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Reconstitution in Proteoliposomes of the Recombinant Human Riboflavin Transporter 2 (SLC52A2) Overexpressed in E. coli

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Reconstitution in Proteoliposomes of the Recombinant Human Riboflavin Transporter 2 (SLC52A2) Overexpressed in E. coli

Lara Console et al. Int J Mol Sci.

Abstract

Background: the SLC52A2 gene encodes for the riboflavin transporter 2 (RFVT2). This transporter is ubiquitously expressed. It mediates the transport of Riboflavin across cell membranes. Riboflavin plays a crucial role in cells since its biologically active forms, FMN and FAD, are essential for the metabolism of carbohydrates, amino acids, and lipids. Mutation of the Riboflavin transporters is a risk factor for anemia, cancer, cardiovascular disease, neurodegeneration. Inborn mutations of SLC52A2 are associated with Brown-Vialetto-van Laere syndrome, a rare neurological disorder characterized by infancy onset. In spite of the important metabolic and physio/pathological role of this transporter few data are available on its function and regulation.

Methods: the human recombinant RFVT2 has been overexpressed in E. coli, purified and reconstituted into proteoliposomes in order to characterize its activity following the [3H]Riboflavin transport.

Results: the recombinant hRFVT2 showed a Km of 0.26 ± 0.07 µM and was inhibited by lumiflavin, FMN and Mg2+. The Riboflavin uptake was also regulated by Ca2+. The native protein extracted from fibroblast and reconstituted in proteoliposomes also showed inhibition by FMN and lumiflavin.

Conclusions: proteoliposomes represent a suitable model to assay the RFVT2 function. It will be useful for screening the mutation of RFVT2.

Keywords: FMN; SLC; proteoliposomes; riboflavin; transport.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Purification and identification of the recombinant human RFVT2. (a) Solubilized bacterial proteins were loaded on a Ni2+-chelating chromatographic column. After column washing, RFVT2 was eluted using a buffer containing 0.1% C12E8, 200 mM NaCl, 10 mM Tris/HCl pH 8.0 and 50mM Imidazole. Fractions of 1 mL were collected and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) on 12% polyacrylamide gel and stained with Coomassie Blue. Lane 1 and 2 pass-through fractions, lane 3–7 washing fractions, lane 8–11 elution fractions; (b) Immunoblotting of the same samples of (a) using the anti-RFVT2 antibody whit a dilution of 1:2000; (c) Immunoblotting of the same samples of (a) using the anti-His antibody whit a dilution of 1:40000. Similar results were obtained in three different experiments.
Figure 2
Figure 2
[3H]Riboflavin uptake into proteoliposomes reconstituted whit recombinant hRFVT2. (a) The transport measurement was started adding 0.1 µM [3H]riboflavin and stopped at the indicated times, as described in Materials and Methods. The values were corrected by subtracting the [3H]riboflavin taken up by diffusion as described in Materials and Methods. (b) Riboflavin uptake in proteoliposomes measured in 60 min at two different protein concentration. The values are means ± S.D. from three experiments.
Figure 3
Figure 3
Dependence of the transport activity of hRFVT2 on the pH. Purified RFVT2 was reconstituted in proteoliposomes. [3H]Riboflavin uptake into proteoliposomes was performed at the indicated pH and stopped after 20 min as described in the Material and Methods section. The values are means ± standard deviation (SD) from three experiments.
Figure 4
Figure 4
Dependence of the riboflavin transport on substrate concentration. [3H]Riboflavin was added to proteoliposomes at the indicated concentration. The transport measurement was stopped after 15 min. Data were plotted according to the Lineweaver–Burk equation. The values are means ± SD from three experiments.
Figure 5
Figure 5
Effect of various compounds on riboflavin uptake. (a) Transport was measured as described in Materials and Methods. 10 µM FAD or 10 µM FMN or 100 µM lumiflavin or 10 mM Pyridoxal 5′-phosphate (PLP) or 1 mM N-Ethylmaleimide (NEM) were added 1 min before the labeled substrate. Percent of residual activity was calculated for each experiment with respect to the control sample (without added inhibitor, referred as 100%); (b) dose-response curve for the inhibition of RFVT2 transport activity by FMN was obtained adding FMN at the indicated concentrations 1 min before the labeled substrate. riboflavin transport was stopped after 30 min. The values are means ± SD from three experiments. Statistics was performed using Student’s t-test (* p < 0.05).
Figure 6
Figure 6
Effect of various divalent ion on riboflavin uptake. Transport was measured as described in Materials and methods. The two concentration of divalent ions were added 1 min before the labeled substrate. Riboflavin transport was stopped after 30 min. Percent of residual activity was calculated for each experiment with respect to the control sample (white bar) without added inhibitor and referred as 100%. The values are means ± SD from three experiments. Statistics was performed using Student’s t-test (* p < 0.05).
Figure 7
Figure 7
Dose-response curve for the inhibition of RFVT2 transport activity by MgCl2 (a) and CaCl2 (b). It was obtained adding the divalent ions at the indicated concentrations 1 min before the labeled substrate. Riboflavin transport was stopped after 30 min. The values are means ± SD from three experiments.
Figure 8
Figure 8
Efflux of riboflavin from proteoliposomes reconstituted with recombinant RFVT2. Proteoliposomes were incubated with [3H]riboflavin for 60 min. After this incubation, the external radiolabeled substrate was removed. The export of riboflavin from proteoliposomes was stopped at the indicated times and the residual intraliposomal radioactivity was measured as described in the Material and Method section. The values are means ± SD from three experiments.
Figure 9
Figure 9
Native RFVT2 from primary human dermal fibroblasts. (a) Western Blot analysis of Native RFVT2. Protein extract from primary human dermal fibroblasts was separated by SDS–PAGE on 12% polyacrylamide gel and subjected to WB analysis using the anti-RFVT2 antibody whit a dilution of 1:2000. 50 µg and 60 µg of total protein extract from fibroblast were loaded in line 1 and 2 respectively; (b) time course of native RFVT2 extracted from primary human dermal fibroblasts and reconstituted in proteoliposomes. The transport measurement was started adding 0.1 µM [3H]riboflavin and stopped at the indicated times, as described in Materials and Methods; (c) Effect of FMN and lumiflavin on riboflavin uptake mediated by native RFVT2. The riboflavin transport was stopped after 120 min. The values are means ± SD from three experiments. Statistics was performed using Student′s t-test (* p < 0.05).
Figure 10
Figure 10
Structural model of RFVT2. (a) Lateral view of the ribbon diagram of the RFVT2. The protein is constituted by 11 transmembrane α-helices and a big intracellular loop which connect the transmembrane segment 6 with the transmembrane segment 7. The homology model was constructed by the SWISS-MODEL tool using the crystallographic structure of human Equilibrative Nucleoside Transporter 1 (ENT1) (PDB code 6OB6) as a template. (b) Columbic surface coloring of a space filled diagram of RFVT2 homology model. Molecular graphics was performed with UCSF Chimera [24].

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