Journal: Nature Communications
Article Title: SPNS2 exports sphingosine-1-phosphate and imports glucose
doi: 10.1038/s41467-026-71659-7
Figure Lengend Snippet: a Immunofluorescence localization of SPNS2 (red) on plasma membrane of SVEC4-10. b Glucose increases S1P secretion by SVEC4-10 cells (n = 3, N = 3). c SVEC4-10 cells were treated with S1P lyase inhibitor A6770 (200 µM) or with SPNS2 inhibitor SLF1081851 (2 µM) and levels of S1P in cells (n = 8–10, N = 5) and medium (n = 9, N = 3) as well as glucose uptake were determined (n = 7, N = 3). d SPNS2 expression in two SPNS2 stably overexpressing SVEC4-10 cell lines generated by CRISPR activation plasmids (CTL1, SPNS2-OE1) or lentiviral activation particles (CTL2, SPNS2-OE2) and in two SPNS2 deleted SVEC4-10 cell lines (SPNS2-KO1 generated with double nickase plasmids, and SPNS2-KO2 via CRISPR/Cas9 knock-out and homology-directed repair plasmids) compared to their controls. e – h S1P levels in cells and medium, and glucose uptake were measured in SPNS2-OE1 ( e ), SPNS2-OE2 ( f ), SPNS2-KO1 ( g ), SPNS2-KO2 ( h ). (n = 6, N = 3). i , j SPNS2-OE1, SPNS2-KO1 cells and their controls were treated with S1P (500 nM), and phosphorylation of p42/44 ( i ) and glucose uptake ( j ) were determined (n = 6, N = 3). k , l Glucose uptake in SPNS2-OE1, SPNS2-KO1 and their control cells treated with insulin (100 nM) or GLUT1 inhibitor BAY-876 (10 nM) (n = 3, N = 3). Data are means ± s.d. b One-way analysis of variance test followed by Šídák’s multiple comparisons test. c – l two-tailed unpaired t-test. Source data are available for this figure in the Source Data file.
Article Snippet: To overexpress TurboGFP-tagged SPNS2 for ligand binding studies, SVEC4-10 cells were transfected with 2 μg of SPNS2 ( NM_001124758 ) Human Tagged ORF Clone in pCMV6-AC-GFP (#RG225940, Origene), 2 μg of Spns2 ( NM_153060 ) Mouse Tagged ORF Clone in pCMV6-AC-GFP (# MG218672 , Origene), or as controls with 2 μg of the pCMV6-AC-GFP vector (# PS100010 , Origene) or 2 μg of glucose transporter GLUT1 (SLC2A1) ( NM_006516 ), or Human Tagged ORF Clone in pCMV6-AC-GFP (#RG222696, Origene).
Techniques: Immunofluorescence, Clinical Proteomics, Membrane, Expressing, Stable Transfection, Generated, CRISPR, Activation Assay, Knock-Out, Phospho-proteomics, Control, Two Tailed Test
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![a Representative image showing localization of SPNS2-TurboGFP and GLUT1-TurboGFP. b Temperature shift melting curves for purified hSPNS2-TurboGFP in the absence (blue) and presence of glucose (black). (n = 3, N = 3). Values are means ± s.d. c Apparent melting temperatures (T m ) for hSPNS2-TurboGFP, purified untagged hSPNS2 and purified mSPNS2-FLAG were calculated from the inflection points of the fitting curves. (n = 3, N = 3). Values are means ± s.e.m. Two-tailed unpaired t-test. d Schematic representation of the scintillation proximity assay (SPA). TurboGFP-tagged transport proteins are bound to scintillation beads. When radiolabeled glucose binds to these bead-bound transport proteins, emitted β-rays are close enough to stimulate the scintillation beads to emit light. Illustration created by Luciana Giono. e SPA signals of [ 3 H]glucose (0.8 μCi) binding to TurboGFP (vector), GLUT1-TurboGFP mSPNS2-TurboGFP, and hSPNS2-TurboGFP and its mutants E433A and T329A immobilized on the surfaces of polyvinyl toluene protein A-coated scintillation beads (500 μg per well) were measured by a scintillation counter. Values are means ± s.e.m. (n = 7; N = 2) and (n = 3; N = 3). One-way analysis of variance test followed by Dunnett’s multiple comparisons test. Source data are available for this figure in the Source Data file.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_9034/pmc13129034/pmc13129034__41467_2026_71659_Fig5_HTML.jpg)
![a , b Identification of key SPNS2 residues involved in glucose transport. a Western blots and representative images of localization of hSPNS2-TurboGFP and its mutants. b Glucose uptake activities of SPNS2 variants with mutations in potential key residues involved in glucose or S1P engagement. hSPN S2 , GLUT1 vector, or the indicated mutants were overexpressed in SPNS2-KO1 cells lacking endogenous SPNS2. Glucose uptake was normalized to SPNS2 expression determined by GFP fluorescence ( n = 5, N = 3). Data are means ± s.e.m. One-way analysis of variance test followed by Dunnett’s multiple comparison test. c – f Direct glucose and S1P transport by SPNS2 proteoliposomes. c Illustration of cell-free preparation of SPNS2 proteoliposomes for functional transport analysis. d Comparable levels of hSPNS2 and its variants by western blots. e , f Proteoliposomes of WT hSPNS2 and variants were loaded without or with glucose ( e ) or S1P ( f ) as indicated and uptake of 1 μM NBD-S1P ( e ) or 1 µM NBD-glucose ( f ) determined. Arbitrary units (a.u.) (n = 3, N = 3). Nonspecific uptake was measured using protein-free liposomes (empty), vector containing proteoliposomes (vector), and T1R1 containing proteoliposomes (control). (n = 3, N = 3). One-way analysis of variance test followed by Dunnett’s multiple comparisons test. g Illustration of SPNS2-mediated export of S1P out of cells while transporting glucose inward. Illustrations in panels c and g created by Luciana Giono. h – j SPNS2-mediated D-[3H]glucose uptake. h Time-dependent specific uptake of D-[ 3 H]glucose into hSPNS2-containing proteoliposomes that were loaded without or with S1P (n = 3–5, N = 3). Nonspecific uptake measured using protein-free liposomes (empty) was subtracted from the specific uptake. i Uptake of [ 3 H]glucose by hSPNS2 or empty liposomes at 40 sec (n = 4, N = 3). j Kinetics of D-glucose uptake by hSPNS2. Specific uptake measured at 40 sec was calculated by subtraction of nonspecific [ 3 H]glucose uptake by empty liposomes and fitted to a non-linear regression analysis using Michaelis–Menten enzyme kinetics plot with K M , V max , and k cat values calculated (n = 4, N = 4). Data are means ± s.e.m. of independent experiments. Source data are available for this figure in the Source Data file.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_9034/pmc13129034/pmc13129034__41467_2026_71659_Fig6_HTML.jpg)