Abstract
Sphingosine-1-phosphate (S1P), a potent signalling lipid secreted by red blood cells and platelets1,2, plays numerous biologically significant roles3,4,5,6. However, the identity of its long-sought exporter is enigmatic. Here we show that the major facilitator superfamily transporter 2b (Mfsd2b), an orphan transporter, is essential for S1P export from red blood cells and platelets. Comprehensive lipidomic analysis indicates a dramatic and specific accumulation of S1P species in Mfsd2b knockout red blood cells and platelets compared with that of wild-type controls. Consistently, biochemical assays from knockout red blood cells, platelets, and cell lines overexpressing human and mouse Mfsd2b proteins demonstrate that Mfsd2b actively exports S1P. Plasma S1P level in knockout mice is significantly reduced by 42–54% of that of wild-type level, indicating that Mfsd2b pathway contributes approximately half of the plasma S1P pool. The reduction of plasma S1P in knockout mice is insufficient to cause blood vessel leakiness, but it does render the mice more sensitive to anaphylactic shock. Stress-induced erythropoiesis significantly increased plasma S1P levels and knockout mice were sensitive to these treatments. Surprisingly, knockout mice exhibited haemolysis associated with red blood cell stomatocytes, and the haemolytic phenotype was severely increased with signs of membrane fragility under stress erythropoiesis. We show that S1P secretion by Mfsd2b is critical for red blood cell morphology. Our data reveal an unexpected physiological role of red blood cells in sphingolipid metabolism in circulation. These findings open new avenues for investigating the signalling roles of S1P derived from red blood cells and platelets.
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Acknowledgements
This study was supported in part by the Singapore Ministry of Health’s National Medical Research Council NMRC/BNIG/2023/2014, National University of Singapore (NUS) start-up, NUS Young Investigator Award grants (to L.N.N.), NRF2016NRF-NRFI001-15 (to D.L.S.), NRFI2015-05 and Biomedical Research Council and Science and Engineering Research Council joint grant BMRC-SERC 112 148 0006 (to M.R.W.), and NMRC/CIRG/1377/2013 grant (to S.-A.E.S.T.) We thank Y. J. Wu for technical assistance with scanning electron microscopy and the Osato laboratory for technical assistance with flow cytometry.
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T.M.V. designed and performed all in vivo and ex vivo experiments; preparation of samples for lipidomics; analysed data, and wrote the paper; A.-N.I. performed quantitative PCR of bone marrow cell populations; J.C.F., F.T., A.C.-G. performed lipidomic analysis. X.R.T., D.M.W., F.Z. generation of plasmids; T.M. provided technical advices with irradiation; S.K. performed P50 and deformability experiments; S.-A.E.S.T. provided reagents; T.S. supervised A.-N.I.; D.L.S. provided reagents; M.R.W. supervised J.C.F., F.T., A.C.-G.; L.N.N. conceived and designed the study and experiments, performed in vitro transport assays, in vivo experiments with T.M.V., analysed all data, and wrote the paper.
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Reviewer Information Nature thanks J. Chun, T. Günther-Pomorski, T. Hla, N. Huang and H. Lodish for their contribution to the peer review of this work.
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Extended data figures and tables
Extended Data Figure 1 Mfsd2b is a plasma membrane protein of haematopoietic cells which does not transport LPC.
a, Computational predicted model of hMfsd2b structure (http://zhanglab.ccmb.med.umich.edu/I-TASSER/). Human Mfsd2b was predicted to have common MFS fold with 12 transmembrane domains. Shown are Asp95 (D95) and Thr157 (T157). b, Expression of Mfsd2b was increased after 5FU treatment. Mfsd2b expression was higher in bone marrow and spleen (top) and peripheral young RBCs (bottom) from 5FU-treated animals. c, Mfsd2b protein is present in RBC ghost membranes. d, Limited trypsin digestion of intact RBCs. Mfsd2b protein visibly disappeared after trypsin incubation. Abbreviation ‘ns’ is non-specific protein band. e, Reticulocyte counts from WT and KO mice after phenylhydrazine treatment. The data are related to Fig. 1c (four mice per genotype). f, Transcriptional analysis of mMfsd2b in indicated cell populations isolated from mouse bone marrow. Expression level of mMfds2b was expressed as the ratio to GAPDH. g, Import assays of HEK293 cells overexpressed with empty plasmid (mock), mMfsd2b, mMfsd2b mutant D85A, or hMfsd2b with indicated lipids. h, Lipidomic analysis of major phospholipids in WT and KO RBCs (eight samples per genotype, two experiments). Total levels of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), sphingomyelin (SM), phosphatidylglycerol (PG), phosphatidylinositol (PI), LPC, and lysophosphatidylethanolamine (LPE) were quantified from 4.46 million washed WT and KO RBCs. The amount of lipid species was normalized to internal standards and expressed as relative levels. Data are mean and s.d. (e) or s.e.m. (h).
Extended Data Figure 2 Reduction of S1P levels in HDL and albumin fractions of KO mice.
a, b, S1P levels from HDL and albumin fractions isolated from WT and KO mice. Each symbol represents one mouse (four WT, six KO). c, d, SDS gel and western blot analysis of HDL proteins from two independent isolation batches, respectively. One microlitre of HDL fraction from two representative mice for each isolation batch was diluted in SDS sample buffer and loaded and analysed on SDS gel or western blot probed with anti-ApoA1. The results showed that HDL isolated from batch 2 was not contaminated with albumin. These samples were used for the S1P analysis shown in a and b. ***P < 0.001, two-tailed unpaired t-test. Data are mean and s.d.
Extended Data Figure 3 RBC export assays with NBD-sphingosine and [3-3H]sphingosine and the requirement of acceptors for S1P release in RBC.
a–f, TLC analysis and quantification of NBD-S1P bands isolated from RBCs supernatant and pellets from export assays at indicated BSA concentrations (three biological replicates per genotype, experiments were repeated twice with similar results). g–j, Export of [3-3H]S1P in RBCs using [3-3H]sphingosine as substrate. Fifty million washed WT and KO RBCs were incubated with 5 μM [3-3H]sphingosine in Tyrode-H buffer containing 0.5% BSA. After 2 h of incubation, lipids from medium (g) and cell pellets (h) were extracted and analysed by TLC. i, j, Quantification of S1P bands from g and h, respectively (six biological replicates per genotype). k, S1P levels in WT and KO RBCs without release. WT and KO RBCs were incubated with 2 μM [3-3H]sphingosine (in ethanol) for 1 h without BSA and quantified. l, m, S1P-preloaded RBCs as in k were incubated with Tyrode-H buffer containing 0.5% BSA for 1 h. S1P from supernatant (l) and cell pellets (m) were quantified (three biological replicates per genotype, experiments were repeated three times with similar results). The data showed that S1P release from RBCs is dependent on BSA. n, o, Phospholipid liposomes and 10% fresh milk can be used to capture S1P release from RBCs (three biological replicates per genotype; experiments were repeated twice with similar results). ***P < 0.001. **P < 0.01, two-tailed unpaired t-test. Data are mean and s.d.
Extended Data Figure 4 Postnatal deletion of Mfsd2b in RBCs reduces S1P export.
a, Genotyping of tail tips collected from WT, whole-body KO, and postnatal KO (Mfsd2b f/f) mice with or without Mx1-Cre. ‘Floxed’ denotes conditional alleles, WT denotes wild-type alleles, and KO denotes whole-body KO alleles. This shows that the Mfsd2b gene was still present in DNA samples extracted from the tails of the mice. b, Expression of Mfsd2b in RBCs was analysed by western blot collected from the indicated mice. Mfsd2b protein was barely visible after 3 weeks of treatment with poly(I:C) to induce Mx1-Cre in haematopoietic cells. Abbreviation ‘n.s.’ is non-specific protein band; Ter119 (glycophorin A) and β-actin antibodies were used as loading control. c, d, S1P export activity was strongly inhibited in RBC from Mfsd2b f/f; Mx1-cre mice. NS, not significant. ***P < 0.001, two-tailed unpaired t-test.
Extended Data Figure 5 Mfsd2b KO mice exhibited a dramatic accumulation of S1P in platelets and mild splenomegaly.
a, Total concentrations of S1P in platelets from male WT (seven mice) and KO mice (eight mice) (data were collected from two experiments). b, Platelet count in WT and KO blood (14 pairs, 3 experiments). c, d, Levels of S1P found in WT platelets and RBCs. Data are extracted from a and Fig. 4m for RBCs. e, Organ weights of WT and KO mice at 8 weeks old (five mice per genotype). Note that spleens of KO mice were significantly enlarged (11 mice per genotype for spleen weight). f, S1P export activity of nucleated cells isolated from the indicated tissues. S1P export was slightly increased in KO splenocytes (five biological replicates per genotype). ***P < 0.001, *P < 0.05, two-tailed unpaired t-test. Data are mean and s.d. (a–d) and s.e.m. (e, f).
Extended Data Figure 6 Heterologous expression of Mfsd2b increased S1P export in cell lines and Mfsd2b is an active exporter for S1P in RBCs.
a, Western blot for expression analysis of mouse SphK1, mMfsd2, hMfsd2b, and hMfsd2b mutants D95A, T157R, and K423A in HEK293 cells. Top panel is probed with anti-Mfsd2b antibody, bottom panel is re-probed with anti-mouse SphK1 antibody. We noted that mouse Mfsd2b protein expressed in HEK293 cells were significantly more glycosylated (arrowheads) compared with human Mfsd2b. This might cause the slightly reduced activity of mMfsd2b compared with hMfsd2b in export assays. b, Localization of human Mfsd2b and corresponding mutants in HEK293 cells. c, d, TLC analysis of S1P from supernatant and cells expressed with mSphK1 (mock) or co-expression of mSphK1 with hMfsd2b, D95A, and T157R mutants. e, Quantification of S1P bands in c. f, Time course of export of S1P from hMfsd2b (WT) and hMfsd2b mutants D95A, K423A in HEK293 cells (three biological replicates for c–f, repeated twice). g, h, Export of S1P is not inhibited by a presence of tenfold excess of S1P in the medium; 20 million RBCs and 1 μM [3-3H]sphingosine were used for export assay (three biological replicates per genotype). ***P < 0.001, one-way ANOVA (f) and two-tailed unpaired t-test (e, g, h). Data are mean and s.d.
Extended Data Figure 7 Mfsd2b KO mice had mild reduction of circulatory lymphocytes and were more susceptible to PAF-induced anaphylactic shock.
a, b, Circulatory numbers of T and B cells from 2-month-old male mice, respectively (11 mice per genotype, 2 experiments). c, Response of 3-month-old male WT and KO mice to the treatment of FTY720, a lymphopenia drug (nine WT and eight KO, two experiments). d, Evans blue extravasation examination of WT and KO mice. There was no significant Evans blue extravasation seen in KO mice. e, Survival curve of 2- to 3-month-old male WT and KO mice to a treatment of PAF (20 μg/kg (body weight)). Mfsd2b KO mice were more significantly sensitive to PAF treatment (11 WT and 9 KO, 2 experiments). P values were calculated using two-tailed unpaired t-tests for a–c and a Mantel–Cox test for e. Data are mean and s.e.m.
Extended Data Figure 8 Mfsd2b KO mice exhibited RBC phenotypes.
a, b, Reticulocytes (eight mice per genotype) and stomatocytes (9 WT and 13 KO) from 3- to 4-month-old male WT and KO mice. c, Scanning electron microscopy of RBCs from 7-month-old female WT and KO mice. We noted that stomatocyte phenotypes were progressive with age. d, e, KO RBCs had normal oxygen carrying capacity expressed as P50 values and normal deformability (11 mice per genotype, 2 experiments). f, Knockout mice had a significantly faster turnover rate than wild-type littermates (five mice per genotype). g, KO RBCs had a faster turnover rate in WT circulation (five mice per genotype). Reciprocal transfusion of CFSE-labelled WT or KO RBCs to KO or WT mice was performed. One day later, the mice were injected with 5FU to induce anaemia. CFSE-labelled RBCs were quantified by flow cytometry. h, Peripheral RBCs of five WT and four KO mice were enumerated at day 12 after 5FU treatment. ***P < 0.001, **P < 0.01, *P < 0.05, two-tailed unpaired t-test. Data are mean and s.d.
Extended Data Figure 9 Mfsd2b KO mice exhibited normal erythropoiesis but were sensitive to stress-induced erythropoiesis.
a, Gating of erythroid populations from a representative sample. b, Frequency of erythroid cell populations in bone marrow of WT and KO mice (seven mice per genotype, three experiments). c, Representative cytospin images of flow-cytometry-sorted erythroid populations as shown in a from WT and KO bone (two mice each genotype). I, proerythroblasts; II, basophilic erythroblasts; III, polychromatic erythroblasts; IV, reticulocytes; V, erythrocytes. d, KO mice were severely anaemic with 5FU treatment. Administration of S1P receptor agonist SEW2871 did not ameliorate the phenotype. Age-matched male WT and KO mice were injected with a single dose of 5FU followed by daily oral gavage with SEW2871. Peripheral blood cells were counted (six mice per genotype, two experiments). e, Body weight at day 11 of female WT and KO mice treated and non-treated with 5FU (eight mice per genotype). f, More than 75% of female KO mice died of severe anaemia at day 12 after 5FU treatment (three WT and five KO). Arrowheads indicate the colour of the liver. g, h, Treatment with 5FU resulted in abnormal RBC morphological changes ranging from transient hyperchromasia to stomatocytes and to haemolysis in both male and female KO RBCs (six mice per genotype). Black arrowheads indicate hyperchromasia; red arrowheads indicate stomatocytes. Note that there was a delay of stomatosis in male KO mice and they recovered from a single dose of 5FU treatment. i, Significant reduction of HCT and increase of MCH in male mice treated with 5FU at day 8 (six WT and eight KO). ***P < 0.001, *P < 0.05, two-tailed unpaired t-test. Data are mean and s.e.m. (d). NS, not significant.
Extended Data Figure 10 Increased accumulation of S1P resulted in haemolysis in KO RBCs under 5FU treatment.
a, Sphingolipid levels in WT and KO RBCs before and after 9 days of 5FU treatment. The dihydrosphingosine level was significantly increased in 5FU-treated KO RBCs compared with untreated KO RBCs. Total level of ceramides in KO RBCs at normal condition was slightly increased compared with that of WT RBCs. The level of ceramides and sphingomyelins in 5FU-treated mice was unaltered. b, Sphingolipid levels in WT and KO plasma before and after 9 days of 5FU treatment. Only the ceramide level was slightly increased in KO plasma. Note that the data of ceramides from mice without treatment have been shown in Fig. 2l. c, d, Levels of major S1P species found in WT and KO RBCs without treatment and in the 5FU-treated condition. DhS1P is the second most abundant S1P species found in RBCs at normal condition. This species was specifically increased under 5FU treatment. e, Comparison of S1P species from KO RBCs at normal condition with 5FU treatment (data extracted from c and d). DhS1P (18:0) level was increased to 2.9-fold in KO RBCs under 5FU treatment (six biological replicates per genotype). f, g, Haemolysis of WT and KO RBCs was examined with the indicated concentrations of sphingosine and dihydrosphingosine, respectively. SphK1 inhibitor PF543 was used to inhibit the synthesis of S1P and DhS1P from their respective substrates. The results showed that accumulation of DhS1P is the main cause of haemolysis in KO RBCs (four biological replicates per genotype). ***P < 0.001, **P < 0.01, *P < 0.05, two-tailed unpaired t-test. P values in f and g were calculated from WT and KO RBCs without PF543. Data are mean and s.d. (f, g).
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Vu, T., Ishizu, AN., Foo, J. et al. Mfsd2b is essential for the sphingosine-1-phosphate export in erythrocytes and platelets. Nature 550, 524–528 (2017). https://doi.org/10.1038/nature24053
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DOI: https://doi.org/10.1038/nature24053
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