Parabutoporin—an antibiotic peptide from scorpion venom—can both induce activation and inhibition of granulocyte cell functions

Peptides 25 (2004) 1079–1084

Parabutoporin—an antibiotic peptide from scorpion venom—can both induce activation and inhibition of granulocyte cell functions Jean Willems a,∗ , Leentje Moerman a , Suzanne Bosteels a , Erik Bruyneel b , Filip Ryniers b , Fons Verdonck a b

a Interdisciplinary Research Center (IRC), KULAK, University Campus, B-8500 Kortrijk, Belgium Department of Radiotherapy and Experimental Cancerology, University Hospital, B-9000 Gent, Belgium

Received 13 January 2004; received in revised form 31 March 2004; accepted 1 April 2004 Available online 1 June 2004

Abstract Parabutoporin (PP) affects motility and NADPH oxidase activity in normal human polymorphonuclear neutrophils and in granulocytic HL-60 cells. These PP-induced interactions utilize a Rac activation pathway. PP induces chemotaxis of neutrophils and HL-60 cells via a pertussis toxin-sensitive way, thus using trimeric G-proteins. The enhanced chemotaxis is also apparent in undifferentiated HL-60 cells which lack functional formyl peptide receptors. On the other hand, PP strongly reduces the superoxide production by the NADPH oxidase complex after either PMA or fMLP activation of granulocytes. These combined results strongly suggest a direct activation of G-proteins and subsequent Rac activation as the basis for the observed effects. The unexpected inhibitory effect of PP, despite Rac activation, on superoxide production in granulocytes is explained by the direct interaction of membrane localized PP which prevents the formation of a functional NADPH oxidase complex. © 2004 Elsevier Inc. All rights reserved. Keywords: Parabutoporin; Granulocytes; Superoxide; Chemotaxis

1. Introduction We have recently isolated and sequenced three bioactive peptides from the venom of South African Scorpions. Parabutoporin (PP, from Parabuthus schlechteri) is 45 amino acids long while opistoporin 1 and 2 (OP1 and OP2 , from Opistophtalmus carinatus) are 44 amino acids long. All three were initially characterized by their antibacterial and antifungal activity [10]. This is mainly directed against Gram-negative bacteria (MIC 1.6–6.5 ␮M) and is related to the fact that all three peptides are ␣ helical cationic peptides [10] which easily accumulate into membranes to form pores [3,4]. In addition, we also found that, at submicromolar concentrations, these peptides, and especially PP, can either stimulate or inhibit the activity of human neutrophils as evidenced by exocytosis, chemotaxis and superoxide production assays [18]. In addition, these peptides can induce transient Ca2+ release from intracellular stores [11]. We had, however, no clear indications about possible mechanisms. Indeed, interaction of peptides with granulo∗

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0196-9781/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2004.04.008

cytes usually results in activation of all functions such as enhanced chemotaxis, superoxide production, phagocytosis and exocytosis (reviewed in [8]). The observation that one peptide, in the same concentration range, can both activate and suppress important neutrophil activities is a very unusual event [18] which drew our attention. In this study, we examined possible explanations for this duality. We have focused on chemotaxis and the production of reactive oxygen intermediates, two granulocyte functions which are mediated by Rac activation [9]. Rac activation can explain the chemotactic effect but cannot be hold responsible for the impaired superoxide production. We looked for inhibition of NADPH oxidase activation by binding of PP. Indeed, an analogous inhibitory action on NADPH oxidase was described for the cationic peptide mastoparan [16,17]. In cell free systems, it can bind to components of the oxidase complex. We have investigated both in vitro and in vivo if a similar inhibition is valid for PP. This effect should be facilitated by the incorporation of PP in the cell membrane due to its amphipatic ␣ helical structure [18]. We looked for additional proofs and, using confocal microscopy, we explained why PP is an excellent inhibitor of NADPH oxidase in intact granulocytes.

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2. Materials and methods 2.1. Parabutoporin PP was initially isolated from the venom of P. schlechteri scorpions. It was purified by HPLC, sequenced and the synthetic 45 amino acid peptide [18] was used throughout this study. 2.2. Blood cells Human granulocytes were obtained from the blood of healthy volunteers and purified after centrifugation on Ficoll-Paque (Pharmacia, Sweden) and hypotonic lysis of contaminating red blood cells [18]. 2.3. Other cells HL-60 cells were obtained from ATCC (VA, USA). Cells were grown in RPMI-1640 medium (Gibco, Scotland) supplemented with 10% FCS, 2 mM glutamine, 100 U/ml penicillin and 100 ␮g/ml streptomycin. HL-60 cells were differentiated to the granulocytic phenotype by incubation with DMSO (1.3% final concentration) and harvested at day 4 after the start of the treatment. Cells used in any assay were washed twice in unsupplemented RPMI-1640 medium before use. 2.4. Assays for superoxide determination and chemotaxis NADPH oxidase activity which generates superoxide was quantitated using a bioluminescence test. Chemotaxis was performed using 24 well Transwell plates (Costar Europe, The Netherlands) having 3 or 5 ␮ filters (for human neutrophils or HL-60 cells, respectively). Both assays were described [18]. In these tests we included some well known inhibitors like pertussis toxin (PTX; G␣i/o inhibitor) or wortmannin (PI3 K inhibitor). For this purpose, adequate concentrations were preincubated for 30 min with the cells before starting the oxidase or chemotaxis assay. Unless otherwise indicated the results of both assays are presented as a percentage ± standard deviation (n ≥ 3) in relation to standard procedures. 2.5. Rac activation assay The level of the active GTP-bound form of Rac was measured essentially as described [1,19]. In brief, cells were lysed with Fisch buffer (10% (w/v) gelatin, 50 mM Tris pH 7.5, 100 mM NaCl, 1% (w/v) NP-40, 2 mM MgCl2 and a cocktail of proteinase and phosphatase inhibitors including 1 ␮g/ml pepstatin, 1 ␮g/ml leupeptin and 1 mM orthovanadate). GTP-bound Rac was precipitated by PAK-1 (p21-activated kinase-1) fused to glutathione-S-transferase precoupled to glutathione-Sepharose beads. The total

amount of Rac in cell lysates and the corresponding GTP-bound fraction was detected by Western blot. In our test 250 ␮l of granulocytes (3×107 /ml) were mixed with either PBS (10 min) or fMLP (1 ␮M final concentration; 1 min) or PMA (1 ␮g/ml; 10 min) or PP (5×10−7 M; 10 min) and then lysed and processed as described above. 2.6. Antibodies Polyclonal antibodies against lactoferrin were raised in rabbits. From pooled bleedings, we prepared immunoglobulin fractions by standard Na2 SO4 precipitation techniques. Purified antibodies were isolated from these immunoglobulin fractions by affinity chromatography on self prepared lactoferrin-Sepharose. Antisera against the p47, p67 or gp91 components of NADPH oxidase were obtained from Santa Cruz Biotechnology (CA, USA). 2.7. Fluorescence microscopy We prepared Alexa Fluor 430-labeled PP using a labeling kit according to the protocol provided by the supplier (Molecular Probes Europe, The Netherlands). We used, in addition, Alexa Fluor 594-labeled cholera Toxin B-chain (Molecular Probes Europe, The Netherlands). For fluorescence microscopy assays cells (106 /ml) were incubated with Alexa Fluor 430 PP (5 × 10−7 M final concentration) and Alexa Fluor 594 cholera toxin B (5 ␮g/ml) for 10 min at room temperature. Cells were imaged using a Zeiss Axiovert 100 M inverted microscope with a 40×/1.3 oil-immersion objective, and equipped with a Zeiss LSM 510 confocal point scanning system (Zeiss GmbH, Jena, Germany) [6]. Alexa Fluor 430 was excited with the 458 or 488 nm line of a 25 m W Argon laser; the Alexa Fluor 594 was excited with the 543 line of a 50 m W HeNe laser. With this combination of fluorophores there was no overlap or ‘bleeding’ through between channels. The pinhole size was set to 1 Airy unit, resulting in an optical slice thickness (Z-direction) of 0.9 ␮M. Pixel width was between 0.21 and 0.36 ␮M. 2.8. Cell extracts Freshly isolated human granulocytes (obtained from the same donor) were frozen immediately after isolation in the presence of 2 mM PMSF and 10 ␮g/ml leupeptin. The pool, containing about 4 × 108 cells was thawed, treated with 15 mM octylglucoside and subjected to three more freeze and thaw cycles. Cell free extracts, obtained after high speed centrifugation, were applied on a PP-Sepharose affinity column (self prepared according to the procedure from Pharmacia, the supplier of CNBr-Sepharose) and reloaded twice using PBS as an eluent. Bound proteins were eluted from the column using 1 mM HCl followed by 3% ammonium bicarbonate. They were then lyophilized

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and subsequently used in an ELISA assay after reconstitution in 1 ml PBS. We coated the proteins (50 ␮l per cup; in triplicate) at different dilutions and post-coated with gelatin (3 mg/ml). The proteins were stained with different antibodies (anti-lactoferrin at 20 ␮g/ml) or antisera (anti p 47, anti p 67 or anti gp 91 at 1/200) followed by alkaline phosphatase labeled second antibodies (goat anti rabbit or rabbit anti goat obtained from Sigma, MI, USA) at 1/200. Optical densities were measured at 405 nm after the addition of the substrate (para nitrophenyl phosphate) using a EL340 microplate reader (Biotek, VT, USA).

Table 1 Parabutoporin-induced chemotactic response of cells expressed as a percentage of activity in relation to either standard 4 × 10−7 M fMLP treatment (human blood cells) or to standard 8 × 10−7 M fMLP treatment (HL 60 cells differentiated for 4 days using DMSO)

2.9. Protein kinase C

HL60 HL60 HL60 HL60

PKC was assayed using a kit obtained from Upstate (Campro, The Netherlands). The prescribed protocol was followed. Different concentrations of PP were included in the test to measure its possible PKC inhibitory effect.

3. Results PP is originally described as an antibiotic peptide which can inhibit the growth of bacteria and fungi [10] and is lytic for eukaryotic cells at micromolar concentrations [10,18]. In addition, it also interacts with granulocytes [11,18]. At submicromolar concentration without significant aspecific lysis (as measured by lactoferrin release—data not shown) it clearly induces the activation of Rac in granulocytes (Fig. 1). The stimulation-index for PP is even slightly higher than that for PMA (1 ␮g/ml) which is often used as a standard [1]. PP shows powerful dose-dependent chemotactic activity with a specificity for granulocytes. We compared its potency—at submicromolar concentrations—with that for standard fMLP treatment. In our assays 4 × 10−7 M of fMLP-induced maximal granulocyte chemotaxis and PP at 8 × 10−7 M scored 73% of this high value (Table 1). This chemotactic effect is largely inhibited by pretreatment of the cells with pertussis toxin (500 ␮g/ml). This is in accordance with other chemoattractants which act via specific receptors coupled to heterotrimeric G-proteins [13,14].

Fig. 1. Rac activation in granulocytes. Cells were treated with PBS, fMLP (1 ␮M), PMA (1 ␮g/ml) or PP (5 × 10−7 M). We show immunoblots of total Rac and GTP-bound (activated) Rac. The histogram (quantitated using Quality One Software) shows the stimulation index in relation to the ratio of activated Rac/total Rac for PBS-treated cells.

Cells

Inducer

PMN PMN PMN PMN PMN

a b

(undifferentiated) (differentiated) (undifferentiated) (differentiated)

PP PP (PTX) PP PP (PTX) PP (wortmannin) (2 × 10−8 M) PP PP PP (PTX) PP (PTX)

Activity (%) 8 8 4 4 4

× × × × ×

10−7 10−7 10−7 10−7 10−7

73 ± 20 19 ± 9a 36 ± 15 9 ± 7a 28 ± 9b

4 4 4 4

× × × ×

10−7 10−7 10−7 10−7

48 ± 13 44; 40 5 10

Significant P < 0.05. Not significant.

Normally, the transduction cascade proceeds via PLC/PKC or PI3 K to activate Rac which plays an important role as well in motility of the granulocytes as in the activation of their NADPH oxidase complex [9]. Using human neutrophils, we observed that the PI3 K inhibitor wortmannin (2 × 10−8 M) had almost no inhibitory effect on PP-induced chemotaxis (Table 1) although it inhibited fMLP-induced chemotaxis as expected, thus favoring the PLC pathway. We also tested the effect of PP on HL-60 cells. Undifferentiated HL-60 cells are devoid of formyl peptide receptors [13] and only react to fMLP after treatment with differentiation-inducing agents such as DMSO. In our assay conditions, 8×10−7 M fMLP showed maximum chemotaxis for DMSO-treated cells. PP, at submicromolar concentrations also scored very well on differentiated HL-60 cells. To our surprise also undifferentiated cells responded very good, giving chemotaxis values even exceeding those for differentiated cells (Table 1). Here too PTX abrogates the effect. Parabutoporin also strongly reduces the capacity of phagocytes to produce superoxide anions via the NADPH oxidase system as evidenced by chemiluminescence-based assays. This reduction is not due to some superoxide dismutase activity of the peptide since it has no influence on the detection of superoxide produced by the classical cell free xanthine oxidase system (data not shown). We observed that, using freshly isolated human neutrophil granulocytes, preincubation of the peptide and subsequent activation by the phorbol ester PMA, a calculated IC50 inhibitory concentration of about 0.4 ␮M could be obtained (Table 2). PP also inhibited the PMA activation of undifferentiated as well as DMSO-treated HL-60 cells at the same extent (Table 2) and pretreatment of the cells with PTX had no influence on the inhibitory capacity of PP. Using fMLP as an activator for granulocytes or differentiated HL-60 cells analogous inhibitory results were obtained (Table 3). These findings might suggest that PP acts as a PKC inhibitor since both PMA (direct PKC activator) and fMLP activate

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Table 2 Inhibition of superoxide production by PP in cells stimulated with PMA Cells

PP (final concentration, M)

Inhibition (%)

PMN

10−6

5 × 10−7 10−7

92 ± 5 53 ± 14 9±6

HL 60 (undifferentiated)

10−6 5 × 10−7 10−7

93 ± 4 41 ± 12 5±5

HL 60 (DMSO-4d)

10−6 5 × 10−7 10−7

90 ± 7 40 ± 7 4

PMN (PTX) HL60 (DMSO-4d, PTX)

10−6 5 × 10−7

86 ± 10 38 ± 8

Inhibition in relation to cells (PMN or HL-60) stimulated with PMA (1 ␮g/ml) in the absence of PP.

NADPH oxidase via PKC [2]. At submicromolar concentration (8×10−7 M), where PP shows almost 80% inhibition, it has no significant PKC-inhibiting activity in an in vitro test (data not shown). Alternatively, the inhibition of superoxide formation might be due to a defective NADPH-oxidase assembly due to binding of PP with one of the oxidase components. Similar influences were described in cell free systems for mastoparan and cationic neuropeptides [15]. To test for this possibility we absorbed cell lysates (obtained after three cycles of freeze and thaw) of octylglucoside treated granulocytes on a PP-Sepharose column. The bound components were eluted and analyzed in a semi-quantitative way in an ELISA-assay using polyclonal anti p47 phox, anti p67 phox and anti gp91 phox antibodies directed against components of the NADPH oxidase complex. Antibodies against lactoferrin, a granulocyte protein (not related to the oxidase) were used as a control. Fig. 2 shows that the PP affinity column retains various components of the NADPH oxidase complex and that in our assay p47 has more affinity than p67 or the gp91 component. Such an inhibition of the oxidase by peptides which interfere with p47 has also been described [15]. Since we observed the inhibitory effect on superoxide production in viable granulocytes, we extended our study and looked for the localization of PP in intact granulocytes (in contrast to cell free systems). We used confocal fluorescence microscopy and hereby incubated granulocytes with fluorescent cholera toxin Table 3 Inhibition of superoxide production by PP in cells stimulated with fMLP Cells

PP (final concentration, M)

Inhibition (%)

PMN

10−6

5 × 10−7 10−7

96 ± 3 51 ± 9 12 ± 5

HL60 (DMSO-4d)

10−6 5 × 10−7 10−7

95 ± 5 43 ± 10 9±3

Fig. 2. Granulocyte cell extracts were bound to PP-Sepharose, subsequently desorbed by treatment with acidic and alkaline buffer solutions, lyophilized and quantitated in an ELISA assay using different antisera directed against NADPH oxidase components. Secondary antibodies were alkaline phosphatase labelled and measured at 405 nm.

B-subunit (Alexa 594-labeled) and fluorescent PP (Alexa 430-labeled). The former is known to accumulate in lipid rafts enriched in glycosphingolipid microdomains of cell membranes [7]. The latter was tested for bioactivity in the superoxide production assay and proved to be as active as the non-labeled peptide (data not shown). We observed a clear colocalisation of both fluorescent peptides in membranes of viable granulocytes at a PP concentration of 5 × 10−7 M (Fig. 3).

4. Discussion Parabutoporin is a 45-mer polycationic ␣-helical peptide [18]. Therefore, its antibiotic activity [10] and its lytic effect on eukaryotic cells [18] are not a surprise. Both are evident in the micromolar range. However, we found that at submicromolar, non lytic, concentrations PP also clearly affects granulocyte functions. Often members of the Rho family of small GTP-binding proteins, especially Rac [5] are involved in granulocyte chemotaxis and NADPH-oxidase activity [9]. We therefore tested PP in Rac activation assays on granulocytes and found that it scored even better than classical stimuli such as fMLP and PMA. Under normal physiological conditions, Rac activation of granulocytes is receptor-initiated and proceeds via heterotrimeric G-proteins and PKC/PLC or PI3 K-involving pathways. The fact that the PP-induced effects were PTX-sensitive and Wortmannin-insensitive indicated that PP effects are G-protein dependent and preferentially use the PLC pathway. Furthermore, undifferentiated HL-60 cells (having no formyl peptide receptors) also respond very well in the chemotaxis assay. They indeed react at least as well as differentiated cells which have normal receptors [13]. It thus looks as if G-proteins are directly activated by PP. Such a receptor-independent activation of G-proteins by ␣ helical

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Fig. 3. Confocal fluorescence microscopy of granulocytes incubated with Alexa Fluor 594 cholera toxin B (red) and Alexa Fluor 430 PP (green). Colocalisation is seen as orange fluorescence.

peptides has been described [12]. Our group also recently found that PP induces a reversible calcium release from intracellular stores and that this effect can be explained by such a direct activation [11]. G-protein triggering and subsequent Rac activation in granulocytes not only stimulates chemotaxis but also should increase superoxide production by the NADPH oxidase complex [5]. In striking contrast with the stimulation of chemotaxis we observed that PP causes a strong reduction of superoxide production after PMA of fMLP stimulation of the granulocytes. This effect of PP was evident at the same submicromolar concentration which activates Rac and promotes chemotaxis. Different explanations could account for this unexpected result. Firstly, superoxide levels could be lowered by simple superoxide dismutase-like activity of PP, but this was not the case. Secondly, in our assay, we used either PMA or fMLP to stimulate granulocytes for superoxide production. Both are direct (PMA) or indirect (fMLP) activators of PKC activity [2]. PKC acts upstream of the NADPH oxidase activation [2]. PP could possibly act as a PKC inhibitor. Testing PP for such PKC inhibitory activity showed no effect in the concentration range at which reduction of superoxide production was noticed. Third, a steric effect induced by the binding of the peptide with one of the oxidase components could result in defective superoxide production. Analogous interactions were described for mastoparan which, in cell free systems, can inhibit NADPH oxidase [15]. Using mastoparan, a tetradecapeptide from wasps, it was shown that its inhibitory effect on NADPH oxidase, in a cell free system, can be explained by the direct binding of the peptide to components of the oxidase complex [16]. The p67-phox reacted best and the peptide motifs in mastoparan which induced the strongest inhibition were described [17]. These motifs (two basic amino acids followed by two apolar amino acids) also are found in PP [18], which has some similarity with mastoparan. However, PP is much longer than mastoparan (45 amino acids compared to 14) and this is an even better candidate to interact with one of the NADPH oxidase components which are located at the inner leaflet of the cell membrane.

To account for these interactions in vivo, PP should accumulate in the cell membrane. This was verified by confocal microscopy and proved to be the case. The membrane localization was not unexpected since PP forms an ␣ helix in apolar media [18] and thus is a very good candidate to penetrate into membranes. In view of its length (45 amino acids), it can most probably reach the innerside of the cell and interact with signaling molecules. At this location, it could, by direct contact, stimulate G-proteins without the need of a specific receptor and also interact with one or more components of the preactivated (by Rac) NADPH oxidase complex. In conclusion, we showed that PP, at submicromolar concentrations, activates Rac in granulocytes most probably after direct activation of G proteins. Rac activation normally is expected to stimulate chemotaxis, exocytosis and superoxide production. Our PP induces a deviant effect since superoxide production is inhibited. We have explained this inhibition by presenting strong arguments in favor of membrane insertion of the PP. Once inserted, the PP can as well trigger G proteins as prevent the necessary assembly of functional NADPH oxidase by blocking the normal effect of Rac and thus account for the dual effect we observed.

Acknowledgments We thank Dr. Karin Sipido (Laboratory of Experimental Cardiology, K.U. Leuven) for excellent help in the microscopy experiments. References [1] Akasaki T, Koga H, Sumimoto H. Phosphoinositide 3-kinase dependent and independent activation of the small GTP-ase Rac 2 in human neutrophils. J Biol Chem 1999;274:18055–9. [2] Dang PM, Fontayne A, Hakim J, El Benna J, Perianin A. Priming of human neutrophil respiratory burst by granulocyte/macrophage colony-stimulating factor (GM-CSF) involves partial phosphorylation of p47phox. J Biol Chem 1999;274:20704–8. [3] Dathe M, Wieprecht T. Structural features of helical antimicrobial peptides: their potential to modulate activity on model membranes and biological cells. Biochim Biophys Acta 1999;1462:71–87.

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