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First published online May 1, 2006
Journal of Experimental Biology 209, 1988-1995 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02193
Immunolocalisation of the D. melanogaster Nramp homologue Malvolio to gut and Malpighian tubules provides evidence that Malvolio and Nramp2 are orthologous
1 Institute of Genetics, The University of Nottingham, Queen's Medical
Centre, Nottingham NG7 2UH, UK
2 School of Biological Sciences, University of Southampton, Bassett Crescent
East, Southampton SO16 7PX, UK
* Author for correspondence (e-mail: chb{at}soton.ac.uk)
Accepted 28 February 2006
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Key words: Nramp, divalent cation transport, Drosophila melanogaster, innate immunity, brain, haemocyte, Malpighian tubule, gut cell
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), is the prototypic member of a family of genes that are
widely conserved (Cellier et al.,
1995), and Nramp1 controls natural resistance and
susceptibility to infection within strains of mice. The Nramp1
phenotype is associated with a non-conserved amino acid substitution
G169D (Malo et al.,
1994); allele D169 is null
(Vidal et al., 1995).
Nramp genes encode highly hydrophobic polytopic integral membrane
transporter proteins, and the mutation outlined above maps to the fourth of 12
putative membrane-spanning domains. Two paralogous genes have been identified
in human and mouse (Vidal et al.,
1993; Barton et al.,
1994; Gruenheid et al.,
1995), although in mouse a third gene fragment, more closely
related to mouse Nramp1 has been described, but not functionally
characterised (Dosik et al.,
1994). Nramp1, isolated for its role in controlling the
growth of intracellular pathogens (Plant
and Glynn, 1974; Bradley,
1974; Gros et al.,
1981), is exclusively expressed in murine macrophages. The
ubiquitously expressed Nramp2/Slc11a2 gene is implicated in
the proton-dependent divalent cation uptake into red blood cells, the uptake
of cations by the duodenal enterocyte, and in the
transferrin–transferrin-receptor-mediated pathway of cellular iron
assimilation (Fleming et al.,
1997; Gunshin et al.,
1997). Nramp2 is a carrier of a non-conserved mutation,
G185R, in microcytic anaemia strains in both mouse (mk)
(Fleming et al., 1997) and rat
(Belgrade) (Fleming et al.,
1998). The unequivocal role of Nramp2 in divalent cation
transport has lead to the suggestion that Nramp1 also transports divalent
cations, and that susceptibility to infection is a manifestation of impaired
macrophage cation transport. However, the precise role of Nramp1 in infection
biology has been difficult to define. Consequently there is continuing
controversy regarding the direction of cation transport and the mechanism of
pathogen growth-control (Kuhn et al.,
1999; Goswami et al.,
2001; Jabado et al.,
2000; Gomes and Appelberg,
1998). This is proposed to be either by divalent cation starvation
(Jabado et al., 2000;
Gomes and Appelberg, 1998) or
through a Fenton chemistry reactive-oxygen species killing process
(Kuhn et al., 1999). What is
clear is that Nramp1 plays a major role in regulating the inflammatory
response within the macrophage; macrophages from D169 or null strains
exhibit a reduced ability to synthesize inflammatory mediators compared with
their G169 allele counterparts, and the action of Nramp1
clearly constitutes a major component of the innate immune response
(Blackwell and Searle, 1999).
Nramp1 may also play a role in homoeostatic cation transport associated with
the salvage and recycling of divalent cations within splenic macrophages and
hepatic Kupffer cells from effete erythrocytes or other apoptotic cells, and
also in cation transport in haemorrhagic disease
(Atkinson and Barton, 1998;
Biggs et al., 2001;
Mulero et al., 2002). It is
not clear if this role is a direct activity of Nramp1 or a
pleiotropic effect of Nramp1G169 macrophages having a more
hostile phagosomal microenvironment and greater ability to degrade ingested
cells (Hackam et al.,
1998).
The use of Drosophila melanogaster is increasing for the
identification and study of the genes that play a role within the innate
immune response, in view of the simplicity with which mutant strains can be
generated and the similarities between flies and mammals in many aspects of
these pathways (Hoffmann et al.,
1999). Work by the Ezekowitz group
(Ramet et al., 2002;
Franc et al., 1999) has
demonstrated the power of this organism in defining key genes associated with
phagocytosis, a prerequisite process in the removal of adventitious pathogenic
organisms, and dead or dying cells. The completion and on-going annotation of
the D. melanogaster genome sequence has indicated that it is unique
amongst many multicellular organisms analysed to date in that only a single
copy of the Nramp gene family, known as Mvl
(Malvolio), is found (Rodrigues
et al., 1995), although there are multiple transcripts and two
distinct polypeptide species differing at the C-terminal region. This finding
begs the question as to whether Mvl represents an Nramp1 or
Nramp2 orthologue, or whether Mvl is a bifunctional
Nramp gene. If the latter scenario appears correct then D.
melanogaster is a suitable organism to study the biology and function of
a primitive Nramp gene that may have key properties from both
mammalian paralogues. Mvl was cloned in a screen for genes that
influence taste perception; a hypomorphic mutation in Mvl, results in
decreased preference to sugar and an increased acceptance of salt
(Rodrigues et al., 1995). This
chemosensory phenotype is unusual for an Nramp gene based our
knowledge from studies on mammalian Nramps. Electrophysiology in
these Mvl mutants indicates no abnormal responses within the
peripheral neurons suggesting that the mutation is likely to influence events
downstream. Observations, using a ß-galactosidase reporter gene assay, in
the specific enhancer trap line used to isolate Mvl, indicate
expression within the peripheral and central nervous system, as well as in
some cells of the haematopoietic system, notably the macrophages of the embryo
and the adult. However, studies were not directed to the tissue localisation
of the Mvl protein or its sub-cellular localisation.
Recent complementation studies showed Nramp2, but not
Nramp1, rescues the transport defect of yeast divalent cation
transport mutants (Pinner et al.,
1997; Tabuchi et al.,
1999). Complementation studies in D. melanogaster, with
an ectopically and ubiquitously expressed human NRAMP1 gene, show no
behavioural or physiological effects in the normal animals, however the taste
defect is rescued in the Mvl mutant strain by NRAMP1
(D'Sousa et al., 1999), and
also by rearing of flies in the presence of millimole concentrations of
divalent cations (Orgad et al.,
1998), indicating that this phenotype is a direct consequence of
sub-optimal cation concentrations. Unfortunately, results on the
complementation by Nramp2/NRAMP2 were not reported. One conclusion
from these data is that Mvl is orthologous with
Nramp1/NRAMP1. However, the more widespread expression of
Mvl than Nramp1, and the more restricted expression of
Mvl compared with Nramp2, does not support this proposal.
We, therefore, set out to investigate Mvl protein expression, using an
in-house prepared anti-Mvl polyclonal antibody, and to compare it with the
ß-galactosidase reporter, using the Mvl enhancer trap line. Our
results revealed expression in most tissues was either of a punctate nature,
diffuse or both and was observed in macrophage-like cells, Malpighian tubules,
testis, brain, the aminoserosa of embryos and the larva and adult alimentary
canal.
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Materials and methods |
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),
and the eluted IgG was found to be reactive against the Mvl–GST fusion
protein, but not native GST. Affinity-purified antibody was used in all
histochemical applications. To confirm that the antibody was reacting against
native Mvl sequences in vivo two overlapping oligopeptides were
produced MSSNE AYAHEP GAGGD GPGGS SGASG GGSQR SNQLH HQQIL; HQQIL LNETT YLKPA
AKQAY FSDEK VLIPD DDSTN VGFSF RK and used to block immunoreactivity. In
control in vitro experiments the peptides blocked reactivity of the
affinity-purified serum against the Mvl-GST, but not the crude serum against
the native GST, as predicted, indicating that the blockade was specific. To
further characterise the anti-Mvl antiserum the ORF from full-length Mvl cDNA
was cloned, by PCR, into the pcDNA3.1 expression plasmid and transfected into
Cos-1 cells, as before. Whole cell extracts from these cells were used in
western blotting experiments, as described before, using ECL (GE Healthcare UK
Ltd, Little Chalfont, UK).
Immunohistochemistry of Drosophila tissue
Oregon R D. melanogaster Meigen embryos were collected and
prepared for immunohistochemistry using standard techniques. Tissues were
dissected in Drosophila saline and fixed in 4% paraformaldehyde. Embryos and
tissues were blocked in 3% normal horse serum (NHS; 3%), and incubated with
anti-Mvl primary antibody (1:1000) overnight. Tissues were washed and
incubated with biotinylated anti-rabbit IgG (1:200; Vector Laboratories,
Peterborough, UK). After washing, tissues were incubated with avidin- and
biotin-conjugated horseradish peroxidase, washed, and processed as recommended
by the manufacturer. Embryos were covered in glycerol (70%) and mounted using
a gelatin:glycerol mountant. Adult and larval brains and testis were treated
in essentially the same way, with an acid wash after fixation to aid antibody
penetration.
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Immunohistochemistry
Embryonic haemocytes
Mvl immunoreactivity was first detected in the syncitial blastoderm in a
punctate pattern of expression (not shown), and later at stage 15 in the
amnioserosa, as previously reported
(Rodrigues et al., 1995). Mvl
immunoreactivity was also detected at this stage, within a subset of cells
that are scattered throughout the haemocoel of the embryo. These cells contain
large phagosomes, are presumed to be phagocytic, and are possibly haemocytes.
The Malvolio-positive staining within these macrophage-like cells was
restricted to an unidentified subcellular compartment, which appeared to be in
close association with the large phagosome
(Fig. 2A,B). This subcellular
staining was blocked with peptide pre-treatment
(Fig. 2C). Haemocyte-like cells
containing large phagosomes, and Mvl immunoreactivity were also seen
throughout pupal stages (data not shown).
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). The location of staining differed between samples, however,
in all preparations examined staining was limited to within the region of the
lower ureter to the main segment (Fig.
4F). The second, small discrete cell type
(Fig. 4G), often had thin
processes projecting into the haemocoel (not shown). These cells were limited
to the upper ureter and lower tubule in all samples examined.
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Adult brain and gonad
Mvl immunoreactivity was observed within cells scattered throughout the
adult brain. These cells were presumed to be neuronal, based on size and
shape. Staining was again punctate and probably confined to a subcellular
compartment (Fig. 5A). Staining
was also found specifically within a sub-region of the gonad of the adult male
fly known as the accessory gland. Cells at the apex of this gland had a
diffuse sub-cellular stain that was blocked with peptides
(Fig. 5C,D).
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), but
also allowed a much greater detailed analysis of Mvl expression.
Study of Mvl is of particular interest, as it represents a prototypic member
of the Nramp gene family, as genome sequencing has revealed only a
single copy of this highly conserved gene family in D. melanogaster
and it is unique amongst many other higher eukaryotes where at least two
orthologues have been described. The D. melanogaster enhancer trap
line, from which Mvl was identified, has an unusual phenotype, of
taste perception, compared with that of the mammalian Nramps. The
relationship between divalent cation transport and the function of the
gustatory pathway is not obvious at present, however, in this study we were
unable to detect expression of Malvolio within either the third antennal
segment, neurons of the maxillary palps or labellar chemosensory neurons,
which had been previously described by reporter gene. This difference could be
due to low level or transient expression of Mvl in these tissues, making
immunological detection difficult. Nramp-mediated cation transport is
associated with both the acquisition of divalent cations from the duodenal
lumen, the transport of divalent cations into cells through the
transferrin–transferrin-receptor pathway of iron uptake and in the
transport of cations associated with defence against intracellular macrophage
pathogens. The transport of cations within macrophages by Nramp1 constitutes a
branch of the innate immune response, but a role is also possible in
homoeostasis and pathology associated with the salvage and recycling of
cations from apoptotic cells, and effete red cells. The data from this current
study support this concept of transport for iron salvage/recycling, as strong
Mvl expression was demonstrated within haemocytes during embryonic and also
pupal stages of the D. melanogaster life cycle, when considerable
tissue remodelling occurs.
A significant and novel finding from this study was the observation that
the Mvl protein was expressed within the D. melanogaster gut at both
larval and adult stages. Expression within the midgut is consistently
associated with two regions, the anterior midgut and the posterior midgut. The
anterior region of the midgut, known as the cuprophilis region is an important
site for nutrient absorption, especially dietary copper
(Filshie et al., 1971;
Dubreuil et al., 1998). The
posterior region of the midgut is equally, if not more, important in nutrient
absorption, and is populated by iron-accumulating cells
(Dimitriadis and Kastritsis,
1983; Poulson and Bowen,
1952). A hallmark of Nramp2 is expression within enterocytes,
where it has a pivotal role in the uptake of iron and other divalent cations
from the duodenal lumen and where it is subjected to regulation by cation
availability. It would be of significant interest to assess if Mvl
expression was also sensitive to dietary iron. There is a precedent for this
as iron loading can rescue the taste defect
(Orgad et al., 1998). Although
not the direct target of the pathogenic sequence alteration, there is an
increased prevalence of NRAMP2 in patients with hereditary haemochromatosis,
an iron overload disease, which is associated with increased uptake of
divalent cations. It would be of interest to overexpress Mvl and
examine whether enhanced iron acquisition also leads to tissue injury, as
observed in untreated haemochromatosis. The decreased expression, or impaired
targeting or function of Nramp2 also contributes to iron deficiency in
microcytic anaemia. Since Mvl also was localised within gut cells, we conclude
that it also participates in the acquisition of iron and other divalent
cations for Drosophila growth and development. Conditional
Mvl mutants could be of value for modelling this human disease. These
data were supportive of an analogous mechanism for the acquisition of divalent
cations from the gut lumen for both flies and mammals. Interestingly, Mvl is
also expressed in the Drosophila fat body, an organ that has been
shown to play a role in iron uptake, storage and release back into the
haemolymph in insects (Huebers et al.,
1988). Since the release of the entire D. melanogaster
genome sequence, it has become clear that whereas many genes involved in iron
homeostasis are conserved between insects and vertebrates, many are not,
indicating important differences in their metabolism of iron
(Nichol et al., 2002).
However, these data are clearly supportive of Mvl providing the conduit for
iron acquisition for all stages of D. melanogaster development.
Patches of anti-Mvl staining were found along the length of the Malpighian
tubules, within large cells that are similar in appearance to the principal
(type 1) cells. In addition, a smaller cell type was also stained in a
discrete area of the tubules. There were small cells of 3–4 µm
diameter, with thin processes that, based on their size, appearance and
location within the lower tubule, are likely to be the neuroendocrine cells
described previously (Sozen et al.,
1997). These cells demonstrated homogeneous staining throughout,
which could either represent cytoplasmic or more probably surface staining.
The patchy and inconsistent staining of the principal cells within the tubules
is likely to reflect differences in the response to dietary iron load between
specimens. Cells of the gut also displayed both punctate and diffuse staining
that could also be surface staining. Given that Mvl shows punctate
intracellular staining in many other cells, and that there are three variants
of Mvl, it is possible that variant Mvl forms could be targeted to
different subcellular structures
(http://flybase.bio.indiana.edu/).
It is also possible that Mvl reflects Nramp2 localisation, showing both plasma
membrane and recycling endosome staining, where it is colocalised with
transferrin. However, to address this issue would require the development of
isotype-specific antibodies, but this is not without difficulty given the
small differences between protein variants.
Expression of Mvl within the Malpighian tubules was novel and suggestive of
this protein playing a role in divalent cation reabsorption. This is similar
to the Nramp2 gene that is proposed to participate in a re-uptake system for
divalent cation at the brush border of kidney proximal tubules
(Canonne-Hergaux and Gros,
2002). Our studies also indicated that Mvl is expressed within the
testis. A study of Nramp2 in phagocytic Sertoli cell lines of the testis shows
Nramp2 association with early and late endosomes
(Jabado et al., 2002).
Following phagocytosis, Nramp2 becomes associated with the phagosome and is
proposed to participate in active transport at the phagosomal membrane in
these cells in providing essential iron for spermatogenesis. Animals that are
hypotransferrinemic, have reduced levels of spermatogenesis indicating the
important role of iron in this and other processes. The localisation of Mvl
within the testis may support an analogous function in providing iron for the
maturing sperm cells.
Western blot analysis of homogenates from embryos, larvae, pupae and adult Drosophila, revealed two distinct bands of approximately 42 kDa and 50 kDa. This compares with the predicted molecular masses of 53.3 kDa and 65.5 kDa. However, hydrophobic proteins often run anomalously in SDS-PAGE and post-translational modification of Nramp1 also influences apparent size. The 50 kDa band appeared in extracts from embryos and pupae. The 42 kDa band appeared in extracts from embryos, larvae and adult, but not pupae. We have shown that at these stages Mvl is expressed in phagocytic haemocytes, and it is possible that it is the 50 kDa Mvl that plays a role in divalent cation transport, during the extensive tissue remodelling at these stages. As the 42 kDa band appeared in extracts from embryos, larvae and adult, but not pupae it is possible that the 42 kDa Mvl is involved in divalent metal uptake, and distribution at these stages, but not transport associated with the phagosome.
These studies have revealed novel sites for Mvl expression that clearly
supports its orthology with Nramp2, however, expression within cells
displaying a phagocytic morphology also supports its orthology with Nramp1. We
conclude that Mvl is a bifunctional divalent cation transporter protein, based
on cell distribution and is likely to have functional characteristics of both
proteins, although it is not clear how the antiport–synport controversy
of Nramp1 (Goswami et al.,
2001; Jabado et al.,
2000) will relate to Mvl function. Furthermore, this study does
not reveal how divalent cation transport is linked to the gustatory
circuit.
Future studies might be directed at examining the DNA regulatory sequences
controlling Mvl expression within particular cellular structures and the
identification of motifs within the protein that target it to particular
sub-cellular regions. Furthermore, given that iron regulatory protein (IRP)
proteins are found in Drosophila
(Muckenthaler et al., 1998)
and that Drosophila ferritin carries an iron response element
(Georgieva et al., 1999) it is
a distinct possibility that other genes, possibly including Mvl, may
carry a functional iron response element to modulate function.
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Acknowledgments |
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