Molecular cloning and sequencing of Japanese pufferfish (Takifugu rubripes) NADPH oxidase cDNAs

Abstract The superoxide-producing NADPH oxidase complex of phagocytes plays a crucial role in host defenses against microbial infection. NADPH oxidase consists of a membrane heterodimeric protein, composed of gp91 phox and p22 phox , and cytosolic proteins, p40 phox , p47 phox and p67 phox . In the present study, cDNAs of all the components of NADPH oxidase were cloned from peripheral white blood cells of the Japanese pufferfish utilizing the reverse transcription-polymerase chain reaction. The sequences of these cDNAs showed that the pufferfish gp91 phox , p22 phox , p40 phox , p47 phox and p67 phox clones contained open reading frames encoding 565, 186, 348, 423 and 495 amino acids, respectively. Comparison of the deduced amino acid sequences showed that the pufferfish gp91 phox , p22 phox , p40 phox , p47 phox and p67 phox sequences shared 68.0, 61.8, 53.8, 54.7 and 41.9% identity with those of human components, respectively. gp91 phox has three potential N-linked glycosylation sites. gp91 phox and p22 phox have six and three hydrophobic regions, respectively, that are predicted to be transmembrane regions. p47 phox and p67 phox have two potential Src homology 3 domains and p40 phox has one. The functional domains are highly conserved in many animals, though the sequence of the components of the pufferfish showed low homology with that of mammals. The Fugu NADPH oxidase genes were expressed in various tissues of unstimulated fish. The level of gp91 phox , p47 phox and p67 phox expression were high only in the blood and kidney, while p22 phox and p40 phox were constitutively expressed in a wide range of tissues. These results suggest that Japanese pufferfish NADPH oxidase components possess functional activities similar to those of human.