An Amphipathic Helical Region of the N-terminal Barrel of Phospholipid Transfer Protein Is Critical for ABCA1-dependent Cholesterol Efflux

John F Oram,Gertrud Wolfbauer,Chongren Tang,W Sean Davidson,John J Albers

doi:10.1074/jbc.m800117200

John F Oram, Gertrud Wolfbauer + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m800117200

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Abstract

Phospholipid lipid transfer protein (PLTP) mimics high-density lipoprotein apolipoproteins in removing cholesterol and phospholipids from cells through the ATP-binding cassette transporter A1 (ABCA1). Because amphipathic alpha-helices are the structural determinants for ABCA1 interactions, we examined the ability of synthetic peptides corresponding to helices in PLTP to remove cellular cholesterol by the ABCA1 pathway. Of the seven helices tested, only one containing PLTP residues 144-163 (p144), located at the tip of the N-terminal barrel, promoted ABCA1-dependent cholesterol efflux and stabilized ABCA1 protein. Mutating methionine 159 (Met-159) in this helix in PLTP to aspartate (M159D) or glutamate (M159E) nearly abolished the ability of PLTP to remove cellular cholesterol and dramatically reduced PLTP binding to phospholipid vesicles and its phospholipid transfer activity. These mutations impaired PLTP binding to ABCA1-generated lipid domains and PLTP-mediated stabilization of ABCA1 but increased PLTP binding to ABCA1. PLTP interactions with ABCA1 also mimicked apolipoproteins in activating Janus kinase 2; however, the M159D/E mutants were also able to activate this kinase. Structural analyses showed that the M159D/E mutations had only minor effects on PLTP conformation. These findings indicate that PLTP helix 144-163 is critical for removing lipid domains formed by ABCA1, stabilizing ABCA1 protein, interacting with phospholipids, and promoting phospholipid transfer. Direct interactions with ABCA1 and activation of signaling pathways likely involve other structural determinants of PLTP.

Highlights

Phospholipid transfer protein (PLTP)3 plays important and diverse roles in lipoprotein metabolism [1, 2]
We showed previously that PLTP can mimic HDL apolipoproteins in binding to ATP-binding cassette transporter A-1 (ABCA1), removing cellular cholesterol and phospholipids, and stabilizing ABCA1 protein [15]
ABCA1-dependent lipid export occurs by a cascade of events involving direct apolipoprotein binding to ABCA1, activation of signaling pathways, apolipoprotein binding to lipid domains formed by ABCA1, and solubilization of these lipids [12, 26, 46, 48, 49]

Summary

EXPERIMENTAL PROCEDURES

Lipoproteins, ApoA-I, Recombinant PLTP, and Peptides— HDL was prepared by sequential ultracentrifugation in the density range 1.125–1.21 g/ml and depleted of apoE and apoB by heparin-agarose chromatography [30]. Cell Surface and ABCA1 Binding—For cell surface binding assay [28, 33, 34], control or mifepristone-treated BHK cells were incubated for 2 h with 2 ␮g/ml 125I-PLTP minus or plus 40 ␮g/ml unlabeled PLTP, chilled on ice, washed twice at 0 °C with PBS/BSA and twice with PBS, and digested with 0.1 N NaOH. For the ABCA1 binding studies, cells were incubated for 2 h with 5 ␮g/ml 125I-PLTP, treated for 30 min at room temperature with PBS containing 1 mg/ml DSP (cross-linking agent), and washed twice with cold PBS containing 20 mM glycine [35, 36]. Cells were washed twice with PBS/BSA, incubated for 4 h with DMEM/BSA with or without 8-BrcAMP plus or minus 10 ␮g/ml apoA-I, PLTP, or peptides, and membrane ABCA1 protein levels were measured by immunoblot analysis [29, 35]. The fractional helical content was calculated using the formula of Chen et al [42] and the MRE

RESULTS

A PLTP WT

DISCUSSION