Biochemical and genetic defects in CGD

In 1978, Segal, Jones and colleagues published a landmark paper describing a novel cytochrome b within phagosomes of neutrophils that was not detect- 

Extensive studies into the association of this cytochrome b with CGD neutrophils were performed by Segal and Jones, and by other workers, in the late 1970s and early 1980s. The cytochrome was completely absent (as determined by the absence of a distinctive absorption spectrum in spectroscopic studies) in almost all cases of X-linked CGD, but present at decreased levels in female relatives of these patients. In almost all cases of autosomal recessive CGD, the cytochrome was present but non-functional, in that it did not become reduced upon cellular activation. This indicated both the heterogeneous nature of the disease and also that some other biochemical defect was responsible for impaired function in these patients. Hence, the search was on for other components of the NADPH oxidase. 

it has been shown that, in the majority of X-linked CGD patients, the abnormality is due to the failure to transcribe the mRNA encoding the large (/J) subunit of the b cytochrome. In these patients, both the heavy /)-chain and the light a-chain are absent, even though the molecular defect appears to be restricted to the heavy chain thus, expression of the heavy chain is somehow necessary for the expression/translation/stabilisation of the 

The second major breakthrough in understanding the defect in CGD neutrophils came through the development of assays in which the NADPH oxidase can be activated in a cell-free system in vitro ( 5.3.2.3). In these systems, activation of the oxidase can be achieved by the addition of cytoplasm to plasma membranes in the presence of NADPH and arachidonic acid (or SDS or related substances). Interestingly, the oxidase cannot be activated in these cell-free systems using extracts from CGD neutrophils however, cytosol and plasma membranes from normal and CGD neutrophils may be mixed, and in most cases activity is restored if the correct mixing pattern is used. For example, as may be predicted, in X-linked CGD it is the membranes that are defective (because the cytochrome b is deficient), whereas in autosomal recessive CGD the cytosol is defective in the cell-free system. 

Nunoi and co-workers (1988) fractionated neutrophil cytoplasm by Mono Q anion-exchange chromatography and obtained three fractions (NCF-1, -2 and -3) that were active in the assembly of the oxidase. Independently, Volpp and colleagues (Volpp, Nauseef Clark, 1988) prepared antiserum from cytosolic factors that eluted from a GTP-affinity column, and this antiserum (Bl) recognised cytoplasmic factors of relative molecular masses 47 kDa and 66 kDa. It was later shown by this group that these cytosolic factors translocated to the plasma membrane during activation. NCF-1 was shown to contain the 47-kDa protein and NCF-2 the 66-kDa protein. Analysis of the defect in the cytosol of autosomal recessive CGD patients revealed that most of these (88%) lacked the 47-kDa protein (p41 -phox), whereas the remainder lacked the 66-kDa protein (p66-phox). Both of these components have now been cloned and recombinant proteins expressed. Interestingly, in the cell-free system, recombinant p47-phox and p66-phox can restore oxidase activity of the cytosol from autosomal recessive CGD patients who lack these components. 

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