Functional consequences of the human DMT1 (SLC11A2) mutation on protein expression and iron uptake

Blood ◽  
2005 ◽  
Vol 106 (12) ◽  
pp. 3985-3987 ◽  
Author(s):  
Monika Priwitzerova ◽  
Guangjun Nie ◽  
Alex D. Sheftel ◽  
Dagmar Pospisilova ◽  
Vladimir Divoky ◽  
...  
Keyword(s):  
Protein Expression ◽  
Iron Uptake ◽  
Chinese Hamster ◽  
Microcytic Anemia ◽  
Homozygous Mutation ◽  
Divalent Metal Transporter 1 ◽  
Divalent Metal Transporter ◽  
Hypochromic Microcytic Anemia ◽  
Functional Consequences ◽  
Uptake Activity

We have previously described a case of severe hypochromic microcytic anemia caused by a homozygous mutation in the divalent metal transporter 1 (DMT1 1285G > C). This mutation encodes for an amino acid substitution (E399D) and causes preferential skipping of exon 12 during processing of the DMT1 mRNA. To examine the functional consequences of this mutation, full-length DMT1 transcript with the patient's point mutation or a DMT1 transcript with exon 12 deleted was expressed in Chinese hamster ovary (CHO) cells. Our results demonstrate that the E399D substitution has no effect on protein expression and function. In contrast, deletion of exon 12 led to a decreased expression of the protein and disruption of its subcellular localization and iron uptake activity. We hypothesize that the residual protein in hematopoietic cells represents the functional E399D DMT1 variant, but because of its quantitative reduction, the iron uptake activity of DMT1 in the patient's erythroid cells is severely suppressed.

DMT1 Mutation in a Patient with Hypochromic Microcytic Anemia: Functional Consequences and Response to Erythropoietin.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3587-3587
Author(s):  
Monika Priwitzerova ◽  
Dagmar Pospisilova ◽  
Guangjun Nie ◽  
Alex D. Sheftel ◽  
Martha P. Mims ◽  
...  
Keyword(s):  
Iron Uptake ◽  
Chinese Hamster ◽  
Microcytic Anemia ◽  
Hemoglobin Level ◽  
Homozygous Mutation ◽  
Divalent Metal Transporter 1 ◽  
Divalent Metal Transporter ◽  
Hypochromic Microcytic Anemia ◽  
Functional Consequences ◽  
Uptake Activity

Abstract We have previously described a case of severe hypochromic microcytic anemia caused by a homozygous mutation in the divalent metal transporter 1 ( DMT1 1285G>C ). This mutation encodes for an amino acid substitution (E399D) and causes preferential skipping of exon 12 during processing of the DMT1 mRNA. To examine the functional consequences of this mutation, full length DMT1 transcript with the patient’s point mutation or a DMT1 transcript with exon 12 deleted was expressed in Chinese hamster ovary (CHO) cells. Our results demonstrate that the E399D substitution has no effect on protein expression and function. In contrast, deletion of exon 12 led to a decreased expression of the protein and disruption of its subcellular localization and iron uptake activity. We hypothesize that the residual protein in hematopoietic cells represents the functional E399D DMT1 variant, but because of its quantitative reduction, the iron uptake activity of DMT1 in the patient’s erythroid cells is severely suppressed. Because of the positive effect of erythropoietin (EPO) on the growth of patient’s BFU-Es in our in vitro studies, we have treated the patient with recombinant human EPO. The dose of 1.2 μg/kg given once a week did not lead to any improvement in hemoglobin level. However, doubling the dose of EPO (2.4 μg/kg) led to an increase in hemoglobin level from 75 to 91 g/L and to an improvement in the patient’s clinical condition.

Identification of a human mutation of DMT1 in a patient with microcytic anemia and iron overload

Blood ◽  
2005 ◽  
Vol 105 (3) ◽  
pp. 1337-1342 ◽  
Author(s):  
Martha P. Mims ◽  
Yongli Guan ◽  
Dagmar Pospisilova ◽  
Monika Priwitzerova ◽  
Karel Indrak ◽  
...  
Keyword(s):  
Iron Overload ◽  
Iron Absorption ◽  
Microcytic Anemia ◽  
Homozygous Mutation ◽  
Divalent Metal Transporter 1 ◽  
Hypochromic Microcytic Anemia ◽  
Nucleotide Mutation ◽  
Human Mutation ◽  
Processing Stability ◽  
Iron Utilization

Abstract Divalent metal transporter 1 (DMT1) is a transmembrane protein crucial for duodenal iron absorption and erythroid iron transport. DMT1 function has been elucidated largely in studies of the mk mouse and the Belgrade rat, which have an identical single nucleotide mutation of this gene that affects protein processing, stability, and function. These animals exhibit hypochromic microcytic anemia due to impaired intestinal iron absorption, and defective iron utilization in red cell precursors. We report here the first human mutation of DMT1 identified in a female with severe hypochromic microcytic anemia and iron overload. This homozygous mutation in the ultimate nucleotide of exon 12 codes for a conservative E399D amino acid substitution; however, its pre-dominant effect is preferential skipping of exon 12 during processing of pre–messenger RNA (mRNA). The lack of full-length mRNA would predict deficient iron absorption in the intestine and deficient iron utilization in erythroid precursors; however, unlike the animal models of DMT1 mutation, the patient is iron overloaded. This does not appear to be due to up-regulation of total DMT1 mRNA. DMT1 protein is easily detectable by immunoblotting in the patient's duodenum, but it is unclear whether the protein is properly processed or targeted.

Effect of DMT1 Mutations at Birth and in the First Years of Life.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3586-3586
Author(s):  
Iolascon Achille ◽  
d’Apolito Maria ◽  
Servedio Veronica ◽  
De Falco Luigia ◽  
Piga Antonio ◽  
...  
Keyword(s):  
Iron Overload ◽  
Iron Uptake ◽  
Iron Absorption ◽  
Transferrin Saturation ◽  
Adult Life ◽  
Microcytic Anemia ◽  
Hepatic Iron ◽  
Liver Iron ◽  
Divalent Metal Transporter 1 ◽  
Divalent Metal Transporter

Abstract Divalent metal transporter 1 (DMT1) is involved in dietary iron uptake on the luminal side of duodenal enterocytes and transfers iron from the endosome to the cytosol in the marrow erythroblasts. Spontaneous (mk mice and Belgrade rats) or acquired (DMT1 -/- mice) inactivation of DMT1 in rodents produces a severe microcytic anemia at birth, caused by inefficient intestinal iron absorption and defective iron utilization in erythroid cells. The first reported patient with DMT1 mutations had microcytic anemia and iron overload in adult life. We here report the hematological phenotype of a newborn with a severe mycrocytic anemia (Hb 4 g/dL, MCV 71 fL) at birth and during the first months of life. Serum iron, transferrin saturation and serum ferritin were 160 microg/L, 100% and 846 ng/ml respectively at 3 months of age. Hepatic iron overload wad documented at the age of 5 years by both non invasive SQUID and liver biopsy. Sequence analysis of genomic DNA of the family revealed that the child was compound heterozygote for two novel DMT1 mutations, inherited by the asymptomatic parents. The first change deleted 3 bp (c.310 - 3_5del CTT) in intron 4 resulting in a splicing abnormality and the skipping of exon 5. The second was C>T 1246 substitution that causes arginine > cysteine replacement at position 416 (p. R416C) in the protein. This missense affects an highly conserved residue in one of the putative transmembrane domains. A striking reduction of the protein in peripheral blood cells of the proband was demonstrated by western blot using an anti-DMT1 antibody. The child required blood transfusions at birth and in the first two months of life. Thereafter, treatment with subcutaneous erythropoietin mantained hemoglobin levels between 7.5–9.5 g/dL, allowing transfusion-independence. The haematological phenotype of this patient highlights the essential role of DMT1 in erythropoiesis. The early and significant hepatic iron accumulation indicates that, as in animal models, DMT1 is dispensable for liver iron uptake. Finally DMT1 inactivation in the gut is likely bypassed by other pathways of iron absorption.

Microcytic anemia and hepatic iron overload in a child with compound heterozygous mutations in DMT1 (SCL11A2)

Blood ◽  
2006 ◽  
Vol 107 (1) ◽  
pp. 349-354 ◽  
Author(s):  
Achille Iolascon ◽  
Maria d'Apolito ◽  
Veronica Servedio ◽  
Flora Cimmino ◽  
Antonio Piga ◽  
...  
Keyword(s):  
Iron Overload ◽  
Iron Uptake ◽  
Mononuclear Cells ◽  
Microcytic Anemia ◽  
Compound Heterozygous ◽  
Hepatic Iron ◽  
Peripheral Blood Mononuclear ◽  
Divalent Metal Transporter 1 ◽  
Divalent Metal Transporter ◽  
Hepatic Iron Overload

Abstract Divalent metal transporter 1 (DMT1) mediates apical iron uptake in duodenal enterocytes and iron transfer from the transferrin receptor endosomal cycle into the cytosol in erythroid cells. Both mk mice and Belgrade rats, which carry an identical DMT1 mutation, exhibit severe microcytic anemia at birth and defective intestinal iron use and erythroid iron use. We report the hematologic phenotype of a child, compound heterozygote for 2 DMT1 mutations, who was affected by severe anemia since birth and showed hepatic iron overload. The novel mutations were a 3-bp deletion in intron 4 (c.310-3_5del CTT) resulting in a splicing abnormality and a C>T transition at nucleotide 1246(p. R416C). A striking reduction of DMT1 protein in peripheral blood mononuclear cells was demonstrated by Western blot analysis. The proband required blood transfusions until erythropoietin treatment allowed transfusion independence when hemoglobin levels between 75 and 95 g/L (7.5 and 9.5 g/dL) were achieved. Hematologic data of this patient at birth and in the first years of life strengthen the essential role of DMT1 in erythropoiesis. The early onset of iron overload indicates that, as in animal models, DMT1 is dispensable for liver iron uptake, whereas its deficiency in the gut is likely bypassed by the up-regulation of other pathways of iron use.

Copper repletion enhances apical iron uptake and transepithelial iron transport by Caco-2 cells

2002 ◽  
Vol 282 (3) ◽  
pp. G527-G533 ◽  
Author(s):  
Okhee Han ◽  
Marianne Wessling-Resnick
Keyword(s):  
Iron Uptake ◽  
Iron Transport ◽  
Cell Monolayer ◽  
Transepithelial Transport ◽  
Apical Surface ◽  
Divalent Metal Transporter 1 ◽  
Divalent Metal Transporter ◽  
Metal Transporter ◽  
Copper Status ◽  
Copper Supplementation

The influence of copper status on Caco-2 cell apical iron uptake and transepithelial transport was examined. Cells grown for 7–8 days in media supplemented with 1 μM CuCl2had 10-fold higher cellular levels of copper compared with control. Copper supplementation did not affect the integrity of differentiated Caco-2 cell monolayers grown on microporous membranes. Copper-repleted cells displayed increased uptake of iron as well as increased transport of iron across the cell monolayer. Northern blot analysis revealed that expression of the apical iron transporter divalent metal transporter-1 (DMT1), the basolateral transporter ferroportin-1 (Fpn1), and the putative ferroxidase hephaestin (Heph) was upregulated by copper supplementation, whereas the recently identified ferrireductase duodenal cytochrome b (Dcytb) was not. These results suggest that DMT1, Fpn1, and Heph are involved in the iron uptake process modulated by copper status. Although a clear role for Dcytb was not identified, an apical surface ferrireductase was modulated by copper status, suggesting that its function also contributes to the enhanced iron uptake by copper-repleted cells. A model is proposed wherein copper promotes iron depletion of intestinal Caco-2 cells, creating a deficiency state that induces upregulation of iron transport factors.

scholarly journals The Divalent Metal Transporter 1 (DMT1) Is Required for Iron Uptake and Normal Development of Oligodendrocyte Progenitor Cells

2018 ◽  
Vol 38 (43) ◽  
pp. 9142-9159 ◽  
Author(s):  
Veronica T. Cheli ◽  
Diara A. Santiago González ◽  
Leandro N. Marziali ◽  
Norma N. Zamora ◽  
María E. Guitart ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Iron Uptake ◽  
Normal Development ◽  
Divalent Metal ◽  
Oligodendrocyte Progenitor Cells ◽  
Divalent Metal Transporter 1 ◽  
Oligodendrocyte Progenitor ◽  
Divalent Metal Transporter ◽  
Metal Transporter

scholarly journals Hepcidin inhibits apical iron uptake in intestinal cells

2008 ◽  
Vol 294 (1) ◽  
pp. G192-G198 ◽  
Author(s):  
Natalia P. Mena ◽  
Andrés Esparza ◽  
Victoria Tapia ◽  
Pamela Valdés ◽  
Marco T. Núñez
Keyword(s):  
Iron Uptake ◽  
Basolateral Membrane ◽  
Intestinal Cells ◽  
Divalent Metal Transporter 1 ◽  
Divalent Metal Transporter ◽  
Cell Monolayers ◽  
Metal Transporter ◽  
Protein Levels ◽  
J774 Cells ◽  
Iron Export

Hepcidin (Hepc) is considered a key mediator in iron trafficking. Although the mechanism of Hepc action in macrophages is fairly well established, much less is known about its action in intestinal cells, one of the main targets of Hepc. The current study investigated the effects of physiologically generated Hepc on iron transport in Caco-2 cell monolayers and rat duodenal segments compared with the effects on the J774 macrophage cell line. Addition of Hepc to Caco-2 cells or rat duodenal segments strongly inhibited apical 55Fe uptake without apparent effects on the transfer of 55Fe from the cells to the basolateral medium. Concurrently, the levels of divalent metal transporter 1 (DMT1) mRNA and protein in Caco-2 cells decreased while the mRNA and protein levels of the iron export transporter ferroportin did not change. Plasma membrane localization of ferroportin was studied by selective biotinylation of apical and basolateral membrane domains; Hepc induced rapid internalization of ferroportin in J774 cells but not in Caco-2 cells These results indicate that the effect of Hepc is cell dependent: in macrophages it inhibits iron export by inducing ferroportin degradation, whereas in enterocytes it inhibits apical iron uptake by inhibiting DMT1 transcription. Our results highlight the crucial role of Hepc in the control of intestinal iron absorption.

Effects of iron regulatory protein regulation on iron homeostasis during hypoxia

Blood ◽  
2003 ◽  
Vol 102 (9) ◽  
pp. 3404-3411 ◽  
Author(s):  
Brian D. Schneider ◽  
Elizabeth A. Leibold
Keyword(s):  
Iron Uptake ◽  
Iron Homeostasis ◽  
Rna Binding ◽  
Late Phase ◽  
Regulatory Protein ◽  
Binding Activity ◽  
Divalent Metal Transporter 1 ◽  
Divalent Metal Transporter ◽  
Iron Regulatory Proteins ◽  
Ft Synthesis

AbstractIron regulatory proteins (IRP1 and IRP2) are RNA-binding proteins that affect the translation and stabilization of specific mRNAs by binding to stem-loop structures known as iron responsive elements (IREs). IREs are found in the 5′-untranslated region (UTR) of ferritin (Ft) and mitochondrial aconitase (m-Aco) mRNAs, and in the 3′-UTR of transferrin receptor (TfR) and divalent metal transporter-1 (DMT1) mRNAs. Our previous studies show that besides iron, IRPs are regulated by hypoxia. Here we describe the consequences of IRP regulation and show that iron homeostasis is regulated in 2 phases during hypoxia: an early phase where IRP1 RNA-binding activity decreases and iron uptake and Ft synthesis increase, and a late phase where IRP2 RNA-binding activity increases and iron uptake and Ft synthesis decrease. The increase in iron uptake is independent of DMT1 and TfR, suggesting an unknown transporter. Unlike Ft, m-Aco is not regulated during hypoxia. During the late phase of hypoxia, IRP2 RNA-binding activity increases, becoming the dominant regulator responsible for decreasing Ft synthesis. During reoxygenation (ReO2), Ft protein increases concomitant with a decrease in IRP2 RNA-binding activity. The data suggest that the differential regulation of IRPs during hypoxia may be important for cellular adaptation to low oxygen tension.

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