Aedes aegypti
ferritin
A cytotoxicprotectoragainstironandoxidative challenge?
Dawn L. Geiser
1,2
, Carrie A. Chavez
3
, Roberto Flores-Munguia
1
, Joy J. Winzerling
1,2
and Daphne Q D. Pham
3
1
Department of Nutritional Sciences, College of Agriculture and Life Sciences and
2
Center for Insect Science, The University
of Arizona, Tucson, AZ, USA;
3
Department of Biological Sciences, University of Wisconsin-Parkside, Kenosha, WI, USA
Diseases transmitted by hematophagous (blood-feeding)
insects are responsible for millions of human deaths world-
wide. In hematophagous insects, the blood meal is important
for regulating egg maturation. Although a high concentra-
tion of iron is toxic for most organisms, hematophagous
insects seem unaffected by the iron load in a blood meal. One
means by which hematophagous insects handle this iron
load is, perhaps, by the expression of iron-binding proteins,
specifically the iron storage protein ferritin. In vertebrates,
ferritin is an oligomer composed of two types of subunits
called heavy and light chains, and is part of the constitutive
antioxidant response. Previously, we found that the insect
midgut, a main site of iron load, is also a primary site of
ferritin expression and that, in the yellow fever mosquito,
Aedes aegypti, the expression of the ferritin heavy-chain
homologue (HCH) is induced following blood feeding. We
now show that the expression of the Aedesferritin light-chain
homologue (LCH) is also induced with blood-feeding, and
that the genes of the LCH and HCH are tightly clustered.
mRNA levels for both LCH-andHCH-genes increase
with iron, H
2
O
2
and hemin treatment, and the temporal
expression of the genes is very similar. These results confirm
that ferritin could serve as the cytotoxicprotector in
mosquitoes against the oxidative challenge of the blood-
meal. Finally, although the Aedes LCH has no iron
responsive element (IRE) at its 5¢-untranslated region
(UTR), the 5¢-UTR contains several introns that are alter-
natively spliced, and this alternative splicing event is different
from any ferritin message seen to date.
Keywords: Aedesaegypti mosquito; light-chain ferritin; iron;
oxidative stress; alternative splicing.
In vertebrates, ferritin is found mainly in the cytoplasm.
Cytoplasmic ferritin is a ubiquitous iron storage protein,
and a main site of synthesis is the liver [1]. Vertebrate
cytoplasmic ferritin contains 24 subunits, made of heavy
(H) and light (L) polypeptide chains that are encoded by
different genes [2]. The H-chain is responsible for the rapid
oxidation and uptake of iron [3], whereas the L-chain
creates the nucleation site for ironand formation of the
iron core ) a complex of iron, phosphate and oxygen
[4,5].
When murine erythroid leukemia cells are stably trans-
fected with the gene coding for the H-chain subunit, the
cellular labile iron pool, as measured by calcein fluorescence,
is significantly lower than that of nontransfected cells [6].
Yet the total cellular iron concentration, as measured by
atomic absorption, and the cellular reductive power, as
measured by glutathione levels, remain unchanged. Fur-
thermore, when the cells are treated with hydrogen peroxide
(H
2
O
2
), an inverse relationship is seen between cell damage
and the level of expression of ferritin. Based on these data,
Epsztejn and colleagues concluded that the vertebrate
ferritin H-chain acts as a regulator of the cellular labile
iron pool and an attenuator of the cellular oxidative
response. They proposed that the first line of defense against
chemically induced oxidative stress is the increase in
expression of the ferritin H-chain [6].
Expression of the ferritin L-chain message is also aug-
mented in oxidative stress-related diseases [7]. Tsuji et al.[8]
determined that oxidants induce the expression of both the
ferritin H- and L-chain messages. Northern blot analyses
indicate that both H
2
O
2
and tert-butylhydroquinone induce
ferritin H and L mRNA in a dose-dependent manner and
that this induction is inhibited by actinomycin D. This
response differs from those seen previously for hormones,
cytokines and certain chemicals that up-regulate transcrip-
tion of only the H-chain gene [9–14]. These data suggested
that transcriptional regulation of the ferritin genes is
important in a cell’s response to oxidative stress. Tsuji and
others postulated that ferritin functions as a cytoprotective
protein whose role is to sequester free iron to minimize
oxidative damage [8,15].
Hematophagous insects receive a toxic level of heme in
their blood meal, yet they are unaffected by the iron load
and the oxidative challenge. For these animals, one defense
against the iron load andoxidative challenge is perhaps, as
in vertebrates, through the iron storage protein ferritin. Like
vertebrate ferritins, insect ferritins are heteromultimers.
One type of subunit, the H-chain homologue (HCH),
shares significant similarity with the vertebrate H-chain and
Correspondence to D. Q D. Pham, Department of Biological
Sciences, University of Wisconsin-Parkside, Kenosha,
WI 53141–2000, USA.
Fax: + 1 262 595 2056, Tel.: + 1 262 595 2172,
E-mail: daphne.pham@uwp.edu
Abbreviations: HCH, heavy-chain homologue; LCH, light-chain
homologue; UTR, untranslated region; IRE, iron
responsive element.
(Received 12 May 2003, accepted 9 June 2003)
Eur. J. Biochem. 270, 3667–3674 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03709.x
contains all residues that form the ferroxidase center
involved with rapid iron uptake (reviewed in [16]). Another
subunit, the L-chain homologue (LCH), shares significant
similarity with the vertebrate L-chain but does not contain
the glutamate residues involved in iron nucleation [16]. In
Drosophila, iron treatment induces both HCH and LCH
messages and subunits [17,18].
As hematophagous insects ingest heme proteins in
volumes several times their body weight, they need
multiple defence mechanisms to accommodate the mas-
sive iron load. These mechanisms must work in conjunc-
tion to avoid the cytotoxic effects of free radicals resulting
from the interaction between heme or free iron and
oxygen [19–21]. In our previous work, we isolated and
sequenced the genomic clone that encodes the Aedes
ferritin HCH, and found that the expression of this gene
is induced by iron treatment and blood feeding [22,23].
We now report the identification and sequence of both
the cDNA and genomic clones for the Aedes ferritin
LCH. We found that iron, H
2
O
2
and hemin treatment
induce the expression of both ferritin LCH and HCH
messages in Aag2 cultured cells. The expression of LCH
also increases significantly with blood feeding in
whole animals. Taken together, these data suggest that
up-regulation in the expression of the ferritin genes
follows iron-treatment or blood-feeding might serve as a
cytoprotective protein that sequesters free iron to mini-
mize iron-mediated oxidative stress in the gut and
hemolymph (blood). We also found that the LCH gene
has no iron responsive element (IRE) and utilizes an
alternative splicing event different from all known ferritin
genes.
Experimental procedures
Identification of the genomic and cDNA clones
encoding the ferritin LCH
During our attempt to obtain a clone that contains the
upstream region of the ferritin HCH gene, we obtained a
4-kb genomic clone predicted to encode the ferritin LCH.
The identification of the genomic clone was performed as
described previously [22]. Once the nucleic acid sequence of
this clone was determined, we identified the cDNA clone by
the following procedure. Total RNA was isolated from
A. aegypti CCL-125 cells (American Type Culture Collec-
tion, Manassas, VA, USA) using the RNeasyÒ Mini Kit
(Qiagen, Valencia, CA, USA), according to the manufac-
turer’s instructions. Purified RNA was treated with DNase I
(Invitrogen) for 15 min at 25 °Candfor10minat70°C.
The total RNA was reverse-transcribed with Super-
Script
TM
II RNase H
–
Reverse Transcriptase (Invitrogen)
according to the manufacturer’s instructions. PCR was
performed using Taq DNA polymerase (Invitrogen) with
primers designed from the genomic sequence: 5¢-TTCA
CCGCCCAGTTTTCCTCA-3¢ and 5¢-CTCCACCTT
GTCCAGGTATTC-3¢ (Fig. 1A, double-line arrows) for
35 cycles (1 min at 94 °C, 1 min at 64 °C, 1 min at 72 °C).
We obtained a 582-bp PCR product that was cloned into
pGEMÒ-T Easy Vector (Promega); the deduced amino
acid sequence matched that of the ferritin LCH gene
sequence. The 3¢-UTR of the cDNA was obtained using
a RLM-RACE library (FirstChoice
TM
RLM-RACE Kit,
Ambion, Austin, TX) made from mRNA isolated from
CCL-125 cells by Poly(A) QuikÒ mRNA Isolation Kit
(Stratagene). The 3¢-RACE was conducted using Expand
Fig. 1. The nucleic acid sequence, deduced amino acid sequence and
schematic representation of the A. aegypti LCH and HCH genes.
(A) The nucleic acid sequence and deduced amino acid sequence of
the genomic clone for the A. aegypti LCH gene (GenBank accession
number AY171561). + 1 HCH, transcriptional start-site of the HCH
gene; + 1 LCH, transcriptional start-site for the LCH gene. Nucleo-
tides with no number, the nucleotide sequence deposited previously for
the HCH gene (GenBank accession number AF126431); nucleotides in
introns, boldface, lower-case letter; nucleotides in exons, upper-case
letters; nucleotides in cassette exon, boldtype and underlined, upper-
case letter; amino acids, italicized, upper-case letters; single-line arrow,
primer for primer-extension analysis; double-line arrows, primers for
RT-PCR; triple-underline arrows, primers for RACE. N-glycosylation
site, dotted box; polyA site, double line; TATA-box, boxed letter;
transcriptional start sites, caret; X, amino acids different from those
reported previously [27]. (B) Schematic representation of the A. aegypti
LCH and HCH genes. +1, transcription initiation site; ATG, start
codon; TAA, stop codon; AATAA, poly adenylation site; boxes,
exons; lines, the introns; numbers of bases in the exons, under the
boxes, and numbers of bases in the introns, between the boxes. The
figure is not drawn to scale.
3668 D. L. Geiser et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Long Template PCR System (Roche) with a primer designed
from the ferritin LCH genomic sequence 5¢-CTGTACC
GCAAGATCTCCGAC-3¢,nestedprimer5¢-GAATAC
CTGGACAAGGTGGAG-3¢ (Fig. 1A, triple-underline
arrows), and an adapter primer provided in the RLM-
RACEKitfor1cyclefor3minat94°C followed by
35 cycles for 30 s at94 °C, 30 s at 60 °C, 3 min at 42 °C, and a
final cycle extension for 7 min at 72 °C. This procedure
gave a 594-bp anda 177-bp product. Initially, the 5¢-end
was obtained using RACE, gene-specific primer, 5¢-GTC
GGAGATCTTGCGGTACAG-3¢ (GS1), nested primer 5¢-
CGGTGGAATTATTATTGTCAGCG-3¢,nestedprimer
5¢-GTTCCCAGGATGAACTTCATG-3¢ (Fig. 1A, triple-
underline arrows), and adapter primers provided by the
RACE kit (Invitrogen). PCR was performed as stated
above for the cDNA clone. Products (Clones R179, R221,
R273 and R329; Fig. 3) were cloned into pGEMÒ-T Easy
Vector and sequenced. After the 5¢-RACE was completed,
primers were designed for the two transcriptional start sites.
These primers (5¢-CTCATAAGCGATCAGATATTCG-3¢
and 5¢-CCCCCCAACGAGTACTCTC-3¢) together with
GS1 primer were used to obtain the alternatively spliced
products (P280, P297, P394, P410, P433, P449, P502 and
P518; Fig. 3).
The genomic clone and all PCR products were sequenced
in both directions by automated cycle sequencing using
Big Dye Terminator Kit (Applied Biosystems Inc.) on an
Applied Biosystems 377 automated DNA sequencer with
ÔXLÕ upgrades (DNA Sequencing Facility, Arizona
Research Laboratories, University of Arizona, Tucson,
AZ, USA). The deduced amino acid sequences were
analyzed using software from the National Center for
Biotechnology Information (http://www.ncbi.nlm.nih.gov/,
BLAST
[24]) and
CEA
(DNA Strider
TM
, France).
Northern blot analyses and primer extension analyses
RNA from Aag2 cultured cells [25] and A. aegypti animals
was isolated by Trizol reagent (Invitrogen) according to the
manufacturer’s suggestions. Northern blot analyses were
performed with standard protocols [26], except that gel
electrophoresis was performed with 20 m
M
NaP
i
buffer and
3% formaldehyde. Primer extension analyses were per-
formed as described previously [22]; the primer used had the
following sequence: 5¢-GCGAGCAAGGCAACGGTTCC
CAGGATGAAC-3¢ (Fig. 1A, single-line arrow). Total
RNA (10 lg) was used for blood-fed animals and 30 lg
was used for sugar-fed animals. A higher amount of total
RNA (30 lg) was used for sugar-fed animals to compensate
for the fact that the abundance of the ferritin message is
lower in sugar-fed animals.
Glycosylation assays
Aedes larval ferritin was isolated and purified as described
previously [27]. The ferritin sample was treated with
O-glycosidase and PNGase F (Glycopro GE50 deglycosy-
lation kit; Prozyme, San Leandro, CA, USA) according to
the manufacturer’s instructions and analyzed (20 lgprotein
per well) by 18.75% homogeneous SDS/PAGE. Proteins
were visualized with Coomassie Blue staining. Fetuin and
horse spleen ferritin (Sigma) were used as positive and
negative controls, respectively. The molecular mass stand-
ards were from Invitrogen.
Animals and cells
The CCL125 and Aag2 cultured cells were maintained as
described previously [25,28]. The animals were a generous
gift from S. C. Johnson Co. (Racine, WI, USA). The
animals from a wild-type stock collected in Orlando,
Florida at the USDA, ARS, CMAVE [29] were reared
and fed as described previously [30]. Males were left on
sugar water for the entire experiment. All adult animals used
were 5-days-old, and larvals used were 4th instar.
Results
The nucleic acid sequence of the cDNA has a 663-bp ORF,
which encodes a predicted 221-amino acid peptide with a
M
r
of 25 kDa (Fig. 1A). The putative methionine start
codon is preceded by several in-frame stop codons, and
the region around the designated start site follows Kozak’s
rule (PuNNATGPu) [31], indicating that this sequence
represents the full-length ORF. The deduced amino acid
sequence shows significant identity to known ferritin
L-chains and is therefore predicted to be a L-chain homo-
logue. Our deduced amino acid sequence deviates by two
residues (Fig. 1A, X) from the N-terminus sequence iden-
tified for the 28-kDa Aedesferritin subunit reported previ-
ously [27]. Similar to other insect ferritin subunits, the
deduced amino acid sequence for the Aedes LCH subunit
also contains a signal peptide (Fig. 1A, amino acids 1–19).
The Aedes LCH gene has 5 exons and 4 introns; several
introns are small (108, 69 and 66 bp) (Fig. 1A,B). Primer
extension analyses indicate that transcriptional initiation for
the LCH gene involves multiple start sites (Fig. 1A, ÔvÕ and
boldtype; Fig. 2). These data are corroborated by the 5¢-end
RACE and RT-PCR data, in which products with multiple
transcriptional start sites were also obtained (Fig. 3). Of the
RACE and RT-PCR products, four had the first intron
removed (Fig. 3, Ô1Õ), two had the second intron removed
(Fig. 3, Ô2Õ), two had the region from the beginning of the first
intron to the end of the second intron removed (Fig. 3, Ô12Õ),
and four had no intron removed (Fig. 3, Ô0Õ). Data from
RACE and RT-PCR showed that both transcriptional
initiation sites were used (Fig. 3, Ô1, 12, 2 and 0Õ). Primer
extension analysis, RACE and RT-PCR results also agree
with the observation that none of the transcription start sites
(Fig. 1A, ÔvÕ) match the consensus sequence for an eukary-
otic transcriptional initiation site (PyPyANT/APyPy) and
that the TATA-box for the LCH gene is noncanonical
[Fig. 1A, boxed (tatattt vs. tatat/aat/a)].
Although the Aedes LCH subunit has very low similarity
with the vertebrate L-chain, a PHD algorithm [32] still
predicts four a-helices for the mosquito chain as seen for
thevertebrateL-chains(Fig.4,ÔˆˆˆÕ). However, predic-
tions using SwissPdbViewer [33,34] (http://www.embl-
heidelberg.de/predictprotein/predictprotein.html) indicate
that the structural differences between the Aedes LCH
subunit and known vertebrate L-chain is significant as no
log trace can be obtained (data not shown). The Aedes LCH
subunit also lacks the cluster of glutamic acids (Fig. 4, Ô›Õ)
that functions as the porphyrin-binding pocket in the
Ó FEBS 2003 Aedesaegyptiferritin response to ironandoxidative challenge (Eur. J. Biochem. 270) 3669
vertebrate ferritin L-chains [35–38]. Only two sites in this
pocket are semiconserved or conserved. The loss of the
glutamic acid cluster indicates that ferrihydrite nucleation is
probably different for the Aedes LCH subunit. In addition,
although four amino acids (Fig. 4, Ô^D#ßÕ)thatmakeupthe
salt bridges (Fig. 4, Ô"^D#ßÕ) are semiconserved or con-
served, the substitutions of these sites in the Aedes LCH
subunit make bridging unlikely. As these salt bridges
maintain the stability of vertebrate ferritins, the loss of
these bridges in the mosquito ferritin suggests either that the
insect ferritin is less stable or is stabilized by other forces.
Current data support the latter hypothesis because insect
ferritins have been shown to be quite stable [27,39,40].
Prosite analysis [41] predicts that the deduced amino acid
sequence of the Aedes LCH gene contains an N-glycosyla-
tion site at position 22–25 NNST (Fig. 1A, dotted box).
This prediction is substantiated by our enzymatic deglyco-
sylation assay; the 28-kDa subunit is deglycosylated
following treatment with FNGaseF, an N-linked deglyco-
sylation enzyme (Fig. 5, lane 8), but not O-glycosidase, an
O-linked deglycosylation enzyme (Fig. 5, lane 7).
Fig. 2. Multiple transcriptional start sites for ferritin LCH gene. Tran-
scripts were analyzed by primer extension analysis. The left panel shows
the autoradiograph of the analysis. G, A, T, C represent standard
sequencing reactions using the oligoprimer (single-line arrow) in
Fig. 1A with termination mix ddG, ddA, ddT or ddC, respectively.
Numbers on the right of the DNA ladder are the number of nucleotides
obtained by numbering from the end of the oligoprimer used in the
analysis; S, primer extension using total RNA from sugar-fed females;
B, primer extension using total RNA from blood-fed females. The
schematic representations of the splicing products are shown on
the right-hand side. Nucleotide sequences, sequences at the 5¢-end of
the splicing products; open box, exon 1; vertical-striped box, exon 2;
checked box, exon 3; black lines, introns. The sizes of the splicing
products are shown on the right. The inset at the bottom represents the
autoradiograph for a shorter electrophoresis run of the same primer
extension analysis and shows the smaller splicing products.
Fig. 3. PCR products showing alternative splicing of the A. aegypti
LCH message. The names of the clones are shown on the left of the
sequence and the intron(s) removed is(are) shown on the right. Product
names starting with ÔRÕ, RACE products; ÔPÕ, RT-PCR products; 1,
intron 1 removed; 12, intron 1, exon 2 and intron 2 removed; 2, intron
2 removed and 0, no intron removed; –, no nucleotide;
ATG, start of
translational start site; line between diamonds, connection site for
exons 3 and 4; :, sequence continued as shown in Fig. 1A; boldtype
nucleotides, primers used in RT-PCR.
3670 D. L. Geiser et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Our data indicate that the LCH message is induced by
iron, H
2
O
2
, hemin and blood feeding (Fig. 6), suggesting
that LCH expression is transcriptionally regulated. When
Aag2 cells were treated with iron, expression of the LCH
gene increased in a dose-dependent manner and reached a
maximum at 100 l
M
(Fig. 6A). Induction was observed
8 h post iron treatment and continued into 16 h post iron
treatment (Fig. 6A). Both H
2
O
2
and hemin treatments
(Figs 6B,C) showed similar expression patterns to the iron
treatment. For a 100 l
M
H
2
O
2
treatment, induction occured
at 16 h post-treatment. For 500 l
M
H
2
O
2
treatment,
induction was seen at 4 h and continued into 16 h post-
treatment. No induction was observed for the H
2
O
2
treatment at concentrations lower than 100 l
M
(data not
shown). For a 10 l
M
hemin treatment, induction was seen
at 4 h post-treatment and increased progressively to 16 h.
Hemin treatment at >10 l
M
resulted in a similar expression
pattern, however, cell viability was so low that no conclu-
sions could be reached (data not shown).
Northern blot analysis was also used to study develop-
mental changes in LCH message expression. LCH mRNA
expression is low in adult males (M) and sugar-fed females
(SF), but is up-regulated in blood-fed females (B) (Fig. 6D).
As both H
2
O
2
and hemin induce LCH mRNA expression,
the induction following blood feeding could reflect an
oxidative challenge caused by the heme in a blood meal.
LCH message does not seem to be induced in 4th instar
larvae,ascomparedtotheactinlevel(Fig.6D,L).
Our Northern analyses indicate that the HCH gene also
responds to iron, H
2
O
2
, and hemin in a very similar manner
to that of the LCH (Fig. 6). The intensity of the response is
slightly higher for the HCH than LCH gene. Interestingly,
the temporal response for both LCH and HCH messages
are nearly identical for all three treatments. From our
previous work, developmental expression of the HCH gene
is also quite similar to that of the LCH gene [23].
Discussion
Our data show that the A. aegypti LCH gene lies adjacent
to and in opposite orientation to the HCH gene (Fig. 1B).
These data are in agreement with those obtained for
Drosophila, where the ferritin genes are also located adjacent
to and in opposite direction from each other [42]. As in
Drosophila, the head-to-head organization of the ferritin
genes also suggests that they are coordinately controlled
[17]. This hypothesis is supported by the similar temporal
expression patterns of these genes (Fig. 6), which suggest
that there is no insulator between the genes because
currently known insulators are found between genes with
independent profiles of expression (reviewed in [43,44]). Our
data further indicate that the Aedes LCH gene contains a
noncanonical TATA-box (Fig. 1A, box). Primer extension
analysis and RACE data show that transcription initiates at
multiple start sites (Figs 2 and 3). This situation parallels
transcriptional initiation seen for TATA-less promoters,
where multiple transcriptional start sites are observed,
perhaps as a result of a random response to the lack of a
strong selector [45–48].
Our data indicates that the expression of the Aedes LCH
message involves an alternative-splicing event like that seen
with the Drosophila HCH message [49]. However, unlike the
Fig. 4. Comparison of A. aegypti LCH with
LCH sequences from other species. The acces-
sion number for the Anopheles gambia LCH is
EAA08169; Calpodes ethlius LCH, AF161710;
Drosophila melanogaster LCH, AF145124;
Manduca sexta LCH, L47123; Nivaparvata
lugens LCH, AJ251147; Homo sapiens
H-chain, AAA35833 and H. sapiens L-chain,
AAA52439. r, ferroxidase center; #"Deß,
salt bridges; ›, porphorin-binding pocket;
ˆˆˆ, alpha helices; gray boxes, semicon-
served and black boxes, conserved.
Ó FEBS 2003 Aedesaegyptiferritin response to ironandoxidative challenge (Eur. J. Biochem. 270) 3671
Drosophila HCH message, the first intron of the LCH
message does not contain an IRE (Fig. 1A), and the
alternative splicing is quite different. Aedes LCH alternative
splicing involves a regulated exon that is included or
excluded from the mRNA ) the cassette exon 2 [50]
(Figs 1B and 3). This cassette exon is located at positions
142–186 and is relatively small (Fig. 1). The ÔsmallnessÕ of
exon 2 (44 bp) should lead to exon skipping [51,52].
Surprisingly, exon skipping does not seem to be a major
event in Aedes as shown by the retention of both exons 2
and 3 (79 bp) (Figs 1 and 3).
All alternatively spliced products are observed for the
LCH message (Figs 2 and 3). This lack of a definitive
splicing choice in the LCH message points to a tissue-
specific regulation (reviewed in [50]). Furthermore, as the
cassette exon is not part of the coding region, its removal
will not alter the protein (Fig. 1A). This observation
suggests that the alternative splicing event could allow for
the use of alternative promoters or promoter elements [50].
The involvement of transcriptional regulation in this process
is further supported by recent observations that the removal
of the 5¢-UTR significantly increases expression of the LCH
gene in both iron-treated and untreated cells in transient
transfection assays (data not shown).
The Northern blot analyses suggest that regulation of the
Aedes LCH gene is at the transcriptional level and are in
agreement with the observation that the Aedes LCH gene
contains no IRE (Figs 1 and 6). In mammals, the IRE
allows translational control of ferritin synthesis. Data from
Drosophila [17,42] and Anopheles [53,54] also show that
these dipteran LCH genes have no IRE yet are induced by
iron. Previously, we found that transcriptional regulation is
important for the HCH gene under iron overload condi-
tions [25]. Now, we report that transcriptional regulation is
probably also important for both HCH and LCH genes
under oxidative challenge as the expression of both HCH
and LCH message increases with H
2
O
2
or hemin treatment
(Fig. 6B,C).
The blood meal of hematophagous insects contains
10 m
M
heme [55]. The concentration of heme increases
as the meal is digested because water is excreted rapidly
through the Malpighian tubules [56]. Yet, these animals
seem unaffected by this oxidative challenge [19]. Previous
works [19–21] indicate that hematophagous insects use
multiple mechanisms to deal with the heme challenge (e.g.
by polymerizing heme into hemozoin or by binding heme to
proteins and thus in nontoxic form). We now show that
another mechanism by which hematophagous insects could
use to ward off oxidative stress andiron challenge is by
inducing the ferritin expression because treatment with iron,
H
2
O
2
or hemin induces expression of both ferritin genes
(Fig. 6A–C).
Notably, the time of response for LCH and HCH is very
similar and may explain how these subunits are assembled
(Fig. 6). Most insect ferritin subunits contain a signal
peptide and are secreted [16]. In fact, insect ferritins have
been isolated from the hemolymph (blood) [18,39,40] and
from medium of Aag2 cultured cells (data not shown).
Fig. 5. Evidence for N-linked glycosylation in ferritin LCH. Purified
ferritin from A. aegypti 4th instar larval was treated with O- and
N-linked deglycosylation enzymes and analyzed by 18.75% homo-
geneous SDS/PAGE. Proteins were visualized with Coomassie Blue
staining. 1, molecular mass standards; 2, fetuin (positive control);
3, fetuin treated with deglycosylation enzymes; 4, horse spleen ferritin
(negative control); 5, horse spleen ferritin treated with deglycosylation
enzymes; 6, A. aegypti ferritin; 7, A. aegyptiferritin treated with
O-glycosidase; 8, A. aegyptiferritin treated with PNGaseF.
Fig. 6. Specific induction of A. aegyptiferritin LCH and HCH mRNA
expression by iron, H
2
O
2
, hemin and bloodfeeding. Actin, message for
the actin gene; LCH, message for the ferritin LCH gene; HCH,mes-
sage for the ferritin HCH gene. (A) Iron treatment. Aag2 cells were
treated with ferrous ammonium sulfate (FAS) as described previously
[25]. Top panel, concentrantion of ferrous ammonium sulfate (FAS)
used; second panel, hours post iron-treatment. (B) H
2
O
2
treatment.
Abbreviations are as A, except that H
2
O
2
was used. (C) Hemin
treatment. Abbreviations are as A, except that hemin was used. (D) In
whole animals. L, 4th instar; M, adult male; S, adult female, sugar fed;
B, adult female, blood fed.
3672 D. L. Geiser et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Previous work indicated that insect ferritins assemble in the
rough endoplasmic reticulum but are not secreted immedi-
ately [40,57]. The similar temporal expression patterns of the
LCH and HCH genes shown here suggest that both types
of subunits were synthesized simultaneously and that the
subunits could enter the lumen of the rough endoplasmic
reticulum about the same time to form the oligomer ferritin
shell.
Acknowledgements
This work was supported by funds from the National Institutes of
Health, National Institute of General Medical Sciences (GM56812 to
J. J. W. and GM55886 to D. Q D. P.), and the Agricultural Experi-
ment Station, College of Agriculture and Life Sciences, University of
Arizona. The authors thank D. Higgs for his input, and H. Meier,
M. C. Meier, T. Jones and S. C. Johnson Co. (Racine, WI, USA) for
providing the Aedesaegypti mosquitoes.
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3674 D. L. Geiser et al. (Eur. J. Biochem. 270) Ó FEBS 2003
. Aedes aegypti ferritin A cytotoxic protector against iron and oxidative challenge? Dawn L. Geiser 1,2 , Carrie A. Chavez 3 , Roberto Flores-Munguia 1 , Joy J. Winzerling 1,2 and Daphne. Our data further indicate that the Aedes LCH gene contains a noncanonical TATA-box (Fig. 1A, box). Primer extension analysis and RACE data show that transcription initiates at multiple start sites. (PyPyANT/APyPy) and that the TATA-box for the LCH gene is noncanonical [Fig. 1A, boxed (tatattt vs. tatat/aat /a) ]. Although the Aedes LCH subunit has very low similarity with the vertebrate L-chain,