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In Vivo Linking of Membrane Lipids and the Anion Transporter Band 3 with Thiourea-modified Amphiphilic Lipid Probes.

Moriyama A, Katagiri N, Nishimura S, Takahashi N, Kakeya H - Sci Rep (2015)

Bottom Line: However, lipid-protein interactions are poorly understood at a molecular level especially in the live cell membrane, due to current limitations in methodology.Cholesterol and a phospholipid were both conjugated to a fluorescent tag through a linker containing thiourea.This method could prove an effective strategy for analyzing lipid-protein interactions in vivo in the live cell membrane.

View Article: PubMed Central - PubMed

Affiliation: Department of System Chemotherapy and Molecular Sciences, Division of Bioinformatics and Chemical Genomics, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.

ABSTRACT
Membrane proteins interact with membrane lipids for their structural stability and proper function. However, lipid-protein interactions are poorly understood at a molecular level especially in the live cell membrane, due to current limitations in methodology. Here, we report that amphiphilic lipid probes can be used to link membrane lipids and membrane proteins in vivo. Cholesterol and a phospholipid were both conjugated to a fluorescent tag through a linker containing thiourea. In the erythrocyte, the cholesterol probe fluorescently tagged the anion transporter band 3 via thiourea. Tagging by the cholesterol probe, but not by the phospholipid probe, was competitive with an anion transporter inhibitor, implying the presence of a specific binding pocket for cholesterol in this ~100 kDa protein. This method could prove an effective strategy for analyzing lipid-protein interactions in vivo in the live cell membrane.

No MeSH data available.


Chemical structures of lipids and lipid probes.
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f1: Chemical structures of lipids and lipid probes.

Mentions: Cholesterol (1), the key lipid species in regulating the membrane order14, likely modulates protein stability and function in general15. There have been reports of cholesteryl ethers with polyethylene glycol (PEG) forming liposomes16 or micelles1718 in aqueous solutions. Such amphiphilic molecules can be used as cholesterol probes with no membrane permeability, e.g. fluorescein-labeled PEG-cholesterol molecules with a PEG repeat of 50 or 200 were shown to distribute to cholesterol-rich domains in the outer leaflet of the plasma membrane1819. We designed and synthesized a cholesterol probe (2) and surveyed the interactions of cholesterol in the sheep erythrocyte (Fig. 1). The cholesterol probe 2 has a short linker for chemically tagging amino acids near the cholesterol binding site. We chose thiourea as the reactive moiety because of its mild reactivity. As expected, the fluorescence of probe 2 was observed mainly on the cell surface, when erythrocytes were treated with the probe (Fig. 2a). The treated erythrocytes were lysed and subjected to SDS-PAGE, followed by fluorescence imaging. We detected tagging of a ~100 kDa protein with high specificity (Fig. 2b). In contrast, the control probe 3, in which cholesterol was replaced with a Boc group, neither bound to the cells nor tagged the protein (Fig. 2a,b). Judging from the molecular weight and the SDS-PAGE pattern, the tagged protein was identified as the anion transporter band 3, which was subsequently confirmed by LC-MS/MS analysis (Figure S1).


In Vivo Linking of Membrane Lipids and the Anion Transporter Band 3 with Thiourea-modified Amphiphilic Lipid Probes.

Moriyama A, Katagiri N, Nishimura S, Takahashi N, Kakeya H - Sci Rep (2015)

Chemical structures of lipids and lipid probes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4663539&req=5

f1: Chemical structures of lipids and lipid probes.
Mentions: Cholesterol (1), the key lipid species in regulating the membrane order14, likely modulates protein stability and function in general15. There have been reports of cholesteryl ethers with polyethylene glycol (PEG) forming liposomes16 or micelles1718 in aqueous solutions. Such amphiphilic molecules can be used as cholesterol probes with no membrane permeability, e.g. fluorescein-labeled PEG-cholesterol molecules with a PEG repeat of 50 or 200 were shown to distribute to cholesterol-rich domains in the outer leaflet of the plasma membrane1819. We designed and synthesized a cholesterol probe (2) and surveyed the interactions of cholesterol in the sheep erythrocyte (Fig. 1). The cholesterol probe 2 has a short linker for chemically tagging amino acids near the cholesterol binding site. We chose thiourea as the reactive moiety because of its mild reactivity. As expected, the fluorescence of probe 2 was observed mainly on the cell surface, when erythrocytes were treated with the probe (Fig. 2a). The treated erythrocytes were lysed and subjected to SDS-PAGE, followed by fluorescence imaging. We detected tagging of a ~100 kDa protein with high specificity (Fig. 2b). In contrast, the control probe 3, in which cholesterol was replaced with a Boc group, neither bound to the cells nor tagged the protein (Fig. 2a,b). Judging from the molecular weight and the SDS-PAGE pattern, the tagged protein was identified as the anion transporter band 3, which was subsequently confirmed by LC-MS/MS analysis (Figure S1).

Bottom Line: However, lipid-protein interactions are poorly understood at a molecular level especially in the live cell membrane, due to current limitations in methodology.Cholesterol and a phospholipid were both conjugated to a fluorescent tag through a linker containing thiourea.This method could prove an effective strategy for analyzing lipid-protein interactions in vivo in the live cell membrane.

View Article: PubMed Central - PubMed

Affiliation: Department of System Chemotherapy and Molecular Sciences, Division of Bioinformatics and Chemical Genomics, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.

ABSTRACT
Membrane proteins interact with membrane lipids for their structural stability and proper function. However, lipid-protein interactions are poorly understood at a molecular level especially in the live cell membrane, due to current limitations in methodology. Here, we report that amphiphilic lipid probes can be used to link membrane lipids and membrane proteins in vivo. Cholesterol and a phospholipid were both conjugated to a fluorescent tag through a linker containing thiourea. In the erythrocyte, the cholesterol probe fluorescently tagged the anion transporter band 3 via thiourea. Tagging by the cholesterol probe, but not by the phospholipid probe, was competitive with an anion transporter inhibitor, implying the presence of a specific binding pocket for cholesterol in this ~100 kDa protein. This method could prove an effective strategy for analyzing lipid-protein interactions in vivo in the live cell membrane.

No MeSH data available.