Delineation of the phagocyte NADPH oxidase through studies of chronic granulomatous diseases of childhood

Delineation

of the phagocyte NADPH oxidase through

studies of chronic granulomatous

diseases of childhood

John I. Callin, Thomas 1. Leto, Daniel Rotrosen, Cheung H. Kwong and Harry 1. Malech National Institute

of Allergy and Infectious

Diseases, Bethesda, Maryland,

USA

The phagocyte NADPH oxidase is a complex system consisting of membrane and cytosolic components that must assemble at the membrane for proper activation. Studies of patients with chronic granulomatous diseases of childhood have enabled the molecular characterization of these components, which has led to studies defining their interaction during NADPH complex assembly. Understanding NADPH oxidase assembly provides an opportunity to develop therapeutics for the regulation of this important reaction of inflammation.

in Immunology

Current Opinion

Introduction When phagocytes are appropriately stimulated by opsonized microorganisms, antigen-antibody complexes, chemotactic factors or other stimuli, an NADPH oxidasedependent respiratory burst is activated resulting in the univalent reduction of molecular oxygen to superoxide anion. This superoxide anion is then converted to hydrogen peroxide by superoxide dismutase [I]. In the presence of iron salts, superoxide anion and hydrogen peroxide may interact further to produce hydroxyl radicals. These toxic oxygen products, particularly hydrogen peroxide, provide the phagocytic cell with a potent mechanism for killing micro-organisms. Recent studies have provided important insights into the components of the NADPH oxidase system required for phagocyte activation. Activation requires cellular components from both the cytosol and the cell membrane (Fig. 1). Following activation, all of the components necessary for superoxide production reside in the phagocyte plasma membrane or phagosome membrane and consist of an electron transport chain driven by a NADPH oxidase. The complete enzyme system appears to involve a klavoprotein and a unique cytochrome b558 [ 2 I. This cytochrome has been purilied and when analyzed by sodium dodecyl sulfate polyacrylamide electrophoresis (SDS PAGE), was found to consist of a highly glycolsylated large 91 kD subunit (gpslpb”“, glycopotein 91 kD phagocyte o&&e) and a small 22kD protein subunit (p2p). Studies of a cell-free system containing plasma membrane and purified proteins have demonstrated that two cytosolic components of 47 kD and 67 kD (p67*) [3,4] form a complex with cytochrome b55s

1991,

4153-56

and interact with a third cytosolic protein, a small GTPbinding protein p21 raCl [5-l. The GDP-dissociation inhibitor rhoGD1 may also be involved in the interaction of p21ra’ with the oxidase complex. Much of what is known about the NADPH oxidase system has come from studies of patients deficient in the system, who have chronic granulomatous diseases of childhood (CGDS).

Cytosol proteins

Membrane protein Cytochrome bSS8

~67~”

IO

Activation

rat-1

II

Large

subunit

Phagosome

Oxygen Assembly

of proteins Electrons a Superoxide anion

Electrons NADPH

Fig.1. Schematic diagram of components (See text for details.)

of the NADPH

oxidase.

Abbreviations AR-autosomal SDS-PACE--sodium

recessive; dodecyl

CGD-chronic sulfate

granulomatous

polyacrylamide

disease of childhood;

gel electrophoresis;

@ Current Biology Ltd ISSN0952-7915

X-x-linked.

53

54

Innate immunity

Chronic granulomatousdiseasesof childhood CGDs are a genetically heterogeneous group of disorders with a common phenotypic defect characterized by failure of phagocytic cells to activate the respiratory burst and produce microbicidal products [&I. CGDs are associated with a serious derangement in the ability to defend against bacterial and fungal infections. There is also excessive granuloma formation, indicating that these oxygen products may be important in negative feedback mechanisms required for normal termination of the iriIammatory response. CGDs occur with a frequency of one out of 1 OOOOOOZ two-thirds of cases are inherited as an X-linked disorder (X-CGD) and a third of the cases are inherited as an autosomal recessive disorder (AR-CGD). In addition to pedigree, the classification of CGDs has been relined by identification of specific abnormalities of the phagocytes associated with different forms of CGDs [7] as indicated in table 1 Table 1. Genetic forms of chronic granulomatous diseases of childhood.*

Defect Abnormal membrane

Abnormal

Chromosomal

Patients

protein

localization

Wo)

gp92@ox*

xp21.1

-60

p22@oxt

16~24

<5

p47Phoxt

7q11.23

-30

p67P”oxt

lq25

-5

cytochrome bs%

Cytosolic abnormalities

‘Indicates X-linked inheritance; tautosomal recessive inheritance.

Cytochromeb,, Cytochrome b558 is an iron-containing heme protein with an electron potential of - 245 mV that is capable of reducing molecular oxygen, and is found in phagocytic cell membranes [2]. Analysis of its reduced minus oxidized difference spectrum reveals several light absorption peaks, including a large peak at 424nm, and a smaller peak at 558 nm. Several years ago, it was found that most patients with X-CGD lack spectral peaks characteristic of cytochrome b,, [8]. This observation suggested that X-CGD might result from a defect in the production of cytochrome bsss. The X-CGD gene, mapped to chromosome band Xp21.1 [PI, was cloned on the basis of its location and tissue-specific expression and was shown to encode the larger subunit of the cytochrome bT5s complex [ l(t14], The cDNA for gp9lphox predicts a protein of 65 kD, which is close to the 58 kD core peptide seen after cleavage of asparagine-linked oligosaccharides from the 91 kD large subunit of cytochrome b,, by endoglycosidase F. The sequence of gp9lpbo3cis not similar to any known cytochrome, and has five hydrophobic regions,

which could be transmernbrane sequences, and live possible sites for N-linked giycosyiation. All patients with X-CGD, those with the common cytochrome b-negative form and an unusual family with cytochrome b present, who have been studied at the molecular level, were found to have a mutation at the X-CGD gene locus (KJ Lomax, C Burch-Whitman, HL Tiffany, JI Gallin, HL Malech, abstract, Proc Nat1 Acud Sci U S A 1988, 85:3314-3323). The p2W subunit gene has also been cloned and found to be expressed in many cell types [ 15 I. Mutations in this gene have been reported to be associated with the rare autosomal recessive cytochrome b-negative form of CGD [16]. In both forms of cytochrome b-negative CGD, X-linked as well as autosomal, both subunits are absent, suggesting that stable expression of each subunit requires expression of the other subunit gene [ 21. Like gp91*, the sequence of p22phaz does not appear to be similar to any known cytochrome. However, a 31. amino-acid region, centered on a potential heme-binding histidine, has a 39% identity with polypeptide I (the heme-binding subunit) of mitochondrial cytochrome c ox&se. Further analysis of the protein sequence has revealed p22pbaz to have other general structural motifs in common with heme-binding proteins. However, it has not been possible to separate cytochrome b5% peptide subunits with the heme group still boun4. Therefore, it is not clear how, or even if, the heme prosthetic group(s) interact with both subunits. An important role of the carboxyl terminus of gp9lpha2 has been demonstrated. Studies using rabbit antibodies against a synthetic peptide corresponding to the carboxyl terminus showed that this subunit lies within the cytoplasmic domain of gp9lphM [17**]. Studies using synthetic peptides to this region of gp91pbox indicated that the active region encompasses a seven amino acid sequence (RGVHFIF; in the one letter amino acid code) near the carboxyl terminus. This domain appears to mediate interaction of cytochrome b55s with other cellular proteins essential for activation of the phagocyte respiz+ toty burst.

Cytosolicfactors Neutrophil cytosol contains several components required for the cell-free activation of superoxide generation, which can be separated by ion exchange chromatography [3,4]. Studies of CGD patients indent&d two of the important cytosolic components. A few individuals with cytochrome b5gs-positive CGD had a neutrophil cytosol defect that was complementary to that seen in all other cytochrome b55s-positive AR-CGD patients examined. Moreover, the defect in the former patients’ neutrophil cytosols could be corrected by a recombinant 67kD factor (p67~) produced by either Escherichi coli or baculovirus-infected cells [18**, 19.1. The neutrophil cytosol defect in the other AR-CGD patients could be corrected with a 47kD recombinant protein factor also produced in either E. coli or baculovirus expression systems [ 19*,20**,21**]. Studies of nearly 100

Delineation of the phagocyte NADPH oxidase Callin, Leto, Rotrosen,

patients with CGD indicated that the most common form of cytochrome b558positive CGD results from a defect in p47pb” whereas a rare form of CGD occurs as a result of deficient p67ph” 171. The chromosomal position of these two cytosolic proteins have been localized to chromosomes 7 and 1 (Table 1) [ 22**].

The cDNA for p67ph” encodes a 526aminoacid

protein [18**], whereas the cDNA for p47pb” encodes a 390amino-acid protein [20**,21**]. p47ph” is a basic protein with an arginine- and serine-rich carboxyl terminus that has a cluster of potential phosphorylation sites. Upon activation of neutrophils, eight distinct p47* phosphoproteins are detected [ 23.1. Phosphorylation of p47Ph is associated with its translocation to the carboxyl terminus of gp91pbaz probably to the RGVHFIF region in the carboxyl terminus of gp9lphoxmentioned above. A temporal correlation between specific p47* phosphorylation events and p47ph” translocation to the membrane was demonstrated 124.1 and suggested a model of ox&se activation in which a series of p47* phosphotylation events occuting in the cytosol precede, and may be required for, p47* interaction with the membrane 124.1. The data suggest p47Wx, but not p67*, binds to gp9lpb” early in activation. In the absence of p67*, a metastable activation intermediate is formed that upon addition of p67pb” and trace amounts of cytosol, results in an active NADPH oxidase. A sequence motif common to both p47* and p67mx is also present in the noncatalytic domain of src-related protein kinases, phopholipase C, non-erythroid 01spectrin, RASp21 GTPase-activating protein (GAP), and actin-binding proteins (myosin I) of dictyostelium acan thameoba, and yeast [18**]. The fuctional role of the conserved sequence motif in these diverse proteins is not clear but may involve interaction with common GTP binding proteins or components of the cytoskeleton. Deletion of the src motifs from recombinant proteins did not result in loss of functional activity in a cell-free ox&se assay system (TL I&o, abstract, J Cell Bioll990, 111:49). Future studies will address the role of these sequence motifs in whole cell systems. The requirement of trace amounts of normal cytosol in the presence of p47* and p67Pbx for NADPH oxidase activation suggested that additional factors were needed for activation [3,lY]. In the guinea pig macrophage this cytosolic component purifies as a heterodimer consisting of 24 kD and 26 kD subunits that were identihed by a combination of microsequencing and immunochemical techniques as a ras-related low molecular weight GTPbinding protein, racl, and the guanine nucleotide exchange inhibitor, rhoGD1, respectively [5**]. Reconstitution studies using recombinant human p47* and p67* indicate that the human counterpart is a complex of 26 kD racl and rhoGD1 that could be resolved only by isoelectric focusing in the presence of urea followed by SDS-PAGE (CH Kwong, HL Malech, TL Leto, abstract, Clin Res 1991, 273). ml is a member of the rho family of ras-related GTP binding proteins that appears to be involved in regula tion of cytoskeletal architecture. Studies using recombi-

Kwong,

Malech

nant racl complexed with either GTP or GDP suggest that racl is the component that mediates the previously observed guanine nucleotide dependence of the cell-free NADPH oxidase sysem [ 5**]. The mechanism by which racl and rhoGD1 contribute to activation of the oxidase is not clear. rhoGD1 forms complexes with the GDPbound but not the GTP-bound forms of rho proteins and appears to modulate translocation of rho proteins from cytosol to membranes [25]. Whether rhoGD1 interacts with racl in an analgous manner has not been studied. Current efforts to define more precisely the mechanisms by which racl participates in assembly of the NADPH oxidase may shed light on the more general roles served by rasrelated proteins in other cell systems.

Conclusion Studiesof phagocytes obtained from patients with CGDs have helped define the protein components of the NADPH oxidase system that are essential for the univalent reduction of oxygen to superoxide anion, which is then converted to hydrogen peroxide. The availability of recombinant membrane and cytosolic components should enable structure-function studies to define the interaction of the components. Future studies that delineate active sites and analyze the structure of the NADPH oxidase components should provide an understanding of how the molecules assemble at the plasma membrane. It may then be possible to design therapeutics that modulate the assembly of NADPH oxidase and thereby regulate this important reaction of the inflammatory response.

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JI Gak, TL Leto, D Rotrosen, CH Kwong, HL Makch, Iaboratoty of Host Defenses, National Institute of AIIergy and Infectious Diseases, Bethesda, Maryland 20892, USA