Isolation and characterization of mosquito ferritin and cloning of a cDNA that encodes one subunit.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 29:293-307 (1995) Isolation and Characterization of Mosquito Ferritin and Cloning of a cDNA That Encodes One Subunit Boris C. Dunkov, Dianzheng Zhang, Kyril Choumarov, Joy J. Winzerling, and John H. Law Department of Biochemistry and the Center for Insect Science, Uriiversity of Arizona, Tucson, Ar-izona Ferritin, an iron storage protein, was isolated from larvae and pupae of Aedes aegypti grown in an iron-rich medium. Mosquito ferritin i s a high molecular weight protein composed of several different, relatively small, subunits. Subunits of molecular mass 24, 26, and 28 kDa are equally abundant, while that of 30 kDa is present only in small amounts. The N-terminal sequence of the 24 and 26 kDa subunits are identical for the first 30 amino acids, while that of the 28 kDa subunit differs. Studies using antiserum raised against a subunit mixture showed that the ferritin subunits were present i n larvae, pupae, and adult females, and were increased in animals exposed to excess iron. The antiserum also was used to screen a cDNA library from unfed adult female mosquitoes. Nine clones were obtained that differed only in a 27 bp insertion in the 3' end. Rapid amplification of cDNA ends (RACE) was used to obtain the complete protein coding sequence. A putative iron-responsive element (IRE) is present in the 5'-untranslated region. The deduced amino acid sequence shows a typical leader sequence, consistent with the fact that most insect ferritins are secreted, rather than cytoplasmic proteins. The sequence encodes a mature polypeptide of 20,566 molecular weight, smaller than the estimated size of any of the subunits. However, the sequence exactly matches the N-terminal sequences of the 24 and 26 kDa subunits as determined by Edman degradation. Of the known ferritin sequences, that of the mosquito is most similar to that of somatic cells of a snail. o 1995 ~ i ~ e y - ~Inc. is, Key words: ferritin, cDNA sequence, Aedes aegypti, mosquito, cloning, expression Acknowledgments: The authors are grateful to Dr. Henry Hagedorn, Department of Entomology and the Center for Insect Science, University of Arizona, for the helpful discussions in the course of this work and to Drs. John Andersen and Ren6 Feyereisen, Department of Entomology and the Center for Insect Science, University of Arizona, for kindly providing the RACE kit. This work was supported by grants from the John D. and Catherine T. MacArthur Foundation, 8900408, and the U.S. Public Health Service, A132595 and GM29328. Received October 26, 1994; accepted February 15, 1995. Address reprint requests to John H. Law, Ph.D., Department of Biochemistry, University of Arizona, Tucson, AZ 85721. The sequence reported in this paper has been deposited in the GenBank data base (accession no. L37082). 0 1995 Wiley-Liss, Inc. 294 Dunkov et al. INTRODUCTION Iron is an essential element for all living organisms. However, ionic iron in the presence of oxygen can form free radicals that are destructive to biological materials. Consequently, ferric ions in biological systems are chelated with small molecules or sequestered by specific proteins. The principal biological iron storage protein is ferritin. This protein consists of 24 small subunits (-20 kDa) arranged in a cage-like sphere in which ferric iron is stored as a crystalline mineral (Crichton and Ward, 1992;Meldrum et al., 1992). Although vertebrate ferritin is a cytoplasmic protein, a glycosylated form occurs at low levels in blood (Campbell et al., 1989; Theil, 1990a). Iron metabolism has been extensively studied in vertebrates, and ferritins have been isolated from a wide variety of organisms, including bacteria and plants (Andrews et al., 1991, 1992). In contrast, iron metabolism in insects has been a relatively neglected field. To date, ferritins from two insect species (Heubers et al., 1988; Nichol and Locke, 1989; Winzerling et al., 1995) have been reported. Locke and Nichol (1992) noted several important differences between vertebrate and insect ferritins. Insect ferritins have more mass (660 kDa) than vertebrate ferritins (440 kDa), exist primarily in the vacuolar system rather than in cytoplasm, are abundant in insect blood (hemolymph), and are usually glycosylated with mannose-rich chains. Hemaphagous insects transmit several vertebrate diseases; thus the study of iron metabolism in these organisms could have value in designing important control strategies. Further, insects that feed on vertebrate blood receive a diet rich in iron, and therefore might have a distinctive iron metabolism. For these reasons, we are investigating iron metabolism of the yellow fever mosquito, Aedes aegypti. We report here the isolation and properties of a ferritin from this insect and we have successfully cloned a cDNA that encodes one of the ferritin subunits. MATERIALS AND METHODS Insect Culture and Iron Loading A. aegypti of the Rockefeller strain were raised at 27"C, 80% humidity with a 12h:12h 1ight:dark photoperiod according to Shapiro and Hagedorn (1982). Larvae were iron loaded by adding 0.05 M FeC13 to the culture water for a final concentration of 0.005 M FeC& (Nichol and Locke, 1990). Late fourth instar larvae or pupae were collected, rinsed, and immediately frozen at -80°C. Isolation of A. aegypti Ferritin A. aegypfi larvae or pupae were ground in 50 mM HEPES, pH 7.4, containing 5 mM phenylthiourea and 1 mM PMSF* (5 ml buffer/g insects) using a *Abbreviations used: ELISA = enzyme-linked immunosorbent assay; FlTC = fluorescein isothiocyanate; IRE = iron-responsive element; IRP = iron-regulatory protein; NFDM = non-fat dry milk; PBS = 0.1 M sodium hosphate, 0.15 M NaCI, pH 7.0; PCR = polymerase chain reaction; PMSF = phenylmethanesul onylfluoride; RACE = rapid amplification of cDNA ends; RT = room temperature; SDS = sodium dodecyl sulfate; TBS = 0.02 M Tris, 0.1 M NaCI, pH 7.5; UTR = untranslated region. P Mosquito Ferritin: Characterization and Cloning 295 Polytron homogenizer (PCU-2, Kinematica, Switzerland), 3 x 30 s at maximum speed. The homogenate was sonicated on a Branson Sonifier 200 (Branson Sonic Power Co., Danbury, CT) for 2 x 45 s, at 50%maximum power to disrupt the membranes of the vacuolar system (Nichol and Locke, 1989) and centrifuged for 15 min at 15,OOOg. The supernatant was filtered through 4 layers of filter paper (Whatman No. 1) and centrifuged at 180,OOOg for 1 h at 15°C in a Beckman (Fullerton, CA) Ti 60 rotor. The resulting pellet was homogenized in 1 ml buffer per g larvae or pupae and the homogenate was heated at 75°C for 15 min (Nichol and Locke, 1989), cooled on ice, and the heat-denatured proteins were removed by centrifugation (15 min at 15,OOOg). The supernatant was layered over a KBr solution saturated at 15°C and centrifuged in a Beckman Ti 60 rotor at 180,OOOg for 22 h at 15°C (Winzerling et al., 1995).Under these conditions some KBr crystals formed on the bottom of the tube. The brown pellet that contained ferritin was suspended in a small volume of 50 mM HEPES buffer, pH 7.4, layered on the saturated KBr solution and centrifuged as above. The final brown pellet was resuspended in 50 mm HEPES, pH 7.4, and the KBr was removed by dialysis. Protein Gel Electrophoresis Native PAGE was performed in 6.0% homogeneous slab gels using the buffer system described by Davis (1964).Electrophoresis of ferritin under nonreducing conditions in SDS 5.0% homogeneous slab gels was done by solubilizing the protein in 80mh4 Tris, pH 6.8,1% SDS, 10%glycerol without boiling. SDS-PAGE (reducing conditions) was performed according to Laemmli (1970) using 12.5% homogeneous slab gels. Gels were stained with Coomassie blue R-250. Native holoferritin also was visualized by Ferene S stain according to Chung (1985). Relative molecular mass was determined from low molecular mass standards (Bio-Rad, Richmond, CA), horse spleen ferritin (Sigma, St. Louis, MO), and Munduca sextu ferritin (prepared as described by Winzerling et al., 1995). Western Blot Fourth instar larvae, pupae, or adults were selected from mass cultures of insects raised on either control diet or diet containing 0.005 M FeC13.Groups of five insects were frozen immediately in liquid nitrogen and stored at -80°C. These samples were homogenized in 25 pl of PBS containing a cocktail of aqueous protein inhibitors (1 pl) (0.15 mg/ml aprotinin, 0.15 mg/ml benzamidine, 0.2 mg/ml leupeptin) and 1 pl of protein inhibitors dissolved in ethanol (100 mM PMSF, 0.2 mg/ml pepstatin A, 10 mg/ml TPCK). The samples were diluted 1:l in SDS-PAGE sample buffer (2x) (Laemmli, 1970) and centrifuged for 15 min at 12,OOOg. The supernatant was brought to 70 yl with SDS-PAGE sample buffer (1x), boiled 5 min, and centrifuged at 12,0009 for 30 s. The supernatant (14 PI, the amount approximately equal to one insect) and control purified ferritin (22 pg) were loaded on a 12.5%homogeneous slab gel and electrophoresis was conducted at 9 mA overnight (RT). The polypeptides were transferred to a nitrocellulose membrane by semi-dry blot (Idea Scientific, Minneapolis, MN). Blots were blocked with 1%NFDM 296 Dunkov et al. in TBS for 30 min, allowed to react for 1 h with rabbit anti-A. negypti ferritin serum (1:2,000) diluted in 1%NFDM-TBS, washed four times with TBS that contained 0.05% Tween 20, and ferritin was visualized with alkaline phosphatase stain using goat anti-rabbit IgG alkaline phosphatase conjugate (BioRad, Inc.). Concanavalin A-binding glycoproteins were detected after nitrocellulose blotting by probing with FITC-labeled concanavalin A (Sigma). Production of Antibodies Ferritin (200 pg) purified from A. aegypti pupae as described above was subjected to SDS-PAGE on a 12.5% homogeneous slab gel, stained with Coomassie blue, and the bands corresponding to the three ferritin subunits were cut from the gel. All bands were homogenized together in a glass-Teflon homogenizer, emulsified in 0.5 ml RIBI Adjuvant (RIBI Immunochem Research, Inc., Hamilton, MT), and injected subcutaneously into a rabbit. Two additional injections were given, one at 2 weeks and one at 13 weeks. The antibody titers were determined by ELISA (Engvall and Perlmann, 1972) at 10 days after the second injection and followed thereafter. The final bleeding was done at 16 weeks. Antibody specificity was determined by immunoblotting (Burnette, 1981) using the Vectastain ABC peroxidase immunodetection kit (Vector Laboratories, Burlingame, CA). The antiserum reacted with 50 ng of ferritin in 1:20,000 dilution. Protein Sequence Determination Mosquito ferritin (40 pg) was loaded on an SDS 12.5%homogeneous minigel and electrophoresis was conducted at 15 mA, RT, until the dye reached the bottom of the gel. The polypeptides were transferred to a nitrocellulose membrane (Applied Biosystems, Foster City, CA) by semi-dry blot. The blot was stained for 1 min with Ponceau red or Coomassie blue R-250, destained 10 min with 1.0% acetic acid, and air-dried. The protein bands were cut from the membrane and sent for N-terminal sequencing; the 24 and 26 kDa bands were sequenced by Harvard Microchemistry Facility (Cambridge, MA), and the 28 kDa band was sequenced by the Macromolecular Facility, Arizona Research Laboratories-Biotechnology Program, University of Arizona, Tucson, AZ. Screening and Isolation of the Recombinant Phage and Subcloning of the Inserts Rabbit anti-A. aegypti ferritin serum was used to screen a female A. aegypti (Bahama strain) cDNA hgtll library kindly provided by Dr. A. A. James (University of California, Irvine, CA). Positive plaques were detected with goat anti-rabbit IgG conjugated to alkaline phosphatase (BioRad). Approximately 140,000 plaques were screened and eleven putative positive clones were rescreened to homogeneity. The inserts from nine positive clones were amplified by PCR using single plaque extracts as a DNA template with primers flanking the EcoRI site in hgtll vector. The PCR products were cloned into pCR I1 vector using the “TA cloning” kit (Invitrogen, Inc., San Diego, CA). Plasmid DNA was purified using the Magic Minipreps kit (Promega Corp., Madison, WI). DNA was sequenced Mosquito Ferritin: Characterization and Cloning 297 using Sequenase (version 2.0) according to manufacturer’s instructions (United States Biochemicals, Cleveland, OH). Rapid Amplification of cDNA Ends (RACE) The 5VTR and part of the coding region of the ferritin cDNA were obtained by a modified RACE procedure (Edwards et al., 19911, using the 5’AmpliFINDER RACE kit (Clontech, Palo Alto, CA).An antisense primer with the sequence, 5’-TGGTGACGGTTGGCACCT-3’ was designed from cDNA sequence (see Fig. 6, nt 568-585). Using this primer, we synthesized cDNA from A. aegypti adult female mRNA. The RNA was hydrolyzed with 6 N NaOH and a single-stranded oligonucleotide anchor was ligated to the 5’ end of the single strand cDNA by T4 RNA ligase. A second pair of primers, one complementary to the anchor and the other with the sequence 5’TGGTCGTAGTCCAGCTTGAAGTGCTTG-3’ (see Fig. 6, nt 539-5651, were used for PCR. The PCR products were subcloned and sequenced as described above. Messenger RNA was purified from adult female mosquitoes using the Micro-Fast Track system (Invitrogen, Inc.). DNA and Protein Sequence Analysis Analysis of DNA and protein sequences was performed using the DNA and protein sequence analysis programs from the GCG Package (Genetics Computer Group, 1991). RESULTS Purification of Ferritin Two properties of the ferritins, the high density of the iron-loaded holoprotein and its thermostability, were employed to purify ferritin from A . aegypti larvae and pupae. Although the high pH of the growth medium caused the formation of insoluble ferric hydroxides, the larvae showed a significant increase in ferritin yield when animals were raised on the culture containing FeC13. This is probably because the iron salts were precipitated on food particles ingested by the larvae. Ferritin resistance to thermal denaturation at 75°C is well documented (Nichol and Locke, 1989). Heat treatment precipitated other proteins from the homogenate and centrifugation produced a clear extract that contained ferritin. Following the first ultracentrifugation through a KBr solution, we obtained a sticky brown pellet containing primarily ferritin. An additional ultracentrifugation resulted in homogeneous ferritin. Native gel electrophoresis of ferritin purified from control larvae showed a single protein band while the preparation from iron-loaded larvae showed two bands of apparent high molecular weight (Fig. la). Staining with Ferene S (not shown) indicated that the protein contained iron. A . aegypti ferritin did not dissociate into subunits when subjected to non-reducing SDSPAGE (not shown). However, SDS-PAGE under reducing conditions revealed that this ferritin consists of 3 major polypeptides of about 28,26, and 24 kDa (Fig. lb) and a minor amount of a polypeptide of about 30 kDa. The 28 kDa and the minor 30 kDa subunits bound FITC labeled con- Dunkov et al. 298 a b kDa k Da 669 94 440 67 330 43 140 30 a b d c e f c 20 kDa 14 28, 26 24/ a b c d e r g a b c d e f Fig. 1. a: Native PAGE of A. aegypti larval ferritin. Mosquitoes were fed a control diet or a diet that contained 5 mM FeCI3. Ferritin was isolated, loaded onto a 6% homogeneous native slab gel, and electrophoresis conducted as described in Materials and Methods. The gel was stained with Coomassie blue R-250. Lane a: high molecular mass standards; lane b: horse spleen ferritin; lane c: M. sexta ferritin; lanes d and e: A. aegypti ferritin from control larvae, and lane f: A. aegypti ferritin from iron-fed larvae. b: SDS-PAGE of A. aegypti larval ferritin. Mosquitoes were fed a diet that contained 5 mM FeC13. Ferritin was isolated, prepared and run on an SDS-PAGE 12.5% homogeneous slab gel a5 described in Materials and Methods. The gel was stained with Coomassie blue R-250. lane a: low molecular mass standards; lanes b,c, and d: A. aegypti ferritin 33, 16, 9 pg, respectively; lane e: adult M. sexta ferritin; lane f: horse spleen ferritin. c: Western blot of the differential expression of ferritin subunits in various A. aegypti life stages. Mosquitoes were raised on a control diet or on a diet containing 5 mM FeCI3 and protein extracts prepared as described in Materials and Methods. Samples were loaded onto a 12.5% homogeneous slab gel and electrophoresis was conducted at 9 mA, overnight, at RT. The polypeptides were transferred to a nitrocellulose membrane by semi-dry blot. The blot was blocked, washed, and visualized with alkaline phosphatase stain as described in Materials and Methods. Lane a: purified pupal ferritin; lane b: control larvae; lane c: iron-fed larvae; lane d: control pupae; lane e: pupae raised on iron-supplemented diet; lane f: control female adult; lane g: female adult mosquitoes raised on iron-supplement diet. canavalin A indicating that they were glycosylated with a mannose-rich oligosaccharide (data not shown). Presence of Ferritin in Adult Mosquitoes Western blotting showed that the rabbit antiserum reacted specificaIly with all three major subunits, if present, in the extracts from larval, pupal, and Mosquito Ferritin: Characterization and Cloning 299 adult animals (Fig. lc). Western blotting also confirmed the presence of ferritin in extracts from adult mosquitoes and showed that adult ferritin had the same subunit composition as the larval and pupal proteins (Fig. lc). Only two of the three subunits were seen in the control larvae and control adult extracts, while iron feeding stimulated synthesis of all three subunits in both of these stages. The 26 kDa protein was the major band in all samples. Rabbit anti-A. aegypti ferritin antiserum did not cross-react with horse spleen or M . sexta ferritins (data not shown). N-Terminal Sequence of the A. aegypti Ferritin Subunits The sequences of the first 20-11 amino acids of the A. aegypti ferritin subunits are shown in Figure 2. The first 30 amino acids from the N-terminal sequences of the 26 and 24 kDa subunits are identical. Comparison of these mosquito ferritin sequences with those of other organisms showed no similarities. Cloning of A. aegypti Ferritin cDNA Figure 3 shows the strategies used to sequence the clones obtained from the cDNA library and via RACE. Partial sequences of all nine cDNA clones differed only in a 27 bp insertion immediately preceding the poly A tail. RACE revealed that all cDNA clones were truncated at an EcoRI site at the 5’ end; the truncation probably occurred during the construction of the cDNA library. One of the clones (F) was completely sequenced using nested sequencing primers. This cDNA sequence and the deduced amino acid sequence are shown in Figure 4. The RACE procedure also yielded two groups of clones, one group that contained the remaining coding sequence and 203 bp of the 5’ untranslated region (nt 1-203, Fig. 4). The N-terminal sequence obtained from the 26 and 24 kDa subunits matched the deduced amino acid sequence (Fig. 4, underlined sequence). Further comparison of the deduced amino acid sequence with the N-terminal sequence also revealed the presence of a signal peptide. Analysis of the 5’UTR of the A. aegypti ferritin cDNA revealed the presence of a stem loop structure (bases 81-115, Fig. 4). This stem loop structure appears to be a putative iron-responsive element (IRE), since its sequence matches the consensus sequence derived from vertebrate IRES (Fig. 5). Multiple sequence alignment with eleven other ferritin sequences (Fig. 6) showed regions of similarity and conservation of the putative ferrooxidase center residues. Pairwise comparison to other ferritin sequences showed that the A . 28 kDa D 1 N N CS TV N FTA Q F S S I A H I G 5 10 15 20 26 kDa E QT V G A T D N Y Q W D S V D D Q[C] L A A L H R Q I N K E F D A - I 1 Y L (K) (Y) (A) 1 5 10 15 20 25 30 35 40 24 kDa E Q T V C A T D N Y Q W D S V D D Q[C] L A A L H R QI N K E 1 5 10 15 20 25 30 Fig. 2 . N-terminal sequences of A. aegypti ferritin subunits. 300 Dunkov et al. 1 0 I 200 I I 400 600 I 800 1 I I 1000 1200 clone 4 clone 3 clone F ATG t Ec9R I --203 POlYA TAA I 627 v/l k 426 20 7 f------------ ____) Fig. 3. Sequencing strategy of the ferritin cDNA clones. The scale denotes nucleotides in base pairs. Clone F represents a cDNA clone obtained by screening an expression library (see Materials and Methods), while clones 3 and 4 were obtained by the RACE technique (see Materials and Methods). The center diagram shows the coding region (627 bpi, part of the 5’ UTR (203 bp), and the 3‘UTR (427 bp). Both strands of the cDNA were sequenced throughout the coding region and 3’ untranslated region. The terminal region of the 5’ untranslated region was sequenced unidirectionally in two independent RACE clones. The horizontal arrows indicate the length and direction of the sequenced fragments. aegypti sequence has greatest similarity to the Limnaea stagnnlis soma ferritin subunit (Fig. 7). DISCUSSION While ferritins have been isolated and characterized from a variety of organisms ranging from plants and bacteria to mammals, relatively little is known about ferritins from invertebrate animals. Sequences are available only for a snail (von Darl and Bottke, 1992), a schistosome (Dietzel et al., 1992), and a crustacean (ferritin-like protein, artemin) (De Graaf et al., 1990). Up to this point, ferritins have been isolated from only two insects, both lepidopterans (Heubers et al., 1988; Nichol and Locke, 1989; Winzerling et al., 1995), but their sequences have not been determined. Insect ferritins have some interesting differences from most other ferritins. They are larger than those reported from other animals in terms of the apparent masses of the subunits, although the size of the ferritin sphere appears to be about the same as ferritins of other organisms (Locke and Nichol, 1992). Furthermore, in many insects, ferritins are glycosylated and they appear in the endoplasmic reticulum of the cells (rather than in the cytoplasm) and in relatively large quantities in hemolymph (Locke and Nichol, 1992). This may indicate some fundamentally different functions for the insect ferritins. One of the snail ferritins, that which occurs in the oocyte, is also a secreted protein that can be found in the hemolymph (Bottke, 1982). Mosquito Ferritin: Characterization and Cloning 1 301 60 GGACTAGTACCGGACACCGATAGAGGTGGAAUTACGACATAGAGGTGGAAGCAATACGA 61 120 T l ' A T K C A T C G C A G C C c T K l G T W ~ A T ~ G T 121 180 TpGRARAcTAGTGAAGcrc;cTAcGAAAATcTAGITcn;T 181 240 T I C G G A A A C C ~ T G G T A G C C A T C A M M K S V F F G V V A I T 241 300 C C G P A C C C A ' P C A ~ A C t A G G A G A C G G C C C A G G V A I L S I Y Q E T A Q A Q E Q T V G A 301 360 C A A C C G A T A A ' I T A C C G A % A T C A G % C ~ C ! G C C T D N Y Q W D S V D D Q C L A A L H R O 361 420 AGA~'ITCGAlGCGXGA'KATCTACCTGUGTASCCGCCTA~ I N K E F D A S I I Y L K Y A A Y F A Q 421 480 481 540 541 600 601 660 661 720 721 780 781 840 841 900 901 960 961 1020 1021 1080 1081 1140 1141 1201 1200 1260 1261 Fig. 4. Nucleotide sequence of the cDNA for A. aegypti ferritin and the deduced amino acid sequence for the protein. The underlined protein sequence matches the N-terminal sequence obtained from the purified 24 and 26 kDa ferritin subunits. The underlined 27 bp nucleotide sequence near the 3'-end of the cDNA was not present in all clones. The putative polyadenylation signal is underlined. We have undertaken a study of iron metabolism in mosquitoes because of the differences in the sources of dietary iron between the male and female adult animals, and between the larvae and adult animals. Larvae are aquatic, and their diet can be expected to contain mainly inorganic iron. Adult males Dunkov et al. 302 G U A G G c U G A u C N N N N N C-G G-U U - A G-U U - A C - N N N N N N C N-N N-N N-N N-N N-N U - A U - A C-G C-G A - U c-c 5' U 3' U - A G-C 5' 3' A. aegypti Consensus IRE Fig. 5. Sequence and structure of the putative IRE of A. aegypti ferritin mRNA and consensus IRE (according to Harford and Klausner, 1990). 1 %ail-S RaM-L m - L %his-1 %his-2 Soy SMil-Y Aaeqypci Artemin * 100 50 ...................................... . . . . . . . . WVRCWFWROCE MIblRWWC3 ...................................... . . . . . . . .W V W W I = C E M ...................................... . . . ..TpIsTFQ vRouuMIcGB MImQMLE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .....nsvsQm@mmEsE Y i l t m ~ I m ~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M E Q VRpmqOcB ........................................................................... IS- II(QIysDvE MVNSLVNLY .......................................................................... KSL .uma.Q. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . KSSSR -PALRIIOIQ-SUVSl'FSGFSPKPN S VSLSFGauyLRlffAs "PLTGVIFK P F r m K K S U IIVPl%WSL W A C E C E SAIWQTHVE .................... ........................... bSWJ LELTULYCSS UY-T VrcOmwlN Q W I .................... . . . . . . . . . . . . . F-. GWAITVATL SIYQECAQAQ m T L N f PmmraqCL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . AT DXFlNICQSAP-P SRIDWPECE -1200 RaM-H RaM-n h - H Snail4 ......................... KPDR .......................... KPm .......................... KPAE .......................... Mm? . . . . . . . . . . . . . . . . . . . . . . . . . GPIX .......................... NnPS LBB FRauIpcICo NATKTIW ..................... Nt ............................. P Fig. 6. Alignment of known ferritin subunit amino acid sequences. Abbreviations and sources: Rana = Kana catesbeiana heavy (H), medium (M), or light (L) ferritin chain (Dickey et al., 1987; Didsbury et al., 1986); Hum = human light (L) or heavy (H) ferritin chain (Boyd et al., 1985; Dorner et al., 1985); Snail = Linmaea stagnalis soma (S) and yolk (Y) ferritin (von Darl and Bottke, unpublished data); Schis = Schistosorna mansoni ferritin chains 1 and 2 (Dietzel et al., 1992); Soy = soyabean (Lescure et al., 1991; Ragland et al., 1990); Aaegypti = A. aegypti ferritin (this paper); and Artemin = artemin from Arfemia salina cysts (De Craaf et al., 1990). The Cterminal portion of Artemin is not shown. * = conserved putative ferrooxidase residues. Mosquito Ferritin: Characterization and Cloning 303 Fig. 7. Pairwise identities of amino acid sequences aligned in Figure 6. Identities were calculated using the program DISTANCES (GCG Package). For each pairwise comparison the sum of identities were divided by the length of the shorter sequence without gaps. feed mainly on nectar, which is probably iron deficient. In contrast, the adult female feeds on vertebrate blood, a diet rich in iron-containing hemoglobin. In order to isolate ferritin from larvae and pupae, we homogenized whole animals that had been fed an iron-rich diet. Sonication was used to release cellular ferritin. While these procedures of protein isolation can be risky because both digestive and cellular proteases are released, the known resistance of many ferritins to proteolysis provided optimism that intact ferritin could be isolated by this method. The isolation procedure presented here varied slightly from another we recently published (Winzerling et al., 1995). This technique takes advantage of the fact that iron-loaded ferritin has greater density than most cellular material, thus it pellets with ultracentrifugal force in a KBr solution, while other proteins float. The resulting product is essentially homogeneous. A. aegypti native ferritin behaves in polyacrylamide gels as a very large molecule, although its exact molecular mass has not been determined. On dissociation under reducing conditions in the presence of SDS, ferritin from iron-fed larvae dissociates into four subunits. Those of masses 24,26, and 28 kDa are almost equally abundant, while that of mass 30 kDa is a minor component. The 28 and 30 kDa subunits are glycosylated, while the 24 and 26 kDa polypeptides apparently are not. The second protein band observed on the native PAGE of preparation from iron-fed larvae probably represents ferritin with different subunit composition, produced under the conditions of iron excess. Both the snail oocyte ferritin and mosquito ferritins are known to be secreted from cells, and both are synthesized with leader sequences. According to von Heijne (1983, 1987), the predicted site of cleavage of the leader peptide for the mosquito subunit would be after the AQA sequence at residues 24-26, while the sequence for the mature subunit begins at residue 28 304 Dunkov et al. (Fig. 4). If this prediction is correct, there must be a subsequent processing step to remove the intervening glutamine residue. The amino acid sequence deduced from the cDNA sequence (209 residues) corresponds to the molecular weight of 23,758. If the leader peptide and the intervening glutamine residue (residues 1-27) are removed, then the mature subunit sequence (182 residues) corresponds to a molecular weight of 20,566; this is smaller than that estimated by SDS-PAGE (24,000). Since the 24 and 26 kDa subunits share the same N-terminal sequence, it is likely that they are products of the same gene. The difference between the molecular weight calculated from the deduced sequence and that predicted from the SDS-PAGE suggests that both subunits may be posttranslationally modified. At present, we have no idea what such a modification might be. However, glycosylation is unlikely, both because neither subunit binds concanavalin A and because the deduced sequence has no predicted glycosylation sites. The 28 kDa subunit is clearly the product of a different gene. We did not have sufficient 30 kDa subunit to determine its N-terminal sequence. Vertebrate ferritins can consist of any combination of three subunits; two very similar subunits designated H or H’ (heavy) and a related, but less similar L (light) subunit. Of the invertebrate ferritins sequenced to date, two have been shown to have two different subunits. The schistosome has sex specificity of subunits, with one predominating in males, and the other in females (Dietzel et al., 1992).The snail has tissue specific ferritins, one is found in the somatic cells and the other is vitellogenic, secreted by the midgut gland and sequestered in the ovary (Bottke, 1982). Whether similar specificity exists in insects is not yet clear. The subunits of the snail somatic ferritin (von Darl and Bottke, 1992)and the schistosome ferritins (Dietzel et al., 1992)show greater similarity to the H chains of vertebrates than to vertebrate L chains. The mosquito ferritin has greater similarity to the human H chain than to the human L chain, but the similaritiesto the three chains of the frog are not very different. Vertebrate H chain subunits have a catalytic site (ferroxidase site) that is responsible for the oxidation of cytoplasmic ferrous ion to ferric ion (Balla et al., 1992; Wade et al., 1991).Thus cytoplasmic iron is converted to the proper form for mineralized storage as it enters the ferritin sphere. With respect to a ferroxidase site, the mosquito ferritin has retained all of the putative binding site residues, marked by asterisks in Figure 6. Waldo et al. (1993) have recently suggested that an iron-tyrosinate complex formed only by vertebrate H chains (not L chains) is involved in rapid biomineralization of iron. The specific tyrosine (or tyrosines) responsible for formation of this complex has not been determined, but these authors indicated that the residue 102 (shown boxed in Fig. 6) might be responsible for complex formation. This residue is present in all vertebrate heavy chains, as well as in the snail somatic ferritin and in the Scm 1 (female predominant) ferritin of the schistosome. However, it is lacking in the snail vitellogenic ferritin, the male predominant schistosome ferritin, and the mosquito ferritin. If this residue is essential, possibly another of the mosquito subunits is responsible for rapid biomineralization in mosquitoes. The fact that insect ferritins are secreted from cells, and also presumably taken up as well, suggests that they function differently from the cytoplas- Mosquito Ferritin: Characterization and Cloning 305 mic ferritins of other species. Locke and Nichol (1992) have speculated that insect ferritins might function in the excretion of iron (and possibly other cations) by exocytosis from midgut cells into the midgut lumen. They also postulate that lumenal ferritin could function in transport of iron through midgut cells, as well as from hemolymph into cells. The availability of molecular tools should facilitatethe experimental testing of these interesting hypotheses. As demonstrated by Western blotting, the amount of ferritin polypeptides is increased by administration of iron in the diet (Fig. lc). In vertebrates, synthesis of ferritin in response to iron is primarily controlled at translation. The ferritin message contains an IRE in the 5'-untranslated region, where an ironregulatory protein (IRP, formerly called IRE-BP) is bound under conditions of iron depletion (Eisenstein and Munro, 1990; Theil, 1990b).IRP blocks translation of the ferritin message, so that ferritin is not synthesized. When iron is present, an iron-sulfur cluster in IRP is assembled, and IRP loses affinity for the IRE and gains aconitase enzymatic activity (Haile et al., 1992). Cytoplasmic aconitase thus functions as an iron sensor for the cell. Several other proteins of iron metabolism are controlled in a similar or reciprocal fashion (OHalloran, 1993). 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