Download Ectopic expression of natural resistance

Biochemical Society Transactions (1998) 26
10
Ectopic expression of natural resistance-amdated
macrophage protein-1 in COS-1 cells modulates iron
accumulation
. .
Table 1.
S199
COS-I &
Cell associated iron levels
Peter G.P. Atkinson and C.Howard Barton
University of Southampton, Division of Biochemistry and Molecular
Biology, Bassett Crescent East, Southampton SO16 7PX, UK
Nrampl (natural resistance-associated macrophage protein) confers
nsistance to infection toward a number of unrelated intracellular
pathogens in the mouse [I]. Based on primary sequence analysis,
Nrampl encodes an integral membrane protein with a putative
transport function [2,3]. This conclusion is consistent with other
members of the highly conserved Nramp gene family. For example,
Nramp2 has been shown to transport a number of metal ions when
expressed in frog oocytes [4], and the Nramp-related yeast genes,
SMFl and SMF2 were found to encode high and low aftinity
manganese transporters respectively [5]. The phagocytic location of
Nrampl [6] suggests that the protein is ideally situated to influence
the micro-environment of the invading pathogen within the
phagosome. Here we show that Nrampl can influence cellular iron
levels. Ectopic expression of Nrampl in COS- 1 cells modulated iron
levels following loading cells with exogenous iron. This important
finding has implications for how Nrampl may confer resistance to
microbial infection in vivo and the normal role of the protein in
macrophage function.
Transient transfections into COS- 1 cells were performed
using lipofectamine (BRL) according to the manufacturer’s
instructions with a pcDNA3 N r a m ~ l allele
’ ~ ~ expression plasmid.
Experiments were controlled by transfecting with the pcDNA3
plasmid alone. Cells were used 48 hours post-transfection for iron
uptake experiments. Iron was presented to the cells in the form of a
low molecular chelate, iron:NTA (nitrilotriacetate) complex using a
1: 1 molar ratio. Transfected COS-1 cells were extensively washed in
RPMI medium alone to remove serum and then incubated in RPMI
medium containing 50pM Fe : NTA for 4 hours at 37’C. Cells were
chilled on ice, extensively washed in ice-cold PBS containing 5mM
EDTA (pH7.4) and harvested into the same buffer. Aliquots were
removed for protein (BCA Pierce) and iron determination. Cellular
iron levels were measured as described [7] using the ferrozine
method.
In three independent experiments, successful Nrampl protein
expression in COS-1 cells was confirmed by Western blotting with an
anti-Nrampl specific anti-serum (data not shown). As expected,
extracts from cells transfected with vector alone failed to show any
reactivity towards the anti-serum (data not shown). COS-I cells,
incubated in serum-free media alone, or in the presence of 50pM
NTA did not contain measurable cellular iron as assayed by the
ferrozine method (data not shown). In contrast, COS-1 cells
accumulated measurable quantities of this metal in the presence of
extracellular iron. However, Nrampl transfected cells were
consistentlyassociated with lower net iron levels when compared to
the experimental controls. These reductions ranged from 24-55%
and averaged 39% over the three experiments (Table I). There was
some inter-experimental variation in iron levels in both control and
Nrampl positive cells, but the differences measured between the
control and experimental were consistent across experiments.
In spite of the major advances in our knowledge of the
genetics and molecular biology of the Nrumpl locus , the function
of the protein at the biochemical level remains enigmatic. However,
by analogy with other Nramp family members, it appears probable
that Nrampl acts as a metal ion transporter of unknown specificity.
We now present evidence demonstrating that Nrampl can modulate
nmol.Wmg proteid4h
Percent (Z)
Experiment 1
Control
64.0
100
Nrarnpl
49.0
76.6
Experiment 2
Conuol
38.5
100
Nrampl
17.4
45.2
Experiment 3
Control
18.5
100
Nrampl
12.1
65.5
the. cellular accumulation of extracellular iron. Ectopic expression of
Nrampl in COS-I cells was consistently associated with a reduction
in the measured iron content when compared to COS-1 cells
transfected with vector alone. A mean reduction in cell associated
iron of nearly 40% was observed over three experiments. This result
is supported by the work of Zwilling and co-workers [8] who noted
a 50% reduction in the cellular iron content in .I-IFN stimulated
macrophages tiom Nramp 1-resistant mice compared to macrophages
from Nrampl-susceptible mice [8]. Based upon its endosomal
location, it is tempting to speculate that in professional phagocytes
Nrampl functions to shuttle internalised iron across the
endosomdphagosomal membrane into the cytosol. Therefore, it
might be speculated that in Nrampl transfected COS-1 cells,
intemalised iron was more efficiently transported into the cytosol
from the endosome than in Nrampl minus controls. Increases in
cytosolic redox active “free” iron would be buffered by fenitin and
other chelating agents, but in the short term the efflux of iron from
the cell may be expected to increase. In the absence of Nrampl, the
movement of iron from the endosome to the cytosol would be
slowed, with iron accumulating within the endosome. Therefore iron
efflux from the cell would not be as highly stimulated, with the net
result being an increase in the total iron associated with the cells not
expressing Nrampl. This hypothesis would also explain why
Nrampl-susceptible macrophages were associated with twice the iron
levels of Nrampl-resistant macrophages [I].Thus both the pleiotropic
effects of Nrampl and its role in host defence can start to be
understood in terms of cellular iron regulation.
This work is supported by grants from the Royal Society and the BBSRC.
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