High level expression of Nramp1^G169 in RAW264.7 cell transfectants: analysis of intracellular iron transport

Abstract

Nramp1 (natural resistance-associated macrophage protein) was positionally cloned as the defective biallelic locus in inbred mouse strains associated with uncontrolled proliferation of obligate intracellular macrophage pathogens. The causative defect was described as G169D within membrane spanning domain 4 of a transporter. The biochemical activity of Nramp1 is implied from sequence conservation with Nramp2. Nramp2 encodes a divalent cation transporter and is the carrier of a defect in models of microcytic anaemia, associated with impaired intestinal iron uptake. Iron sequestration has been proposed as an antimicrobial mechanism. Therefore, such an activity for Nramp1 is consistent with model systems. Here we showed that Nramp1 directs iron transport within the macrophage. We describe stable, high-level Nramp1G169 allele-derived polypeptide expression in Balb/c Nramp1D169 RAW264.7 cells. Transfectants express levels, comparable to those in Nramp1G169-resistant macrophages, of a 90-100x103 MW Nramp1 polypeptide. Expression of the Nramp1 polypeptide correlates with lower cellular iron loads and a reduced chelatable iron pool following challenge with iron: nitrilotriacetate. Pulse chase experiments support an enhanced iron flux in expressing cells. These data are supported using the fluorescent iron probe calcein. In Nramp1G169-expressing cells we observed an increased iron flux into the cytoplasm from a calcein-inaccessible cellular location. These data suggest Nramp1, in resting macrophage cells, mobilizes iron, from an intracellular vesicle, which is destined for cell secretion. We propose that under these conditions Nramp1 plays a role in a salvage pathway of iron recycling.

Evolution of Differences in Transport Function in Slc11a Family Members

“…A key question is whether antiport activity co-evolved in the vertebrate line to support life on land, or whether it evolved in birds and mammals specifically as a mechanism for resistance again intracellular pathogens? The influence of Slc11a1 on efficiency of iron recycling by macrophages (6,55) supports the hypothesis that Slc11a1 may have evolved with land-dwelling vertebrates to facilitate iron recycling from effete red cells. Hence, the influence of Slc11a1 on resistance to intracellular infection may be a by-product of, rather than the driving force for, evolution of the Slc11a1 subfamily.…”

“…They compared phagosomes isolated from macrophages stably expressing wild type Slc11a1 Gly 469 versus mutant/functionally null Slc11a1 Asp 469 , and observed greater import of iron into phagosomes from wild type macrophages. They also found that phagosomes isolated from wild type cells pre-labeled with 55 Fe-citrate before phagocytosis contained up to four times more iron compared with phagosomes from mutant cells, and that treatment of wild type macrophages with the lysomotrophic agents chloroquine or ammonium chloride reduced the import of iron significantly (23,24). Multiple groups have compared intracellular iron levels and determined that mutant macrophages have higher cytoplasmic iron than wild type macrophages (6,54,55,57,58).…”

“…They also found that phagosomes isolated from wild type cells pre-labeled with 55 Fe-citrate before phagocytosis contained up to four times more iron compared with phagosomes from mutant cells, and that treatment of wild type macrophages with the lysomotrophic agents chloroquine or ammonium chloride reduced the import of iron significantly (23,24). Multiple groups have compared intracellular iron levels and determined that mutant macrophages have higher cytoplasmic iron than wild type macrophages (6,54,55,57,58). Macrophages expressing wild type Slc11a1 also have a greater capacity to recycle iron (6), in particular when iron is acquired by Fc receptor-mediated phagocytic uptake of transferrin/anti-transferrin complexes that are delivered to Slc11a1-positive late endosomes and lysosomes, and not when iron is delivered to early endosomes by transferrin-receptor-mediated uptake.…”

“…By insertion of an HA tag into the extracellular loop between TMD7 and TMD8 Forbes and Gros (56) found that Slc11a1 was mis-targeted to the plasma membrane of Chinese hamster ovary cells, allowing direct study of transport function at the plasma membrane. Using either the metal-sensitive fluorophors calcein and Fura2, or 55 Fe 2ϩ and 54 Mn 2ϩ radioisotopes, these authors demonstrated uptake of Fe 2ϩ , Mn 2ϩ , and Co 2ϩ into HA-tagged Slc11a1-transfected cells. Slc11a1 transport was dependent on time, temperature, and acidic pH, with metal ion transport occurring down the proton gradient.…”

Allelic association between the NRAMP1 gene and susceptibility to tuberculosis in Guinea–Conakry

“…This appears to be the first association of a non-MHC gene and an infectious disease supported in family studies, but with increasing attention to the analysis of family-based association studies (Gruenheid et al 1997). Localized on the phagolysosomal membrane, it is probably involved in regulation of cellular iron levels (Atkinson & Barton, 1999) and may affect the pH of the phagolysosome (Hackam et al 1999). Because Nramp1 genotypes in mice determine the susceptibility\resistance phenotype through a single amino acid substitution, a G169D change, NRAMP1, the human homologue, is a candidate gene for mycobacterial disease.…”