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

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 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 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-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 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 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 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) 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 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¢,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 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-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 Aedes aegypti ferritin response to iron and oxidative 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 Aedes aegypti ferritin response to iron and oxidative 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 and iron 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. 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. 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. 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