Orphan Transporter Research Articles

Illuminating the function of the orphan transporter, SLC22A10, in humans and other primates

SLC22A10 is an orphan transporter with unknown substrates and function. The goal of this study is to elucidate its substrate specificity and functional characteristics. In contrast to orthologs from great apes, human SLC22A10, tagged with green fluorescent protein, is not expressed on the plasma membrane. Cells expressing great ape SLC22A10 orthologs exhibit significant accumulation of estradiol-17β-glucuronide, unlike those expressing human SLC22A10. Sequence alignments reveal a proline at position 220 in humans, which is a leucine in great apes. Replacing proline with leucine in SLC22A10-P220L restores plasma membrane localization and uptake function. Neanderthal and Denisovan genomes show proline at position 220, akin to modern humans, indicating functional loss during hominin evolution. Human SLC22A10 is a unitary pseudogene due to a fixed missense mutation, P220, while in great apes, its orthologs transport sex steroid conjugates. Characterizing SLC22A10 across species sheds light on its biological role, influencing organism development and steroid homeostasis.

The effect of genetic variants of SLC22A18 on proliferation, migration, and invasion of colon cancer cells

Solute carrier family (SLC) transporters are expressed in the digestive system and play important roles in maintaining physiological functions in the body. In addition, SLC transporters act as oncoproteins or tumor-suppressor proteins during the development, progression, and metastasis of various digestive system cancers. SLC22A18, a member of the SLC22 gene family, is an orphan transporter with an unknown endogenous substrate. Previous study revealed that SLC22A18 is downregulated in colorectal cancer tissues and that it acts as a suppressor in colorectal cancer, although the effects of SLC22A18 variants on colon cancer cell proliferation, migration, and invasion are unknown. Therefore, in this study, we identified SLC22A18 variants found in multiple populations by searching public databases and determined the in vitro effects of these missense variations on transporter expression and cancer progression. Our results indicated that three missense SLC22A18 variants—p.Ala6Thr, p.Arg12Gln, and p.Arg86His—had significantly lower cell expression than the wild type, possibly owing to intracellular degradation. Furthermore, these three variants caused significantly higher proliferation, migration, and invasion of colon cancer cells than the wild type. Our findings suggest that missense variants of SLC22A18 can potentially serve as biomarkers or prognostic tools that enable clinicians to predict colorectal cancer progression.

MFSD7c functions as a transporter of choline at the blood–brain barrier

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.

Hiat1 as a new transporter involved in ammonia regulation

The orphan transporter hippocampus-abundant transcript 1 (Hiat1) was first identified in the mammalian brain. Its specific substrate specificity, however, has not been investigated to date. Here, we identified and analyzed Hiat1 in a crustacean, the green crab Carcinus maenas. Our phylogenetic analysis showed that Hiat1 protein is conserved at a considerable level between mammals and this invertebrate (ca. 78% identical and conserved amino acids). Functional expression of Carcinus maenas Hiat1 in Xenopus laevis oocytes demonstrated the capability to transport ammonia (likely NH4+) in a sodium-dependent manner. Furthermore, applying quantitative polymerase chain reaction, our results indicated a physiological role for Carcinus maenas Hiat1 in ammonia homeostasis, as mRNA abundance increased in posterior gills in response to elevated circulating hemolymph ammonia upon exposure to high environmental ammonia. Its ubiquitous mRNA expression pattern also suggests an essential role in general cellular detoxification of ammonia. Overall, our results introduce a new ubiquitously expressed ammonia transporter, consequently demanding revision of our understanding of ammonia handling in key model systems from mammalian kidneys to crustacean and fish gills.

Untargeted metabolomics identifies the potential role of monocarboxylate transporter 6 (MCT6/SLC16A5) in lipid and amino acid metabolism pathways.

Monocarboxylate transporter 6 (MCT6; SLC16A5) is an orphan transporter protein with expression in multiple tissues. The endogenous function of MCT6 related to human health and disease remains unknown. Our previous transcriptomic and proteomic analyses in Mct6 knockout (KO) mice suggested that MCT6 may play a role in lipid and glucose homeostasis, but additional evidence is required. Thus, the objective of this study was to further explore the impact of MCT6 on metabolic function using untargeted metabolomic analysis in Mct6 KO mice. The plasma from male and female mice and livers from male mice were submitted for global metabolomics analysis to assess the relative changes in endogenous small molecules across the liver and systemic circulation associated with absence of Mct6. More than 782 compounds were detected with 101 and 51 metabolites significantly changed in plasma of male and female mice, respectively, and 100 metabolites significantly changed in the livers of male mice (p < .05). Significant perturbations in lipid metabolism were annotated in the plasma and liver metabolome, with additional alterations in the amino acid metabolism pathway in plasma samples from male and female mice. Elevated lipid diacylglycerol and altered fatty acid metabolite concentrations were found in liver and plasma samples of male Mct6 KO mice. Significant reduction of N‐terminal acetylated amino acids was found in plasma samples of male and female Mct6 KO mice. In summary, the present study confirmed the significant role of MCT6 in lipid and amino acid homeostasis, suggesting its contribution in metabolic diseases.

Mammalian monocarboxylate transporter 7 (MCT7/Slc16a6) is a novel facilitative taurine transporter

Monocarboxylate transporter 7 (MCT7) is an orphan transporter expressed in the liver, brain, and in several types of cancer cells. It has also been reported to be a survival factor in melanoma and breast cancers. However, this survival mechanism is not yet fully understood due to MCT7’s unidentified substrate(s). Therefore, here we sought to identify MCT7 substrate(s) and characterize the transport mechanisms by analyzing amino acid transport in HEK293T cells and polarized Caco-2 cells. Analysis of amino acids revealed significant rapid reduction in taurine from cells transfected with enhanced green fluorescent protein-tagged MCT7. We found that taurine uptake and efflux by MCT7 was pH-independent and that the uptake was not saturated in the presence of taurine excess of 200 mM. Furthermore, we found that monocarboxylates and acidic amino acids inhibited MCT7-mediated taurine uptake. These results imply that MCT7 may be a low-affinity facilitative taurine transporter. We also found that MCT7 was localized at the basolateral membrane in polarized Caco-2 cells and that the induction of MCT7 expression in polarized Caco-2 cells enhanced taurine permeation. Finally, we demonstrated that interactions of MCT7 with ancillary proteins basigin/CD147 and embigin/GP70 enhanced MCT7-mediated taurine transport. In summary, these findings reveal that taurine is a novel substrate of MCT7 and that MCT7-mediated taurine transport might contribute to the efflux of taurine from cells.

Identification of novel potential interaction partners of UDP-galactose (SLC35A2), UDP-N-acetylglucosamine (SLC35A3) and an orphan (SLC35A4) nucleotide sugar transporters

Nucleotide sugar transporters (NSTs) are ER and Golgi-resident members of the solute carrier 35 (SLC35) family which supply substrates for glycosylation by exchanging lumenal nucleotide monophosphates for cytosolic nucleotide sugars. Defective NSTs have been associated with congenital disorders of glycosylation (CDG), however, molecular basis of many types of CDG remains poorly characterized. To better understand the biology of NSTs, we identified potential interaction partners of UDP-galactose transporter (SLC35A2), UDP-N-acetylglucosamine transporter (SLC35A3) and an orphan nucleotide sugar transporter SLC35A4 of to date unassigned specificity. For this purpose, each of the SLC35A2-A4 proteins was used as a bait in four independent pull-down experiments and the identity of the immunoprecipitated material was discovered using MS techniques. From the candidate list obtained, we selected a few for which the interaction was confirmed in vitro using the NanoBiT system, a split luciferase-based luminescent technique. NSTs have been shown to interact with two ATPases (ATP2A2, ATP2C1), Golgi pH regulator B (GPR89B) and calcium channel (TMCO1), which may reflect the regulation of glycosylation by ion homeostasis, and with basigin (BSG). Our findings provide a starting point for the NST interaction network discovery in order to better understand how glycosylation is regulated and linked to other cellular processes. SignificanceDespite the facts that nucleotide sugar transporters are a key component of the protein glycosylation machinery, and deficiencies in their activity underlie serious metabolic diseases, biology, function and regulation of these essential proteins remain enigmatic. In this study we have advanced the field by identifying sets of new potential interaction partners for UDP-galactose transporter (SLC35A2), UDP-N-acetylglucosamine transporter (SLC35A3) and an orphan transporter SLC35A4 of yet undefined role. Several of these new interactions were additionally confirmed in vitro using the NanoBiT system, a split luciferase complementation assay. This work is also significant in that it addresses the overall challenge of discovering membrane protein interaction partners by a detailed comparison of 4 different co-immunoprecipitation strategies and by custom sample preparation and data processing workflows.

Glutamine Uptake via SNAT6 and Caveolin Regulates Glutamine-Glutamate Cycle.

SLC38A6 (SNAT6) is the only known member of the SLC38 family that is expressed exclusively in the excitatory neurons of the brain. It has been described as an orphan transporter with an unknown substrate profile, therefore very little is known about SNAT6. In this study, we addressed the substrate specificity, mechanisms for internalization of SNAT6, and the regulatory role of SNAT6 with specific insights into the glutamate–glutamine cycle. We used tritium-labeled amino acids in order to demonstrate that SNAT6 is functioning as a glutamine and glutamate transporter. SNAT6 revealed seven predicted transmembrane segments in a homology model and was localized to caveolin rich sites at the plasma membrane. SNAT6 has high degree of specificity for glutamine and glutamate. Presence of these substrates enables formation of SNAT6-caveolin complexes that aids in sodium dependent trafficking of SNAT6 off the plasma membrane. To further understand its mode of action, several potential interacting partners of SNAT6 were identified using bioinformatics. Among them where CTP synthase 2 (CTPs2), phosphate activated glutaminase (Pag), and glutamate metabotropic receptor 2 (Grm2). Co-expression analysis, immunolabeling with co-localization analysis and proximity ligation assays of these three proteins with SNAT6 were performed to investigate possible interactions. SNAT6 can cycle between cytoplasm and plasma membrane depending on availability of substrates and interact with Pag, synaptophysin, CTPs2, and Grm2. Our data suggest a potential role of SNAT6 in glutamine uptake at the pre-synaptic terminal of excitatory neurons. We propose here a mechanistic model of SNAT6 trafficking that once internalized influences the glutamate–glutamine cycle in presence of its potential interacting partners.

ABC Transporter DerAB of Lactobacillus casei Mediates Resistance against Insect-Derived Defensins.

Bce-like systems mediate resistance against antimicrobial peptides in Firmicutes bacteria. Lactobacillus casei BL23 encodes an "orphan" ABC transporter that, based on homology to BceAB-like systems, was proposed to contribute to antimicrobial peptide resistance. A mutant lacking the permease subunit was tested for sensitivity against a collection of peptides derived from bacteria, fungi, insects, and humans. Our results show that the transporter specifically conferred resistance against insect-derived cysteine-stabilized αβ defensins, and it was therefore renamed DerAB for defensin resistance ABC transporter. Surprisingly, cells lacking DerAB showed a marked increase in resistance against the lantibiotic nisin. This could be explained by significantly increased expression of the antimicrobial peptide resistance determinants regulated by the Bce-like systems PsdRSAB (formerly module 09) and ApsRSAB (formerly module 12). Bacterial two-hybrid studies in Escherichia coli showed that DerB could interact with proteins of the sensory complex in the Psd resistance system. We therefore propose that interaction of DerAB with this complex in the cell creates signaling interference and reduces the cell's potential to mount an effective nisin resistance response. In the absence of DerB, this negative interference is relieved, leading to the observed hyperactivation of the Psd module and thus increased resistance to nisin. Our results unravel the function of a previously uncharacterized Bce-like orphan resistance transporter with pleiotropic biological effects on the cell.IMPORTANCE Antimicrobial peptides (AMPs) play an important role in suppressing the growth of microorganisms. They can be produced by bacteria themselves-to inhibit competitors-but are also widely distributed in higher eukaryotes, including insects and mammals, where they form an important component of innate immunity. In low-GC-content Gram-positive bacteria, BceAB-like transporters play a crucial role in AMP resistance but have so far been primarily associated with interbacterial competition. Here, we show that the orphan transporter DerAB from the lactic acid bacterium Lactobacillus casei is crucial for high-level resistance against insect-derived AMPs. It therefore represents an important mechanism for interkingdom defense. Furthermore, our results support a signaling interference from DerAB on the PsdRSAB module that might prevent the activation of a full nisin response. The Bce modules from L. casei BL23 illustrate a biological paradox in which the intrinsic nisin detoxification potential only arises in the absence of a defensin-specific ABC transporter.

Evidence for Urate Uptake Through Monocarboxylate Transporter 9 Expressed in Mammalian Cells and Its Enhancement by Heat Shock.

Background: Monocarboxylate transporter 9 (MCT9), an orphan transporter member of the solute carrier family 16 (SLC16), possibly reabsorbs uric acid in the renal tubule and has been suggested by genome-wide association studies to be involved in the development of hyperuricemia and gout. In this study we investigated the mechanisms regulating the expression of human (h) MCT9, its degradation, and physiological functions.Methods and Results: hMCT9-FLAG was stably expressed in HEK293 cells and its degradation, intracellular localization, and urate uptake activities were assessed by pulse-chase analysis, immunofluorescence, and [14C]-urate uptake experiments, respectively. hMCT9-FLAG was localized on the plasma membrane as well as in the endoplasmic reticulum and Golgi apparatus. The proteasome inhibitors MG132 and lactacystine increased levels of hMCT9-FLAG protein expression with enhanced ubiquitination, prolonged their half-life, and decreased [14C]-urate uptake. [14C]-urate uptake was increased by both heat shock (HS) and the HS protein inducer geranylgeranylacetone (GGA). Both HS and GGA restored the [14C]-urate uptake impaired by MG132.Conclusions: hMCT9 does transport urate and is degraded by a proteasome, inhibition of which reduces hMCT9 expression on the cell membrane and urate uptake. HS enhanced urate uptake through hMCT9.

Deficiency of MFSD7c results in microcephaly-associated vasculopathy in Fowler syndrome.

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.

Molecular characterization of the orphan transporter SLC16A9, an extracellular pH- and Na+-sensitive creatine transporter

Human monocarboxylate transporters (hMCTs) mediate the transport of monocarboxylates across plasma membranes. One such transporter, hMCT9, has been shown to be related to serum uric acid levels and the risk of renal overload gout. However, the functional characteristics of hMCT9 remain unknown. The aim of this study was to investigate the expression and localization of hMCT9 using a Xenopus laevis oocyte heterologous expression system and characterize its transport properties. Kinetic analysis of hMCT9-mediated creatine uptake revealed that uptake consisted of two components, with apparent Km values of 237 mm (low-affinity) and 23.7 mm (high-affinity), respectively. The transport activity of hMCT9 was dependent on the extracellular pH and activity sharply increased with increasing pH. Under Na+-free conditions, hMCT9-mediated creatine uptake was reduced by one-half, indicating that hMCT9 is a Na+-sensitive transporter. Moreover, carbonyl cyanide 3-chlorophenylhydrazone (a protonophore) inhibited hMCT9 activity, whereas valinomycin (a K+-ionophore) did not inhibit the transporter. These results suggest that hMCT9 is susceptible to changes in H+ gradients. A cis-inhibition assay of hMCT9-and hMCT12-mediated creatine transport revealed that cyclocreatine, creatine, guanidineacetate, and 3-guanidinopropionate are recognized by the transporter, and 4-guanidinobutyrate and guanidinoethyl sulfonate selectively inhibited hMCT9 activity. These findings demonstrate that hMCT9 is an extracellular pH- and Na+-sensitive creatine transporter.

Unraveling the functional role of the orphan solute carrier, SLC22A24 in the transport of steroid conjugates through metabolomic and genome-wide association studies.

Variation in steroid hormone levels has wide implications for health and disease. The genes encoding the proteins involved in steroid disposition represent key determinants of interindividual variation in steroid levels and ultimately, their effects. Beginning with metabolomic data from genome-wide association studies (GWAS), we observed that genetic variants in the orphan transporter, SLC22A24 were significantly associated with levels of androsterone glucuronide and etiocholanolone glucuronide (sentinel SNPs p-value <1x10-30). In cells over-expressing human or various mammalian orthologs of SLC22A24, we showed that steroid conjugates and bile acids were substrates of the transporter. Phylogenetic, genomic, and transcriptomic analyses suggested that SLC22A24 has a specialized role in the kidney and appears to function in the reabsorption of organic anions, and in particular, anionic steroids. Phenome-wide analysis showed that functional variants of SLC22A24 are associated with human disease such as cardiovascular diseases and acne, which have been linked to dysregulated steroid metabolism. Collectively, these functional genomic studies reveal a previously uncharacterized protein involved in steroid homeostasis, opening up new possibilities for SLC22A24 as a pharmacological target for regulating steroid levels.

Characterization and Proteomic-Transcriptomic Investigation of Monocarboxylate Transporter 6 Knockout Mice: Evidence of a Potential Role in Glucose and Lipid Metabolism.

Monocarboxylate transporter 6 [(MCT6), SLC16A5] is an orphan transporter with no known endogenous substrates or physiological role. Previous in vitro and in vivo experiments investigated MCT6 substrate/inhibitor specificity in Xenopus laevis oocytes; however, these data remain limited. Transcriptomic changes in the livers of mice undergoing different dieting schemes have suggested that Mct6 plays a role in glucose and lipid metabolism. The objectives of this study were 1) to develop a novel knockout (KO) mouse model (Mct6-/-) using CRISPR/Cas9 technology, 2) to characterize the KO animal model by examining physiological and biochemical parameters, and 3) to understand the physiological role of MCT6 in vivo through global proteomic and liver transcriptomic profiling. mRNA tissue analysis demonstrated knockout of Mct6, which showed greater than 90% knockdown of Mct6 (Slc16a5) gene expression in all major tissues analyzed when normalized to Mct6+/+ mice. Proteomic analyses identified greater than 4000 unique proteins in kidney, liver, and colon tissues, among which 51, 38, and 241 proteins were significantly altered, respectively (for each tissue), between Mct6+/+ and Mct6-/- mice. Additionally, Mct6-/- mice demonstrated significant changes in 199 genes in the liver compared with Mct6+/+ mice. In silico biological pathway analyses revealed significant changes in proteins and genes involved in glucose and lipid metabolism-associated pathways. This study is the first to provide evidence for an association of Mct6 in the regulation of glucose and lipid metabolism. SIGNIFICANCE STATEMENT: This paper focuses on elucidating the innate biological role of an orphan transporter in vivo, which has not been investigated thus far. Using efficient and high-throughput technologies, such as CRISPR/Cas9 gene editing, liquid chromatography-tandem mass spectrometry-based proteomic and RNA-sequencing transcriptomic analyses, our laboratory provides the first existence and characterization of a Mct6 knockout mouse model. The evidence gathered in this paper, as well as other laboratories, support the importance of MCT6 in regulating a variety of glucose and lipid metabolic pathways, which may indicate its significance in metabolic diseases.

Imaging inflammation using an activated macrophage probe with Slc18b1 as the activation-selective gating target

Activated macrophages have the potential to be ideal targets for imaging inflammation. However, probe selectivity over non-activated macrophages and probe delivery to target tissue have been challenging. Here, we report a small molecule probe specific for activated macrophages, called CDg16, and demonstrate its application to visualizing inflammatory atherosclerotic plaques in vivo. Through a systematic transporter screen using a CRISPR activation library, we identify the orphan transporter Slc18b1/SLC18B1 as the gating target of CDg16.

Identification of a Novel Nucleobase-Ascorbate Transporter Family Member in Fish and Amphibians

Nucleobase-Ascorbate Transporter (NAT) family includes ascorbic acid, nucleobases, and uric acid transporters: With broad evolutionary distribution. In vertebrates, four members have been previously recognized, the ascorbate transporters Slc23a1 and Slc3a2, the nucleobase transporter Slc23a4 and an orphan transporter Slc23a3. Using phylogenetic and synteny analysis, we identify a fifth member of the vertebrate slc23 complement (slc23a5), present in neopterygians (gars and teleosts) and amphibians, and clarify the evolutionary relationships between the novel gene and known slc23 genes. Further comparative analysis puts forward uric acid as the preferred substrate for Slc23a5. Gene expression quantification, using available transcriptomic data, suggests kidney and testis as major expression sites in Xenopus tropicalis (western clawed frog) and Danio rerio (zebrafish). Additional expression in brain was detected in D. rerio, while in the Neoteleostei Oryzias latipes (medaka) slc23a5 expression is restricted to the brain. The biological relevance of the retention of an extra transporter in fish and amphibians is discussed.

Expanded genetic screening in Caenorhabditis elegans identifies new regulators and an inhibitory role for NAD+ in axon regeneration.

The mechanisms underlying axon regeneration in mature neurons are relevant to the understanding of normal nervous system maintenance and for developing therapeutic strategies for injury. Here, we report novel pathways in axon regeneration, identified by extending our previous function-based screen using the C. elegans mechanosensory neuron axotomy model. We identify an unexpected role of the nicotinamide adenine dinucleotide (NAD+) synthesizing enzyme, NMAT-2/NMNAT, in axon regeneration. NMAT-2 inhibits axon regrowth via cell-autonomous and non-autonomous mechanisms. NMAT-2 enzymatic activity is required to repress regrowth. Further, we find differential requirements for proteins in membrane contact site, components and regulators of the extracellular matrix, membrane trafficking, microtubule and actin cytoskeleton, the conserved Kelch-domain protein IVNS-1, and the orphan transporter MFSD-6 in axon regrowth. Identification of these new pathways expands our understanding of the molecular basis of axonal injury response and regeneration.

Insights into major facilitator superfamily domain-containing protein-2a (Mfsd2a) in physiology and pathophysiology. What do we know so far?

Major facilitator superfamily domain-containing protein-2a (Mfsd2a) which was considered as an orphan transporter has recently gained attention for its regulatory role in the maintenance of proper functioning of the blood-brain barrier. Besides the major role of Mfsd2a in maintaining the barrier function, increasing evidence has emerged with regard to the contributions of Mfsd2a to various biological processes such as transport, cell fusion, cell cycle, inflammation and regeneration, managing tumor growth, functioning of other organs with barrier functions or responses to injury. The purpose of this article is to review the different roles of Mfsd2a and its involvement in the physiological and pathophysiological processes primarily in the central nervous system and throughout the mammalian body under the lights of the current literature.

Involvement of an Orphan Transporter, SLC22A18, in Cell Growth and Drug Resistance of Human Breast Cancer MCF7 Cells

The SLC22A18 gene, which encodes an orphan transporter, is located at the 11p15.5 imprinted region, an important tumor suppressor gene region. However, the role of SLC22A18 in tumor suppression remains unclear. Here, we investigated the involvement of SLC22A18 in cell growth, invasion, and drug resistance of MCF7 human breast cancer cell line. Western blot analysis indicated that SLC22A18 is predominantly expressed at intracellular organelle membranes. Quantitative proteomics showed that knockdown of SLC22A18 significantly altered the expression of 578 (31.0%) of 1867 proteins identified, including proteins related to malignancy and poor prognosis of breast cancer. SLC22A18 knockdown (1) increased MCF7 cell growth concomitantly with a >7-fold increase of annexin A8 (involved in cell growth and migration; a predictor of poor prognosis), (2) induced spherical morphology of MCF7 cells concomitantly with a nearly 3-fold increase of CD44 (involved in regulation of malignant phenotypes), and (3) increased chemosensitivity to vinca alkaloids concomitantly with a >80% reduction of doublecortin-like kinase 1 (involved in regulation of microtubule polymerization). Our results suggest that SLC22A18 may act as a tumor suppressor by regulating the expression levels of cell growth–related proteins, and vinca alkaloids might show therapeutic efficacy against low-SLC22A18–expressing breast cancer.

Mfsd2b is essential for the sphingosine-1-phosphate export in erythrocytes and platelets.

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.