Functional properties of transfected human DMT1 iron transporter

Recently, mutation of the DMT1 gene has been discovered to cause ineffective intestinal iron uptake and abnormal body iron metabolism in the anemic Belgrade rat and mk mouse. DMT1 transports first-series transition metals, but only iron turns on an inward proton current. The process of iron transport was studied by transfection of human DMT1 into the COS-7 cell line. Native and epitope-tagged human DMT1 led to increased iron uptake. The human gene with the Belgrade rat mutation was found to have one-fifth of the activity of the wild-type protein. The pH optimum of human DMT1 iron uptake was 6.75, which is equivalent to the pH of the duodenal brush border. The transporter demonstrates uptake without saturation from 0 to 50 microM iron, recapitulating earlier studies of isolated intestinal enterocytes. Diethylpyrocarbonate inhibition of iron uptake in DMT1-transfected cells suggests a functional role for histidine residues. Finally, a model is presented that incorporates the selectivity of the DMT1 transporter for transition metals and a potential role for the inward proton current.     

Regulation of reticuloendothelial iron transporter MTP1 (Slc11a3) by inflammation

Acute and chronic inflammation cause many changes in total body iron metabolism including the sequestration of iron in phagocytic cells of the reticuloendothelial system. This change in iron metabolism contributes to the development of the anemia of inflammation. MTP1, the duodenal enterocyte basolateral iron exporter, is also expressed in the cells of the reticuloendothelial system (RES) and is likely to be involved in iron recycling of these cells. In this study, we use a lipopolysaccharide model of the acute inflammation in the mouse and demonstrate that MTP1 expression in RES cells of the spleen, liver, and bone marrow is down-regulated by inflammation. The down-regulation of splenic expression of MTP1 by inflammation was also observed in a Leishmania donovani model of chronic infection. The response of MTP1 to lipopolysaccharide (LPS) requires signaling through the LPS receptor, Toll-like receptor 4 (TLR4). In mice lacking TLR4, MTP1 expression is not altered in response to LPS. In addition, mice lacking tumor necrosis factor-receptor 1a respond appropriately to LPS with down-regulation of MTP1, despite hyporesponsiveness to tumor necrosis factor-alpha signaling, suggesting that this cytokine may not be required for the LPS effect. We hypothesize that the iron sequestration in the RES system that accompanies inflammation is because of down-regulation of MTP1.     

Carboxyl-terminus determinants of the iron transporter DMT1/SLC11A2 isoform II (-IRE/1B) mediate internalization from the plasma membrane into recycling endosomes

Mutations in DMT1 (Nramp2 and Slc11a2) impair iron metabolism and cause microcytic anemia. DMT1 is expressed at the duodenal brush border where it controls uptake of dietary iron and is present at the plasma membrane and in recycling endosomes of most cells, where it is necessary for acquisition of transferrin-associated iron. The goal of this study was to identify signal(s) in the cytoplasmic segments of DMT1 responsible for its subcellular targeting and internalization from the plasma membrane into recycling endosomes. We introduced mutations in the amino terminus (DeltaNT), carboxyl terminus (DeltaCT), as well as in NPAY28-31, YSCF62-65, and YLLNT555-559 motifs of a DMT1 construct bearing an exofacial epitope tag, which allowed labeling of the transporter at the cell surface for kinetic studies. Mutants were stably expressed in LLC-PK1 kidney cells and were studied for transport activity, subcellular localization, cell-surface and recycling pool distribution, and internalization from the plasma membrane. Kinetic studies showed that carboxyl-terminus mutants (DeltaCT and DeltaYLLNT) had an increased fraction of the "recycling pool" that was expressed at the cell surface because of impaired internalization from the plasma membrane. Further cell-surface-labeling and immunofluorescence studies in intact cells showed that the DeltaYLLNT and DeltaCT mutants were targeted to the lysosomal compartment upon internalization. These results suggest that the major signal for internalization and recycling of DMT1 isoform II (-IRE/1B) resides in its carboxyl terminus and that removal of this signal leads to a default lysosomal targeting.     

Dynamic traffic through the recycling compartment couples the metal transporter Nramp2 (DMT1) with the transferrin receptor

Nramp2 (natural resistance-associated macrophage protein 2, also called DMT1 and Slc11a2) is a proton-dependent cation transporter, which plays a central role in iron homeostasis. To study the subcellular distribution and dynamics of the transporter, we generated a construct encoding the long splice variant of Nramp2 (isoform II) tagged with the hemagglutinin epitope on a predicted extracellular loop. Cells stably transfected with this construct revealed the presence of Nramp2 in both the plasma membrane and in an endomembrane compartment. By labeling the exofacial epitope with a pH-sensitive fluorescent indicator, we were able to establish that this variant of Nramp2 resides in a vesicular compartment with an acidic lumen (pH 6.2) and that acidification was maintained by vacuolar-type ATPases. Dual labeling experiments identified this compartment as sorting and recycling endosomes. Kinetic studies by surface labeling with 125I-labeled antibodies established that the fraction of endomembrane Nramp2 was approximately equal to that on the cell surface. The two components are in dynamic equilibrium: surface transporters are internalized continuously via a clathrin and dynamin-dependent process, whereas endosomal Nramp2 is recycled to the plasmalemma by a phosphatidylinositol 3-kinase-dependent exocytic process. Depletion of cholesterol had no discernible effect on Nramp2 internalization, suggesting that rafts or caveolae are not essential. Because the pH at the cell surface and in endosomes differs by >or=1 unit, the rates of transport of Nramp2 at the surface and in endomembrane compartments will differ drastically. Their subcellular colocalization and parallel trafficking suggest that Nramp2 and transferrin receptors are functionally coupled to effect pH-dependent iron uptake across the endosomal membrane.     

Two isoforms of a divalent metal transporter (DMT1) in Schistosoma mansoni suggest a surface-associated pathway for iron absorption in schistosomes

We describe two homologues of the mammalian divalent metal transporter (DMT1) for Schistosoma mansoni, a pathogenic intravascular parasite of humans. Schistosomes have a high nutritional and metabolic demand for iron. Nucleotide sequences of the parasite homologues, designated SmDMT1A and -B, are identical in all but the 5'-regions. The predicted amino acid sequences share at least 60% identity with DMT1 (=Nramp2) of humans, mice, and rats, and at least 55% identity with Nramp1 from mice, humans and Caenorhabditis elegans. SmDMT1A is expressed in differentiating eggs, miracidia, cercariae, schistosomula, and adults, whereas SmDMT1B is expressed in all but the miracidium and occurs at lower levels than SmDMT1A in differentiating eggs and cercariae. An iron-responsive element, present at the 3'-untranslated region of many DMT1 molecules, is not present in schistosome mRNAs studied here. A Western blot analysis of adult worm preparations using a homologous rabbit serum raised against a schistosome DMT1 peptide and a heterologous serum raised against mammalian DMT1, revealed a band approximating 115 kDa. By immunofluorescence microscopy, the schistosome DMT1s localize primarily to the tegument. Iron uptake assays demonstrated that SmDMT1s were able to rescue yeast growth in ferrous iron-transport deficient yeast (fet3fet4). The results suggest that schistosomes express molecules for ferrous iron transport in their tegument, suggesting trans-tegumental transport as one means of iron acquisition for these parasites.     

Cellular localization and developmental changes of the different isoforms of divalent metal transporter 1 (DMT1) in the inner ear of rats

Divalent metal transporter 1 (DMT1) is generally considered to be the major transmembrane protein responsible for the uptake of a variety of divalent cations. Four isoforms of DMT1 have been identified in mammalian cells encoded by a single gene that differ both in their N- and C-terminal sequences with two mRNA isoforms possessing an iron response element (IRE) motif downstream from the stop codon on the message. Two distinct promoter sites regulate production of the 1A or 1B isoforms (translation starts at exon 2) for both the +IRE or –IRE species of the transporter resulting in the generation of four distinct configurations of this protein. Prior studies from our laboratory using cochlear organotypic cultures isolated from postnatal day three rats (P3) have demonstrated that Mn causes significant and selective damage to sensory hair cells and auditory nerve fibers and spiral ganglion neurons in a time and concentration dependent manner. Since DMT1 plays a critical role in controlling the uptake of a variety of essential and toxic metals into the cochlea, we compared the distribution and developmental changes of the 1A, +IRE and ?IRE isoforms in rat inner ear. Results reveal that all three isoforms of DMT1 are selectively expressed in different cell populations within the cochlea and, additionally, demonstrate their cellular and subcellular distribution changes with development.     

The natural resistance-associated macrophage protein 1 Slc11a1 (formerly Nramp1) and iron metabolism in macrophages

Slc11a1 (solute carrier family 11 member 1) (formerly Nramp1) modulation of iron metabolism in macrophages plays an important role in early phase macrophage activation, and therefore host innate immunity. This review focuses on the role of Nramp1 in intramacrophage iron metabolism, with emphasis on the two prevailing mechanisms of Nramp1 modulation of iron metabolism in macrophages.     

Mitochondria represent another locale for the divalent metal transporter 1 (DMT1)

The divalent metal transporter (DMT1) is well known for its roles in duodenal iron absorption across the apical enterocyte membrane, in iron efflux from the endosome during transferrin-dependent cellular iron acquisition, as well as in uptake of non-transferrin bound iron in many cells. Recently, using multiple approaches, we have obtained evidence that the mitochondrial outer membrane is another subcellular locale of DMT1 expression. While iron is of vital importance for mitochondrial energy metabolism, its delivery is likely to be tightly controlled due to iron's damaging redox properties. Here we provide additional support for a role of DMT1 in mitochondrial iron acquisition by immunofluorescence colocalization with mitochondrial markers in cells and isolated mitochondria, as well as flow cytometric quantification of DMT1-positive mitochondria from an inducible expression system. Physiological consequences of mitochondrial DMT1 expression are discussed also in consideration of other DMT1 substrates, such as manganese, relevant to mitochondrial antioxidant defense.     

Human NRAMP2/DMT1, which mediates iron transport across endosomal membranes, is localized to late endosomes and lysosomes in HEp-2 cells

NRAMP2 (natural resistance-associated macrophage protein 2)/DMT1 (divalent metal transporter 1) is a divalent metal transporter conserved from prokaryotes to higher eukaryotes that exhibits an unusually broad substrate range, including Fe(2+), Zn(2+), Mn(2+), Cu(2+), Cd(2+), Co(2+), Ni(2+), and Pb(2+), and mediates active proton-coupled transport. Recently, it has been shown that the microcytic anemia (mk) mouse and the Belgrade (b) rat, which have inherited defects in iron transport that result in iron deficiency anemia, have the same missense mutation (G185R) in Nramp2. These findings strongly suggested that NRAMP2 is the apical membrane iron transporter in intestinal epithelial cells and the endosomal iron transporter in transferrin cycle endosomes of other cells. To investigate the cellular functions of NRAMP2, we generated a polyclonal antibody against the N-terminal cytoplasmic domain of human NRAMP2. The affinity-purified anti-NRAMP2 N-terminal antibody recognized a 90-116-kDa membrane-associated protein, and this band was shifted to 50 kDa by deglycosylation with peptide N-glycosidase F. Subcellular fractionation revealed that NRAMP2 co-sedimented with the late endosomal and lysosomal membrane proteins and LAMP-1 (lysosome-associated membrane protein 1), but not with the transferrin receptor in early endosomes. The intracellular localization of endogenous NRAMP2 and recombinant green fluorescent protein (GFP)-NRAMP2 was examined by immunofluorescence staining and by native fluorescence of GFP, respectively. Both endogenous and GFP-NRAMP2 were detected in vesicular structures and were colocalized with LAMP-2, but not with EEA1 (early endosome antigen 1) or the transferrin receptor. These results indicated that NRAMP2 is localized to the late endosomes and lysosomes, where NRAMP2 may function to transfer the endosomal free Fe(2+) into the cytoplasm in the transferrin cycle.     

The significance of the mutated divalent metal transporter (DMT1) on iron transport into the Belgrade rat brain

Brain iron transport and distributional pattern of divalent metal transporter I (DMT1) were studied in homozygous Belgrade rats (b/b) which suffer from a mutation in the DMT1 gene. In adult rats, brain uptake of transferrin-bound iron injected intravenously (i.v.) was significantly lower compared with that in heterozygous Belgrade (+/b) and Wistar rats, whereas transferrin uptake was identical. The difference in iron uptake was not apparent until 30 min after injection. The brain iron concentration was lower, and neuronal transferrin receptor-immunoreactivity higher, in adult b/b rats, thus confirming their iron-deficient stage. Antibodies targeting different sites on the DMT1 molecule consistently detected DMT1 in neurones and choroid plexus at the same level irrespective of strain, but failed to detect DMT1 in brain capillary endothelial cells (BCECs), or macro- or microglial cells. The absence of DMT1 in BCECs was confirmed in immunoblots of purified BCECs. DMT1 was virtually undetectable in neurones of rats aged 18 post-natal days irrespective of strain. Neuronal expression of transferrin receptors and DMT1 in adult rats implies that neurones at this age acquire iron by receptor-mediated endocytosis of transferrin followed by iron transport out of endosomes mediated by DMT1. The existence of the mutated DMT1 molecule in neurones suggests that the low cerebral iron uptake in b/b rats derives from a reduced neuronal uptake rather than an impaired iron transport through the blood-brain barrier.     

Mitochondria represent another locale for the divalent metal transporter 1 (DMT1)

The divalent metal transporter (DMT1) is well known for its roles in duodenal iron absorption across the apical enterocyte membrane, in iron efflux from the endosome during transferrin-dependent cellular iron acquisition, as well as in uptake of non-transferrin bound iron in many cells. Recently, using multiple approaches, we have obtained evidence that the mitochondrial outer membrane is another subcellular locale of DMT1 expression. While iron is of vital importance for mitochondrial energy metabolism, its delivery is likely to be tightly controlled due to iron''s damaging redox properties. Here we provide additional support for a role of DMT1 in mitochondrial iron acquisition by immunofluorescence colocalization with mitochondrial markers in cells and isolated mitochondria, as well as flow cytometric quantification of DMT1-positive mitochondria from an inducible expression system. Physiological consequences of mitochondrial DMT1 expression are discussed also in consideration of other DMT1 substrates, such as manganese, relevant to mitochondrial antioxidant defense.     

Cloning and functional characterization of the Anopheles albimanus DMT1/NRAMP homolog: implications in iron metabolism in mosquitoes

In addition to its wide role in metabolism, iron in insects has been implicated in vitellogenesis and the immune response. The NRAMP family comprises a well-conserved family of divalent cation transporters in metazoans. To gain insight on the role of NRAMP in Anopheles albimanus, we cloned a cDNA encoding a 571-residue protein (AnaNRAMP) with the structural features defining the NRAMP family. AnaNRAMP mRNA induced (59)Fe(2+) incorporation when injected into Xenopus oocytes. Western blot analysis revealed that AnaNRAMP is expressed in the head, midgut and at high levels in Malpighian tubules of unfed female mosquito. Upon blood feeding, AnaNRAMP levels were reduced in the midgut whereas they increased in the Malpighian tubules. Using immuno-localization by transmission electron microscopy, AnaNRAMP was localized in the membrane of the intra-cellular concretions or spherites of the Malpighian tubule principal cells. Taken together, our results suggest an important role of AnaNRAMP in iron transport and indicate a role of the mosquito Malpighian tubule as an important organ for iron homeostasis.     

Discovery of benzylisothioureas as potent divalent metal transporter 1 (DMT1) inhibitors

Inhibition of intestinal brush border DMT1 offers a novel therapeutic approach to the prevention and treatment of disorders of iron overload. Several series of diaryl and tricyclic benzylisothiourea compounds as novel and potent DMT1 inhibitors were discovered from the original hit compound 1. These compounds demonstrated in vitro potency against DMT1, desirable cell permeability properties and a dose-dependent inhibition of iron uptake in an acute rat model of iron hyperabsorption. Tricyclic compounds increased the in vitro potency by up to 16-fold versus the original hit. Diaryl compounds 6b and 14a demonstrated significant iron absorption inhibition in vivo with both 25 and 50 mg/kg doses. The diaryl and tricyclic compounds described in this report represent promising structural templates for further optimization.     

SLC11A1 (NRAMP1) polymorphisms and tuberculosis susceptibility: updated systematic review and meta-analysis

Background

Natural resistance associated macrophage protein 1 (NRAMP1), encoded by the SLC11A1 gene, has been described to regulate macrophage activation and be associated with infectious and autoimmune diseases. The relation between SLC11A1 polymorphisms and tuberculosis susceptibility has been studied in different populations.

Methods

We systematically reviewed published studies on SLC11A1 polymorphisms and tuberculosis susceptibility until September 15, 2010 and quantitatively summarized associations of the most widely studied polymorphisms using meta-analysis.

Results

In total, 36 eligible articles were included in this review. In Meta-analysis, significant associations were observed between tuberculosis risk and widely studied SLC11A1 polymorphisms with summarized odds ratio of 1.35 (95%CI, 1.17–1.54), 1.25 (95% CI, 1.04–1.50), 1.23 (95% CI, 1.04–1.44), 1.31 (95%CI, 1.08–1.59) for 3′ UTR, D543N, INT4, and 5′ (GT)n, respectively. Heterogeneity between studies was not pronounced, and the associations did not remarkably vary in the stratified analysis with respect to study population and study base.

Conclusions

The association between SLC11A1 polymorphisms and tuberculosis susceptibility observed in our analyses supports the hypothesis that NRAMP1 might play an important role in the host defense to the development of tuberculosis.     

Identification of a tyrosine-based motif (YGSI) in the amino terminus of Nramp1 (Slc11a1) that is important for lysosomal targeting

In macrophages, Nramp1 (Slc11a1) is expressed in lysosomes and restricts replication of intracellular pathogens by removing divalent metals (Mn2+ and Fe2+) from the phagolysosome. Nramp2 (DMT1, Slc11a2) is expressed both at the duodenal brush border where it mediates uptake of dietary iron and ubiquitously at the plasma membrane/recycling endosomes of many cell types where it transports transferrin-associated iron across the endosomal membrane. In Nramp2, a carboxyl-terminal cytoplasmic motif ((555)YLLNT(559)) is critical for internalization and recycling of the transporter from the plasma membrane. Here we studied the subcellular trafficking properties of Nramp1 and investigated the cis-acting sequences responsible for targeting to lysosomes. For this, we constructed and studied Nramp1/Nramp2 chimeric proteins where homologous domains of each protein were exchanged. Chimeras exchanging the amino-(upstream TM1) and carboxyl-terminal (downstream TM12) cytoplasmic segments of both transporters were stably expressed in porcine LLC-PK1 kidney cells and were studied with respect to expression, maturation, stability, cell surface targeting, transport activity, and subcellular localization. An Nramp2 isoform II chimera bearing the amino terminus of Nramp1 was not expressed at the cell surface but was targeted to lysosomes. This lysosomal targeting was abolished by single alanine substitutions at Tyr15 and Ile18 of a (15)YGSI(18) motif present in the amino terminus of Nramp1. These results identify YGSI as a tyrosine-based sorting signal responsible for lysosomal targeting of Nramp1.