Structural basis for the recognition of oxidized phospholipids in oxidized low density lipoproteins by class B scavenger receptors CD36 and SR-BI

Abstract

Specific oxidized phospholipids (oxPC(CD36)) accumulate in vivo at sites of oxidative stress and serve as high affinity ligands for scavenger receptors class B (CD36 and SR-BI). Recognition of oxPC(CD36) by scavenger receptors plays a role in several pathophysiological processes. The structural basis for the recognition of oxPC(CD36) by CD36 and SR-BI is poorly understood. A characteristic feature of oxPC(CD36) is an sn-2 acyl group that incorporates a terminal gamma-hydroxy (or oxo)-alpha,beta-unsaturated carbonyl. In the present study, a series of model oxidized phospholipids were designed, synthesized, and tested for their ability to serve as ligands for CD36 and SR-BI. We demonstrated that intact the sn-1 hydrophobic chain, the sn-3 hydrophilic phosphocholine or phosphatidic acid group, and the polar sn-2 tail are absolutely essential for high affinity binding. We further found that a terminal negatively charged carboxylate at the sn-2 position suffices to generate high binding affinity to class B scavenger receptors. In addition, factors such as polarity, rigidity, optimal chain length of sn-2, and sn-3 positions and negative charge at the sn-3 position of phospholipids further modulate the binding affinity. We conclude that all three positions of oxidized phospholipids are essential for the effective recognition by scavenger receptors class B. Furthermore, the structure of residues in these positions controls the affinity of the binding. The present studies suggest that, in addition to oxPC(CD36), other oxidized phospholipids observed in vivo may represent novel ligands for scavenger receptors class B.

FIGURE 1.

A negative carboxylate group at the terminal of the sn-2 position of phospholipid confers significant binding activity to CD36 and SR-BI. The synthetic phospholipids were analyzed for their ability to compete for the binding of ¹²⁵I-NO₂-LDL (5 μg/ml) to CD36 and SR-BI transfected 293 cells as described under experimental procedures. Binding abilities of the synthetic phospholipids to both receptors were determined by assessing the concentrations of synthetic phospholipids (presented as an equimolar mixture of synthetic phospholipids and PAPC) required to block 50% of ¹²⁵I-NO₂-LDL binding (IC₅₀). Results represent the mean ± S.E. of three independent experiments. ^#, p < 0.0001 for comparison versus KOdiA-PC, PSPC, KDdiA-PC, and PDPC; *, p < 0.05 for comparison versus KOdiA-PC and **, p < 0.05 for comparison versus KDdiA-PC.

FIGURE 2.

Phospholipids with neutral polar functional groups in the distal end of sn-2 position of phospholipids have weak binding activity. The synthetic phospholipids were analyzed for their ability to compete for the binding of ¹²⁵I-NO₂-LDL (5 μg/ml) to CD36 and SR-BI transfected 293 cells as in Fig. 1. Results represent the mean ± S.E. of three independent experiments. *, p < 0.05 for comparison versus P6HHPC, P8HOPC, P8AOPC, and P9MNPC.

FIGURE 3.

All three parts of oxidized phospholipids are indispensable for the recognition by the CD36 and SR-BI. The synthetic phospholipids were analyzed for their ability to compete for the binding of ¹²⁵I-NO₂-LDL (5 μg/ml) to CD36 and SR-BI transfected 293 cells as in Fig. 1. Results represent the mean ± S.E. of three independent experiments. *, p < 0.001 for comparison versus DPPA, AcSPC, LysoPC, and PSG; **, p < 0.05 for comparison versus PSPC.

FIGURE 4.

Chain length at sn-2 and -3 positions modulates the binding affinity of oxidized phospholipids. A, longer chain length at sn-3 position provides better binding activity. Results represent the mean ± S.E. of three independent experiments. *, p < 0.05 for comparison versus PSPC. B, SR-BI is more sensitive to the chain length at sn-2 position than CD36. The synthetic phospholipids were analyzed for their ability to compete for the binding of ¹²⁵I-NO₂-LDL (5 μg/ml) to CD36- and SR-BI-transfected 293 cells as in Fig. 1. Results represent the mean ± S.E. of three independent experiments. *, p < 0.05 for comparison versus PGPC, PSPC, and PDPC.

FIGURE 5.

The rigidity of γ-oxo-α,β-unsaturated carbonyl in oxPC_CD36 increases the binding activities to both receptors. The synthetic phospholipids were analyzed for their ability to compete for the binding of ¹²⁵I-NO₂-LDL (5 μg/ml) to CD36- and SR-BI-transfected 293 cells as in Fig. 1. Results represent the mean ± S.E. of three independent experiments. *, p < 0.05 for comparison versus PPPC and PMPC; **, p < 0.05 for comparison versus PMPC.

FIGURE 6.

Model synthetic phospholipids bind directly to CD36 and SR-BI peptides containing binding site for oxidized LDL. Binding of POPC vesicles containing 20 mol% of synthetic phospholipids and tracer amount of [³H]DPPC to the indicated glutathione-Sepharose-bound GST fusion proteins (GST-CD36_118–182, GST-CD36_164A166A, or GST-SR-BI_183–205) was assessed as described under “Experimental Procedures.” Results represent the mean ± S.E. of three independent experiments. *, p < 0.01 for comparison versus PSPC, PSPH, PDPC, and PSPA; ^#, p < 0.05 and ^##, p < 0.01.

FIGURE 7.

Model synthetic phospholipids interfere with foam cell formation. Murine thioglycollate-elicited peritoneal macrophages were isolated from mice of indicated phenotype, cultured, and incubated with NO₂-LDL (25 μg/ml) and [¹⁴C]oleate (1.5 μCi/ml) in the presence or absence of indicated synthetic phospholipids (30 μm). A and B, [¹⁴C]cholesteryl ester synthesis assay was carried out as described under “Experimental Procedures.” C and D, IC₅₀ of synthetic phospholipids correlate with the capacity to inhibit cholesteryl ester accumulation in macrophages. *, p < 0.05; **, p < 0.01 versus NA (no addition).