Jeanette H. W. Leusen^1,2, Arthur J. Verhoeven¹ and Dirk Roos^1,3

¹ Central Laboratory of the Netherlands Red Cross Blood Transfusion Service and Laboratory for Experimental and Clinical Immunology, University of Amsterdam, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands.

²Department of Pediatrics, Emma's Children Hospital, Academic Medical Center, Amsterdam, The Netherlands.

Received 04/15/96; Accepted 05/05/96; On-line 07/01/96

Four oxidase components have been identified through studies with CGD cells (14-20): two membrane-bound components, gp91-phox and p22-phox, which together comprise cytochrome b₅₅₈, and two cytosolic components called p47-phox and p67-phox. Upon cell activation, the latter two proteins, together with a third cytosolic p40-phox component, translocate to cytochrome b₅₅₈ in the plasma membrane to form the active enzyme (21, 22). Also, two low-molecular-weight GTP-binding proteins are involved in the activation of the NADPH oxidase, viz. Rap 1A and rac. The components of the NADPH oxidase and the assembly of this enzyme in the plasma membrane of a phagocyte are depicted in Fig. 1.

Figure 1. Assembly of the NADPH oxidase in phagocytic cells. In resting phagocytes, the cytochrome b₅₅₈ subunits p22-phox and gp91-phox, in association with Rap1A, are located in the membranes of specific granules and of secretory vesicles. Upon cell activation, these organelles fuse with the plasma membrane, which results in expression of cytochrome b₅₅₈ in this membrane. At the same time, a complex of p47-phox, p67-phox and p40-phox translocates from the cytosol to the plasma membrane and forms a complex with cytochrome b₅₅₈. This translocation is facilitated by simultaneous redistribution of rac to the plasma membrane.

4.1. Cytochrome b₅₅₈

The membrane-bound gp91-phox/p22-phox heterodimer is designated cytochrome b₅₅₈ because of its optical spectrum with an a absorbance peak at 558 nm (23-25). This protein is also known as cytochrome b_-245 for its unusual low midpoint potential at -245 mV (26). Both a flavin (27-29) and two heme redox centers (30) are contained within the cytochrome heterodimer. Electrons supplied by NADPH are transferred to oxygen as follows (25,31)

In resting neutrophils, this flavocytochrome is localized in the membranes of specific granules and of secretory vesicles (26, 32-34). During cell activation, fusion of these organelles with the plasma membrane leads to re-allocation of the oxidase (34, 35).

The cDNA's of both subunits of cytochrome b₅₅₈ have been cloned and sequenced, and the genes encoding these proteins have been localized and characterized (15, 36-38). The alpha subunit p22-phox contains 195 amino acids, with hydrophobic helices in the Nterminal half of the protein that could serve as membrane-spanning domains (38). The CYBA gene encoding p22-phox is located on chromosome 16 (15).

The beta subunit gp91-phox of cytochrome b₅₅₈ contains 570 amino acids, with four or five transmembrane helices and five potential N-linked glycosylation sites in the amino-terminal region (37, 39, 40). The topology of gp91-phox has been determined with anti-peptide antibodies and partial proteolysis (40). This study showed that residues 150-172 are exposed on the outside, and an 18-kD C-terminal fragment is cytosolic. Furthermore, there is evidence that residue 240 is glycosylated, thus extracellular (A.W. Segal, unpublished results). The CYBB gene for gp91-phox is located on the X chromosome (37, 39). Mutations in this gene account for all cases of X-linked CGD (X91 CGD), which is the most common form of the disease (about 70% of all patients). In most cases of X-linked CGD, gp91-phox cannot be detected on immunoblot; this is referred to as X91⁰ CGD. In some cases, residual gp91-phox protein and oxidase activity are present (X91^- CGD) (41), and in a few exceptional cases the (non-functional) protein is present in normal amounts (X91⁺ CGD) (42-44). In p22-phox-affected CGD patients, the protein is typically absent (15, 38, 45), except in one patient with A22⁺ CGD (46). The mutation in the CYBA gene of this last patient is predicted to lead to a proline-toglutamine substitution at residue 156 in p22-phox. This residue resides in a proline-rich region that could be an important counter structure for src homology region 3 (SH3) domains (47-49) (see Fig. 2). The functional defect in this patient has been studied in ref. (50). Both subunits of cytochrome b₅₅₈ are usually missing in A22⁰ CGD as well as in X91⁰ CGD (24, 38, 51, 52). This indicates that these subunits stabilize each other (53). It is not known, however, which regions in p22-phox and gp91-phox are involved in this mutual stabilization.

4.2. Rap1A

A 22-kD low-molecular-weight GTP-binding protein co-purifies with cytochrome b₅₅₈ and remains associated with the cytochrome even after immunoaffinity purification on matrices composed of antibodies to cytochrome b₅₅₈ (54, 55). This protein was identified as Rap1A by immunostaining. Rap1A has been shown to bind specifically to cytochrome b₅₅₈, with a one-to-one stoichiometry. In neutrophils, Rap1A becomes phosphorylated by the cAMP-dependent kinase PKA (56). A serine residue (¹⁸⁰Ser) at the COOH-terminal region of Rap1A has been identified as a site of phosphorylation by PKA (56), and phosphorylation of Rap1A abrogates the interaction with cytochrome b₅₅₈ (57). It is possible that (phosphorylation of) Rap1A regulates the deactivation of the NADPH oxidase.

MGDTFIRHIALLGFEKRFVPSQHYVYMFLVKWQDLSEKWYRRFTEIYEFHKTLKE
MFPIEAGAINPENRIIPHLPAPKWFDGQRAAENRQGTLTEYCSTLMSLPTKISRCP
HLLDFFKVRPDDLKLPTDNQTKKPETYLMPKDGKSTATDITGPIILQTYRAIADYEKT
SGSEMALSTGWVVEVVEKSESGWWFCQMKAKRGWIPASFLEPLDSPDETEDPE
PNYAGEPYVAIKAYTAVEGDEVSLLEGEAVEVIHKLLDGWWVIRKDDVTGYFPSMYL
                  303/304             320     328
QKSGQDVSQAQRQIKRGAPRRSSIRNAHSIHQRSRKRLSQDAYRRNSVRRFLQQR
        345 348                   370      379
RRQARPGPQSPGSPLEEERQTQRSKPQPAVPPRPSADLILNRCSESTKRKLASAV

Figure 2. Putative phosphorylation sites in p47-phox. The 390 amino-acid sequence of p47-phox is given. In the last quarter of the protein, six putative PKC phosphorylation sites are localised (marked in red) and two putative MAP kinase sites (marked in blue). The consensus sequence for a PKC site is R-X-S-X-(R-R), with R for arginine or another positively charged amino acid (i.e. lysine) and X for a non-charged amino acid. The last two positively charged amino acids are not essential for a PKC site. The consensus sequence for a MAP kinase site is P-X-S/T-P or even X-P, with P for proline, S/T for serine or threonine and X for any amino acid. Whether these sites actually become phosphorylated is discussed in the text. The importance of the C-terminal sequence (marked in purple) is to be discussed in the text.

4.3 p47-phox

The majority of patients with CGD due to a deficiency of cytosolic NADPH oxidase components lacks detectable levels of p47-phox by immunoblotting; this defectaccounts for about 30% of all patients with CGD (58). Segel et al. have shown that a 47-kD phosphoprotein is absent in the neutrophils from certain patients with autosomal recessive inheritance of CGD who have normal levels of cytochrome b₅₅₈ (59). Subcellular fractionation techniques have shown that this 47-kD phosphoprotein (now recognized as p47-phox) is present in the cytosol and in the plasma membrane, but not in granule fractions of PMA-stimulated neutrophils (60, 61). An explanation for this dual location was provided by Heyworth et al., who showed that phosphorylation of p47-phox initially occurs in the cytosol, before transloction of p47-phox to the membrane, and continues after membrane association (62). Two-dimensional electrophoresis revealed that the two most acidic isoforms are not found in stimulated neutrophils from CGD patients lacking cytochrome b₅₅₈ (63, 64). Normal cells, as well as cells that contain normal levels of dysfunctional cytochrome b₅₅₈ (bearing a ⁴¹⁵Pro--->His substitution in gp91-phox) contain all phosphorylated isoforms (64). Thus, the final phosphorylations of p47-phox occur only when an intact cytochrome b₅₅₈ is present and are likely to take place after translocation of p47-phox to the membrane.

The cDNA for p47-phox has been cloned, and the gene has been localized and characterized (18, 19). The NCF1 gene encoding p47-phox is located on chromosome 7 (65); mutations in this gene cause the A47 type of CGD. Recombinant p47-phox as well as purified cytosol fractions containing p47-phox are able to restore oxidase-supporting activity in cytosol from A47 CGD neutrophils (17-19, 66-68). The deduced amino-acid sequence (390 residues) of p47-phox contains at least six potential serine phosphorylation sites for protein kinase C (PKC), in good agreement with the apparent number of phosphorylated isoforms seen on two-dimensional electrophoresis. However, the actual kinase responsible for p47-phox phosphorylation in intact cells is not yet known. A schematic representation of the phos-phorylation sites in p47-phox is shown in Fig. 2.

Furthermore, the amino-acid sequence of p47-phox contains two SH3 motifs and at least one proline-rich region (Fig. 3). These domains probably play a role in the interaction between the cytosolic components and in the assembly of the NADPH oxidase (see below).

4.4. p67-phox

A minority of patients with cytosol-deficient CGD are lacking p67-phox, as determined by immunoblotting; this defect accounts for less than 5% of all cases of CGD (58). A full-length cDNA has been obtained and sequenced (20); p67-phox is encoded by the NCF2 gene that is located on chromosome 1 (65). The predicted 526-amino-acid sequence of p67-phox also contains two SH3 domains an at least one proline-rich region, as depicted in Fig. 3. Translocation experiments have revealed that p67-phox is unable to translocate to the plasma membrane in cells of p47-phox-deficient patients (69). In contrast, p47-phox appeared to translocate normally in cells of a p67-phox-deficient donor (69-71). However, we observed otherwise (almost no translocation of p47-phox in p67-phox deficient patients, J. Leusen et al., manuscript submitted).

4.5. p40-phox

A third cytolic phox protein was shown to reside in a complex with p67-phox in the cytosol of resting neutrophils (72, 73). The cDNA of this 40-kD protein has also been cloned, and the predicted protein of 339 amino acids contains one SH3 domain (73). Although p40-phox is not required for activity in the cell-free assay (see Section 5.1 'Cell-free system'), it is thought to play a role in stabilizing p67-phox in intact cells.

4.6. p21-rac

The last cytosolic protein required for the activity of the NADPH oxidase is a rasrelated protein called rac (74-78). This protein family has a wide tissue distribution; in neutrophils p21-rac2 is the most abundant rac protein, but p21-rac1 is also present (79). Probably, rac functions by changing from an inactive, GDP-bound state to an active, GTP-bound state in which it can mediate the activation of the NADPH oxidase. Fine-tuning of this process may be mediated by the regulation of GTP/GDP exchange of rac by GDP-dissociation inhibitor (GDI) protein and GDP-dissociation stimulator (GDS) protein (75-78, 80).

Figure 3. SH3 domains and proline-rich regions in the components of the NADPH oxidase. The membrane-bound p22-phox, the light subunit of cytochrome b₅₅₈, contains one proline-rich region, a putative counter structure for one of the SH3 domains in the cytosolic proteins. P47-phox and p67-phox each contain two SH3 domains and one proline-rich region, p40-phox contains only one SH3 domain.

CGD patients with decreased NADPH oxidase activity due to mutations in rac or GTP/GDP exchange-regulating proteins are not known, probably due to the fact that these proteins are involved in several other (essential) cellular functions, such as vesicular transport and cytoskeleton dynamics. Mutations in such proteins may be incompatible with life.