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J. Biol. Chem., Vol. 281, Issue 1, 313-323, January 6, 2006

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Characterization of a Defensin from the Oyster Crassostrea gigas

RECOMBINANT PRODUCTION, FOLDING, SOLUTION STRUCTURE, ANTIMICROBIAL ACTIVITIES, AND GENE EXPRESSION^*

Yannick Gueguen12, Amaury Herpin¶13, André Aumelas||, Julien Garnier, Julie Fievet, Jean-Michel Escoubas4, Philippe Bulet**, Marcelo Gonzalez, Christophe Lelong3, Pascal Favrel3, and Evelyne Bachère

From the Ifremer, CNRS, Université de Montpellier II, UMR 5171, Génome Populations Interactions Adaptation, 2 Place E. Bataillon, CC80, F-34095 Montpellier cedex 5, France, the Laboratoire de Biologie et Biotechnologies Marines, Institut de Biologie Fondamentale et Appliquée, UMR 100 Ifremer, Université de Caen, Physiologie et Ecophysiologie des Mollusques Marins, 14032 Caen cedex, France, the ^||Centre de Biochimie Structurale, CNRS UMR 5048, INSERM U554, CNRS UMR5048, Université Montpellier I, 29 rue de Navacelles, F-34090 Montpellier Cedex, France, ^**Atheris Laboratories, Case Postale 314, CH-1233 Bernex-Geneva, Switzerland, and the ^¶SARS International Centre for Marine Molecular Biology, High Technology Centre, 5008 Bergen, Norway

In invertebrates, defensins were found in arthropods and in the mussels. Here, we report for the first time the identification and characterization of a defensin (Cg-Def) from an oyster. Cg-def mRNA was isolated from Crassostrea gigas mantle using an expressed sequence tag approach. To gain insight into potential roles of Cg-Def in oyster immunity, we produced the recombinant peptide in Escherichia coli, characterized its antimicrobial activities, determined its solution structure by NMR spectroscopy, and quantified its gene expression in vivo following bacterial challenge of oysters. Recombinant Cg-Def was active in vitro against Gram-positive bacteria but showed no or limited activities against Gram-negative bacteria and fungi. The activity of Cg-Def was retained in vitro at a salt concentration similar to that of seawater. The Cg-Def structure shares the so-called cystine-stabilized - motif (CS-) with arthropod defensins but is characterized by the presence of an additional disulfide bond, as previously observed in the mussel defensin (MGD-1). Nevertheless, despite a similar global fold, the Cg-Def and MGD-1 structures mainly differ by the size of their loops and by the presence of two aspartic residues in Cg-Def. Distribution of Cg-def mRNA in various oyster tissues revealed that Cg-def is mainly expressed in mantle edge where it was detected by mass spectrometry analyses. Furthermore, we observed that the Cg-def messenger concentration was unchanged after bacterial challenge. Our results suggest that Cg-def gene is continuously expressed in the mantle and would play a key role in oyster by providing a first line of defense against pathogen colonization.

Received for publication, October 5, 2005 , and in revised form, October 20, 2005.

^* This study was part of a collaborative project supported by the European Commission, DG XII, in the program International Co-operation with Developing Countries, INCO-DC, Contract ICA4-CT-2001-10023 (IMMUNAQUA). Work in the authors' laboratories was supported in part by the Ifremer, the CNRS, the INSERM, and the University of Montpellier II and Caen. This study was also part of a French program MOREST funded by Ifremer, the Regions Basse-Normandie, Bretagne, Pays de la Loire and Poitou-Charentes and by the Conseil Général du Calvados. Atheris Laboratories was supported by the Swiss Federal Office for Education and Science contract 02.0360. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AJ565499 [GenBank] and CAJ19280 [GenBank] .

The atomic coordinates and structure factors (code 2B68) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

The on-line version of this article (available at http://www.jbc.org) contains Table S1.

¹ Both authors contributed equally to this work.

³ Financially supported by the Conseil Régional de Basse-Normandie, Agence de l'Eau Seine Normandie and FEDER 4474 (program PROMESSE).

⁴ Present address: Ecologie microbienne des insectes et interactions hôte-pathogène, UMR INRA Université de Montpellier II, 2 Place E. Bataillon, CC80, F-34095 Montpellier cedex 5, France.

² To whom correspondence should be addressed: Tel.: 33-46-714-4707; Fax: 33-46-714-4622; E-mail: ygueguen{at}ifremer.fr.

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