Lipopolysaccharide primes the respiratory burst of Atlantic salmon SHK-1 cells through protein kinase C-mediated phosphorylation of p47phox.
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 gp91phox and p22phox, and the cytosolic proteins, p40phox, p47phox and p67phox. In the present study, we clone and sequence the full-length cDNAs coding for the Atlantic salmon (Salmo salar) phagocyte NADPH oxidase components, p47phox, p67phox and gp91phox, using a homology cloning approach. The sequences of these cDNAs showed that the S. salar p47phox, p67phox and gp91phox genes contained single open reading frames, which encoded predicted proteins of 413, 504 and 565 amino acids, respectively. Comparison of the deduced amino acid sequences showed that the S. salar p47phox, p67phox and gp91phox sequences shared 51, 45 and 68% identity with those of human components, respectively. Despite this relatively low homology between salmon and mammalian NADPH oxidase subunits, their functional domains are highly conserved. We also found that the mRNA levels of p47phox, p67phox and gp91phox expression were higher in immune-related tissues, such as kidney, spleen and gill. In addition, infection of the salmon macrophage cell line SHK-1 with Piscirickettsia salmonis induced the expression of p47phox, but had no effect on p67phox and gp91phox expression. Finally, we show for the first time in fish that activation of macrophages with lipopolysaccharide promotes the activation of protein kinase C, which in turn phosphorylates p47phox, leading to NADPH oxidase activation and reactive oxygen species generation. Collectively, these results suggest that the mechanisms of activation of phagocyte NADPH oxidase are well conserved from fish to mammals.
Olavarría
Víctor H
VH
Department of Biochemistry, Faculty of Science, University Austral, Campus Isla Teja, Valdivia, Chile.
Gallardo
Lorena
L
Figueroa
Jaime E
JE
Mulero
Victoriano
V
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D004789:Enzyme Activation; D020869:Gene Expression Profiling; D007113:Immunity, Innate; D008070:Lipopolysaccharides / Q000276:immunology*; D008262:Macrophage Activation; D008264:Macrophages / Q000276:immunology* / Q000378:metabolism; D008969:Molecular Sequence Data; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D016366:Open Reading Frames; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D017382:Reactive Oxygen Species; D016897:Respiratory Burst; D020317:Salmo salar / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013481:Superoxides / Q000378:metabolism
D008070:Lipopolysaccharides; D010750:Phosphoproteins; D017382:Reactive Oxygen Species; C079789:neutrophil cytosol factor 67K; D013481:Superoxides; D019255:NADPH Oxidases; D011493:Protein Kinase C