MD-2 binds cholesterol

Abstract

Cholesterol is a structural component of cellular membranes, which is transported from liver to peripheral cells in the form of cholesterol esters (CE), residing in the hydrophobic core of low-density lipoprotein. Oxidized CE (OxCE) is often found in plasma and in atherosclerotic lesions of subjects with cardiovascular disease. Our earlier studies have demonstrated that OxCE activates inflammatory responses in macrophages via toll-like receptor-4 (TLR4). Here we demonstrate that cholesterol binds to myeloid differentiation-2 (MD-2), a TLR4 ancillary molecule, which is a binding receptor for bacterial lipopolysaccharide (LPS) and is indispensable for LPS-induced TLR4 dimerization and signaling. Cholesterol binding to MD-2 was competed by LPS and by OxCE-modified BSA. Furthermore, soluble MD-2 in human plasma and MD-2 in mouse atherosclerotic lesions carried cholesterol, the finding supporting the biological significance of MD-2 cholesterol binding. These results help understand the molecular basis of TLR4 activation by OxCE and mechanisms of chronic inflammation in atherosclerosis.

Keywords: Cholesterol; LPS; MD-2; TLR4.

Figure 1. Cholesterol binds to MD-2

A, Recombinant, His-tagged MD-2 was purified from HEK293 cells, and 2 µg MD-2 was incubated with ³H-cholesterol. MD-2 was then isolated on a Ni-NTA agarose column eluted with imidazole, and the amounts of MD-2-associated ³H counts were measured. The material isolated from HEK293 cells transfected with an empty vector carrying His-tag was used as a negative control. Mean±SEM; n=3; ***, p < 0.005. B-D, Binding of ³H-cholesterol (1 µM) to MD-2 (2 µg) was competed with increasing concentrations of unlabeled cholesterol (B), LPS (Kdo2-lipidA, panel C), or OxCE-BSA and BSA (D). The competitors were preincubated with MD-2 for 1 hour at room temperature before adding ³H-cholesterol. Then, the experiment was performed as in panel A. Mean±SEM; n=3-4; *, p < 0.05; ***, p < 0.005.

Figure 2. Cholesterol is associated with MD-2 in human plasma and in mouse atherosclerotic lesions

A and C, MD-2 was measured in human lipoprotein-deficient plasma (LPDP, panel A) and in lysates of mouse atherosclerotic lesions (C) using a sandwich ELISA. B and D, Unesterified cholesterol was measured using a standard enzymatic/chromogenic assay in immunoprecipitates of LPDP (B) and atherosclerotic lesions (D), which were pulled down with a non-specific IgG, a specific MD-2 antibody, or the MD-2 antibody followed by a MβCD treatment. Mean±SEM; n=3-4. *, p < 0.05.