Low NADPH oxidase activity in Epstein-Barr-virus-immortalized B-lymphocytes is due to a post-transcriptional block in expression of cytochrome b558.
The NADPH oxidase of phagocytes is known to be expressed in Epstein-Barr-virus-transformed B-lymphocytes, albeit at levels only approx. 5% of those found in neutrophils. We have investigated the basis of this low level of expression and find that all four specific components of the NADPH oxidase are expressed in B-lymphocytes, but only p47-phox protein attains levels equivalent with those found in neutrophils. This component was shown to phosphorylate and translocate to the membrane normally on activation. The other cytosolic component, p67-phox, did show a deficit, and by supplementing a B-cell cytosol extract with recombinant p67-phox, this was shown to account for the somewhat reduced activity of B-cell cytosol in a cell-free oxidase system. The cell-free analysis also clearly located the major deficiency in superoxide-generating capacity of B-lymphocytes to the membrane. Western blotting of membrane proteins revealed major reductions in the amount of cytochrome b558. Analysis of the levels of mRNA for both subunits of cytochrome b558, however, showed levels greater than expected. Significantly more mRNA for gp91-phox was present in B-cells than in undifferentiated HL60 cells, although it was not quite as abundant as in differentiated HL60 cells, which are capable of producing large amounts of superoxide. We conclude that the failure of B-lymphocytes to generate amounts of superoxide equivalent to those generated by neutrophils is primarily due to a post-transcriptionally determined block to the accumulation of cytochrome b558.
Chetty
M
M
Department of Medicine, Rayne Institute, University College London, U.K.
Thrasher
A J
AJ
Abo
A
A
Casimir
C M
CM
D001402:B-Lymphocytes / Q000201:enzymology* / Q000821:virology; D015153:Blotting, Western; D002461:Cell Line, Transformed; D003573:Cytochrome b Group / Q000235:genetics*; D004854:Herpesvirus 4, Human; D006801:Humans; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009252:NADPH Dehydrogenase / Q000235:genetics / Q000378:metabolism; D019255:NADPH Oxidases; D009504:Neutrophils / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D012333:RNA, Messenger / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D003573:Cytochrome b Group; D010750:Phosphoproteins; D012333:RNA, Messenger; C079789:neutrophil cytosol factor 67K; D013481:Superoxides; C036888:cytochrome b558; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D009252:NADPH Dehydrogenase