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The turbot (Scophthalmus maximus) Myeloperoxidase: characterization and functional studies

AuthorsFontenla, F.; Noia, M.; Piazzon de Haro, María Carla ; Valle, A.; Leiro, José-Manuel; Lamas, Jesús
Immune system
Issue Date16-Jun-2019
Citation3rd Conference on Fish and Shellfish Immunology (2019)
AbstractMyeloperoxidase (MPO) is a major enzyme that is mainly present in fish neutrophils. This enzyme is well characterised in mammals but little is known about its structure and function in fish. In this study, we sequenced the turbot MPO and studied some of its functions in turbot. The 5690 bp turbot myeloperoxidase gene contains an ORF with 14 exons. In addition to the 13 introns of the ORF, there is one intron of 134 nucleotides located in the 5´UTR region. The untranslated 5’ and 3´ regions have 111 bp and 970 bp respectively. The coding sequence contains 2301 nucleotides that encode a polypeptide of 767 aa with a predicted molecular mass of 86.21 kDa. BLASTp analysis revealed that turbot MPO displays high similarity to the MPO of other fish species (identity varied between 60 and 82%) and lower than those of mammals (identity 50%) and reptiles (identity 47%). Turbot MPO was found to have several conserved domains such as the signal peptide, propeptide (118 aa) and light (113 aa) and heavy chains (591 aa). Other important sites for regulation of MPO activity are also present in the turbot molecule, including distal haem cavities I and II and proximal haem cavities I and II. Several catalytic, haem linkage and cysteine residues, a Ca+2-binding motif and also eight potential N-linked glycosylation sites were identified. Western blot analysis and use of an anti-turbot MPO polyclonal antibody revealed that turbot MPO exists in its mature form as a homodimer of about 150 kDa in the anterior kidney, spleen, peritoneal fluid and serum, indicating that the protein loses the propeptide during maturation. The MPO transcripts were most strongly expressed in the anterior kidney, gill, white blood cells and spleen, and they were most weakly expressed in liver, muscle, heart and brain. Immunofluorescence was used to identify cells compatible with neutrophils containing MPO + granules in the anterior kidney, spleen, blood, gill and intestine. In an in vitro stimulation test, anterior kidney leukocytes (HKL) were isolated on a Percoll gradient and stimulated with purified MPO obtained by affinity chromatography. Incubation of HKL with turbot MPO generates positive regulation of the proinflammatory cytokines TNF-α and IL12-β, the cytokine IFNγ, the chemokine IL8 and the subunits of the CD11b / CD18 integrins, suggesting that, in addition to its microbicidal activity, the MPO may act as a mediator of the immune response in fish.
DescriptionTrabajo presentado en 3rd International Conference on Fish and Shellfish Immunology (ISFSI), celebrada en Gran Canaria (España), del 16 al 20 de junio de 2019
Appears in Collections:(IATS) Comunicaciones congresos
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