Deletion mutagenesis of p22phox subunit of flavocytochrome b558: identification of regions critical for gp91phox maturation and NADPH oxidase activity.
The heterodimeric flavocytochrome b558, comprised of the two integral membrane proteins p22phox and gp91phox, mediates the transfer of electrons from NADPH to molecular oxygen in the phagocyte NADPH oxidase to generate the superoxide precursor of microbicidal oxidants. This study uses deletion mutagenesis to identify regions of p22phox required for maturation of gp91phox and for NADPH oxidase activity. N-terminal, C-terminal, or internal deletions of human p22phox were generated and expressed in Chinese hamster ovary cells with transgenes for gp91phox and two other NADPH oxidase subunits, p47phox, and p67phox. The results demonstrate that p22phox-dependent maturation of gp91phox carbohydrate, cell surface expression of gp91phox, and the enzymatic function of flavocytochrome b558 are closely correlated. Whereas the 5 N-terminal and 25 C-terminal amino acids are dispensable for these functions, the N-terminal 11 amino acids of p22phox are required, as is a hydrophilic region between amino acids 65 and 90. Upon deletion of 54 residues at the C terminus of p22phox (amino acids 142-195), maturation and cell surface expression of gp91phox was still preserved, although NADPH oxidase activity was absent, as expected, due to removal of a proline-rich domain between amino acids 151-160 that is required for recruitment of p47phox. Antibody binding studies indicate that the extreme N terminus of p22phox is inaccessible in the absence of cell permeabilization, supporting a model in which both the N- and C-terminal domains of p22phox extend into the cytoplasm, anchored by two membrane-embedded regions.
Zhu
Yanmin
Y
Herman B. Wells Center for Pediatric Research, Department of Pediatrics (Hematology/Oncology), Microbiology/Immunology, and Medical and Molecular Genetics, James Whitcomb Riley Hospital for Children, Indianapolis, Indiana 46202, USA.
Marchal
Christophe C
CC
Casbon
Amy-Jo
AJ
Stull
Natalie
N
von Löhneysen
Katharina
K
Knaus
Ulla G
UG
Jesaitis
Algirdas J
AJ
McCormick
Sally
S
Nauseef
William M
WM
Dinauer
Mary C
MC
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D003573:Cytochrome b Group / Q000235:genetics*; D017353:Gene Deletion; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D003573:Cytochrome b Group; D008562:Membrane Glycoproteins; C036888:cytochrome b558; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C086986:CYBA protein, human