| Title | Built for the kill. Studies on the neutrophil NADPH oxidase |
|---|---|
| Period | 01 / 2000 - 04 / 2004 |
| Status | Completed |
| Dissertation | Yes |
| Research number | OND1270753 |
| Phagocytic cells (granulocytes, eosinophils, monocytes and macrophages) kill microorganisms, mainly bacteria and fungi, by ingestion (phagocytosis) and production of superoxide. The physiological importance of this mechanism is demonstrated by the clinical syndrome chronic granulomatous disease (CGD), a severe congenital immunodeficiency syndrome characterized by absent or reduced phagocytic superoxide production. The molecular basis of this disease is a dysfunction of the NADPH oxidase, the phagocyte-specific enzyme responsible for the generation of superoxide. The active oxidase is composed of several subunits, four of which, gp91-phox, p22-phox, p47-phox and p67-phox, are essential for its function. A mutation in any of these four subunits will lead to CGD. Gp91-phox and p22-phox form together the membrane-integrated cytochrome b558, while p47-phox and p67-phox are cytosolic. The activation of the enzyme is a complex cascade-like sequence of events, resulting in translocation of p47-phox and p67-phox to the membrane and formation of the active enzyme; the activated oxidase then transfers electrons from intracellular NADPH onto extracellular oxygen, thereby generating superoxide. The glycoprotein gp91-phox is the central component of the complex, because it contains all the the parts of the electron transport chain, namely the binding site for NADPH (together with p67-phox), FAD, two hemes and a binding site for O2, as well as several sites of interaction with the cytosolic oxidase components. CYBB, its gene, is isolated on chromosome X and mutations of this locus result therefore in the X-linked form of CGD. Although a three-dimensional model of the cytosolic part of gp91-phox has been developed, based on partial homology with a plant enzyme, little factual evidence exists to corroborate this hypothetical construct. Aim of this project is therefore to characterize more precisely the different functional domains of gp91-phox, such as, for example, the binding sites of NADPH and FAD. For this purpose, specific mutations are to be introduced in CYBB, expressed in myeloid cells and analyzed as to their functional consequences. We have first recreated by site-directed mutagenesis four mutations previously found in CGD patients. These mutations lead to the rare X91+ phenotype, in which normal amounts of a dysfunctional protein are expressed. We have first expressed these mutated proteins in the erythroleukemic K562 cells. These cells constitutively express p22-phox; hence, co-expression of gp91-phox may be expected to lead to stable expression of the whole cytochrome b558 molecule. We found, however, that introduction of wild-type gp91-phox leads to efficient transduction (95% of the cells) but with expression levels too low to permit analysis of FAD or NADPH. Moreover, because K562 cells lack the p47-phox and p67-phox components of the NADPH oxidase, the oxidase activity can only be tested in a cell-free assay with K562 membranes and neutrophil cytosol. This is a rather artificial system that lacks a large number of regulatory interactions operative in intact cells. In this cell-free system with K562 membranes expressing wild-type cytochrome b558, we observed about 40% of the superoxide generation found in a similar system with neutrophil membranes (compared on the basis of the cytochrome b558 content). To overcome these problems, we have transduced the K562 cells with p47-phox and p67-phox, to create an intact-cell system for testing the gp91-phox mutants. We have also studied PLB985 cells, a myeloid cell line that can be induced to differentiation and expression of NADPH oxidase components, with an NADPH oxidase activity of about 60% of that found in neutrophils. In a mutant of these cells, obtained from Dr. M. Dinauer (Indianapolis, IN, USA), the CYBB gene has been disrupted, rendering a cell line fit for testing gp91-phox mutants at a high expression level in a whole-cell context. We are now expressing the originally created mutants, as well as several new ones with mutations in the putative FAD-binding regions, in the p47-p67-transfected K562 cells and in the Delta-gp91-PLB-985 cells. We are also setting up a very sensitive FAD assay, based on chemiluminescence emitted in a luciferase reaction. In future, we will try to create new gp91-phox mutants at random, select these on the basis of gp91-phox protein expression without NADPH oxidase activity, and test their effects on FAD and NADPH binding. |
| Robin van Bruggen onderzocht verschillende aspecten van de NADPH-oxidase van neutrofielen. NADPH-oxidase is een zeer belangrijk enzym voor het doden van micro-organismen. Dit enzym zet zuurstof om in superoxide, een reactieve vorm van zuurstof. De superoxide reageert met opgenomen micro-organismen, wat leidt tot vernietiging van deze organismen. Van Bruggen gaat dieper in op de manier waarop de NADPH-oxidase geactiveerd wordt en hoe de pathogene salmonellabacterie de activering van dit enzym tegengaat. |
| Secretariat | Sanquin Research |
|---|
| Supervisor | Prof.dr. D. Roos |
|---|---|
| Researcher | M. de Boer |
| Project leader | Dr. C. Meischl |
| Doctoral/PhD student | Dr. R. van Bruggen |
| A70000 | Public health and health care |
|---|---|
| C10000 | Biotechnology |
| D21300 | Biochemistry |
| D21400 | Genetics |
| D23220 | Internal medicine |
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