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Chemistry and Physics of Lipids 164 (2011) 78–82

Contents lists available at ScienceDirect

Chemistry and Physics of Lipids

journa l homepage: www.e lsev ier .com/ locate /chemphys l ip

ultiple phospholipid substrates of phospholipase C/sphingomyelinase HR2

rom Pseudomonas aeruginosa

avid J. Lópeza, M. Isabel Colladob, Maitane Ibargurena, Adriana I. Vasil c, Michael L. Vasil c,élix M. Goni a, Alicia Alonsoa,∗

Unidad de Biofísica (Centro Mixto CSIC-UPV/EHU), and Departamento de Bioquímica, Universidad del País Vasco, Barrio Sarriena s/n, 48940 Leioa, Bilbao, SpainServicio General de Resonancia Magnética Nuclear, Universidad del País Vasco, Bilbao, SpainDepartment of Microbiology, University of Colorado Denver, Anschutz Medical Center, Aurora, CO, USA

r t i c l e i n f o

rticle history:eceived 24 June 2010eceived in revised form 2 November 2010

a b s t r a c t

The activity of phospholipase C/sphingomyelinase HR2 (PlcHR2) from Pseudomonas aeruginosa was char-acterized on a variety of substrates. The enzyme was assayed on liposomes (large unilamellar vesicles)composed of PC:SM:Ch:X (1:1:1:1; mol ratio) where X could be PE, PS, PG, or CL. Activity was measured

ccepted 2 November 2010vailable online 10 November 2010

eywords:hospholipase C/sphingomyelinase HR2

seudomonas aeruginosa

directly as disappearance of substrate after TLC lipid separation. Previous studies had suggested thatPlcHR2 was active only on PC or SM. However we found that, of the various phospholipids tested, only PSwas not a substrate for PlcHR2. All others were degraded, in an order of preference PC > SM > CL > PE > PG.PlcHR2 activity was sensitive to the overall lipid composition of the bilayer, including non-substratelipids.

hospholipase Cphingomyelinase

. Introduction

PlcHR2 is a phospholipase C/sphingomyelinase from Pseu-omonas aeruginosa that belongs to the PLC/phosphatase super-amily (Stonehouse et al., 2002). Members of this group includeroteins from Mycobacterium tuberculosis, Bordetella spp., Fran-isella tularensis and Burkholderia pseudomallei. PlcHR2 is aeterodimer composed of a catalytic subunit (PlcH, 78 117 Da) andchaperone subunit (PlcR2, 17 117 Da). The latter is believed to be

nvolved in the secretion of the complex (Shen et al., 1987). PlcHR2as been described to affect angiogenesis by inducing cytotoxic-

ty in endothelial cells (Vasil et al., 2009). A SM synthase activityas also been observed on the same protein (Luberto et al., 2003),lthough whether both activities are mechanistically related isnknown.

As a phospholipase C/sphingomyelinase, preliminary experi-ents suggested that PlcHR2 was active on PC and SM, but not

n other phospholipid classes (Stonehouse et al., 2002). This con-lusion was reached on the basis of assays in which the lipids were

Abbreviations: Cer, ceramide; Ch, cholesterol; CL, cardiolipin; DAG, diacylglyc-rol; LUV, large unilamellar vesicles; SM, sphingomyelin; PC, phosphatidylcholine;E, phosphatidylethanolamine; PG, phosphatidylglycerol; PS, phosphatidylserine;LC, phospholipase C; PlcHR2, phospholipase C/sphingomyelinase HR2.∗ Corresponding author. Fax: +34 94 6013360.

E-mail address: alicia.alonso@ehu.es (A. Alonso).

009-3084/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.chemphyslip.2010.11.001

© 2010 Elsevier Ireland Ltd. All rights reserved.

present in the form of mixed micelles with detergents, or with shortfatty acyl-chain phospholipids. More recent data have describedreliable methods for the assay of PlcHR2 lipase activities on sub-strates in bilayer form, and in the absence of detergents (Monteset al., 2007; Ibarguren et al., 2010; Montes et al., 2008). In par-ticular, the enzyme was assayed on vesicles of a rather complexcomposition, namely SM:PC:PE:Ch (1:1:1:1; mol ratio), to whichthe end-products DAG and/or Cer were occasionally added (Monteset al., 2007; Ibarguren et al., 2010). It was found that, at least atthe initial stages of the reaction, only PC and SM were hydrolyzed(Montes et al., 2007) in agreement with the previous observations(Stonehouse et al., 2002). Moreover, DAG was seen to enhance theenzyme activity, while Cer acted as an inhibitor (Ibarguren et al.,2010). We report here that under certain conditions, that may occurin the in vivo situation, PlcHR2 can cleave phospholipids other thanPC or SM.

2. Materials and methods

2.1. Materials

PlcHR2 was purified as previously described (Stonehouse et al.,

2002). Egg PE, egg PC, egg PG and DAG were purchased fromLipid Products (South Nutfield, UK). CL, PS, egg SM, egg Cerand cholesterol were from Avanti Polar Lipids (Alabaster, AL). N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) waspurchased from Apollo Scientific Ltd. (Cheshire, UK). Other

D.J. López et al. / Chemistry and Physics of Lipids 164 (2011) 78–82 79

Fig. 1. Differential PlcHR2 phospholipase activity measured at 37 ◦C. 0.5 �g/ml PlcHR2 were assayed at 37 ◦C on 0.3 mM LUV composed of (A) PC:SM:PE:Ch (1:1:1:1; molr (D) PCp eparad 7.4. Ao .05. (Fa

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atio), (B) PC:SM:PE:Ch (1:1:1:1) + 10% DAG, (C) PC:SM:PE:Ch (1:1:1:1) + 10% Cer,roportions than graph A after 10 min incubation. PC (�), SM (�) and PE (�) were sensitometry. All experiments were performed in 25 mM HEPES, 150 mM NaCl, pHf the amounts of phospholipids after 10 min incubation. **p < 0.01; *p < 0.05; —p > 0fter enzyme addition.

eagents, silica G-60 plates and solvents for chromatographicssays were from Merck (Darmstadt, Germany).

.2. Liposome preparation

Large unilamellar vesicles (LUV) of diameters 100–150 nm wererepared by the extrusion method (Mayer et al., 1986) usinguclepore filters 0.1 �m pore diameter at room temperature, in5 mM HEPES, 150 mM NaCl, pH 7.4. Final lipid concentration was.3 mM. Phospholipid concentration was measured in terms of

ipid phosphorous (Bartlett, 1959). Quantitative analysis of the lipidomposition of our LUV preparations, as described by Ruiz-Argüellot al. (1996), showed that it did not differ significantly from thenitial lipid mixture.

.3. PlcHR2 activity on LUVs measured by TLC

Phospholipase activity on LUVs was assayed either by quan-itative thin layer chromatography (TLC) or by determining

:SM:PE:Ch (1:1:1:1) + 5% DAG + 5% Cer and (E) PC:SM:PE:Ch:DAG:Cer at the sameted by thin layer chromatography and the intensity of each spot was measured byverage values ± SEM (n = 3). The inset in each graph represents the Student’s t-test) TLC chromatographic spots corresponding to the conditions of (A) at 0 and 15 min

water-soluble phosphorous release. LUVs of different compositionswere diluted to 0.3 mM in 25 mM HEPES, 150 mM NaCl, pH 7.4 andincubated at 37 ◦C with 0.5 �g/ml PlcHR2 at different times. Lipidextraction was performed using a modification of the Folch method(Folch et al., 1957). For the TLC experiments, 200 �l liposome sus-pension was mixed with 1 ml chloroform:methanol:hydrochloricacid (66:33:1, v:v:v) and vortexed thoroughly. The tubes werecentrifuged at 14 000 × g in an Eppendorf centrifuge in order toseparate two phases. The lower organic phase was evaporatedunder nitrogen flow, resuspended in 80 �l chloroform and sepa-rated on TLC silica G60 plates using chloroform:methanol:aceticacid:water (60:50:1:4, v/v/v/v) as solvent. Plates were charredwith 10% sulfuric acid (v/v) followed by heating at 110 ◦C for

15 min. Quantification of lipids was performed by measuring theoptical densities of each spot using a GS-800 densitometer fromBio-Rad Laboratories (Hercules, CA). Note that densitometer mea-surements were compared between spots corresponding to thesame lipid at each time and not between lipids. The initial amount

80 D.J. López et al. / Chemistry and Physics of Lipids 164 (2011) 78–82

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ig. 2. Nuclear magnetic resonance analysis of PlcHR2 phospholipase activity. (A) RUV previous to the enzyme incubation. Vesicle composition was initially PC:SM:Pn that particular assay. (B) Representative NMR spectrum showing the chemical snder each lipid indicate their amount relative to PG in that particular assay.

f each lipid was quantified as lipid phosphorus with a molybdateeagent.

.4. PlcHR2 activity on LUVs measured by NMR

5 mM LUV of PC:SM:PE:Ch (1:1:1:1; mol ratio) were incu-ated in 25 mM HEPES, 150 mM NaCl, pH 7.4 with 8.3 �g/mllcHR2 for 30 min, at 37 ◦C. Lipids were extracted in chloro-orm:methanol:buffer (2:1:0.6; v:v) and the organic phase wasupplemented with egg-PG at the same proportion than the indi-idual lipids. The lipid mixture was evaporated under a streamf N2 and completely devoid of chloroform traces under vacuumor 2 h. The lipid film was resuspended in 750 �l deuterated chlo-oform:methanol:200 mM EDTA-Cs (500:200:50; v:v) (Menesesnd Glonek, 1988; Sachedina et al., 1991). The organic phase wasetrieved, dehydrated with a 4 A molecular sieve and then trans-erred to 5 mm NMR tubes. Data acquisition was performed in aruker AV500 spectrometer (Rheinstetten, Germany) operating at02.4 MHz for 31P, with a 5 mm wide-band probe and a gradient

n the Z-axis, at 25 ◦C. The experiments were performed with thegig sequence (Bruker) and a delay time of 15 s between scans. Dataere processed with a 1 Hz exponential factor.

.5. Statistics

Unless otherwise indicated, data are average values of threendependent measurements ± standard error (SEM). Student’s t-

entative NMR spectrum showing the chemical shift of the different components of1:1:1:1; mol ratio). Numbers under each lipid indicate their amount relative to PGf the different components of LUV after 30-min incubation with PlcHR2. Numbers

test was used in order to assess the significance of observeddifferences.

3. Results and discussion

In order to determine the substrate specificity of PlcHR2, theenzyme was incubated with LUV composed of SM:PC:PE:Ch atequimolar ratios. The reaction was allowed to proceed for a ratherlong time, i.e. 15 min. Aliquots were retrieved at various times, eachof the phosphorous containing lipids were separated by TLC andthen quantified as detailed under Section 2. At 37 ◦C, the controlmixture (Fig. 1(A)) shows that PC was rapidly hydrolyzed with vir-tually no latency period. SM hydrolysis ensued after about 2 min.Then, at about 10 min after enzyme addition, some PE hydrolysiswas observed, that proceeded steadily until, at 15 min, the reac-tion was stopped (Fig. 1). Representative TLC chromatographicspots are shown in Fig. 1(F), corresponding to mixtures 0 and15 min after enzyme addition. In our previous study (Montes et al.,2007) the reported enzyme activities correspond mostly to initialenzyme rates, as appropriate for the kinetic measurements thatwere intended there, and the hydrolytic reactions were stopped

after 10 min at the latest, when PE had been hardly attacked by theenzyme, hence our mistaken conclusions about enzyme specificity.

Addition of 10 mol% DAG to the bilayer composition at thetime of LUV preparation did not change the maximum slope (i.e.the initial slope after the lag time) of the PlcHR2 total hydrolytic

D.J. López et al. / Chemistry and Physics of Lipids 164 (2011) 78–82 81

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ig. 3. Differential PlcHR2 phospholipase activity measured at 28 C. 0.5 �g/ml Plcatio), (B) PC:SM:PE:Ch (1:1:1:1) + 10% DAG (C) PC:SM:PE:Ch (1:1:1:1) + 10% Cer any thin layer chromatography and the intensity of each spot was measured by densin = 3). The inset in each graph represents the Student’s t-test of the amounts of pho

ctivity (Ibarguren et al., 2010), although the lag time for enzyme-ependent vesicle aggregation, that is strictly dependent on theydrolytic activity (Basánez et al., 1996), was decreased by onerder of magnitude (Ibarguren et al., 2010). The time-course ofydrolysis of the DAG-containing bilayers can be seen in Fig. 1(B).nder these conditions PC and SM appear to be degraded largelys in the absence of DAG. PE however parallels closely the patternf SM degradation for the whole duration of the experiment.

Both 10 mol% Cer and a mixture of 5 mol% Cer + 5 mol% DAG hadhe effect of decreasing PlcHR2 hydrolysis rate (Ibarguren et al.,010). When the time-course of hydrolysis of the individual lipids

s measured (Fig. 1(C and D)) PE is again the lipid most affected byhe presence of the inhibitory end-product, remaining undegradedntil about 10 min after enzyme addition.

From the data in Fig. 1(A–D) it can be concluded that PE is indeedegraded by PlcHR2, that its hydrolysis starts at a later stage thanhat of SM or PC, and that its latency period is particularly sensitiveo the presence of the end-products DAG and Cer, the former short-ning and the latter prolonging the lag time. The above data alsohow that, under these conditions, PC is degraded by the enzymereferentially over SM, and indeed, PE.

Hydrolysis of PC, SM and PE by PlcHR2 in LUV composed ofC:SM:PE:Ch (1:1:1:1) was also assayed by 31P NMR. The enzymeeaction was stopped by addition of chloroform:methanol (2:1,/v). A known amount of egg PG was added to the organic extract asquantification standard. 31P NMR spectra of the lipids extracted

t 0 and 30 min after enzyme addition are shown in Fig. 2. Notehe relative areas under each peak. All three phospholipids haveeen degraded, but to a different extent, again in decreasing orderf preference PC > SM > PE.

Some experiments were directed at finding conditions underhich preferential hydrolysis of phospholipids would be changed.

n one case the experiments were performed at 28 ◦C, instead of7 ◦C. As seen in Fig. 3, under these conditions PE is not degraded in0 min. PC and SM are both degraded to a similar extent, but clearly

ere assayed at 37 C on 0.3 mM LUV composed of (A) PC:SM:PE:Ch (1:1:1:1; molC:SM:PE:Ch (1:1:1:1) + 5% DAG + 5% Cer. PC (�), SM (�) and PE (�) were separated

ry. All experiments were performed in 25 mM HEPES, pH 7.4. Average values ± SEMlipids after 15 min incubation. **p < 0.01; *p < 0.05; —p > 0.05.

less than at 37 ◦C, the proportion of PC and SM cleaved after 15 minat 28 ◦C being respectively ≈ 50% and 65% the amounts hydrolyzedat 37 ◦C. Also at 28 ◦C the end-products DAG or Cer fail to have anyclear effect, except perhaps a small activation of PC hydrolysis byDAG (Fig. 3(D)). Thus the general trend appears to be that, whenthe enzyme is working at low rates, the preference for PC and SMis more marked. The decreased rates may also explain the lack ofobservable effects of activators/inhibitors.

In a different experiment, the possibility was tested that PEhydrolysis was elicited after 10 min at 37 ◦C because of the par-ticular composition of the sample under those conditions. To testthis hypothesis, LUV were prepared with a composition mimickingthe above system after 10 min, according to the data in Fig. 1(A), i.e.PC:PE:SM:Ch:DAG:Cer (2.48:11.75:7.28:15:15.77:7.72; mol ratio).These LUV were incubated at 37 ◦C, PlcHR2 was added and the reac-tion monitored for a further 15-min period. The results are shownin Fig. 1(E). PC and SM hydrolysis proceeds more slowly than underthe original conditions (Fig. 1(A), 10–15 min time range), while PEremains unaltered. We conclude that either PE hydrolysis is notrelated to the presence of a certain bilayer composition, or elsethat LUV preparation under the conditions of Fig. 1(E) does not ade-quately mimic the conditions of Fig. 1(A) after 10 min. Under theconditions of Fig. 1(A), lateral (domains) or transbilayer (asymme-try) heterogeneities may occur that are not reproduced in Fig. 1(E).A similar case of enzyme-induced heterogeneities was describedfor a combination of Bacillus cereus phospholipase C and B. cereussphingomyelinase (Ruiz-Argüello et al., 1998).

The capacity of PlcHR2 to hydrolyse phospholipids other than PCor SM was further explored with mixtures of the kind SM:PC:X:Ch(1:1:1:1), where X was PE, PS, PG or CL. The reaction was allowed to

proceed for several hours to ensure that phospholipid degradationwas maximal. The results are summarized in Fig. 4, in which theamounts of unreacted (undegraded) phospholipid are represented.The various phospholipids are degraded in the order of preferenceCL > PE > PG > PS, the latter remaining virtually unaltered. It is note-

82 D.J. López et al. / Chemistry and Phys

Fig. 4. PlcHR2 hydrolysis of different phospholipid species. LUV of a compositionSM:PC:Ch:X (1:1:1:1; mol ratio), where X could be either PE, PS, PG, or CL, weretreated with PlcHR2 and the lipids in the reaction mixture were extracted, sepa-rT(

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class of microbial phosphocholine-specific phospholipases C. Mol. Microbiol. 46,661–676.

Vasil, M.L., Stonehouse, M.J., Vasil, A.I., Wadsworth, S.J., Goldfine, H., Bolcome 3rd,

ated by TLC, and quantified. All mixtures contained initially 15 nmol of each lipid.he figure represents the amounts of unreacted phospholipids. Average values ± SDn = 3).

orthy that the degradation of SM and PC is influenced by the thirdhospholipid: PC and SM are hydrolysed to a lesser extent whenS is present, and the opposite occurs in the presence of PE. It isot uncommon to find phospholipases whose hydrolytic activity is

nfluenced by the overall lipid composition of the bilayer, includ-ng the presence of non-substrate lipids (Contreras et al., 2004; Pirett al., 2005; Ahyayauch et al., 2005; Nagahama et al., 2007).

We conclude that PlcHR2 from Pseudomonas aeruginosa couldse a variety of phospholipids as substrates. Of those studiedere, the order of preference was PC > SM > CL > PE > PG. PS wasot hydrolyzed. The rates of hydrolysis of the individual phos-holipids were influenced by the overall lipid composition of theilayer.

cknowledgements

This work was supported in part by the Spanish Ministerio deducación y Ciencia (BFU2008-01637/BMC) (A.A), Etortek (07/26)A.A.), (BFU 2007-62062) (F.M.G.), Basque Government (IT461-07)F.M.G.) and NIH (HL062608) (M.L.V). D.J.L. and M.I. were graduatetudents supported by the Basque Government.

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