci. 4: 1061–1071.Suche in Google Scholar
Dolashka P., Dolashki A., Van Beeumen J., Floetenmeyerd M., Velkova L., Stevanovic S. & Voelter W. 2016. Antimicrobial activity of molluscan hemocyanins from helix and rapana snails. Curr. Pharm. Biotechnol. 17: 263–270.10.2174/1389201016666150907113435Suche in Google Scholar
Dolashka P., Zal F., Dolashki A., Molin L., Traldi P. & Salvato B. 2012. ESI-MS and MALLS analysis of quaternary structure of molluscan hemocyanins. J. Mass Spectrom. 47: 940–947.10.1002/jms.2967Suche in Google Scholar
Dolashka P., Moshtanska V., Borisova V., Dolashki A., Stevanovic S., Dimanov T. & Voelter W. 2011. Antimicrobial proline-rich peptides from the hemolymph of marine snail Rapana venosa.Peptides 32: 1477–1483.10.1016/j.peptides.2011.05.001Suche in Google Scholar
Dolashka-Angelova P., Dolashki A., Savvides S.N., Hristova R., Van Beeumen J., Voelter W., Devreese B., Weser U., Di Muro P., Salvato B. & Stevanovic S. 2005a. Structure of hemocyanin subunit CaeSS2 of the crustacean Mediterranean crab Carcinus aestuarii.J. Biochem. 138: 303–312.10.1093/jb/mvi130Suche in Google Scholar
Dolashka-Angelova P., Dolashki A., Stevanovic S., Hristova R., Atanasov B., Nikolov P. & Voelter W. 2005b. Structure and stability of arthropodan hemocyanin Limulus polyphemus. Spectrochim. Acta –Part A Mol. Biomol. Spectrosc. 61: 1207–1217.10.1016/j.saa.2004.06.043Suche in Google Scholar
Dolashki A., Radkova M., Todorovska E., Ivanov M., Stevanovic S., Molin L., Traldi P., Voelter W. & Dolashka P. 2015. Structure and characterization of Eriphia verrucosa hemocyanin. Mar. Biotechnol. 17: 743–752.10.1007/s10126-015-9653-9Suche in Google Scholar
Figueroa-Soto C.G., de la Barca A.M.C., Vazquez-Moreno L., Higuera-Ciapara I. & Yepiz-Plascencia G. 1997. Purification of hemocyanin from white shrimp (Penaeus vannamei Boone) by immobilized metal affinity chromatography. Comp. Biochem. Physiol. Part B Biochem Mol. Biol. 117: 203–208.10.1016/S0305-0491(96)00321-5Suche in Google Scholar
Freimoser F.M., Jakob C.A., Aebi M. & Tuor U. 1999. The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay is a fast and reliable method for colorimetric determination of fungal cell densities. Appl. Environ. Microbiol. 65: 3727–3729.10.1128/AEM.65.8.3727-3729.1999Suche in Google Scholar PubMed PubMed Central
García-Carreño F.L., Cota K. & Del Toro M.A.N. 2008. Phenoloxidase activity of hemocyanin in whiteleg shrimp Penaeus vannamei: conversion, characterization of catalytic properties, and role in postmortem melanosis. J. Agric. Food Chem. 56: 6454–6459.10.1021/jf800839xSuche in Google Scholar PubMed
Herbinière J., Braquart-Varnier C., Grève P., Strub J.M., Frère J., Van Dorsselaer A. & Martin G. 2005. Armadillidin: a novel glycine-rich antibacterial peptide directed against gram-positive bacteria in the woodlouse Armadillidium vulgare (Terrestrial Isopod, Crustacean). Dev. Comp. Immunol. 29: 489–499.10.1016/j.dci.2004.11.001Suche in Google Scholar
Hoq M.I., Seraj M.U. & Chowdhury S. 2003. Isolation and characterization of antibacterial peptides from the mud-crab, Scylla serrata. Pakistan J. Biol. Sci. 6: 1345–1353.10.3923/pjbs.2003.1345.1353Suche in Google Scholar
Horn E.C. & Kerr M.S. 1969. The hemolymph proteins of the blue crab, callinectes sapidus –I. Hemocyanins and certain other major protein constituents. Comp. Biochem. Physiol. 29: 493–508.10.1016/0010-406X(69)91602-8Suche in Google Scholar
Huang W.S., Wang K.J., Yang M., Cai J.J., Li S.J. & Wang G.Z. 2006. Purification and part characterization of a novel antibacterial protein scygonadin, isolated from the seminal plasma of mud crab, Scylla serrata (Forskal, 1775). J. Exp. Mar. Biol. Ecol. 339:37–42.10.1016/j.jembe.2006.06.029Suche in Google Scholar
Jayasankar V. & Subramoniam T. 1999. Antibacterial activity of seminal plasma of the mud crab Scylla serrata (Forskal). J. Exp. Mar. Biol. Ecol. 236: 253–259.10.1016/S0022-0981(98)00203-2Suche in Google Scholar
Jayasree S. 2001. Biological properties of a natural agglutinin in the hemolymph of Indian white prawn, Penaeus indicus H. Milne Edwards. Aquaculture 194: 245–252.10.1016/S0044-8486(00)00528-7Suche in Google Scholar
Jiravanichpaisal P., Lee S.Y., Kim Y.A., Andrén T. & Söderhäll I. 2007. Antibacterial peptides in hemocytes and hematopoietic tissue from freshwater crayfish Pacifastacus leniusculus:characterization and expression pattern. Dev. Comp. Immunol. 31: 441–455.10.1016/j.dci.2006.08.002Suche in Google Scholar
Kawabata S.I., Tokunaga F., Kugi Y., Motoyama S., Miura Y., Hirata M. & Iwanaga S. 1996. Limulus factor D, a 43-kDa protein isolated from horseshoe crab hemocytes, is a serine protease homologue with antimicrobial activity. FEBS Lett. 398: 146–150.10.1016/S0014-5793(96)01224-0Suche in Google Scholar
Kawabata T., Yasuhara Y., Ochiai M., Matsuura S. & Ashida M. 1995. Molecular cloning of insect pro-phenol oxidase: a copper-containing protein homologous to arthropod hemocyanin. Proc. Natl. Acad. Sci. USA 92:7774–7778.10.1073/pnas.92.17.7774Suche in Google Scholar PubMed PubMed Central
Khoo L., Robinette D. & Noga E. 1999. Callinectin, an antibacterial peptide from blue crab, Callinectes sapidus, hemocytes. Mar. Biotechnol. 1: 44–51.10.1007/PL00011750Suche in Google Scholar PubMed
Kingston H. & Jessie L. 1989. Introduction to microwave sample preparation: theory and practice. Anal. Chem. 61: 330A–330A.10.1021/ac00180a709Suche in Google Scholar
Laemmli U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.10.1038/227680a0Suche in Google Scholar
Lamy J., Lamy J., Sizaret P.Y., Billiald P., Jolles P., Jolles J., Feldmann R.J. & Bonaventura J. 1983. Quaternary structure of Limulus polyphemus hemocyanin. Biochemistry 22:5573–5583.10.1021/bi00293a019Suche in Google Scholar
Lee S.Y., Lee B.L. & Söderhäll K. 2003. Processing of an antibacterial peptide from hemocyanin of the freshwater crayfish Pacifastacus leniusculus. J. Biol. Chem. 278: 7927–7933.10.1074/jbc.M209239200Suche in Google Scholar
Majumder M., Chattopadhyay T., Guha A.K. & Chatterjee B.P. 1997. Inhibition of bacterial respiration by a low-molecular weight lectin, scyllin, from Scylla serrata crab hemolymph. Indian J. Biochem. Biophys. 34: 87–89.Suche in Google Scholar
Mangum C.P. & Greaves J. 1996. Hemocyanins of the genus Uca: structural polymorphisms and native oligomers. J. Exp. Mar. Biol. Ecol. 199: 1–15.10.1016/0022-0981(95)00154-9Suche in Google Scholar
Markl J. & Decker H. 1992. Molecular structure of the arthropod hemocyanins. Adv. Comp. Environ. Physiol. 13: 325–376.10.1007/978-3-642-76418-9_12Suche in Google Scholar
Martin J.L.M. 1975. Recherches sur le métabolisme des métaux chez les Crustacés décapodes marins?: leurs rapports avec la mue et la reproduction. Thèse doctorat: Sciences naturelles, Université d’Aix-Marseille 2.Suche in Google Scholar
Maurer H.R. 1971. Disc Electrophoresis and Related Techniques of Polyacrylamide Gel Electrophoresis. Walter de Gruyter, Berlin, 222 pp.Suche in Google Scholar
Meiyalagan V. & Arumugam M. 2015. Detection and preliminary characterization of antibacterial protein(s) in the serum of mud crab, Scylla serrata. Invertebrate Survival Journal 12: 287–295.Suche in Google Scholar
Nagai T. & Kawabata S.I. 2000. A link between blood coagulation and prophenol oxidase activation in arthropod host defense. J. Biol. Chem. 275: 29264–29267.10.1074/jbc.M002556200Suche in Google Scholar
Pan J.Y., Zhang Y.L., Wang S.Y. & Peng X.X. 2008. Dodecamer is required for agglutination of Litopenaeus vannamei hemocyanin with bacterial cells and red blood cells. Mar. Biotechnol. 10:645–652.10.1007/s10126-008-9115-8Suche in Google Scholar
Paul R. & Pirow R. 1998. The physiological significance of respiratory proteins in invertebrates. Zool. (Jena) 100: 298–306.Suche in Google Scholar
Pisuttharachai D., Yasuike M., Aono H., Yano Y., Murakami K., Kondo H., Aoki T. & Hirono I. 2009. Characterization of two isoforms of Japanese spiny lobster Panulirus japonicus defensin cDNA. Dev. Comp. Immunol. 33: 434–438.10.1016/j.dci.2008.11.007Suche in Google Scholar
Redfield A.C., Coolidge T. & Mary AS. 1928. The respiratory proteins of the blood. I. The copper content and the minimal molecular weight of the hemocyanin of Limulus polyphemus. J. Biol. Chem. 76: 185–195.10.1016/S0021-9258(18)84119-9Suche in Google Scholar
Rolland J.L., Abdelouahab M., Dupont J., Lefevre F., Bachère E. & Romestand B. 2010. Stylicins, a new family of antimicrobial peptides from the Pacific blue shrimp Litopenaeus stylirostris.Mol.Immunol. 47:1269–1277.10.1016/j.molimm.2009.12.007Suche in Google Scholar
Schnapp D., Kemp G.D. & Smith V.J. 1996. Purification and characterization of a proline-rich antibacterial peptide, with sequence similarity to bactenecin-7, from the haemocytes of the shore crab, Carcinus maenas.Eur. J. Biochem. 240:532–539.10.1111/j.1432-1033.1996.0532h.xSuche in Google Scholar
Sheena N., Ajith T.A., Mathew A.T. & Janardhanan K.K. 2003. Antibacterial activity of three macrofungi, Ganoderma lucidum, Navesporus floccosa and Phellinus rimosus occurring in South India. Pharm. Biol. 41:564–567.10.1080/13880200390501226Suche in Google Scholar
Smith V.J. & Chisholm J.R. 1992. Non-cellular immunity in crustaceans. Fish Shellfish Immunol. 2:1–31.10.1016/S1050-4648(06)80024-0Suche in Google Scholar
Smith V.J. & Chisholm J.R. 2001. Antimicrobial proteins in crustaceans. Adv. Exp. Med. Biol. 484:95–112.10.1007/978-1-4615-1291-2_10Suche in Google Scholar PubMed
Smith V.J., Fernandes J.M.O., Kemp G.D. & Hauton C. 2008. Crustins: enigmatic WAP domain-containing antibacterial proteins from crustaceans. Dev. Comp. Immunol. 32:758–772.10.1016/j.dci.2007.12.002Suche in Google Scholar PubMed
Sperstad S.V., Haug T., Vasskog T. & Stensvåg K. 2009. Hyastatin, a glycine-rich multi-domain antimicrobial peptide isolated from the spider crab (Hyas araneus) hemocytes. Mol. Immunol. 46:2604–2612.10.1016/j.molimm.2009.05.002Suche in Google Scholar PubMed
Stensvåg K., Haug T., Sperstad S.V., Rekdal Ø., Indrevoll B. & Styrvold O.B. 2008. Arasin 1, a proline-arginine-rich antimicrobial peptide isolated from the spider crab, Hyas araneus. Dev. Comp. Immunol. 32:275–285.10.1016/j.dci.2007.06.002Suche in Google Scholar PubMed
Takagi T. & Nemoto T. 1980. Tachypleus tridentatus hemocyanin. Separation and characterization of monomer subunits and studies on sulfhydryl groups. J. Biochem. 87:1785–1793.10.1093/oxfordjournals.jbchem.a132923Suche in Google Scholar PubMed
Tamura K., Stecher G., Peterson D., Filipski A. & Kumar S. 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30:2725–2729.10.1093/molbev/mst197Suche in Google Scholar PubMed PubMed Central
Yan F., Zhang Y., Jiang R., Zhong M., Hu Z., Du H., Lun J., Chen J. & Li Y. 2011. Identification and agglutination properties of hemocyanin from the mud crab (Scylla serrata). Fish Shellfish Immunol. 30:354–360.10.1016/j.fsi.2010.11.004Suche in Google Scholar PubMed
Yedery R.D. & Reddy K.V.R. 2009. Purification and characterization of antibacterial proteins from granular hemocytes of Indian mud crab, Scylla serrata. Acta Biochim. Pol. 56:71–82.10.18388/abp.2009_2518Suche in Google Scholar
Zahri S., Nouri-Ganbalani G. & Agahi S. 2010. Hemocyanin subunits composition as a molecular marker for identification of intra-specific variations of Gammarus. J. Cell Mol. Res.2:56–60.Suche in Google Scholar
Zhang Y., Yan F., Hu Z., Zhao X., Min S., Du Z., Zhao S., Ye X. & Li Y. 2009. Hemocyanin from shrimp Litopenaeus vannamei shows hemolytic activity. Fish Shellfish Immunol. 27:330–335.10.1016/j.fsi.2009.05.017Suche in Google Scholar PubMed
- Abbreviations
- ppm
parts per million
©2016 Institute of Molecular Biology, Slovak Academy of Sciences
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- Cellular and Molecular Biology
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- Cellular and Molecular Biology
- Application of violet pigment from Chromobacterium violaceum UTM5 in textile dyeing
- Cellular and Molecular Biology
- Chemical composition and antioxidant activity of some important betel vine landraces
- Cellular and Molecular Biology
- Development of conventional and real time PCR assay for detection and quantification of Rhizoctonia solani infecting pulse crops
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