Cloning and expression of the gene encoding the porcine NADPH oxidase light-chain subunit (p22-phox).
In previous studies, we showed that interleukin-4 (IL-4) suppressed porcine (p) macrophage superoxide production and that the mechanism of suppression involved down-regulation of the superoxide-generating enzyme NADPH oxidase heavy-chain 91-kDa subunit mRNA (gp91-phox) expression. In order to examine the effect of IL-4 on expression of the gene encoding the porcine NADPH oxidase light-chain 22-kDa subunit (p22-phox), we cloned the p22-phox cDNA from a macrophage library. The p22-phox cDNA is 786 bp in length and contains a 576-bp open reading frame which predicts a primary translation product of 192 amino acids (aa). Comparison of the porcine and human 22-phox cDNAs showed a high degree of similarity between the two species in their nucleotide (85%) and deduced aa (83%) sequences. as well as in their hydropathy profiles. Notable features, including a high proline content and an iron-coordinating His94, are conserved in both the porcine and human 22-Phox. A single species of mRNA of about 1 kb was detected in macrophages. The mRNA levels remained unchanged in cells treated with lipopolysaccharide (LPS) or with IL-4 at various concentrations from 0-50 ng/ml. Prolonged treatment with LPS or IL-4 did not enhance the effect of these substances on p22-phox mRNA expression. The effect of IL-4 on p22-phox mRNA expression was also compared with another immunosuppressive cytokine, transforming growth factor-beta 1 (TGF beta 1). No change in mRNA expression was found in the cells with or without TGF beta 1 treatment. The results indicated that the heavy and light chains of NADPH oxidase are independently regulated by IL-4 in macrophages.
Department of Veterinary PathoBiology, College of Veterinary Medicine, University of Minnesota, St. Paul 55108.
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Zhou
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Murtaugh
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D015971:Gene Expression Regulation, Enzymologic; D015847:Interleukin-4 / Q000494:pharmacology; D007700:Kinetics; D008264:Macrophages / Q000378:metabolism; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D009252:NADPH Dehydrogenase / Q000235:genetics*; D019255:NADPH Oxidases; D010750:Phosphoproteins / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013552:Swine; D016212:Transforming Growth Factor beta / Q000494:pharmacology
D018076:DNA, Complementary; D026901:Membrane Transport Proteins; D010750:Phosphoproteins; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D015847:Interleukin-4; D019255:NADPH Oxidases; C086986:CYBA protein, human; D009252:NADPH Dehydrogenase