[HTML][HTML] Two CGD families with a hypomorphic mutation in the activation domain of p67phox

D Roos, JD van Buul, ATJ Tool, JD Matute… - Journal of clinical & …, 2014 - ncbi.nlm.nih.gov
D Roos, JD van Buul, ATJ Tool, JD Matute, CM Marchal, BH Hayee, MY Köker, M de Boer
Journal of clinical & cellular immunology, 2014ncbi.nlm.nih.gov
Methods Leukocyte NADPH oxidase activity, expression of oxidase components and gene
sequences were measured with standard methods. The mutation found in the patients'
NCF2 gene was expressed as Ala202Val-p67 phox in K562 cells to measure its effect on
NADPH oxidase activity. Translocation of the mutated p67 phox from the cytosol of the
patients' neutrophils to the plasma membrane was measured by confocal microscopy and by
Western blotting after membrane purification. Results The exceptional feature of the A67 …
Methods
Leukocyte NADPH oxidase activity, expression of oxidase components and gene sequences were measured with standard methods. The mutation found in the patients’ NCF2 gene was expressed as Ala202Val-p67 phox in K562 cells to measure its effect on NADPH oxidase activity. Translocation of the mutated p67 phox from the cytosol of the patients’ neutrophils to the plasma membrane was measured by confocal microscopy and by Western blotting after membrane purification.
Results
The exceptional feature of the A67 CGD patients reported here is that the p. Ala202Val mutation in the activation domain of p67 phox was clearly hypomorphic: substantial expression of p67 phox protein was noted and the NADPH oxidase activity in the neutrophils of the patients was 20–70% of normal, dependent on the stimulus used to activate the cells. The extent of Ala202Val-p67 phox translocation to the plasma membrane during cell activation was also stimulus dependent. Ala202Val-p67 phox in K562 cells mediated only about 3% of normal oxidase activity compared to cells transfected with the wild-type p67 phox.
Conclusion
The mutation found in NCF2 is the cause of the decreased NADPH oxidase activity and the (mild) clinical problems of the patients. We propose that the p. Ala202Val mutation has changed the conformation of the activation domain of p67 phox, resulting in reduced activation of gp91 phox.
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