RESUMEN
AIMS: Circulating levels of sphingosine 1-phosphate (S1P), an HDL-associated ligand for endothelial cell (EC) protective S1P receptor-1 (S1PR1), are reduced in disease states associated with endothelial dysfunction. Yet as S1PR1 has high affinity for S1P and can be activated by ligand-independent mechanisms and EC-autonomous S1P production, it is unclear if relative reductions in circulating S1P impact endothelial function. It is also unclear how EC S1PR1 insufficiency, whether induced by ligand deficiency or by S1PR1-directed immunosuppressive therapy, affects different vascular subsets. METHODS AND RESULTS: We here fine-map the zonation of S1PR1 signalling in the murine blood and lymphatic vasculature, superimpose cell type-specific and relative deficiencies in S1P production to define ligand source- and dose-dependence, and correlate receptor engagement to essential functions. In naïve blood vessels, despite broad expression, EC S1PR1 engagement was restricted to resistance-size arteries, lung capillaries and high-endothelial venules (HEV). Similar zonation was observed for albumin extravasation in EC S1PR1 deficient mice, and brain extravasation was reproduced with arterial EC-selective S1pr1 deletion. In lymphatic EC, S1PR1 engagement was high in collecting vessels and lymph nodes and low in terminal capillaries that drain tissue fluids. While EC S1P production sustained S1PR1 signaling in lymphatics and HEV, hematopoietic cells provided â¼90% of plasma S1P and sustained signaling in resistance arteries and lung capillaries. S1PR1 signaling and endothelial function were both surprisingly sensitive to reductions in plasma S1P with apparent saturation around 50% of normal levels. S1PR1 engagement did not depend on sex or age, but modestly increased in arteries in hypertension and diabetes. Sphingosine kinase (Sphk)-2 deficiency also increased S1PR1 engagement selectively in arteries, which could be attributed to Sphk1-dependent S1P release from perivascular macrophages. CONCLUSIONS: This study highlights vessel subtype-specific S1PR1 functions and mechanisms of engagement and supports the relevance of S1P as circulating biomarker for endothelial function.
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Human feline leukaemia virus subgroup C receptor-related proteins 1 and 2 (FLVCR1 and FLVCR2) are members of the major facilitator superfamily1. Their dysfunction is linked to several clinical disorders, including PCARP, HSAN and Fowler syndrome2-7. Earlier studies concluded that FLVCR1 may function as a haem exporter8-12, whereas FLVCR2 was suggested to act as a haem importer13, yet conclusive biochemical and detailed molecular evidence remained elusive for the function of both transporters14-16. Here, we show that FLVCR1 and FLVCR2 facilitate the transport of choline and ethanolamine across the plasma membrane, using a concentration-driven substrate translocation process. Through structural and computational analyses, we have identified distinct conformational states of FLVCRs and unravelled the coordination chemistry underlying their substrate interactions. Fully conserved tryptophan and tyrosine residues form the binding pocket of both transporters and confer selectivity for choline and ethanolamine through cation-π interactions. Our findings clarify the mechanisms of choline and ethanolamine transport by FLVCR1 and FLVCR2, enhance our comprehension of disease-associated mutations that interfere with these vital processes and shed light on the conformational dynamics of these major facilitator superfamily proteins during the transport cycle.
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Colina , Etanolamina , Proteínas de Transporte de Membrana , Humanos , Sitios de Unión , Transporte Biológico , Cationes/química , Cationes/metabolismo , Membrana Celular/metabolismo , Membrana Celular/química , Colina/metabolismo , Colina/química , Etanolamina/metabolismo , Etanolamina/química , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Modelos Moleculares , Conformación Proteica , Receptores Virales/metabolismo , Receptores Virales/química , Especificidad por Sustrato , Triptófano/metabolismo , Triptófano/química , Tirosina/metabolismo , Tirosina/química , MutaciónRESUMEN
Accumulation of sphingolipids, especially sphingosines, in the lysosomes is a key driver of several lysosomal storage diseases. The transport mechanism for sphingolipids from the lysosome remains unclear. Here, we identified SPNS1, which shares the highest homology to SPNS2, a sphingosine-1-phosphate (S1P) transporter, functions as a transporter for lysolipids from the lysosome. We generated Spns1-KO cells and mice and employed lipidomic and metabolomic approaches to reveal SPNS1 ligand identity. Global KO of Spns1 caused embryonic lethality between E12.5 and E13.5 and an accumulation of sphingosine, lysophosphatidylcholines (LPC), and lysophosphatidylethanolamines (LPE) in the fetal livers. Similarly, metabolomic analysis of livers from postnatal Spns1-KO mice presented an accumulation of sphingosines and lysoglycerophospholipids including LPC and LPE. Subsequently, biochemical assays showed that SPNS1 is required for LPC and sphingosine release from lysosomes. The accumulation of these lysolipids in the lysosomes of Spns1-KO mice affected liver functions and altered the PI3K/AKT signaling pathway. Furthermore, we identified 3 human siblings with a homozygous variant in the SPNS1 gene. These patients suffer from developmental delay, neurological impairment, intellectual disability, and cerebellar hypoplasia. These results reveal a critical role of SPNS1 as a promiscuous lysolipid transporter in the lysosomes and link its physiological functions with lysosomal storage diseases.
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Modelos Animales de Enfermedad , Enfermedades por Almacenamiento Lisosomal , Lisosomas , Ratones Noqueados , Animales , Femenino , Humanos , Masculino , Ratones , Hígado/metabolismo , Lisofosfolípidos/metabolismo , Enfermedades por Almacenamiento Lisosomal/metabolismo , Enfermedades por Almacenamiento Lisosomal/genética , Enfermedades por Almacenamiento Lisosomal/patología , Lisosomas/metabolismo , Esfingolípidos/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismoRESUMEN
Mutations in the orphan transporter MFSD7c (also known as Flvcr2), are linked to Fowler syndrome. Here, we used Mfsd7c knockout (Mfsd7c-/-) mice and cell-based assays to reveal that MFSD7c is a choline transporter at the blood-brain barrier (BBB). We performed comprehensive metabolomics analysis and detected differential changes of metabolites in the brains and livers of Mfsd7c-/-embryos. Particularly, we found that choline-related metabolites were altered in the brains but not in the livers of Mfsd7c-/- embryos. Thus, we hypothesized that MFSD7c regulates the level of choline in the brain. Indeed, expression of human MFSD7c in cells significantly increased choline uptake. Interestingly, we showed that choline uptake by MFSD7c is greatly increased by choline-metabolizing enzymes, leading us to demonstrate that MFSD7c is a facilitative transporter of choline. Furthermore, single-cell patch clamp analysis showed that the import of choline by MFSD7c is electrogenic. Choline transport function of MFSD7c was shown to be conserved in vertebrates, but not in yeasts. We demonstrated that human MFSD7c is a functional ortholog of HNM1, the yeast choline importer. We also showed that several missense mutations identified in patients exhibiting Fowler syndrome had abolished or reduced choline transport activity. Mice lacking Mfsd7c in endothelial cells of the central nervous system suppressed the import of exogenous choline from blood but unexpectedly had increased choline levels in the brain. Stable-isotope tracing study revealed that MFSD7c was required for exporting choline derived from lysophosphatidylcholine in the brain. Collectively, our work identifies MFSD7c as a choline exporter at the BBB and provides a foundation for future work to reveal the disease mechanisms of Fowler syndrome.
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Barrera Hematoencefálica , Células Endoteliales , Síndrome del Ovario Poliquístico , Trastornos Urinarios , Animales , Humanos , Ratones , Transporte Biológico , Encéfalo , ColinaAsunto(s)
Cisplatino , Enfermedades del Sistema Nervioso Periférico , Humanos , Cisplatino/efectos adversos , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Enfermedades del Sistema Nervioso Periférico/tratamiento farmacológico , Enfermedades del Sistema Nervioso Periférico/prevención & control , Transducción de SeñalRESUMEN
MFSD7b belongs to the Major Facilitator Superfamily of transporters that transport small molecules. Two isoforms of MFSD7b have been identified and they are reported to be heme exporters that play a crucial role in maintaining the cytosolic and mitochondrial heme levels, respectively. Mutations of MFSD7b (also known as FLVCR1) have been linked to retinitis pigmentosa, posterior column ataxia, and hereditary sensory and autonomic neuropathy. Although MFSD7b functions have been linked to heme detoxification by exporting excess heme from erythroid cells, it is ubiquitously expressed with a high level in the kidney, gastrointestinal tract, lungs, liver, and brain. Here, we showed that MFSD7b functions as a facilitative choline transporter. Expression of MFSD7b slightly but significantly increased choline import, while its knockdown reduced choline influx in mammalian cells. The influx of choline transported by MFSD7b is dependent on the expression of choline metabolizing enzymes such as choline kinase (CHKA) and intracellular choline levels, but it is independent of gradient of cations. Additionally, we showed that choline transport function of Mfsd7b is conserved from fly to man. Employing our transport assays, we showed that missense mutations of MFSD7b caused reduced choline transport functions. Our results show that MFSD7b functions as a facilitative choline transporter in mammalian cells.
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Colina , Proteínas de Transporte de Membrana , Mutación Missense , Animales , Humanos , Colina/metabolismo , Hemo , Mamíferos , Proteínas de Transporte de Membrana/genéticaRESUMEN
Protein Spinster homolog 2 (Spns2) is a sphingosine-1-phosphate (S1P) transporter that releases S1P to regulate lymphocyte egress and trafficking. Global deletion of Spns2 (Spns2-/-) has been shown to reduce disease severity in several autoimmune disease models. To examine whether Spns2 could be exploited as a drug target, we generated and characterized the mice with postnatal knockout of Spns2 (Spns2-Mx1Cre). Our results showed that Spns2-Mx1Cre mice had significantly low number of lymphocytes in blood and lymphoid organs similar to Spns2-/- mice. Lymph but not plasma S1P levels were significantly reduced in both groups of knockout mice. Our lipidomic results also showed that Spns2 releases different S1P species into lymph. Interestingly, lymphatic vessels in the lymph nodes (LNs) of Spns2-/- and Spns2-Mx1Cre mice exhibited morphological defects. The structures of high endothelial venules (HEV) in the LNs of Spns2-Mx1Cre mice were disorganized. These results indicate that lack of Spns2 affects both S1P secretion and LN vasculatures. Nevertheless, blood vasculature of these Spns2 deficient mice was not different to controls under homeostasis and vascular insults. Importantly, Spns2-Mx1Cre mice were resistant to multiple sclerosis in experimental autoimmune encephalomyelitis (EAE) models with significant reduction of pathogenic Th17 cells in the central nervous system (CNS). This study suggests that pharmacological inhibition of Spns2 may be exploited for therapeutic applications in treatment of neuroinflammation.
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Lisofosfolípidos , Esfingosina , Animales , Proteínas de Transporte de Anión/metabolismo , Linfocitos/metabolismo , Lisofosfolípidos/metabolismo , Ratones , Ratones Noqueados , Enfermedades Neuroinflamatorias , Esfingosina/metabolismoRESUMEN
Sphingosine-1-phosphate (S1P) is a potent lipid mediator that is secreted by several cell types. We recently showed that Mfsd2b is an S1P transporter from hematopoietic cells that contributes approximately 50% plasma S1P. Here we report the characterization of compound deletion of Mfsd2b and Spns2, another S1P transporter active primarily in endothelial cells. Global deletion of Mfsd2b and Spns2 (global double knockout [gDKO]) results in embryonic lethality beyond embryonic day 14.5 (E14.5), with severe hemorrhage accompanied by defects of tight junction proteins, indicating that Mfsd2b and Spns2 provide S1P for signaling, which is essential for blood vessel integrity. Compound postnatal deletion of Mfsd2b and Spns2 using Mx1Cre (ctDKO-Mx1Cre) results in maximal 80% reduction of plasma S1P. ctDKO-Mx1Cre mice exhibit severe susceptibility to anaphylaxis, indicating that S1P from Mfsd2b and Spns2 is indispensable for vascular homeostasis. Our results show that S1P export from Mfsd2b and Spns2 is essential for developing and mature vasculature.
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Anafilaxia , Proteínas de la Membrana/metabolismo , Anafilaxia/metabolismo , Animales , Proteínas de Transporte de Anión/metabolismo , Transporte Biológico , Células Endoteliales/metabolismo , Homeostasis , Lisofosfolípidos/metabolismo , Ratones , Esfingosina/metabolismoRESUMEN
Methamphetamine (METH) is a major public health and safety problem in the United States. Chronic METH abuse is associated with a 2-fold-higher risk of HIV infection and, possibly, additional infections, particularly those that enter through the respiratory tract or skin. Cryptococcus neoformans is an encapsulated opportunistic yeast-like fungus that is a relatively frequent cause of meningoencephalitis in immunocompromised patients, especially in individuals with AIDS. C. neoformans melanizes during mammalian infection in a process that presumably uses host-supplied compounds such as catecholamines. l-3,4-Dihydroxyphenylalanine (l-Dopa) is a natural catecholamine that is frequently used to induce melanization in C. neoformans. l-Dopa-melanized cryptococci manifest resistance to radiation, phagocytosis, detergents, and heavy metals. Using a systemic mouse model of infection and in vitro assays to critically assess the impact of METH on C. neoformans melanization and pathogenesis, we demonstrated that METH-treated mice infected with melanized yeast cells showed increased fungal burdens in the blood and brain, exacerbating mortality. Interestingly, analyses of cultures of METH-exposed cryptococci supplemented with l-Dopa revealed that METH accelerates fungal melanization, an event of adaptation to external stimuli that can be advantageous to the fungus during pathogenesis. Our findings provide novel evidence of the impact of METH abuse on host homeostasis and increased permissiveness to opportunistic microorganisms.
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Criptococosis , Cryptococcus neoformans , Infecciones por VIH , Metanfetamina , Sepsis , Animales , Antifúngicos/farmacología , Antifúngicos/uso terapéutico , Criptococosis/microbiología , Modelos Animales de Enfermedad , Humanos , Levodopa/farmacología , Levodopa/uso terapéutico , Mamíferos , Melaninas , Metanfetamina/farmacología , Ratones , Saccharomyces cerevisiaeRESUMEN
The brain exchanges nutrients and small molecules with blood via the blood-brain barrier (BBB). Approximately 20% energy intake for the body is consumed by the brain. Glucose is known for its critical roles for energy production and provides substrates for biogenesis in neurons. The brain takes up glucose via glucose transporters GLUT1 and 3, which are expressed in several neural cell types. The brain is also equipped with various transport systems for acquiring amino acids, lactate, ketone bodies, lipids, and cofactors for neuronal functions. Unraveling the mechanisms by which the brain takes up and metabolizes these nutrients will be key in understanding the nutritional requirements in the brain. This could also offer opportunities for therapeutic interventions in several neurological disorders. For instance, emerging evidence suggests a critical role of lactate as an alternative energy source for neurons. Neuronal cells express monocarboxylic transporters to acquire lactate. As such, treatment of GLUT1-deficient patients with ketogenic diets to provide the brain with alternative sources of energy has been shown to improve the health of the patients. Many transporters are present in the brain, but only a small number has been characterized. In this review, we will discuss about the roles of solute carrier (SLC) transporters at the blood brain barrier (BBB) and neural cells, in transport of nutrients and metabolites in the brain.
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Encefalopatías/metabolismo , Encéfalo/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Animales , Astrocitos/metabolismo , Barrera Hematoencefálica/metabolismo , Humanos , Ácido Láctico/metabolismoRESUMEN
We recently discovered that Mfsd2b, which is the S1P exporter found in blood cells. Here, we report that Mfsd2b is critical for the release of all S1P species in both resting and activated platelets. We show that resting platelets store S1P in the cytoplasm. After activation, this S1P pool is delivered to the plasma membrane, where Mfsd2b is predominantly localized for export. Employing knockout mice of Mfsd2b, we reveal that platelets contribute a minor amount of plasma S1P. Nevertheless, Mfsd2b deletion in whole body or platelets impairs platelet morphology and functions. In particular, Mfsd2b knockout mice show significantly reduced thrombus formation. We show that loss of Mfsd2b affects intrinsic platelet functions as part of remarkable sphingolipid accumulation. These findings indicate that accumulation of sphingolipids including S1P by deletion of Mfsd2b strongly impairs platelet functions, which suggests that the transporter may be a target for the prevention of thrombotic disorders.
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Plaquetas/metabolismo , Lisofosfolípidos/metabolismo , Proteínas de la Membrana/metabolismo , Esfingosina/análogos & derivados , Trombosis de la Vena/patología , Animales , Plaquetas/citología , Plaquetas/efectos de los fármacos , Citoplasma/metabolismo , Modelos Animales de Enfermedad , Fibrinolíticos/farmacología , Fibrinolíticos/uso terapéutico , Humanos , Masculino , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Pruebas de Función Plaquetaria , Esfingosina/metabolismo , Trombosis de la Vena/sangre , Trombosis de la Vena/diagnóstico , Trombosis de la Vena/tratamiento farmacológicoRESUMEN
Sphingosine-1-phosphate (S1P) is a potent lipid mediator that exerts its activity via activation of five different G protein-coupled receptors, designated as S1P1-5. This potent lipid mediator is synthesized from the sphingosine precursor by two sphingosine kinases (SphK1 and 2) and must be exported to exert extracellular signaling functions. We recently identified Mfsd2b as the S1P transporter in the hematopoietic system. However, the sources of sphingosine for S1P synthesis and the transport mechanism of Mfsd2b in erythrocytes remain to be determined. Here, we show that erythrocytes efficiently take up exogenous sphingosine and that a de novo synthesis pathway in part provides sphingosines to erythrocytes. The uptake of sphingosine in erythrocytes is facilitated by the activity of SphK1. By converting sphingosine into S1P, SphK1 indirectly increases the influx of sphingosine, a process that is irreversible in erythrocytes. Our results explain for the abnormally high amount of sphingosine accumulation in Mfsd2b knockout erythrocytes. Furthermore, we show that Mfsd2b utilizes a proton gradient to facilitate the release of S1P. The negatively charged residues D95 and T157 are essential for Mfsd2b transport activity. Of interest, we also discovered an S1P analog that inhibits S1P export from erythrocytes, providing evidence that sphingosine analogs can be used to inhibit S1P export by Mfsd2b. Collectively, our results highlight that erythrocytes are efficient in sphingosine uptake for S1P production and the release of S1P is dependent on Mfsd2b functions.
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Eritrocitos/metabolismo , Lisofosfolípidos/metabolismo , Proteínas de la Membrana/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Animales , Transporte Biológico , Vías Biosintéticas , Ratones , Ratones Endogámicos C57BL , Modelos MolecularesRESUMEN
Lipids are abundant and play essential roles in human health and disease. The main functions of lipids are building blocks for membrane biogenesis. However, lipids are also metabolized to produce signaling molecules. Here, we discuss the emerging roles of circulating lysophospholipids. These lysophospholipids consist of lysoglycerophospholipids and lysosphingolipids. They are both present in cells at low concentration, but their concentrations in extracellular fluids are significantly higher. The biological functions of some of these lysophospholipids have been recently revealed. Remarkably, some of the lysophospholipids play pivotal signaling roles as well as being precursors for membrane biogenesis. Revealing how circulating lysophospholipids are produced, released, transported, and utilized in multi-organ systems is critical to understand their functions. The discovery of enzymes, carriers, transporters, and membrane receptors for these lysophospholipids has shed light on their physiological significance. In this review, we summarize the biological roles of these lysophospholipids via discussing about the proteins regulating their functions. We also discuss about their potential impacts to human health and diseases.
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Proteínas Portadoras/metabolismo , Lisofosfolípidos/fisiología , Animales , Biomarcadores/análisis , Biomarcadores/metabolismo , Proteínas Portadoras/genética , Humanos , Lisofosfolípidos/química , Transducción de SeñalRESUMEN
Several missense mutations in the orphan transporter FLVCR2 have been reported in Fowler syndrome. Affected subjects exhibit signs of severe neurological defects. We identified the mouse ortholog Mfsd7c as a gene expressed in the blood-brain barrier. Here, we report the characterizations of Mfsd7c-KO mice and compare these characterizations to phenotypic findings in humans with biallelic FLVCR2 mutations. Global KO of Mfsd7c in mice resulted in late-gestation lethality, likely due to CNS phenotypes. We found that the angiogenic growth of CNS blood vessels in the brain of Mfsd7c-KO embryos was inhibited in cortical ventricular zones and ganglionic eminences. Vascular tips were dilated and fused, resulting in glomeruloid vessels. Nonetheless, CNS blood vessels were intact, without hemorrhage. Both embryos and humans with biallelic FLVCR2 mutations exhibited reduced cerebral cortical layers, enlargement of the cerebral ventricles, and microcephaly. Transcriptomic analysis of Mfsd7cK-KO embryonic brains revealed upregulation of genes involved in glycolysis and angiogenesis. The Mfsd7c-KO brain exhibited hypoxia and neuronal cell death. Our results indicate that MFSD7c is required for the normal growth of CNS blood vessels and that ablation of this gene results in microcephaly-associated vasculopathy in mice and humans.
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Barrera Hematoencefálica , Corteza Cerebral , Regulación del Desarrollo de la Expresión Génica , Proteínas de la Membrana/deficiencia , Microcefalia , Neovascularización Fisiológica/genética , Animales , Barrera Hematoencefálica/embriología , Barrera Hematoencefálica/patología , Corteza Cerebral/irrigación sanguínea , Corteza Cerebral/embriología , Corteza Cerebral/patología , Modelos Animales de Enfermedad , Glucólisis/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Microcefalia/embriología , Microcefalia/genética , Microcefalia/patología , SíndromeRESUMEN
Sphingosine-1-phosphate (S1P) is an essential, bioactive lysophospholipid mediator that regulates various physiological functions such as lymphocyte trafficking, inflammation and behavioural characteristics of the vascular system. S1P signalling is mediated via a family of five GPCRs, which are expressed in various cell types and tissues. S1P concentration is maintained in a gradient through the activity of S1P degrading enzymes, and this gradient is critical for lymphocyte egress. To exert its extracellular signalling roles, S1P must be secreted out of the cells by protein transporters. The recent discovery of S1P transporters has shed light on the sources of S1P. However, these transporters still need to be clarified as they are important in defining the S1P gradient for lymphocyte recirculation and the source of S1P for maintenance of blood vessels. Here, we review the current understanding of S1P sources, highlighting the roles of S1P transporters with an emphasis on haematopoietic cells as a major source of circulatory S1P.
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Plaquetas/metabolismo , Eritrocitos/metabolismo , Lisofosfolípidos/metabolismo , Transducción de Señal , Esfingosina/análogos & derivados , Animales , Humanos , Esfingosina/metabolismoRESUMEN
Sphingosine-1-phosphate (S1P), a potent signalling lipid secreted by red blood cells and platelets, plays numerous biologically significant roles. 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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Plaquetas/metabolismo , Eritrocitos/metabolismo , Lisofosfolípidos/metabolismo , Proteínas de la Membrana/metabolismo , Esfingosina/análogos & derivados , Anemia/genética , Anemia/metabolismo , Animales , Transporte Biológico , Forma de la Célula , Recuento de Eritrocitos , Eritrocitos/citología , Eliminación de Gen , Células HEK293 , Humanos , Lisofosfolípidos/sangre , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal , Esfingosina/sangre , Esfingosina/metabolismoRESUMEN
Eye photoreceptor membrane discs in outer rod segments are highly enriched in the visual pigment rhodopsin and the ω-3 fatty acid docosahexaenoic acid (DHA). The eye acquires DHA from blood, but transporters for DHA uptake across the blood-retinal barrier or retinal pigment epithelium have not been identified. Mfsd2a is a newly described sodium-dependent lysophosphatidylcholine (LPC) symporter expressed at the blood-brain barrier that transports LPCs containing DHA and other long-chain fatty acids. LPC transport via Mfsd2a has been shown to be necessary for human brain growth. Here we demonstrate that Mfsd2a is highly expressed in retinal pigment epithelium in embryonic eye, before the development of photoreceptors, and is the primary site of Mfsd2a expression in the eye. Eyes from whole body Mfsd2a-deficient (KO) mice, but not endothelium-specific Mfsd2a-deficient mice, were DHA-deficient and had significantly reduced LPC/DHA transport in vivo Fluorescein angiography indicated normal blood-retinal barrier function. Histological and electron microscopic analysis indicated that Mfsd2a KO mice exhibited a specific reduction in outer rod segment length, disorganized outer rod segment discs, and mislocalization of and reduction in rhodopsin early in postnatal development without loss of photoreceptors. Minor photoreceptor cell loss occurred in adult Mfsd2a KO mice, but electroretinography indicated visual function was normal. The developing eyes of Mfsd2a KO mice had activated microglia and up-regulation of lipogenic and cholesterogenic genes, likely adaptations to loss of LPC transport. These findings identify LPC transport via Mfsd2a as an important pathway for DHA uptake in eye and for development of photoreceptor membrane discs.
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Ácidos Docosahexaenoicos/metabolismo , Lisofosfatidilcolinas/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Células Fotorreceptoras/metabolismo , Angiografía , Animales , Transporte Biológico Activo/fisiología , Ácidos Docosahexaenoicos/genética , Lisofosfatidilcolinas/genética , Proteínas de Transporte de Membrana/genética , Ratones , Ratones Noqueados , Microglía/metabolismo , Imagen Óptica , Simportadores , Regulación hacia ArribaRESUMEN
Major facilitator superfamily domain containing 2A (MFSD2A) was recently characterized as a sodium-dependent lysophosphatidylcholine transporter expressed at the blood-brain barrier endothelium. It is the primary route for importation of docosohexaenoic acid and other long-chain fatty acids into fetal and adult brain and is essential for mouse and human brain growth and function. Remarkably, MFSD2A is the first identified major facilitator superfamily member that uniquely transports lipids, implying that MFSD2A harbors unique structural features and transport mechanism. Here, we present three three-dimensional structural models of human MFSD2A derived by homology modeling using MelB- and LacY-based crystal structures and refined by biochemical analysis. All models revealed 12 transmembrane helices and connecting loops and represented the partially outward-open, outward-partially occluded, and inward-open states of the transport cycle. In addition to a conserved sodium-binding site, three unique structural features were identified as follows: a phosphate headgroup binding site, a hydrophobic cleft to accommodate a hydrophobic hydrocarbon tail, and three sets of ionic locks that stabilize the outward-open conformation. Ligand docking studies and biochemical assays identified Lys-436 as a key residue for transport. It is seen forming a salt bridge with the negative charge on the phosphate headgroup. Importantly, MFSD2A transported structurally related acylcarnitines but not a lysolipid without a negative charge, demonstrating the necessity of a negatively charged headgroup interaction with Lys-436 for transport. These findings support a novel transport mechanism by which lysophosphatidylcholines are "flipped" within the transporter cavity by pivoting about Lys-436 leading to net transport from the outer to the inner leaflet of the plasma membrane.