Cloning of murine gp91phox cDNA and functional expression in a human X- linked chronic granulomatous disease cell line

Abstract

The phagocyte cytochrome b558, a heterodimer comprised of gp91phox and p22phox, is a flavocytochrome that mediates the transfer of electrons from NADPH to molecular oxygen in the respiratory burst oxidase. The human gene encoding the glycosylated gp91phox subunit is the site of mutations in X-linked chronic granulomatous disease (CGD). Reverse transcriptase-polymerase chain reaction was used to obtain a full- length clone for the murine gp91phox cDNA, which was 87% identical to the human gp91phox cDNA. The encoded murine protein had 39 amino acids out of 570 that differed from the human, many of which were conservative substitutions. Nonconservative replacements occurred in hydrophilic regions outside of domains previously implicated in binding to NADPH, flavin, and the cytosolic oxidase subunit p47phox. Some substitutions altered potential N-glycosylation sites, which is likely to explain why the glycosylated murine protein migrates with an apparent molecular mass of 58 kD instead of 91 kD as seen for the human protein. Expression of murine gp91phox in a human myeloid cell line with a null gp91phox allele using a mammalian expression plasmid or a retroviral vector rescued stable expression of the p22phox subunit and fully reconstituted respiratory burst activity. This suggests that the murine gp91phox subunit forms a functional cytochrome b558 heterodimer with human oxidase subunits, consistent with the high degree of identity between the mouse and human proteins in domains implicated in cytochrome function.

Granulocyte Colony-Stimulating Factor Prior to Nonmyeloablative Irradiation Decreases Murine Host Hematopoietic Stem Cell Function and Increases Engraftment of Donor Marrow Cells

“…There was variability in the fraction of oxidase‐positive neutrophils detected over time in individual recipients, similar to our previous studies using this vector [16, 18, 24]. A declining fraction of oxidase‐positive neutrophils was also seen after 6 months in some mice.…”

“…Transduction of BM cells by coculture with a high titer MSCV‐m91 Neo GP + E86 ecotropic packaging line was performed as previously described [23, 24]. Briefly, BM was harvested from 6–12‐week‐old male X‐CGD mice 3 days after intraperitoneal injection with 150 mg/kg 5‐fluoruracil (Adrucil; Pharmacia, Kalamazoo, MI, http://www.gelifesciences.com), cultured for 2 days in αMEM containing 30% fetal calf serum, 100 U/ml recombinant human interleukin‐6 and 100 ng/ml recombinant murine stem cell factor (Peprotech, Rocky Hill, NJ, http://www.peprotech.com), and then cultured for 2 additional days on mitomycin‐C (Bedford Laboratories, Bedford, OH, http://www.bedfordlabs.com) ‐treated packaging cells in the same media plus 8 μg/ml Polybrene (Sigma‐Aldrich, St. Louis, http://www.sigmaaldrich.com).…”

Upregulation of p67phoxand gp91phoxin aortas from angiotensin II-infused mice

“…In the rat aorta, further studies have corroborated our finding that the adventitia is a major site of vascular O 2 Ϫ production and NAD(P)H oxidase component expression under basal conditions (34) and have demonstrated the involvement of adventitial O 2…”

“…Western blots of aortic homogenates from vehicle-infused mice treated with monoclonal antibody against human gp91 phox exhibited a cross-reactive band of ϳ77 kDa (Fig. 3A), a value less than that reported for human gp91 phox but higher than that expressed from the mouse phagocyte gp91 phox clone (2); these discrepancies are likely related to varying degrees of glycosylation (2). The intensity of this band was increased in homogenates from ANG II-infused mice, whereas with ANG II plus losartan-treated mice, the intensity was not different from that in sham treatment (Fig.…”

Prednisolone augments superoxide formation in porcine pulmonary artery endothelial cells through differential effects on the expression of nitric oxide synthase and NADPH oxidase

“…Both mouse and human deglycosylated gp91 phox are 54 kDa. This difference is due to less glycosylation sites in the mouse sequence (Bjorgvinsdottir et al ., 1996). In initial Western blots, both human and porcine neutrophil lysates ran as a smear starting from 90 to 50 kDa.…”