Kellie J Hall

Matthew L Jones

Alastair W Poole
email: a.poole@bris.ac.uk

C signaling

Keywords: protein kinase C, Src family kinase, phosphorylation, thrombin, PAR-1, PAR-4 GPCR, G protein-coupled receptor, PKC, protein kinase C, ADP, adenosine

INTRODUCTION

The protein kinase C (PKC) family comprises 10 isozymes grouped into 3 classes: conventional (α, γ, βI, βII), novel (δ, ε, η/L, θ) and atypical (ζ, ι/λ) [START_REF] Newton | Protein kinase C: structural and spatial regulation by phosphorylation, cofactors, and macromolecular interactions[END_REF], of which human platelets express predominantly 4 isoforms, α, β, δ and θ [2-8]. The PKC family has long been known to be involved in a number of platelet processes, most importantly aggregation and secretion, where stimulation of platelets with diacyl glycerol (DAG) or phorbol ester can induce aggregation, and agonist-induced secretion can be prevented by pharmacological inhibition of a broad range of PKC isoforms [9-12]. Individual PKC isoforms however are likely to play distinct roles, and we have recently shown PKCδ to play a critical role negatively regulating collagen-induced platelet aggregation through inhibition of VASPmediated filopodia formation [START_REF] Pula | PKCδ regulates collagen-induced platelet aggregation through inhibition of VASP-mediated filopodia formation[END_REF].

Activation mechanisms for protein kinase C (PKC) family members involve the summation or synergy between multiple input signals, including classically cytosolic free calcium and diacylglycerol. These signals have in turn been thought to regulate recruitment of PKCs to the plasma membrane, where the kinase is able to be fully activated and where it encounters many of its substrates. Very recently it has been elegantly shown for example that PKCα is capable of very rapidly moving to and from the plasma membrane [14], controlled by rapid changes in cell calcium, demonstrating PKC to be a key readout sensor for the wide variety of transient calcium responses seen in many cell types.

Additionally however PKC isoforms undergo a series of phosphorylation events on serine/threonine residues, and for PKCδ in particular also on tyrosines [3, 15-29]. Human PKCδ contains 20 tyrosine residues, several of which have been shown to be phosphorylated including Tyr52, Tyr155, Tyr187, Tyr311, Tyr332, Tyr525 and Tyr565 [START_REF] Steinberg | Distinctive activation mechanisms and functions for protein kinase Cδ[END_REF]. These residues lie primarily either within the catalytic domain or the hinge region of PKCδ, and are generally not conserved between other PKC family members, making regulation by tyrosine phosphorylation relatively specific for PKCδ. PKCδ has been shown to be phosphorylated on tyrosine in human platelets in responses to agonists [31-33], including thrombin, and it has recently been shown that Tyr311 is phosphorylated in response to PAR1 and PAR4 activatory peptides [33]. Tyr311 lies within the hinge region of PKCδ, between the C1 and catalytic domains, and sits within an optimal Src substrate consensus sequence. Phosphorylation of this residue in other cell types has been linked to increased kinase activity and altered downregulation or degradation of the kinase [34], although the role of this phosphorylation in platelets has not been directly addressed.

EXPERIMENTAL Materials

Anti-phosphotyrosine monoclonal antibody 4G10, anti-phospho-Src (Tyr-416) clone9A6, anti-Src antibody and recombinant protein tyrosine phosphatase 1B (PTP-1B) were from Upstate Biotechnology Inc. (Lake Placid, NY). Phospho-specific antibodies against specific tyrosine sites on PKCδ and anti-GPIb (anti-CD42b antibody) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti-PKCδ antibody was from BD Biosciences (Oxford, UK). Anti-actin antibody was purchased from Lab Vision Corp (Suffolk, UK). Horseradish peroxidase-conjugated secondary antibodies, enhanced chemiluminescence (ECL) kits and [γ-32 P]ATP were purchased from Amersham Biosciences.

Collagen was purchased from Nycomed (Munich, Germany) and Complete mini protease inhibitor tablets were from Roche Applied Science. Monoclonal anti β-tubulin antibody, monoclonal anti-α-tubulin antibody, protein phosphatase inhibitor cocktail mixture I, bovine thrombin, ADP and Protein-A sepharose were from Sigma-Aldrich. The PAR-1 specific agonist SFLLRN and PAR-4 specific agonist AYPGKF were from Bachem (Merseyside, UK). The PKC inhibitor GF109203X, Phorbol 12-myristate 13-acetate (PMA), U73122 and PP2 was from Tocris (Avonmouth, UK). Piceatannol and PP1 were from Alexis Corp (Nottingham, UK). Protein kinase Cδ peptide substrate, PP3 and LFM-A13 were purchased from Calbiochem (LaJolla, CA). Anti-phosphothreonine (Thr-505) PKCδ antibody was from Cell Signalling Technology (New England Biolabs, Hitchin, UK). All other reagents were of analytical grade.

Preparation and Stimulation of Human Platelets

Human blood was drawn from healthy, drug-free volunteers on the day of the experiment into the anticoagulant sodium citrate (1:20 (v/v) dilution of a 4% solution). Acid citrate dextrose (120 mM sodium citrate, 110 mM glucose, 80 mM citric acid, used at 1:7 v/v) was used as a further anticoagulant. Platelet-rich plasma (PRP) was prepared by centrifugation at 180 x g for 20 min, and platelets were then isolated by centrifugation for 10 min at 550 x g in the presence of prostaglandin E1 (40 ng/ml). The pellet was resuspended to a density of 4 x 10 8 platelets/ml in a modified Tyrode's-HEPES buffer (145 mM NaCl, 2.9 mM KCl, 10 mM HEPES, 1 mM MgCl2, 5 mM glucose, pH 7.3). Indomethacin (10 µM) was added to this platelet suspension, and a 30-min rest period was allowed before stimulation. All platelet stimulation occurred in the presence of EGTA (1 mM). Platelets were preincubated with different inhibitors or vehicle solution (DMSO) for 10 min at 37 °C, and stimulated in an aggregometer (Chrono-Log Corp., Labmedics Ltd., Manchester, UK) at 37 °C, with continuous stirring at 800 rpm. Reactions were halted by either addition of 5xSDS sample buffer (24 mM Tris, pH 6.8, 10% (v/v) glycerol, 0.8% (v/v) SDS, 6 mM 2-mercaptoethanol, and 0.04% bromophenol blue) to produce whole cell lysates (WCL) or by addition of 2xNonidet P-40 lysis buffer for immunoprecipitation (as described below).

Immunoprecipitation of PKCδ

Reactions were stopped by lysis of platelets with an equal volume of 2x Nonidet P-40 lysis buffer (NP-40) (100 mM Tris, 300 mM NaCl, 20mM EDTA, 1mM Na 3 VO 4 and 2% (v/v) Nonidet P-40 substitute, pH 7.5), plus complete protease inhibitors . Lysates were precleared with protein-A-Sepharose beads for 1 hour. Antibody-protein-A complexes were formed by incubation of protein-A-Sepharose with 1 µg of antibody for 1 hour at room temperature.

Precleared lysates were added to the antibody-protein-A complexes and incubated at 4 °C with constant rotation overnight. Immunoprecipitates were washed 3 times with 1x NP-40 before addition of 5 x SDS sample buffer and boiling for 5 minutes.

Electrophoresis of Proteins and Western Blotting

Proteins were resolved by electrophoresis in 9-12% SDS-PAGE gels. Samples were then transferred to polyvinylidene difluoride membranes, blocked with 5-10% bovine serum albumin, and incubated for 1 h or overnight at room temperature with the appropriate primary antibody. Membranes were then washed before incubation with the appropriate secondary antibody followed by thorough washing. Bound peroxidase activity was detected using enhanced chemiluminescence (ECL).

In Vitro Kinase Assays

PKCδ was immunoprecipitated from Nonidet P-40 lysates as described and washed 3 times with 1x Nonidet P-40 wash buffer containing 0.5mM Na 3 VO 4 . Some thrombin treated samples were dephosphorylated by exposure to recombinant PTP-1B (1µg, specific activity 13nmol•min -1 •µg -1 using p-nitrophenylphosphate) for 10 mins at 37ºC. All immunoprecipitates were then washed 3 times with 1x Nonidet P-40 wash buffer containing 1mM Na 3 VO P]-ATP). After incubation at 30ºC for 30 mins, the reaction was terminated by addition of 10% phosphoric acid and samples were added to 3cm squares of P81 ion exchange chromatography paper. The P81 papers were washed 5 times in 0.5% phosphoric acid, followed by a wash in acetone. Papers were then dried and labelled PKCδ peptide substrate was quantified by liquid scintillation counting.

Subcellular fractionation of human platelets

Basal and stimulated platelets were resuspended in ice-cold 2x sonication buffer (final concentration 320mM Sucrose, 4mM HEPES, 0.5mM Na 3 VO 4 , pH 7.4) containing complete protease inhibitors, and sonicated for 4 x 20s pulses. Intact platelets were removed by centrifugation at 1500 x g for 10mins before centrifugation at 100,000 x g for 60min. The supernatant was removed (cytosolic fraction) and the pellet (particulate fraction) was resuspended in Tris-HCl buffer containing 10mM Tris, 158mM NaCl, 1mM EGTA, 0.5mM Na 3 VO 4 , 0.1% SDS, 1% sodium deoxycholate, 1% Triton-X100, pH7.2, with complete protease inhibitors. Protein concentrations were quantified using the BCA-assay (Sigma-Aldrich). Equal protein concentrations of the fractions were either resolved by SDS-PAGE and western blotted for tubulin or GPIb to confirm fractionation had occurred, or PKCδ was immunoprecipitated from each fraction as described above, resolved by SDS-PAGE and western blotted using anti-PKCδ or phospho-specific antibodies.

Data Analysis

Analysis of statistical significance was performed using one-way ANOVA with Bonferroni Post test if p<0.05. In all cases, p<0.05 was considered significant.

RESULTS

Thrombin induces phosphorylation of PKCδ on Y311 and Y565 in human platelets

PKCδ has been shown to be phosphorylated on tyrosine in multiple cell types including platelets [START_REF] Murugappan | Differential role of PKC-δ isoform in agonist-induced dense granule secretion in human platelets[END_REF][31][START_REF] Moussazadeh | Protein kinase C-delta activation and tyrosine phosphorylation in platelets[END_REF][START_REF] Murugappan | Molecular mechanism and functional implications of thrombin-mediated tyrosine phosphorylation of PKCδ in platelets[END_REF]. A recent report had shown that PAR1 and PAR4 peptides were able to induce phosphorylation of Y311 [33], a residue shown by others to be important in regulating proteolytic degradation of PKCδ [34]. It was now important to understand the interplay between phospholipase C signalling and tyrosine phosphorylation of PKCδ, and in particular whether these two events were interdependent or unrelated, and how they contributed to the activity of PKCδ. In the first instance it was important to determine whether thrombin could induce phosphorylation of PKCδ on Y311 and other putative tyrosine phosphorylation sites.

Both thrombin and collagen were able to induce phosphorylation of PKCδ on tyrosine (Fig. 1A) and thrombin induced a transient phosphorylation of both Y311 and Y565 (Fig. 1B), which was apparent at an early time point (20 s) but had substantially disappeared by 3 min. This was paralleled by a time course of phosphorylation on these residues in response to peptide activation of PAR1 and PAR4 receptors (Fig. 1C), with PAR1 activation profile being highly transient, and PAR4 being more sustained. A time course of phosphorylation on Y311 and Y565 showed that for collagen these events occurred more slowly and achieved maximal phosphorylation by 3 min (Fig. 1C(ii)). It has been demonstrated that Y52, Y155, Y332 and Y525 may also be phosphorylated in other cell types [15, 22, 27, 35-37]. Figure 2 shows that, in response to thrombin, collagen or PAR1/4 peptides, these tyrosines are not phosphorylated, although pervanadate used as a positive control is able to induce phosphorylation of these sites.

Phospholipase C signaling is necessary and sufficient to induce phosphorylation of

PKCδ on Y311 and Y565 downstream of Src family kinases

Platelets express a variety of non-receptor tyrosine kinases that may be responsible for phosphorylation of PKCδ on Y311 and Y565. Figure 3A shows that the Src family kinase (SFK) inhibitors PP1 and PP2, but not the inactive analogue PP3 nor the Syk inhibitor piceatannol nor the Btk inhibitor LFM-A13, are able to block phosphorylation on these two sites induced by thrombin. Phosphorylation of PKCδ on Y311 and Y565 is therefore downstream of SFKs, but also requires phospholipase C (PLC) activity, since it is blocked by the PLC inhibitor U73122.

Importantly, allosteric activation of PKCδ by the phorbol ester PMA, which bypasses receptor activation, is also able to induce phosphorylation of Y311 and Y565 in a Src kinasedependent manner (Fig. 3B). Because receptor activation is avoided, phosphorylation induced by PMA is not dependent upon PLC activity, and is not blocked by U73122. It was possible therefore either that SFK activity was dependent upon PMA, and in the case of thrombin was dependent upon diacyl glycerol, or that these lipid signals recruit PKCδ to the membrane where a basally active SFK may phosphorylate it, or a combination of both. We therefore assessed whether PMA or thrombin were able to activate SFK by using a phosphospecific antibody to pY416(Src), which recognises the autophosphorylation site in SFKs and is therefore a marker of activity in the cell. Figure 4 shows a basal SFK activity, which is ablated by PP1, and which is increased by approximately 2 fold by stimulation with thrombin. There is still substantial thrombin-dependent SFK activation in platelets pre-treated with U73122, the PLC inhibitor, and PMA alone is not able to induce SFK activation. These data suggest that basal activity of Src is sufficient to allow full phosphorylation of PKCδ on Y311 and Y565, and therefore that cellular localisation of PKCδ is a more critical regulatory step than regulation of Src kinase activity per se.

Phosphorylation of PKCδ on Y311 and Y565 is not required for membrane recruitment, whereas membrane recruitment is required for phosphorylation on these residues

The data from Figs 3&4 were consistent with an activation process where PLC signaling products lead to recruitment of PKCδ to the membrane, whereupon SFKs are able to phosphorylate it. If this were the case then SFKs should not be required for membrane recruitment. Figure 5A shows that PKCδ translocates rapidly but transiently from cytosol to membrane compartments, in a manner that parallels its tyrosine phosphorylation (Fig. 1).

Importantly, phosphorylation of Y311 and Y565 occur only in membrane-associated fractions, and not in PKCδ localised to the cytosol, consistent with recruitment to the membrane being required for phosphorylation. Phosphorylation on tyrosine was however not required for translocation, since blockade of phosphorylation by PP1 had no effect upon membrane recruitment of the kinase (Fig. 5B). Conversely, membrane recruitment of PKCδ did require PLC activity, since blockade of PLC by U73122 substantially reduced its translocation.

PMA, which bypasses receptor-mediated PLC activation, also induced membrane recruitment of PKCδ (Fig. 6), and also induced tyrosine phosphorylation of Y311 and Y565 only when Regulation of PKCδ in platelets by tyrosine phosphorylation the kinase was localised to the membrane. The time courses of membrane recruitment and phosphorylation on Y311 or Y565 show that membrane recruitment is rapid, being maximal by 30 s and sustained over 5 min, yet phosphorylation does not take place until later time points (Fig. 6A). Again, PP1 abolished tyrosine phosphorylation of PKCδ, but had no effect upon membrane translocation (Fig. 6B). In summary therefore these data are consistent with a model whereby PKCδ translocates to the membrane and is subsequently phosphorylated on Y311 and Y565 in a SFK-dependent manner.

Tyrosine phosphorylation of PKCδ potentiates its kinase activity

From Figs 5 & 6 it can be concluded that tyrosine phosphorylation of PKCδ has no role to play in translocating the kinase from cytosol to membrane, and in fact membrane recruitment is a requirement for tyrosine phosphorylation. It was therefore important to ascertain whether tyrosine phosphorylation of Y311 and Y565 played a role in regulating the kinase activity of PKCδ. Figure 7A(i) shows an in vitro kinase assay, demonstrating that thrombin stimulation of platelets leads to an increased activity of PKCδ. This increased activity is partially but non-significantly inhibited when platelets were pretreated with PP1, which abolished tyrosine phosphorylation of Y311 and Y565 (Fig. 7A(ii)). Importantly, because of other potential effects of SFK inhibition in the cell, we were able to dephosphorylate PKCδ in vitro using the tyrosine phosphatase PTP1b (Fig. 7A(ii)). Dephosphorylation in vitro also partially but nonsignificantly reduced the activity of PKCδ (Fig. 7A(i)), to the same extent as PP1 when applied to platelets. This suggested that phosphorylation on Y311 and Y565 may partially contribute to and potentiate kinase activation of PKCδ downstream of thrombin.

In order to demonstrate that the activity of PKCδ in platelets, rather than in vitro, was similarly partially dependent upon tyrosine phosphorylation by SFKs, Fig. 7B shows data demonstrating phosphorylation of PKCδ on Thr505. Autophosphorylation on this site provides a read-out of kinase activity in the cell, and Fig. 7B shows that thrombin induces a marked increase in phosphorylation of this residue, thereby confirming activation of the kinase by this agonist in platelets. Pre-treatment of platelets with PP1, the SFK inhibitor, partially inhibits activity of the kinase, paralleling data from in vitro kinase assay shown in Although it had previously been shown that PKCδ is phosphorylated on tyrosine in human platelets in response to thrombin [3, 32], only one report has so far identified any sites of phosphorylation in these cells [33]. [START_REF] Murugappan | Molecular mechanism and functional implications of thrombin-mediated tyrosine phosphorylation of PKCδ in platelets[END_REF] had shown that Tyr311 to be phosphorylated in response to activation by PAR1-and PAR4-selective peptide agonists, although the authors do not demonstrate phosphorylation on any other tyrosine residues [START_REF] Murugappan | Molecular mechanism and functional implications of thrombin-mediated tyrosine phosphorylation of PKCδ in platelets[END_REF].

Here we have extended this initial observation by demonstrating that the physiological agonist thrombin is able to induce phosphorylation on Tyr311, but additionally that phosphorylation also occurs at Tyr565, although not Tyr52, Tyr155, Tyr332 or Tyr525. The reason for the discrepancy between our study and that of [START_REF] Murugappan | Molecular mechanism and functional implications of thrombin-mediated tyrosine phosphorylation of PKCδ in platelets[END_REF] [START_REF] Murugappan | Molecular mechanism and functional implications of thrombin-mediated tyrosine phosphorylation of PKCδ in platelets[END_REF], who did not observe phosphorylation of Tyr565, is not known at present. Throughout this study the phosphorylation status of Tyr565 paralleled phosphorylation of Tyr311, and we were not able to distinguish any circumstances in which phosphorylation of one residue would occur in the absence of the other. Blake et al. (1998) had shown that although PKCδ could be phosphorylated on multiple Tyr residues, Tyr311 phosphorylation was a 'master' site, in that mutation of this residue to a non-phosphorylatable Phe ablated phosphorylation of PKCδ on all other tyrosines. This may be the case in the platelet system also, where Tyr311 may be required for phosphorylation of Tyr565, but we were not able to determine this within the present study. The time courses of phosphorylation of each residue are very rapid and do not substantially differ from each other, although at least for PAR4 peptide activation (Fig. 1C) Tyr311 phosphorylation is maximal by 20s, whereas for Tyr565 phosphorylation is not maximal until 1 min. This may suggest that phosphorylation of Tyr311 may precede that of Tyr565, least in response to activation of PAR4, and therefore that Tyr311 phosphorylation may be a pre-requisite for phosphorylation of Tyr565.

We show that Tyr311 phosphorylation is downstream of Src family kinases (Fig. 3A) in response to thrombin. This is consistent with [START_REF] Murugappan | Molecular mechanism and functional implications of thrombin-mediated tyrosine phosphorylation of PKCδ in platelets[END_REF] who showed Src kinase-dependent phosphorylation of this residue in response to PAR1 and PAR4 activation [START_REF] Murugappan | Molecular mechanism and functional implications of thrombin-mediated tyrosine phosphorylation of PKCδ in platelets[END_REF], and is consistent with the fact that this residue sits within an excellent Src phosphorylation consensus sequence. We had shown previously that PKCδ selectively complexes with the tyrosine kinase Fyn in activated conditions [31], and this may therefore be the Src family kinase responsible for phosphorylating PKCδ in response to thrombin.

Phospholipase C activity was also required for phosphorylation on both Tyr311 and Tyr565, since phosphorylation was blocked by the PLC inhibitor U73122. This suggested either that the Src kinase responsible for phosphorylating PKCδ was activated downstream of PLC, or that PKCδ recruitment to the plasma membrane and allosteric regulation was required to allow phosphorylation. We showed in Fig. 4A however that although thrombin was able to induce activation of Src family kinases, this was largely independent of PLC activity, since U73122 did not significantly decrease Src activity in response to thrombin. This suggests that the ablation of phosphorylation of PKCδ on Tyr311 and Tyr565 in the presence of U73122 may be because Src family kinases are able to phosphorylate PKCδ when allosterically modulated by diacylglycerol. It needs also to be mentioned that U73122 has been shown also to have actions other than inhibition of PLC, including inhibition of PLD [START_REF] Bosch | U73122 and U73343 inhibit receptor-mediated phospholipase D activation downstream of phospholipase C in CHO cells[END_REF] and stimulation of calcium release [START_REF] Muto | The putative phospholipase C inhibitor U73122 and its negative control, U73343, elicit unexpected effects on the rabbit parietal cell[END_REF], and therefore it is possible that inhibition of translocation of PKCδ and its phosphorylation on tyrosine by this compound may be through an off-target inhibitory mechanism. The observation that the phorbol ester PMA also induced phosphorylation of both tyrosine residues on PKCδ indicated that membrane localisation and allosteric regulation were the important signals required to allow tyrosine phosphorylation. Consistent with this model, PMA had no effect upon Src kinase activity per se. Also consistent with this model is that basal activity of Src is sufficient to allow full phosphorylation of PKCδ on Y311 and Y565. This suggests therefore that cellular localisation of PKCδ is a more critical regulatory step for controlling it tyrosine phosphorylation than regulation of Src kinase activity per se. We would therefore propose from the data that phosphorylation of PKCδ on Tyr311 and Tyr565 occurs in a Src kinase-dependent manner in response to thrombin, only when PKCδ is allosterically activated. This would suggest that tyrosine phosphorylation may only occur when PKCδ has translocated to the particulate fraction. This was indeed shown to be the case in Fig. 5A, where we show that thrombin induces a transient membrane translocation of the kinase, and that PKCδ is phosphorylated on Tyr311 and Tyr565 only when translocated to the membrane. Indeed, at late time points (3 min) when PKCδ had re-entered the cytosolic fraction, the kinase was also no longer phosphorylated on tyrosine. These data suggested therefore that the early translocation to the membrane would occur independent of tyrosine phosphorylation, and that phosphorylation occurred subsequent to the translocation event.

Using PP1, which blocks tyrosine phosphorylation, Figs 5B & 6 showed that indeed tyrosine phosphorylation was not required for translocation to the membrane, whether induced by thrombin or PMA. These data would suggest that, in the case of PMA which does not significantly increase activity of Src above basal levels, translocation to the plasma membrane for sustained periods is sufficient to allow slow phosphorylation of Src on Y311 and Y565 (Fig. 6A). For the thrombin, which induces a doubling in Src activity (Fig. 4), the kinetics of phosphorylation of PKCδ on tyrosine are markedly more rapid (Fig. 5A). However, although U73122 substantially reduces translocation of PKCδ to the membrane, it does not abolish it (Fig. 5). Despite some translocation, phosphorylation of Y311 and Y565 is completely ablated (Fig. 5), and this may therefore suggest that translocation alone may not be sufficient to allow phosphorylation, at least over a 1 min time period in response to thrombin. It is still not clear also whether tyrosine dephosphorylation is required in order to allow PKCδ to detach from the membrane at late time points of activation, or whether PKCδ is dephosphorylated rapidly after membrane detachment. Similarly, the nature of the tyrosine phosphatase responsible for this dephosphorylation event is not presently known.

Finally, since tyrosine phosphorylation of PKCδ did not regulate its translocation to the membrane, it was important to address whether it may regulate its activity. By in vitro kinase assay and assay of autophosphorylation on Thr505 of PKCδ, we were able to show that blockade of its tyrosine phosphorylation or removal of phosphates from tyrosines in vitro was able to diminish partially the activity of the kinase (Fig. 7). This reduction in activity was non-significant, compared to thrombin-stimulated levels of activity, however there was a clear trend towards reduction in activity which was consistent between experiments. These data were paralleled by data for phosphorylation of PKCδ on Thr505, where a non-significant Regulation of PKCδ in platelets by tyrosine phosphorylation reduction in the presence of PP1 was shown (Fig. 5B), although a consistent trend to inhibition was seen in experiments. It should be noted that, unlike other isoforms of PKC, phosphorylation on Thr505 in the activation loop of the kinase has been shown not to be essential for PKCδ activation [START_REF] Stempka | Requirements of protein kinase cdelta for catalytic function. Role of glutamic acid 500 and autophosphorylation on serine 643[END_REF]41]. Phosphorylation on this site is also not necessarily as a result of autophosphorylation, as PKD1 has been shown also to phosphorylate this site [START_REF] Good | Protein kinase C isotypes controlled by phosphoinositide 3-kinase through the protein kinase PDK1[END_REF].

Phosphorylation of Thr505 may therefore not reflect activity of PKCδ, but it is clear from a comparison of data shown in Fig 7A and 7B that the responses to thrombin in the presence and absence of PP1 are parallel, and therefore likely to be supportive of each other. They suggest that phosphorylation of PKCδ on Tyr311 and/or Tyr565 may potentiate activation of the kinase, consistent with a previous report for Y311 phosphorylation in cardiac myocytes [START_REF] Rybin | Stimulus-specific Differences in Protein Kinase Cδ Localization and Activation Mechanisms in Cardiomyocytes[END_REF], possibly by stabilising the kinase in the active conformation achieved after allosteric regulation by diacylglycerol binding to the C1 domain. In this way, PKCδ takes input signals from multiple sources, where translocation to the membrane is permissive for phosphorylation of two tyrosine residues which synergise with PLC-dependent signals to activate fully the kinase, and in this way this convergence of signals on PKCδ makes it a coincidence detector for these signalling events in the platelet. A, platelets were stimulated for the indicated time points with thrombin (0.5 units/ml), and B, platelets were preincubated (10 min) with DMSO (0.1%), PP1 (10 µM) or U73122 (10 µM) before stimulation with thrombin (0.5 units/ml) for 1 min. Cytosolic and particulate fractions were isolated as described under "Experimental Procedures". For A and B, i and ii, PKCδ was immunoprecipitated from cytosol and particulate fractions and samples were western blotted for i, pY311 or ii, pY565. Membranes were stripped and reprobed for PKCδ as indicated. For A, Platelets were stimulated for the indicated times with PMA (100 nM) and B, Platelets were preincubated (10 min) with DMSO (0.1%) or PP1 (10µM) before stimulation with PMA (100 nM) for 5 min at 37 ºC. Cytosolic and particulate fractions were isolated as described under "Experimental Procedures". For A and B PKCδ was immunoprecipitated from cytosol or particulate fractions, and samples were western blotted for (i), pY311 or (ii), pY565.

Membranes were stripped and reprobed for PKCδ as indicated. In (iii), particulate and cytosolic fractions were lysed in Laemmli sample buffer and separated by SDS-PAGE.

Samples were western blotted, as indicated, for α-tubulin as a cytosolic marker and GPIb as a membrane marker to ensure complete fractionation of platelets.

Data shown are representative of three independent experiments.

Biochemical

  Journal Immediate Publication. Published on 15 Jun 2007 as manuscript BJ20070244 © 2007 The Authors Journal compilation © 2007 Biochemical Society Regulation of PKCδ in platelets by tyrosine phosphorylation

Biochemical

  Journal Immediate Publication. Published on 15 Jun 2007 as manuscript BJ20070244 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Fig. 7A .

 7A Fig. 7A. As positive controls for the assay, PKCδ activity is also shown to be dependent upon PLC activity, as shown by an ablation of Thr505 phosphorylation in the presence of the U73122. The phorbol ester PMA is also able to directly induce activation of PKCδ and phosphorylation on Thr505 (Fig. 7B).

Biochemical

  Journal Immediate Publication. Published on 15 Jun 2007 as manuscript BJ20070244 © 2007 The Authors Journal compilation © 2007 Biochemical Society Regulation of PKCδ in platelets by tyrosine phosphorylation

Biochemical

  Journal Immediate Publication. Published on 15 Jun 2007 as manuscript BJ20070244 © 2007 The Authors Journal compilation © 2007 Biochemical Society Regulation of PKCδ in platelets by tyrosine phosphorylation

Figure 1 .

 1 Figure 1. PKCδ is phosphorylated on tyrosines 311 and 565 downstream of PAR1 and PAR4 activation. A, PKCδ was immunoprecipitated (IP) from 1% Nonidet P-40 lystates of platelets stimulated with 0.1U/ml thrombin (1 min) or 30µg/ml collagen (3 mins). Proteins were separated by SDS-PAGE, transferred to polyvinylidene difluoride membrane and western blotted (WB) with monoclonal antiphosphotyrosine antibody 4G10. Blots were reprobed with anti-PKCδ antibody as indicated. B, PKCδ was immunoprecipitated from 1% Nonidet P-40 lysates of basal platelets or platelets stimulated with 0.5U/ml Thrombin for the time periods indicated. Samples were western blotted using phospho-specific antibodies directed against the residues i, Y311 (pY311) or ii, Y565 (pY565) of PKCδ. Samples were reprobed for PKCδ as indicated. C, PKCδ was immunoprecipitated from 1% Nonidet P-40 lysates of basal platelets or platelets stimulated with either (i) PAR1 or PAR4 agonist peptides SFLLRN (50 µM) or AYPGKF (500 µM) respectively, or (ii) collagen (30 µg/ml), for the indicated time periods. Samples were western blotted for pY311 or pY565 as indicated. Membranes were stripped and reprobed for PKCδ to ensure equal loading. Data shown are representative of three independent experiments.

Figure 2 .

 2 Figure 2. Collagen, SFLLRN, AYPGKF and Thrombin do not induce phosphorylation of PKCδ on tyrosine residues 52, 155, 332 or 525 in platelets.PKCδ was immunoprecipitated from either basal or stimulated platelets (collagen 50µg/ml 3 minutes, thrombin 0.5unit/ml 1 minute, SFLLRN 100µM 20 seconds or AYPGKF 500µM 1 minute), or platelets treated with 200µM pervanadate for 1 hour at 37ºC. Immunoprecipitates were resolved by SDS-PAGE and phosphorylation of PKCδ was detected by Western Blotting using phospho-specific antibodies directed against residues Y52 (pY52), Y155 (pY155), Y332 (pY332) or Y525 (pY525). Membranes were stripped and reprobed for PKCδ to ensure equal loading. Data shown are representative of three independent experiments.

Figure 3 .

 3 Figure 3. Tyrosine phosphorylation of PKCδ on Y311 and Y565 is inhibited by PP1, PP2 and U73122.Platelets were preincubated (10 min) with either DMSO (0.1%), PP1 (10 µM), PP2 (10 µM), PP3 (10 µM), U73122 (10 µM), piceatannol (10 µg/ml) or LFM-A13 (40 µM) as indicated before stimulation with thrombin (A, 0.5 U/ml) for 1 min or PMA (B, 100 nM) for 5 min.

Figure 4 .

 4 Figure 4. Thrombin but not PMA stimulate an increase in Src kinase activity which is inhibited by PP1 but not U73122.Platelets were preincubated (10 mins) with DMSO (0.1%), PP1 (10 µM) or U73122 (10 µM) before stimulation with thrombin (0.5 units/ml) for 1 min or PMA (100 nM) for 5 min.Platelets were lysed in Laemmli sample buffer, proteins were separated by SDS-PAGE and transferred to PVDF membrane. In A samples were western blotted using anti-pTyr 416 Src antibody and in C membranes were reprobed using anti-Src antibody to ensure equal loading.In B, bands from (A) were quantified by densitometry and represented in the histogram as a percentage of maximal Src phosphorylation in response to thrombin. Data is presented as mean ± S.E.M (n=3) (*, p<0.01 compared to DMSO alone; **, p<0.001 compared to DMSO alone; ***, p<0.001 compared to thrombin pretreated with DMSO).

Figure 5 .

 5 Figure 5. Thrombin stimulates transient translocation of PKCδ to the particulate fraction, which is downstream of PLC but not Src kinase activation.

A

  and B, iii, equal protein concentrations of particulate and cytosolic fractions were lysed in laemmeli sample buffer and separated by SDS-PAGE. Samples were western blotted, as indicated, for α-tubulin or β-tubulin as cytosolic markers and GPIb as a membrane marker to ensure complete fractionation of platelets. Data shown are representative of three independent experiments.

Figure 6 .

 6 Figure 6. PMA stimulates translocation of PKCδ to the particulate fraction which is not inhibited by PP1.

Figure 7 .

 7 Figure 7. Tyrosine phosphorylation potentiates the kinase activity of PKCδ.A (i), PKCδ was immunoprecipitated from platelets preincubated with DMSO (0.1%) or PP1 (10 µM) before stimulation with thrombin (0.5 units/ml) for 1 min. Immunoprecipitates from thrombin-stimulated platelets were dephosphorylated, where indicated, using PTP1b and an in vitro kinase assay performed as described under "Experimental Procedures". Kinase activity is expressed as fold increase relative to unstimulated platelets. Data shown are mean ± S.E.M (*, p<0.05 compared to unstimulated platelets). A (ii), PKCδ immunoprecipitates from above were separated on SDS-PAGE and transferred to PVDF membrane. Samples were western blotted for pY311 (left panel) or pY565 (right panel) to ensure dephosphorylation occurred and reprobed for PKCδ as indicated to ensure equal loading. B, Platelets were preincubated (10 mins) with DMSO (0.1%), PP1 (10 µM) or U73122 (10 µM) before stimulation with thrombin (0.5 units/ml) for 1 min or PMA (100 nM) for 5 min. Platelets were lysed in Laemmli sample buffer, proteins were separated by SDS-PAGE and transferred to PVDF membrane. In B (i) samples were western blotted using anti-pThr505 PKCδ antibody (arrow corresponds to PKCδ) and reprobed in (iii) using anti-PKCδ and anti-actin antibodies to ensure equal loading. The apparent reduction in immunoreactivity of PKCδ in samples taken from PMA-stimulated platelets (far right lane) has been noted previously[START_REF] Rybin | Immunoblotting PKC-δ: a cautionary note from the benc[END_REF], and is likely not to reflect a true reduction in protein loading but an altered ability of PKCδ to bind antibody, possibly as a result of multiple phosphorylations. Anti-actin reprobe was therefore also included to demonstrate equal loading of the gel. In B (ii) bands from B (i) were

Fig. 2

 2 Fig.2

Biochemical Journal Immediate Publication. Published on 15 Jun 2007 as manuscript BJ20070244 © 2007 The Authors Journal compilation © 2007 Biochemical Society Regulation

  of PKCδ in platelets by tyrosine phosphorylation

Biochemical Journal Immediate Publication. Published on 15 Jun 2007 as manuscript BJ20070244 © 2007 The Authors Journal compilation © 2007 Biochemical Society

	Regulation of PKCδ in platelets by tyrosine phosphorylation
	30mM MgCl 2 , 200µCi/ml [γ-
	4 , and
	once in Kinase Assay Buffer (100mM NaCl, 20mM HEPES, 5mM MgCl 2 , 5mM MnCl 2 , pH
	7.2). Immunoprecipitates were resuspended in 20µl of kinase assay buffer with 10µg of PKCδ
	peptide substrate. Reactions were started by addition of 10µl of ATP Buffer (0.15mM ATP,

  32

Biochemical Journal Immediate Publication. Published on 15 Jun 2007 as manuscript BJ20070244 © 2007 The Authors Journal compilation © 2007 Biochemical Society Regulation of PKCδ in platelets by tyrosine phosphorylation

Biochemical Journal Immediate Publication. Published on 15 Jun 2007 as manuscript BJ20070244 © 2007 The Authors Journal compilation © 2007 Biochemical Society

ACKNOWLEDGEMENTS

KJH is supported by an MRC studentship. The work was supported by programme and project grants from the British Heart Foundation (grant nos. PG/04/097/17620 & RG/05/015).


Jörg Andrä

Jörg Howe

Patrick Garidel

Manfred Rössle

Walter Richter

José Leiva-Léon

Ignacio Moriyon

Rainer Bartels

Thomas Gutsmann

Klaus Brandenburg

José Leiva- Leónǁ

Ignacio Moriyonǁ

Mechanism of interaction of optimized Limulus-derived cyclic peptides with endotoxins: thermodynamical, biophysical and microbiological analysis

Keywords: LPS, lipopolysaccharide, FTIR Fourier-transform infrared spectroscopy, FRET fluorescence resonance energy transfer spectroscopy, MNC mononuclear cells, LALF Limulus-anti-lipopolysaccharide factor Antimicrobial peptide, LPS neutralization, cytokine induction, Limulus test, SAXS, FTIR, DSC, FFTEM Biophysics of cyclic peptide-endotoxin interaction

come   L'archive ouverte pluridisciplinaire

INTRODUCTION

Cell wall-bound molecules of bacteria such as lipopolysaccharide (LPS) may be released into the environment by the attack of the immune system or simply due to cell division, and may there interact with serum or membrane proteins such as soluble or membrane-bound lipopolysaccharide-binding protein (LBP) and CD14 [START_REF] Tobias | Lipopolysaccharide binding protein-mediated complexation of lipopolysaccharide with soluble CD14[END_REF][START_REF] Hailman | Lipopolysaccharide (LPS)-binding protein accelerates the binding of LPS to CD14[END_REF][START_REF] Gutsmann | Interaction between lipopolysaccharide (LPS), LPS-binding protein (LBP), and planar membranes[END_REF]. In a following step, this interaction may then promote to specific receptors such as TLR4/MD2, which eventually leads to cell activation [START_REF] Akashi | Human MD-2 confers on mouse Toll-like receptor 4 species-specific lipopolysaccharide recognition[END_REF][START_REF] Beutler | Endotoxin, toll-like receptor 4, and the afferent limb of innate immunity[END_REF]. At low and moderate concentrations of LPS the following inflammation reaction is beneficial for the host, but at higher LPS concentrations the cascade of molecular and cellular events may lead to the endotoxic shock syndrome with high mortality particularly in critical care units [START_REF] Rietschel | Bacterial endotoxins as inducers of septic shock[END_REF][START_REF] Heumann | Molecular basis of host-pathogen interaction in septic shock[END_REF]. It is known that lipid A, the hydrophobic moiety anchoring LPS into the outer membrane of Gram-negative bacteria, constitutes the 'endotoxic principle' of LPS. Enterobacterial lipid A consists of a diglucosamine backbone phosphorylated at positions 1 and 4' to which mainly six acyl chains are linked at positions 2,3 and 2',3'. Furthermore, in all LPS chemotypes to lipid A two special monosaccharides, 2keto-3-deoxyoctonate (Kdo) are linked, which are negatively charged due to their carboxylate groups. Thus, a necessary condition for LPS neutralization is the presence of positive charges, which can be achieved by the use of cationic peptides [START_REF] Andrä | Mechanisms of endotoxin neutralization by synthetic cationic compounds[END_REF]. Previously, we have synthesized peptides based on the Limulus-anti-LPS-factor (LALF) from the horseshoe crab Limulus polyphemus [START_REF] Andrä | Biophysical characterization of the interaction of Limulus polyphemus endotoxin neutralizing protein with lipopolysaccharide[END_REF]. These partial structures containing the LPS-binding domain or parts of it were found to have high endotoxin-binding and neutralizing activities, comparable to that of the parent recombinant rLALF protein (also called endotoxin-neutralizing protein ENP) [START_REF] Paus | Production of recombinant endotoxin neutralizing protein in Pichia pastoris and methods for its purification[END_REF],

and to be non-toxic for erythrocytes or cultured human monocytes [START_REF] Vallespi | A Limulus antilipopolysaccharide factor-derived peptide exhibits a new immunological activity with potential applicability in infectious diseases[END_REF].

Based on these peptides, we have synthesized a number of cyclic peptides and have performed a detailed biophysical characterization of their interaction with mutant Ra LPS from Salmonella minnesota strain R60, which has well-defined chemical structure and corresponds to the active unit in the heterogeneous mixtures of wild-type LPS [START_REF] Jiao | Characterization of the lipid A component of genuine smooth-form lipopolysaccharide[END_REF]. In some cases also deep rough mutant Re LPS (from S. minnesota strain R595) as well as the 'endotoxic principle' lipid A were used. Information was obtained with regard to changes caused by peptide binding in LPS/lipid A aggregate structure, phase transition behaviour (temperature and enthalpy), mobilities of the acyl chains in the single phases, the binding epitopes, in particular the negative charges in lipid A, and the binding stoichiometry.

Correlation of these results with data on the cytokine-inhibiting capacity of the peptides and their antimicrobial activity clearly established that the neutralizing activity of the peptides was linked to their affinity for LPS and to their ability to incorporate into target cell membranes. These data allow an understanding of the mechanism of the endotoxin:peptide interaction.

EXPERIMENTAL Lipids

Lipopolysaccharides from rough mutant Re and Ra from Salmonella minnesota (strains R595 and R60, respectively) were extracted by the chloroform/petrol ether method [START_REF] Galanos | A new method for the extraction of R lipopolysaccharides[END_REF] from bacteria grown at 37°C, purified, and lyophilized. Free lipid A was isolated by acetate buffer treatment of LPS R595. After isolation, the resulting lipid A was purified and converted into its triethylamine salt. Results of all the standard assays performed on the purified LPS and lipid A (analysis of the amount of glucosamine, total and organic phosphate, and the distribution of the fatty acid residues) were in good agreement with the chemical properties expected for LPS R595 and R60, whose molecular structure has already been solved [START_REF] Holst | Core structures of enterobacterial lipopolysaccharides[END_REF].

Preparation of endotoxin aggregates

LPS or lipid A was solubilized in the appropriate buffer (lipid concentration 1 to 10 mM, depending on the applied technique), extensively vortexed, sonicated for 30 min in a water bath, and subjected to several temperature cycles between 20 and 60 °C. Finally, the lipid suspension was incubated at 4 °C for at least 12 h before use.

Peptides

Peptides containing the described LPS-binding domain of LALF [15] termed cLALF9,12, 15,16, 18, and 20 (structures see Fig. 1) were synthesized with an amidated C-terminus by the solid-phase peptide synthesis technique on an automatic peptide synthesizer (model 433 A; Applied Biosystems) on the standard Fmoc-amide resin according to the fastmoc synthesis protocol of the manufacturer. The N-terminal Fmoc-group was removed from the peptideresin and the peptide was deprotected and cleaved with 90 % trifluoroacetic acid (TFA), 5 % anisole, 2 % thioanisole, 3 % dithiothreitol for 3 h at room temperature. After cleavage the suspension was filtered and the soluble peptides were precipitated with ice-cold diethylether followed by centrifugation and extensive washing with ether. Peptides were purified by RP-HPLC using an Aqua-C18 column (Phenomenex). Elution was done by using a gradient of 0-70% acetonitrile in 0.1 % (TFA). Cyclation via cysteine residues was achieved by incubating the peptides in 10 % dimethylsulfoxid (DMSO) for 24 h at room temperature. The peptides were then again purified by reversed-phase HPLC to purities above 95%. Purity was determined by matrix-assisted laser-desorption-time-of-flight mass spectrometry (MALDI-TOF MS, Bruker). In some cases also peptide cLALF22, corresponding to the complete LPSbinding domain of LALF [START_REF] Hoess | Crystal structure of an endotoxin-neutralizing protein from the horseshoe crab, Limulus anti-LPS factor, at 1.5 A resolution[END_REF], was used. It differs from cLALF20 by the additional amino acids HY between the GC and the RI residues (third and fourth amino acid from the Nterminus [Fig. 1]).

Fourier-transform infrared spectroscopy (FTIR)

The infrared spectroscopic measurements were performed on an IFS-55 spectrometer (Bruker). Samples, dissolved in 20 mM Hepes buffer, pH 7.0, were placed in a CaF 2 cuvette with a 12.5 µm teflon spacer. Temperature-scans were performed automatically between 10 and 70 °C with a heating-rate of 0.6 °C/ min. Every 3 °C, 50 interferograms were accumulated, apodized, Fourier transformed, and converted to absorbance spectra.

Differential Scanning Calorimetry (DSC)

DSC measurements were performed with VP-DSC and MC-2 calorimeters (MicroCal, Inc., Northhampton, MA, USA) at a heating and cooling rate of 1 °C•min -1 . The DSC samples were prepared by dispersing a known amount (~3 mg/ml) in 10 mM PBS buffer at pH 7.4. The samples were hydrated in the liquid crystalline phase by vortexing. Prior to the measurements the DSC samples were stored for a defined time at 4 ºC (see before). The measurements were performed in the temperature interval from 5 °C to 95 °C. In the figures only the temperature range is shown where phase transitions were observed. Five consecutive heating and cooling scans checked the reproducibility of the DSC experiments of each sample [START_REF] Blume | From Macromolecules to Man[END_REF]. The accuracy of the DSC experiments was ± 0.1 °C for the main phase transition temperatures and ± 1 kJ•mol -1 for the main phase transition enthalpy. The DSC data were analysed using the Origin software. The phase transition enthalpy was obtained by integrating the area under the heat capacity curve [START_REF] Garidel | Divalent cations affect chain mobility and aggregate structure of lipopolysaccharide from Salmonella minnesota reflected in a decrease of its biological activity[END_REF].

Isothermal Titration Calorimetry (ITC)

Microcalorimetric measurements of peptide binding to endotoxins were performed on a MCS isothermal titration calorimeter (Microcal Inc.) at 37 °C as recently described [START_REF] Brandenburg | Biophysical investigations into the interaction of lipopolysaccharide with polymyxins[END_REF]. Endotoxin samples (0.05 to 0.15 mM) -prepared as described above -were dispensed into the microcalorimetric cell (volume 1.3 ml) and peptide solutions in the concentration range 0.5 to 5 mM were filled into the syringe compartment (volume 100 µl), each after thorough degassing of the suspensions. After temperature equilibration, the peptides were titrated in 3 µl portions every 5 min into the lipid-containing cell under constant stirring, and the measured heat of interaction after each injection measured by the ITC instrument was plotted versus time. As addition of the peptides into pure buffer solution gave only negligible heat of dilution, the total heat signal from each experiment was determined as the area under the respective single peaks and plotted versus the [peptide]:[lipid] molar ratio. Since the instrument works in temperature equilibrium at a constant 'current feedback' corresponding to a power of approximately 74 µW, the occurrence of an exothermic reaction leads to a lowering of this current, whereas an endothermic reaction causes an increase. All titration curves were repeated at least twice.

X-ray diffraction

X-ray diffraction measurements were performed at the European Molecular Biology Laboratory (EMBL) outstation at the Hamburg synchrotron radiation facility HASYLAB using the double-focusing monochromator-mirror camera X33 [START_REF] Koch | X-ray diffraction and scattering on disordered systems using synchrotron radiation[END_REF]. Diffraction patterns in the range of the scattering vector 0.1 < s < 4.5 nm -1 (s = 2 sin θ/λ, 2θ scattering angle and λ the wavelength = 0.15 nm) were recorded at 40 °C with exposure times of 1 min using an image plate detector with online readout (MAR345, MarResearch, Norderstedt/Germany). The saxis was calibrated with Ag-Behenate which has a periodicity of 58.4 nm. The diffraction patterns were evaluated as described previously [START_REF] Brandenburg | Investigation into the acyl chain packing of endotoxins and phospholipids under near physiological conditions by WAXS and FTIR spectroscopy[END_REF] assigning the spacing ratios of the main scattering maxima to defined three-dimensional structures. The lamellar and cubic structures are most relevant here. They are characterized by the following features:

(1) Lamellar: The reflections are grouped in equidistant ratios, i.e., 1, 1/2, 1/3, 1/4, etc. of the lamellar repeat distance d l (2) Cubic: The different space groups of these non-lamellar three-dimensional structures differ in the ratio of their spacings. The relation between reciprocal spacing s hkl = 1/d hkl and lattice constant a is

s hkl = [(h 2 + k 2 + l 2 ) / a ] 1/2
(hkl = Miller indices of the corresponding set of plane).

Freeze-fracture transmission electron microscopy (FFTEM)

LPS and LPS:peptide samples were prepared as described above around 90 % water content (3-5 mg/ 30 µl). For freeze-fracturing, the samples, copper sandwich profiles, and instruments for manipulation were incubated at room temperature or at 40 °C. A small amount of the sample was sandwiched between two copper profiles as used for the double-replica technique and frozen by plunging the sandwiches immediately into liquified propane cooled in liquid nitrogen. Fracturing and replication were performed at -150 °C in a BAF 400T freeze-fracture device (BAL-TEC, Balzers, Liechtenstein) equipped with electron guns an a film sheet thickness monitor. The replicas were placed on copper grids, cleaned with a chloroformmethanol mixture, and examined in an EM900 electron microscope (Zeiss, Oberkochen,

Germany).

Fluorescence resonance energy transfer spectroscopy (FRET)

The fluorescence resonance energy transfer (FRET) technique was used as a probe dilution assay [START_REF] Schromm | Lipopolysaccharide binding protein (LBP) mediates CD14independent intercalation of lipopolysaccharide into phospholipid membranes[END_REF] to obtain information on the intercalation of the peptides in the absence and presence of LPS into liposomes made from phosphatidylserine (PS). For the FRET experiments, liposomes were double-labeled with NBD-PE and Rh-PE in chloroform

[PS]:[NBD-PE]:[Rh-PE] at 100:1:1 molar ratios. The solvent was evaporated under a stream of nitrogen, the lipids resuspended in PBS, mixed thoroughly, and sonicated with a Branson sonicator for 1 min (1 ml solution). Subsequently, the preparation was temperature-cycled at least twice between 4 °C and 56 °C, each cycle followed by intense vortexing for a few min, and then stored at 4 °C for at least 12 h prior to measurement. A preparation of 900 µl of the double-labeled liposomes (10 -5 M) at 37 °C was excited at 470 nm (excitation wavelength of NBD-PE), and the intensities of the emission light of the donor NBD-PE (531 nm) and acceptor Rh-PE (593 nm) were measured simultaneously on the fluorescence spectrometer SPEX F1T11 (SPEX Instr., Edison, NY). The peptides cLALF (final concentration 1 µM) were added to liposomes after 50 s, and LPS after 100 s (cLALF:LPS 1:1 molar). Furthermore, coincubated (LPS + cLALF) samples were added after 50 s. Since FRET spectroscopy is used here as a probe dilution assay, intercalation of molecules causes an increase of the distance between donor and acceptor in the liposomes and, thus, leads to a reduced energy transfer. This again causes an increase of the donor and decrease of the acceptor intensities. For a qualitative analysis of experiments, the quotient of the intensities of the donor dye and the acceptor dye are plotted against time (denoted in the following as the FRET signal). The data shown are representative for three independent experiments.

Stimulation of human mononuclear cells by LPS

Mononuclear cells (MNC) were isolated from heparinized blood of healthy human donors as described previously [START_REF] Brandenburg | Cross-linked hemoglobin converts endotoxically inactive pentaacyl endotoxins into a physiologically active conformation[END_REF]. The cells were resuspended in medium (RPMI 1640) and their number was equilibrated at 5x10 6 cells/ml. For stimulation, 200 µl MNC (1x10 6 cells) were transferred into each well of a 96-well culture plate. LPS and peptide:LPS mixtures were incubated for 30 min at 37 °C, and added to the cultures at 20 µl per well. The cultures were incubated for 4 h at 37 °C under 5 % CO 2 . Supernatants were collected after centrifugation of the culture plates for 10 min at 400 x g and stored at -20 °C until determination of TNFα content. Immunological determination of TNFα was carried out in a Sandwich ELISA using a monoclonal antibody against TNF (clone 6b from Intex AG, Switzerland) and was described earlier in detail [START_REF] Brandenburg | Cross-linked hemoglobin converts endotoxically inactive pentaacyl endotoxins into a physiologically active conformation[END_REF].

Assays for cytotoxicity

Cytotoxicity of the peptides was determined against HeLa cells in medium by a MTT (3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay as described previously [START_REF] Andrä | Enhancement of endotoxin neutralization by coupling of a C12-alkyl chain to a lactoferricin-derived peptide[END_REF].

Assay for hemolytic activity

Hemolytic activity of the peptides against human erythrocytes was determined as described previously [START_REF] Andrä | Enhancement of endotoxin neutralization by coupling of a C12-alkyl chain to a lactoferricin-derived peptide[END_REF]. Briefly, peptides were incubated with a freshly prepared erythrocyte suspension from human blood (5 % of normal hematocrit) in 10 mM phosphate buffered saline, pH 7.2, for 30 min at 37 °C. Released hemoglobin was determined photometrically at 412 nm.

Assays for antibacterial activities

The antibacterial activity of the peptides was determined by microdilution susceptibility assays performed i) in standard Mueller-Hinton broth or ii) in 20 mM Hepes buffer, pH 7.0 supplemented with 10% LB broth. (BioMérieux, Marcy l´Etoile, France) for 1 to 3 days depending on the growth rate of the bacteria, suspended in 2 ml of 0.9 % saline and adjusted to 10 8 CFU/ml. The bacterial suspension was diluted 100-fold in Mueller-Hinton broth, and 0.1 ml of this new dilution (10 5 CFU) was added to the peptide dilutions. The microtitre plates were incubated at 37 ºC for 24 h. The minimal inhibitory concentration (MIC) of each peptide against a given bacterial strain was regarded as the minimal concentration of the peptide which prevents the growth of that organism 24 h after inoculation. The bactericidal effect of the peptides was determined, after 24 h incubation at 37 °C, by plating 10 µl of the content of non-cloudy wells onto Mueller-Hinton agar plates. Plates were incubated at 37 ºC for 1 to 5 days, and a viable count was performed. The minimal bactericidal concentration (MBC) of each peptide for a given bacterial strain was regarded as the minimal concentration of the peptide which kills 99.9 % of the colony-forming units (CFU) present in the final inoculum.

1) Assay in

2) Assays in Luria-Bertani (LB) medium / buffer. Peptides (dissolved in 20 mM Hepes, pH 7.0) were two-fold diluted in the same buffer in 96-well microtitre plates (Greiner, Germany).

Subsequently, a suspension of log-phase bacteria in LB was added (10 µl, containing 10 4 CFU) to the peptide solution (90 µl). The plates were incubated overnight in a wet chamber at 37 °C under constant shaking and bacterial growth was monitored by measuring the absorbance at 620 nm in a microtiter plate reader (Rainbow, Tecan, Crailsheim, Germany).

The MIC was defined as the lowest peptide concentration, at which no bacterial growth was measurable. Portions of each well (10 µl) were diluted with 90 µl Hepes buffer, plated out in duplicate on LB-agar plates, incubated overnight at 37 °C, and bacterial colonies were counted. The MBC was defined as the peptide concentration at which no colony growth was observed. Experiments were performed at least twice in duplicates.

RESULTS

Various physical techniques were applied to characterize the interaction of LPS Ra, in some cases also of LPS Re and lipid A as endotoxic principle, with the peptides.

Gel to liquid crystalline phase transition behavior

Biochemical Journal Immediate Publication. Published on 14 May 2007 as manuscript BJ20070279

The influence of the peptides on the gel to liquid crystalline β↔α phase behaviour of the acyl chains of LPS Ra, as studied by DSC, showed for all peptides strong interaction with the LPS aggregates (Fig. 2). For all peptides, the endothermic melting enthalpy of the hydrocarbon chains of LPS decreases with increasing peptide concentration and vanished at equimolar ratios. The peptides cLALF15 and 20 acted strongest, for which the endotherm disappears already at a [LPS]:[peptide] 2:1 molar ratio. This became evident more clearly, when the enthalpy change ∆H of the acyl chain melting transition was plotted versus the [LPS Ra]:[peptide] molar ratio (Fig. 3). From these data it can be concluded that, beside the electrostatic attraction between the positive charges of the peptide and the negative charges of LPS, a hydrophobic interaction also takes place, leading to a complete abolishment of the chain melting. This chain melting-abolishing effect can be interpreted in two ways, as fluidization of the hydrocarbon chains already at low temperatures or as maintaining of the gel phase up to high temperatures. To differentiate between these possibilities, FTIR was applied by monitoring the peak position of the symmetric stretching vibration of the hydrocarbon chains ν s (CH 2 ) versus temperature. This is plotted in Fig. 4 for the interaction of LPS Ra with cLALF9, 15, 18, and 20. As it is known that the peak position of ν s (CH 2 ) lies around 2850 cm -1 in the gel and around 2852.5 to 2853 cm -1 in the liquid crystalline phases [START_REF] Brandenburg | Biophysical characterization of the interaction of high-density lipoprotein (HDL) with endotoxins[END_REF], it can be deduced from Fig. 4 that the action of the peptides led to a fluidization of the acyl chains in the gel phase, connected with a strong reduction of the phase transition temperature T c . In particular at 37 °C, a dramatic increase in the wavenumber values to more than 2853 cm -1 took place. Nevertheless, the change of the wavenumbers over a broad range 10-40 °C for the [LPS]:[peptide] mixtures at 1:0.5 and 1:1 molar ratio is a hint that in the DSC scans this transition cannot be observed enthalpically.

Binding experiments

Biochemical Journal Immediate Publication. Published on 14 May 2007 as manuscript BJ20070279

Isothermal titration calorimetry is the method of choice to determine the enthalpy change caused by the peptide:LPS binding and its stoichiometry. For this, 0.05 mM LPS Ra dispersions were titrated with 2 mM peptide solutions. One original titration is shown in Fig. 5A for LPS Ra and cLALF20 showing an exothermic reaction (decrease of feedback power to compensate the temperature increase in the sample cell). A saturation is observed after the

Ultrastructural analysis

It has beeen shown that non-lamellar aggregate structures for LPS (in particular the lipid A part of LPS) are essential for the expression of biological activity [START_REF] Brandenburg | Physicochemical properties of bacterial glycopolymers in relation to bioactivity[END_REF]. We thus applied synchrotron radiation small-angle X-ray diffraction to determine the aggregate structure of LPS in the presence of the peptides. For the latter, we used deep rough mutant LPS Re with a short core oligosaccharide rather than LPS Ra with the complete core oligosaccharide [START_REF] Rietschel | Bacterial endotoxin: Chemical constitution, biological recognition, host response, and immunological detoxification[END_REF],

since the spectra of the former are more readily resolvable and interpretable [29,[START_REF] Seydel | Structural polymorphisms of rough mutant lipopolysaccharides Rd to Ra from Salmonella minnesota[END_REF]. The diffraction patterns for pure LPS Re were indicative for the existence of a mixed unilamellar/cubic structure as described earlier [29] (data not shown). In the presence of the peptides cLALF18 and 20, the diffraction patterns in the temperature range 5 to 60 °C were characteristic for the existence of multilamellar structures, deduced from the occurrence of reflections at equidistant ratios (Fig. 6A). For the LPS:cLALF18 complex, a closer look to the patterns at 40 °C (Fig. 6B) showed a main periodicity at 6.62 nm and three further reflections corresponding to the higher orders, and some smaller peaks. These were more readily expressed for the LPS:cLALF20 complex, showing a second lamellar periodicity at 5.32 nm and its second order at 2.64 nm (Fig. 6B, bottom).

Regarding the periodicities, it has been shown for LPS Re that these values lie in the range 5.90 to 6.40 in cases in which this LPS adopts a multilamellar structure (in the presence of Mg 2+ or at low water content, [29] ). Therefore, the addition of cLALF18 apparently leads to an increase in lamellar stacking periodicity, whereas that of cLALF20 does not, or is even decreased regarding the value of the smaller periodicity.

Also, the aggregate structures of free lipid A (as 'endotoxic principle') in the presence of cLALF18 and 20 were elucidated (data not shown). Similar to the case of LPS Re, the reflections at equidistant ratios indicated multilamellar structures. The value of the periodicities (6.49 nm), however, were very high regarding the fact that the values for pure lipid A are around 4.9 to 5.3 nm [START_REF] Brandenburg | Phase diagram of lipid A from Salmonella minnesota and Escherichia coli rough mutant lipopolysaccharide[END_REF]. Thus, in this case a strong increase of the water layer between neighbouring bilayers due to peptide binding took place (swelling of the interlamellar repeat).

For the system LPS Re:cLALF18 (2.5:1 molar as in the X-ray diffraction experiments), also freeze-fracture electron microscopy was used. Micrographs are displayed in Fig. 7A-C. For pure LPS, vesicle-like structures are found in the size range 100 to 200 nm (Fig. 7A). The structures, however, often are not closed and formed something like 'open eggshells'. When the peptide was added to the LPS, the suspension became turbid with subsequent precipitation. The supernatant (Fig. 7B) as well as the precipitate (Fig. 7C) were investigated.

It can be clearly seen that in the supernatant very large vesicle-like structures and multilamellar stacks are observed apparently due to the formation of fused 'eggshells', with diameters up to 500 nm. For the precipitate, the morphology completely changes with the occurrence of giant stacks. These stacks corresponded to the multilamellar structures found in the X-ray experiments, with distances between neighboring stacks in the range 6.3 to 7.5 nm.

Fig. 8C shows also that in cross-fractured areas stacks with more than 100 single bilayer lamellae can be found.

Intercalation into phospholipid liposomes

A possible intercalation of the peptides or peptide:LPS mixtures into phospholipid membranes were analysed by FRET. As shown in Fig. 8 for cLALF9 and cLALF18, the peptides alone (added at t = 50 s) were able to intercalate into PS liposomes (increase of FRET signal). When LPS was added subsequently at t = 100 s, there was an additional incorporation, in contrast to added LPS in the absence of the peptide, for which no incorporation was observed (data not shown, see [START_REF] Schromm | Lipopolysaccharide binding protein (LBP) mediates CD14independent intercalation of lipopolysaccharide into phospholipid membranes[END_REF]). This means that the peptides acted as a kind of transport molecule similar to that observed for LBP [START_REF] Schromm | Lipopolysaccharide binding protein (LBP) mediates CD14independent intercalation of lipopolysaccharide into phospholipid membranes[END_REF].

Finally, when peptides and LPS (peptide:LPS) were incubated before addition to the liposomes, the data indicate a reduced, but still existing membrane incorporation.

Similar results were obtained for all other peptides investigated (data not shown). In some experiments, instead of PS, phospholipids corresponding to the composition of the macrophage membrane (PL Mφ ) were used. The results were qualitatively the same as with pure PS liposomes, only the observed signal intensities were lower (data not shown).

Inhibition of LPS-induced stimulation of human mononuclear cells

We tested the ability of the peptides to inhibit cell stimulation by LPS Ra from S. minnesota strain R60. Cells were stimulated by LPS alone or preincubated with various amounts of peptide (Fig. 9). All peptides interacted with LPS as reflected by a reduction of cell activation.

Peptide cLALF12 was able to completely block LPS-induced cell activation (1 ng/ml LPS) at 1 µg/ml. Polymyxin B, the gold standard for cationic LPS-neutralizing peptides, exhibited the same activity at a 10-fold lower concentration.

Cytotoxicity and hemolytic activity

The potential harmful effects of the peptides to human cells were estimated by assessing their cytotoxicity against HeLa cells and their potency to lyse human erythrocytes. At a concentration of 100 µg/ml none of the peptides were cytotoxic and the hemolytic activity was 15 % or below (Table 1).

Antibacterial activity

The antibacterial activity of the peptides was determined first against the LPS-mutant strains S. minnesota R595 and R60 (used for the preparation of the LPS with defined structures used in the above-described experiments) grown in LB, a low ionic strength medium that bolsters peptide antimicrobial activity. These experiments suggested that none of the new peptides outperformed the parent peptide cLALF22 (Table 1) but because of the low MICs obtained under these conditions and with these sensitive LPS mutant strains, we performed additional and more stringent experiments. To this end, we tested the antibacterial activity in Mueller-Hinton broth, which is rich in divalent cations, and using a smooth-type Escherichia coli strain and a clinical isolate of Pseudomonas aeruginosa, a bacterium with an outer membrane and LPS markedly stabilized by divalent cations. Under these more stringent conditions, the parent peptide cLALF22 outperformed again all the derivatives. This was particularly clear with P. aeruginosa highlighting the lack of an effective displacement of divalent cations from outer membrane and LPS targets by the other peptides (Table 1). It should be mentioned, however, that some of the variants exhibited activity against S. aureus. With respect to the most resistant bacterium, i.e. P. aeruginosa, cLALF9 was the peptide with highest activity beside cLALF22.

DISCUSSION

The development of suitable antimicrobial peptides for a therapy of septic shock and also the fight against local infections is still a challenge in human health care regarding the increase of infectious diseases worldwide and problems such as the emergence of resistances [START_REF] Hancock | Antimicrobial and host defense peptides asw new anti-infective therapeutic strategies[END_REF]. For these purposes, the development of drugs based on natural endotoxin-binding structures seems a promising approach. This approach comprises, for example, the use of lactoferrinbased [START_REF] Andrä | Enhancement of endotoxin neutralization by coupling of a C12-alkyl chain to a lactoferricin-derived peptide[END_REF] and of LALF (Limulus-anti-LPS factor)-based peptides [START_REF] Andrä | Biophysical characterization of the interaction of Limulus polyphemus endotoxin neutralizing protein with lipopolysaccharide[END_REF]. For synthetic peptides based on the sequence of endotoxin-binding proteins like LALF, it was shown before, that some are in principle suitable for application in anti-sepsis research, in particular as they have a sufficiently long half-life in serum [START_REF] Dankesreiter | Synthetic endotoxin-binding peptides block endotoxin-triggered TNF-α production by macrophages in vitro and in vivo and prevent endotoxin-mediated toxic shock[END_REF]. Previously, we have published biophysical data on the interaction of some synthetic cyclic peptides with LPS Re, and reported that certain physico-chemical parameters of the LPS Re:peptide interaction correlate with the ability to neutralize LPS [START_REF] Andrä | Cyclic antimicrobial peptides based on Limulus antilipopolysaccharide factor for neutralization of lipopolysaccharide[END_REF]. On the basis of these findings, we have constructed several new cyclic peptides (cLALF 9, 12, 15, 16, 18 and 20). These peptides were designed on the basis of obtaining the hydrophobic cluster at the C-terminus and stepwise shortening the peptides from the N-terminus (Fig. 1). We have performed a comprehensive biophysical and biological study of their interaction with the well-characterized endotoxins LPS Re and LPS Ra from S. minnesota strains R595 and R60. Except for the ultrastructural studies in which LPS Re was used, the latter was studied because it has been shown that the endotoxically active unit in natural smooth form LPS corresponds to an LPS with complete core oligosaccharide [START_REF] Jiao | Characterization of the lipid A component of genuine smooth-form lipopolysaccharide[END_REF].

Our results indicate a strong interaction of the peptides both with bacteria and with isolated LPS. The antibacterial activity of the peptides was clearly more marked on LPS deficient Salmonella mutants in low ionic strength broth than on wild-type clinical isolates in the divalent-cation rich Mueller-Hinton broth. In fact, under the more stringent conditions only the parent peptide exhibited activity on P. aeruginosa (Tab. 1). It has to be noted, however, that this bacterium is notoriously refractory to bactericidal peptides under these conditions, and this highlights the activity of cLALF22. On the other hand, the peptides were not cytotoxic and only slightly hemolytic (Tab. 1). In a similar way, they were able to inhibit the LPS-induced cytokine production of human mononuclear cells (Fig. 9). Very interesting is the dependence of the antibacterial action on the presence of divalent cations (Mueller-Hinton broth). The latter are known to stabilize bacterial outer membranes as well as isolated LPS aggregates [START_REF] Andrä | Cyclic antimicrobial peptides based on Limulus antilipopolysaccharide factor for neutralization of lipopolysaccharide[END_REF]. Therefore, the action of those peptides which essentially attack the LPS layer of the outer membrane is impeded, which explains the reduction of the MIC/MBC values (Tab. 1).

The binding of the peptides with LPS shows the following features, which may be relevant for an understanding of the binding to isolated LPS as well as to LPS as constituent of the outer bacterial membrane.

The binding led to a strong fluidization of the hydrocarbon chains of LPS (Fig. 4) with a disappearance of the phase transition enthalpy (Fig. 2,3) similar to the observations made for the well-known decapeptide polymyxin B (PMB) [START_REF] Brandenburg | Biophysical investigations into the interaction of lipopolysaccharide with polymyxins[END_REF]. Concomitantly, a change of the aggregate structures of LPS and lipid A, being unilamellar or mixed unilamellar/inverted cubic in pure form, into multilamellar stacks (Figs. 6 and not shown) were observed, also like PMB. This process is strongly exothermic (Fig. 5A,B) due to the electrostatic attraction between the negative charges of LPS/lipid A and the positive charges of the peptides (in particular amino acids Arg=R and Lys=K, Fig. 1). The enthalpic values (maximum values more than 40 kJ/mol, see Fig. 5B) also compare well with those from the interaction of PMB and the nonapeptide PMBN with LPS [START_REF] Brandenburg | Temperature dependence of the binding of endotoxins to the polycationic peptides polymyxin B and its nonapeptide[END_REF].

The electron miocrographs (Fig. 7) give a clear impression of the underlying morphology, with nearly spherical vesicle-like structures ('open eggshells', Fig. 7A) for pure LPS, and very large 'giant' liposomal structures in the supernatant (Fig. 7B), apparently due to fusions of the single 'eggshells'. In the precipitate, huge stacks were found (Fig. 7C) corresponding to the multilamellae deduced from SAXS experiments. As already proposed in a preceding paper, the degree of multilamellarization may be a direct measure for the ability of a given peptide to inhibit the LPS-induced cytokine production in mononuclear cells [START_REF] Andrä | Enhancement of endotoxin neutralization by coupling of a C12-alkyl chain to a lactoferricin-derived peptide[END_REF]. According to theoretical considerations by Bouwstra et al. [START_REF] Bouwstra | Small angle X-ray scattering: Possibilities and limitations in characterization of vesicles[END_REF], from the shapes of the reflections at equidistant ratios statements about the number of lamellae and size distributions of particles should be possible. Unfortunately, this is apparently valid only for relatively simple situations for pure phospholipids. For LPS, in cases in which multilamellar aggregates are formed (at low water content and/or high Mg 2+ concentrations [29,30]), we did not succeed to get these informations (unpublished data). Furthermore, due to the fact that for LPS:peptide mixtures we did not see spherical liposomes but rather relatively different forms in the supernatant and completely different morphologies in the precipitate, the data analysis of Bouwstra and coworkers is not applicable. Therefore, SAXS measurements alone are not sufficient for a complete analysis, and the number of lamellae must be estimated by a different technique, i.e., by analysing the FFTEM pictures. For cLALF18, Fig. 7C is indicative that stacks with very high numbers of lamellae are formed, and these can be enumerated (most of them are > 100).

It is tempting to assume that this is relevant for the interpretation of biological data, since the interaction of LPS with binding-proteins such as CD14 and LBP is reduced for stacks with high number of lamellae as compared to the unilamellar system in the absence of the peptide.

This will be a task for the future.

We have found here a significant increase of the aggregate sizes of LPS (Fig. 7c) concomitant with a multilamellarization (Fig. 6) in contrast to the findings of Rosenfeld et al [37], which report that peptides such as human LL-37 and magainin would lead to a dissociation of the LPS aggregates thus preventing it to react with LBP or cellular receptors. These authors, however, use more indirect techniques such as flow cytometry with FITC-labelled LPS which does not represent a direct technique as shown here with the FFTEM method and furthermore, they have used chemically ill-defined smooth-form LPS which usually consists of a mixture of fractions differing in the length of the sugar chains and the acylation of the lipid A part [START_REF] Jiao | Characterization of the lipid A component of genuine smooth-form lipopolysaccharide[END_REF].

It must be emphasized that except for uncharacterized wild type LPS (S-LPS) in older papers [START_REF] Shands | Bacterial endotoxins[END_REF] no electron micrographs of chemically characterized endotoxins have ever been published. For lipid A, previously we have characterized the non-lamellar structures using SAXS and FFTEM [39]. A combination of SAXS and cryo-TEM was applied to analyse the interaction of granulysin-derived peptides with endotoxins, in particular with LPS Ra from S.

minnesota [START_REF] Chen | Biophysical analysis of the interaction of granulysin-derived peptides with enterobacterial endotoxins[END_REF]. The authors found for pure LPS Ra fibrillary structures with cylindrical forms with a diameter of the fibrilles of 12 nm, whereas in the presence of a granulysin-derived peptide regularly-stacked ribbons emerged, which would correspond to the multilamellar stacks of LPS Re as seen here.

It is important to note that the results of the combined EM and SAXS measurements presented here allow the unequivocal elucidation of the underlying aggregate structures, which should be of uttermost use in the interpretation of the inhibition activity of the peptides.

For an understanding of the interaction mechanism, the FRET data (Fig. 8), which exhibit an intercalation of the peptides into liposomal membranes, are important. This may be of relevance for their antibacterial activity (Tab. 1) as well for the inhibition of the LPS-induced cytokine expression (Fig. 9). For the former, because the bacterial membranes may thus be heavily disturbed in their function, and for the latter, since the uptake of the peptide into the membranes of mononuclear cells can be assumed to be of relevance for their ability to secret cytokines. There is considerable evidence, that the cell activation by LPS starts via a membrane step, induced by binding proteins such as LBP and CD14 [START_REF] Hailman | Lipopolysaccharide (LPS)-binding protein accelerates the binding of LPS to CD14[END_REF][START_REF] Pugin | Lipopolysaccharide activation of human endothelial and epithelial cells is mediated by lipopolysaccharide-binding protein and soluble CD14[END_REF][START_REF] Tapping | Cellular binding of soluble CD14 requires lipopolysaccharide (LPS) and LPS-binding protein[END_REF][START_REF] Ulevitch | Recognition of gram-negative bacteria and endotoxin by the innate immune system[END_REF][44]. The detailed mechanisms, however, are not completely resolved. It was proposed that LBP accelerates the binding of LPS to CD14, and/or that LBP leads to a disintegration, possibly monomerization, of LPS aggregates [START_REF] Hailman | Lipopolysaccharide (LPS)-binding protein accelerates the binding of LPS to CD14[END_REF][START_REF] Tobias | Lipopolysaccharide (LPS)-binding proteins BPI and LBP form different types of complexes with LPS[END_REF], which would imply that the presence of monomers or small aggregates of endotoxin are necessary for endotoxic activity in accordance to findings of Takayama ands co-workers [START_REF] Takayama | Monomeric Re lipopolysaccharide from Escherichia coli is more active than the aggregated form in the Limulus amebocyte lysate assay and in inducing Egr-1 mRNA in murine peritoneal macrophages[END_REF]. In contrast, Mueller et al [START_REF] Mueller | Endotoxin: physical requirements for cell activation[END_REF][START_REF] Mueller | Aggregates are the biologically active units of endotoxins[END_REF] showed that aggregates are the biologically active units of endotoxins. We have found that the binding of LBP to LPS leads to a multilamellarization of LPS which is rather indicative of an increase rather than a decrease of aggregate sizes as found here for peptide binding (unpublished results).

Independently of this question, it can be stated that LPS incorporated into target cell membranes represents a strong disturbance of the membrane architecture due to the conical geometry (shape) of its lipid A moiety, which triggers cell activation by leading to a conformational change of membrane proteins responsible for cell signalling such as TLR4 [START_REF] Beutler | Endotoxin, toll-like receptor 4, and the afferent limb of innate immunity[END_REF][START_REF] Qureshi | Endotoxin-tolerant mice have mutations in toll-like receptor 4 (Tlr4)[END_REF], MaxiK [START_REF] Blunck | New insights into endotoxin-induced activation of macrophages: Involvement of a K + channel in transmembrane signaling[END_REF], or a complete receptor cluster [START_REF] Triantafilou | A CD14-independent LPS receptor cluster[END_REF]52]. Since the peptides incorporate also into the membrane, they may be able to change the conical shape of LPS by inducing a lamellar geometry as it was seen in the X-ray diffraction experiments for LPS Re (Fig. 6) and lipid A (not shown).

CONCLUSION

The ability of the peptides to act antimicrobially, to inhibit the LPS-induced cytokine secretion without exhibiting cytotoxicity and hemolysis make them or derivatives thereof potential candidates in sepsis therapy. In the present paper the biophysical prerequisites for effective endotoxin neutralization were described: An exothermic reaction between LPS and peptides leading to a fluidization of the lipid A acyl chains with concomitant disappearence of the gel to liquid crystalline phase transition, a multilamellarization of the aggregate structure of LPS with a very high number of bilayer stacks, and an incorporation of the peptides into target phospholipids as model of immune cell membranes. Antibacterial activity is given as MIC (MBC) in µg/ml and was determined by microdilution assays in (1) 10% LB / 90 % buffer, and in (2) Müller-Hinton broth (see Materials and Methods). Cytotoxicity against HeLa cells and lysis of human erythrocytes (hRBC) was determined for 100 µg/ml peptide and was calculated relative to an untreated control.

Figure legends

Fig. 1: Amino acid sequences of the synthesized cyclic LALF peptides. The cyclation is done between the two cysteine residues as indicated for cLALF20.

  Mueller-Hinton broth. Susceptibility testing was performed following the recommendations of the Clinical Standards Institute (CLSI, formerly NCCLS) [Standards, 2000 [25]. Peptides (2 mg/ml in 4 mM HEPES, pH 7.2) were two-fold diluted in Mueller-Hinton Broth (ref. 275730, pH 7.3, Difco Laboratories, Le Pont de Claix, France) in 96-well microtitre plates (TPP, Trasadingen, Switzerland) to obtain concentrations from 512 to 0.25 µg/ml in a volume of 100 µl. Bacteria were grown on Mueller-Hinton agar plates

  10 th injection (after approximately 2500 s). When plotting the integrated areas of the enthalpy curves (enthalpy change ∆H c ) versus [LPS]:[peptide] molar ratios (Fig.5B), it can be deduced that (i), for all peptides the negative enthalpy changes ∆H c corresponds to exothermic reactions; and that (ii), the titrations indicate a two-step process. Thus, for cLALF15 minima in the curve were observed at [LPS]:[peptide] = 0.3 and 1.6, respectively. Furthermore, binding saturation took place at highest molar ratio for cLALF9 ([LPS]:[peptide] = 2) and was lowest for cLALF20 ([LPS]:[peptide] < 1). The saturation values for the other peptides were intermediate between these two.

Fig. 2 :

 2 Fig. 2: DSC heating-scans of LPS R60 in the presence of different concentrations of peptides

Fig. 3 :

 3 Fig. 3: Calorimetrically determined enthalpy change ∆H c versus [LPS]:[peptide] molar ratio

Fig. 4 :Fig. 5 : 30 x 3

 45303 Fig. 4: Gel to liquid crystalline phase behaviour of the hydrocarbon chains in the lipid A

Fig. 6 :Fig. 7 :Fig 8 :

 678 Fig. 6: Synchrotron radiation small-angle X-ray diffraction patterns of [LPS]:[peptide] mixtures ([LPS]:[cLALF] = 2.5:1 molar. The scattering vector s = 1/d = 2 sinθ / λ (d = spacing ratio, θ = scattering angle, λ= wavelenth = 0.15 nm) is plotted versus the

Fig. 9 :

 9 Fig. 9: Concentration-dependent inhibition of the biological activity of LPS Ra from S.

  Fig.2

  Fig. 5B

  Fig. 7A

Fig. 7B

  Fig. 7B

Fig. 7C

  Fig. 7C

Fig. 9

 9 Fig.8

Table 1 : Antibacterial, cytotoxic, and hemolytic activity of cLALF peptides

 1 52 Triantafilou, M., Brandenburg, K., Kusumoto, S., Fukase, K., Mackie, A.,Seydel, U., and Triantafilou, K. (2004) Combinational clustering of receptors following stimulation by bacterial products determines LPS responses. Biochem.J. 381, 527-536

		Sal.	Sal.	E. coli	P.	S.	HeLa	hRBC
		min.	min.	ATCC	aeruginosa 2	aureus 2	(%	(%
		R595 1	R60 1	25922 2			cytotox)	hemoglobin
								release)
	CLALF9	4 (8)	8 (16) 64 (64)	256 (256)	128 (256)	0	9
	CLALF12 4 (16) 8 (32) 64 (128) >256 (>256) 64 (128)	0	11
	CLALF15 4 (32) 8 (32) 32 (32) >256 (>256) 64 (64)	0	14
	CLALF16 4 (16)	16 (32)	64 (64) >256 (>256)	128 (128)	0	15
	CLALF18 4 (16) 8 (32)	256 (256)	>256 (>256)	256 (256)	0	11
	CLALF20 8 (16)	16 (64)	64 (128) >256 (>256)	>256 (>256)	0	6
	CLALF22 2 (4)	8 (16) 32 (32)	32 (64)	n.d.	0	19

Biochemical Journal Immediate Publication. Published on 14 May 2007 as manuscript BJ20070279 © 2007 The Authors Journal compilation © 2007 Biochemical Society

© 2007 The Authors Journal compilation © 2007 Biochemical Society

Acknowledgements

We are indebted to G. von Busse, U. Diemer, C. Hamann, B. Fölting, K. Stephan, and R. Kaiser for technical assistance in the IR spectroscopic, LAL, FRET, DSC, TNFα and FFTEM measurements, respectively. This work has been carried out with financial support from the Commission of the European Communities, specific RTD programme "Quality of Life and Management of Living Resources", QLK-CT-2002-01001", 'Antimicrobial endotoxin neutralizing peptides to combat infectious diseases', and from Deutsche Forschungsgemeinschaft SFB 617, 'Molecular mechanisms of epithelial defense' (project A17). Work at the University of Navarra was supported by a grant from the Ministerio de Sanidad y Consumo of Spain (FIS-PI050768).