OxPAPC stabilizes liquid-ordered domains in biomimetic membranes

Abstract

Long-chain polyunsaturated fatty acids (PUFAs) are prone to nonenzymatic oxidation in response to differing environmental stressors and endogenous cellular sources. There is increasing evidence that phospholipids containing oxidized PUFA acyl chains control the inflammatory response. However, the underlying mechanism(s) of action by which oxidized PUFAs exert their functional effects remain unclear. Herein, we tested the hypothesis that replacement of 1-palmitoyl-2-arachidonyl-phosphatidylcholine (PAPC) with oxidized 1-palmitoyl-2-arachidonyl-phosphatidylcholine (oxPAPC) regulates membrane architecture. Specifically, with solid-state ²H NMR of biomimetic membranes, we investigated how substituting oxPAPC for PAPC modulates the molecular organization of liquid-ordered (L_o) domains. ²H NMR spectra for bilayer mixtures of 1,2-dipalmitoylphosphatidylcholine-d₆₂ (an analog of DPPC deuterated throughout sn-1 and -2 chains) and cholesterol to which PAPC or oxPAPC was added revealed that replacing PAPC with oxPAPC disrupted molecular organization, indicating that oxPAPC does not mix favorably in a tightly packed L_o phase. Furthermore, unlike PAPC, adding oxPAPC stabilized 1,2-dipalmitoylphosphatidylcholine-d₆-rich/cholesterol-rich L_o domains formed in mixtures with 1,2-dioleoylphosphatidylcholine while decreasing the molecular order within 1,2-dioleoylphosphatidylcholine-rich liquid-disordered regions of the membrane. Collectively, these results suggest a mechanism in which oxPAPC stabilizes L_o domains-by disordering the surrounding liquid-disordered region. Changes in the structure, and thereby functionality, of L_o domains may underly regulation of plasma membrane-based inflammatory signaling by oxPAPC.

Figure 1

²H NMR spectra for 50 wt % aqueous dispersions in 50 mM Tris buffer (pH 7.5) of DPPC-d₆₂/CHOL (50:50 mol) (left column), DPPC-d₆₂/CHOL/PAPC (45:45:10 mol) (middle column), and DPPC-d₆₂/CHOL/oxPAPC (45:45:10 mol) (right column). Spectra are symmetrized about the central frequency to enhance signal/noise. Plateau values $S_{CD}^p$ for the order parameter in the upper portion of the chains in DPPC-d₆₂ measured from the spectra can be found in Table 1. To see this figure in color, go online.

Figure 2

²H NMR spectra for a 50 wt % aqueous dispersion in 50 mM Tris buffer (pH 7.5) of DPPC-d₆₂/CHOL (50:50 mol) at 37°C (top panel) and of DPPC-d₆₂/DOPC/CHOL (40:40:20 mol) at 23°C (bottom panel). Arrows highlight spectral features. In the top panel, the outer arrows (black) designate methylene groups in the upper portion (plateau region of order) of the chains, while the inner arrows (black) are due to the terminal methyl groups of the chains of DPPC-d₆₂ in the L_o environment formed by DPPC-d₆₂/CHOL. Separate signals for the terminal methyl groups on the sn-1 and -2 chains of DPPC-d₆₂ are discernible under close examination. In the bottom panel, the arrows designate the corresponding features associated with the ordered L_o (dashed blue) and disordered L_d (red) domains formed by DPPC-d₆₂/DOPC/CHOL. Separate signals for the terminal methyl groups on the sn-1 and -2 chains of DPPC-d₆₂ assigned to L_o domains are discernible under close examination. The spectra are symmetrized about the central frequency to enhance signal/noise. To see this figure in color, go online.

Figure 3

²H NMR spectra for 50 wt % dispersions in 50 mM Tris buffer (pH 7.5) of DPPC-d₆₂/DOPC/CHOL (40:40:20 mol) (left column), DPPC-d₆₂/DOPC/CHOL/PAPC (36:36:18:10 mol) (middle column), and DPPC-d₆₂/DOPC/CHOL/oxPAPC (36:36:18:10 mol) (right column). Spectra are symmetrized about the central frequency to enhance signal/noise. To see this figure in color, go online.

Figure 4

Plateau values $S_{CD}^p$ for the order parameter in the upper portion of the chains for DPPC-d₆₂ in L_o and L_d environments in DPPC-d₆₂/DOPC/CHOL (40:40:20 mol) (black square), DPPC-d₆₂/DOPC/CHOL/PAPC (36:36:18:10 mol) (red circle), and DPPC-d₆₂/DOPC/CHOL/oxPAPC (36:36:18:10 mol) (blue triangle). The dependence upon temperature of $S_{CD}^p$ (top panel) and of the difference between their value ${\Delta S}_{CD}^p$ in L_o and L_d domains (bottom panel) are plotted. Dashed vertical lines represent the midpoint between the temperatures where spectra transition from two components to a single component, which serves as our definition of the mixing temperature ($T_m$). Closed symbols represent mixtures with phase coexistence and open symbols correspond to mixtures with a single liquid-crystalline phase. A table of the $S_{CD}^p$ values can be found in Table S1. To see this figure in color, go online.

Figure 5

The fractional amount of DPPC-d₆₂ in L_o (blue) and L_d (red) environments in DPPC-d₆₂/DOPC/CHOL (40:40:20 mol) (left), DPPC-d₆₂/DOPC/CHOL/PAPC (36:36:18:10 mol) (middle), and DPPC-d₆₂/DOPC/CHOL/oxPAPC (36:36:18:10 mol) (right) at 23°C. Estimates of the fractional amount were determined from the relative integrated intensity of signals assigned to terminal methyl groups in depaked spectra as outlined in earlier work (16). A reproducibility of ±5% applies. The depaked spectra are shown in Figure S2. To see this figure in color, go online.