Mutation at histidine 338 of gp91(phox) depletes FAD and affects expression of cytochrome b558 of the human NADPH oxidase.
Defective NADPH oxidase components prevent superoxide (O-2) generation, causing chronic granulomatous disease (CGD). X-linked CGD patients have mutations in the gene encoding the gp91(phox) subunit of cytochrome b558 and usually lack gp91(phox) protein completely (X91(0)). gp91(phox) is considered to be a flavocytochrome that contains binding sites for NADPH, FAD, as well as heme. We here report a rare X-linked CGD patient whose neutrophils entirely failed to produce O-2, but presented a diminished expression of gp91(phox) containing about one-third of the heme present in normal individuals by Soret absorption. Translocation of cytosolic factors p67(phox) and p47(phox) was normal. However, the FAD content in his neutrophil membranes was as low as that of X91(0) patients, suggesting complete depletion of FAD in his gp91(phox). This was in agreement with the finding that a single base substitution (C1024 to T) changed His-338 to Tyr in gp91(phox) in a predicted FAD-binding domain of the flavocytochrome model. The loss of FAD could not be corrected even after addition of reagent FAD or a FAD-rich dehydrogenase fraction isolated from normal neutrophils to the patient's membranes, in a reconstitution in vitro with normal cytosol. These results indicate that His-338 is a very critical residue for FAD incorporation into the NADPH oxidase system. This is the first such mutation found in CGD.
National Children's Medical Research Center, Setagaya-ku, Tokyo, 154-8509, Japan.
L S
LS
Yoshida
F
F
Saruta
K
K
Yoshikawa
O
O
Tatsuzawa
S
S
Tsunawaki
D000595:Amino Acid Sequence; D001665:Binding Sites; D001692:Biological Transport; D002675:Child, Preschool; D003573:Cytochrome b Group / Q000096:biosynthesis; D003600:Cytosol / Q000378:metabolism; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis*; D008040:Genetic Linkage; D006105:Granulomatous Disease, Chronic / Q000235:genetics*; D006418:Heme / Q000032:analysis; D006639:Histidine / Q000235:genetics*; D006801:Humans; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics*; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D009252:NADPH Dehydrogenase / Q000032:analysis; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000096:biosynthesis; D009504:Neutrophils / Q000201:enzymology; D010750:Phosphoproteins / Q000032:analysis; D017386:Sequence Homology, Amino Acid; D012729:Sex Chromosome Aberrations / Q000235:genetics*; D013481:Superoxides / Q000378:metabolism; D014960:X Chromosome
D003573:Cytochrome b Group; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D010750:Phosphoproteins; D013481:Superoxides; D005182:Flavin-Adenine Dinucleotide; D006418:Heme; D006639:Histidine; C036888:cytochrome b558; D005287:Ferredoxin-NADP Reductase; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C086986:CYBA protein, human; D009252:NADPH Dehydrogenase