Báo cáo khoa học: Aedes aegypti ferritin A cytotoxic protector against iron and oxidative challenge? ppt

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

Aedes aegypti ferritin

A cytotoxic protector against iron and oxidative challenge?

Dawn L Geiser1,2, Carrie A Chavez3, Roberto Flores-Munguia1, Joy J Winzerling1,2and Daphne Q.-D Pham3

1

Department of Nutritional Sciences, College of Agriculture and Life Sciences and2Center for Insect Science, The University

of Arizona, Tucson, AZ, USA;3Department 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 Aedes ferritin 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- and HCH-genes increase with iron, H2O2 and hemin treatment, and the temporal expression of the genes is very similar These results confirm that ferritin could serve as the cytotoxic protector 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: Aedes aegypti 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 iron and formation of the

iron core) a complexof 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

(H2O2), 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 H2O2and tert-butylhydroquinone induce ferritin Hand 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 and oxidative 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)

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, H2O2 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 aegyptiCCL-125 cells (American Type Culture

Collec-tion, Manassas, VA, USA) using the RNeasy 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 25C and for 10 min at 70 C

The total RNA was reverse-transcribed with

Super-ScriptTMII 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

GTCCAGGTATTC-3¢ (Fig 1A, double-line arrows) for

35 cycles (1 min at 94C, 1 min at 64 C, 1 min at 72 C)

We obtained a 582-bp PCR product that was cloned into

pGEM-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 (FirstChoiceTMRLM-RACE Kit, Ambion, Austin, TX) made from mRNA isolated from CCL-125 cells by Poly(A) Quik 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-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.

Long Template PCR System (Roche) with a primer designed

from the ferritin LCH genomic sequence 5¢-CTGTACC

GCAAGATCTCCGAC-3¢, nested primer 5¢-GAATAC

CTGGACAAGGTGGAG-3¢ (Fig 1A, triple-underline

arrows), and an adapter primer provided in the

RLM-RACE Kit for 1 cycle for 3 min at 94C followed by

35 cycles for 30 s at 94C, 30 s at 60 C, 3 min at 42 C, and a

final cycle extension for 7 min at 72C This procedure

gave a 594-bp and a 177-bp product Initially, the 5¢-end

was obtained using RACE, gene-specific primer, 5¢-GTC

GGAGATCTTGCGGTACAG-3¢ (GS1), nested primer

5¢-CGGTGGAATTATTATTGTCAGCG-3¢, nested primer

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 pGEM-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]) andCEA(DNA StriderTM, 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 mMNaPibuffer 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

Aedeslarval 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 lg protein

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

Mrof 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 Aedes ferritin 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-boxfor 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 the vertebrate L-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

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#Õ) that make up the

salt bridges (Fig 4, Ô"^D#Õ) 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 posiN-glycosyla-tion 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 mixddG, 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.

Our data indicate that the LCH message is induced by

iron, H2O2, 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 lM(Fig 6A) Induction was observed

 8 h post iron treatment and continued into 16 h post iron

treatment (Fig 6A) Both H2O2 and hemin treatments

(Figs 6B,C) showed similar expression patterns to the iron

treatment For a 100 lMH2O2treatment, induction occured

at 16 h post-treatment For 500 lM H2O2 treatment,

induction was seen at 4 h and continued into 16 h

post-treatment No induction was observed for the H2O2

treatment at concentrations lower than 100 lM (data not

shown) For a 10 lMhemin treatment, induction was seen

at 4 h post-treatment and increased progressively to 16 h

Hemin treatment at >10 lMresulted 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 H2O2and hemin induce LCH mRNA expression,

the induction following blood feeding could reflect an

oxidative challenge caused by the heme in a blood meal

LCHmessage does not seem to be induced in 4th instar

larvae, as compared to the actin level (Fig 6D,L)

Our Northern analyses indicate that the HCH gene also

responds to iron, H2O2, 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; #"De,

salt bridges; ›, porphorin-binding pocket;

ˆˆˆ, alpha helices; gray boxes,

semicon-served and black boxes, consemicon-served.

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 LCHgene 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 H2O2or hemin treatment (Fig 6B,C)

The blood meal of hematophagous insects contains

10 mM 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 and iron challenge is by inducing the ferritin expression because treatment with iron,

H2O2 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 aegypti ferritin treated with

O-glycosidase; 8, A aegypti ferritin treated with PNGaseF.

Fig 6 Specific induction of A aegypti ferritin 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.

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

LCHand 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 Aedes aegypti mosquitoes.

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