American Journal of Hematology 37:263-266 (1991)

The Respiratory Burst Oxidase and the Molecular Basis of Chronic Granulomatous Disease Bernard M. Babior Department of Molecular and Experimental Medicine, Research Institute of Scripps Clinic, La Jolia, California

INTRODUCTION

in resting phagocytes but is activated when the cells are Chronic granulomatous disease (CGD) is an inherited stimulated. This enzyme catalyzes the reaction disorder of phagocyte function characterized by recurrent 2 O2 + NADPH +2 02- + NADPH+ + HS severe bacterial and fungal infections that usually begin in early childhood. These infections, most frequently due to Staphylococcus aureus or an enterobacillus, are very The 02-produced in this reaction is the precursor of the difficult to treat. In earlier days, CGD patients usually active microbicidal oxidants, an elaborate mixture of died during the first or second decade from uncontrolla- reactive oxidizing radicals (e.g., OH') and oxidized ble sepsis. With the introduction of antibiotic prophylaxis halogens (e.g., OC1- and NH2C1) that are produced in for CGD, the course of the disease changed, with a longer reactions that follow the O,--forming reaction [3-81. Rapid advances have recently been made in the life expectancy and fewer hospitalizations but more understanding of the respiratory burst oxidase at a complications from imperfectly suppressed infections, molecular level, largely as a consequence of the simulincluding strictures of hollow organs and slow destructaneous discovery by several groups of a method to tion of the lungs, and more fungal disease, particularly activate the enzyme in homogenates from resting phagofrom Aspergillus. Very recently, the combination of cytes [9-121. The following is a brief review of this topic, antibiotic prophylaxis and long-term y-interferon has first from a biochemical point of view, and then within been adopted as the treatment for CGD, a change that the context of CGD. promises to effect a great improvement in the outlook for patients with this disorder. The clinical aspects of CGD have recently been reviewed [l]. RESPIRATORY BURST OXIDASE The basic problem with CGD phagocytes is that they The respiratory burst oxidase has turned out to be a are unable to express the respiratory burst. This is a surprisingly complex enzyme composed of many polymetabolic event characteristic of professional phagopeptides, probably not all yet identified. In activated cytes' that results in the production of highly reactive cells, the catalytic activity of the oxidase is located in the oxidizing agents used by the phagocytes as microbicidal plasma membrane, as are all the oxidase components agents. The respiratory burst occurs whenever phagocytes are exposed to an appropriate stimulus and is recognized to date. In resting cells, however, the oxidase components are distributed between the plasma memdefined by the production of large amounts of superoxide brane and the cytosol, and activation is accomplished in (02J and H202, accompanied by sharp increases in part by the transfer of the cytosolic components to the oxygen uptake (supplying the oxygen from which 02plasma membrane. The physical separation of the oxiand H202 are made) and hexose monophosphate shunt dase components in resting phagocytes is undoubtedly a activity (the cell's source of NADPH). In the clinical safety measure to prevent the generation of dangerous laboratory, the respiratory burst is usually detected by the microbicidal oxidants under inappropriate circumstances. NBT test, a test in which neutrophils are activated in the The individual components currently recognized are as presence of nitroblue tetrazolium, a water-soluble yellow follows: dye. Under these conditions, normal neutrophils generate 02-,which converts NBT to a dark blue insoluble 1. Cytochrome b558.This heme-containing species, material, but CGD cells do not generate 02- and first recognized as an oxidase component by Segal and therefore leave the dye unchanged. The key to the respiratory burst is the respiratory burst Received for publication August 2, 1990; accepted February 14, 1991, oxidase, a membrane-associated enzyme that is dormant 'B lymphocytes also express a respiratory burst, but it is much smaller than the respiratory burst of phagocytes [2].

0 1991 Wiley-Liss, Inc.

Address reprint requests to Dr. Bernard M. Babior, Department of Molecular and Experimental Medicine, Research Institute of Scripps Clinic, La Jolla, CA 92037.

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Concise Review: Babior

odonium, a potent inactivator of the respiratory burst oxidase.

associates [13], is intrinsic to the membrane. It is composed of two kinds of subunits, a 91K lycoprotein (gp91ph0x)2and a 22K polypeptide ( ~ 2 Ox), 2 ~ which associate tightly together and copurify on chromatography [ 14,151. Also copurifying with cytochrome b558 is a guanine nucleotide-binding protein whose relationship to the oxidase is uncertain [16]. Both subunits of the cytochrome have been cloned and sequenced [ 17,181. It is generally held that cytochrome b558 is the terminal component of the oxidase, transferring single electrons to oxygen to form 0,- [ 13,191. 02-production can occur, however, in the absence of this cytochrome [20,21]. It is likely, although not certain, that 02-production under these circumstances results from electron transfer via a nonphysiological route. 2. P47ph". This component is a highly basic protein (PI > 10) found in the cytosol in resting phagocytes but transferred to the plasma membrane when the cells are activated [22]. The message for this protein has recently been cloned and sequenced [23,24]. In the resting cell, p47phoxis unphosphorylated, but activation results in the uptake of additional phosphates to achieve a total of 7-8 phosphates/molecule in the fully phosphorylated polypeptide, all of which are attached to serine residues [25-281. It is likely that the phosphorylation of this protein has something to do with its participation in oxidase activation. 3. P67ph". The component ~ 6 7 ~ is ~ also ' " found in the cytosol of resting phagocytes and like p47phox, it is transferred to the plasma membrane when the cells are activated [22]. Its message too has been cloned and sequenced [29]. By sequence comparison, p67phoxis not related to any other protein in the database, but it contains regions that resemble portions of the cytoskeletal protein fodrin. It may be that connnections between the oxidase and the phagotype cytoskeleton, already demonstrated by ourselves [30] and b Jesaitis' group [31], are mediated in part through p67P Ox. 4. Other components. The respiratory burst oxidase is likely to contain at least two other components: the NADPH-binding component and a9avoprotein [32,33]. The former, like p47phoxand p67phox,is found in the cytosol of resting cells but is transferred to the membrane upon activation [34,35]. It has not yet been identified, but there is evidence to suggest that it is neither p47phox[36] nor ~ 6 7 ~ ~[35]. " " In addition, although the NADPHbinding component might be expected to contain a flavin, there appears to be a separate flavoprotein component that resides in the membrane [33,37]. This component may have been identified by Cross et al. [38] as a 48K protein that is labeled by radioactive diphenylenei-

1. X-linked CGD. The gene that encodes gp91PhoX, the large subunit of cytochrome bss8, resides on the Xchromosome [17,44,45]. X-linked CGD is caused by a mutation affecting that gene [ 17,461, Since X-linked genetic diseases result from mutations in a single allele while autosomal recessive diseases require mutations in both alleles, it is not surprising that most cases of CGD are of the X-linked variety [47]. In almost all cases of X-linked CGD, cytochrome b,,, is missing from the phagocytes, as demonstrated both spectrophotometrically [47] and by immunoblotting, which shows that both subunits of the cytochrome are absent [15,48]. These cases are generally referred to as cytochrome-negative CGD. Messenger RNA is usually missing as well, but the responsible genetic abnormality has not been identified.

'The oxidase polypeptides are designated by terminology that specifies their molecular weight ( M J and their nature as components of the phagocyte oxidase. Thus, gp9 Iphox, the M , 91K glycoprotein component of the respiratory burst oxidase.

'G proteins are guanine nucleotide-binding regulatory proteins that receive signals from membrane receptors and communicate them to effector systems in the cell.

5

x

There is no reason to believe that the above list represents a complete catalog of respiratory burst oxidase components. The role played by guanosine triphosphate (GTP) in the activation of the respiratory burst oxidase [39] strongly suggests the involvement of a G protein3 in oxidase activity. The ability of certain fatty acids and other lipid anions to activate the oxidase in a cell-free system [9-12,401 suggests that activation in whole cells may involve the release of an anionic lipid, possibly through the action of a phospholipase. In this connection, a case has recently been made for the involvement of a phospholipase D in oxidase activation in whole cells [41]. The reversibility of oxidase activation [42,43] implies the existence of enzymatic machinery to accomplish the deactivation process. Thus, it seems likely that several additional oxidase components remain to be discovered. CHRONIC GRANULOMATOUS DISEASE AND THE RESPIRATORY BURST OXIDASE

CGD has been found to result from lesions affecting each of the four known oxidase proteins (i.e., p47pho", p67phox,and p22phoXand gp9lPhoX,the two subunits of cytochrome b558).Indeed, it was through their absence in one or another type of CGD that these proteins were identified as components of the respiratory burst oxidase. Clinically the various types of CGD resemble each other closely, but they differ in their mode of inheritance and in whether cytochrome bS5, is present in the affected phagocytes. The features of the various types of CGD are described below, and summarized in Table 1.

Concise Review: Respiratory Burst Oxidase and CGD TABLE 1. Classification of CGD

Affected component

Mode of inheritance

Frequency (%)

Cytochrome b558

gp91PhoX p22phox p47phox p67phox

X-linked Autosomal recessive Autosomal recessive Autosomal recessive

56 6 33 5

Usually absent Usually absent Present Present

In a few patients, X-linked CGD is associated with a functionally defective cytochrome that is present in normal amounts (so-called X-linked cytochrome-positive CGD). In two brothers with this form of CGD, the disease was shown to result from a pro -+ his substitution at position 451 of the gp91PhoXmolecule [49]. 2. Autosomal recessive cytochrome-negative CGD. Autosomal recessive cytochrome-negative CGD appears to result from a mutation affecting the autosomal gene encoding p22ph0x,the small subunit of cytochrome b558 [50].This form of CGD is phenotypically identical to the X-linked cytochrome-negative disease: phagocytes show no cytochrome spectrum [51], and p22phoXand gp91PhoX are both absent from the cells [15]. It accounts for only a small minority of CGD cases. 3. Autosomal recessive cytoc~rome-posit~veCGD . Most cases of this type of CGD are due to a deficiency in p47phoX,the phosphoprotein component of the respiratory burst oxidase. Deficiency of this protein was initially suspected from studies of protein phosphorylation in neutrophils from affected patients [27,52,53] and was later confirmed by immunoblots performed with an antibody that recognized several proteins in neutrophil cytosol, among them p47phoXand ~ 6 7 ~ [54]. ~ ” ” The immunoblots also revealed a few cases that resulted from a deficiency of ~ 6 7 ~ ~ ” ” . All CGD cases studied to date fall into one of the foregoing categories. It may be anticipated, however, that from time to time a patient will appear with a form of CGD that does not fall into one of those categories. Study of such a patient could furnish important new insights into the operation of the respiratory burst oxidase. ACKNOWLEDGMENT

This work was supported in part by grants AI-24227 and AI-28479 from the U.S. Public Health Service. REFERENCES I . Forrest CB, Forehand JR, Axtell RA, Roberts RL, Johnston RB Jr: Clinical features and current management of chronic granulomatous

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