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The NADPH oxidase is specifically activated in the wall of the phagocytic vacuole in response to a number of different stimuli, for example the immature peptide secreted from bacteria at sites of infection fMLP, opsonised particles and the PKC agonist PMA. Once activated the NADPH oxidase catalyses electron transfer from the NADPH donor via a membrane associated cytochrome b to molecular oxygen, resulting in the one electron reduction of oxygen producing superoxide anion (O2’). Superoxide is

specifically discharged into the lumen of the phagocytic vacuole, where it is rapidly converted into secondary species such as hydrogen peroxide (H2O2), hydroxyl radical

(OH) and hypochlorous acid (HOCl). These highly reactive oxygen metabolites are capable of killing microorganisms and in collaboration with the contents of the granules are the principal factors of the host defence mechanism of neutrophils. Maturation of the phagocytic vacuole occurs from the fusion with intracellular granules during the process of degranulation, which accompanies the respiratory burst. The contents of the granules are released into the phagocytic vacuole and the resulting phagolysosome is highly acidic and rich in hydrolytic enzymes (Segal and Abo, 1993).

Prevention of uncontrolled release of superoxide metabolites from phagocytes may be brought about by the complex nature of this enzyme system. The neutrophil NADPH oxidase which lies dormant in resting cells is a multicomponent enzyme that consists of five essential components; four phagocytic oxidase {phox) proteins, gp9ff^^^ and p22^^^^ (which comprise the membrane associated cytochrome b), p47^*®'^ and (which reside in the cytosol of resting cells) and the Ras-related small molecular weight GTP- binding protein Rac 1/2 (also found in the cytosol of resting cells). Each of the phox proteins was identified by an absence or loss of function in different forms of chronic granulomatous disease (CGD). The involvement of Rac protein was established by investigation of NADPH oxidase activity in 'vitro.

1.5.1. Chronic granulomatous disease (CGD).

CGD which occurs in about 1 in 250 000 individuals results from a heterogeneous group of inherited molecular disorders. Biochemically CGD is characterised by the inability of phagocytic cells including neutrophils, eosinophils, monocytes, and macrophages, to produce superoxide, leaving patients highly susceptible to infections. Histologically CGD is recognised by the appearance of granulomata, which result from the fusion of

phagocytic leukocytes that have engulfed but are unable to destroy invading pathogens. In the various forms of CGD described, mutations occur in the genes encoding one or other of the phox components resulting in a reduction or complete loss of NADPH oxidase activity. The genetic defects may result in either an absence or partial loss of protein expression or production of a dysfunctional protein. Furthermore the two subunits of the cytochrome b, gp91^*°^ and p22^^''^ appear to stabilise each other. In X- linked CGD where gp91^^^^ is absent, a loss of p22^^®^ is also noted, and vice versa in autosomal recessive CGD when p22^^^^ is undetectable. The underlying genetic defects affecting the phox proteins are heterogeneous, and a wide variety of mutations have been characterised as detailed in a review by Roos et al., (1996).

I.5.I.I. X-linked CGD.

The most common form of CGD, found in approximately 60 % of cases is X-linked and is caused by mutations in the CYBB gene encoding the large subunit of the cytochrome b, . Mutations in the CYBB gene can occur from, deletions, insertions, splice site mutations, and missense or nonsense mutations, although in one patient a combination of a deletion and an insertion has been characterised. Deletions which range in size from a single base-pair to 5 000 kb, and insertions which are usually of a single base-pair, generally lead to mRNA instability and a lack of gp91^^°^ protein and result in the most severe form of CGD in which NADPH oxidase activity is completely lost. Splice site mutations in which a complete exon in the gp91^*'^^ mRNA is missing account for approximately 16 % of X-linked CGD patients. gp91^^"'' protein is undetectable in all but one of these cases, where only part of exon 12 is missing. In this case the protein is probably dysfunctional due to a 10 amino acid deletion which is located to within the 20 amino acid region to which the NADPH binding site has been mapped. Nonsense mutations usually induce a stop codon and therefore almost always lead to a total lack of gp9 1/^^ox contrast missense mutations, which affect the level of gp9ff*®^ protein and

the function of the cytochrome 6 in a variety of ways, often lead to the expression of a stable but non-functional protein.

Mutations that result in the production of non-functional gp9ff^''^ protein have been used to characterise the biochemical properties of gp9ff^^^ and the cytochrome b . Three patients with a proline to histidine mutation at position 415 were shown to exhibit reduced 2-azido-NADPH binding, which indicated that gp9ff*'^'^ was the enzymatic

subunit of the cytochrome b (Segal et al., 1992). Investigation of a solubilised mutant cytochrome b, with a mutation in gp91^^'’^ at residue 54 of arginine to serine, demonstrated a shift in optical absorbance properties, a reduction in the midpoint potential of one of the two heme groups involved and also a lack of electron transport fi’om FAD, indicating that electron transfer fi*om FAD to oxygen in this oxidase enzyme requires involvement of two heme groups in series (Cross et al., 1995). In both cases the cytosolic components p47^^‘^^ and p67^^''^ translocated to the membrane as required for formation of the active NADPH oxidase enzyme complex, although the activity o f which was markedly reduced. Thus such mutations have enabled useful investigations of NADPH oxidase function.

1.5.1.2. Autosomal recessive CGD.

Mutations in the CYBA gene encoding the small subunit of the cytochrome b , p22^^°^ account for a rare subgroup of autosomal recessive CGD (Dinauer et al., 1990). A variety of mutations account for this phenotype, including deletions, insertions, splice site mutations and missense mutations. With one exception p22^^"^ protein levels are undetectable, either due to expression of an unstable protein or to a decreased interaction with gp91^^"^. Mutation of p22^^°^ at position 156, pro line to glutamine (P156Q) which yields a stable protein probably results in a loss of superoxide production due to a lack of translocation of the cytosolic components to the membrane, as was characterised using in vitro assays (Dinauer et al., 1991; Leusen et al., 1994).

Autosomal recessive CGD is more commonly due to deficiencies in either of the cytosolic proteins p47^^‘'^ or . The majority of patients deficient of p47^^^^ are homozygous for a dinucleotide deletion that occurs at a tandem GTGT repeat. This deletion predicts premature termination of translation after the synthesis of a 50 amino acid peptide. A few patients are heterozygous for this GT deletion in combination with a point mutation. Only a few mutations in the gene encoding p67^^‘^^ have been identified, most of which despite normal amounts of mRNA result in lack of p67^^^^ protein. Of those characterised a single base-pair deletion in the 5’ splice site in intron III results in lack of exon 3, whereas a dinucleotide insertion in exon 5 predicts loss of protein due to premature termination during synthesis. A point substitution in exon 3 of G233 to A predicts replacement of residue 78 from a glycine to glutamate residue and is suggested to result in production of an unstable protein (de Boer et al., 1994). In one patient with

half the normal amount of a GAA codon deletion, predicted to result in deletion of lysine 58, was found in one allele whereas the other allele had an 11-13 kb deletion. It is likely that the in frame deletion of lysine 58 results in the expression of a non­ functional protein, and that protein is not produced from the second allele.

1.5.2. In vitro reconstitution of the NADPH oxidase.

Information regarding the function of the phagocytic oxidase components was acquired from the analysis of cells from patients with CGD, and the ability to reconstitute NADPH oxidase activity in a cell-free system. Electron transport can be reproduced in a mixture of cytosol and membranes from unstimulated cells by the addition of amphiphiles such as AA, (Bromberg et al., 1984; Cumette et al., 1985; McPhail et al., 1984) or SDS (Bromberg et al., 1985). Activity of the NADPH oxidase can be monitored by the level of superoxide production estimated by measuring cytochrome c reduction in the presence ofNADPH.

1.5.3. The neutrophil NADPH oxidase components.

The components are found in three distinct complexes in resting neutrophil cells, either in association with membranes or in the cytosol. gp91^^°^ and p2 2^^'''' are tightly associated

and make up the membrane associated heterodimeric cytochrome b. p47^^*^^ and p67^^"^ exist in the cytosol in a high molecular mass complex which contains the majority of

and some p47^*‘"^ , although the latter is also found free within the cytosol. A third cytosolic phox component p40^*""^ has also been identified and is found to exist in complex with p67^^"’'' in resting cells. This component is not essential for oxidase activity reconstituted in vitro and its function in vivo is not clear. Rac 1/2 is found in the cytosol of resting cells in complex with RhoGDI (a guanine nucleotide dissociation inhibitor). Upon activation of the respiratory burst the cytosolic phox components and Rac 1/2 translocate to the membrane, where they form an active enzyme complex with the cytochrome b .

1.5.3.1 The cytochrome b.

The heterodimeric membrane associated cytochrome b which is comprised of the two components gp9U^^'' and , present with a stoichiometry of 1:1, was identified as a

component of the respiratory burst oxidase by Allison’s group (Segal et al., 1978). It has been shown that gp9U^‘"^ (P-subunit) is an FAD-containing flavoprotein dehydrogenase

with redox function that can be reconstituted by relipidation in vitro (Segal et al., 1992; Abo et al., 1992). This 6-type flavocytochrome has a typical optical absorption

maximum of 558-559 nm but an unusually low midpoint potential at -245 mV, hence the cytochrome b is also referred as flavocytochrome bss% or 6 .2 4 5 (DeLeo and Quinn, 1996).

Redox titration studies of COS-7 expressing gp91^*''^ and p22^*'’'^ indicate that gp91^^''^ contains the binding sites for both heme groups present (Yu et al., 1998). The cytochrome 6 is also thought to contain the NADPH binding site. Indeed in one rare

form of X-linked CGD, a missence mutation occurs in a region of with weak homology to the NADPH binding site of the ferrodoxin NADP+ reductase family. In this form of CGD normal amounts of gp91^^°^ are present and translocation of the cytosolic factors to the membrane occurs upon stimulation; however superoxide is not produced (Dinauer et al., 1989; Roos et al., 1996).

Cellular staining with anti-cytochrome 6 antibodies and subcellular fractionation studies

have shown that ~ 2 0 % o f the cytochrome 6 is located within the plasma membrane with

-80 % present in the membranes of the specific granules of resting neutrophils. However in neutrophils that had phagocytosed Staphylococcus aureus, the cytochrome 6 was

found mainly in the membrane of the phagocytic vacuole (Jesaitis et al., 1990). A role for the Ras related GTPase RaplA as a chaperone for cytochrome 6 translocation upon

activation of the NADPH oxidase has been suggested from its ability to associate and colocalise with the cytochrome 6 (Quinn et al., 1989 and 1992).

is a heavily glycosylated 570 residue poly-peptide that has three transmembrane helices (Imajoh-Ohmi et al., 1992). The stability of gp91^*'''' depends upon the simultaneous expression of p22^^^^ . In CGD cells deficient o f p22^*^^ , gp91^^®^ is absent despite the presence of ample mRNA (Parkos et al., 1988).

is a transmembrane protein with extracellular and cytosolic domains. The cytosolic domain (amino acids 132-195) is well conserved between human and mouse sequences (Sumimoto et al., 1989) and contains two polyproline domains, amino acids 149-162 and 174-195 (Parkos et al., 1988; Sumimoto et al., 1989). Mutation of one of the pro line residues in the first polyproline domain (P156Q) results in a form of CGD (Dinauer et al., 1991).

1.5.3 2. The cytosolic component .

-g ^ jiigUy basic protein of 390 amino acids that represents approximately 0.5 % of neutrophil cytosolic protein (Leto et al., 1991). This protein contains two SH3 domains (amino acids 151-214 and 227-284) and a number of proline rich sequences (amino acids 70-83, 300-346 and 360-370) (Volpp et al., 1991; Leto et al., 1990), see appendix A. 6 for sequence and domain structure of p47^*®^. In addition to the proline-

rich regions the C-terminal half of p4 7^^^'' is highly basic and contains multiple

serine/threonine phosphorylation sites. Indeed extensive phosphorylation of p47^*"'^ occurs upon stimulation of the oxidase (see below).