Identification and characterization of myeloperoxidase in orange-spotted grouper (Epinephelus coioides)

Abstract

Cryptocaryon irritans is an important protozoan ciliate, which has led to heavy economic losses in marine aquaculture. Previous studies have indicated that C. irritans infection could induce the migration of neutrophils to infection sites. Myeloperoxidase (MPO) mainly exists in the cytoplasmic granules of the neutrophil and performs its function by a unique enzymatic capacity to produce hypohalous acid and other toxic oxidants. To determine the involvement of MPO and neutrophils against C. irritans infection in the host, we amplified MPO cDNA (EcMPO) from orange-spotted grouper (Epinephelus coioides). The open reading frame (ORF) of EcMPO encodes a putative polypeptide of 770 amino acids and has typical structural characteristics of mammalian MPO, including a signal peptide, a propeptide, a light chain, a heavy chain, and a peroxidase domain. Bioinformatics analysis has demonstrated that the most important functional sites in mammalian MPO were also conserved in grouper and other piscine MPO, implying the functional conservation of this protein during evolution. A rabbit anti-MPO recombinant protein polyclonal antibody was produced, which could recognize the native MPO protein. The expression of EcMPO was higher in the lympho-hematopoietic organs, such as head kidney, trunk kidney, spleen, but lower in muscle, heart, and brain. After infection with C. irritans, the EcMPO transcript was significantly up-regulated at specific time points in the infection sites (skin and gill) and systemic immune organs (head kidney and spleen); The number of EcMPO positive cells first increased and then decreased in the gill, but was still higher than the control after 7 days. These results demonstrated that EcMPO and its positive cells may be involved in anti-C. irritans infection in the grouper, which is attributed to the innate immune mechanisms of the host against parasite infection.