Formation of asymmetric vesicles via phospholipase D-mediated transphosphatidylation

Accepted Manuscript Formation of asymmetric vesicles via phospholipase D-mediated Transphosphatidylation

Rina Takaoka, Haruko Kurosaki, Hiroyuki Nakao, Keisuke Ikeda, Minoru Nakano PII: DOI: Reference:

S0005-2736(17)30321-8 doi:10.1016/j.bbamem.2017.10.011 BBAMEM 82611

To appear in: Received date: Revised date: Accepted date:

4 June 2017 3 October 2017 9 October 2017

Please cite this article as: Rina Takaoka, Haruko Kurosaki, Hiroyuki Nakao, Keisuke Ikeda, Minoru Nakano , Formation of asymmetric vesicles via phospholipase D-mediated Transphosphatidylation. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Bbamem(2017), doi:10.1016/ j.bbamem.2017.10.011

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Formation of Asymmetric Vesicles via Phospholipase D-mediated Transphosphatidylation Rina Takaoka,† Haruko Kurosaki,† Hiroyuki Nakao,‡ Keisuke Ikeda,‡ Minoru Nakano*,‡ Faculty of Pharmaceutical Sciences and ‡Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan

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Abstract

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Most biomembranes have an asymmetric structure with regard to phospholipid distribution

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between the inner and outer leaflets of the lipid bilayers. Control of the asymmetric distribution plays a pivotal role in several cellular functions such as intracellular membrane fusion and cell

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division. The mechanism by which membrane asymmetry and its alteration function in these transformation processes is not yet clear. To understand the significance of membrane asymmetry

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on trafficking and metabolism of intracellular vesicular components, a system that experimentally

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reproduces the asymmetric nature of biomembranes is essential. Here, we succeeded in obtaining asymmetric vesicles by means of transphosphatidylation reactions with phospholipase D (PLD),

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which acts exclusively on phosphatidylcholine (PC) present in the outer leaflet of vesicles. By

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treating PC vesicles with PLD in the presence of 1.7 M serine and 0.3 M ethanolamine, we obtained asymmetric vesicles that are topologically similar to intracellular vesicles containing

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phosphatidylserine and phosphatidylethanolamine in the cytosolic leaflet. PLD and other unwanted compounds could be removed by trypsin digestion followed by dialysis. Our established technique has a great advantage over conventional methods in that asymmetric vesicles can be provided at high yield and high efficiency, which is requisite for most physicochemical assays.

Keywords: asymmetric vesicles; phosphatidylcholine; phospholipase D; flip-flop

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ACCEPTED MANUSCRIPT 1. Introduction Most eukaryotic cellular phospholipids are synthesized in the endoplasmic reticulum (ER) and are transferred to the plasma membrane (PM) or various organelles via lipid transfer proteins and vesicular transport. While lipids included in the ER are distributed equally to both

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cytosolic/luminal leaflets, other organelles’ membranes have an asymmetric structure [1]. For

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example, phosphatidylserine (PS) and phosphatidylethanolamine (PE) in the PM are localized

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exclusively in the cytosolic leaflet by the action of amino-phospholipid translocases that flip these lipids inward at the expense of ATP hydrolysis. Indeed, a breakdown in the asymmetry and

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concomitant exposure of PS toward the extracellular leaflet are symptoms of apoptosis [2, 3]. The

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asymmetric distribution and its alteration are also reportedly connected to the process of intracellular vesicle fusion and cell division [4]. Therefore, maintenance and control of the

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membrane asymmetry play important roles in cellular life and death.

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The mechanism by which membrane asymmetry and its alteration function in the process of membrane transformation remains to be clarified. To address this knowledge gap, it is necessary

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to develop technologies that will produce asymmetric vesicles that mimic the structure and

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characteristics of the biomembranes. Cyclodextrin-mediated phospholipid exchange between vesicles with different compositions would produce the asymmetric vesicles [5, 6], but this

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procedure generates a large amount of undesired vesicles that need to be removed in a complicated manner. Engineering of µm-sized asymmetric vesicles has been attained by submerging a droplet of phospholipid-containing w/o emulsion into an aqueous phase through a phospholipid monolayer spread at the oil–water interface [7]. Alternatively, asymmetric vesicles can be produced by microfluidic jetting into asymmetric bilayers created by the contact of two w/o emulsion droplets with different phospholipid compositions [8]. Particles obtained by these sophisticated methods are advantageous when observing vesicles under optical microscopy. However, this is not suitable for 2

ACCEPTED MANUSCRIPT vesicle preparation in large quantities. Furthermore, the vesicles would be inevitably contaminated by oil [8]. Phospholipase D (PLD) is an enzyme that catalyzes the hydrolysis of phospholipids. Treatment of vesicles consisting of phosphatidylcholine (PC) with PLD from Streptomyces sp. has

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been shown to convert all PC molecules located at the outer leaflet into phosphatidic acid (PA),

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resulting in the formation of “PCIN/PAOUT” asymmetric vesicles [9, 10]. PLD is also known to

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catalyze the transphosphatidylation reaction with a primary alcohol to yield variety of phospholipids, such as PS and PE [11, 12], but the reaction has been generally carried out in the

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presence of an organic solvent to increase the reaction efficiency. Only a few studies have reported

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the transphosphatidylation reaction on vesicle membranes [13-15], but they have paid no attention to the creation of the membrane asymmetry. Because PS and PE are major components of the

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cellular phospholipids, we aimed at producing “PCIN/PS·PEOUT” asymmetric vesicles by applying

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transphosphatidylation to vesicles (Figure 1).

Figure 1. Schematic representation of the production of “PCIN/PS·PEOUT” asymmetric vesicles by PLD-mediated transphosphatidylation.

2. Experimental Section 2.1. Materials 3

ACCEPTED MANUSCRIPT PLD from Streptomyces sp., trypsin from porcine pancreas (25mg/mL trypsin solution in 0.9% NaCl), peroxidase from horseradish, and cholesterol were purchased from Sigma-Aldrich (St. Louis, MO). 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (PC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (PS) were purchased from NOF Corporation

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(Tokyo, Japan). 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (PE) was obtained from

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Avanti Polar Lipids (Alabaster, AL, USA). Choline oxidase from Alcaligenes sp.,

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4-aminoantipyrine, ethanolamine, and heptaethylene glycol dodecyl ether were purchased from WAKO (Osaka, Japan). N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline sodium salt

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(DAOS) and calcein were purchased from Dojindo (Kumamoto, Japan). Triton X-100 and L-serine

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were obtained from Nacalai Tesque (Kyoto, Japan) and Peptide Institute, Inc. (Osaka, Japan),

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respectively. All other chemicals used were of the highest reagent grade.

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2.2. Preparation of Vesicles

To prepare large unilamellar vesicles, the required amounts of a methanol–chloroform

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solution of phospholipids and cholesterol were placed in a round-bottomed glass flask. After

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evaporation of the organic solvents, the residues were dried overnight in vacuum. The dried lipid was hydrated with Tris-HCl buffer (10 mM Tris, 150 mM NaCl, and 0.01 g/mL NaN3; pH 7.4). The

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lipid suspension was vortexed, freeze-thawed seven times, and extruded through a 100 nm pore polycarbonate filter using LiposoFast (Avestin, Ottawa, Canada). The diameter of vesicles was confirmed to be approximately 130 nm by dynamic light scattering (DelsaMax CORE, Beckman Coulter Inc., Indianapolis, IN, USA). The concentration of phospholipids was determined by phosphorus assay [16].

2.3. Treatment of Vesicles with PLD 4

ACCEPTED MANUSCRIPT To vesicles containing 3.0 mM PC in Tris-HCl buffer (10 mM Tris, 150 mM NaCl, and 0.01 g/mL NaN3; pH 7.4), 1.65 U/mL PLD from Streptomyces sp. was added in the presence or absence of 1.7 M serine and/or 0.3 M ethanolamine, and the mixture was incubated at 37°C. Although addition of serine and/or ethanolamine substantially changed pH, its adjustments were

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not made unless otherwise mentioned (see details in Results and Discussion). At the indicated time

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the amount of released choline by the method described below.

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points, aliquots of the mixture were withdrawn, and the PLD reaction progress was determined as

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2.4. Quantification of Choline

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Quantification of choline was carried out by an enzymatic assay. The sample was added to a reaction solution consisting of 4.0 U/mL choline oxidase, 2.5 U/mL peroxidase, 0.73 mM

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DAOS, and 0.73 mM 4-aminoantipyrine in HEPES buffer (10 mM HEPES, 150 mM NaCl, and

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0.01 g/mL NaN3; pH 7.4). After incubation for 10 min at 37°C, the concentration of a generated

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blue dye was measured by optical absorption at 505 nm.

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2.5. Determination of Phospholipid Composition After incubating the PLD-treated sample for 5 h (or 24 h), phospholipids in the sample

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were extracted by methanol/chloroform (1:2 in vol.) solution. After evaporation of the organic solvents, the residues were dried overnight in a vacuum. The dried lipids were dissolved in a mixture of n-hexane/2-propanol/acetate buffer (0.2 M acetate; pH 4.3) (3:8:1 in vol.). Phospholipids in the sample were separated and quantified by HPLC (LC-2000Plus, Jasco Co., Tokyo, Japan) with a silica gel column (Inertsil SIL-100A 5 µm, 4.6 mm × 250 mm, GL Sciences Inc., Tokyo, Japan). The sample was eluted by a solution with the same composition as above

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ACCEPTED MANUSCRIPT (n-hexane/2-propanol/acetate buffer) at a flow rate of 1.0 mL/min at 70°C and detected by its absorbance at 205 nm.

2.6. Isolation of Asymmetric Vesicles

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After a 5-h incubation period, 25mg/mL trypsin solution (in 0.9% NaCl) was added to

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the PLD-treated sample to a final concentration of 0.75 mg/mL in order to digest and deactivate

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PLD. After incubation at 37°C for 30 min, the mixture was dialyzed 4 times against Tris-HCl buffer at 4°C by using a Float A-Lyzer G2 (MWCO: 1000KD, Spectrum Laboratories, Inc., Ohtsu,

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Japan) to remove trypsin, digested PLD, and low molecular weight compounds (choline, serine,

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and ethanolamine). The isolated sample was assayed for residual PLD activity and trypsin concentration. The former was tested by adding newly prepared 3.0 mM PC vesicles into the

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sample and detecting PC hydrolysis using the choline assay as described above. The latter was

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evaluated from tryptophan fluorescence from the samples before and after dialysis, which was measured on a JASCO FP-8300 (Tokyo, Japan) with an excitation wavelength of 290 nm. The

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diameter of vesicles was determined by dynamic light scattering (DelsaMax CORE).

2.7. Verification of the Membrane Asymmetry

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The isolated vesicles were incubated with 1.65 U/mL PLD at 37°C in order to hydrolyze PC exposed in the outer leaflet. After 1-7 days of incubation, portions of the mixture were withdrawn and the concentration of released choline was determined by the enzymatic assay described above. Total PC concentration was determined by quantifying choline released as the vesicles were incubated with 1.65 U/mL PLD in the presence of a membrane-solubilizing agent (1.0 % heptaethylene glycol dodecyl ether).

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ACCEPTED MANUSCRIPT 3. Results and Discussion 3.1. Formation of Asymmetric Vesicles by PLD PLD from Streptomyces sp. (1.65 U/mL) was added to 3.0 mM PC vesicles (d~130 nm) in Tris-HCl buffer at 37°C. Both hydrolysis and transphosphatidylation of PC produce choline. To

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track the degree of progress of the PLD reaction, we monitored the production of choline by an

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enzymatic method with choline oxidase. The choline production reached ca. 40% of total PC at 7 h

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after addition of PLD and became constant thereafter (Figure 2). Taking into account the presence of multilamellar species, which reduces the fraction of phospholipids existing in the outer leaflet of

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the outermost layer, this result suggests that almost all the PC molecules in the outer leaflet of

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vesicles were converted but those in the inner leaflet were not, which agrees with the previous report [10]. We have previously shown by neutron scattering that PC does not flip, while PA (in PA

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bilayers) exhibits flip-flop with t1/2 of ca. 7 h at pH 7.4 and 37°C [9]. Hence, it was expected that PA

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molecules produced at the outer leaflet flip into the inner leaflet, and that the flip of PA forces PC to turn out of the interior, thereby converts all the PC molecules. However, both the previous studies

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[9, 10] and the present data showed that the PLD reaction was arrested after nearly half the total

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lipids were converted. This suggests that because PC molecules do not flop, they remain in the inner leaflet and give no space for PA to flip inward, resulting in the formation of asymmetric

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vesicles. The presence of 1.7 M serine or 30 mol% cholesterol quickened the onset of the plateau but did not change the plateau value. It has been previously shown that incorporation of oleic acid esters (glycerol monooleate or methyl oleate) increases exposure of the PC headgroup and facilitates PC hydrolysis mediated by this enzyme [10]. It is conceivable that incorporation of cholesterol also brought about a similar effect, i.e., increase in the distance between PC headgroups and thereby their exposure to the enzyme. The effect of serine to enhance the PLD reaction may be ascribed to the decrease in pH of the reaction solution, as described later. 7

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Figure 2. Choline production from 3.0 mM PC vesicles containing 0 or 30 mol% cholesterol after treatment with 1.65 U/mL PLD at 37°C in the absence or presence of 1.7 M serine. Error bars represent S.D. of triplicates.

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Lipid compositions of vesicles after a 5-h treatment with PLD were determined by HPLC. PC was hydrolyzed into PA in the absence of a primary alcohol, i.e., serine or ethanolamine

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(Figure 3A). In the presence of 1.7 M serine, however, the reaction yielded PS, whose amount

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exceeded that of PA (Figure 3B), which clearly demonstrates that transphosphatidylation occurs

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with the addition of serine. Conversely, in the presence of 0.3 M ethanolamine, transphosphatidylation to form PE proceeded only slightly (Figure 3C). We could not track choline

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production in the presence of ethanolamine because this compound seems to act as a weak

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substrate for choline oxidase, and hence, influence the quantification of choline (data not shown). Importantly, HPLC data revealed that no PA was produced when ethanolamine was added (Figure 3C). This result indicates that both hydrolysis as well as transphosphatidylation of PC were prevented on the conditions used. Interestingly, however, incorporation of 1.7 M serine with 0.3 M ethanolamine restored PLD activity, which produced both PS and PE equally and more abundantly than PA (Figure 3D). This result shows that the presence of serine prevents ethanolamine from inhibiting PLD activity and encourages transphosphatidylation. Restoration of PLD activity is certainly due to the presence of serine, but not its reactant product, PS, because incorporation of PS 8

ACCEPTED MANUSCRIPT into vesicles did not restrain the inhibitory effect of ethanolamine (Figure S1 in the Supplementary Material). A 24-h incubation also provided a phospholipid composition similar to the 5-h incubation (Figure 3E). Most importantly, ca. 50% of total phospholipids remained unchanged as PC, which obviously represents the asymmetric “PCIN/PS·PEOUT” structure of the vesicles after PLD

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treatment.

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The buffer used in this study (10 mM Tris-HCl) is considered incapable of keeping pH

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unchanged when 0.3 M ethanolamine and/or 1.7 M serine are added. We found that in the experimental condition 0.3 M ethanolamine increased pH to 11.4 and 1.7 M serine decreased to 6.1,

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and that 0.3 M ethanolamine plus 1.7 M serine increased to 8.7. We examined the PLD reaction in

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the presence of 0.3 M ethanolamine and under the control of pH at 7.4 by adding HCl, and indeed, observed that both hydrolysis (to produce PA) and transphosphatidylation (to produce PE)

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proceeded (Figure 3F). It is, therefore, concluded that the inhibition of PLD reaction by

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ethanolamine was due to the increase in pH. In the presence of both ethanolamine and serine, PLD promoted the transphosphatidylation under a slightly basic condition (pH 8.7). Hence, in the

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following experiments to prepare “PCIN/PS·PEOUT” asymmetric vesicles, PLD reactions were carried

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out without neutralizing the pH.

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Figure 3. Phospholipid compositions of vesicles after PLD reactions. PC vesicles (3.0 mM) were treated with 1.65 U/mL PLD at 37°C for 5 h in the absence (A) or presence of (B) 1.7 M serine, (C) 0.3 M ethanolamine, or (D) 1.7 M serine plus 0.3 M ethanolamine. (E) The same as (D) but treated for 24 h. (F) The same as (C) but pH was set at 7.4. Phospholipid compositions of the vesicles were determined by HPLC. Error bars in (A)–(C), (E), and (F) denote maximum and minimum values of duplicates and those in (D) represent S.D. of quadruplicates.

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3.2. Isolation of Asymmetric Vesicles

After the transphosphatidylation reaction these samples contain PLD as well as alcohols

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(serine, ethanolamine, and choline). Although longer incubation times did not change the

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phospholipid composition (Figure 3E), we found that PE (but not PS) can be a substrate for PLD (from Streptomyces sp.) and is hydrolyzed to PA in the absence of serine and ethanolamine (Figure

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S2 in the Supplementary Material). This fact indicates that the enzyme used to prepare the

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asymmetric vesicles needs to be removed, because it progressively alters the lipid compositions. Dialysis of the samples using a dialysis membrane with molecular-weight cut-off of 1,000 kDa

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failed to remove PLD (Figure S3 in the Supplementary Material), whose molecular weight is ca. 60 kDa. This is likely due to binding of the protein to vesicles. PLD did not lose its activity even when heated to 95°C (data not shown). Therefore, we digested PLD with 0.75 mg/mL trypsin (37°C, 30 min) and subsequently dialyzed it against buffer (4°C, 1 d). By this procedure, PLD activity was completely removed (Figure S3 in the Supplementary Material). Fluorescence spectra of the trypsin-treated samples showed that fluorescence from tryptophan residues disappeared by dialysis,

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ACCEPTED MANUSCRIPT which confirmed the removal of polypeptide components, including trypsin, PLD, and their digested fragments (Figure S4 in the Supplementary Material). Lipid compositions of the vesicles isolated by the above procedures were determined by HPLC (Figure 4). By comparing Figure 3D and 4A, it is clear that the phospholipid composition is

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not altered by the isolation procedure. The presence of cholesterol did not largely alter the

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phospholipid composition but slightly decreased the amount of PA (4.5, 1.6, 1.5, and 2.1 mol% of

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total phospholipids for 0, 10, 20, and 30 mol% cholesterol, respectively) (Figure 4). Dynamic light scattering (DLS) data suggested that the diameter of the isolated vesicles ranged from 90–200 nm,

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creation and isolation of the asymmetric vesicles.

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which was unchanged from that of the vesicles as prepared, and that no aggregation occurred in the

Figure 4. Phospholipid compositions of isolated vesicles. PC vesicles (3.0 mM PC) containing (A) 0, (B) 10, (C) 20, and (D) 30 mol% cholesterol were treated with 1.65 U/mL PLD for 5 h at 37°C in the presence of 1.7 M serine and 0.3 M ethanolamine and subsequently treated with 0.75 mg/mL trypsin for 30 min and then dialyzed. Phospholipid compositions of the isolated vesicles were determined by HPLC. Error bars denote maximum and minimum values of duplicates. Insets are histograms of particle size distribution of the isolated vesicles determined by DLS.

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ACCEPTED MANUSCRIPT 3.3. Verification of Membrane Asymmetry It is important to verify the membrane asymmetry of the isolated vesicles and to know how long it is retained. Disruption of the asymmetry would lead to an exposure of PC molecules from the inner to the outer leaflet. Hence, we attempted to detect the amount of PC transferred to

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the outer leaflet by using PLD, which hydrolyzes only PC located at the outer leaflet. As shown in

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Figure 5, for isolated vesicles without cholesterol, treatment with PLD for 1 day yielded choline,

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whose amount corresponded to 27% of the total PC, and longer incubation (up to 7 days) increased the production of choline slightly (~35%). These results imply that the exposure of PC molecules to

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the outer leaflet occurs specifically in the isolation of the vesicles. Removal of a large amount of

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solutes (serine, ethanolamine, etc.) by dialysis would bring about marked changes in the osmotic pressure toward vesicle membranes, which may promote the flip-flop of phospholipids. On the

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other hand, PC exposure of the asymmetric vesicles rarely took place after they were isolated (from

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1 to 7 days), because PC has been reported not to exhibit spontaneous flip-flop [9, 17].

Figure 5. Choline production from isolated vesicles. Asymmetric vesicles containing 0, 10, 20, and 30 mol% cholesterol were prepared and isolated as described in Figure 4, and were further treated with 1.65 U/mL PLD for 1, 3, 5, and 7 days at 37°C. The concentrations of released choline and total PC were determined as described in section 2.7. Error bars denote maximum and minimum values of duplicates.

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ACCEPTED MANUSCRIPT Cholesterol is known to enhance membrane rigidity [18] and inhibit the spontaneous flip-flop of phospholipids [9]. In the present study, vesicles containing a higher amount of cholesterol attained longer lasting membrane asymmetry (Figure 5). At 30 mol% cholesterol, only 4.4% of the total PC was exposed to the outer leaflet in the early stage, and further exposure was

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not observed in 7 days of incubation. Therefore, incorporation of cholesterol was found to be

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effective in keeping vesicles asymmetric.

4. Conclusions

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We established a method to create asymmetric vesicles consisting of PC, PS, PE,

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with/without cholesterol by using the transphosphatidylation reaction catalyzed by PLD. From 3.0 mM PC vesicles, asymmetric vesicles were produced at 74% yield. Phospholipid composition of

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the obtained vesicles were roughly PC : PS : PE = 2 : 1 : 1 and more than 95% of PC molecules

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were localized to the inner leaflet of vesicles with 30 mol% cholesterol, suggesting almost perfect “PCIN/PS·PEOUT” asymmetry. Such high quantity and quality cannot be achieved by other known

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methods of obtaining asymmetric vesicles. Because this bacterial enzyme is known to proceed with

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transphosphatidylation with several primary alcohols, including glycerol [19], asymmetric vesicles containing phosphatidylglycerol and other phospholipids on the outer leaflet could be also

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prepared. The asymmetric vesicles obtained by this procedure would, therefore, be utilized to reproduce membrane-related biological events, such as protein binding and membrane fusion, and to clarify the physicochemical issues how membrane asymmetry influences these events.

Acknowledgements This study was supported by JSPS KAKENHI Grant numbers JP26287098, JP16K18860, and JP17H02941, and by Takeda Science Foundation. 13

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[19] J.O. Rich, Y.L. Khmelnitsky, Phospholipase D-catalyzed transphosphatidylation in anhydrous organic solvents, Biotechnol Bioeng, 72 (2001) 374-377.

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ACCEPTED MANUSCRIPT Formation of Asymmetric Vesicles via Phospholipase D-mediated Transphosphatidylation Rina Takaoka,† Haruko Kurosaki,† Hiroyuki Nakao,‡ Keisuke Ikeda,‡ Minoru Nakano*,‡ Faculty of Pharmaceutical Sciences and ‡Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan

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Highlights

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Novel method to produce asymmetric vesicles with phospholipase D is executed. PC in the outer leaflet is enzymatically converted to PE or PS. Incorporation of cholesterol is effective in long-lasting membrane asymmetry.

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