1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC, 1-stearoyl-2-linoleoyl-sn-glycero-3-phospho-L-serine, SL-PS, 1,2-linoleoyl-sn-glycero-3-phospho-L-serine, LL-PS, 1,2-diheptadecanoyl-sn-glycero-3-phospho-L-serine, PS (C_17:0/C_17:0), 1-stearoyl-2-hydroxy-sn-glycero-3-phospho-L-serine, (C_18:0-lyso-PS), 1-tridecanoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (C_13:0-lyso-PS) were purchased from Avanti Polar Lipids Inc (Alabaster, AL) and were of the highest purity available. Recombinant human lipoprotein-associated phospholipase A₂ (Lp-PLA₂) was obtained from GlaxoSmithKline Co (Collegeville, PA). Cytochrome c (cyt c), diethylenetriaminepentaacetic acid, PLA₁ from Thermomyces lanuginosus, DTPA, H₂O₂ and all organic solvents (HPLC grade) were purchased from Sigma-Aldrich (St. Louis, MO). High purity lyso PS molecular species were obtained after their hydrolysis by porcine pancreas PLA₂ (Sigma-Aldrich, St. Louis, MO, USA) and subsequent separation by 2D-HPTLC. HPTLC silica G plates were purchased from Whatman (Schleicher & Schuell, England). Heptadecanoic acid (C_17:0) was obtained from Matreya LLC (Pleasant Gap, PA). 9S-hydroperoxy-10E,12Z-octadecadienoic acid, 9S-hydroxy-10E,12Z-octadecadienoic acid, 13-oxo-9Z,11E-octadecadienoic acid, 13S-hydroxy-9Z,11E-octadecadienoic acid, 13S-hydroperoxy-9Z,11E-octadecadienoic acid, 9S-hydroxy-10E,12Z-octadecadienoic-9,10,12,13 d₄ acid, 9(10)epoxy-12Z-octadecenoic acid, 12(13)epoxy-9Z-octadecenoic acid were purchased from Cayman Chemical Co (Ann Arbor, Michigan, USA).

DOPC/PS (500 μM, at a ratio of 1:1) liposomes were prepared by sonication in 50 mM HEPES buffer in the presence of 100 μM DTPA pH 7.4. Liposomes, containing PS (250 μM) were incubated with cyt c (5 μM) and H₂O₂ (100 μM) during 30 min or 3 h at 37°C. At the end of incubation, lipids were extracted by Folch procedure (38) with minor modifications.

Liposomes containing DOPC/PS were treated with Ca²⁺-independent secreted Lp-PLA₂ (0.26 - 2.6 μg protein/sample) in 50 mM HEPES pH 8.2, containing 100 μM DTPA for 30 min at 37°C. After incubation with Lp-PLA₂, lipids were extracted and used for structural analysis by LC/ESI-MS.

Liposomes containing DOPC/PS were treated with PLA₁ (2.4 - 24 μg protein/sample) in 50 mM HEPES pH 7.4, containing 100 μM DTPA for 30 min at 37°C. After incubation with PLA₁ lipids were extracted and used for structural analysis by LC-ESI-MS.

was performed on a Dionex HPLC system (utilizing the Chromeleon software) consisting of a Dionex UltiMate 3000 mobile phase pump, equipped with an UltiMate 3000 degassing unit; UltiMate 3000 autosampler (sampler chamber temperature was set at 4°C), 5 μL sample loop. Dionex HPLC system was coupled to ion trap mass spectrometer (FinniganTM LXQ™ with the Xcalibur operating system, Thermo Electron, San Jose, CA). The instrument was operated in the negative ion mode. For optimization of MS conditions and preparation of tune files, standards (2 pmol/μL) were injected by direct infusion through a syringe pump (flow rate 10 μL/min) into the HPLC solvent flow (flow rate 200 μL/min). The electrospray probe was operated at a voltage differential of 5.0 kV in the negative ion mode. Source temperature was maintained at 150°C. Spectra were acquired in negative ion mode using full range zoom (400-1600 m/z and 210 - 400 m/z for FFA) scans. Tandem mass spectrometry (MS/MS analysis) of individual PS species was used to determine the fatty acid composition. MSⁿ analysis was carried out with relative collision energy ranging from 20-40% and with activation q value at 0.25 for collision-induced dissociation (CID) and q value at 0.7 for pulsed-Q dissociation (PQD) technique. MS/MS analysis was performed using isolation width of 1 m/z, 5 micro-scans with maximum injection time 1000 ms. For selected reaction monitoring (SRM) experiments the optimum collision energy was determined for each m/z parent daughter ion pair (30 kV).

Normal-phase LC conditions were as described by Malavolta et al.(39) with slight modifications. Phospholipids (5 μL) were separated on a normal phase column (Luna 3 μm Silica (2) 100A, 150×2 mm, (Phenomenex, Torrance, CA)) with flow rate 0.2 mL/min. The column was maintained at 30°C. The analysis was performed using gradient solvents (A and B). Solvent A was chloroform:methanol:ammonium hydroxide (28%), 80:19.5:0.5 (v/v). Solvent B was chloroform:methanol:water:ammonium hydroxide, 60:34:5:0.5 (v/v). The column was eluted during the first 3 min isocratic at 10% solvent B, 3 – 15 min with a linear gradient from 10% solvent B to 37% solvent B, 15 – 23 min linear gradient to 100% solvent B, and then 23 – 45 min isocratic at 100% solvent B, 47 - 57 min isocratic at 10% solvent B for equilibrium column. Reference standards of lyso PS for LC-ESI-MS analysis were obtained after hydrolysis of SL-PS and LL-PS by PLA₂ and following their separation by 2-D-HPTLC.

FFA were separated on a reverse phase column (Luna 3 μm C₁₈ (2) 100A, 150×2 mm, (Phenomenex)) at a flow rate of 0.2 mL/min. The column was maintained at 30°C. The analysis was performed using gradient solvents (A and B) containing 5 mM ammonium acetate. Solvent A was tetrahydrofuran/methanol/water/CH₃COOH, 25:30:44.9:0.1 (v/v). Solvent B was methanol/water, 90:10 (v/v). The column was eluted during the first 3 min isocratically at 50% solvent B, from 3 to 23 min with a linear gradient from 50% solvent B to 98% solvent B, then 23 – 40 min isocratically using 98% solvent B, 40-42 min with a linear gradient from 98% solvent B to 50% solvent B, 42 - 48 min isocratically using 50% solvent B for equilibration of the column.

Three dimensional structures of SLPS, LLPS, SL-PSox and LL-PSox were generated using Marvin Sketch 5.3.6.(40) Hydroxy- and hydroperoxy-groups were placed at the sn-2 positions of SL-PS and LL-PS, yielding 9-hydroxy, 9-hydroperoxy, 9-hydroxy-14hydroxy, 9-hydroxy-12,13-epoxy, 9-hydroperoxy-14-hydroperoxy, 13-hydroxy, and 13-hydroperoxy species. Oxidatively-fragmented PS species containing either –oxo- or carboxy groups at the C₉ position (the end of truncation) were also created. These different species of oxidized PS were docked to the crystal structure of Lp-PLA₂ (PDBid: 3F9C using AutoDock Vina(41, 42) available at http://vina.scripps.edu. The lipid and Lp-PLA₂ 3D structures were converted from pdb into pdbqt format using MGL Tools.(43) The Lp-PLA₂ structure was treated as the receptor and was kept rigid during docking. In contrast, rotatable bonds in the lipid structures imparted flexibility on the ligands. A grid box was centered at the −14.59, −9.92, −32.08 coordinates with 70Å units in x, y and z directions to cover the active site of Lp-PLA₂. Support for the chosen location of the grid box comes from a prediction of putative binding pockets by using Pocket-Finder software (http://www.modelling.leeds.ac.uk/ pocketfinder). Using this independent approach, the active site was predicted to be the largest putative ligand binding site. Docking with Autodock Vina(42) resulted in 9 lowest energy conformations. Of these conformations, we concentrated on those binding poses where the sn-2 ester bond of the acyl chain was found in proximity to the active site. For docking purposes, proximity was defined as distance less than 5 Å to either residue Ser₂₇₃ or His₃₁₅ of the catalytic triad.

We characterized cyt c/H₂O₂ catalyzed oxidation of two PS species: SL-PS, and LL-PS, containing non-oxidizable stearic acid (C_18:0) in the sn-1 position and oxidizable linoleic acid (C_18:2) in the sn-2 position, or linoleic acid in both sn-1 and sn-2 positions, respectively. A complex mixture of oxidation products, containing 1, 2, 3 and 4 oxygens was documented by LC-MS profiles (Fig. 1A), MS¹ (Fig.1B) and MS² spectra (data not shown) of SL-PSox. Peroxidation of SL-PS (30 min at 37°C) resulted in the formation of several oxygenated products at m/z 802, 818, 834 and 850 corresponding to the following species: C_18:0/C_18:2+O, C_18:0/C_18:2+2O, C_18:0/C_18:2+3O and C_18:0/C_18:2+4O (Fig. 1C). Detailed MS² analysis demonstrated that the PS [M-H]⁻ ions at m/z 802, 818, 834 and 850 contained mono-hydroxy, mono-hydroperoxy, di-hydroxy and di-hydroperoxy groups attached to either C₉ or C₁₃ carbons of linoleic acid (Table 1). Oxygenated molecular species of SL-PSox with m/z 800 containing oxidized isomers of 9-oxo- and 13-oxo-linoleic acid were also observed (data not shown). The molecular species with two oxygens (m/z 818) – corresponding to C_18:0/C_18:2+2O was dominant and its amount was estimated to be 9.4 ± 1.0 mol% of SL-PS.

DOPC liposomes containing oxidized species of either SL-PS or LL-PS were treated with human recombinant Lp-PLA₂ and the hydrolysis products were analyzed by LC-ESI-MS. Typical LC-ESI-MS base-profiles of lyso-PS formed during hydrolysis of SL-PS with Lp-PLA₂ are presented in Fig. 2 Aab. Hydrolysis of SL-PS by Lp-PLA₂ yielded non-oxidized 1-stearoyl-2-lyso-PS (m/z 524) (Fig. 2Aa-c) and a mixture of free linoleic acid (m/z 279) along with a wide spectrum of its oxygenated metabolites (Fig. 3Aa). In line with previous reports, (19-21) significantly higher contents of hydrolysis products were found in the presence of peroxidized molecular PS species compared with non-oxidized PS (Fig. 2Ba-c). We found that Lp-LA₂-driven accumulation of lyso-PS was dependent on incubation time with enzyme (Fig. 2Ac). Further, the hydrolysis rate grew proportionally to the increased level of SL-PS oxidation (Fig. 2Ad) whereby oxidatively truncated species were preferable substrates for Lp-PLA₂ (Fig. 2Bc).Under our experimental conditions, no hydrolysis of non-oxidized DOPC was observed. This is consistent with the exclusive role of PS – compared to other classes of phospholipids as an activator of PLA₂.(44) Expectedly, PLA₁-induced hydrolysis of peroxidized SL-PS caused the release of stearic acid (m/z 283) and a mixture of lyso-PS with non oxygenated (m/z 520) and oxygenated linoleic acid (m/z 534, 536, 550, 552, 566, 568, 584) (data not shown).

Efficiency of Lp-PLA₂ hydrolysis of PSox species was proportional to the amount of oxygens present in the acyl chains (Fig 2Bc) and increased in the order: SL-PS (m/z 786) << SL-PSox (m/z 802) < SL-PSox (m/z 818) < SL-PSox (m/z 834) << SL-PSox (m/z 694). Quantitatively, the amounts of hydrolyzed SL-PS corresponded to the accumulated lyso-PS species. Because oxidatively fragmented PC species have been reported as substrates for Lp-PLA₂ (45) we were anxious to examine the effectiveness of the enzyme towards truncated species of PSox. To this end, we increased incubation time (up to 3h) for DOPC/SL-PS liposomes in the presence of cyt c/H₂O₂. This resulted in the significantly increased amounts of the major oxidized SL-PS species at m/z 818 (up to 40 Mol % of PS) and simultaneously to the appearance of relatively small amounts of oxidatively fragmented PS molecular species at m/z 678 and 694 corresponding to 1-stearoyl-2-(9-oxononanoyl)-sn-glycero-3-phosphoserine (ON-PS, 1 Mol % of PS) and 1-stearoyl-2-azelayl-sn-glycero-3-phosphoserine (A-PS, 3 Mol % of PS). In spite of their low abundance, these truncated PSox species were hydrolyzed with higher efficiency compared to other oxidized PS species (Fig. 2Bc). We further performed MS³ analysis to determine which of the oxidatively modified PS acyls - oxygenated at the C₉ or C₁₃ carbon atoms - is a preferable substrate for the enzyme. MSⁿ analysis of non-truncated SL-PSox, at m/z 818 (Fig. 2Cb) and m/z 802 (Fig. 2Cc) showed preferential cleavage of fatty acid residues containing hydroperoxy- and hydroxy-group at the C₉ position. This preference was also observed for the oxidatively fragmented species of PSox (Fig.2Ca). Accordingly, the remaining, non-hydrolyzed, SL-PSox (m/z 802) contained only 13-hydroxy-linoleic acid in the sn-2 position (Fig. 2Cd). The hydrolysis by Lp-PLA₂ – while overall effective towards SL-PSox – was still not 100% complete.

Characterization of the released oxidatively modified linoleic acid derivatives revealed the presence of molecular ions with m/z 295 that were identified as 9-hydroxy-, 13-hydroxy-linoleic acid and mixture of epoxy-derivatives of 9,10 epoxy- and 12,13 epoxy-linoleic acid (Fig. 3Ba, 3Bb). The amount of 9-hydroxy-isomers of linoleic acid hydrolyzed by Lp-LPA₂ was significantly higher (51.3 vs 18.1 pmol/nmol lysoPS, for SL-PS) compared with its 13-hydroxy-isomers (Fig, 3Bc). The same tendency was observed for 9,10 epoxy- and 12,13 epoxy- linoleic acid (m/z 295) (25.2 vs 5.3 pmol/nmol lysoPS, for SL-PS), respectively (Fig, 3Bc). Among the oxygenated FFA (with one oxygen), 9-oxo- and 13-oxo-linoleic acid (m/z 293) were found (data not shown). Cleaved oxygenated linoleic acid containing two oxygens was represented by 9-hydroperoxy-linoleic acid and a mixture of dihydroxy- (8,13-, 9,14-hydroxy- and 9-hydroxy-, 12,13-epoxy-) derivatives of linoleic acid, respectively (Fig. 3Ca, 3Cb, 3Cc).

Cyt c-driven oxidation of LL-PS yielded a large variety of oxidized species (Fig. 4AB, Table 2). LL-PS molecular species with oxidized linoleic acid residues containing 1, 2, 3 and 4 oxygens at m/z 798, 814, 830 and 846, respectively, were detected in the MS spectra (Fig. 4B). Quantitatively, significantly greater accumulation of oxidized products was found among modified molecular species of LL-PS compared to those formed from SL-PS (Fig. 4C). Expectedly, oxygenation of LL-PS occurred in both sn-1 and sn-2 positions whereby oxygenated species with oxygen(s) at C₉ and C₁₃ carbon atoms of linoleic acid were formed. Oxidation of linoleic acid in the sn-1 position was less pronounced compared with that localized in the sn-2 position. Interestingly, oxygenation in sn-1 position was observed only after the addition of two oxygens in sn-2 position in molecular species of LL-PSox (Table 2).

Typical LC-MS profiles of lyso-PS are presented in Fig 5Aa,b. Hydrolysis of oxidized LL-PS by Lp-PLA₂ revealed the presence of two types of lyso-PS products with either non-oxygenated (Fig. 5Ba) or oxygenated linoleic acid in sn-1 position (Fig. 5Bb). Non-oxidized 1-linoleoyl-2-lyso-PS with m/z 520 represented a dominant product, with smaller amounts of oxygenated lyso-PS species containing only one and two oxygens in the fatty acid moieties (m/z 534, 536 and 550, 552, respectively) (Fig. 5Bc). The total amount of accumulated lyso-PS was 18.1 ± 1.3 Mol % of LL-PS, whereby the contents of non-oxidized-lyso-PS (1-linoleoyl-2-lyso-PS) and oxidized lyso-PS (1-oxidized linoleoyl-2-lyso-PS) were estimated as 13.5 ± 0.9 and 4.6 ± 0.3 Mol % of LL-PS, respectively (Fig. 5Bd). LL-PSox species were hydrolyzed with higher efficiency (Fig. 5Ca) yielding higher contents of hydrolysis products of PSox (Fig. 5Cb) compared with non-oxidized PS (Fig. 5Cc).

Similarly to SL-PSox, the preferable cleavage of oxygenated fatty acid residues containing hydroxy- and hydroperoxy groups in the C₉ position was observed for LL-PSox (data not shown). Interestingly, among products of LL-PSox hydrolysis, “heavily” oxygenated linoleic acid with 1-4 oxygens at C₉ or C₁₃ carbon atoms (m/z 293, 295, 309, 311, 325, 327, 343) were detected (Fig. 6A). MS² analysis of the liberated oxygenated linoleic acid revealed the presence of 9-hydroxy- and 13-hydroxy-, (Fig. 6Ba, Bb), 8,13-dihydroxy- (Fig. 6Ca, 6Cb) and 9,14-dihydroxy- (Fig. 6Ca, 6Cc) and 9-hydroperoxy-derivatives (data not shown). Quantitatively, the level of oxidized linoleic acid was significantly higher than the content of non-oxidized linoleic acid whereby hydroperoxy-molecular species were dominant. Overall, 170.1 ± 16.6 pmol of oxidized and non-oxidized linoleic acid per nmol of LL-PS was released due to hydrolysis of oxidized LL-PS by Lp-PLA₂. The contents of released oxidized and non-oxidized linoleic acid were 154.4 ± 13.1 and 15.7 ± 2.7 pmol/nmol LL-PS, respectively. These estimates are in good agreement with the data on Lp-PLA₂ catalyzed accumulation of lyso-PS (see above).

To further verify the localization of oxygenated linoleic acid residues generated by Lp-PLA₂ hydrolysis, we additionally utilized PLA₁. In this case, the hydrolysis of oxidized LL-PS caused the release of non-oxidized plus oxidized linoleic acid (containing 1 and 2 oxygens only) (Fig. 7A) and oxidized species of lyso-PS containing oxygenated linoleic acid with 1-4 oxygens (m/z 536, 552, 568 and 584, respectively) (Fig. 7B). A lyso-PSox hydrolysis product with m/z 536 (containing 13-hydroxy-linoleic acid with m/z 295) originated from two oxygenated molecular species of LL-PSox - one with m/z 814 (containing trace amount of oxidized linoleic acid (m/z 295) in sn-1 and sn-2 position) and the other one with m/z 830 (containing oxidized linoleic acid (m/z 295 and m/z 311) in sn-1 and sn-2 positions, respectively) (Fig. 7C).

In an effort to better understand the specificity of Lp-PLA₂ catalysis, we performed docking studies of a total of 8 different species of PS and its oxidation products at different positions including C₉, C₁₃ and C₁₄ as well as truncated species containing either oxo- or carboxy-groups at C₉ position. Specifically, 9-hydroxy, 9-hydroperoxy, 9,14-dihydroxy, 9-hydroxy-12,13-epoxy, 9,14-dihydroperoxy, 13-hydroxy, 13-hydroperoxy, truncated C₉-oxo and C₉-carboxy sn-2 species of both SL-PS and LL-PS were docked to the crystal structure of Lp-PLA₂ (PDBid : 3F9C, chain A)(41) to provide models for how different species interact with the protein that would help gain mechanistic insight into the molecular basis for the experimental data obtained. The active site of Lp-PLA₂ is characterized by a catalytic triad composed of residues Ser₂₇₃, Asp₂₉₆ and His₃₅₁ (Fig. 8A), which accomplish hydrolysis of the sn-2 ester bond. Thus, we analyzed in detail those docking poses in which the sn-2 ester bond of the different oxidized species of SL-PS and LL-PS was located in proximity (within 5Å) to Ser₂₇₃ and His₃₅₁. In particular, we looked for poses in which the side chain ‘O’ of Ser₂₇₃ was close to the sn-2 carbonyl ‘O’ and the sn-2 ester ‘O’ atom faced the closest His₂₅₁ ‘N’ atom (Fig. 8A, dashed lines). Table 3 lists the lowest binding energies, the number of poses satisfying this proximity criterion and the above-mentioned distances to Ser₂₇₃ and His₃₅₁ for the different oxidized species. The binding poses corresponding to -C₉-oxo, -C₉-carboxy, -9-hydroxy, -13-hydroperoxy, -9,14-dihydroxy of SL-PS and -9-hydroperoxy of both LL-PS and SL-PS species are shown in Fig. 8. In most cases, the phospholipid head groups of SL-PS and LL-PS bind close to a positively charged Lys₃₇₀ (Fig. 8). This residue was shown previously to play a role in binding of HDL(46) and LDL(47) to LpPLA₂. The -OH of the serine head-group of PS in most cases points towards Lys₃₇₀ (Fig. 8B-D), although more rarely it points away from Lys₃₇₀ (Fig. 8E), albeit still in proximity. This proximity, particularly when the serine head-group is facing Lys₃₇₀, leaves only one reasonable choice for the location of the sn-1 acyl chain, which is to be buried in a hydrophobic pocket (Fig. 8). In general, the position of the sn-1 chain in the hydrophobic pocket is independent of the lipid species analyzed. The sn-1 acyl chain binding site is similar to the proposed platelet activating factor (PAF) binding site.(41)

What is the mechanism by which oxidized lipids bind in proximity to the catalytic active site? Although the energies predicted for the different lipid species did not differ drastically, non-oxidized PS does bind less preferentially as compared to oxidized species of both SL-PS and LL-PS (Table 3). Furthermore, a relatively larger number of conformations were observed in proximity of the catalytic site (Table 3). A detailed analysis of the environment of the OH and OOH groups in the different high-ranking poses suggests that a number of interactions by these groups with the protein contribute favorably to binding (Table 4). In particular, OH and OOH groups were frequently observed to interact with aromatic residues in the binding pocket, in particular Phe₃₂₂ (in contact in poses for *C9-O, *C9-OOH, 9OOH, 9OH-14OH, 9OOH 14OOH) and Tyr₃₂₄ (for *C9OOH, 9OH, 9OOH, 9OH-14OH, 9OOH-14OOH), but sometimes Phe₁₁₀ (for *C9-OOH, 9OH-14OH), Tyr₃₂₁ (9OH-14OH) or Trp₂₉₈ (for *C9-OOH, 9OH-14OH) were involved. Polar interactions also play a role in some conformations, such as with Arg₂₁₈ (for *C9-O, *C9-OOH, 9-OOH), Gln₂₁₁ (for *C9-O), and Glu₂₁₄ (for *C9-O, *C9-OOH, 9OOH, 9OH-14OH). There are also intramolecular interactions within the lipids observed, such as with the PS-phosphate (*C9-O, *C9-OOH, 9OH, 9OOH) or the PS-amino group (9OH-14OH). Thus predicted stabilization of oxidized lipids through molecular contacts is in line with the known specificity of Lp-PLA₂ towards oxidized phospholipids (45) and provides mechanistic insight in to the underlying reasons for the stabilization. Further, the sn-2 ester bond in both LL-PS and SL-PS was positioned in proximity to both Ser₂₇₃ and His₃₅₁ in the case of oxidatively fragmented species at the C₉ position as compared to C₁₃ position (Table 3). This suggests that one should expect the truncated sn-2 species to be preferred substrates, followed by C₉-oxygenated non-truncated isomers, compared to C₁₃-isomers - in line with the effectiveness of the hydrolysis of different oxidized PS species observed experimentally.