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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.


Reactivity of thiourea with amine.(a) Time course analysis of the reaction converting compound 5 to 6. Compound 5 was incubated with n-propylamine (3473 eq) and analyzed by LC-MS. UV chromatograms at 488 nm are shown. (b) Chemical structures of model compounds 5 and 6. The thiourea compound 5 was converted to the guanidine compound 6 in the presence of excess n-propylamine.
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f3: Reactivity of thiourea with amine.(a) Time course analysis of the reaction converting compound 5 to 6. Compound 5 was incubated with n-propylamine (3473 eq) and analyzed by LC-MS. UV chromatograms at 488 nm are shown. (b) Chemical structures of model compounds 5 and 6. The thiourea compound 5 was converted to the guanidine compound 6 in the presence of excess n-propylamine.

Mentions: Band 3 is the most abundant protein in the erythrocyte membrane (30% in human erythrocytes)20. No other protein bands tagged by the cholesterol probe 2 were detectable. The tagging level saturated after 24 h incubation in the presence of 400 nM of probe 2, and a linear correlation between the tagging level and the probe concentration was observed below 800 nM using an 8 h incubation period (Figure S2). The tagging level was tentatively estimated to be 0.073% when incubated with 400 nM of probe 2 for 8 h (Figure S2). This low-level tagging was likely due to mild-reactivity of thiourea (see Fig. 3). The abundance of endogenous cholesterol in the erythrocyte membrane can also contribute to the low efficiency; the quantity of cholesterol in human erythrocytes is 36 nmol/10 e8 cells21, whereas 0.4 nmol of probe 2 was incubated with 10 e8 cells in our experiments. Significant competition would therefore have been present between the abundant endogenous cholesterol and the cholesterol probe. In addition, there is a possibility that probe 2 can access band 3 in a specific conformation only, or in a specific complex; band 3 is known to exist in at least three populations, including the ankyrin-associated complex, the junctional complex, and as free dimers2223, and within each class are subpopulations with further structural heterogeneity24.


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)

Reactivity of thiourea with amine.(a) Time course analysis of the reaction converting compound 5 to 6. Compound 5 was incubated with n-propylamine (3473 eq) and analyzed by LC-MS. UV chromatograms at 488 nm are shown. (b) Chemical structures of model compounds 5 and 6. The thiourea compound 5 was converted to the guanidine compound 6 in the presence of excess n-propylamine.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Reactivity of thiourea with amine.(a) Time course analysis of the reaction converting compound 5 to 6. Compound 5 was incubated with n-propylamine (3473 eq) and analyzed by LC-MS. UV chromatograms at 488 nm are shown. (b) Chemical structures of model compounds 5 and 6. The thiourea compound 5 was converted to the guanidine compound 6 in the presence of excess n-propylamine.
Mentions: Band 3 is the most abundant protein in the erythrocyte membrane (30% in human erythrocytes)20. No other protein bands tagged by the cholesterol probe 2 were detectable. The tagging level saturated after 24 h incubation in the presence of 400 nM of probe 2, and a linear correlation between the tagging level and the probe concentration was observed below 800 nM using an 8 h incubation period (Figure S2). The tagging level was tentatively estimated to be 0.073% when incubated with 400 nM of probe 2 for 8 h (Figure S2). This low-level tagging was likely due to mild-reactivity of thiourea (see Fig. 3). The abundance of endogenous cholesterol in the erythrocyte membrane can also contribute to the low efficiency; the quantity of cholesterol in human erythrocytes is 36 nmol/10 e8 cells21, whereas 0.4 nmol of probe 2 was incubated with 10 e8 cells in our experiments. Significant competition would therefore have been present between the abundant endogenous cholesterol and the cholesterol probe. In addition, there is a possibility that probe 2 can access band 3 in a specific conformation only, or in a specific complex; band 3 is known to exist in at least three populations, including the ankyrin-associated complex, the junctional complex, and as free dimers2223, and within each class are subpopulations with further structural heterogeneity24.

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.