title: Molecular characterization, gene structure and antibacterial activity of a g-type lysozyme from the European sea bass (Dicentrarchus labrax L.).
abstract: In fish, the first line of defense is represented by the innate immune system and the lysozyme is one of the molecules involved in this mechanism of protection. Three types of lysozymes have been identified in metazoan, the c-type (chicken or conventional), the g-type (goose-type) and the i-type (invertebrate type). They are all involved in the hydrolysation of the bacterial cell wall. Our work has been focused on the molecular characterization, expression analysis by real-time PCR, both at basal condition and after in vivo challenges, and 3D structural studies on the g-type lysozyme from sea bass (Dicentrarchus labrax L.). Moreover, a recombinant sea bass lysozyme has been produced in Escherichia coli and used to investigate the activity of the enzyme at different pH and temperatures and to perform antibacterial assays against typical fish pathogens. The cloned sea bass cDNA for g-type lysozyme (accession number FN667957) consists of 742 bp and translates for a putative protein of 188 amino acids. The molecular weight is 20.251, 41Da with a theoretical pI of 8.53, two cysteine residues along the sequence and no putative signal peptide. These features of the enzyme are in agreement with the expected characteristics of a proper g-type lysozyme, except for the cysteine residues that in fish are quite variable in number. An alignment between known g-type lysozyme sequences evidences that the amino acid residues thought to be involved in the enzyme catalysis (Glu(71), Asp(84) and Asp(95) in sea bass) are quite well conserved between mammalian, avian and fish sequences. The sea bass g-type lysozyme gene is composed of four exons and three introns and this gene structure is more compact compared to other known fish lysozyme homologues. Modeling of 3D structure has been performed on the template structure of g-type lysozyme from Atlantic cod. The catalytic site appears well conserved when compared with known structures of fish g-type lysozymes (cod and salmon). The basal expression of lysozyme transcripts is highest in gills, followed by head kidney and peripheral blood leukocytes. The lysozyme expression is up regulated in head kidney leukocytes both after challenge with the fish bacterial pathogen Photobacterium damselae subsp. piscicida. The lytic activity, determined using as substrate Micrococcus lysodeikticus, was optimal at pH 5.5 and at a temperature of 30°C. In conclusion, these results suggest that the identified g-type lysozyme should be involved in the innate immune responses of sea bass.
authors.affiliation: Department for Innovation in Biological, Agro-food and Forest systems, University of Tuscia, Viterbo, Italy. Electronic address: fbuono@unitus.it.
authors.forename: Francesco
authors.initials: F
authors.lastname: Buonocore
authors.affiliation: Department for Innovation in Biological, Agro-food and Forest systems, University of Tuscia, Viterbo, Italy.
authors.forename: Elisa
authors.initials: E
authors.lastname: Randelli
authors.affiliation: Department for Innovation in Biological, Agro-food and Forest systems, University of Tuscia, Viterbo, Italy.
authors.forename: Pamela
authors.initials: P
authors.lastname: Trisolino
authors.affiliation: Institute of Food Sciences, CNR, Laboratory of Bioinformatics and Computational Biology, CNR, Avellino, Italy.
authors.forename: Angelo
authors.initials: A
authors.lastname: Facchiano
authors.affiliation: Institute of Protein Biochemistry, CNR, Napoli, Italy.
authors.forename: Donatella
authors.initials: D
authors.lastname: de Pascale
authors.affiliation: Department for Innovation in Biological, Agro-food and Forest systems, University of Tuscia, Viterbo, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Scapigliati
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001492:Bass / Q000235:genetics / Q000276:immunology* / Q000382:microbiology*; D003001:Cloning, Molecular; D029941:Fish Proteins / Q000737:chemistry / Q000145:classification / Q000235:genetics* / Q000276:immunology*; D015971:Gene Expression Regulation, Enzymologic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry / Q000145:classification / Q000235:genetics* / Q000276:immunology*; D010802:Phylogeny; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
chemical_list: D000900:Anti-Bacterial Agents; D029941:Fish Proteins; D011994:Recombinant Proteins; D009113:Muramidase
keywords: 3D structure; Antibacterial activity; Expression analysis; Lysozyme; Sea bass