Expression of a p67(phox) homolog in Caco-2 cells giving O(2)(-)-reconstituting ability to cytochrome b(558) together with recombinant p47(phox).
Human normal and transformed (Caco-2) colon tissues as well as guinea pig gastric mucosal cells express Nox1, which is a homolog of the phagocyte NADPH oxidase subunit, gp91(phox) of membrane-bound cytochrome b(558). It was reported that Nox1-transfection to NIH 3T3 cells could provide O(2)(-)-generating ability, independently of regulatory cytosolic factors (Rac2, p67(phox), and p47(phox)) that are obligatory in the phagocyte oxidase system. Here, we detected and sequenced a p67(phox) homolog in Caco-2 almost identical to the neutrophil sequence, except for three nucleotide substitutions, two of which changed lysines 181 and 328 to arginines. Investigation of its ability to support O(2)(-)-generation in cell-free reconstitution experiments combining with neutrophil cytochrome b(558) showed O(2)(-)-generation, provided that recombinant p47(phox) was added. This result demonstrates that the intrinsic p67(phox) homolog of Caco-2 was able to function as a phagocyte p67(phox) for cytochrome b(558). The requirement of p47(phox) addition suggested that this component was absent in Caco-2 cells. Caco-2 membranes, used as a source of Nox1 in place of cytochrome b(558), did not show significant O(2)(-)-generation, which was mainly explained by their very little Nox1 expression.
Yoshida
L
L
Department of Infectious Diseases, National Research Institute for Child Health and Development, 3-35-31, Taishido, Setagaya, 154-8567, Tokyo, Japan.
Nishida
S
S
Shimoyama
T
T
Kawahara
T
T
Rokutan
K
K
Tsunawaki
S
S
D018938:Caco-2 Cells; D002474:Cell-Free System; D003573:Cytochrome b Group / Q000378:metabolism*; D006801:Humans; D026901:Membrane Transport Proteins; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D009252:NADPH Dehydrogenase / Q000378:metabolism; D000074624:NADPH Oxidase 1; D019255:NADPH Oxidases; D010750:Phosphoproteins / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins; D017386:Sequence Homology, Amino Acid; D013481:Superoxides / Q000378:metabolism*
D003573:Cytochrome b Group; D026901:Membrane Transport Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; C079789:neutrophil cytosol factor 67K; D013481:Superoxides; C036888:cytochrome b558; D009247:NADH, NADPH Oxidoreductases; D000074624:NADPH Oxidase 1; D019255:NADPH Oxidases; C086986:CYBA protein, human; C060505:neutrophil cytosolic factor 1; D009252:NADPH Dehydrogenase