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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.

234, 531–536 (1997)

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Synthetic Peptides Corresponding to Various Hydrophilic Regions of the Large Subunit of Cytochrome b558 Inhibit Superoxide Generation in a Cell-Free System from Neutrophils Mi-Yeoun Park, Shinobu Imajoh-Ohmi, Hiroyuki Nunoi, and Shiro Kanegasaki1 The Institute of Medical Science, University of Tokyo, Minatoku, Tokyo 108, Japan

Received April 16, 1997

Cytochrome b558 is a component of the superoxidegenerating system in neutrophils and plays key roles in both the assembly of a functional complex with cytosolic proteins and shuttling an electron from NADPH to molecular oxygen. To determine the role of predicted hydrophilic domains of gp91-phox, a glycosylated subunit of the cytochrome, we synthesized peptides corresponding to the regions and tested whether they affected superoxide generation in the cell-free system obtained from human neutrophils. Among twelve peptides tested, six peptides, four of which correspond to previously unreported regions, inhibited superoxide generation in the cell-free system. All of the active peptides were effective when added to the system before activation with sodium dodecyl sulfate. Four peptides, including two peptides corresponding to two newly identified regions, inhibited the translocation of the cytosolic components, p47-phox and p67-phox. The extent of inhibition on translocation of these components varied depending on the peptide used. q 1997 Academic Press

The superoxide-generating system in neutrophils consists of cytochrome b558 in the membrane, and a few cytosolic proteins that include proteins designated as p47- and p67-phox (phox: phagocyte oxidase). The cytochrome is composed of a glycosylated 91 kDa subunit and a 22 kDa subunit, termed gp91-phox and p22-phox, respectively. cDNA for all phox proteins have been cloned and sequenced (1-7). During activation of the 1

To whom correspondence should be addressed. Abbreviations used: SDS, sodium dodecylsulfate; GTPgS, guanosine 5*-(g-thio)triphosphate; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; SOD, superoxide dismutase; BSA, bovine serum albumin; PVDF, polyvinylidene difluoride; HRP, horseradish peroxidase; IC, inhibitory concentration; SH3, src homology region 3.

respiratory burst, p47- and p67-phox translocate from the cytosol to the membrane where the cytochrome functions structurally to coordinate the interactions with cytosolic proteins. A small GTP-binding protein, Rac-p21 is also known to tanslocate to the membrane and is essential for the superoxide-generating activity (8-11). According to studies (12-16) of the assembly of a functional complex in neutrophils from chronic granulomatous disease, which lack either p47-phox or p67phox, it was suggested that p47-phox binds to the cytochrome initially and that p67-phox-binding depends on the presence of p47-phox. Cytochrome b558 is considered to be the terminal oxidase that shuttles an electron from NADPH in the cytoplasm to molecular oxygen at the surface of the cells. Electron flow activation in the cytochrome seems to be regulated through the association of p47-phox, p67phox and probably Rac. The two subunits of the cytochrome are closely associated and can only be dissociated under extreme conditions (i.e. in sodium dodecyl sulfate: SDS) that also destroy the cytochrome spectrum, resulting in loss of the non-covalently bound heme. Consequently, it is uncertain which of the subunits actually carries the heme and also whether there is more than one heme. By comparing sequential homology with several flavoproteins, the large subunit of the cytochrome is considered to contain FAD and NADPH binding sites (5-7). By hydrophobisity and surface-probability analyses, several membrane-spanning regions and extracellular and intracellular regions have been predicted (17), although the transmembrane orientation of specific regions remain uncertain. Several regions of the cyochrome have been identified as potential sites for interaction with p47-phox (13, 18-21). To investigate the interaction of the specific components, Rotrosen et al. (19) used synthetic peptides corresponding to the COOH-terminus of gp91phox and an antibody that recognized this region. They found that these inhibited superoxide-generation and

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the interaction between gp91-phox and p47-phox. Based on the topological study of cytochrome b558 , Nakanishi et al. (20) studied the interaction between cytosolic components and the cytochrome. They reported that the COOH-termini of both subunits interact with p47-phox. The proline-rich domain of p22-phox, near the COOH-terminus, was shown to interact with one of two SH3 domains of p47-phox (15,16). By using a random-sequence peptide phage display library analysis, DeLeo et al. (22) showed that two additional sites, namely the predicted cytosolic loop of gp91-phox encompassing residues 86-93 and a domain near the cytosolic C-terminal tail encompassing residues 450-457, were involved in interaction of p47-phox with gp-91phox. To understand the interaction of cytochrome b558 with cytosolic components as well as the topology of the cytochrome, we focused on the predicted hydrophilic domains of gp91-phox (17), synthesized peptides corresponding to these regions and tested to determine whether they could affect the supeoxide-generation in a cell-free system obtained from human neutrophils. Among twelve peptides tested, we found six peptides, four of which correspond to previously unreported regions inhibited superoxide-generation in the cell-free system. All of the active peptides were effective when added to the system before but not after activation with SDS. Four peptides including two peptides corresponding to two novel regions, inhibited the translocation of p47-phox and p67-phox. MATERIALS AND METHODS Materials used. Based on the primary structure deduced from the cDNA sequence of the large subunit of cytochrome b558 (1), the hydrophilicity index and surface probability were calculated, respectively, according to Kyte and Doolittee (23) and Emini et al (24). Peptides were synthesized on a PerSeptive Biosystems 9050 peptide synthesizer, deprotected and purified as described previously (20). NADPH and GTPgS were purchased from Sigma, SDS and N-octyl b-glycoside from Dojindo Laboratory, Kumamoto. Other chemicals were of the highest grade obtainable. Isolation of neutrophils and preparation of the cell-free system. Human neutrophils were isolated as described previously (25). Plasma membrane and cytosolic fractions were prepared from cavitated neutrophils by sequential centrifugation (25). In most of the experiments, solublized membrane with N-octyl b-glycoside was used for the assay of superoxide generation. Assay of superoxide generation in the cell free system. The assay was performed in a 96-well microtiter plate with a final volume of 100 ml/well as described previously (25). SOD-sensitive reduction of acetylated ferricytochrome c was used as a measure of superoxide generation. The reaction mixture contains 200 mM acetylated cytochome c, 10 mM FAD, 1 mM EGTA, 1 mM MgCl2 , 10 mM guanosine 5-[g-thio] triphosphate, membrane and cytosol fractions equivalent to 2 1 106 cells in 10 mM HEPES buffer pH 7.0. The control well contained 250 units of superoxide dismutase. After incubation for 2 min at 25 7C, 100 mM SDS was added to the mixture for activation and after 3 min, 200 mM NADPH to initiate the reaction. The change of adsorbance at 550 nm was recorded continuously by a Bio Rad

microplate reader. The maximum linear rate of the reaction was quantified using Kinetic Collector 2.0. Inhibition of superoxide generation in the cell free system by synthetic peptides. Peptides dissolved in the assay buffer containing 10 mM HEPES, pH7.0, 10 mM ATP, 75 mM NaCl, 2 mM sodium azide, 170 mM sucrose, 1 mM MgCl2 , and 0.5 mM EGTA were added to the reaction mixture described above before or after the addition of SDS. The potential of the synthetic peptides to inhibit superoxide generation was quantified by measuring the concentration of the peptides required for 50% inhibition of the generation in the absence of peptides (a half inhibitory concentration; IC50). Control value usually showed 1.5-2.0 nmol/min/well at 25 7C. Assay of translocation of cytosolic proteins to membrane. Translocation of p47-phox and p67phox to the plasma membrane was performed according to the methods described by Verhoeven et al. (26) and Park et. al. (21) with slight modifications. In brief, neutrophil plasma membrane (eq. 1 1 108 cells) and cytosol (eq. 5 1 107 cells) were mixed in 1 ml of the sample buffer containing 10 mM HEPES,10 mM ATP, 75 mM NaCl , 2 mM sodium azide, 170 mM sucrose, 1 mM MgCl2, and 0.5 mM EGTA / pH7.0 and the mixture was incubated at 287C for 2 min. Then, 10 mM GTPgS and 100 mM SDS were added and the mixture was incubated at 257C for 10 min and layered onto discontinuous sucrose gradients composed of 0.5 ml of 15% (w/v) layered over 0.5 ml of 50% sucrose, both in sample buffer. To assay inhibition of the translocation by synthetic peptides, the peptides were added just before addition of SDS. The translocation mixture was centrifuged at 100,000 1g for 20 min at 257C in a Hitachi CP100H ultracentrifuge using a swing bucket rotor (RP55S-125). Immediately after centrifugation, approximately 0.5 ml of fluid was withdrawn from the top of the gradient, then an equal volume on the 50% sucrose cushion, containing the plasma membrane, was carefully removed. The fraction containing the plasma membrane was suspended in 2% sucrose in the buffer for superoxide generation (see above) and centrifuged at 16, 000 rpm for 20 min at 47C in a microcentrifuge (Sakura Seiki Co., Tokyo). The pellet was suspended in 200 ml buffer for superoxide generation. A portion of the pellet (100 ml) was subjected to immunoblotting analysis. Another portion (50 ml) was used for the assay of superoxide generation without adding the cytosolic fraction. Polyacrylamide gel electrophoresis and blotting. The isolated membrane fraction was treated with ice-cold trichloroacetic acid (10%). After centrifugation at 16, 000 rpm for 20 min at 47C, the pellet was suspended in Laemmli buffer containing 20% (w/v) SDS, 10% (v/v) b mercaptoethanol, and 10% glycerol in 125 mM Tris-HCl, pH 6.8. The sample was heated at 80 7C for 20 min. Polyacrylamide gel electrophoresis was carried out according to Laemmli (27). Proteins were transferred onto a polyvinylidene difluoride (PVDF: Milipore Co.) membrane using a Bio-craft apparatus (BE-300). The buffer used contained 25 mM Tris, pH 8.0, 192 mM glycine, 1% (w/v) SDS, and 20% (v/v) methanol. The protein-blotted membrane was treated with 5% (w/v) BSA dissolved in Tris-buffered saline containing 0.5M NaCl and 10 mM Tris, pH 8.0 for blocking. Subsequently, the membrane was incubated overnight at 47C with a mixture of primary antibodies (anti-p47KN and anti-p67Kc corresponding to the residues 81-101 of p47-phox and 508-526 of p67-phox, respectively), and then with HRP-labeled goat anti-rabbit protein A. The proteins were visualized by exposure to Fuji X-Ray film after treating the PVDF membrane with the detection reagents (RPN 2106, Amersham International, Buckighamshire). Quantification of the densitometric areas was performed using Super Image Scanner with NIH Image 1.55 software.

RESULTS Effect of synthetic peptides corresponding to various hydrophilic regions of the large subunit of cytochrome

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FIG. 1. The possible orientation of gp91-phox and list of synthetic peptides used. The orientation of gp91-phox predicted from cDNA sequence (column), selected regions for the peptide synthesis (boxes below the column), and sequences of such peptides are shown. These regions cover hydrophilic domains of gp91-phox except for those around 151-173 and 370-399, which were predicted to be exposed to the outside of the cell (17). In the upper column, a helical ( ) and b sheet structures ( ) are shown and in the lower, hydophobic (ø) and hydrophilic regions (h). Predicted binding sites for FAD (j) and NADPH (…) are shown in the middle. The secondary structure was calculated according to Chou-Fasman (28) and Granier-OsguthorpeRobson (29) and only the structures coinciding with each other are shown. Numbers above the column indicate the number of amino acid residues.

b558 on superoxide-generation in a cell-free system from human neutrophils. Based on hydrophilicity index and the surface probability in the primary structure of the large subunit of cytochrome b558 (17), hydrophilic regions shown in Fig. 1 were selected and peptides corresponding to these regions were synthesized chemically. The effect of these peptides on the superoxide generating activity in a cell-free system consisting of the membrane and cytosolic fractions obtained from human neutrophils was analyzed. As shown in Fig. 2, some peptides added to the system before addition of SDS, inhibited NADPH-dependent superoxide generation in a dose-dependent manner. The concentrations of the peptides examined that exhibited 50% inhibition of activity in the cell free system assayed in the absence of peptides (IC50) are summarized in Table 1. Peptides with sequences corresponding to amino acid residues 27-46, 87-100, 282296, 304-321, 434-455 and 559-565 inhibited superoxide generation significantly. In contrast, peptides corresponding to residues 221-236, 231-252, 250-270, 329-350, 484-502 and 526-548 exhibited little or no inhibition. The inhibition was observed when the peptides were added before but not after activation of the cell free system with SDS (data not shown). The results suggest that the peptides inhibited the interaction between cytochrome b558 and cytosolic proteins. Inhibition of translocation of the cytosolic components to the membrane by synthetic peptides corresponding to various regions of the large subnit of cytochrome b558 . Translocation of the cytosolic compo-

FIG. 2. Inhibition of superoxide generation in a cell-free system by synthetic peptides corresponding to the hydrophilic domains of gp91-phox. Indicated concentrations of each synthetic peptide were added to the reaction mixture consisting of solubilized membrane and cytosolic fractions (equivalent to 2 1 106 cells in 100 ml) and the mixture was incubated at 377C for 2 min. The mixture was then activated with 100 mM SDS. After 3 min, the mixture received 200 mM NADPH to initiate the reaction and the change of adsorbance at 550 nm was recorded continuously at 25 7C. The results were expressed as % of activity, where the activity without peptide was taken as 100%. The peptides shown in this figure were those corresponding to residues 27-46 (- j -), 87-100 (- h -), 282-296 (- s -), 304321 (- l -), and 434-455 (- n -). The results were expressed as % of remaining activity. Data shown are the average of three independent experiments.

nents to the membrane was performed and the effect of the synthetic peptides on the translocation was examined. Each synthetic peptide was added to the reaction mixture containing plasma membrane and cytosol TABLE 1

Effect of Synthetic Peptides Corresponding to Various Hydrophilic Regions of gp91-phox on Superoxide Generation in a Cell-Free Systema from Neutrophils Inhibition (IC50b mM) Peptide No.

Residues

Membrane

Solubilized membrane

1 2 3 4 5 6 7 (7* 8 9 10 11 12

27-46 87-100 221-236 231-252 250-270 282-296 304-321 306-319 329-350 434-455 484-502 526-549 559-565

58 { 2 75 { 3 ú300 ú300 260 75 { 5 90 { 6 65 { 4 ú300 75 { 2 ú300 ú300 80 { 3

34 { 4 40 { 2 ú300 ú300 ú300 30 { 2 35 { 3 20 { 3) ú300 25 { 3 ú300 ú300 53 { 2

Data shown are the averages of three independent experiments. a A half inhibitory concentration. b As membrane fraction, either isolated membrane or solubilized membrane was used.

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DISCUSSION

FIG. 3. Effect of synthetic peptides corresponding to the hydrophilic domains of gp91-phox on translocation of p47- and p67-phox to membrane. Plasma membrane (eq. 1 1 108 cells) and cytosol (eq. 5 1 107 cells) from human neutrophils in 1 ml sample buffer were incubated at 25 7C for 10 min in the presence of 100 mM SDS. Each peptide (100 mM) was added 2 min before the addition of SDS. The mixtures were then layered onto discontinuous sucrose gradients. After centrifugation of the mixture at 100,000 1g for 20 min at 25 7C, the fraction containing the plasma membrane was removed and subjected to immunoblotting analysis. The results of peptides corresponding to residues 559-565 (lane 3), 27-46 (lane 5), 87-100 (lane 6), and 434-455 of gp91-phox (lane 7) are shown. As controls, the mixtures without peptide (lane 1), without peptide and SDS (lane 2), and with peptide corresponding to residues 82-95 of p22-phox (lane 4, see reference 25) are also shown. Data shown represent one of three independent experiments.

In this paper, we showed that six peptides representing various regions of the large subunit of cytochrome b558 (gp91-phox) inhibited superoxide-generation in a cell free system from neutrophils. Among them, four peptides corresponding to amino acid residues 27-46, 282-296, 304-321 and 434-455 represent novel regions that may be responsible for the inhibition of superoxide-generation. (A weak inhibitory activity was reported of a peptide corresponding to amino acid residues 451-463-ref. 22) All of the active peptides exhibited an inhibitory effect when they were added before but not after the system was assembled. Since these peptides were only effective in the cell free system, these peptides must interfere with some kind of interaction between the cytochrome and cytosolic components during assembly. Results also suggest that the corresponding regions of gp91-phox are located on the cytoplasmic side of the membrane and interact with the cytosolic components (See Fig. 1). The proposed cytoplasmic regions include the first and second regions

2 min before the addition of SDS and incubated at 257C for 10 min in the presence of GTPgS. The membrane was isolated by discontinuous gradient centrifugation and analyzed by SDS-polyacrylamide gel electrophoresis followed by immunoblotting. Among the six peptides that inhibited superoxidegeneration, four peptides corresponding to residues 27-46, 87-100, 434- 455 and 559-565 were found to inhibit the translocation at concentrations above 50 mM. As shown in Fig 3, peptides corresponding to residues 27-46 and 434-455 inhibited the translocation of p47-phox more than that of p67-phox, whereas the peptide corresponding to residues 87-100 inhibited p67-phox translocation more extensively than p47-phox. This observation did not change when different concentrations of peptides (50, 100 and 200 mM) were used and was reproducible in 3 experiments at a concentration of 100 mM. To investigate the correlation between the extents of inhibition of translocation and of superoxide-generating activity, we assayed the superoxide-generating ability of the membrane obtained after translocation. The assay mixture contained the isolated membrane from the translocation mixture, GTPgS, NADPH and was stimulated again with SDS. As shown in Fig. 4, the superoxide generation activities and degrees of association of p47-phox and p67-phox correlated well with each other except in the case of the peptide corresponding residues 27-46. In the latter case, significant amounts of p47-phox and p67-phox were associated to the membrane whereas superoxide-generating activity was almost completely inhibited by this peptide.

FIG. 4. Correlation between inhibition of translocation of cytosolic components to the membrane and remaining superoxide-generating activity in the membrane. The translocation experiments were carried out under the conditions described in the legend to Fig. 3. A portion of the fraction containing the plasma membrane was subjected to immunoblotting analysis. Densitometric quantification of the blotting was performed using a Super Image scanner with NIH Image 1.55 software. Peptides 1, 2, 9, and 12 and peptide S82 represent the data obtained with peptides corresponding to residues 2746, 87-100, 434-455, and 559-565 of gp91-phox and 82-95 of p22phox, respectively (see Fig. 1). Another portion of the fraction was used for the assay of superoxide generation without adding fresh cytosolic fraction but in the presence of SDS and NADPH. The results were expressed as % cytosolic proteins associated with membrane and % remaining superoxide-generating activity where the results of the reaction mixture without peptide (PC) and without peptide and SDS (NC) were taken as 100 and 0%, respectively. Data shown are the average of two independent experiments.

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of predicted cytosolic loops (residues 27-46 and 87-100), one region containing an alpha helical configuration (residues 304-321 or 306-319), one region encompassing a predicted NADPH binding site (residues 434455) and one site close to the carboxyl end (residues 559-565). Among the six peptides that inhibited superoxidegeneration, four peptides, including two peptides representing two newly identified regions (residues 2746 and 434-455), inhibited the translocation of p47phox and p67-phox to the plasma membrane where cytochrome b558 resided. It was shown by using a random-sequence peptide phage display library analysis that residues 77-93 are important for the assembly of a functional unit and that a peptide encompassing this region inhibited superoxide generation (22). We confirmed this observation using a peptide corresponding to residues 87-100 and further observed that the peptide inhibited the translocation of the cytosolic proteins to the plasma membrane. We also confirmed that peptides corresponding to residues 559-565 inhibited the activity and translocation of the cytosolic components (19, 21). It is interesting to note that among the peptides, inhibited the translocation of the cytosolic proteins, some peptides inhibited the translocation of p47-phox more that of p67-phox (peptides 27-46 and 434-455), whereas other peptide inhibited the translocation of p67-phox (peptide 87-100) more than that of p47phox. However, a peptide corresponding to the C terminal region of gp91-phox inhibited the translocation of both cytosolic proteins equally. The results suggest that not only p47-phox associates with the cytochrome at multiple sites during activation but also p67-phox also associates with the cytochrome firmly and that these interactions are essential for exhibiting the superoxide-generating activity. Incidentally, it was recently shown from two groups that superoxide-generaing activity in a cell free system could be reconstituted with higher concentrations of p67-phox and Rac without p47-phox (30, 31). In addition, although it was shown that the translocation of p67-phox depended on p47-phox and that the aminoterminal SH3 domain of p47-phox interacts preferentially with the small subunit of the cytochrome p22phox, it was suggested that SH3-mediated interaction was only partially responsible for the assembly of the functional complex and activation (15, 16). The extents of inhibition of translocation by most of the synthetic peptides and of the remaining superoxide-generating activity in the membrane were found to be well correlated. However, in the case of the peptide representing residues 27-46, significant amounts of p47-phox and p67-phox were still associated to the membrane under the conditions where superoxide-generating activity was almost completely inhibited. This region seems to form the first cytosolic loop near the

N-terminal region. The present results suggest that, although an association with cytosolic components may take place without this region, the interaction between this region of gp91-phox and one of cytosolic components is still important for the electron transferring activity of the cytochrome. All of the peptides that inhibited superoxide-generation in the cell-free system contain three to five basic amino acids. It is possible that these peptides bind to cytosolic components by electrostatic interaction and inhibited the assembly of a functional complex or superoxide-generating activity. It was reported that any polybasic peptide with more than 5 basic residues could inhibit activity (32). However, it was also shown that an eleven-residue peptide containing six lysine residues and a number of polybasic peptides failed to inhibit superoxide generating activity in a cell-free system (32, 33). Therefore, the inhibition by the peptides shown in this study is considered to be specific to the sequences. ACKNOWLEDGMENT We thank Martin Dahl for his advice on English expression in the manuscript.

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