WO1999019350A1

WO1999019350A1 - Use of elicitins as lipid carriers

Info

Publication number: WO1999019350A1
Application number: PCT/FR1998/002203
Authority: WO
Inventors: Vladimir Mikes; Jean-Pierre Blein; Marie-Louise Milat; Michel Ponchet; Pierre Ricci
Original assignee: Institut National De La Recherche Agronomique (Inra)
Priority date: 1997-10-15
Filing date: 1998-10-14
Publication date: 1999-04-22
Also published as: AU9545598A; FR2769630A1; EP1023321A1; FR2769630B1

Abstract

The invention concerns the use of elicitins as lipid carriers, in particular steroids, such as sterols.

Description

USE OF ELICITINS FOR THE TRANSPORT OF LIPIDS

The invention relates to the use of proteins of the elicitin family for the fixation and transport of lipid molecules. Elicitins are extracellular proteins, which have been demonstrated in certain species of phytopathogenic fungi of the class Oomycetes [cf. reviewed by P. RICCI, "Induction of the Hypersensitive Response and Systemic Acquired Resistance by Fungal Proteins: the Case of Elicitins", published in Plant-Microbes Interactions, Volume 3, edited by G. STACEY and NT KEEN, (editions CHAPMAN and HALL ) pp. 53-75, (1997)].

Elicitins, when inoculated with a host plant, have been shown to play an essential role in inducing a hypersensitivity reaction manifested by: the formation of localized necrotic lesions, located not only at the point d 'inoculation, but also at the level of leaf territories distant from it; and or

- activation in the plant of defense mechanisms leading to the acquisition of systemic resistance with a broad spectrum. The inducing activity can vary according to the type of elicitin, and according to the plant concerned.

Elicitins were first identified in several species of the genus Phytophthora. Closely related proteins, both structurally and functionally, were then demonstrated in Pythium vexans [HUET et al.,

Molecular Plants / Microbes Interactions, 8: 2, pp. 302-310

(1995)]. The common characteristics between the elicitins of Phytophthora and the similar proteins of Pythium, define a family of proteins, which will be called hereinafter “family of elicitins”. This family includes small, highly water-soluble proteins, of around 10 kDa, which, in their mature secreted form, consist of a single polypeptide chain of around a hundred amino acids, rich in hydrophobic residues and containing 6 cysteine residues , forming 3 intramolecular disulfide bridges, which give the protein a globular tertiary structure.

The term “rich in hydrophobic residues” is understood here to mean a protein in which these hydrophobic residues (in which the cysteine residues also involved in disulfide bridges, and the tyrosine residues are included) represent at least 50% of the total amino acid residues, the apolar amino acids representing at least 40% of the total amino acids.

In proteins of the elicitin family, these hydrophobic residues generally represent between 50 and 60% approximately of the total amino acid residues, and the apolar amino acids represent between 40 and 50% approximately of the total amino acid residues.

Among the elicitins isolated from fungi of the genus Phytophthora, there are acid elicitins (or α-elicitins), the pi of which is between 3 and 5, such as capsicin and parasiticin, isolated from Phytophthora capsici and Phytophthora respectively. parasi tica, and basic elicitins (β-elicitins), whose pi is between 7 and 9, such as the cryptogeine of Phytophthora cryptogea and the cinnamomine of Phytophthora cinnamomi. The elicitins which have been identified in Pythium vexans have a pi comparable to that of the α-elicitins of Phytophthora.

Experiments carried out in tobacco have shown that the basic elicitins of Phytophthora are active at concentrations 50 to 100 times more weak, with regard to the induction of distal necrosis, than acid elicitins. The acid elicitins of Pythium vexans have an activity which appears to be greater than that of the acid elicitins of Phytophthora (ΗUET et al., Publication cited above).

The proposed mode of action of elicitins involves binding to a specific membrane receptor, which, via protein phosphorylation, and a network of second messengers, causes a cascade of events causing localized cell destruction , and in parallel, the induction of resistance mechanisms.

Until now, no function of elicitins, apart from the activity on plants mentioned above, was known, and the only uses which have been proposed for these proteins relate to the protection of plants against parasites.

The inventors have now found that proteins of the elicitin family have the property of fixing lipid molecules, such as steroids, and in particular sterols.

The subject of the present invention is the use of at least one protein from the elicitin family for capturing and fixing lipids, in an in vitro system or in a plant, as well as for obtaining a medicament. able to capture and fix lipids.

According to a preferred embodiment of the present invention, said lipids are steroids, and in particular sterols.

For the purposes of the present invention, the term “protein of the elicitin family” means not only the proteins already characterized in Phytophthora and Pythium, but also any protein having the structural characteristics and functional of the elicitin family, as defined above. Such proteins can in particular be obtained from Oomycetes, such as Phytophthora or Pythium. According to the invention, elicitins can be used to extract the lipid molecules mentioned above from a medium containing them. This can be done by bringing the medium concerned into contact with a preparation containing one or more protein (s) from the elicitin family, then separation of the medium depleted in lipid molecules, and from the elicitins which have fixed these molecules. This separation can optionally be facilitated by using elicitins linked to a solid support, for example a chromatography column, microbeads, membranes, etc.

Elicitins can also be used to obtain lipid-lowering drugs, preferably cholesterol-lowering drugs. The inventors have also found that elicitins also have the property of catalyzing the transfer of lipid molecules such as steroids, and in particular sterols to, or from micelles, or else lipid bilayers, such as liposomes or natural or artificial membranes.

According to a preferred embodiment of the present invention, at least one protein of the elicitin family is used to stimulate the transfer of the lipid molecules mentioned above, in an in vitro system or in a plant.

For example, this transfer can be carried out, in one step, by adding the elicitin (s) to a medium containing the structures (micelles and / or lipid bilayers) “donor” and “acceptor”, or else in several steps , by adding the elicitin (s), to a medium containing the “donor” structures, then by separating from said medium the elicitin (s) having fixed the lipid molecules, and by adding them to a medium containing the “acceptor” structures. According to another preferred embodiment of the present invention, at least one protein of the elicitin family is used for obtaining a medicament capable of transferring lipid molecules, such as steroids and in particular sterols.

The transfer activity is more or less important depending on the elicitin concerned, and this, independently of its fixing properties of lipid molecules. It is thus possible, depending on whether it is desired, in the intended use, to favor the fixation of the sterols or their transfer, to choose the most appropriate elicitin.

The respective fixing and transfer capacities of the sterols can easily be evaluated by a person skilled in the art by implementing the experimental protocols described in detail in the examples below.

Unlike the lipid transfer proteins capable of transferring sterols, known up to now, [FISCHER et al., Biochemistry, 24, pp. 3322-3331, (1985); SCHROEDER et al., J. Biol. Chem. , 265, pp. 151-157, (1990); SCHROEDER et al., Lipids, 25, pp. 669-674, (1990); OODFORD et al., Chem. Phys. Lipids, 76, pp. 73-84, (1995); FROLOV et al., J. Biol. Chem., 271, pp. 16075-16083, (1996)] which are all intra-cellular proteins, elicitins are secreted, extra-cellular proteins. This facilitates their obtaining and their purification from culture media, which can either be cultures of fungi producing them naturally, or cell cultures transformed by elicitin genes.

The present invention will be better understood with the aid of the additional description which follows, which refers to examples illustrating the properties of elicitins in the setting of sterol fixation and transfer.

I: LIPID FIXING PROPERTIES: Products: A basic elicitin, cryptogeine, and 3 acid elicitins, cactorin, capsicine, and parasiticin, were prepared according to the protocol described by BONNET et al., Eur. J. Plant. Pathol., 102, pp. 181-192, (1996), dissolved in water, and stored at -30 ° C until use.

The phospholipids, 2-p-toluidinonaphthalene-6-sulfonate (TNS) and the sterols are marketed by SIGMA. The sterols and TNS are dissolved in ethanol. Buffer I: 175 mM annitol, 0.5 mM

CaCl ₂ , 175 mM K ₂ S0 ₄ , and 5 mM MES, pH7 Fluorescence measurements:

The fluorescence measurements are carried out with stirring, at 25 ° C. using a SHIMAZU RF 5001 PC spectrofluorimeter, in a fluorinated tank, in

2 ml of buffer I. The fluorescence intensity is expressed in arbitrary units (u.a.).

For DHE (dehydroergosterol) and TNS, the excitation wavelengths were fixed at 325 and 355 nm respectively, and the emission wavelengths at 370 and 430 nm.

The fluorescence of elicitins alone is negligible. EXAMPLE 1: DEMONSTRATION OF THE INTERACTION OF ELICITINS WITH DEHYDROERGOSTEROL.

Interaction detection

The interaction of elicitins with DHE was detected by observing changes in the fluorescence emission spectrum of DHE in the presence of the various elicitins in buffer I.

In the absence of elicitins, the emission maximums for DHE after excitation at 325 nm are 370, 402, and 424 nm. The addition of each of the elicitins in the same buffer causes a marked increase in the maximum emission at 370 nm. The shape of the fluorescence emission spectra emitted from all the elicitin-DHE complexes is similar. These results are illustrated in FIG. 1, which represents the fluorescence emission spectra of the DHE-elicitin mixture. The fluorescence spectra are recorded 5 minutes after the addition of elicitin (2 μM) to DHE (1 μM) in buffer I. The excitation wavelength is 325 nm.

1. DHE without elicitin;

2. DHE + capscein;

3. DHE + parasiticin; . DHE + cactorin; 5. DHE + cryptogeine.

The modifications in the fluorescence emission spectrum of DHE in the presence of elicitins show that DHE interacts with these proteins. Kinetics of the interaction, and pH effect The kinetics of the DHE-elicitin interaction have been studied for each of the elicitins mentioned above.

The results are illustrated in FIG. 2A, which represents the kinetics of the elicitin-DHE interaction, under the following conditions: 1.6 μM of DHE and 2 μM of the elicitin tested are added to buffer I; the excitation wavelength is 325 nm, and the emission is measured at 370 nm.

These results show that the kinetics differ somewhat depending on the elicitin concerned. Under the experimental conditions described above, the initial rate of interaction is high in the case of cryptogeine and cactorin, and reaches a plateau after 100 seconds and 200 seconds respectively. The fluorescence gradually increases for capsicin and parasiticin, and reaches the plateau after 5 minutes.

The effect of pH on the interaction between DHE and 2 elicitins, a basic, cryptogeine, and an acid, capsicine, was analyzed.

The fluorescence kinetics of the elicitin / DHE interaction was determined in buffer I, adjusted to different pH between 4.5 and 8.

Figure 2B illustrates the effect of pH on the interaction of cryptogein O or capsicin • with DHE.

The fluorescence intensity is recorded 100 seconds (in the case of cryptogeine) or else 300 seconds (in the case of capsicine) after the addition of elicitin to DHE.

These results show that the fluorescence intensity of the DHE-cryptogeine complex changes significantly between pH 4.5 and pH 5.5, then remains constant. A similar effect is observed for capsicin, despite its different isoelectric point, and slower kinetics. DHE / elicitin binding parameters

The increased fluorescence of DHE upon binding to elicitins is used to determine the parameters of DHE binding. DHE is added to buffer I, at concentrations varying from 0.2 to 3.8 μM. The fluorescence intensity (F ₀ ) is measured.

2 μM of each elicitin are then added and the fluorescence intensity (F) of the mixture is measured after stabilization.

The difference ΔF = F-Fo is determined, which makes it possible to establish titration curves representing ΔF as a function of the DHE concentration.

On the other hand, the titration of a constant concentration (0.25 μM) of DHE, by each elicitin until reaching a fluorescence plateau makes it possible to determine the amount of bound DHE.

To determine the dissociation constants and the number of binding sites, curves representing 1 / [bound DHE] as a function of 1 / [free DHE] were plotted on the basis of the following equation:

I / C _b = (Kd / NA). 1 / C _f + 1 / NA in which

C _b and C _f are respectively the concentrations of bound DHE and of free DHE,

K _d is the dissociation constant,

A is the concentration of acceptor, and N the number of binding sites.

The dissociation constants of the elicitin-DHE complex (Kd), the stoichiometry of the interaction (N) and the relative intensity of fluorescence of the bound DHE (Qb) are represented in Table I below:

TABLE I

These results show that all of the elicitins studied have the same binding stoichiometry, that is to say one molecule of sterol per molecule. elicitin, and dissociation constants of the same order of magnitude (between 0.11 and 0.58 μM).

These binding parameters can be compared with those of several intracellular animal proteins known for their ability to fix lipids. For example, the sterol transport protein SCP2 [SCHROEDER et al., J. Biol. Chem., 265, pp. 151-157, (1990)] fixes the DHE with an apparent dissociation constant of 1.2 to 1.5 μM, and a stoichiometry of about 0.8 to 1 molecule of sterol per molecule of protein. The sterol transfer protein SCP, whose structure is different from the previous one, fixes the DHE with a dissociation constant of 0.88 μM and a similar stoichiometry [FISCHER et al, Biochemistry, 24, pp. 3322-3331, (1985)]. A rat liver protein, which has the property of fixing fatty acids, seems to have a higher affinity with a dissociation constant of 0.29 μM, and a stoichiometry of 0.9 binding site per protein molecule; on the other hand, bovine serum albumin has a lower affinity (Kd = 2.9 μM) and 6 to 7 binding sites per protein molecule [SCHROEDER et al., publication cited above].

These results show that the elicitins tested have binding parameters of the same order as those of the proteins capable of binding the sterols previously known.

EXAMPLE 2 INTERACTION OF CRYPTOGEIN WITH PHYTOSTEROLS.

The binding of non-fluorescent sterols, which is the case of plant-based sterols, to elicitins, was measured indirectly by observing their action on the fluorescence of the complexes formed between elicitins and a fluorescent probe, TNS.

The fluorescence of TNS alone, in solution in water, is negligible. When adding cryptogeine there is an increase in the fluorescence at 430 nm. The interaction of TNS with cryptogeine is faster than that of DHE. The study of the cryptogeine / TNS binding parameters shows a dissociation constant of 1.45 ± 0.07 μM, and a stoichiometry of 1.4 ± 0.03 binding sites per molecule of cryptogeine. Cryptogeine therefore has a lower affinity for TNS than for DHE.

The influence of different sterols on the fluorescence of the cryptogein-TNS mixture has been studied. FIG. 3 represents the extinction kinetics curves, by different sterols, of the fluorescence of TNS fixed to cryptogeine, established under the following conditions:

To a 7 μM solution of TNS in buffer I, 2.5 μM of cryptogeine is added, and the mixture is incubated for 2 minutes. 1.2 μM of sterol to be tested is then added, and the fluorescence is measured for 200 seconds. The excitation wavelength and the emission wavelength are 355 and 430 nm respectively.

The addition of a sterol to the preincubated cryptogeine with TNS induces a significant decrease in the fluorescence of TNS at 430 nm, which stabilizes after 1 to 3 minutes. The kinetics of this extinction are rapid in the case of cholesterol, and slower in the case of campesterol, stigmasterol, and β-sitosterol or DHE.

To study the mechanism inducing this decrease in fluorescence, cryptogeine was preincubated with stigmasterol and the titration curves of the complex by TNS were compared with those of cryptogein without stigmasterol.

The forms of these titration curves are different, while the maximum values obtained in the presence of an excess of TNS are similar. This suggests as competition the most likely mechanism TNS and the sterol, and a displacement of the bound TNS, resulting from a higher association constant of the sterol.

FIG. 3A represents the fluorescence titration curves of cryptogeine with TNS: O cryptogeine alone (2.5 μM);

• cryptogeine preincubated for 5 minutes with stigmasterol (2.5 μM).

FIG. 3B represents the titration curves for the extinction of the fluorescence of TNS fixed to cryptogeine by different sterols. The TNS-cryptogeine complex was titrated in the presence of cholesterol O, campesterol A., β-sitosterol M, dehydroergosterol • and stigmasterol Q, in the buffer I. The intensity of the fluorescence of TNS is measured after stabilization signal, that is after 2 or 3 minutes.

It has been verified that no direct interaction between sterols and TNS occurs. For example, when cholesterol alone (3 μM) is added to TNS

(7 μM), no change in fluorescence is observed.

These results show that all the sterols studied bind to cryptogeine with similar dose-response curves, but none of them can completely quench the fluorescence of TNS. EXAMPLE 3: ISOLATION OF A CRYPTOGEIN-STIGMASTEROL COMPLEX.

In order to verify that the fluorimetry results reflect the formation of a sterol-elicitin complex, a stigmasterol-cryptogein complex was prepared, then isolated by gel filtration.

Cryptogeine was dissolved in buffer I (970 μg for 948 μl of buffer) and a solution of stigmasterol in ethanol was added slowly (52 μl of a 1 mg / ml solution). After incubation for 20 min, the cryptogein-stigmasterol complex is separated from the excess sterol by gel filtration, on a column. SEPHADEX G-25 (PHARMACIA) of 125 mg balanced with buffer I. The elution of the fractions containing the protein is carried out with buffer I, and followed by measurement of absorption in UV at 280 nm; the excess sterol adsorbed on the SEPHADEX column is eluted by adding ethanol to buffer I.

Table II below groups together the results of two independent experiments carried out with a respective initial protein / sterol ratio of initial protein-sterol ratio 1/1, 6 ^a and 1/1, 3 ^b .

Table II

a: initial protein / sterol ratio = 1/1, 6 b: initial protein / sterol ratio = 1/1, 3

Fraction 1 contains the stigmasterol-cryptogein complex. All of the initial cryptogeine is recovered in this fraction. The cryptogeine-stigmasterol molar ratio in the complex is between 1.08 and 0.93. These results confirm the stoichiometry measured by fluorimetry.

II - LIPID TRANSFER PROPERTIES.

EXAMPLE 4: STIMULATION BY THE ELICITINS OF THE TRANSFER OF STEROLS BETWEEN MICELLES.

To test the sterol transfer activity of elicitins, the fluorescence of DHE was measured in mixed micelles formed from 2 types of micelles containing DHE respectively, or else a non-fluorescent sterol.

In water, DHE occurs in the form of micelles with a critical micellar concentration of 0.025 μM. The quantum fluorescence yield of DHE in the form of micelles is low, due to self-extinction caused by the interaction between DHE molecules. When non-fluorescent sterol micelles are added to DHE micelles, spontaneous transfer of the sterols causes mixed micelles to form. This leads to dilution and separation of the DHE molecules, which results in an increase in the fluorescence of DHE.

FIG. 4 represents the transfer of the sterols between micelles of DHE and micelles of stigmasterol, spontaneous, or else stimulated by elicitins, and evaluated by measuring the fluorescence of DHE.

The micelles are prepared in buffer II: 10 mM MES (pH 7), 0.02% Na azide.

The “donor” micelles contain 0.63 μM of DHE, the “acceptor” micelles contain 3 μM of stigmasterol, in buffer II.

The stimulated micellar transfer is induced by adding 0.5 μM of the elicitin to be tested.

At the start of the experiment, all of the DHE molecules are present in “donor” micelles. When an excess of stigmasterol micelles is added to DHE micelles in buffer II, the fluorescence of DHE gradually increases (Figure 4, bottom curve), reflecting the spontaneous exchange of sterols between the micelles, which leads to dilution DHE. This is confirmed by mixing the 2 sterols in ethanol beforehand, and adding them at the same concentrations as above: the fluorescence of DHE in mixed micelles is 8 times greater than that observed in micelles containing only DHE (Figure 4, “stigmasterol-DHE” curve). Addition of cryptogeine to the donor (DHE) and acceptor mixture (stigmasterol) significantly stimulates exchange, and the fluorescence of DHE reaches a maximum after about 4 minutes. The other elicitins tested are also capable of promoting the exchange of sterols between the micelles, but to a lesser extent. Cactorin is less effective than cryptogein, and the effect of capsicin is very weak.

The increase in fluorescence (9 arbitrary units in the presence of cryptogeine) observed in this experiment after addition of the elicitins to the mixture of DHE and stigmasterol micelles well reflects the dilution of DHE in the stigmasterol micelles, and not the fluorescence of the complex. DHE- elicitin, which under the same conditions, represents only 2 arbitrary units in the presence of cryptogeine. In addition, the contribution of the DHE-elicitin complex to total fluorescence is negligible, since there is competition between stigmasterol and DHE for binding to cryptogeine, and the experiments were carried out in the presence of an excess of stigmasterol.

Similar results have been obtained using cholesterol, in place of stigmasterol, in "acceptor" micelles.

EXAMPLE 5: STIMULATION BY THE ELICITINS OF THE TRANSFER OF STEROLS BETWEEN ARTIFICIAL MEMBRANES.

Preparation of liposomes:

Two types of liposomes were prepared: "donor" liposomes containing phosphatidylcholine, phosphatidylserine, and DHE, "acceptor" liposomes containing phosphatidylcholine, phosphatidylserine, and cholesterol.

The phospholipids and sterols are dissolved in chloroform. For each test, 4.4 mg of phosphatidylcholine, 0.8 mg of phosphatidylserine and 1.5 mg of sterol (cholesterol or DHE) are mixed; the solvents are evaporated under nitrogen. Then 1 ml of exchange buffer (buffer II) is added to the preparation. The mixture is vortexed under nitrogen, then sonicated for 3 times 5 minutes at 40 ° C. The liposome suspension is centrifuged (150,000 g, 1 hour), and the supernatant containing the unilamellar vesicles is used for the measurements. The yield of unilamellar vesicles, estimated on the basis of the fluorescence of DHE before and after centrifugation, is approximately 60%. Study of sterol transfer.

The molecular transfer of DHE between the liposomes is visualized by the increase in the steady state fluorescence polarization of the DHE in the donor / acceptor mixture. The fluorescence polarization measurements are carried out at 25 ° C. with a KONTRON SFM-25 spectrofluorimeter equipped with polarizers.

The intensities of the horizontal and vertical components are determined for 10 minutes. Values obtained in the absence of DHE are subtracted.

Thanks to the transfer of energy between the DHE molecules in the “donor” liposomes, the basic fluorescence polarization is low (around 0.21). The variations in polarization during the mixing of “donor” and “acceptor” liposomes, in the absence or in the presence of elicitin, are shown in FIG. 5.

These results are obtained under the following conditions: The ratio between “donor” liposomes and “acceptor” liposomes is 1/9.

The total concentration of vesicles in buffer II is 140 μg / ml, and the concentration of elicitin is 1 μM. d = “donor” vesicles + cryptogeine;

O = spontaneous transfer; • = “donor” vesicles + “acceptor” vesicles + cryptogeine;

A = “donor” vesicles + “acceptor” vesicles + capscein; M = "donor" vesicles + vesicles

"Acceptor" + cactorin;

Δ = "donor" vesicles + vesicles

"Acceptor" + parasiticin.

These results show that: - when 1 μM of cryptogeine is added to 140 μg per ml of “donor” liposomes alone, no change in polarization is observed.

- spontaneous sterol transfer causes an increase in fluorescence polarization from 0.20 to 0.28. the addition of elicitins to the mixture of “donor” and “acceptor” liposomes stimulates the exchange of sterols between the liposomal membranes.

As in the case of micelle transfer experiments, among the elicitins tested, it is cryptogeine which most effectively stimulates the exchange of sterol between artificial membranes. Cactorin is less effective, and parasiticin and capsicin are only weakly so. The presence of phosphatidylserine increases the negative surface charge, which can considerably increase the exchange stimulated by a basic sterol transport protein.

Claims

1) Use of at least one protein from the elicitin family for the capture and fixation of lipid molecules, in an in vitro system or in a plant

2) Use of at least one protein from the elicitin family to obtain a medicament capable of capturing and fixing the lipid molecules.

3) Use according to any one of claims 1 or 2, characterized in that said lipid molecules are steroids, in particular sterols.

4) Use according to any one of claims 1 to 3, characterized in that it further comprises the transfer of said lipid molecules by said protein of the elicitin family.