NADPH oxidase of Epstein-Barr-virus immortalized B lymphocytes. Effect of cytochrome b(558) glycosylation.
The phagocyte NADPH oxidase is known to be expressed in Epstein-Barr virus (EBV) immortalized B lymphocytes. But even if its molecular composition and its catalytic mechanisms are similar, the activity measured in B cells is very low compared to that of neutrophils. This could be explained by the low expression of cytochrome b558, the membrane redox component, but also by a defect in the activation process. This work is focused on gp91-phox glycosylation in B lymphocytes to assess its role in the complex assembly upon activation. Atomic force microscopy (AFM) combined with immunochemical approaches were used to investigate the effect of the glycosylation on the structure of cytochrome b558 inserted into liposomes, on the reconstituted oxidase activity in vitro, and to directly monitor interaction forces between specific antibodies and the hemoprotein in its native or deglycosylated state. The results show that in EBV-B cells, gp91-phox glycosylation is higher than in neutrophils. The interaction force measured between the monoclonal antibody 11C12, known to inhibit O(-2) production in B lymphocytes, and the hemoprotein is increased after deglycosylation. This suggested that the epitope region recognized by this antibody is partly hidden in B cells, and that this region could be involved in the conformational change that occurs in the hemoprotein during the complex assembly. The high glycosylation of gp91-phox in B cells associated with the lipidic environment could lead to additional structural constraints in the membrane-bound hemoprotein that partly blocked the hemoprotein in its inactive state.
Paclet
M H
MH
GREPI, Laboratoire d'Enzymologie, CHU Albert Michallon BP 217, 38043 Grenoble cedex 9, France.
Coleman
A W
AW
Burritt
J
J
Morel
F
F
D001402:B-Lymphocytes / Q000201:enzymology*; D015153:Blotting, Western; D002461:Cell Line, Transformed; D003573:Cytochrome b Group / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D004854:Herpesvirus 4, Human / Q000502:physiology*; D006801:Humans; D018625:Microscopy, Atomic Force; D019255:NADPH Oxidases / Q000378:metabolism*
D003573:Cytochrome b Group; C036888:cytochrome b558; D019255:NADPH Oxidases