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FIGURE 1. Physical interaction between GLUT9 isoforms and ITM2B-myc or TMEM85-myc: (A) Western blot analyses of the lysates of transiently transfected HEK 293T cells, co-expressing GLUT9a/b and ITM2B/TMEM85-myc, using anti-GLUT9 antibody and anti-myc antibody respectively. (B) Western blot analyses of the lysates of microinjected oocytes co-expressing GLUT9a/b and ITM2B/TMEM85-myc. Each oocyte was microinjected with 12.5 ng of GLUT9a/b cRNA or a mixture containing 12.5 ng of GLUT9a/b cRNA and 12.5 ng of ITM2B/TMEM-myc cRNA. GAPDH protein band for each sample acts as a loading control. (C) Co-immunoprecipitation of GLUT9a/b with ITM2B/TMEM85-myc, using sepharose bead conjugated mouse anti-Myc antibody, from lysates of co-transfected HEK 293T cells co-expressing GLUT9a/b and ITM2B/TMEM85-myc. GLUT9 isoforms were detected by Western blotting using rabbit anti-GLUT9 antibody. (D) Co-immunoprecipitation of GLUT9a/b with ITM2B/TMEM85-myc, from lysates of Xenopus laevis oocytes co-expressing GLUT9a/b and ITM2B/TMEM85-myc. (E) Left panel: Western blot analyses of the lysates (60 μg total protein/lane) of HEK 293T and Caco-2 for endogenous GLUT9 and ITM2B proteins using anti-GLUT9 antibody and anti-ITM2B antibody respectively. Right panel: Endogenous GLUT9 was co-immunoprecipitated with endogenous ITM2B from the lysates of HEK 293T or Caco-2 cells using mouse anti-ITM2B antibody and sepharose (R) bead conjugated anti-mouse IgG antibody, F(ab’)2 fragment. GLUT9 was detected by immunoblotting using rabbit anti-GLUT9 antibody and ITM2B by rabbit anti-ITM2B antibody. IB, immunoblotting; IP, immunoprecipitation.
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FIGURE 2. ITM2B and SLC2A9 gene expression in human renal proximal tubule cells: (A) Immunohistochemistry of human kidney section with anti-ITM2B antibody at 20X magnification. ITM2B protein expression (dark brown) is detected in renal proximal tubule epithelial cells. (B) RT-PCR detection of ITM2B and GAPDH mRNAs in human kidney, human proximal tubule epithelial cells (PTC-05), HEK 293T, Caco-2, and HeLa cells; GAPDH mRNA expression acts as a control. (C) RT-PCR detection of mRNA expression of GLUT9a and GLUT9b in PTC-05 cells. (D) RT-PCR detection of TMEM85 mRNA in human kidney with no expression in the cell lines tested here. (E) Cell membrane localization of ITM2B-myc protein in transiently transfected HEK 293T cells by confocal immunofluorescence microscopy using Alexa Fluor 488 conjugated mouse anti-myc antibody. (F) Cell membrane localization of GLUT9a and GLUT9b proteins in transiently transfected HEK 293T cells by confocal immunofluorescence microscopy using rabbit anti-GLUT9 antibody and Alexa Fluor 594 conjugated anti-rabbit IgG, F(ab’) fragment. (G) Cell membrane localization of GLUT9a and ITM2B-myc proteins in transiently transfected HEK 293T cells co-expressing both proteins by confocal immunofluorescence microscopy: GLUT9 protein (red) was detected using rabbit anti-GLUT9 antibody and Alexa Fluor 594 conjugated anti-rabbit IgG and ITM2B-myc (green) was detected using Alexa Fluor 488 conjugated mouse anti-myc antibody. The rightmost panel shows the merged images of ITM2B-myc and GLUT9a protein localization.
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FIGURE 3. ITM2B inhibits [14C]-urate uptake and stimulates [14C]-urate efflux mediated by human GLUT9 isoforms. (A) The [14C]-urate uptake activity of GLUT9a/b expressed alone or co-expressed with ITM2B/TMEM85 in transiently transfected HEK 293T cells (4 × 106) was measured in an isotonic uptake medium containing 20 μM [14C]-urate for 1 h at ∼25°C. (B) The time course plot of [14C]-urate uptake mediated by GLUT9a/b expressed alone or co-expressed with ITM2B in transiently transfected HEK 293T cells (4 × 106). (C) ITM2B inhibits [14C]-urate uptake mediated by GLUT9a/b following a non-competitive inhibition (mixed) model. The Eadie-Hofstee plot of [14C]-urate uptake mediated by GLUT9a/b expressed alone or co-expressed with ITM2B in transiently transfected HEK 293T cells (4 × 106). V, the [14C]-urate uptake rate in pmol/4 × 106 cells/h; V/S, [14C]-urate uptake rate per concentration (mM) of [14C]-urate (S). (D) The time course plot of [14C]-urate uptake mediated by GLUT9a/b expressed alone or co-expressed with ITM2B in Xenopus laevis oocytes measured in an isotonic uptake medium containing 40 μM [14C]-urate for 1h at ∼25°C. (E) The [14C]-urate uptake activity of GLUT9a expressed alone or co-expressed with ITM2B, TMEM85 or a transmembrane protein (SLC26A6) in oocytes was measured in membrane-polarized oocytes (ND96 medium). (F) The [14C]-urate uptake activity of GLUT9b expressed alone or co-expressed with ITM2B, TMEM85 or SLC26A6 in oocytes was measured in membrane-polarized oocytes. (G) ITM2B inhibits [14C]-urate uptake mediated by GLUT9a/b following a non-competitive inhibition (mixed) model. The Eadie-Hofstee plot of [14C]-urate uptake mediated by GLUT9a/b expressed alone or co-expressed with ITM2B in oocytes. (H) ITMB stimulates urate efflux mediated by GLUT9 isoforms. Each oocyte was microinjected with 50 nl of cRNA solution containing 12.5 ng of GLUT9a/b cRNA or a mixture of 12.5 ng of GLUT9a/b cRNA and 12.5 ng of ITM2B/TMEM85/SLC26A6 cRNA. The [14C]-urate efflux mediated by GLUT9a/b expressed alone or co-expressed with ITM2B in oocytes was measured in membrane-polarized oocytes for 1 h at ∼25°C. For the [14C]-urate efflux experiment, each oocyte was pre-injected with 50 nl of 1500 μM [14C]-urate. ∗P < 0.001 compared with urate efflux in absence of ITM2B.
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FIGURE 4. ITM2B and GLUT9 are N-glycosylated transmembrane proteins; effect of ITM2B on N-glycosylation of GLUT9 isoforms in oocytes. (A) Western blot analysis of the lysate of transfected HEK 293T expressing ITMB-myc protein or N-glycosylation-deficient mutant ITM2B (N170Q)-myc protein, digested with and without N-glycanase (N-gly) in vitro. (B) Western blot analysis of the lysate of Xenopus oocytes expressing ITMB-myc protein, digested with and without N-glycanase (N-gly) in vitro. (C) Western blot analysis of the lysate of Xenopus oocytes expressing either isoform of GLUT9 (GLUT9a/b) alone or co-expressed with ITM2B-myc, digested with and without N-glycanase (N-gly) in vitro. GAPDH protein band for each sample acts as a loading control. (D) Western blot analysis of the lysate of Xenopus oocytes expressing either isoform of GLUT9 or their respective N-glycosylation-deficient mutants (GLUT9a-N90Q or GLUT9b-N61Q), generated by site-directed mutagenesis of the shared residues Asn-90 in GLUT9a and Asn-61 in GLUT9b. The molecular sizes of GLUT9 isoforms were compared before and after digestion with N-glycanase (N-gly). (E) Cell membrane localization of N-glycosylation-deficient mutants, GLUT9a-N90Q and GLUT9b-N61Q, in transiently transfected HEK 293T cells, was detected by confocal immunofluorescence microscopy using rabbit anti-GLUT9 antibody and Alexa Fluor 594 conjugated anti-rabbit IgG. ITM2B-myc or its mutant and GLUT9 isoforms or its mutants were detected by rabbit anti-myc antibody and rabbit anti-GLUT9 antibody respectively. IB, immunoblotting.
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FIGURE 5. ITM2B differentially affects the N-linked glycosylation level of GLUT9 isoforms in oocytes. (A) Western blot analysis of lysates of oocytes expressing GLUT9a alone, or co-expressing GLUT9a and varying amount of ITM2B or fixed amount of other control transmembrane proteins as shown. GAPDH protein band acts as a loading control. For the expression of GLUT9a alone, each oocyte was microinjected with 25 ng of GLUT9a cRNA. For co-expression of GLUT9a with ITM2B, each oocyte was microinjected with a mixture containing 25 ng of GLUT9a cRNA and 5–25 ng of ITM2B cRNA. For co-expression of GLUT9a with TMEM85 or SLC26A6, each oocyte was microinjected with a mixture of 25 ng each of GLUT9a cRNA and TMEM85 or SLC26A6 cRNA. (B) Western blot analysis of lysates of oocytes expressing GLUT9b alone, or co-expressing GLUT9b and varying amount of ITM2B or fixed amount of other control transmembrane proteins as shown. GAPDH protein band acts as a loading control. (C) Western blot analysis of lysates of transiently transfected HEK 293T cells expressing GLUT9a/b, or their respective N-glycosylation-deficient mutants or co-expressing ITM2B-myc with GLUT9a/b, or their respective N-glycosylation-deficient mutants. (D) Western blot analysis of lysates of oocytes expressing GLUT9a/b, or their respective N-glycosylation-deficient mutants or co-expressing ITM2B-myc with GLUT9a/b, or their respective N-glycosylation-deficient mutants. GLUT9 isoforms were detected by immunoblotting using rabbit anti-GLUT9 antibody and ITM2B-myc and TMEM85-myc were detected using rabbit anti-myc antibody. IB, immunoblotting.
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FIGURE 6. ITM2B physically interacts with N-glycosylation-deficient mutants of GLUT9 isoforms and efficiently inhibits their urate transport function. (A) Co-immunoprecipitation of GLUT9a/b or their respective N-glycosylation-deficient mutant (GLUT9a-N90Q or GLUT9b-N61Q) with ITM2B/TMEM85-myc, using mouse anti-Myc antibody conjugated with sepharose beads, from lysates of transiently co-transfected HEK 293T cells co-expressing GLUT9a/b or their respective mutants and ITM2B/TMEM85-myc. (B) Co-immunoprecipitation of GLUT9a/b or their respective N-glycosylation-deficient mutant with ITM2B/TMEM85-myc. The co-immunoprecipitated GLUT9 isoforms or their respective mutants were immunodetected by Western blotting using rabbit anti-GLUT9 antibody and ITM2B/TMEM85-myc was detected using rabbit anti-Myc antibody. (C) The [14C]-urate uptake activity of GLUT9 isoforms or their respective N-glycosylation-deficient mutants, expressed alone or co-expressed with ITM2B/TMEM85, measured in ND96 medium. Each oocyte was microinjected with 50 nl of cRNA solution containing 12.5 ng cRNA of GLUT9a/GLUT9b or the cRNA of their respective mutant or a mixture containing 12.5 ng of GLUT9a/GLUT9b cRNA or the cRNA of their respective mutant and 12.5 ng of cRNA of ITM2B/TMEM85-myc. IP, immunoprecipitation.
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FIGURE 7. ITM2B inhibits the urate transport activity of N-terminal deletion mutants of GLUT9 isoforms. (A) Predicted membrane topology model of the isoforms of GLUT9 (GLUT9a and GLUT9b) with 12 hydrophobic transmembrane domains are shown at the left panels. The N-glycosylation site (confirmed by mutational analyses shown in Figure 4D) in the first extracellular loop was highlighted green. The amino acid (aa) sequences of the N-terminal cytoplasmic part of the GLUT9a/b (540/511 aa) and their respective N-terminal deletion mutants (495/490 aa) with starting amino acid M (marked blue) were shown at the right panels with the first transmembrane hydrophobic domains as marked bold red. (B) The [14C]-urate uptake activity of GLUT9a, GLUT9b, N-terminal deletion mutants of GLUT9a and GLUT9b, expressed alone or co-expressed with ITM2B in Xenopus oocytes, was measured in ND96 medium. Each oocyte was microinjected with 50 nl of cRNA solution containing 12.5 ng cRNA of GLUT9a/b or their respective N-terminal deletion mutants. For co-expression of GLUT9 or its mutants with ITM2B, each oocyte was microinjected with 50 nl of cRNA solution containing 12.5 ng of GLUT9a/b or their respective N-terminal deletion mutants and 12.5 ng of ITM2B cRNA. ∗P < 0.001 compared with the respective urate uptake mediated by GLUT9a(540aa) or GLUT9b(511aa).
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FIGURE 8. ITM2B mutants associated with familial Danish dementia (FDD), retinal dystrophy (FRD) or N-glycosylation (ITM2B-N170Q) exhibit attenuated inhibition of urate uptake by GLUT9 isoforms. (A) Western blot analyses of lysates of oocytes expressing GLUT9a/b or co-expressing GLUT9a/b and ITM2B-myc or myc-tagged mutants of ITM2B associated with familial British dementia (FBD), FDD, FRD or N-glycosylation (N170Q) in Xenopus oocytes. GAPDH protein band acts as a loading control. (B) The [14C]-urate uptake activity of GLUT9 isoforms expressed alone or co-expressed with ITM2B or its mutants associated with FBD, FDD, FRD or N-glycosylation (N170Q), was measured in ND96 medium. ∗P < 0.001 compared with urate uptake in presence of normal ITM2B. (C) The [14C]-urate transport activity of GLUT9 isoforms expressed alone or co-expressed with ITM2B or mutants of ITM2B, was measured in membrane-depolarized oocytes (in Na+-free medium containing 98 μM KCl). Note that ITM2B showed proportionate inhibition in both membrane-polarized and depolarized oocytes (Na+-free medium containing 98 μM KCl). ∗P < 0.001 compared with urate uptake mediated by GLUT9 in presence of normal ITM2B. (D) ITMB stimulates urate efflux mediated by GLUT9 isoforms but the ITM2B(N170Q) does not. For [14C]-urate efflux experiment in Xenopus laevis oocytes, each oocyte was pre-injected with 50 nl of 1500 μM [14C]-urate and then subjected to urate efflux for 1h in ND96 medium. ∗P < 0.001 compared with urate efflux mediated by GLUT9 in absence of ITM2B. NS, statistically non-significant. (E) Co-immunoprecipitation of GLUT9a/b with myc-tagged ITM2B or its mutants, from lysates of oocytes co-expressing GLUT9a/b and ITM2B-myc or its mutants. Each oocyte was microinjected with 50 nl of cRNA solution containing 12.5 ng of GLUT9a/b cRNA or a mixture of cRNAs containing 12.5 ng of GLUT9a/b cRNA and 12.5 ng of cRNA of ITM2B or its mutants. (F) Co-immunoprecipitation of GLUT9a with ITM2B-myc or ITM2B(N170Q)-myc, from lysates of oocytes co-expressing GLUT9a and ITM2B-myc or ITM2B(N170Q)-myc. GLUT9 isoforms were immunodetected by Western blotting using rabbit anti-GLUT9 antibody and ITM2B-myc or myc-tagged ITM2B mutant was immunodetected using rabbit anti-Myc antibody. IB, immunoblotting; IP, immunoprecipitation.
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