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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.


Related in: MedlinePlus

Competitive tagging of band 3 by lipid probes.Erythrocytes were pre-treated with a transporter inhibitor DIDS (100 μM) for 30 min, then time-dependent tagging was examined using the cholesterol probe 2 (a) or the DPPE probe 8 (b). The concentration of the probes was 400 nM. The fluorescence intensities relative to that obtained after 24 h incubation in the absence of DIDS are shown. The tagging level in the absence (black solid line, Figures S2 and S4) or in the presence (red dashed line) of DIDS is shown. Means ± SD of 3–6 experiments are shown.
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f4: Competitive tagging of band 3 by lipid probes.Erythrocytes were pre-treated with a transporter inhibitor DIDS (100 μM) for 30 min, then time-dependent tagging was examined using the cholesterol probe 2 (a) or the DPPE probe 8 (b). The concentration of the probes was 400 nM. The fluorescence intensities relative to that obtained after 24 h incubation in the absence of DIDS are shown. The tagging level in the absence (black solid line, Figures S2 and S4) or in the presence (red dashed line) of DIDS is shown. Means ± SD of 3–6 experiments are shown.

Mentions: Biophysical analyses had predicted the strong binding of cholesterol to band 3, an interaction that may regulate the structure and transporter activity of this protein252627. So far however, nothing has been reported regarding the location of the binding pocket. To investigate the tagging site of the cholesterol probe 2, a competitive tagging assay using 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), an anion transporter inhibitor, was examined. When erythrocytes were treated with DIDS, we found that tagging by cholesterol probe 2 was significantly suppressed (85% inhibition after 24 h incubation) (Fig. 4a). DIDS is known to form covalent bonds with two lysine residues, e.g. Lys 539 and Lys851 in human band 3, although Lys851 is only reactive at high pH (>8)28. In sheep erythrocytes, Lys539 is conserved (Lys557 in sheep), whereas Lys851 is replaced by Arg869, suggesting that Lys557 reacted with thiourea in the cholesterol probe 2 (Fig. 5, S1). Alternatively, tagging by cholesterol probe 2 may be inhibited allosterically by DIDS. In fact, band 3 is expected to be fixed in an outward conformation by stilbene compounds such as DIDS29, which likely inhibit anion transport activity in an allosteric manner30. It is noted that cysteine, another nucleophilic amino acid residue, was not necessary for tagging band 3 since pretreatment of erythrocytes with excess amount of N-ethylmaleimide did not inhibit it (Figure S3).


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)

Competitive tagging of band 3 by lipid probes.Erythrocytes were pre-treated with a transporter inhibitor DIDS (100 μM) for 30 min, then time-dependent tagging was examined using the cholesterol probe 2 (a) or the DPPE probe 8 (b). The concentration of the probes was 400 nM. The fluorescence intensities relative to that obtained after 24 h incubation in the absence of DIDS are shown. The tagging level in the absence (black solid line, Figures S2 and S4) or in the presence (red dashed line) of DIDS is shown. Means ± SD of 3–6 experiments are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Competitive tagging of band 3 by lipid probes.Erythrocytes were pre-treated with a transporter inhibitor DIDS (100 μM) for 30 min, then time-dependent tagging was examined using the cholesterol probe 2 (a) or the DPPE probe 8 (b). The concentration of the probes was 400 nM. The fluorescence intensities relative to that obtained after 24 h incubation in the absence of DIDS are shown. The tagging level in the absence (black solid line, Figures S2 and S4) or in the presence (red dashed line) of DIDS is shown. Means ± SD of 3–6 experiments are shown.
Mentions: Biophysical analyses had predicted the strong binding of cholesterol to band 3, an interaction that may regulate the structure and transporter activity of this protein252627. So far however, nothing has been reported regarding the location of the binding pocket. To investigate the tagging site of the cholesterol probe 2, a competitive tagging assay using 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), an anion transporter inhibitor, was examined. When erythrocytes were treated with DIDS, we found that tagging by cholesterol probe 2 was significantly suppressed (85% inhibition after 24 h incubation) (Fig. 4a). DIDS is known to form covalent bonds with two lysine residues, e.g. Lys 539 and Lys851 in human band 3, although Lys851 is only reactive at high pH (>8)28. In sheep erythrocytes, Lys539 is conserved (Lys557 in sheep), whereas Lys851 is replaced by Arg869, suggesting that Lys557 reacted with thiourea in the cholesterol probe 2 (Fig. 5, S1). Alternatively, tagging by cholesterol probe 2 may be inhibited allosterically by DIDS. In fact, band 3 is expected to be fixed in an outward conformation by stilbene compounds such as DIDS29, which likely inhibit anion transport activity in an allosteric manner30. It is noted that cysteine, another nucleophilic amino acid residue, was not necessary for tagging band 3 since pretreatment of erythrocytes with excess amount of N-ethylmaleimide did not inhibit it (Figure S3).

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.


Related in: MedlinePlus