Hyphantria cunea ferritin heavy chain homologuecDNA sequence and mRNA expression.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 56:21–33 (2004) Hyphantria cunea Ferritin Heavy Chain Homologue: cDNA Sequence and mRNA Expression Hong Ja Kim,1 Chi Young Yun,2 Hyang Mi Cheon,1 Boa Chae,1 In Hee Lee,3 Seun Ja Park,1 Young Jin Kang,1 and Sook Jae Seo1* We have sequenced a cDNA clone encoding a 26-kDa ferritin subunit, which was heavy chain homologue (HCH), in fall webworm, Hyphantria cunea. The HCH cDNA was obtained from the screening of a cDNA library using a PCR product. H. cunea ferritin is composed of 221 amino acid residues and their calculated mass is 26,160 Da. The protein contains the conserved motifs for the ferroxidase center typical for heavy chains of vertebrate ferritin. The iron-responsive element sequence with a predicted stem-loop structure is present in the 5¢-untranslated region of ferritin HCH mRNA. The sequence alignment of ferritin HCH shows 68.9 and 68.7% identity with Galleria mellonella HCH (26 kDa ferritin) and Manduca sexta HCH, respectively. While G type insect ferritin vertebrate light chain homologue (LCH) is distantly related to H. cunea ferritin HCH (17.2– 20.8%), the Northern blot analysis revealed that H. cunea ferritin HCH was ubiquitously expressed in various tissues and all developmental stages. The ferritin expression of midgut is more responsive to iron-fed, compared to fat body in H. cunea. Arch. Insect Biochem. Physiol. 56:21–33, 2004. © 2004 Wiley-Liss, Inc. KEYWORDS: Hyphantria cunea; ferritin; cDNA; cloning; expression INTRODUCTION Iron is an essential element for all living organisms and ferritin is an important iron storage protein. Ferritin is a large hollow sphere with a molecular mass of 440 kDa in vertebrates (Harrison and Arosio, 1996). In vertebrates, ferritin polymers have 24 subunits of two types, heavy (H) and light (L), which are encoded by separate genes (Munro, 1993). The H subunit contains a ferroxidase center involved in rapid iron uptake and oxidation (Lawson et al., 1989). The L subunit is responsible for the formation of the iron core (Levi et al., 1992). In most animals, ferritin spheres are found in the cytosol, where they serve to store ferric ions. On the other hand, most insects store iron in ferritin found in the vacuolar compartment and can secrete it through the Golgi complex and secretory vesicles (Nichol et al., 2002). Insect ferritins are larger (>600 kDa) than vertebrate ferritins, and have larger subunits (24–32 kDa) (Huebers et al., 1988; Nichol and Locke, 1989; Dunkov et al., 1995; Winzerling et al., 1995; Capurro et al., 1996). Insect ferritins were cloned and sequenced from several species (Dunkov et al., 1995; Pham 1 Division of Life Science, Gyeongsang National University, Jinju, Korea 2 Department of Biology, Daejeon University, Daejeon, Korea 3Department of Life Science, Hoseo University, Asan, Korea Kim and Yun contributed equally to this work. Contract grant sponsor: Korea Science Engineering Foundation (ABRL Program) and Brain Korea 21 Project; Contract grant number: R14-2002-056-01001-0. Abbreviations used: A. aegypti = Aedes aegypti; A. germari = Apriona germari; C. ethlius = Calpodes ethlius; D. melanogaster = Drosophila melanogaster; G. mellonella = Galleria mellonella; H. cunea = Hyphantria cunea; M. sexta = Manduca sexta; PAGE = polyacrylamide gel electrophoresis; PCR = polymerase chain reaction. *Correspondence to: Sook Jae Seo, Division of Life Science, College of Natural Sciences, Gyeongsang National University, Jinju, 660-701, Korea. E-mail : firstname.lastname@example.org Received 18 April 2003; Accepted 24 December 2003 © 2004 Wiley-Liss, Inc. DOI: 10.1002/arch.10141 Published online in Wiley InterScience (www.interscience.wiley.com) 22 Kim et al. et al., 1996; Charlesworth et al., 1997; Nichol and Locke, 1999; Du et al., 2000; Kim et al., 2001b, 2002). Insect ferritin subunits resemble those of vertebrates and fall into two classes, according to the overall sequence similarity to vertebrate H or L chains. Although the vertebrate H chain has a greater molecular mass than the L chain, the smaller subunits of ferritins in insects are the homologues of the vertebrate H chains with the conservation of seven metal-binding residues found in the H chains (Charlesworth et al., 1997). The larger ferritin subunits (G) are homologues of the vertebrate L chains. In D. melanogaster, a pair of subunits with apparent 25 and 26 kDa are products of the same gene, and are homologues of the vertebrate H chain, while the 28-kDa subunit is a product of a different gene and this subunit likely serves the role of the vertebrate L chain (Dunkov and Georgieva, 1999). Ferritin from the tobacco hornworm M. sexta is composed of four bands (Pham et al., 1996). Three, including the G subunit, share the same N terminus, whereas the nonglycosylated S subunit has a different N terminus. The HCH and LCH of insect ferritin have been cloned and sequenced for Aedes (Dunkov et al., 1995), Drosophila (Charlesworth et al., 1997; Georgieva et al., 2002), Manduca (Pham et al., 1996), Calpodes (Nichol and Locke, 1999), Galleria (Kim et al., 2001b, 2002), and two tick species (Kopá ek et al., 2003). The fall webworm, Hyphantria cunea, is a polyphagus pest that eats about 160 species of broadleaf trees, and damages the roadside and garden trees around urban areas rather than forest on mountains in Korea (Lee and Chung, 1998). Thus, it can be good as a model of iron metabolism or pest control research for polyphagus insects, according to insect diversity. We have also acquired ferritin gene for further study on iron metabolism since it was purified and some properties of ferritin from H. cunea characterized (Kim et al., 1996; Yun et al., 1998). Here we report that we have cloned and sequenced cDNA encoding ferritin HCH from H. cunea, and compared their deduced amino acid sequences with other insect ferritins. This subunit is a homologue of vertebrate H chain. H. cunea ferritin HCH has seven conserved amino acids common to vertebrate H chain that are required for metal binding. We showed the presence of conserved cysteine residues in the N-terminus and putative iron response element from ferritin HCH of H. cunea. 3¢-Untranslated region (UTR) of this subunit cDNA has several non-canonical putative polyadenylation signals, besides one typical polyadenylation signals. We also report that ferritin HCH of H. cunea is expressed during the developmental stages from larvae to adult and ubiquitously in all tissues tested with the exception of testis. MATERIALS AND METHODS Animals Fall webworm, H. cunea, was reared on an artificial diet (Ito and Tanaka, 1960) at 27°C and 75% relative humidity with a photoperiod of 16 L:8 D. For iron-feeding, larvae were fed on an artificial diet containing 50 mM ferric chloride (FeCl3) for 16 h. Collection and Processing of Hemolymph and Tissues Hemolymph was collected into cold test tubes by cutting forelegs of 4-day-old last instar larvae. A few crystals of phenylthiourea were added to the tubes to prevent melanization. Hemolymph was centrifuged at 10,000g for 10 min to remove hemocytes and cell debris, and the supernatant was stored at –70°C until used. Purification of Ferritin From H. cunea Hemolymph For the improvement of purification yield of ferritin, the procedure by Kim et al. (1996) was modified. The purification of ferritin from H. cunea hemolymph was accomplished by heat treatment at 75°C, KBr density gradient ultracentrifugation (TST 41.14 rotor, Kontron Co.), and TSK-gel DEAE650M anion exchange column (2.15 ´ 20 cm) chromatography using FPLC system (Tosho Co.). After ultracentrifugation at 200,000g for 20 h, ferritin fraction, the brownish precipitate at the bottom of test tube, was removed. The precipitate was Archives of Insect Biochemistry and Physiology H. cunea Ferritin HCH resuspended in 0.02 M Tris-HCl buffer (pH 8.3), and concentrated by removing ammonium sulfate using desalting column (Bio-Rad, Richmond, CA). The preparation in turn was subjected to anion exchange chromatography on TSK-gel DEAE-650 M column (Toyopearl Co.) equilibrated with 0.02 M Tris-HCl buffer (pH 8.3), and eluted between 0.02– 0.3 M buffer gradient at a flow rate of 5 ml/min and with a fraction volume of 5 ml. Electrophoresis Native-polyacrylamide gel electrophoresis (PAGE) was conducted on 4–24.5% gradient polyacrylamide gel as described by Davis (1964). SDS-PAGE was carried out on a 7.5–15.0% gradient separating gel at 15 mA, according to the procedure of Laemmli (1970). The gels were stained in Coomassie Brilliant Blue R-250 after electrophoresis. Determination of Molecular Masses of Proteins Native molecular mass of ferritin was measured on 4–24.5% gradient polyacrylamide gel using thyroglobulin (669 kDa), horse ferritin (440 kDa), catalase (232 kDa), lactate dehydrogenase (140 kDa), and albumin (67 kDa) as standard molecular mass marker proteins (Pharmacia Biotech. Co.). The molecular masses of ferritin subunits were measured on 7.5–15.0% gradient SDS gel as described by Lambin et al. (1976). Standard molecular mass marker proteins used were phosphorylase b (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa), and lysozyme (14.4 kDa) from Bio-Rad. Primer Synthesis, PCR, and Subcloning of PCR Product Degenerate primers between 17 and 20 nucleotides in length were designed from the N-terminal and consensus sequence from HCH insect ferritins. The sense primer (17 mer) was 5¢-TGYCAYATHAAYCCNGT-3¢ (24–29 amino acids) and the antisense primer was 5¢-TGNCCYTTRTAYTGYTCYT-3¢ (181–187 amino acids). cDNA was amplified from cDNA library using May 2004 23 degenerate primers and Taq DNA polymerase. The PCR cycles were as follows; 3 min at 94°C; 35 cycles of 30 s at 94°C/1 min at 40°C/1 min at 72°C; 3 min at 72°C. The amplified PCR product was separated on a 1% agarose gel and a 0.5-kb fragment was obtained. This fragment was excised from the agarose gel, purified, ligated into a T-vector, and amplified in XL1 Blue competent cells. Screening of H. cunea Ferritin cDNA From cDNA Library The cDNA library from the fat body of last instar larvae in H. cunea was kindly provided by Dr. Ho Yong Park (Korea Research Institute of Bioscience and Biotechnology, Daejeon). For screening, 50,000 plaques were plated on 15-cm-diameter agar plates. Nitrocellulose filters (Schleicher & Schuell, Dassel, Germany) were taken from the plates and hybridized at high stringency (65°C and 0.1 ´ SSC) with the DIG-labeled PCR product according to the manufacturer’s recommendation (Boehringer Mannheim Corp., Indianapolis, IN). The positive clones were rescreened at low density on 9-cm-diameter agar plates. Colony hybridization yielded 5 positive clones from the library. Positive plaques were individually isolated in phage buffer by the plate lysate method followed by lambda DNA extraction (Sambrook et al., 1989). The insert of the positive clone was subcloned into an EcoRI site of pBluescript SK(+). DNA Sequencing The manual nucleotide sequence analysis of both DNA strands was performed by the dideoxy chain termination method (Sanger et al., 1977). Six percent acrylamide gels, 35S-radiolabeled nucleotides, and sequenase (US Biochemical, Cleveland, OH) were used, according to the recommendation of the manufacturer. Analysis of Sequence Data The EMBL DataBank was searched with BLAST. Editing and analysis of the DNA sequence data 24 Kim et al. were performed with DNASTAR software (DNASTAR Inc., WI). Northern Blot Tissues were dissected from 4- and 5-day-old last instar larvae (fat body, epidermis, midgut, Malpighian tubule, and testis) and from 8-dayold pupae (ovary). Total RNA (15 mg) from tissues were denatured and subjected to electrophoresis in a 1.2% agarose gel containing 2.2 M formaldehyde. Following electrophoresis, gels were rinsed in 10 ´ SSC and transferred to nitrocellulose (Schleicher & Schuell) in 10 ´ SSC. Blots were prehybridized with 1.5 ´ SSPE, 7% SDS, 10% PEG, 0.1 mg/ml sonicated denatured salmon sperm DNA, and 0.25 mg/ml BSA for 4 h at 65°C. Hybridization was performed for 18 h at 65°C in the prehybridization buffer with 5 ´ 105 cpm/ml of 32P-labeled probe (whole ferritin cDNA), which was prepared by the method of random priming (Feinberg and Vogelstein, 1983). The filter was then washed twice with 1 ´ SSC, 0.1% SDS at 65°C for 15 min, and subsequently two more times for 15 min with 0.1 ´ SSC, 0.1% SDS at 65°C before exposure to X-ray film at –70°C. rRNA was used as an internal loading control. RESULTS AND DISCUSSION Purification of Ferritin HCH in H. cunea On a 4–24.5% gradient polyacrylamide gel, H. cunea ferritin purified from last instar larval hemolymph appears as a single band of about 660 kDa, much larger than horse spleen ferritin, 440 kDa (Fig. 1A). When the purified ferritin was denatured and separated by 7.5–15.0% SDS-PAGE, it appears to be composed of three subunits: 26, 31, and 32 kDa (Fig. 1B); 26 and 32 kDa subunits are major, but the 31-kDa subunit is minor. Molecular mass of ferritin in vertebrates is reported to be approximately 440 kDa and consists of 24 subunits with the molecular mass range of 18–24 kDa. However, insect ferritins and their subunits are reported to be larger than those of vertebrate in molecular mass. C. ethlius ferritin has a molecular mass of Fig. 1. Shown are 4–24.5% native-PAGE (A) and 7.5– 15% SDS-PAGE (B) of ferritin purified from H. cunea hemolymph. A: Lane 1: Horse spleen ferritin; lane 2: ferritin purified from H. cunea hemolymph (about 660 kDa). B: Lane 1: Standard molecular mass marker proteins (BioRad); lane 2: H. cunea ferritin subunits (26, 31, and 32 kDa). more than 600 kDa and is composed of two major subunits (24 and 31 kDa) and two minor subunits (26 and 28 kDa) (Nichol and Locke, 1989). G. mellonella ferritin was determined to have a molecular mass of 630 kDa, and consists of two major polypeptides of 26 and 32 kDa and one minor polypeptide of 30 kDa (Kim et al., 2001a, 2002). Previously the N-terminal sequence of H. cunea ferritin HCH was determined as XPQXHINPV (Yun et al., 1998), which was in agreement with sequenced data. Unfortunately, attempts to perform N-terminal sequencing of the two other ferritin subunits (31 and 32 kDa) failed to produce the result due to almost overlapping of two bands. In an N-terminal sequence of ferritin HCH, the second X was conserved cysteine residue in the N-termini of other insect ferritins. Therefore, the sense primer was produced from the amino acid sequence of CHINPV for the cloning of cDNA encoding ferritin HCH. Archives of Insect Biochemistry and Physiology H. cunea Ferritin HCH Cloning and Sequencing of Ferritin HCH cDNA in H. cunea Lepidopteran hemolymph ferritin is composed of several subunits (Nichol and Locke, 1989; Kim et al., 2001b). We have sequenced the cDNA encoding the smaller hemolymph ferritin subunit 26 kDa of H. cunea, which was HCH. The cDNA library from fall webworm was screened with a PCRgenerated DIG-labeled probe of 0.5 kb. Degenerate primers for the PCR-generated cDNA probe were from the N-terminal and consensus sequence of insect ferritins, which are homologues of the vertebrate H chain. From a total of 50,000 screened 25 plaques, five positive plaques were isolated. Insert lengths were checked after plasmid isolation by digestion with EcoRI and XhoI. One insert was about 1.3 kb, consistent with a full-length cDNA clone for H. cunea ferritin HCH. The length of cDNA for H. cunea ferritin HCH was 1,293 bp (Fig. 2). Within the cDNA sequences was an open reading frame of 663 bp encoding a protein of 221 amino acids with an estimated molecular mass of 25,160 Da. The remaining sequences were untranslated regions. The deduced amino acid sequence encoded by the cDNA matching the N-terminal sequence of ferritin was preceded by a sequence encoding 20 amino acids showing secreted-type ferritin. Ma- Fig. 2. Nucleotide sequences and deduced amino acid sequences of a cDNA encoding ferritin HCH from H. cunea. The iron-responsive element (IRE) is bold-underlined in which the consensus loop is overlined. The signal peptide sequence is in italic type and the N-terminal sequence of matured ferritin is underlined. An asterisk marks the translation stop codon. The polyadenylation signal is boxed and the potential phosphorylation sites are circled. May 2004 26 Kim et al. ture protein without signal peptide was an estimated molecular mass of 23,100 Da. The apparent molecular weight of small ferritin subunit in H. cunea based on SDS-PAGE was approximately 26 kDa. The difference in its molecular weight could be accounted for by posttranslational modifications such as glycosylation and phosphorylation. A computer analysis of our deduced amino acid sequence does not reveal potential glycosylation sites, but phosphorylation sites (Fig. 2). We observed the presence of cysteine residues conserved in the N-terminus of H. cunea ferritin (Fig. 3). In addition to the formation of disulfide bonds, cysteine can be modified by post-translational fatty acid acylation by thioesterification (Schultz et al., 1988; Nichol and Locke, 1999). This kind of linkage acylation could assist in ferritin retention in the vacuolar system when iron levels are low (Nichol and Locke, 1999). The N-terminal extension of the G subunit, or LCH, of insect ferritin contains all three cysteine residues (positions 3, 11, and 23), while two conserved cysteines were found around N-terminus in the S subunit, or HCH, of insect ferritin including H. cunea ferritin HCH. Since the proportion of S subunits increases after iron loading at the same time as holoferritin particles appear in the Golgi and secretory vesicles, the delay of protein exit from ER should be related to G subunit in C. ethlius (Nichol and Locke, 1999). However, further studies are needed for the role of two ferritin types against over-loaded iron. Analysis of the 5¢-UTR of ferritin HCH cDNA revealed the presence of a stem loop structure in H. cunea (Figs. 2 and 4). This stem loop structure appears to be a putative iron-responsive element (IRE). A comparison of IRE in the H. cunea ferritin HCH with IREs from other insect ferritins shows that the consensus structure is well preserved, and the bases of the loop and the c bulge are conserved (Fig. 4). The synthesis of vertebrate ferritin is regulated at the translational level by means of a stem loop in the 5¢-UTR having a consensus CAGUG loop, which binds one of two iron regulatory proteins: IRP1 and IRP2 (Hentze and Kuhn, 1996; Eisenstein and Blemings, 1998). The CAGUGN bases of the loop and the bulge C are required for IRP1 binding (Theil, 1994; Huang et al., 1996), and in addition, the total length of the stem, as well as the flanking regions, can influence the IRP1/IRE interaction (Zhang et al., 2001). Zhang et al. (2001) reported that the human IRP1 repressed translation of the M. sexta HCH and LCH. Fig. 3. Cysteine residues (in box) are conserved in the N-termini of S (top) and G (bottom) type subunit of insect ferritin. N-termini of insect ferritins were aligned using the PAUP program, respectively. The sequence sources: Hc Fer HCH from Hyphantria cunea (26 kDa ferritin), Gm Fer HCH (26 kDa ferritin, AAG41120) and LCH (32 kDa ferritin, AF161709) from Galleria mellonella, Ms Fer HCH (AAK39636) and Ms Fer LCH (AAF44717) from Manduca sexta ferritins, Ce Fer HCH (AAD50238) and Ce Fer LCH (AAD50240) from Calpodes ethlius ferritins S and G, Ag Fer HCH (AAM44043) and Ag Fer LCH (AAM44044) from Apriona germari ferritins 1 and 2, Dm Fer HCH (U91524) from Drosophila melanogaster ferritin. Archives of Insect Biochemistry and Physiology H. cunea Ferritin HCH 27 Fig. 4. Comparison of the putative IREs of ferritin HCH from different insect species. See Figure 3 for abbreviations. Ir Fer HCH, Ixodes ricinus ferritin S (AAC19131); Aa Fer HCH, Aedes agypti ferritin (AY0641060). These results suggest that the translational control of ferritin synthesis by IRP/IRE interaction in insects is very similar to that of vertebrates. But the transcriptional control, in addition to the translational control, has been reported from yellow fever mosquito cells (Aag2 clone), in which iron treatment induces a threefold increase in ferritin mRNA and protein by 16 h (Pham et al., 1999). In the present work with H. cunea, one-typical putative polyadenylation signal (AATAAA) (Fig. 2) and six non-canonical putative polyadenylation signals—AATATA, AATTAA, ATTAAA, AAGAAA, AATATA, and AATAAT—were found (data not shown), and, among them, a last signal located 15 nucleotides May 2004 upstream from poly(A) tail was considered to be most functional. Typical (AATAAA) and modified putative polyadenylation signals were found in cDNA encoding HCH and/or LCH subunits from M. sexta (Pham et al., 1996; Zhang et al., 2001), C. ethlius (Nichol and Locke, 1999), N. lugens (Du et al., 2000), A. aegypti (Dunkov et al., 2002), and G. mellonella (Kim et al., 2001b). Ferritin HCH cDNA in A. aegypti showed the presence of four overlapping non-canonical putative polyadenylation signals proximally to the coding region (Dunkov et al., 2002), and its alternative use of the two polyadenylation signals is confirmed by Northern blot using the probe sequence between the proximal and 28 Kim et al. distal polyadenylation signals (Dunkov et al., 2002). D. melanogaster also produced HCH and LCH messages of different lengths by using two polyadenylation sites (Georgieva et al., 1999, 2002). Georgieva et al. (1999, 2002) reported that Drosophila HCH represented at least four different lengths of mRNAs. That model consists of alternative splicing of the 5¢-UTR of ferritin mRNA containing IRE and the use of two alternative polyadenylation sites. The shorter messages in D. melanogaster lacking an IRE and/or carrying a short 3¢-UTR were also predominant in the midgut and their abundance increased after iron supplementation of the diet. Dunkov et al. (2002) also demonstrated that iron enrichment of the diet results in increased abundance of HCH messages in A. aegypti larvae of messages with short 3¢-UTR particularly. Ferritin heavy chain messages of different lengths resulting from alternative polyadenylation have been described in human (Dhar and Joshi, 1993; Percy et al., 1998). Thus, alternative polyadenylation appears to be a common feature of ferritin mRNAs in vertebrates and insects. It has been reported that differences in the length of the 3¢-UTR could affect the stability, translational efficiency, or localization of various mRNAs (Jackson, 1993). In H. cunea, this kind of change has not been observed. Alignment Sequence Comparison Among Insect Ferritins Figure 5 shows an alignment of the amino acid sequences of H. cunea ferritin HCH. The degree of similarity in the compared regions is quantified in Table 1. To retrieve protein sequences similar to that of H. cunea ferritin HCH, relevant databases were searched using the BLAST (Altschul et al., 1990) and FASTA programs. When the protein sequence data were compared to those of insect ferritins, significant identities were found to Galleria ferritin HCH (26 kDa, 68.9%), M. sexta HCH (68.7%), and other HCH of insect ferritins (40.7– 59.3%). G type insect ferritins, vertebrate L chain homologues are somewhat distantly related to H. cunea ferritin HCH (17.2–20.8%) (Table 1 and Fig. 6). Therefore, reconstruction of the phylogenetic relationships based on amino acid distances revealed two subclades corresponding to the HCH and LCH of insect ferritin, respectively (Fig. 6). Since the percentages of similarity for the H. cunea ferritin HCH to both human heavy (22.5%) and light chains (20.6%) are close in values, no conclusion researching the nature of the H. cunea ferritin HCH can be drawn from this analysis. However, the seven ferroxidase center residues of H chain homologue ferritins are well conserved in the H. cunea ferritin HCH as well as other HCH of insect ferritins from different orders (Fig. 5). This putative ferroxidase center could enable the insect ferritins to incorporate free iron rapidly, allowing ferrous to ferric conversion for iron uptake (Harrison and Arosio, 1996). In non-iron-loaded larvae, the S subunit of C. ethlius is a minor component hemolymph ferritin produced by fat body. In the midgut, however, the S subunit is much more abundant, which enables the midgut to take up iron more rapidly (Nichol and Locke, 1999). TABLE 1. Sequence Homology in the Insect Ferritin Members* Percent identity 1 2 3 4 5 6 7 8 9 10 11 68.7 68.7 82.9 59.3 74.4 70.1 53.8 53.6 52.1 52.4 51.2 51.7 51.7 48.8 47.3 40.7 39.2 41.6 38.8 36.4 39.5 20.8 20.9 22.3 19.0 15.1 21.0 20.1 19.5 19.9 19.4 18.5 17.9 19.5 17.7 67.4 18.6 18.0 20.4 17.6 15.1 19.0 17.2 78.9 68.3 17.2 17.5 19.0 17.6 17.5 18.5 19.1 36.6 35.3 35.7 1 2 3 4 5 6 7 8 9 10 11 Hc Gm Ms Ce Ag Dm Aa Gm Ce Ms Ag Fer HCH Fer HCH Fer HCH Fer HCH Fer HCH Fer HCH Fer HCH Fer LCH Fer LCH Fer LCH Fer LCH *Identities were determined by pairwise alignment using MEGALIGN. See Figures 3 and 4 for abbreviations. Archives of Insect Biochemistry and Physiology H. cunea Ferritin HCH Fig. 5. Multiple amino acid alignment of the insect ferritin subunits. Conserved residues for ferroxidase are boxed. Circle above RGD/E residues indicates an integrin attachment site. Tyr residue sites required for the Fe(III)- May 2004 29 Tyr complex are marked by a triangle. Only one of three Tyr residues is conserved in H. cunea ferritin HCH. Identical residues are marked by asterisk. 30 Kim et al. Fig. 6. Distance-based phylogenetic analysis of H. cuena ferritin HCH and other insect ferritins. The phenogram is based on the alignment shown in Figure 5 and distances are approximate. The letter designation for each of fer- ritin is on the right. G, giga; S, small. A dense line and arrows mark the division between G and S type ferritins. Abbreviations are described in Figures 3 and 4. Other common characteristics of both HCH and LCH of insect ferritin is the conservation of an integrin attachment site consisting of Arg (R), Gly (G), and Asp (D), though aspartic acid is substituted by glutamic acid (E) in most HCH of insect ferritin (marked by circle, Fig. 5). Slight variations of the RGD sequence for cell adhesion have been reported (Plow et al., 1985; Ghiso et al., 1992; Piali et al., 1995; Underwood et al., 1995). The integrinbinding sequence motifs other than RGD share one common amino acid, the aspartic acid (or sometimes the closely related glutamic acid) residue (Ruoslahti, 1996). The aspartic acid may be important because of its potential to contribute to divalent cation binding; one hypothesis regarding the binding of ligands to integrins postulates that the ligand provides a coordination site for divalent cation binding (Edwards et al., 1988). An integrin attachment site could enable ferritin to bind to the cell matrix. That means the characteristics may have a more important function in iron uptake (Nichol and Locke, 1999). Multiple sequence alignment indicates that the H. cunea ferritin HCH like other insect HCH has only one of three Tyr residues (Fig. 5, marked by triangle) required for the formation of the Fe (III)Tyr complex in vertebrate H ferritins (Waldo et al., 1993). In D. melanogaster HCH and most insect LCH, two conserved Tyr residues are found. If this residue is essential, possibly another subunit, LCH of H. cunea ferritin, should be responsible for rapid biomineralization of iron in H. cunea. The pairwise identity between HCH and LCH of insect ferritin is very low. Though they share some identical residues (Fig. 5, marked by asterisk), more of the similarity between HCH and LCH of insect ferritin is accounted for by their common ferritin ancestry than by their insect ancestry (Nichol and Locke, 1999). Tissue Expression of Ferritin HCH in H. cunea Total RNA from various tissues (Fig. 7A and C) and developmental stages (Fig. 7B) of H. cunea were analyzed by Northern blot. Various tissues including gut shows ubiquitous expression of ferritin mRNA with the exception of testis in H. cunea. The expression of ferritin cDNA in epidermis is interesting. Locke and Nichol (1992) reported that iron was involved in several steps of cuticle formation; hydroxylases for tanning, melanization, and wound responses; peroxidases for protein cross linking; and perhaps in hardening and reactions with melanin itself. It was reported that mRNA expression of M. sexta ferritin occurs in midgut, fat body, and Archives of Insect Biochemistry and Physiology H. cunea Ferritin HCH Fig. 7. Northern blot analysis of ferritin HCH mRNA using cDNA coding region as probe in H. cunea. A,B: The expression of H. cunea ferritin HCH in the various organs and developmental stages, respectively. C: The comparative expression of fat body and midgut from control (–) and larvae raised on FeCl3-supplemented diet (+). Te, testis; Mg, midgut; Fb, fat body; Ov, ovary; Ep, epidermis; Mt, Malpighian tubule; L, 4-day-old last instar larvae; PP, prepupae; P4, 4-day-old pupae; P8, 8-day-old pupae; Ad, adult. Total RNA was extracted from fat body of different developmental stage (B). rRNA was used as an internal loading control. For details see Materials and Methods. hemocyte, and among these organs midgut is the major expression site (Pham et al., 1996). In H. cunea, we observed the slight degradation of gut mRNA resulting in dispersed band by Northern blot. Midgut, fat body, ovary, and Malpighian tubule show similar intensity by Northern blot. However, the gut is more responsive to being iron-fed. In the iron-fed group, the prominent increase was observed in gut rather than that of the fat body May 2004 31 (Fig. 7C). Iron increased the amount of spliced and shortened transcripts of Drosophila ferritin in a tissue-specific manner. In the larvae, this change takes place in the gut, but not in the fat body. This is particularly interesting because only increased synthesis and secretion of ferritin from the gut could result in an increased iron excretion (Georgieva et al., 1999). In H. cunea, no difference of mRNA depending on tissue or iron-fed was observed. The expression of ferritin HCH in H. cunea occurs at all developmental stages with a similar intensity suggesting that iron is required at all developmental stages. In C. ethlius, hemolymph holoferritin is sequestered with storage proteins by the premetamorphic fat body to form crystals in granules that disappear in the pupa during adult development (Locke et al., 1991). At the cessation of larval feeding in M. sexta, Fe is rearranged from all tissues to the fat body (Huebers et al., 1988). In adult, rearrangement and formation of organs might make cell metabolism requiring iron activated. Whether HCH and LCH of insect ferritin show different kinds of roles with various tissues or stages will require further study. 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