Computer-identified nuclear localization signal in exon 1A of the transporter DMT1 is essentially ineffective in nuclear targeting.

Divalent metal transporter 1 (DMT1; also called DCT1, Nramp2, or SLC11A2) has multiple isoforms that localize differently in many cell types. DMT1 +IRE species (encoded by mRNA with an iron-responsive element) are limited to the plasma membrane and cytosolic vesicles. In neural cells, -IRE isoforms of DMT1 (encoded by mRNA lacking an IRE) localize to the nucleus, plasma membrane, and cytosolic vesicles. In considering nuclear compartmentalization of -IRE isoforms, we hypothesized that the newly identified exon 1A in the N-terminus of this transporter might contain a nuclear localization signal. DNA constructs starting with exon 1A and ending with exons encoding alternative isoforms were made and transiently transfected into HEK293T and PC12 cells as well as rat sympathetic neurons. None of the constructs appeared in the nucleus despite the presence of exon 1A. Antibody specific for exon 1A was also used in both immunostaining and Western blots to investigate localization of exon 1A expressed both endogenously and ectopically in cells. Again, nuclear localization of DMT1 containing exon 1A was not observed. Our data suggest that exon 1A is neither sufficient nor necessary for DMT1 to appear in the nucleus.

Distribution of divalent metal transporter 1 and metal transport protein 1 in the normal and Belgrade rat.

Iron accumulation in the brain occurs in a number of neurodegenerative diseases. Two new iron transport proteins have been identified that may help elucidate the mechanism of abnormal iron accumulation. The Divalent Metal Transporter 1 (DMT1), is responsible for iron uptake from the gut and transport from endosomes. The Metal Transport Protein 1 (MTP1) promotes iron export. In this study we determined the cellular and regional expression of these two transporters in the brains of normal adult and Belgrade rats. Belgrade rats have a defect in DMT1 that is associated with lower levels of iron in the brain. In the normal rat, DMT1 expression is highest in neurons in the striatum, cerebellum, thalamus, ependymal cells lining the third ventricle, and vascular cells throughout the brain. The staining in the ependymal cells and endothelial cells suggests that DMT1 has an important role in iron transport into the brain. In Belgrade rats, there is generalized decrease in immunodetectable DMT1 compared to normal rats except in the ependymal cells. This decrease in immunoreactivity, however, was absent on immunoblots. The immunoblot analysis indicates that this protein did not upregulate to compensate for the chronic defect in iron transport. MTP1 staining is found in most brain regions. MTP1 expression in the brain is robust in pyramidal neurons of the cerebral cortex but is not detected in the vascular endothelial cells and ependymal cells. MTP1 staining in Belgrade rats was decreased compared to normal, but similar to DMT1 this decrease was not corroborated by immunoblotting. These results indicate that DMT1 and MTP1 are involved in brain iron transport and this involvement is regionally and cellularly specific.

Cellular distribution of iron in the brain of the Belgrade rat.

In this study, we investigated the cellular distribution of iron in the brain of Belgrade rats. These rats have a mutation in Divalent Metal Transporter 1, which has been implicated in iron transport from endosomes. The Belgrade rats have iron-positive pyramidal neurons, but these are fewer in number and less intensely stained than in controls. In the white matter, iron is normally present in patches of intensely iron-stained oligodendrocytes and myelin, but there is dramatically less iron staining in the Belgrade rat. Those oligodendrocytes that stained for iron did so strongly and were associated with blood vessels. Astrocytic iron staining was seen in the cerebral cortex for both normal rats and Belgrade rats, but the iron-stained astrocytes were less numerous in the mutants. Iron staining in tanycytes, modified astrocytes coursing from the third ventricle to the hypothalamus, was not affected in the Belgrade rat, but was affected by diet. The results of this study indicate that Divalent Metal Transporter 1 is important to iron transport in the brain. Iron is essential in the brain for basic metabolic processes such as heme formation, neurotransmitter production and ATP synthesis. Excess brain iron is associated with a number of common neurodegenerative diseases. Consequently, elucidating the mechanisms of brain iron delivery is critical for understanding the role of iron in pathological conditions.

Evidence for and consequences of chronic heme deficiency in Belgrade rat reticulocytes.

The Belgrade rat has a microcytic, hypochromic anemia inherited as an autosomal recessive trait (gene symbol b). Transferrin-dependent iron uptake is defective because of a mutation in Nramp2 (now DMT1, also called DCT1), the protein responsible for endosomal iron efflux. Hence, Belgrade reticulocytes are iron deficient. We show that a chromatographic method is able to measure the amount of 'free' heme in reticulocytes. Most of the 'free' heme is the result of biosynthesis. Succinylacetone, an inhibitor of heme synthesis, decreases the level of 'free' heme and cycloheximide, an inhibitor of globin synthesis, increases the 'free' heme level. In a pulse-chase experiment with 59Fe-transferrin, the 'free' heme pool behaves as an intermediate, with a half-life of just over 2 h. Belgrade reticulocytes contain about 40% as much 'free' heme as do heterozygous or homozygous reticulocytes. This deficiency of 'free' heme slows initiation of translation in Belgrade reticulocytes by increasing the level of an inhibitor of initiation. Thus the Belgrade rat makes a whole animal model available with chronic heme deficiency.

Non-transferrin-bound iron uptake in Belgrade and normal rat erythroid cells.

Belgrade (b) rats have an autosomal recessive, microcytic, hypochromic anemia. Transferrin (Tf)-dependent iron uptake is defective because of a mutation in DMT1 (Nramp2), blocking endosomal iron efflux. This experiment of nature permits the present study to address whether the mutation also affects non-Tf-bound iron (NTBI) uptake and to use NTBI uptake compared to Tf-Fe utilization to increase understanding of the phenotype of the b mutation. The distribution of 59Fe2+ into intact erythroid cells and cytosolic, stromal, heme, and nonheme fractions was different after NTBI uptake vs. Tf-Fe uptake, with the former exhibiting less iron into heme but more into stromal and nonheme fractions. Both reticulocytes and erythrocytes exhibit NTBI uptake. Only reticulocytes had heme incorporation after NTBI uptake. Properly normalized, incorporation into b/b heme was approximately 20% of +/b, a decrease similar to that for Tf-Fe utilization. NTBI uptake into heme was inhibited by bafilomycin A1, concanamycin, NH4Cl, or chloroquine, consistent with the endosomal location of the transporter; cellular uptake was uninhibited. NTBI uptake was unaffected after removal of Tf receptors by Pronase or depletion of endogenous Tf. Concentration dependence revealed that NTBI uptake into cells, cytosol, stroma, and the nonheme fraction had an apparent low affinity for iron; heme incorporation behaved like a high-affinity process, as did an expression assay for DMT1. DMT1 serves in both apparent high-affinity NTBI membrane transport and the exit of iron from the endosome during Tf delivery of iron in rat reticulocytes; the low-affinity membrane transporter, however, exhibits little dependence on DMT1.

Nramp2 is mutated in the anemic Belgrade (b) rat: evidence of a role for Nramp2 in endosomal iron transport.

The Belgrade (b) rat has an autosomal recessively inherited, microcytic, hypochromic anemia associated with abnormal reticulocyte iron uptake and gastrointestinal iron absorption. The b reticulocyte defect appears to be failure of iron transport out of endosomes within the transferrin cycle. Aspects of this phenotype are similar to those reported for the microcytic anemia (mk) mutation in the mouse. Recently, mk has been attributed to a missense mutation in the gene encoding the putative iron transporter protein Nramp2. To investigate the possibility that Nramp2 was also mutated in the b rat, we established linkage of the phenotype to the centromeric portion of rat chromosome 7. This region exhibits synteny to the chromosomal location of Nramp2 in the mouse. A polymorphism within the rat Nramp2 gene cosegregated with the b phenotype. A glycine-to-arginine missense mutation (G185R) was present in the b Nramp2 gene, but not in the normal allele. Strikingly, this amino acid alteration is the same as that seen in the mk mouse. Functional studies of the protein encoded by the b allele of rat Nramp2 demonstrated that the mutation disrupted iron transport. These results confirm the hypothesis that Nramp2 is the protein defective in the Belgrade rat and raise the possibility that the phenotype shared by mk and b animals is unique to the G185R mutation. Furthermore, the phenotypic characteristics of these animals indicate that Nramp2 is essential both for normal intestinal iron absorption and for transport of iron out of the transferrin cycle endosome.

Transferrin and the transferrin cycle in Belgrade rat reticulocytes.

Belgrade rats have an autosomal recessive anemia with hypochromia and microcytosis. Iron uptake into reticulocytes is approximately 20% of normal, but transferrin uptake is unimpaired. We have systematically compared the transferrin cycle in Belgrade versus normal reticulocytes to locate the defect more precisely. Belgrade transferrin was functionally normal as purified transferrin or whole plasma. Transferrin affinity of Belgrade receptors was indistinguishable from normal, but Belgrade reticulocytes had twice as many receptors. Belgrade transferrin endocytosis was 1.5 times faster than normal, whereas exocytosis is about twice as fast. Initially Belgrade reticulocytes internalize iron at an unimpaired rate, but they lag behind normal by 5 min. During reincubation, they release 25-33% of iron taken up during a 30-min preincubation, whereas normal cells do not lose a detectable fraction. Unexpectedly, transferrin cycle time was unchanged. Hence another kinetic step of the cycle is slower, compensating for increases in Belgrade endocytosis and exocytosis. After one cycle, Belgrade reticulocytes retain only half of the iron that entered, but over 90% of iron entering normal cells remains within. Iron unloading is ineffective inside the Belgrade vesicle; 85% of iron that entered on transferrin returned to the medium after exocytosis, whereas only 45% of iron entering normal reticulocytes exits. Ineffective utilization of iron in or near Belgrade endosomes accounts for the Belgrade defect.

Iron distribution in Belgrade rat reticulocytes after inhibition of heme synthesis with succinylacetone.

We have used succinylacetone (4,6-dioxoheptanoic acid), a specific inhibitor of delta-aminolevulinic acid dehydrase, to gain insight into the defect in iron metabolism in the Belgrade anemia. The Belgrade rat has an inherited microcytic, hypochromic anemia associated with poor iron uptake into developing erythroid cells. Succinylacetone inhibits heme synthesis, leading to nonheme iron accumulation in mitochondria and cytosol of normal reticulocytes. When succinylacetone is used to inhibit Belgrade heme synthesis, iron from diferric transferrin does not accumulate in the stromal fraction that contains mitochondria, nor does 59Fe accumulate in the nonheme cytosolic fraction. Hence, the defect in the Belgrade rat reticulocyte occurs in the endocytic vesicle or in a step subsequent to iron transit from the vesicle but before the nonheme cytosolic or mitochondrial iron fractions. Therefore, the mutation affects either the release of iron from transferrin or iron transport from the vesicle to the mitochondrion.

Ferric-salicylaldehyde isonicotinoyl hydrazone, a synthetic iron chelate, alleviates defective iron utilization by reticulocytes of the Belgrade rat.

The Belgrade rat has a hypochromic, microcytic anemia inherited as an autosomal recessive mutation. Although transferrin binds normally to reticulocytes and internalizes normally, iron accumulation into cells and heme is much slower than normal. We have investigated the role of the transferrin cycle in this mutant by bypassing transferrin iron delivery with the iron chelate ferric salicylaldehyde isonicotinoyl hydrazone (Fe-SIH). Fe-SIH increases iron uptake into heme by Belgrade reticulocytes, restoring it almost to normal levels. This increase indicates that Fe-SIH delivers iron to a step in iron utilization that is after the Belgrade defect. Depleting reticulocytes of transferrin did not alter these observations. Failure to achieve above normal rates of iron incorporation could indicate damage due to chronic intracellular iron deficiency. Also, iron delivery by Fe-SIH restored globin synthesis to near-normal levels in Belgrade reticulocytes. The rates of glycine incorporation into porphyrin and heme in Belgrade reticulocytes incubated with Fe2-transferrin or Fe-SIH paralleled the rates of iron incorporation into heme. These data are consistent with the concept that iron availability limits protoporphyrin formation in rat reticulocytes. The protoporphyrin used for heme synthesis is provided by de novo synthesis and not by a pool of pre-existing protoporphyrin. The Belgrade defect occurs in the movement of iron from transferrin to a step prior to the ferrous state and insertion into heme. This defect diminishes the synthesis of heme and, consequently, that of protoporphyrin and globin.

Tissue iron deposition in untransfused beta-thalassemic mice.

Homozygous beta-thalassemic mice show many of the features seen in human beta-thalassemia, such as decreased hemoglobin, hematocrit, and red blood cell count as well as increased reticulocyte count. They also exhibit splenomegaly and a decrease in osmotic fragility of red cells. beta-thalassemic mice were examined for spontaneous iron overload at ages ranging from 20 to 595 days. Accumulation of iron was shown to occur in the spleen, liver, and kidneys but not in the heart. Sections of spleen, liver, kidney, and heart were stained for iron and subjectively scored. Image analysis microscopy was used to examine sections of spleen and liver. Nonheme iron in the four tissues was quantitated using the bathophenanthroline sulfonate colorimetric assay. An increase in tissue iron occurred primarily in the spleen, even before weaning, despite the low iron content of milk. Iron accumulation in the absence of blood transfusion is of interest because iron overload is the major cause of death in human beta-thalassemia.

Differential synthesis of beta-major and beta-minor globin proteins in murine erythroleukemia cells is regulated at the transcriptional level.

We compared the transcription and translation of globin genes in three mouse erythroleukemia cell lines under different conditions in which there is differential expression of beta-major (beta ma) and beta-minor (beta mi). Transcription was measured by pulse labeling of whole cell RNA with [3H]uridine, and translation by labeling whole cell protein synthesis with [3H]leucine. Induction with dimethyl sulfoxide led to increased transcription of alpha, beta ma, and beta mi genes, and the proportion of beta mi RNA synthesized was similar to the proportion of beta mi globin protein produced, whether the cells produced 30-40% beta mi (lines 745 and 9M) or greater than 80% beta mi (clone 25-66). Induction with hemin did not lead to increased globin gene transcription in any line, although globin protein synthesis did increase. Thus, the mechanism of beta globin chain induction depends on the inducing agent, and not on the cell line or the type of beta globin gene product. The relative proportion of beta mi and beta ma globin chain proteins reflects the relative transcription of the two beta genes, whether or not transcription increased following induction.

Diminished acquisition of iron by reticulocytes from mice with hemoglobin deficit.

Reticulocyte iron and transferrin uptake was studied in hemoglobin deficit (gene symbol, hbd), an autosomal recessive trait in the mouse characterized by hypochromic microcytic anemia, reticulocytosis, hyperferremia, and increased red-cell-free protoporphyrin. Reticulocyte-rich red cells were incubated in vitro in a mixture of 125I-labeled diferric mouse transferrin and 59Fe-labeled iron-saturated mouse plasma. At 37 degrees C, the uptake of transferrin by reticulocytes from affected animals (15 ng/micrograms RNA) was the same as that of reticulocytes from control animals. However, the uptake of iron by affected reticulocytes (0.11 ng/micrograms RNA) was significantly lower than that by control reticulocytes (0.24). At 4 degrees C, transferrin binding by affected and control reticulocytes was again indistinguishable. The deficiency in the uptake of iron by affected reticulocytes was not observed on incubation at 4 degrees C. Scatchard analysis of transferrin receptors on hbd/hbd and control reticulocytes showed no difference in pKD and a slight elevation in number of receptors per reticulocyte for hbd/hbd animals. These findings suggest that hbd/hbd reticulocytes have a defect in iron acquisition that is distal to the binding of transferrin to the cell membrane receptor. This defect is similar to one already described in the anemia of the Belgrade laboratory rat.

Post-synthetic modification in vivo of the major hemoglobin beta chain in the rat.

Rat hemoglobins are unusually heterogeneous among mammals. This heterogeneity provides multiple opportunities for asynchronies in synthesis and degradation. We have examined rat hemoglobin turnover after an intravenous injection of 2-14C-glycine. After analyzing incorporation into the seven nonallelic globin chains of adult rats, we found the percentage of the major beta chain decreases over the red cell lifespan while the percentage of one of the minor beta chains increases. This suggests that a post-synthetic modification event converts a portion of the latter into the former.

Hemoglobin deficit: an inherited hypochromic anemia in the mouse.

The character and pathogenesis of hemoglobin deficit (gene symbol, hbd), an autosomal recessive trait in the mouse, were studied. The main hematological features of hemoglobin deficit are anemia, red cell hypochromia and microcytosis, and reticulocytosis. The absence of raised fecal urobilinogen excretion and frank hyperbilirubinemia and bilirubinuria suggests that excess hemolysis is not the primary cause of the anemia. The raised plasma iron concentration and the failure of the anemia to respond to parenteral iron treatment indicate that the anemia is not due to iron deficiency. The absence of siderocytes and sideroblasts suggests that anemia is probably not due to ferrochelatase deficiency. Thalassemia is excluded by the finding of balanced reticulocyte globin chain synthesis. The markedly elevated levels of free red cell protoporphyrin taken together with the other findings already noted suggest that the anemia of hemoglobin deficit is due to a defect in the erythroid cell iron procurement mechanisms leading in turn to diminished heme and hemoglobin synthesis.

Structural analysis of the minor human hemoglobin components: Hb AIa1, Hb AIa2 and Hb AIb.

Human hemolysate contains several minor hemoglobin components, including Hb AIa1, Hb AIa2, Hb AIb and Hb AIc which are post-translational modifications of the major component, Hb A0. Hb AIc is known to contain glucose attached to the N terminus of the beta chains by a ketoamine linkage. We separated the alpha and beta globin chains from purified Hb AIa1, Hb AIa2 and Hb AIb by ion-exchange chromatography. The beta chains were reducible by sodium borohydride and gave a positive thiobarbituric acid test. These results indicated that they are modified by ketoamine-linked carbohydrate. In addition, phosphate analysis revealed 1.5 phosphate residue associated with each beta AIa1 chain and 1 phosphate residue with each beta AIa2 chain. Hb AIa1, Hb AIa2 and Hb AIb were all found to be contaminated by non-globin proteins. Protein-sequencing approaches demonstrated that the N termini of beta AIa1, beta AIa2 and beta AIb were blocked. In support of this conclusion, analysis of tryptic digests of beta AIa2 and B AIb revealed modified N-terminal peptides. We conclude that, like Hb AIc, components Hb AIa1, Hb AIa2 and Hb AIb also contain a sugar moiety linked to the N terminus of the beta chain.

Primary structure of the major beta-chain of rat haemoglobins.

The amino acid sequence of the major beta-chain, (II)beta, from rat haemoglobins was established with an automated sequencer. Amino acid heterogeneities were found that appear to result from allelic variation at particular residues. We applied several new or unusual techniques in determining the sequence: (1) reaction of the polypeptide with dansylaziridine for detection of cysteine; (2) blockage of the N-terminal residue and the epsilon-amino group of lysine residues with 1-fluoro-2-nitro-4-trimethylammoniobenzene iodide and subsequent identification of the modified lysine phenylthiohydantoin by absorbance at 420nm; (3) identification of histidine phenylthiohydantoin by its blue fluorescence under long-wave u.v. light; (4) cleavage of the chain into two or three fragments and subsequent sequencing without purification [a detailed statement giving the major phenylthiohydantoins assigned at each step for each sequence run before their alignment in individual sequences has been deposited as Supplementary Publication SUP 50084 (10 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5]; (5) separation of fragments produced by CNBr cleavage by cation-exchange chromatography; (6) peptide sequencing after attachment of the peptide to cytochrome c. The amino acid sequence was confirmed by amino acid compositions of the complete chain, of CNBr fragments 1 and 3, and of 11 purified tryptic peptides.