Role of Acyl Chain Composition of Phosphatidylcholine in Tafazzin

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Role of Acyl Chain Composition of Phosphatidylcholine in Tafazzin-Mediated Remodeling of Cardiolipin in Liposomes Masato Abe, Yoshiki Sawada, Shinpei Uno, Shuhei Chigasaki, Masahide Oku, Yasuyoshi Sakai, and Hideto Miyoshi Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00941 • Publication Date (Web): 01 Nov 2017 Downloaded from http://pubs.acs.org on November 4, 2017

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Biochemistry

Role of Acyl Chain Composition of Phosphatidylcholine in Tafazzin-Mediated Remodeling of Cardiolipin in Liposomes

Masato Abe, Yoshiki Sawada, Shinpei Uno, Shuhei Chigasaki, Masahide Oku, Yasuyoshi Sakai, and Hideto Miyoshi* Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan

Corresponding Author *E-mail, [email protected].

Tel: +81-75-753-6119. Fax: +81-75-753-6408.

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ABBREVIATIONS CL, cardiolipin; DLCL, dilyso-cardiolipin; DPH, 1,6-diphenyl-1,3,5-hexatriene; ELS, evaporative light scattering; ESI-MS, electrospray ionization mass spectrometry; GST, glutathione Stransferase; HPLC, high performance liquid chromatography; LPC, lyso-phosphatidylcholine; MLCL, monolyso-cardiolipin; PC, phosphatidylcholine; Tm, gel-to-liquid crystalline phase transition temperature.


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ABSTRACT Remodeling of the acyl chain compositions of cardiolipin (CL) species by the transacylase tafazzin is an important process for maintaining optimal mitochondrial functions. The results of mechanistic studies on the tafazzin-mediated transacylation from phosphatidylcholine (PC) to monolyso-CL (MLCL) in artificial lipid membranes are controversial.

The present study

investigated the role of the acyl chain composition of PC in the Saccharomyces cerevisiae tafazzinmediated remodeling of CL by examining the structural factors responsible for the superior acyl donor ability of dipalmitoleoyl (16:1) PC over dipalmitoyl (16:0) PC. To this end, we synthesized systematic derivatives of dipalmitoleoyl PC; for example, the location of the cis double bond was migrated from the 9-position toward either end of the acyl chains (the 5 or 13-position), the cis double bond in the sn-1 or sn-2 position or both was changed to a trans form, and palmitoleoyl and palmitoyl groups were exchanged in the sn-1 and sn-2 positions, maintaining similar PC fluidities. Analyses of the tafazzin-mediated transacylation from these PCs to sn-2’-MLCL(18:118:1/18:1-OH) in liposomal membrane revealed that tafazzin strictly discriminates the molecular configuration of the acyl chains of PCs, including their glycerol positions (sn-1 or sn-2); however, the effects of PC fluidity on the reaction may not be neglected.

On the basis of the findings

described herein, we discuss the relevance of the so-called thermodynamic remodeling hypothesis that presumes no acyl selectivity of tafazzin.


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INTRODUCTION Cardiolipin (CL), a phospholipid with two phosphate polar head groups and four acyl chains, is a major phospholipid in the inner mitochondrial membrane (1, 2). CL is critical for optimal mitochondrial functions, such as oxidative phosphorylation (ATP synthesis), translocation of substrates and apoptosis (3-5).

The biosynthetic pathway of CL, which takes place in

mitochondria, has been extensively investigated in yeast Saccharomyces cerevisiae (6-11). Following the de novo synthesis of CL on the matrix side of the inner mitochondrial membrane, remodeling of acyl chains rule the final molecular composition of mature CL (12).

In this process,

once synthesized immature CL is deacylated to monolyso-CL (MLCL) by CL-specific lipase Cld1 (12, 13). The reacylation of MLCL is then catalyzed solely by tafazzin in yeast, which takes an acyl

chain

from

another

phospholipids,

such

as

phosphatidylethanolamine (PE), and adds it to MLCL (14).

phosphatidylcholine

(PC)

and

Previous studies revealed the high

selectivity of the hydrolysis of yeast mitochondrial CLs by Cld1 (10, 15), which may be essential for producing mature CLs in combination with tafazzin.

Mutations in tafazzin in humans cause

Barth syndrome, resulting in cardiac and skeletal myopathy and respiratory chain defects (16, 17). Claypool and colleagues established a series of Barth syndrome-related tafazzin mutants in S. cerevisiae mitochondria (18, 19). Tafazzin has been considered to exhibit little acyl specificity (selectivity) among phospholipid substrates; namely, the enzyme randomly reacts with actually all phospholipids and MLCL species in mitochondria (12, 14).

Therefore, in light of the diversity of the acyl compositions of CLs in

different organisms or even in tissues within the same organism, an important question is how tafazzin brings about specific patterns of acyl chain compositions in mature CLs; for example, tetralinoleoyl (18:2)-CL is a major CL in mammalian heart, and oleoyl (18:1) and palmitoleoyl (16:1) are major acyl groups in yeast mitochondrial CLs (20). To answer this question, Schlame et al. (21) proposed the so-called thermodynamic remodeling hypothesis based on the findings obtained from experiments using artificial lipid membranes and isolated Drosophila melanogaster tafazzin. According to this hypothesis, tafazzin only functions in non-bilayer-type disordered lipid membranes, but not in liposomes (ordered lipid bilayer membrane), and its acyl specificity is caused by packing properties of membranes in the final equilibrium state.

In other words, multiple transacylation among PC, CL, and their monolyso-

species (LPC and MLCL) will change the acyl compositions of CLs until their free energy is minimal, at which point the acyl composition is optimal for specifically curved membranes. 4 ACS Paragon Plus Environment

For

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example, they reported that tetralinoleoyl-CL (CL(18:2-18:2/18:2-18:2)), which has a striking shape asymmetry (relatively small volume of polar head group and multiple unsaturated acyl chains), is only formed under conditions that favor the hexagonal lipid phase (negatively curved lipid monolayer) (21).

However, there are several issues that are difficult to reconcile with the

thermodynamic remodeling hypothesis (see the Discussion for details).

In particular, it is

paradoxical that hexagonal (21) and micellar (22) phases both support efficient tafazzin-mediated transacylation because the two lipid phases are characterized by opposite curvatures of the lipidwater interface.

In this context, while there are curve-shaped domains (cristae) in the inner

mitochondrial membrane, a high resolution static

31

P NMR (283.4 MHz) study with rat heart

mitochondria led to the question of whether the inner membrane contains domains that are sufficiently curved to be categorized as disordered non-bilayer membrane (23). We recently demonstrated that isolated glutathione S-transferase (GST)-tagged yeast tafazzin efficiently catalyzes transacylation from PCs having various acyl groups to MLCLs even in liposomal membrane (large unilamellar vesicles) (24).

Tafazzin elicited unique acyl chain

specificity against PCs: linoleoyl (18:2)  oleoyl (18:1) ≒ palmitoleoyl (16:1)  palmitoyl (16:0).

In these reactions, tafazzin predominantly removed the sn-2 acyl chain of PCs and

transferred it into the sn-1 and sn-2 positions of sn-1’-MLCL and sn-2’-MLCL, respectively, at almost equivalent rates.

The acyl chain compositions of sn-1’- and sn-2’-MLCLs did not

significantly affect their ability as acyl acceptors.

Additionally, we showed that sn-2-MLCL and

sn-2/sn-2’-dilyso-CL (DLCL) have inherent abilities to work as an acyl donor to sn-2-LPC and acyl acceptor from PC, respectively, in the tafazzin-mediated in vitro transacylation; however, it remains unclear whether sn-2-MLCL and sn-2/sn-2’-DLCL actually work as an acyl donor and acceptor, respectively, in CL remodeling in mitochondria.

Collectively, we concluded that in

contrast to the thermodynamic remodeling hypothesis, acyl selectivity in the tafazzin-mediated reaction is one of the factors that are responsible for the acyl compositions of mature CLs (24). In order to obtain further insights into the catalytic mechanism of tafazzin, it is valuable to elucidate the reasons for the significantly different acyl donor abilities between PC(16:1-16:1) and PC(16:0-16:0) because the two PCs are only differentiated by the presence (or absence) of the cis double bond.

Since the gel-to-liquid crystalline phase transition temperature (Tm) values of

PC(16:1-16:1) and PC(16:0-16:0) bilayer are remarkably different (-36 and 41˚C, ref. 25), we wanted to establish whether the different abilities are attributed to a difference in liposomal membrane fluidities or the structural elements of the acyl chains. 5 ACS Paragon Plus Environment

If the latter is the primary causal

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factor, further questions may have to be addressed: for example, i) which cis double bond (the sn1 or sn-2 acyl chain or both) does tafazzin strictly recognize? and ii) in this process, does tafazzin recognize the location of the cis double bond (the 9-position) along the acyl chain?

To clarify

these points, we herein synthesized a series of PCs (Figure 1) and investigated the transacylation reaction from these PCs to MLCL (sn-2’-MLCL(18:1-18:1/18:1-OH)) mediated by isolated yeast tafazzin in liposomes composed of the phospholipids concerned.

The present study

unambiguously demonstrates that the efficient tafazzin reaction takes places even in liposomes. Analyses of the tafazzin reaction from the view point of a structure/acyl donor ability relationship reveal that tafazzin strictly discriminates the molecular configuration of the acyl chains of PCs, including their glycerol positions (sn-1 or sn-2); however, the effects of PC fluidity on the reaction cannot be neglected.


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Figure 1.

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Structures of PC, MLCL, and CL species studied in the present study.

The

synthetic procedures for these lipid species are described in the Supporting Information. Concerning the abbreviated designation of CL species, four acyl chains are arranged to make the chiral carbon of the central glycerol to have the R configuration. The synthetic product of sn-2’-MLCL(18:1-18:1/18:1-OH) is a racemic mixture of (S) sn-2- and (R) sn-2’-MLCL.

EXPERIMENTAL PROCEDURES Materials 1,2-Dioleoyl-sn-glycero-3-phosphatidylcholine,

1,2-dilinoleoyl-sn-glycero-3-

phosphatidylcholine, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine, and 1,2-dipalmitoleoylsn-glycero-3-phosphatidylcholine were purchased from NOF Corp. (Tokyo, Japan), Santa Cruz Biotechnology (Santa Cruz, CA), Wako Pure Chemical Ltd. (Tokyo, Japan), and Avanti Polar Lipid Inc. (Alabaster, AL), respectively. Synthesis of PC and CL analogues The PC and CL analogues and sn-2’-MLCL(18:2-18:2/18:2-OH) synthesized in the present study are shown in Figure 1.

The synthetic procedures for these lipid species are described in

Supporting Information. Preparation of S. cerevisiae tag-free tafazzin S. cerevisiae GST-tagged tafazzin was expressed in Escherichia coli Rosetta (DE3)pLysS (Merck Millipore, Japan) strains and isolated according to the previously reported procedures (24) with slight modifications.

Cells were harvested, washed with 50 mL of PBSE (137 mM NaCl, 27

mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, and 1 mM EDTA, pH 7.4), and pressed in 20 mL of PBSE containing cOmplete™ EDTA-free (Sigma-Aldrich, Japan) using a One Shot Model Cell disruption system (Constant Systems, UK). The sample was mixed with 0.1% (w/v) Triton X100 (final concentration), incubated at an ambient temperature for 10 min, and centrifuged at 2,000 x g at 4 °C for 5 min.

The supernatant fraction was retrieved and mixed with 2.0 mL of

Glutathione Sepharose 4B resin (GE Healthcare, Japan) at room temperature for 30 min. The column was washed twice with 2.0 mL of PBSE and with cleavage buffer (150 mM NaCl, 1 mM EDTA, 1 mM DTT, and 50 mM Tris-HCl, pH 7.6). Captured GST-tagged tafazzin was treated with 320 U/mL (final concentration) of PreScission protease (GE Healthcare, Japan) in the 8 ACS Paragon Plus Environment

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cleavage buffer at 4 °C for 16 h. This reaction mixture was then applied to Glutathione Sepharose 4B resin to obtain tag-free tafazzin.

The purified enzyme was stored in buffer (150 mM NaCl, 1

mM EDTA, 0.1% (w/v) Triton X-100, and 50 mM Tris-HCl, pH 7.6) at -78 ˚C. Preparation of liposomes and fluorescence polarization Liposomes (large unilamellar vesicles) made of PC (an acyl donor) and sn-2’-MLCL(18:118:1/18:1-OH) (an acyl acceptor) at a definite molar ratio were prepared by the extrusion method using a 100 nm pore polycarbonate filter with a LiposoFast device (Avestin, Ottawa, Canada) in buffer (50 mM Tris-HCl, pH 7.4), as described previously (24). Unless otherwise noted, the PC: sn-2’-MLCL(18:1-18:1/18:1-OH) ratio was set as 8.5:1.5.

The size of liposomes was determined

by dynamic light scattering measurements with ELSZ-2Plus (Otsuka Electronic, Osaka, Japan) at 37 °C. The concentrations of PCs in liposomal preparations were measured using an enzyme assay kit for choline (Wako, Osaka, Japan). Fluorescence measurements were performed with a Shimadzu RF-5000 spectrofluorimeter equipped with a sample heater/cooler. The fluorescence polarization of DPH (rDPH), incorporated in liposomal membrane, was determined in order to evaluate the gel-to-liquid crystalline phase transition temperature (Tm) of liposomes according to the procedure reported by Fukuda et al. (26). Liposomes were suspended in 2.0 mL of buffer (50 mM Tris-HCl, pH 7.4).

The concentration of

the lipid mixture and DPH/lipid ratio were set to 100 µM and 1:200, respectively.

Excitation and

emission wavelengths were 360 and 434 nm, respectively. Temperature was increased from 10 to 50 ˚C in increments of 2.5 ˚C, and the sample was equilibrated for 3 min before polarization was measured. Measurement of transacylation activity of tafazzin in liposomes The tafazzin-mediated transacylation was started by the addition of isolated tag-free tafazzin (4.0 µg) to PC/sn-2’-MLCL(18:2-18:2/18:2-OH) liposomes in 85 µL of reaction buffer (50 mM Tris-HCl, pH 7.4) at 37 °C; the final tafazzin and lipid concentrations were 1.1 µM and 0.39 mM, respectively.

Unless otherwise noted, the final concentration of Triton X-100, which was

introduced into a reaction mixture with the enzyme, was 0.04% (w/v).

We confirmed by dynamic

light scattering measurements that the particle size distribution of liposomes is maintained under the assay conditions used, as described in the text (e.g. Figure 2A). To evaluate the consumption of sn-2’-MLCL(18:1-18:1/18:1-OH) and production of CLs, we 9 ACS Paragon Plus Environment

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adopted a reverse-phase ion-pair HPLC analysis (SCL-10, Shimadzu, Japan) equipped with an evaporative light scattering detector (model 300S, Softa Co. USA), which is convenient for detecting lipids that have no strong chromophore (27, 28).

The RP-18 GP Aqua 5 µm ODS

column (150 × 4.6 mm, KANTO Chemical, Japan) was connected with a guard cartridge (5C18AR-II, 10 × 4.6 mm, Nacalai Tesque, Japan), both of which were incubated at 50°C in the column oven.

In order to accurately quantify lipids, the reaction samples were directly subjected to the

HPLC analysis without an extraction step using an organic solvent. HPLC analytical conditions were described in detail in the previous study (24).


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Figure 2.

Tafazzin-mediated acyl transfer from PC to sn-2’-MLCL in liposomes.

Liposomes

were prepared from a mixture of PC(16:0-16:0), PC(16:1-16:1), PC(18:1-18:1), or PC(18:2-18:2) and sn-2’-MLCL(18:1-18:1/18:1-OH) at a 8.5:1.5 molar ratio. (A) The particle size distribution profile of liposomes composed of PC(16:1-16:1) and sn-2’-MLCL(18:1-18:1/18:1-OH) was determined by dynamic light scattering measurements.

The measurement was performed at

37 °C under the same experimental conditions as those used for the tafazzin reaction (50 mM Tris-HCl, pH 7.4, 1.1 µM tafazzin, 0.04% Triton X-100 (w/v)).

Distribution profiles were

estimated based on light scattering intensity (upper) or particle numbers (lower); these profiles depend on how the measured data are weighted: by scattering intensity or by numbers. (B) The time-course for the production of CL using PC(18:2-18:2) (closed circles), PC(18:1-18:1) (open circles), PC(16:1-16:1) (open diamonds), or PC(16:0-16:0) (closed diamonds) as an acyl donor. The reaction buffer (85 µL, 50 mM Tris-HCl, pH 7.4) contained 33 nmol of total lipids (a final lipid concentration of 0.39 mM). The reaction was initiated by the addition of purified tag-free tafazzin (4.0 µg) (a final tafazzin concentration of 1.1 µM, 0.04% Triton X-100). Data shown are mean values ± S.D. (n = 3–4). (C) Production of CL(18:1-18:1/18:1-16:1) and CL(18:118:1/18:1-16:0) in the direct competition using liposomes composed of PC(16:1-16:1), PC(16:016:0), and sn-2’-MLCL(18:1-18:1/18:1-OH) at a 4.25:4.25:1.5 molar ratio. (D) The reversephase ion-pair HPLC analysis (detected by ELS detector) of the tafazzin-mediated transacylation between PC(16:1-16:1) and sn-2’-MLCL(18:1-18:1/18:1-OH).

The production of CL(18:1-

18:1/18:1-16:1) (~32 min, m/z 713.8 [M - 2NH4]2-), CL(18:1-16:1/18:1-16:1) (~30 min, m/z 699.8 [M - 2NH4]2-), and CL(18:1-18:1/18:1-18:1) (~34 min, m/z 727.8 [M - 2NH4]2-) were confirmed by synthetic standard samples and/or ESI-MS. The peak at ~24 min indicates the consumption of sn-2’-MLCL(18:1-18:1/18:1-OH).

In ELS detection, scattering intensities vary

depending on the lipids. RESULTS Syntheses of various PCs as acyl donors We used abbreviated designations for the PC and CL derivatives studied in this work to characterize their acyl compositions with discrimination between the sn-1 and sn-2 glycerol positions.

Acyl chains are abbreviated by x:y code in which x and y indicate the number of carbon

atoms and double bonds, respectively.

The former and latter codes in the parentheses represent

the acyl groups in the sn-1 and sn-2 positions, respectively; for example, PC(16:0-16:1) means PC 11 ACS Paragon Plus Environment

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having palmitoyl and palmitoleoyl groups in the sn-1 and sn-2 positions, respectively, and sn-2’MLCL(18:1-18:1/18:1-OH) means MLCL that has three oleoyl groups in the sn-1, sn-2, and sn-1’ positions, but lacks an acyl chain in the sn-2’ position of one glycerol moiety. A series of PC derivatives (Figure 1) were synthesized based on the following design concepts. The synthetic methods for the PCs are described in Supporting Information.

The sn-2, not sn-1,

acyl chain in PCs is predominantly removed and added to MLCLs by tafazzin (24). Taking this into consideration, to examine whether the superior efficiency of PC(16:1-16:1) over PC(16:016:0) as an acyl donor is due to the presence of palmitoleoyl (16:1) solely in the sn-2 position or in both glycerol positions, we synthesized PC(16:1-16:0) and PC(16:0-16:1) as hybrids of PC(16:116:1) and PC(16:0-16:0).

In order to investigate the effects of changing the location of the cis

double bond along the acyl chains, we synthesized PC(16:15-16:15)and PC(16:113-16:113), which have the cis double bond in the 5- and 13-positions, respectively, of both acyl chains. PC(16:1trans-16:1), PC(16:1-16:1trans), and PC(16:1trans-16:1trans) were synthesized as geometrical isomers of PC(16:1-16:1) to know whether tafazzin strictly recognizes a difference in the molecular shapes due to geometrical isomerism.

Furthermore, we synthesized three ether-

type derivatives: PC(18:1Et-18:1), PC(18:1-18:1Et), and PC(18:1Et-18:1Et), in which either or both of the ester linkages of the glycerol backbone was replaced by an ether linkage to block the hydrolysis of the concerned alkyl chain(s) by tafazzin. Optimization of assay conditions for tafazzin-mediated transacylation in liposomes We previously investigated transacylation from PCs to MLCLs catalyzed by isolated S. cerevisiae GST-tagged tafazzin in liposomes (24).

As confirmed by dynamic light scattering

measurements, the liposomal structure was maintained under the previous experimental conditions (PC/MLCL molar ratio of 9:1, 0.08% (w/v) Triton X-100) (24).

Nevertheless, to exclude the

potential effect of the GST tag, tag-free enzyme was used in the present study, which was prepared by removing GST from once purified GST-tagged tafazzin (see the Experiment Procedures).

In

addition, we attempted to decrease the concentration of Triton X-100 in the liposomal assay system as low as possible to reduce its effects on the liposomal structure (22).

Therefore, we investigated

the liposomal structure and tafazzin activity by changing concentration of Triton X-100 (Figure S1) and PC:MLCL molar ratio (9:1 or 8.5:1.5).

As a result, the content of Triton X-100 and

PC:MLCL molar ratio were set as 0.04% (w/v) and 8.5:1.5, respectively, throughout the present study.

If ~300 molecules of Triton X-100 bind to tafazzin (22), this leads to a Triton X12 ACS Paragon Plus Environment

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100/phospholipid ratio of ~0.7:1.0 (mol/mol). Since the three factors (i.e. tafazzin form, concentration of Triton X-100, and PC:MLCL molar ratio) were changed from the previous experimental conditions, we reexamined the acyl specificity of PCs in the tafazzin reaction observed in the previous study (24).

We confirmed by dynamic

light scattering measurements that the particle size distribution profiles of liposomes are maintained under the assay conditions used, as shown in Figure 2A, taking liposome composed of PC(16:116:1) and sn-2’-MLCL(18:1-18:1/18:1-OH) as an example. Transacylation from PC(18:2-18:2), PC(18:1-18:1), PC(16:1-16:1), or PC(16:0-16:0) to sn-2’MLCL(18:1-18:1/18:1-OH) was investigated.

Using the synthetic standard compounds (Figure

1), the production of CLs and consumption of sn-2’-MLCL(18:1-18:1/18:1-OH) were simultaneously measured by reverse-phase ion pair HPLC analysis equipped with an evaporative light scattering (ELS) detector

(24).

Although the progression of the tafazzin reaction may be

monitored solely by assessing the consumption of sn-2’-MLCL(18:1-18:1/18:1-OH), we did not do so because we needed to check whether each tafazzin reaction produces a mixture of CLs, which were formed by multiple transacylation.

In addition, sn-2’-MLCL(18:1-18:1/18:1-OH), but not

sn-1’-MLCL(18:1-18:1/OH-18:1) was used as an acyl acceptor throughout this study to avoid acyl automigration from the sn-2 to sn-1 position during the preparation of liposomes and/or under the assay conditions (24). As shown in Figure 2B, we confirmed the acyl chain selectivity almost similar to that observed in the previous study (24), whereas the degree of selectivity was somewhat reduced in the present study (PC(18:2-18:2) ≒ PC(18:1-18:1) ≒ PC(16:1-16:1) > PC(16:0-16:0)).

In particular, the

production of CL(18:1-18:1/18:1-16:0) from PC(16:0-16:0) was significantly greater than that observed in the previous study (< 0.3 nmol). While we cannot currently address the reason for this difference, it may be due to the different assay conditions used between the previous and present studies; especially, the enzyme form (GST-tagged tafazzin vs. tag-free tafazzin). To further specify the acyl selectivity of tafazzin between PC(16:1-16:1) and PC(16:0-16:0), we conducted direct competition between the two PCs in the same liposomes.

Namely, we

analyzed the CL species produced by the tafazzin-mediated transacylation using liposomes composed of PC(16:1-16:1), PC(16:0-16:0), and sn-2’-MLCL(18:1-18:1/18:1-OH) at a 4.25:4.25:1.5 molar ratio. A possible difference in membrane fluidities between the liposomes composed

of

PC(16:1-16:1)/sn-2’-MLCL(18:1-18:1/18:1-OH)

and

PC(16:0-16:0)/sn-2’-

MLCL(18:1-18:1/18:1-OH) mixture in the above experiments can be excluded by this direct 13 ACS Paragon Plus Environment

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competition assay. After 40-min reaction, CL(18:1-18:1/18:1-16:1) was predominantly formed (2.8 ± 0.3 nmol), whereas the formation of CL(18:1-18:1/18:1-16:0) was negligibly small (Fig. 2C), indicating a clear preference for PC(16:1-16:1) over PC(16:0-16:0) as an acyl donor.

The

difference in acyl donor efficiencies was much greater than that observed in liposomes composed of a single PC (Figure 2B). This may be because the difference in apparent reaction velocities between PC(16:1-16:1) and PC(16:0-16:0) under the competitive conditions is determined not only by the difference in their inherent rate constants, but is also amplified by a difference in their binding affinities to the enzyme. Multiple transacylation between PC and CL species In the above tafazzin-mediated reactions between PC(18:2-18:2) or PC(16:1-16:1) and sn-2’MLCL(18:1-18:1/18:1-OH), small amounts of CL species, which were formed by multiple transacylation, were detected besides the anticipated (predominant) CL product, which had not been observed in the previous study (24).

For example, in the reaction between PC(16:1-16:1)

and sn-2’-MLCL(18:1-18:1/18:1-OH), CL(18:1-16:1/18:1-16:1) (retention time of ~30 min) and CL(18:1-18:1/18:1-18:1) (~34 min) were detected besides the anticipated product CL(18:118:1/18:1-16:1) (~32 min) (Figure 2D).

The ratio of CL(18:1-18:1/18:1-16:1):CL(18:1-

18:1/18:1-18:1):CL(18:1-16:1/18:1-16:1) was approximately 8:1:1, and it changed little with the progression of the tafazzin reaction. reaction was plotted in Figure 2B.

The amount of the predominant CL product only in each

Needless to say, only CL(18:1-18:1/18:1-18:1) was formed in

the reaction between PC(18:1-18:1) and sn-2’-MLCL(18:1-18:1/18:1-OH).

These results are

worthwhile to argue whether tafazzin freely shuffles acyl chains among all PCs, CLs, and their monolyso species formed during the reaction (see the Discussion). Gel-to-liquid crystalline phase transition in liposomes composed of the PC/MLCL mixture Liposomal membrane fluidity is one of the important factors regulating the tafazzin reaction because it affects the binding of tafazzin to the membrane surface as well as the binding of individual lipid molecules to the catalytic site of the enzyme.

The gel-to-liquid crystalline phase

transition temperature (Tm) values of PC(16:1-16:1) and PC(16:0-16:0) bilayer are remarkably different (-36 and 41 ˚C, respectively, ref. 25).

Therefore, we cannot rule out the possibility that

apparent acyl specificity in the tafazzin-mediated transacylation (PC(16:1-16:1) > PC(16:0-16:0)) 14 ACS Paragon Plus Environment

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Biochemistry

merely reflects a difference in membrane states between liposomes composed of PC(16:1-16:1) or PC(16:0-16:0) and sn-2’-MLCL(18:1-18:1/18:1-OH) at the experimental temperature (37 ˚C). To clarify this point, we assessed the Tm values of two types of liposomes by measuring the fluorescence polarization (r) of 1,6-diphenyl-1,3,5-hexatriene (DPH) incorporated into the liposomal membrane as a function of temperature (DPH/lipid molar ratio of 1/200).

The

average Tm value of liposomes composed of PC(16:0-16:0) alone, measured as a control, was 42 ˚C (Figure 3, open circles), in agreement with previous studies (25, 29).

Figure 3.

Regarding liposomes composed of PC(16:0-16:0) and

phase transition in liposomes.

sn-2’-MLCL(18:1-18:1/18:1-OH) at a 8.5:1.5 molar

fluorescence polarization (r) of 1,6-

ratio, the sharp change in rDPH shifted to lower

diphenyl-1,3,5-hexatriene (DPH) in

temperature and became broader (Figure 3, closed

liposomal membrane was measured

circles), suggesting an increase in the temperature range

as

of the gel/liquid-ordered phase (26).

Liposomes

The average Tm

a

Gel-to-liquid crystalline

function were

of

The

temperature.

prepared

from

value of liposomes was 31 ˚C, indicating that the

PC(16:0-16:0) alone (open circles) or

liposomal membrane is almost in the liquid crystalline

a mixture of PC(16:0-16:0) and sn-

phase under the assay conditions (37 ˚C).

2’-MLCL(18:1-18:1/18:1-OH) with

Since the Tm values of PC(16:1-16:1) and PC(18:1-

a molar ratio of 8.5:1.5 (closed

18:1) bilayer are -36 and -17 ˚C, respectively, and phase

circles). The DPH/lipid molar ratio

transition does not markedly change depending on

was set as 1:200.

different polar head groups including CL (25, 30), the

mean values ± S.D. (n = 3).

Data shown are

liposomal membrane composed of PC(16:1-16:1) and sn-2’-MLCL(18:1-18:1/18:1-OH) (8.5:1.5) can be considered to be in the liquid crystalline phase at 37 ˚C.

Although 0.04% Triton X-100 is

included in the assay buffer, surfactants loosen the membrane structure.

Taken together,

liposomes composed of PC(16:1-16:1) or PC(16:0-16:0) and sn-2’-MLCL(18:1-18:1/18:1-OH) are both in the liquid crystalline phase under the experimental conditions.

Therefore, a difference in

the liposomal membrane states is not a critical cause for the different acyl donor abilities between PC(16:1-16:1) and PC(16:0-16:0).

The result of the direct competition assay (above) supports 15

ACS Paragon Plus Environment

Biochemistry

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this notion.

Page 16 of 35

Since PC(16:0-16:0) has the most packed acyl chain pair among the PCs examined

in this study, it is reasonable to consider that liposomes composed of other PC and sn-2’MLCL(18:1-18:1/18:1-OH) are all in the liquid crystalline phase under the experimental conditions. Effects of the presence of cis double bond in the sn-1 or sn-2 position In the liquid crystalline phase, acyl chain fluidity (packing) slightly varies reflecting the Tm value: the lower the Tm value, the more fluid the acyl chain (31, 32). Differences in the Tm values of PC pairs, which have two different acyl chains in an opposite substitution pattern in the sn-1 and sn-2 positions, are within ~10 degrees (25, 30). Taking these into consideration, the acyl fluidities of PC(16:1-16:0) and PC(16:0-16:1) can be considered to be similar to each other; however, their fluidities are lower and higher than those of PC(16:1-16:1) and PC(16:0-16:0), respectively. We note that since the acyl donor efficiencies of most PCs examined in the present study including PC(16:0-16:0) were considerably lower than those of effective acyl donors such as PC(18:2-18:2) and PC(18:1-18:1), we did not kinetically analyze them, as was performed in the previous study (24). Alternatively, their initial reaction velocities (at 2, 5, 10, and 20 min) in the tafazzin-mediated transacylation were compared throughout this study. The acyl donor efficiencies of PC(16:1-16:0) and PC(16:0-16:1) were almost similar to that of PC(16:1-16:1) (Figure 4). We previously showed that when both acyl chains in PCs are identical, the sn-2 acyl chain is predominantly removed and added to MLCL by tafazzin (24).

If tafazzin

solely recognizes the sn-2 acyl chain moiety, the efficiencies of PC(16:0-16:1) and PC(16:1-16:0) may be similar to those of PC(16:1-16:1) and PC(16:0-16:0), respectively; however, this was not the case. Therefore, the sn-1 acyl chain may also contribute to acyl donor efficiency in some way. It is worth noting that in the tafazzin-mediated reaction between PC(16:1-16:0) and sn-2’MLCL(18:1-18:1/18:1-OH), CL(18:1-18:1/18:1-16:0) and CL(18:1-18:1/18:1-16:1) were formed at a ratio of ~3:1 (Figure 4C).

The sum of the two products is plotted in Figure 4A.

In contrast,

the reaction between PC(16:0-16:1) and sn-2’-MLCL(18:1-18:1/18:1-OH) predominantly provided CL(18:1-18:1/18:1-16:1) (Figures 4A and 4D). These results suggest that if the sn-2 acyl chain is saturated (i.e. a poor acyl donor), tafazzin removes the acyl group from the sn-1 position, albeit less effectively.

This notion will be supported by the result obtained using

PC(16:1-16:1trans), as described later.


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Biochemistry

Figure 4.

Tafazzin-mediated transacylation from PC to MLCL in liposomes.

Liposomes

were prepared from a mixture of different PC and sn-2’-MLCL(18:1-18:1/18:1-OH) at a 8.5:1.5 molar ratio. The reaction buffer (85 µL, 50 mM Tris-HCl, pH 7.4) contained 33 nmol of total lipids (a final lipid concentration of 0.39 mM).

The reaction was initiated by the addition of

purified tag-free tafazzin (4.0 µg, final concentration of 1.1 µM). The amounts of CL products at time points 2, 5, 10, and 20 min are shown. as 0.04% (w/v).

The final concentration of Triton X-100 was set

(A) and (B) The sum of CL products formed from the reaction between the

indicated PC and sn-2’-MLCL(18:1-18:1/18:1-OH).

(C) Production of CL(18:1-18:1/18:1-

16:0) and CL(18:1-18:1/18:1-16:1) from the reaction between PC(16:1-16:0) and sn-2’MLCL(18:1-18:1/18:1-OH).

(D) Production of CL(18:1-18:1/18:1-16:0) and CL(18:1-

18:1/18:1-16:1) from the reaction between PC(16:0-16:1) and sn-2’-MLCL(18:1-18:1/18:1OH). Data shown are mean values ± S.D. (n = 3–4).


Biochemistry

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Page 18 of 35

Effects of changing the location of the cis double bond along acyl chains A critical difference in structure between PC(16:1-16:1) and PC(16:0-16:0) is the presence (or absence) of a single cis double bond in the 9-position of acyl chains.

Since previous studies

suggested that the presence of a cis double bond (-electron system)in lipid acyl chains is favorable for hydrophobic interaction between acyl chains and hydrophobic amino acid residues in proteins (e.g. the formation of a specific CL-cytochrome c complex, refs. 33 and 34), we cannot rule out the possibility that there is a specific hydrophobic interaction between the cis double bond in palmitoleoyl (16:1) group and some residues of tafazzin.

If this is the case, the presence of the

cis double bond at a definite location (the 9-position) may be favorable for acyl donor ability. To examine this point, we synthesized PC(16:15-16:15)and PC(16:113-16:113). Using a series of synthetic PC(18:0-18:1) derivatives, in which the position of the cis or trans double bond was systematically varied along the sn-2 acyl chain, Nagahama et al. (35) revealed that membrane fluidity, evaluated by Tm values, is maximal when the double bond is located at the middle of the sn-2 acyl chain (9-position), and progressively decreases with the migration of the double bond toward either end of the chain irrespective of the geometrical difference (cis or trans). Wang et al. (36) reported similar findings using different sets of PCs.

Referring to these earlier

studies, the acyl chain fluidities of PC(16:15-16:15)and PC(16:113-16:113) are similar to each other, and lower than that of PC(16:1-16:1).

Nevertheless, the acyl fluidities of the two PCs

are considerably higher than that of saturated PC(16:0-16:0) (25, 35). We

measured

the

tafazzin-mediated

transacylation

between

PC(16:113-16:113) and sn-2’-MLCL(18:1-18:1/18:1-OH).

PC(16:15-16:15)or

The acyl donor efficiencies of

both PCs were remarkably lower than those of PC(16:1-16:1) and PC(16:0-16:0): the amounts of CL(18:1-18:1/18:1-16:15) and CL(18:1-18:1/18:1-16:113) produced after a 40-min reaction were less than 0.2 nmol (Figure 4B).

These results cannot be explained by differences in PC

fluidities. Unexpectedly, the both reactions produced 0.5 ~ 0.8 nmol of CL(18:1-18:1/18:1-18:1) after a 40-min reaction.

Taking the amounts of PCs and sn-2’-MLCL(18:1-18:1/18:1-OH)

consumed into consideration, this is possibly because when PC is an inefficient acyl donor, tafazzin exchanges an acyl chain between different sn-2’-MLCL(18:1-18:1/18:1-OH) to provide CL(18:118:1/18:1-18:1) and dilyso-CL (sn-2/sn-2’-DLCL(18:1-OH/18:1-OH)).

This possibility will be

examined in detail in the section for the ether-type PCs. The above results clearly indicate that the location of the cis double bond along the acyl chains is an important factor for acyl donor ability.

This finding may lead to the following two 18


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Biochemistry

likelihoods: one is that there is a specific hydrophobic interaction between the cis double bond (electron system) in palmitoleoyl group and tafazzin, and the other is that the bend in the middle region of palmitoleoyl group owing to the cis double bond is an important structural factor for acyl donor ability.

The results obtained using the trans isomers of PC(16:1-16:1) rule out the former,

as described in the next section.

We cannot currently address the reason why the migration of the

cis double along the acyl chains resulted in the drastic decrease in acyl donor ability. Effects of changing the geometry of the double bond in the acyl chains When the total number of carbon atoms and position of the double bond along the acyl chain are both identical, the acyl fluidity of the trans isomer is lower than that of the cis isomer, whereas the presence of a trans double bond slightly enhances acyl fluidity over the corresponding saturated acyl chain: the fluidity order of cis double bond > trans double bond > saturated (35, 37). We investigated the effects of introducing a trans double bond into the sn-1 or sn-2 acyl chain of PC using PC(16:1trans-16:1) or PC(16:1-16:1trans), respectively.

The acyl chain fluidities of these

PCs are similar to each other, but lower than that of PC(16:1-16:1). The tafazzin-mediated reaction between PC(16:1trans-16:1) and sn-2’-MLCL(18:1-18:1/18:1OH) predominantly produced CL(18:1-18:1/18:1-16:1), and the acyl donor efficiency of PC(16:1trans-16:1) was slightly lower than that of PC(16:1-16:1) (Figure 4B).

Interestingly, the

reaction between PC(16:1-16:1trans) and sn-2’-MLCL(18:1-18:1/18:1-OH) provided CL(18:118:1/18:1-16:1trans) and CL(18:1-18:1/18:1-16:1) at a ratio of ~1:1; the former and the latter were formed by removal of the sn-2 and sn-1 acyl chain, respectively, of PC(16:1-16:1trans) by tafazzin. The sum of the amount of two products was shown in Figure 4B, which was considerably lower than that of the product formed from PC(16:1trans-16:1).

These results indicate that when the

sn-2 acyl chain is a poor acyl donor, tafazzin removes the acyl group from the sn-1 position, as observed for PC(16:1-16:0).

It is therefore likely that the selectivity of tafazzin against the

glycerol positions (sn-1 vs. sn-2) of PCs flexibly changes depending on the acyl composition. While the acyl chain fluidity of PC(16:1trans-16:1trans) is considerably higher than that of saturated PC(16:0-16:0) (35, 37), the acyl donor efficiency of PC(16:1trans-16:1trans) was significantly lower than that of PC(16:0-16:0) (Figure 4B).

It is worth noting that the reaction

between PC(16:1trans-16:1trans) and sn-2’-MLCL(18:1-18:1/18:1-OH) produced small amount of CL(18:1-18:1/18:1-18:1) (0.5 ~ 0.8 nmol) besides CL(18:1-18:1/18:1-16:1trans) (< 0.2 nmol) after a 40-min reaction, as observed for PC(16:15-16:15)and PC(16:113-16:113). This is 19 ACS Paragon Plus Environment

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Page 20 of 35

possibly because tafazzin exchanged an acyl chain between different sn-2’-MLCL(18:1-18:1/18:1OH) to provide CL(18:1-18:1/18:1-18:1) and dilyso-CL (sn-2/sn-2’-DLCL(18:1-OH/18:1-OH)), as described in the next section. Altogether, the above results indicate not only that acyl donor efficiency is not determined solely by PC fluidity (as indicated in the above section), but also that tafazzin strictly discriminates the geometrical difference in the double bond (cis or trans) in the sn-2 acyl chain.

The trans

double bond is unfavorable for acyl donor ability compared not only to the cis double bond but also to the saturated chain. Effects of changing from an ester to an ether linkage in the glycerol backbone We investigated the effects of changing an ester linkage in the glycerol backbone to an ether linkage on the tafazzin reaction using PC(18:1Et-18:1), PC(18:1-18:1Et), or PC(18:1Et-18:1Et). In this section, we chose PC(18:1-18:1) as the reference PC.

Previous studies using different

techniques showed that in the liquid crystalline phase, acyl chain packing is little affected by this structural change in the glycerol backbone of PCs (38, 39). Since the sn-2 acyl chain in PCs is predominantly removed by tafazzin (24), we considered that PC(18:1Et-18:1), but not PC(18:1-18:1Et) and PC(18:1Et-18:1Et), may be able to function as an efficient acyl donor.

However, the tafazzin-mediated transacylation between PC(18:1Et-18:1),

PC(18:1-18:1Et), or PC(18:1Et-18:1Et) and sn-2’-MLCL(18:1-18:1/18:1-OH) produced almost the same amount of CL(18:1-18:1/18:1-18:1) (Figure 5A).

It is impractical to consider that

tafazzin can break ether bonds in the glycerol backbone of PCs. To get a hint on this seemingly peculiar result, we examined the consumption of the ether-type PCs and sn-2’-MLCL(18:1-18:1/18:1-OH) in the three reactions, and found that the PCs were not consumed at all and the amounts of sn-2’-MLCL(18:1-18:1/18:1-OH) consumed were approximately 2-fold those of CL(18:1-18:1/18:1-18:1) formed in all reaction points (Figure 5B). These results strongly suggest that when PC is an inefficient acyl donor, tafazzin removes an acyl chain from sn-2’-MLCL(18:1-18:1/18:1-OH) and adds it to another sn-2’-MLCL(18:1-18:1/18:1OH) to provide CL(18:1-18:1/18:1-18:1) and sn-2/sn-2’-DLCL(18:1-OH/18:1-OH); namely, tafazzin exchanges an acyl chain between different sn-2’-MLCL(18:1-18:1/18:1-OH).

In support

of this notion, we detected the formation of sn-2/sn-2’-DLCL(18:1-OH/18:1-OH) by a reversephase ion pair HPLC analysis (24) and ESI-MS (m/z 463.4 [M - 2NH4]2-) (Figure 5C). 20 ACS Paragon Plus Environment

In this

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Biochemistry

context, it should be mentioned that tafazzin can mediate

acyl

exchange

between

sn-2’-

MLCL(18:1-18:1/18:1-OH) and LPC(18:1-OH) to

provide

sn-2/sn-2’-DLCL(18:1-OH/18:1-

OH) and PC(18:1-18:1) in liposomes (24). The production of CL(18:1-18:1/18:1-18:1) in the

reaction

using

PC(16:113-16:113),

PC(16:15-16:15) or

PC(16:1trans-

16:1trans) may be similarly explained.

Our

results indicate that the ether-type analogues hardly function as acyl donors; hence, the entire structure of the glycerol diester skeleton of PC must be an essential structure to form an active transition state in the tafazzin-PC complex.


Biochemistry

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Figure 5.

Page 22 of 35

Tafazzin-mediated transacylation from PC(18:1Et-18:1), PC(18:1-18:1Et), or

PC(18:1Et-18:1Et) to MLCL.

Liposomes were prepared from a mixture of PC(18:1Et-18:1),

PC(18:1-18:1Et), or PC(18:1Et-18:1Et) and sn-2’-MLCL(18:1-18:1/18:1-OH) at a 8.5:1.5 molar ratio. The reaction buffer (85 µL, 50 mM Tris-HCl, pH 7.4) contained 33 nmol of total lipids (a final lipid concentration of 0.39 mM). The reaction was initiated by the addition of purified tagfree tafazzin (4.0 µg, a final concentration of 1.1 µM). The final concentration of Triton X-100 was set as 0.04% (w/v).

The production of CL(18:1-18:1/18:1-18:1) and consumption of sn-2’-

MLCL(18:1-18:1/18:1-OH) are shown in panels (A) and (B), respectively. sn-2/sn-2’-DLCL(18:1-OH/18:1-OH) is shown in panel (C).

The accumulation of

Data shown are mean values ± S.D.

(n = 3).

DISCUSSION We investigated the mechanism responsible for remodeling of the acyl chain composition of CL mediated by isolated S. cerevisiae tafazzin using liposomes composed of various PC/MLCL mixtures.

While the assay conditions used were slightly modified from those previously

described (24), the present study corroborated the two key findings previously obtained: i) the tafazzin-mediated transacylation from PC to MLCL can take place in liposomes (ordered lipid bilayer) and ii) PC(16:1-16:1) is significantly superior to PC(16:0-16:0) as an acyl donor. The result of the direct competition test between PC(16:1-16:1) and PC(16:0-16:0) in the same liposomes, which was conducted for the first time in the present study, strengthens the latter point. The present study was designed to address the structural factors responsible for the difference in the acyl donor efficiencies between PC(16:1-16:1) and PC(16:0-16:0) using a series of synthetic analogues.

Note that alteration in the chemical structure of the acyl moiety changes not only the

molecular shape of the lipid as a substrate for tafazzin, but also acyl chain fluidity, which concurrently influences bulk membrane properties such as the gel-to-liquid crystalline phase transition (25, 31, 32) and the distance between the polar head groups (40).

Since the effects of

these two factors (i.e. changes in the molecular shape and fluidity of the acyl chain) on the tafazzin reaction are virtually indivisible, sufficient attention needs to be paid for interpretation of the present results.

An increase in acyl chain fluidity is generally favorable for the enzyme reactions

that take place in biomembranes using the lipid component as a substrate(s) because individual lipid molecules have to reach the catalytic site of the enzyme in its optimal conformation. Even 22 ACS Paragon Plus Environment

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Biochemistry

in these cases, we cannot rule out the possibility that some structural element of acyl chains makes an important contribution to enzyme reactions, as demonstrated for the human lecithin-cholesterol acyltransferase reaction in artificial lipid membranes (32, 41, 42). The results obtained in the present study demonstrate that tafazzin strictly discriminates the molecular configuration of the acyl chains of PCs including their glycerol positions (sn-1 or sn-2) and that the preferential effect of this structural element on acyl donor ability overrides that of PC fluidity in some cases.

For example, the results obtained using the trans isomers of PC(16:1-

16:1) indicated that tafazzin discriminates not only a geometrical difference in the double bond (cis or trans), but also its location in the glycerol positions (sn-1 or sn-2) in a strict sense.

The

presence of the -electron system itself in the 9 position of the acyl chains made no beneficial contribution to acyl donor ability. The bend in the middle region of the sn-2 acyl chain due to the presence of the cis double bond is an important structural element for acyl donor ability. The results obtained using PC(16:15-16:15)and PC(16:113-16:113) strongly support this notion. A possible favorable role of the bend in the middle region of the acyl chains is that this configuration interferes with the highly ordered packing of acyl chains (32), which enables individual lipids to effectively reach the catalytic site of tafazzin from bulk membrane phase, as suggested for the human lecithin-cholesterol acyltransferase reaction (32, 41).

However, this does

not necessarily mean that acyl donor ability is determined solely by PC fluidity because tafazzin discriminates the cis double bond in different glycerol positions (sn-1 or sn-2) in a strict sense, as described above. Moreover, the results obtained using PC(16:1-16:0) and PC(16:1-16:1trans) as an acyl donor indicated that although tafazzin predominantly removes the sn-2 acyl chain of PCs when both glycerol positions are substituted by an efficient acyl group (24), if the sn-2 position is substituted by a poor acyl donor, the enzyme also removes the acyl group from the sn-1 position, albeit less effectively.

This finding highlights the flexibility of the catalytic activity of tafazzin against PC

substrates. The ether-type PC analogues completely lost their acyl donor ability, indicating that tafazzin closely recognizes the entire structure of the glycerol diester backbone, not only the sn-2 ester moiety of PCs. The diester skeleton must be a critical structure to form an active transition state in the tafazzin-PC complex, which may enable the hydrolysis at both glycerol positions mentioned above. Some configuration of the sn-2 acyl chain, which is affected by the bend, might regulate the optimal binding of the glycerol backbone of PC to the catalytic site in tafazzin. Taken together, 23 ACS Paragon Plus Environment

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the present results reveal that tafazzin strictly recognize the molecular configuration of the acyl chains of PCs, including their glycerol positions (sn-1 or sn-2); however, the contribution of acyl chain fluidity to the reaction may not be neglected. Schlame et al. (22) recently investigated the tafazzin-mediated transacylation from PCs to sn2’-MLCL(18:2-18:2/18:2-OH) (Avanti Polar Lipids, Inc.) using isolated GST-tagged yeast tafazzin in liposomes, micelles, or liposome/micelle mixed state. They concluded that tafazzin catalyzes bidirectional exchanges of acyl chains not only between the initial PC and MLCL pair but also between all potential intermediate PC and CL species to attain species distribution with the lowest free energy.

In other words, the acyl specificity of the tafazzin reaction results from the curved-

membrane packing stability of the final equilibrium state.

This notion basically follows the

concept in the original thermodynamic remodeling hypothesis proposed in their earlier study (21). If the multiple exchange (shuffling) of acyl chains freely occurs, as proposed, in the tafazzin reaction between PC(18:1-18:1) or PC(16:1-16:1) and sn-2’-MLCL(18:2-18:2/18:2-OH) for a sufficient period of time, then a predominant product is presumed to be the most stable CL(18:218:2/18:2-18:2) in disordered curved lipid monolayer; however, this was not the case.

For

example, the reaction between PC(16:1-16:1) and sn-2’-MLCL(18:2-18:2/18:2-OH) (9:1 molar ratio) produced a mixture of CL(18:2-18:2/18:2-16:1), CL(18:2-16:1/18:2-16:1), and CL(18:218:2/18:2-18:2) at a ratio of approximately 4:1:2 (Figure 3 in ref. 22). Thus, the formation of a mixture of CL products, which contains sub-stable CL species at considerably high ratio, seems to be difficult to reconcile with the result anticipated from the thermodynamic remodeling hypothesis. We agree that the multiple exchange of acyl chains occurs in the tafazzin-mediated transacylation, as shown in Figure 2C; however, the contribution of multiple exchange to the overall profile of CL products is not significant in our case. Moreover, Schlame et al. (22) analyzed the tafazzin-mediated transacylation between PC and MLCL based on the so-called Haldane relationship (43), which was elaborated later from several points of view (44-46), in order to separate the kinetic and thermodynamic factors that determine the reaction. (Note that these authors paid particular attention to the thermodynamic property of the tafazzin reaction because they assumed that the reaction only occurs in curved lipid monolayer, in which the shape asymmetry of CL products is a critical factor for membrane packing stability in an equilibrium state.)

As a result, they concluded that apparent acyl specificity (e.g. PC(16:1-

16:1) > PC(16:0-16:0)) in the tafazzin reaction is due to the thermodynamic properties of lipids, but not the kinetic properties of the enzyme.

It is worth noting that the Haldane relationship (often 24


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Biochemistry

referred to as reversible Michaelis-Menten kinetics, ref. 46) was basically derived for reversible one substrate/one product reactions (Scheme 1), and extended later to reversible multiple substrates/multiple products reactions, for example, by Noor et al. (46).

To describe the latter

reactions, Noor et al. focused on a simplified case in which the enzyme only exists in one of the three distinct states: free, all substrates bound, or all products bound, as shown in Scheme 2. The tafazzin-mediated transacylation reaction (tafazzin + PC + MLCL

tafazzin + LPC + CL)

seemingly corresponds to the model represented by Scheme 2, in which the steady state is established by the forward and backward reactions of one set of substrates (S1 and S2) and products (P1 and P2). However, according to the proposed mechanism (22), the tafazzin-mediated reaction is not so simple; it may be described by Scheme 3.

Once formed products (P1 and P2) can become

substrates (S3 and S4) that are further converted to different products (P3 and P4) by the enzyme (a reaction step marked by #). To be concrete, if multiple exchange of acyl chains takes place in the tafazzin reaction, as proposed, once produced CL1 and LPC1, from the reaction between the initial MLCL1 and PC1 pair, are further converted to different products (MLCL2 and PC2) by acyl chain exchange in a different glycerol position of CL1 with LPC1. More complicatedly, such a reaction randomly cycles among all potential PCs, CLs, and their monolyso species formed during the reaction. Collectively, if the tafazzin-mediated transacylation proceeds according to this scenario, the overall reaction is too complex to be analyzed by the modified Haldane relationship (46), which

was derived for the reversible reactions with one set of substrates and products. It is important to note that some of the findings reported by Schlame et al. (22), which are valuable for exploring the catalytic mechanism of tafazzin, are inconsistent with our previous 25 ACS Paragon Plus Environment

Biochemistry

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findings (24) and present results. points.

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The issues in that study are summarized in the following three

First, the tafazzin-mediated transacylation in the PC/MLCL mixture (9:1 molar ratio) only

took pace in micelles (disordered curved lipid monolayer), not in liposomes (ordered lipid bilayer). Second, the extent of the conversion of MLCL to CL after the reaction reached equilibrium was considerably low even for efficient acyl donors such as PC(18:2-18:2) and PC(18:1-18:1). For example, the amounts of CLs produced from the PC(18:2-18:2)/ sn-2’-MLCL(18:2-18:2/18:2-OH) and PC(18:1-18:1)/sn-2’-MLCL(18:2-18:2/18:2-OH) pairs were ~50 and ~20%, respectively, of the initial sn-2’-MLCL(18:2-18:2/18:2-OH) (Figure 1 in ref. 22).

Third, the transacylation

between PC and MLCL produced a complex mixture of CLs, which were formed by multiple transacylation among PC and CL species, as described above. In contrast, GST-tagged and tag-free tafazzin both functioned as a transacylase in liposomes composed of various pairs of PC/MLCL in our studies.

The amounts of CLs produced in GST-

tagged tafazzin-mediated reactions between PC(18:2-18:2) or PC(18:1-18:1) and MLCLs were ~80% of initial MLCLs (24).

In addition, the reaction between PCs and sn-1-MLCL or sn-2-

MLCL predominantly provided anticipated CLs, which were acylated at the free glycerol OH of MLCLs by an acyl group in the sn-2 position of PCs (24).

Although a mixture of CL products

was formed by tag-free tafazzin in the present study (Figure 2C), the anticipated CL that formed from the initial pair of PC and MLCL was a predominant product.

Potential reasons for these

discrepancies will be discussed below in relation to the effects of Triton X-100 on liposome/micelle morphology transition. Liposome/micelle morphology is sensitively affected by the content of Triton X-100 in the assay buffer.

Schlame et al. (22) discussed the effects of Triton X-100 on liposome/micelle

morphology transition based on the study by Dennis (47), but did not directly examine the state transition for their PC/MLCL mixtures using appropriate techniques.

Dennis investigated the

effects of Triton X-100 on liposome/micelle transition using egg PC or PC(16:0-16:0) alone, not PC/CL mixtures. Judging from the content of Triton X-100 in the assay buffer (0.12% in Figure 1 and 0.18% in Figure 2 of ref. 22), micelles may have been the dominant state in their assays. We confirmed by dynamic light scattering measurements that when high concentrations of Triton X100 (> 0.12%) are used, the lipid mixture fails to form homogenous large unilamellar vesicles and the population of (possibly) micelle-like morphology with a diameter of ~20–30 nm increased, as shown in Figure 6.

In contrast, the particle size distribution of liposomes was maintained under

the assay conditions in our previous (0.08% Triton X-100, ref. 24) and present (0.04% Triton X26 ACS Paragon Plus Environment

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Biochemistry

100, Figure 2A) studies.

It is important to emphasize

that in order to validate the thermodynamic remodeling hypothesis (21, 22), a critical point to be clarified is whether tafazzin-mediated transacylation only occurs in disordered curved lipid monolayer, and not in liposomes.

The previous (24) and present studies

unambiguously

indicated

that

efficient

tafazzin

reaction takes place in liposomal membrane and, hence, do not support the hypothesis.

Large unilamellar

vesicles with a diameter >100 nm have lipid packing density close to planar bilayer membrane (48). Furthermore, the following point also must be discussed in relation to the lipid mixture morphology. In the study by Schlame et al. (22), the amounts of CLs produced in the GST-tagged tafazzin reaction between PCs

and

sn-2’-MLCL(18:2-18:2/18:2-OH)

were

considerably lower than those in our previous study (24), as mentioned above.

To clarify whether this

was because those authors measured the tafazzin reaction in micelles (not in liposomes), we reexamined the tag-free tafazzin-mediated transacylation between PC(16:1-16:1) and sn-2’-MLCL(18:1-18:1/18:1-OH)

Figure

6.

distribution

The profile

particle of

size

liposomes

composed of PC(16:1-16:1) and sn-2’MLCL(18:1-18:1/18:1-OH).

The

experimental conditions were same as those in Figure 2A, except that the final

in a buffer containing a higher concentration of Triton

concentration of Triton X-100 was

X-100

0.12% (w/v).

(0.18%).

Dynamic

light

scattering

measurements indicated that under the experimental conditions used, the lipid mixture failed to form homogenous liposomes, similar to the profile shown in

Relatively strong

scattering intensities around 400 ~ 1000 nm (upper panel) may be due to aggregation of micelles.

The profile

As a result, the average amount of

slightly varied in each experiment when

CL(18:1-18:1/18:1-16:1) produced after a 40-min

high concentrations of Triton X-100 (>

reaction was 2.3 mol (~45% of an initial sn-2’-

0.12%) were used possibly because of

MLCL(18:1-18:1/18:1-OH)), which was considerably

non-uniform aggregation of micelles.

Figure 6.

lower than that produced in liposomes (3.2 nmol, ~65%, Figure 2B). Therefore, we cannot rule 27 ACS Paragon Plus Environment

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out the possibility that different lipid mixture morphology (micelles vs. liposomes) may be one of the causes for the significant difference in the reaction yields between the earlier study (22) and our studies. Another important potential cause for these discrepancies is a difference in the procedure used to isolate tafazzin.

Schlame et al. (22) omitted any detergent from the buffer in the final two

purification steps in order to perform several assays that include no Triton X-100, whereas we used the enzyme storage buffer containing 0.1% (w/v) Triton X-100.

Notably, we were unable to

prepare active tafazzin by omitting Triton X-100 from the storage buffer probably because of significant aggregation of the hydrophobic enzyme.

Once aggregated GST-tagged tafazzin, which

was previously stored without Triton X-100, may be re-solubilized by the addition of high concentrations of Triton X-100 (>0.12%) (22). However, liposomes change to micelles in the presence of such high concentrations of Triton X-100; consequently, Schlame et al. might have observed the activity of GSTtagged tafazzin only in micelles (22).

Concerning the effects of Triton X-100, we were unable to

detect the activity of tafazzin when the final concentrations of Triton X-100 in the assay buffer were reduced

Role of Acyl Chain Composition of Phosphatidylcholine in Tafazzin

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