Molecular evidence for the role of a ferric reductase in iron transport

Duodenal cytochrome b (Dcytb) is a haem protein similar to the cytochrome b561 protein family. Dcytb is highly expressed in duodenal brush-border membrane and is implicated in dietary iron absorption by reducing dietary ferric iron to the ferrous form for transport via Nramp2/DCT1 (divalent-cation transporter 1)/DMT1 (divalent metal-transporter 1). The protein is expressed in other tissues and may account for ferric reductase activity at other sites in the body.     

二价金属离子转运蛋白1——一个新发现的重要铁转运蛋白

二价金属离子转运蛋白1(divalent metal transporter 1,DMT1)的发现是近年铁代谢研究领域最重大的一项突破.DMT1是哺乳类跨膜铁转运蛋白.这种蛋白质广泛分布于人体各组织.DMT1 mRNA有两种形式,一种含有IRE(iron response element),而另一种则不含此结构.DMT1的功能主要是介导小肠上皮细胞的铁吸收以及参与铁从内吞小体移位到胞浆的过程.DMT1介导的铁转运是一个主动的和H⁺依赖的过程.DMT1也参与其他二价金属如Zn²⁺、Mn²⁺、Co²⁺、Cd²⁺、Cn²⁺、Ni²⁺和Pb²⁺的转运.小肠DMT1的表达受饮食或组织铁控制.第四跨膜区是DMT1的重要功能区.此区基因发生点突变（G185R）是导致不可逆性缺铁性贫血的原因.在帕金森氏病人的黑质发现DMT1表达异常增加,因而DMT1可能也与某些神经退行性疾病的形成有关.     

Schistosoma mansoni: topochemical features of cercariae, schistosomula, and adults

The teguments of developing and mature cercariae, recently transformed, and 1-wk-old schistosomula and adult worms were examined for the ultrastructural location of macromolecular carbohydrates and polyelectrolytes. The surface of mature cercariae within sporocysts and cercariae released from the snail is covered by a filamentous coat which reacts with cytochemical reagents for the demonstration of vicinal glycols, but neither the coat nor the surface of the tegument plasmalemma binds cationic colloidal iron at low pH.Upon penetrating mammalian skin, the cercaria sheds its surface coat; the tegument surface of newly transformed schistosomula, older schistosomula and adult worms stains en bloc with acidic colloidal iron, as does the tegument plasmalemma of mature cercariae if the overlying filamentous coat is first removed by physicochemical means. The cercarial coat thus serves to mask anionic groups at the surface of the tegument plasmalemma which become functionally exposed after penetration of the mammalian host. The distribution of colloidal iron binding sites coincides with those for the carbohydrate-complexing phytohemmagglutnin, concanavalin A, which suggests that these membrane-fixed anions are acid mucopolysaccharides, glycoproteins or glycolipids. Carbohydrate-containing material was also localized within membrane-bound vesicles of the tegument matrix and perikarya of developing cercariae and postcercarial schistosomes, suggesting that surface mucosubstances contributing to the tegument glycocalyx of these worms are elaborated, at least in part, by the tegument itself.     

The iron transporter DMT1

Divalent metal transporter 1 (DMT1) is the first mammalian transmembrane iron transporter to be identified. In 1997, parallel experiments from two groups provided compelling evidence of its function. Fleming and colleagues identified mutations in DMT1 (formerly known as Nramp2 and DCT1) in mice and rats with defects in intestinal iron absorption and red blood cell iron utilization. Gunshin and co-workers (H Gunshin, B MacKenzie, UV Berger, Y Gunshin, MF Romero, WF Boron, S. Nussberger, JL Gollan, MA Hediger, Cloning and characterization of a mammalian proton-coupled metal-ion transporter, Nature 388 (1997) 482-488.) isolated DMT1 through an expression cloning strategy looking for mRNAs that stimulated iron uptake by Xenopus oocytes. Taken together, these data indicate that the twelve transmembrane domain protein DMT1 transfers iron across the apical surface of intestinal cells and out of transferrin cycle endosomes. Human DMT1 may be a good target for pharmacological intervention in patients with iron overload disorders attributable to increased iron absorption.     

Iron uptake and Nramp2/DMT1/DCT1 in human bronchial epithelial cells

The capacity of natural resistance-associated macrophage protein-2 [Nramp2; also called divalent metal transporter-1 (DMT1) and divalent cation transporter-1 (DCT1)] to transport iron and its ubiquitous expression make it a likely candidate for transferrin-independent uptake of iron in peripheral tissues. We tested the hypothesis that non-transferrin-bound iron uptake by airway epithelial cells is associated with Nramp2/DMT1/DCT1 and that exposure to iron can increase Nramp2/DMT1/DCT1 mRNA and protein expression and transport of this metal. Exposure of BEAS-2B cells to ferric ammonium citrate (FAC) resulted in a decrease in Fe(3+) concentration in the supernatant that was dependent on time and initial iron concentration. In the presence of internalized calcein, FAC quenched the fluorescent signal, indicating intracellular transport of the metal. The Nramp2/DMT1/DCT1 mRNA isoform without an iron-response element (IRE) increased with exposure of BEAS-2B cells to FAC. RT-PCR demonstrated no change in the mRNA for the isoform with an IRE. Similarly, Western blot analysis for the isoform without an IRE confirmed an increased expression of this protein after FAC exposure, whereas the isoform with an IRE exhibited no change. Finally, immunohistochemistry revealed an increase in the isoform without an IRE in the rat lung epithelium after instillation of FAC. Comparable to mRNA and protein increases, iron transport was elevated after pretreatment of BEAS-2B cells with iron-containing compounds. We conclude that airway epithelial cells increase mRNA and expression of the Nramp2/DMT1/DCT1 without an IRE after exposure to iron. The increase results in an elevated transport of iron and its probable detoxification by these cells.     

Nramp 2 (DCT1/DMT1) expressed at the plasma membrane transports iron and other divalent cations into a calcein-accessible cytoplasmic pool

Nramp2, also known as DMT1 and DCT1, is a 12-transmembrane (TM) domain protein responsible for dietary iron uptake in the duodenum and iron acquisition from transferrin in peripheral tissues. Nramp2/DMT1 produces by alternative splicing two isoforms differing at their C terminus (isoforms I and II). The subcellular localization, mechanism of action, and destination of divalent cations transported by the two Nramp2 isoforms are not completely understood. Stable CHO transfectants expressing Nramp2 isoform II modified by addition of a hemaglutinin epitope in the loop defined by the TM7-TM8 interval were generated. Immunofluorescence with permeabilized and intact cells established that Nramp2 isoform II is expressed at the plasma membrane and demonstrated the predicted extracytoplasmic location of the TM7-TM8 loop. Using the fluorescent, metal-sensitive dye calcein, and a combination of membrane-permeant and -impermeant iron chelators, Nramp2 transport was measured and quantitated with respect to kinetic parameters and at steady state. Iron transport at the plasma membrane was time- and pH-dependent, saturable, and proportional to the amount of Nramp2 expression. Iron uptake by Nramp2 at the plasma membrane was into the nonferritin-bound, calcein-accessible so-called "labile iron pool." Ion selectivity experiments show that Nramp2 isoform II can also transport Co(2+) and Cd(2+) but not Mg(2+) into the calcein-accessible pool. Parallel experiments with transfectants expressing the lysosomal Nramp1 homolog do not show any divalent cation transport activity, establishing major functional differences between Nramp1 and Nramp2. Monitoring the effect of Nramp2 on the calcein-sensisitve labile iron pool allows a simple, rapid, and nonisotopic approach to the functional study of this protein.     

<Emphasis Type="Italic">Trichobilharzia regenti</Emphasis>: Antigenic structures of intravertebrate stages

Like several other bird schistosomes, neurotropic schistosome of Trichobilharzia regenti can invade also mammals, including humans. Repeated infections cause cercarial dermatitis, a skin inflammatory reaction leading to parasite elimination in non-specific mammalian hosts. However, in experimentally primo-infected mice, the worms escape from the skin and migrate to the central nervous system. In order to evade host immune reactions, schistosomes undergo cercaria/schistosomulum transformation accompanied with changes of surface antigens. The present study is focused on localization of the main antigens of T. regenti; cercariae, schistosomula developed under different conditions and adults were compared. Antigens were localized by immunofluorescence and ultrastructural immunocytochemistry using sera of mice repeatedly infected with T. regenti. Detected antibody targets were located in glycocalyx and penetration glands of cercariae and in tegument of cercariae, schistosomula and adults. Shedding of cercarial glycocalyx significantly reduced surface reactivity; further decrease was reported during ongoing development of schistosomula. Spherical bodies, probably transported from subtegumental cell bodies to worm surface, were identified as the most reactive tegumental structures. Based on similar results for schistosomula developed in specific, non-specific hosts and in vitro, it seems that the ability of T. regenti to decrease the surface immunoreactivity during ontogenesis is independent on the host type.     

Recent progress in structure-function analyses of Nramp proton-dependent metal-ion transporters.

The natural resistance-associated macrophage protein (Nramp) homologs form a family of proton-coupled transporters that facilitate the cellular absorption of divalent metal ions (Me2+, including Mn2+, Fe2+, Co2+, and Cd2+). The Nramp, or solute carrier 11 (SLC11), family is conserved in eukaryotes and bacteria. Humans and rodents express 2 parologous genes that are associated with iron disorders and immune diseases. The NRAMP1 (SLC11A1) protein is specific to professional phagocytes and extrudes Me2+ from the phagosome to defend against ingested microbes; polymorphisms in the NRAMP1 gene are associated with various immune diseases. Several isoforms of NRAMP2 (SLC11A2, DMT1, DCT1) are expressed ubiquitously in recycling endosomes or specifically at the apical membrane of epithelial cells in intestine and kidneys, and can contribute to iron overload, whereas mutations impairing NRAMP2 function cause a form of congenital microcytic hypochromic anemia. Structure-function studies, using various experimental models, and mutagenesis approaches have begun to reveal the overall transmembrane organization of Nramp, some of the transmembrane segments (TMS) that are functionally important, and an unusual mechanism coupling Me2+ and proton H+ transport. The approaches used include functional complementation of yeast knockout strains, electrophysiology analyses in Xenopus oocytes, and transport assays that use mammalian and bacterial cells and direct and indirect measurements of SLC11 transporter properties. These complementary studies enabled the identification of TMS1 and 6 as crucial structural segments for Me2+ and H+ symport, and will help develop a deeper understanding of the Nramp transport mechanism and its contribution to Me2+ homeostasis in human health and diseases.     

Cloning and characterization of the OsNramp family from Oryza sativa,a new family of membrane proteins possibly implicated in the transport of metal ions

The mammalian Nramp1 protein is an integral membrane protein expressed exclusively in macrophages, where it plays a critical role in the ability of these cells to destroy ingested microbes. The bactericidal mechanism of action of Nramp1 remains unknown. We report the identification and characterization of cDNA clones corresponding to three homologues of the mammalian Nramp1 gene from the genome of Oryza sativa, OsNramp1, OsNramp2, and OsNramp3. These three genes encode a novel group of highly similar hydrophobic polypeptides sharing between 64% and 75% sequence similarity, that show similar hydropathy profiles, and predicted secondary structure, including the same number, position, and sequence characteristics (including conserved charges) of transmembrane domains. Together, these define a highly conserved membrane associated hydrophobic core. The three plant proteins show a remarkable degree of sequence similarity with their mammalian counterpart (60% to 70% similarity), including primary and secondary structure elements previously described in ion transporters and channels. Expression studies in normal plant tissues indicate that while OsNramp1 is expressed primarily in roots, and OsNramp2 is primarily expressed in leaves, OsNramp3 is expressed in both tissues. The recent discovery that the yeast Nramp homologue SMF1 functions as a manganese transporter raises the exciting possibility that OsNramp encodes a family of metal ion transporters in plants.     

Differential expression of LacdiNAc,fucosylated LacdiNAc,and Lewis x glycan antigens in intramolluscan stages of Schistosoma mansoni

We report the expression of 3 well-characterized adult Schistosoma mansoni glycan antigens among molluscan stages of the parasite. These antigens are LacdiNAc (LDN; GalNAcbeta1-4GlcNAc-R), fucosylated LacdiNAc (LDNF; GalNAc[Fucal-3]beta1-4GlcNAc-R), and Lewis x (Le(x); Gal[Fucalpha1-3]beta1-4GlcNAc-R). The presence of the glycans was determined by both immunoblot and immunohistological methods using monoclonal antibodies that specifically recognize each glycan epitope. Immunoblot analyses reveal that LDN and LDNF epitopes are expressed on many different glycoproteins, including eggs, mother sporocysts, daughter sporocysts, and cercariae, although LDN expression among daughter sporocysts is greatly reduced. LDN and LDNF epitopes are localized on the tegument and in the intrasporocyst cell masses of both in vitro-derived and in vivo-derived mother sporocysts and in the daughter sporocysts derived on day 16 after infection. Unexpectedly, high levels of LDN and LDNF glycans were detected in the infected, but not in the uninfected, snail hemolymph, suggesting that the infecting larvae secrete LDN and LDNF glycoconjugates into the snail hosts. In contrast, the expression of Le(x) antigen among the molluscan stages is highly restricted. Le(x) is present on a few high-molecular weight glycoproteins in eggs and cercariae but is undetectable in mother and daughter sporocysts. Taken together with our earlier studies on vertebrate stages of S. mansoni, these results show that LDN and LDNF glycans are conserved during schistosome development. The study further extends the evidence that Le(x) is a developmentally regulated antigen in schistosomes.     

The soybean NRAMP homologue,GmDMT1, is a symbiotic divalent metal transporter capable of ferrous iron transport

Iron is an important nutrient in N2-fixing legume root nodules. Iron supplied to the nodule is used by the plant for the synthesis of leghemoglobin, while in the bacteroid fraction, it is used as an essential cofactor for the bacterial N2-fixing enzyme, nitrogenase, and iron-containing proteins of the electron transport chain. The supply of iron to the bacteroids requires initial transport across the plant-derived peribacteroid membrane, which physically separates bacteroids from the infected plant cell cytosol. In this study, we have identified Glycine max divalent metal transporter 1 (GmDmt1), a soybean homologue of the NRAMP/Dmt1 family of divalent metal ion transporters. GmDmt1 shows enhanced expression in soybean root nodules and is most highly expressed at the onset of nitrogen fixation in developing nodules. Antibodies raised against a partial fragment of GmDmt1 confirmed its presence on the peribacteroid membrane (PBM) of soybean root nodules. GmDmt1 was able to both rescue growth and enhance 55Fe(II) uptake in the ferrous iron transport deficient yeast strain (fet3fet4). The results indicate that GmDmt1 is a nodule-enhanced transporter capable of ferrous iron transport across the PBM of soybean root nodules. Its role in nodule iron homeostasis to support bacterial nitrogen fixation is discussed.     

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.     

The effects of pH and the iron redox state on iron uptake in the intestine of a marine teleost fish, gulf toadfish (Opsanus beta)

In the marine teleost intestine the secretion of bicarbonate increases pH of the lumen (pH 8.4 -9.0) and importantly reduces Ca2+ and Mg2+ concentrations by the formation of insoluble divalent ion carbonates. The alkaline intestinal environment could potentially also cause essential metal carbonate formation reducing bioavailability. Iron accumulation was assessed in the Gulf toadfish (Opsanus beta) gut by mounting intestine segments in modified Ussing chambers fitted to a pH-stat titration system. This system titrates to maintain lumen pH constant and in the process prevents bicarbonate accumulation. The luminal saline pH was clamped to pH 5.5 or 7.0 to investigate the effect of proton concentrations on iron uptake. In addition, redox state was altered (gassing with N2, addition of dithiothreitol (DTT) and ascorbate) to evaluate Fe3+ versus Fe2+ uptake, enabling us to compare a marine teleost intestine model for iron uptake to the mammalian system for non-haem bound iron uptake that occurs via a ferrous/proton (Fe2+/H+) symporter called Divalent Metal Transporter 1 (DMT1). None of the redox altering strategies affected iron (Fe3+ or Fe2+) binding to mucus, but the addition of ascorbate resulted in a 4.6-fold increase in epithelium iron accumulation. This indicates that mucus iron binding is irrespective of valency and suggests that ferrous iron is preferentially transported across the apical surface. Altering luminal saline pH from 7.0 to 5.5 did not affect ferric or ferrous iron uptake, suggesting that if iron is entering via DMT1 in marine fish intestine this transporter works efficiently under circumneutral conditions.     

Divalent metal transport in the green microalga <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> is mediated by a protein similar to prokaryotic Nramp homologues

Information about the molecular mechanisms of metal transport in algae is scarce, despite the significant status these organisms have in aquatic ecosystems. In the present study, we describe the cloning and functional characterization of a divalent metal transporter (named DMT1) in the green microalga Chlamydomonas reinhardtii Dangeard. The longest open reading frame of the cloned DMT1 cDNA encodes a protein of 513 amino acids with 11 putative transmembrane domains. The protein belongs to the Nramp family of divalent metal transporters and shows surprisingly higher similarity to some prokaryotic than to eukaryotic polypeptides. Especially the N-terminus, which is longer than of every other homologue considered in this study, displays – uniquely among selected eukaryotic Nramps – exclusively prokaryotic characteristics. Functional complementation experiments in yeast strains with impaired metal transport systems, revealed that C. reinhardtii DMT1 has a broad specificity, acting in the transport of several divalent metals (manganese, iron, cadmium, copper), but excluding zinc. Published online December 2004     

A spontaneous, recurrent mutation in divalent metal transporter-1 exposes a calcium entry pathway

Divalent metal transporter-1 (DMT1/DCT1/Nramp2) is the major Fe²⁺ transporter mediating cellular iron uptake in mammals. Phenotypic analyses of animals with spontaneous mutations in DMT1 indicate that it functions at two distinct sites, transporting dietary iron across the apical membrane of intestinal absorptive cells, and transporting endosomal iron released from transferrin into the cytoplasm of erythroid precursors. DMT1 also acts as a proton-dependent transporter for other heavy metal ions including Mn²⁺, Co²⁺, and Cu², but not for Mg²⁺ or Ca²⁺. A unique mutation in DMT1, G185R, has occurred spontaneously on two occasions in microcytic (mk) mice and once in Belgrade (b) rats. This mutation severely impairs the iron transport capability of DMT1, leading to systemic iron deficiency and anemia. The repeated occurrence of the G185R mutation cannot readily be explained by hypermutability of the gene. Here we show that G185R mutant DMT1 exhibits a new, constitutive Ca²⁺ permeability, suggesting a gain of function that contributes to remutation and the mk and b phenotypes.     

The ultrastructural architecture of the adult Schistosoma japonicum tegument

The tegument of the adult blood fluke Schistosoma japonicum is in direct contact with the host blood and immune systems. A comprehensive understanding of the ultrastructure of the tegument is crucial to the understanding of how the parasite maintains itself within the mammalian host. Important functions such as nutritional uptake and immune evasion are suspected functions of the tegument and this review discusses these aspects and presents some insights into some of these crucial functions. Transmission electron microscopy has allowed the identification of ultrastructural features of the adult S. japonicum, some of which differ from the reported features of other schistosome species. Morphological differences within the tegument of the adult S. japonicum are noted between sexes, among different regions of the worms and between aspects along the length of the parasite. Differences included variations in the ultrastructure, size and number of tegumental bodies and mitochondria within the matrix, and differences in the relative area of the apical surface of the tegument. Functions of the various components of the tegument matrix and specialised functions of different regions of the male and female parasites are discussed based on ultrastructural findings and previously reported biochemical and molecular data.