Glucocerebroside inhibits NADPH oxidase activation in cell-free system

Biochimica et Biophysica Acta 1688 (2004) 197 – 203 www.bba-direct.com

Glucocerebroside inhibits NADPH oxidase activation in cell-free system Patryk Moskwa a, Anita Palicz b, Marie-He´le`ne Paclet c, Marie-Claire Dagher d, Melinda ErdoVVs b, La´szlo´ Maro´di b,*, Erzse´bet Ligeti a b

a Department of Physiology, Semmelweis University, Budapest, Hungary Department of Infectology and Pediatric Immunology, Medical and Health Science Center, University of Debrecen, H-4012, Debrecen, Hungary c GREPI, Laboratoire d’Enzymologie, CHU Albert Michallon, Grenoble, France d Laboratoire DBMS/BBSI, CEA Grenoble, France

Received 13 June 2003; received in revised form 4 December 2003; accepted 4 December 2003

Abstract We reported earlier that monocytes and macrophages from patients with type I Gaucher disease have a decreased capacity to generate superoxide anion (O2 ) on stimulation with opsonized S. aureus or formyl-methionyl-leucyl-phenylalanine. In this study, various forms of the cell-free assay system were used to probe the hypothesis that glucocerebroside (GC) accumulating in Gaucher patients’ phagocytes may interfere with the activation of NADPH oxidase. Xanthine/xanthine oxidase assay was applied to explore the possibility that GC may scavenge O2 . We found that addition of GC to the crude, semirecombinant or fully purified cell-free systems inhibited activation of NADPH oxidase in a concentration-dependent manner. The inhibitory effect of GC could be overcome by increased concentrations of p47phox and p67phox. In contrast, O2 generation was not decreased by GC added to the assembled, catalytically active enzyme complex. In the xanthine/ xanthine oxidase system, GC had no effect on the generation of O2 . These data indicate that assembly of the respiratory burst oxidase of phagocytic cells may be a possible target of the pathologic actions of GC. D 2004 Elsevier B.V. All rights reserved. Keywords: NADPH oxidase; Glucocerebroside; Gaucher disease

1. Introduction In patients with Gaucher disease glucocerebroside (GC) accumulates in lysosomes of monocytes and tissue macrophages resulting in hepatosplenomegaly, lymphadenopathy, anemia, thrombocytopenia, and bone lesions [1,2]. In addition, these patients have increased susceptibility to bacterial and viral infections which may be due to decreased bactericidal capacity and superoxide anion (O2 ) production by lipid-laden mononuclear phagocytes [3,4]. O2 production by phagocytic cells is initiated by activation of the NADPH oxidase consisting of membranebound (flavocytochrome b558) and cytosolic (p47phox, p67phox, p40phox and Rac) components [5,6]. In resting cells, the membrane and cytosolic NADPH oxidase com-

* Corresponding author. Tel.: +36-52-416-841; fax: +36-52-430-323. E-mail address: [email protected] (L. Maro´di). 0925-4439/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbadis.2003.12.002

ponents are separated from each other, so the enzyme is in an inactive state. Specific stimuli acting on plasma membrane receptors can lead to the activation of NADPH oxidase through multi-step intracellular signaling [6,7]. Previously we and others [3,4] reported the impairment of O2 production initiated by opsonized bacteria or chemotactic stimuli in monocytes and macrophages of Gaucher patients. However, the potential step(s) of the intracellular signaling process affected by accumulation of GC remained to be elucidated. In this study, we probed the hypothesis that GC may inhibit activation of NADPH oxidase. We studied the effect of GC on the activation of NADPH oxidase in different cellfree systems. We found that incubation of membrane and cytosolic components with GC inhibited lipid-induced activation of NADPH oxidase in a concentration-dependent manner. These data suggest that excess GC accumulation in monocytes and macrophages in Gaucher patients may interfere with the final step of the signaling cascade, the assembly of the respiratory burst oxidase.

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2. Materials and methods 2.1. Materials Aprotinin, arachidonic-acid (AA), bovine serum albumin (BSA), components of the LB medium, dimethyl sulfoxide (DMSO), EGTA, ferricytochrome c (horse heart, type VI), flavin adenine dinucleotide (FAD), GC, glutathione, glutathione-agarose, leupeptin, NADPH, pepstatin, phenylmethylsulfonyl fluoride (PMSF), 1,2-didecanoyl-sn-glycerol 3-phosphate (phosphatidic acid, PA), N-2-hydroxyethylpiperazine-NV-2-ethanesulfonic acid (HEPES), sucrose, xanthine, xanthine-oxidase, and 1-oleoyl-2-acetylglycerol (OAG) were purchased from Sigma (St. Louis, MO). Ficoll Paquek Plus was obtained from Amersham Pharmacia Biotech (Buckinghamshire, UK). Superoxide dismutase (SOD) and GTPgS were from Boehringer Mannheim, isopropyl h-D-1-thiogalactopyranoside (IPTG) was from Promega. 2.2. Expression vectors and recombinant oxidase components The E. coli clones producing p47phox and p67phox were a generous gift of Dr. F. Wientjes. Recombinant p47phox and p67phox were produced as glutathione-S-transferase (GST)fusion proteins in E. coli and purified as described [8]. Cytochrome b558 was purified and relipidated as described [9]. Prenylated Rac was isolated from the membrane fraction of Sf9 cells by extraction with 1% Chaps and purified as described [10]. 2.3. Isolation of neutrophils and subcellular fractions Neutrophil granulocytes were isolated from buffy coat by dextran sedimentation and centrifugation through Ficoll Paquek Plus [11]. Cells were treated with diisopropyl fluorophosphate (DFP) for 10 min on ice, resuspended in sonication buffer (0.34 M sucrose, 10 mM HEPES, 1 mM EGTA 1 mM PMSF, 100 AM leupeptin in phosphate buffered saline (pH 7.0)) and broken by sonication [12]. Sonicates were centrifuged (200  g, 10 min) to remove unbroken cells and nuclei, layered onto a 17%/40% (w/v) discontinuous sucrose gradient and centrifuged (100,000  g for 60 min) [12 –14]. Cytosolic fractions were collected from the top layer down to the 17%/40% interface and membrane fractions were collected from the 17%/40% interface. Fractions were stored at 70 jC. Protein concentration was determined by the method of Bradford using BSA as standard [15]. 2.4. Measurement of O2 generation in cell-free systems The rate of superoxide generation was determined as the SOD-inhibitable reduction of ferricytochrome c mea-

sured at 550 nm in a Labsystems iEMS Microplate Reader [12]. In the crude system, membranes (5 Ag) and cytosol (50 Ag) were incubated in the presence of 125 AM cytochrome c in a final volume of 80 Al PBS containing 1 mM MgCl2 and 20 AM FAD. Arachidonic acid (180 AM) or PA (375 AM) plus OAG (250 AM) were applied as amphiphiles and GTPgS was added in 1 AM concentration. O2 production was initiated by the addition of 200 AM NADPH and followed for 20 min. The initial linear portion of the absorption curves (lasting for 10– 15 min) was used for calculation of the rate of O 2 production. Parallel samples were run in the presence of 100-Ag SOD. The absorption coefficient of ferricytochrome c of 21 000 M 1 cm 1 was used for calculation of O2 production. In the semirecombinant system, membrane (5 Ag) and recombinant p47phox (0.5 Ag; 87 nM) and recombinant p67phox (0.5 Ag; 67 nM) were used instead of cytosol. The membrane fraction isolated from human PMN contained sufficient Rac as addition of either prenylated or non-prenylated Rac to the system did not influence the maximal rate of O2 production. The incubation was carried out in the presence of 180 AM AA or in the presence of 375 AM PA plus 250 AM OAG and 125 AM cytochrome c in a final volume of 80 Al. GTPgS was present in 1 AM concentration. O2 was initiated by addition of 200 AM NADPH and followed for 20 min. In the fully purified system, 0.005-Ag purified and relipidated cytochrome b558 (0.6 nM) was incubated with 0.5-Ag p47phox (87 nM), 0.5-Ag p67phox (67 nM), 0.12-Ag GTPgS-loaded prenylated Rac (60 nM), 125 AM OAG, 187.5 AM PA and 125 AM cytochrome c in a final volume of 80 Al. O2 production was initiated by the addition of 200 AM NADPH and followed for 20 min. Conditions different from those described above are indicated in the figure legend. GC was dissolved in DMSO; its volume was kept at 1 Al and parallel solvent controls were run in all experiments. For loading of Rac with GTPgS, 1.8 Ag of the small GTPase was incubated in a final volume of 22 Al of PBS containing 4 mM EDTA and 30 AM GTPgS for 10 min at 25 jC. Subsequently, 20 mM MgCl2 was added and the protein was kept on ice [16]. 2.5. Xanthine-oxidase assay The assay was performed as described [14] with slight modification. Xanthine (0.16 mM), xanthine-oxidase (0.095 U/ml) and ferricytochrome c (80 AM) were incubated with 250 AM NADPH in Krebs – Ringer phosphate buffer with dextrose at 37 jC in a thermostated spectrophotometer under stirring condition. The amount of O2 generated was measured by the SODsensitive reduction of ferricytochrome c and expressed as nmol O2 /min/mg xanthine oxidase [17].

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3. Results 3.1. Inhibition of NADPH oxidase activation by GC In the crude activation system, various amounts of GC were added to the membrane and cytosol fractions in the presence of 1 AM GTPgS. Activation was induced by 180 AM AA and O2 generation was subsequently initiated by 200 AM NADPH. O2 generation was detected for 20 min, during the assembly of the active NADPH oxidase complex. As shown in Fig. 1 (panel A), GC inhibited O2 generation in a concentration-dependent manner. The inhibitory effect of GC could be due to direct interaction with the NADPH oxidase components. However, it could also involve other components present in the cytosol or in the membrane. Therefore, in the next step we applied a semirecombinant in vitro system that is composed of the membrane fraction (with associated Rac) and recombinant p47phox and p67phox proteins, which replace completely the cytosolic fraction. Similarly to the crude system, GC was added before 180 AM AA, and O2 generation was elicited by NADPH. The results indicated that similarly to the crude system, in the presence of various concentrations of GC, O2 generation was also decreased in the semirecombinant system (Fig. 1, panel B). We investigated the effect of GC in the fully purified system where the membrane fraction was replaced by relipidated cytochrome b558 and GTPgS-loaded Rac, corresponding thus to a canonical cell-free system containing all four essential components of the NADPH oxidase in purified or recombinant form. Also in this system, GC brought about a concentration-dependent decrease of O2 generation. These data suggest that GC may interact with one or several components of the O2 -generating oxidase. In cell-free systems, AA has a narrow optimal concentration range, which is influenced by protein concentration, ionic strength, or the presence of other lipids [18,19]. Thus, GC may sequester AA and thereby mimic inhibition of

Fig. 1. Effect of different concentrations of (GC) and ceramide (Cer) on amphiphile-induced NADPH oxidase activity in crude (A), semirecombinant (B) and fully purified (C) cell-free systems. At concentrations indicated, GC or Cer was added before 180 AM AA in (A) and (B) or before 187.5 AM PA plus 125 AM OAG in (C). Results are expressed as the rate of superoxide production. Mean F S.E.; n = 4 independent experiments carried out in duplicates.

Fig. 2. Effect of glucocerebroside on NADPH oxidase activity induced by varying concentrations of arachidonic acid (AA) in the crude cell-free system. Results represent the rate of O2 generated in the absence (open columns) or presence (shaded columns) of 105 AM GC added before AA. Mean F S.E.; n = 5 independent experiments carried out in duplicates.

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Fig. 3. Effect of glucocerebroside on NADPH oxidase activity induced by different concentrations of PA and OAG in the crude cell-free system. Results represent the rate of superoxide generated in the absence (open columns) or presence (shaded columns) of GC added before the amphiphiles. Mean F S.E.; n = 4 independent experiments carried out in duplicates.

NADPH oxidase activity. Therefore, we examined the possibility that GC induces a shift of the optimal concentration of AA. To this end, we tested the effect of GC in the presence of various AA concentrations, ranging from 0 to 350 AM. As shown in Fig. 2, at all concentrations tested, O2 generation was lower in the presence of 105 AM GC than in its absence. In addition, the optimal concentration of AA remained between 150 and 200 AM and was not influenced by the presence of GC. As shown earlier, the combination of PA and diacylglycerol induces high level of O2 production in various forms of the in vitro activation system [20,21]. The advantage of this lipid combination is the broad optimal concentration range. We performed experiments with two different concentrations of PA and OAG as shown in Fig. 3. We found that addition of GC before the amphiphiles resulted in significant decrease in the detectable amount of O2 independent of the applied concentration of the amphiphiles. We obtained similar results both in the crude (Fig. 3) and in the semirecombinant and fully purified systems (data not shown). In healthy individuals, GC is metabolized to ceramide and glucose. Therefore, we raised the question whether ceramide, the lipid with the closest structure to that of GC, inhibits O2 generation. Ceramide was applied under the same condition as GC in all three types of the in vitro activation system. In concentrations up to 1.12 mM (i.e. five times higher than the highest concentration applied for GC), ceramide did not inhibit O2 generation (Fig. 1). Glucose, the other degradation product of GC, did not affect the rate of O2 generation either (data not shown). Thus, the inhibitory effect on activation of phagocytic NADPH oxidase is related to GC and not to its degradation products. All these data suggest that GC is not acting by sequestering the amphiphile required to activate the cell-free system but it may specifically inhibit components of the NADPH oxidase.

3.2. Mechanism of inhibition of NADPH oxidase by GC In the preceding experiments we demonstrated that the presence of GC during the activation phase of NADPH oxidase inhibited O2 generation. This effect of GC could be due to interference with either the assembly of the NADPH components or with electron transfer via the active enzyme complex. In the following experiments, GC was added at a time point when the active NADPH oxidase complex was already assembled. According to our previous experience, in the fully purified system, activation of the enzyme is completed in 10 min [12]. In the experiment shown in Fig. 4, GC was added after a 10-min incubation period, simultaneously with NADPH. The results show that up to a concentration of 105 AM, GC did not affect the rate of

Fig. 4. Effect of glucocerebroside (GC) on PA plus OAG-induced NADPH oxidase activity in the fully purified cell-free system. GC was added 10 min after the amphiphiles. Mean + S.E.; n = 4 independent experiments carried out in duplicates.

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Fig. 5. Effect of preincubation of the membrane fraction with GC on activation of the NADPH oxidase. The membrane fraction was preincubated with 210 AM GC or its solvent for 10 min and then diluted to the reaction mixture of the semirecombinant system as described in Materials and methods. Non-pretreated membranes were used in the presence of the indicated concentration of GC. Mean F S.E. of four independent experiments carried out in duplicates.

O2 production in this system (Fig. 4). Similar results were obtained in the crude and the semirecombinant systems (data not shown). Thus, GC does not influence the catalytic activity of the enzyme complex; it may rather interfere with the assembly of NADPH oxidase subunits. We performed experiments to investigate whether GC affects primarily the membrane-localized or the cytosolic components of the NADPH oxidase. To this end, we first designed an experiment to determine whether GC is accumulated in the membrane fraction. As shown in Fig. 5, the concentrated membrane fraction was preincubated for 10 min with 210 AM GC and then diluted to the usual reaction mixture containing the cytosolic components and the cytochrome c. In this way, during the activation of the enzyme, GC was present at a concen-

tration of 13 AM, well below its effective concentration (column 3). The GC-pretreated membranes (column 5) produced the same amount of O2 as the control membranes (column 1) whereas the presence of 210 AM GC during the activation significantly reduced the detectable O2 production. Thus, GC is not likely to accumulate in the membrane fraction but has to be present in the activation phase to exert its inhibitory action. Next we tested whether the inhibitory effect of GC can be overcome by an increase of the amount of p47phox or p67phox applied in the fully purified system (Fig. 6). GC at a concentration of 105 AM reduced O2 production to 63% of the initial rate. A five- or tenfold increase in the applied amount of p47phox resulted in a rise of O2 production to 79% and 83% of the initial value. Augmentation of the amount of p67phox had similar effect whereas addition of bovine serum

Fig. 6. Effect of increased amounts of cytosolic components on the inhibitory action of GC. NADPH oxidase was activated in the fully purified system under conditions detailed in Materials and methods (control) or with fivefold (+) or tenfold (++) higher amount of p47phox or p67phox. O2 production is expressed as the ratio (%) of values obtained in the presence of 105 AM GC and in its absence. Mean F S.E. of three experiments carried out in duplicates.

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albumin to the system in comparable amounts did not interfere with the inhibitory action of GC (not shown). 3.3. Effect of GC on O2 generation in the xanthine/xanthine oxidase system To explore the possibility of any O2 scavenging effect of GC, we used the xanthine/xanthine oxidase assay system. The results showed that addition of GC (10 – 100 AM) to the mixture of xanthine, xanthine oxidase, cytochrome c, and NADPH failed to reduce the amount of O2 released (192 + 9 and 185 + 7 nmol/min/mg O2 generated in the presence and absence of 100 AM GC, respectively; n = 3). These data suggest that GC may not scavenge reactive oxygen radicals.

4. Discussion In Gaucher disease, deficient activity of the lysosomal hydrolase, glucocerebrosidase, results in accumulation of undegraded GC in mononuclear phagocytes. The progressive deposition of GC in macrophages in different organs is responsible for signs and symptoms of the disease. However, fundamental questions remain, regarding how macrophage functions altered by GC contribute to the course of the disease. Macrophages play a key role in host defense against various extracellular and intracellular organisms. The most striking and consistent immunologic feature in Gaucher patients is the decreased capacity of mononuclear phagocytes to kill viable microorganisms [3,4]. Deficiency in bactericidal capacity by these cells may be due to decreased O2 production [3,4]. However, the biochemical basis of the decreased O2 production by Gaucher monocytes and macrophages has not been previously studied in detail. To define possible molecular targets of GC in phagocytic cells, we investigated the effect of this compound in different cell-free systems. In this study, we demonstrated that GC inhibited activation of NADPH oxidase in mixtures of membrane and cytosolic components of the oxidase in a concentration-dependent manner. Several lines of evidence from this work point to GC as a specific inhibitor of NADPH oxidase. In the crude and semirecombinant systems, AA was used to initiate NADPH oxidase activity. AA may activate NADPH oxidase by direct interaction with the p47phox enzyme component [22,23]. One possible explanation for the inhibitory effect of GC in the cell-free systems we used could be that GC as a hydrophobic compound might also interact with AA. However, we showed here that increasing the concentration of AA in the cell-free system did not overcome the inhibitory effect of GC, and GC was inhibitory for NADPH oxidase activation over a wide range of AA concentration tested. In addition, GC was also inhibitory when PA and OAG were used to induce NADPH oxidase, independent of the concentration of these amphiphiles. Ceramide, a related com-

pound, was not inhibitory in the cell-free system, suggesting specificity in the effect of GC. In contrast to the activation phase, we could not detect any decrease in O2 production when GC was added to the assembled NADPH oxidase. These data suggest that GC has no inhibitory effect on the catalytic function of the enzyme complex. GC does not accumulate in the membrane phase but its effect can be overcome by increasing the amount of the cytosolic phox components. We thus propose that through physico-chemical interactions, GC may prevent the proper alignment of NADPH oxidase components that is required for optimal O2 production. Details of the binding of cytosolic subunits to GC and potential interference with their translocation have to be investigated in separate studies. In patients with Gaucher disease, accumulation of GC in various subcellular compartments may interfere with the assembling process of the NADPH oxidase, providing an explanation for earlier observations on impairment of O2 production and possibly contributing to the decreased bacterial killing activity of patients’ mononuclear phagocytes. As shown previously, enzyme replacement therapy resulted in partial restoration of both the O2S production and the bactericidal capacity [4].

Acknowledgements The authors are indebted to Ms. Edit Fedina and Erzse´bet Seres-Horva´th for excellent technical assistance. Grants from the Hungarian Research Fund (OTKA T37755 and Ts040865 to E.L., T038095 to L.M.), the Hungarian Ministry of Health (ETT) and the French – Hungarian intergovernmental project ‘‘Balaton’’ supported this work.

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