Expression of a [formula omitted] homolog in Caco-2 cells giving O 2−-reconstituting ability to cytochrome b558 together with recombinant [formula omitted

Abstract

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.