Impact of Truncated Oxidized Phosphatidylcholines on Phospholipase A2 Activity in Mono- and Polyunsaturated Biomimetic Vesicles

Abstract

The interplay between inflammatory and redox processes is a ubiquitous and critical phenomenon in cell biology that involves numerous biological factors. Among them, secretory phospholipases A₂ (sPLA₂) that catalyze the hydrolysis of the sn-2 ester bond of phospholipids are key players. They can interact or be modulated by the presence of truncated oxidized phosphatidylcholines (OxPCs) produced under oxidative stress from phosphatidylcholine (PC) species. The present study examined this important, but rarely considered, sPLA₂ modulation induced by the changes in biophysical properties of PC vesicles comprising various OxPC ratios in mono- or poly-unsaturated PCs. Being the most physiologically active OxPCs, 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC) have been selected for our study. Using fluorescence spectroscopy methods, we compared the effect of OxPCs on the lipid order as well as sPLA₂ activity in large unilamellar vesicles (LUVs) made of the heteroacid PC, either monounsaturated [1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)], or polyunsaturated [1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PDPC)] at a physiological temperature. The effect of OxPCs on vesicle size was also assessed in both the mono- and polyunsaturated PC matrices. Results: OxPCs decrease the membrane lipid order of POPC and PDPC mixtures with PGPC inducing a much larger decrease in comparison with POVPC, indicative that the difference takes place at the glycerol level. Compared with POPC, PDPC was able to inhibit sPLA₂ activity showing a protective effect of PDPC against enzyme hydrolysis. Furthermore, sPLA₂ activity on its PC substrates was modulated by the OxPC membrane content. POVPC down-regulated sPLA₂ activity, suggesting anti-inflammatory properties of this truncated oxidized lipid. Interestingly, PGPC had a dual and opposite effect, either inhibitory or enhancing on sPLA₂ activity, depending on the protocol of lipid mixing. This difference may result from the chemical properties of the shortened sn-2-acyl chain residues (aldehyde group for POVPC, and carboxyl for PGPC), being, respectively, zwitterionic or anionic under hydration at physiological conditions.

Keywords: PDPC; PGPC; POPC; POVPC; lipid order; sPLA2 activity; vesicle size.

Figure 1

Chemical structures of phospholipids: (A) phosphatidylcholine (PC) molecules depending on the degree of fatty acid unsaturation at sn-2 position: monounsaturated lipid POPC (OA, C18:1) and polyunsaturated one PDPC (DHA, C22:6); (B) oxidatively modified phosphatidylcholines differing in the functional group at the free end of the short fatty acid chain at sn-2 position: POVPC (with aldehyde group) and PGPC (with carboxyl group) and their precursor polyunsaturated lipid PAPC (AA, C20:4); (C) glycerophosphoethanolamine lipid analog with BODIPY^® dye-labeled sn-2 acyl chain and dinitrophenyl quencher-modified head group: PED6 (fluorogenic phospholipase A₂ substrate).

Figure 2

Kinetic curves of PED6 cleavage by sPLA₂ in POPC and PDPC vesicles. FL = (F_530/F_{530, initial}) − 1 as F₅₃₀ was the fluorescence intensity at 530 nm at time t whereas F_{530, initial} represented the sample fluorescence intensity before enzyme addition. Vesicles were formed by hydration at room temperature, RT, (A) and by heating/cooling cycles, HC (B). sPLA₂ was added at t = 0 min as PC/PED6 and (PC + PED6)/enzyme ratio were 10/1 mol/mol and 8000/1 mol/mol accordingly. FL was read by kinetic setting of 1 min intervals during 2.5 h at 37 °C. The graphs show the FL values measured every 5 min. Error bars corresponded to standard deviations from 12 curves (3 different experiments as each sample is measured 4 times). One-way ANOVA method for means comparison was performed. The data were drawn from a normally distributed population, and the population means were significantly different at the 0.05 level (Supplementary Figure S2).

Figure 3

Kinetic curves of PED6 cleavage by sPLA₂ as PED6 was incorporated into POPC and POPC/OxPC (A) as well as PDPC and PDPC/OxPC (B) vesicles. FL = (F_530/F_{530, initial}) − 1 as F₅₃₀ was the fluorescence intensity at 530 nm at time t whereas F_{530, initial} represented the sample fluorescence intensity before enzyme addition. Vesicles were formed by hydration at room temperature (RT) and by heating/cooling cycles (HC). sPLA₂ was added at t = 0 min as PC/PED6 and (PC + PED6)/enzyme ratio are 10:1 mol/mol and 8000:1 mol/mol accordingly. FL signal increase was correspondent to sPLA₂ activity elevation. FL was read by kinetic setting of 1 min intervals for 2.5 h at 37 °C. On the graphs, FL values of every 5 min were presented for clarity. Error bars corresponded to standard deviations from 12 curves (3 different experiments as each sample is measured 4 times). One-way ANOVA method for means comparison was performed Figure S3). The lines presented linear extrapolations of the initial linear part of the normalized kinetic curves: solid lines for vesicles hydrated at RT and dashed ones for vesicles hydrated with HC cycles. The slopes corresponded to the enzymatic reaction rates.

Figure 4

Effect of OxPCs on sPLA₂ activity in PED6-labeled POPC (A1,B1) and PDPC (A2,B2) vesicles presented as absolute (A) and relative (B) changes. ΔFL was defined as the difference between FL intensity for OxPC-containing vesicles and control PC ones (FL_{PC (POPC or PDPC)}). The relative change of FL, ΔFL/FL_PC was expressed in percentages for POPC (B1) and for PDPC (B2). RT—room temperature protocol for lipid mixing; HC—heating/cooling cycles for lipid mixing.

Figure 5

Linear regression of the initial part of the kinetic curves of normalized fluorescence intensity, FL, at 530 nm [FL = F₅₃₀/F_{530, initial} − 1] = a + bt, where a and b denoted the y-intercept and the slope, respectively. The intercept yielded the value of the normalized fluorescence intensity FL = F₅₃₀/F_{530, initial} − 1 at t = 0 min (A1,A2). The enzymatic reaction rate was determined as the slope of the kinetic curves (dFL/dt [min⁻¹]) (B1,B2). RT—room temperature protocol for lipid mixing; HC—heating/cooling cycles for lipid mixing.

Figure 6

Effect of the degree of FA unsaturation at sn-2 position of PCs (POPC and PDPC) and OxPC (POVPC or PGPC) on lipid order in PC and PC/OxPC LUVs measured by Laurdan spectroscopy: (A) Laurdan GP in control POPC (green dashed line) and PDPC (red dashed line) LUVs, and in binary OxPC-containing (15 and 30 mol %) ones with lipid/Laurdan ratio 200/1 mol/mol hydrated at room temperature, RT, (A1), and by heating/cooling cycles, HC, (A2). The lines are added only for clarity. The data represented the means of 9 measurements at 37 °C. Error bars corresponded to the standard deviations. One-way ANOVA method for means comparison was performed. The data were drawn from a normally distributed population and showed statistically significant difference in GP values between pure POPC and PDPC vesicles hydrated at RT, based on Tukey test with p < 0.05 *, (A1). No statistically significant differences (ns) in GP values between POPC and PDPC vesicles hydrated by HC cycles were observed (A2). Different letters below bars indicated the statistically significant differences between LUV compositions at both studied protocols of lipid mixing. (B) ΔGP/GP_PC (%) quantified the reduction in membrane lipid order in presence of OxPC compared to control vesicles without OxPC. RT mixing (B1) and HC mixing (B2). ΔGP was defined as the difference between averaged GP values for binary OxPC-containing vesicles and control PC ones (GP_PC).

Figure 7

Average vesicle size of large unilamellar vesicles (LUVs) composed of PC/OxPC mixtures as PC is POPC ((A), green points) or PDPC ((B), red points) whereas OxPC is POVPC (orange points) or PGPC (dark gray points). Two protocols of lipid mixing were used to form LUVs: RT (filled circles) was lipid mixing at room temperature (25 °C) and HC (open circles) was lipid mixing through heating/cooling cycles. Three independent vesicle size experiments were carried out (vesicle preparations) as each point on the graph averaged three measurements from one experiment.