Natural Resistance-associated Macrophage Protein 2 Research Articles

Interactions and accumulation differences of metal(loid)s in three sea cucumber species collected from the Northern Mediterranean Sea

This study was conducted on Holothuria polii, Holothuria tubulosa, and Holothuria mammata collected from five stations with different depths in the Northern Mediterranean Sea. The body walls and guts of these holothurians were examined in terms of interactions of 10 metals (iron (Fe), copper (Cu), manganese (Mn), zinc (Zn), chromium (Cr), cobalt (Co), vanadium (V), nickel (Ni), cadmium (Cd), and lead (Pb)) and one metalloid (arsenic (As)) using a multivariate analysis, and interspecies differences were determined. The multivariate analysis of variance (MANOVA) revealed significant differences between the species in terms of metal(loid) accumulations. The principal component analysis (PCA) showed a more association between H. tubulosa and H. polii with regard to the accumulation. The cluster analysis (CA) located Pb concentrations of the guts to the farthest place from all elements regardless of the species. A correlation analysis displayed that the element concentrations of the guts were more closely related to each other compared with those of the walls. The most inconsistent element in terms of correlations was the gut Fe contents. Accordingly, while Fe concentrations of H. mammata and H. tubulosa were correlated with all elements (except Pb) in divalent metal transporter 1 (DMT1) (divalent cation transporter 1 (DCT1) or natural resistance-associated macrophage protein 2 (NRAMP2)) belonging to the NRAM protein family, this was not the case in H. polii. Consequently, significant relationships between accumulated metal(loid)s that changed by tissues and sea cucumber species were observed.

Role of the Cellular Prion Protein in the Neuron Adaptation Strategy to Copper Deficiency

Copper transporter 1 (CTR1), cellular prion protein (PrP(C)), natural resistance-associated macrophage protein 2 (NRAMP2) and ATP7A proteins control the cell absorption and efflux of copper (Cu) ions in nervous tissues upon physiological conditions. Little is known about their regulation under reduced Cu availability, a condition underlying the onset of diffused neurodegenerative disorders. In this study, rat neuron-like cells were exposed to Cu starvation for 48 h. The activation of Caspase-3 enzymes and the impairment of Cu,Zn superoxide dismutase (Cu,Zn SOD) activity depicted the initiation of a pro-apoptotic program, preliminary to the appearance of the morphological signs of apoptosis. The transcriptional response related to Cu transport proteins has been investigated. Notably, PrP(C) transcript and protein levels were consistently elevated upon Cu deficiency. The CTR1 protein amount was stable, despite a two-fold increase in the transcript amount, meaning the activation of post-translational regulatory mechanisms. NRAMP2 and ATP7A expressions were unvaried. The up-regulated PrP(C) has been demonstrated to enhance the cell Cu uptake ability by about 50% with respect to the basal transport, and so sustain the Cu delivery to the Cu,Zn SOD cuproenzymes. Conclusively, the study suggests a pivotal role for PrP(C) in the cell adaptation to Cu limitation through a direct activity of ion uptake. In this view, the PrP(C) accumulation observed in several cancer cell lines could be interpreted as a molecular marker of cell Cu deficiency and a potential target of therapeutic interventions against disorders caused by metal imbalances.

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.

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.

Analysis of a positional candidate gene for inflammatory bowel disease: NRAMP2.

Genome scans have identified a region spanning 40 cM on the long arm of chromosome 12 as a susceptibility locus for inflammatory bowel disease (IBD). This locus contains several candidate genes for IBD, one of which is the gene for the natural resistance associated macrophage protein 2 (NRAMP2). This protein is a divalent cation transporter and is expressed in many cells and tissues. The putative role of this protein in innate immunity prompted us to investigate a possible relationship between NRAMP2 and IBD. We assessed the frequency of four restriction fragment length polymorphisms (IVS2+11A/G, IVS4+44C/A, 1254T/C, and IVS15Ex16-16C/G) in a group of 155 Crohn's disease (CD) patients, 114 ulcerative colitis (UC) patients, and 189 healthy controls. Linkage analysis was performed in a group of 70 families with multiple members suffering from IBD. We searched for additional intragenic markers and mutations by sequence analysis of the natural resistance-associated macrophage 2 gene of 33 CD patients, with a positive family history for IBD. We identified one novel restriction fragment length polymorphism in intron 15 of the gene. The frequency of the rare allele is: 0.08 in our control population. An increased frequency of this allele was found in CD patients but this difference did not reach statistical significance. A weak association between CD and homozygosity for the G allele of the IVS2+11A/G was found (OR [odds ratio] = 2.2, CI [confidence interval] = 1.3-3.9, chi2 = 8.4, p = 1.013). Nonparametric linkage analysis and transmissions disequilibrium tests did not provide evidence for linkage of NRAMP2 to IBD, UC, or CD. Sequence analysis of the exons and the iron-responsive element in a panel of 33 CD patients did not reveal any mutations in NRAMP2. Our association, linkage, and sequence analysis in IBD shows that the putative genetic risk factor on chromosome 12 likely is not NRAMP2. The weak association between the G/G genotype of IVS2+11A/G and CD may be due to linkage disequilibrium with a nearby disease-causing gene.