MD-2 binds cholesterol

https://doi.org/10.1016/j.bbrc.2016.01.126Get rights and content

Highlights

  • MD-2, the TLR4 co-receptor, binds cholesterol.

  • LPS competes with cholesterol for MD-2 binding.

  • Oxidized cholesterol ester-protein adducts compete with cholesterol for MD-2 binding.

  • MD-2-associated cholesterol is found in plasma and atherosclerotic lesions.

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.

Introduction

Endogenous lipids, proteins and nucleic acids often undergo, under pathological conditions, oxidation, fragmentation and other types of modification and form damage-associated molecular patterns (DAMPs)1 [1]. DAMPs activate innate immune receptors and induce inflammatory responses, which can be harmful if unconstrained and prolonged, such as in atherosclerosis, a chronic inflammation of large arteries leading to cardiovascular disease (CVD). We have demonstrated that oxidized cholesterol esters (OxCE), which are abundant in atherosclerotic lesions and in blood of patients with CVD, are such DAMPs, which induce inflammatory responses in macrophages via toll-like-receptor-4 (TLR4) [2], [3], [4], [5]. OxCE induce TLR4 dimerization, activation of downstream kinases and transcription factors, resulting in secretion of inflammatory cytokines, cytoskeletal effects and macrophage lipid accumulation. Yet, molecular mechanisms of OxCE activation of TLR4 remain unclear.

Myeloid differentiation-2 (MD-2; also known as LY96) is a co-receptor of TLR4 and is required for bacterial lipopolysaccharide (LPS) binding and subsequent dimerization of two TLR4/MD-2/LPS molecular complexes. MD-2 has a β-cup fold structure composed of two antiparallel β sheets forming a hydrophobic pocket, with positively charged residues located near the opening rim of the cavity [6], [7]. The hydrophobic pocket accommodates fatty acid chains of LPS lipid A, and positively charged residues at its opening bind negatively charged phosphate groups of lipid A. In the molecule of cholesterol, a hydrocarbon chain linked to one side of the steroid form an elongated hydrophobic structure, which may dock in the hydrophobic pocket of MD-2, and a hydroxyl group linked to the other side of the steroid may stabilize cholesterol at the positively charged entrance to the pocket. The goal of this study was to provide experimental evidence to the hypothesis that MD-2 binds cholesterol. If confirmed, this hypothesis will help understand the molecular basis of TLR4 activation by OxCE and mechanisms of chronic inflammation in atherosclerosis.

Section snippets

MD-2 expression and purification

MD-2 was expressed and purified from either human embryonic kidney (HEK) 293 cells or Spodoptera fruigiperda (Sf9) insect cells, with both preparations tested to bind cholesterol. HEK293 cells, cultured in DMEM supplemented with 10% FBS and 50 μg/ml gentamicin (Calbiochem), were transfected with either MD-2 subcloned into pSecTag2A (Invitrogen) or an empty vector, using GenJet In Vitro DNA transfection reagent (SignaGen Laboratories). After 3 days, cells and medium were harvested, lyzed,

Results and discussion

His-tagged MD-2 was expressed in HEK293 cells and purified using a Ni-NTA column. Material from HEK293 cells transfected with an empty vector served as a negative control. Radiolabeled cholesterol bound MD-2 in a concentration-dependent and saturable manner (Fig. 1A). An apparent Kd was calculated as 155 nM (R2 = 0.93). To confirm the specificity of binding, the 3H-cholesterol binding was competed with non-labeled cholesterol, with an apparent Ki of 399 nM (R2 = 0.86) (Fig. 1B). Kdo2-lipidA,

Acknowledgments

This study was supported by grants HL124174, HL055798 and HL088093 (Y.I.M.) from the National Institutes of Health, and SDG14710028 (S.-H.C.) from the American Heart Association.

Cited by (14)

  • Macrophage-derived myeloid differentiation protein 2 plays an essential role in ox-LDL-induced inflammation and atherosclerosis

    2020, EBioMedicine
    Citation Excerpt :

    As an accessory protein, MD2 helps TLR4 to link the ligands (e.g. LPS [44] and saturated fatty acid [39]), leading to inflammatory responses. In addition, oxidized cholesterols [37] and phospholipids (ox-PL) [38], the components of ox-LDL, have been reported to bind MD2 and activate MD2/TLR4 signaling. These studies indicated that the capability of ox-LDL in inducing TLR4 activation likely resulted from its direct interaction with MD2 protein.

  • Oxidized cholesteryl esters and inflammation

    2017, Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
    Citation Excerpt :

    It is unlikely that unesterified cholesterol binding to MD-2 activates TLR4. However, both LPS and, importantly, OxCE-modified BSA compete with cholesterol for MD-2 binding [45]. Future structural studies will test the hypothesis that the polyoxygenated fatty acyl chain in BEP-CE and/or components of OxCE-protein conjugates provide additional interaction surfaces, which, in combination with cholesterol anchoring in the MD-2 hydrophobic pocket, provide sufficient interfaces for OxCE-induced TLR4 dimerization, which was observed experimentally [27].

  • Context-Dependent Role of Oxidized Lipids and Lipoproteins in Inflammation

    2017, Trends in Endocrinology and Metabolism
    Citation Excerpt :

    It is unlikely that unesterified cholesterol binding to MD-2 activates TLR4. However, both LPS and, importantly, OxCE-modified bovine serum albumin (BSA) compete with cholesterol for MD-2 binding [20]. Therefore, it is plausible to suggest that the polyoxygenated fatty acyl chain in BEP-CE and/or components of OxCE–protein conjugates provide additional interaction surfaces, which, in combination with cholesterol anchoring in the MD-2 hydrophobic pocket, contribute to OxCE-induced TLR4 dimerization, which has been observed experimentally [16].

View all citing articles on Scopus
View full text