[Show abstract][Hide abstract] ABSTRACT: Mitochondrial function depends on iron-containing enzymes and proteins, whose maturation requires available iron for biosynthesis of iron-sulfur clusters and heme. Little is known about how mitochondrial iron homeostasis is maintained, although the recent discovery of a mitochondrial ferritin in mammals and plants has uncovered a potential key player in the process. Here, we show that Drosophila melanogaster expresses mitochondrial ferritin from an intron-containing gene. It has high similarity to the mouse and human mitochondrial ferritin sequences and, as in mammals, is expressed mainly in testis. This ferritin contains a putative mitochondrial targeting sequence and an epitope-tagged version localizes to mitochondria in transfected cells. Overexpression of mitochondrial ferritin fails to alter both total-body iron levels and iron that is bound to secretory ferritins. However, the viability of iron-deficient flies is compromised by overexpression of mitochondrial ferritin, suggesting that it may sequester iron at the expense of other important cellular functions. The conservation of mitochondrial ferritin in an insect species underscores the importance of this iron-storage molecule.
Proceedings of the National Academy of Sciences 05/2006; 103(15):5893-8. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The identification, purification and characterization of a new postlarval specific hemolymph protein from Manduca sexta is described. Incorporation of [35S]methionine into Manduca sexta hemolymph proteins in vivo was investigated as a function of development. A major protein band of Mr ≈ 50,000 was highly labeled during the prepupal and adult stage but not in feeding larvae. This postlarval protein (PLP) was isolated from adult male hemolymph and its chemical and immunological properties determined. PLP is a basic protein (pI ∼8.6). Electrophoresis under denaturing conditions reveals a subunit Mr ≈ 50,000 while the native protein has an apparent Mr ∼ 85,000 by gel permeation chromatography. Anti-PLP serum recognized PLP but not other hemolymph proteins on immunoblots. In vitro translation of fat body mRNA followed by immunoprecipitation revealed that fat body is the site of PLP synthesis. Quantitation of PLP levels in hemolymph throughout development was performed and suggests PLP may play a role in adult development of M. sexta.
Archives of Insect Biochemistry and Physiology 02/2005; 9(2):81 - 90. · 1.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The mature flightless grasshopper Barytettix psolus shows a very small adipokinetic response when injected with extracts of its own corpora cardiaca, although the fat body contains enough lipid for a strong response. When these extracts were injected into Melanoplus differentialis, a grasshopper capable of flight, or the moth Manduca sexta, much stronger adipokinetic responses were observed. Upon analysis of B. psolus extracts by HPLC, two components with adipokinetic activity were obtained. The major component appears to be identical to locust adipokinetic hormone (AKH) I. Extracts of B. psolus corpora cardiaca also activated fat body glycogen phosphorylase in B. psolus. This activation, however, did not result in an increase in hemolymph sugar, probably because of low levels of glycogen in the fat body.B. psolus hemolymph contains a high-density lipophorin (HDLp) consisting of the apolipophorins (apoLp) I and II and lipid. Both apoproteins are glycosylated. The hemolymph also contains apoLp-III, although this apoprotein apparently does not associate with HDLp to form a low-density lipophorin (LDLp) following AKH or corpora cardiaca extract injections. When B. psolus lipophorin and AKH were injected into Schistocerca americana, lipophorin took up lipids and combined with apoLp-III, forming LDLp. ApoLp-III from B. psolus injected into S. americana can also form LDLp, demonstrating that the components are functional. A lipid transfer particle isolated from M. sexta and injected into B. psolus does not improve the adipokinetic response. Thus, it appears that the adipokinetic response of B. psolus is not deficient because of the lack of AKH or functional lipophorins, but may be caused by the lack of a full response to AKH by fat body or the deficiency in hemolymph of some as yet unknown factor.
Archives of Insect Biochemistry and Physiology 02/2005; 9(4):255 - 268. · 1.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Three major hemolymph proteins of Papilio polyxenes larvae were isolated and characterized. Density gradient ultracentrifugation of hemolymph resulted in flotation of the major lipoprotein, lipophorin. P. polyxenes larval lipophorin is composed of two apoproteins, apolipophorin-I and apolipophorin-II, plus a mixture of lipids, to give a density of 1.13 g/ml. Immunoblotting experiments using antisera directed against Manduca sexta apolipophorin-I and apolipophorin-II, respectively, revealed cross-reactivity of apoLp-I with Manduca sexta apoLp-I, and apoLp-II with M. sexta apoLp-II.Gel permeation chromatography of the subnatant obtained following density gradient ultracentrifugation revealed the presence of a major protein peak which was shown to contain three major serum proteins, two of which were isolated and characterized. One of these proteins was purified by lectin affinity chromatography. Both proteins have native molecular weights in the range of 450,000 and appear to be hexamers of a single subunit type. Major serum protein-1 is nonglycosylated and has a subunit molecular weight of 75,000. Major serum protein-2 is glycosylated and has a subunit molecular weight of 74,000. Amino acid analysis of this protein revealed a tyrosine plus phenylalanine content of 20 mole percent, characteristic of the arylphorin class of insect storage proteins. Using antibodies against M. sexta larval hemolymph proteins, both the P. polyxenes major serum proteins were shown to be immunologically related to serum proteins of other lepidopteran species.
Archives of Insect Biochemistry and Physiology 02/2005; 3(6):539 - 550. · 1.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The major insect hemolymph lipoprotein, lipophorin, was isolated from adults of eight insect species representing seven insect orders. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to compare their respective apoprotein components. In all species examined lipophorin was composed of at least two apoproteins, apolipophorin I (Mr ∼ 250,000) and apolipophorin II (Mr ∼ 78,000), and two species had a third apoprotein, apolipophorin III (Mr ∼ 17,000). The density of each isolated lipophorin was determined from the refractive index of KBr following density gradient centrifugation. Immunoblotting with anti-larval Manduca sexta apolipophorin I and II of the apoproteins separated by SDS-PACE indicated cross reactivity between anti-M sexta apoLp-ll and apoLp-ll in all species tested. Anti-M sexta apoLp-l exhibited no cross reactivity for any species tested. Fluorescent lectin staining of the apoproteins separated on SDS-PAGE gels revealed the presence of covalently bound carbohydrate residues.
Archives of Insect Biochemistry and Physiology 02/2005; 1(4):375 - 383. · 1.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: All insects appear to have a transport lipoprotein in the hemolymph (blood) that is responsible for moving hydrophobic materials through aqueous compartments. This has been called lipophorin because it is believed to be a reversible transport shuttle. Since most insects undergo some degree of metamorphosis from larval stages to the adult, the need to transport hydrophobic materials or the nature of these materials may change in the course of the life span. This is especially marked in the case of the holometabolous insects – those which undergo drastic change of form, e.g. caterpillar-pupa-adult moth. We have found that lipophorin undergoes a molecular metamorphosis paralleling that of the insect.
[Show abstract][Hide abstract] ABSTRACT: Mosquitoes and all other insects so far examined have an abundant haemolymph transferrin (Tsf). The exact function of these proteins has not been determined, but they may be involved in iron transport, in oogenesis and in innate immune defence against parasites and pathogens. The Tsf gene of Aedes aegypti has been cloned and sequenced. It contains a single small intron, which contrasts it to vertebrate Tsf genes that contain up to sixteen introns. The promoter region of the gene is rich in putative NF-kappaB binding sites, which is consistent with the postulated role of Tsf in insect innate immunity. Tsf message levels are very low in embryos and early larvae, but high in late larvae, pupae and adults. Western blotting experiments revealed high levels of Tsf protein in pupae and adults. Late larvae and ovaries of blood-fed mosquitoes have little intact protein, but two prominent proteolytic degradation products. These may represent biologically active peptides, as has been shown for other organisms. Tsf message is down-regulated by inorganic iron in the diet or environment, but up-regulated by a blood meal in the adult female. The up-regulation following a blood meal may, in part, be due to the decrease in juvenile hormone (JH) that is known to follow blood feeding. Treatment of blood-fed females with methoprene, an analogue of JH, resulted in decrease of the Tsf message.
[Show abstract][Hide abstract] ABSTRACT: Ferritin was purified from iron-fed Drosophila melanogaster extracts by centrifugation in a gradient of potassium bromide. On polyacrylamide gel electrophoresis, the product showed two protein bands corresponding to the ferritin monomer and dimer. Electrophoresis following dissociation with SDS and 2-mercaptoethanol revealed three strong bands of approximately 25, 26, and 28 kDa. N-terminal amino acid sequences were identical for the 25-kDa and 26-kDa subunits, but different for the 28-kDa subunit. Conserved ferritin PCR primers were used to amplify a 360-bp cDNA product, which was used to isolate a clone from a D. melanogaster cDNA library that contained the complete coding sequence for a ferritin subunit. Additional 5' sequence obtained by the RACE method revealed the presence of a putative iron regulatory element. The PCR product was also used to locate the position of the ferritin subunit gene at region 99F on the right arm of the third chromosome. The deduced amino acid sequence of the D. melanogaster ferritin subunit contained a signal sequence and resembled most closely ferritin of the mosquito Aedes aegypti. The evolution of ferritin sequences is discussed.
European Journal of Biochemistry. 07/2004; 247(2):470 - 475.
[Show abstract][Hide abstract] ABSTRACT: Genes encoding ferritins were isolated and cloned from cDNA libraries of hard tick Ixodes ricinus and soft tick Ornithodoros moubata. Both tick ferritins are composed of 172 amino-acid residues and their calculated mass is 19,667.2 Da and 19,974.5 Da for I. ricinus and O. moubata, respectively. The sequences of both proteins are closely related to each other as well as to the ferritin from another tick species Dermacentor variabilis (>84% similarity). The proteins contain the conserved motifs for ferroxidase center typical for heavy chains of vertebrate ferritins. The stem-loop structure of a putative iron responsive element was found in the 5' untranslated region of ferritin mRNA of both ticks. Antibodies against fusion ferritin from O. moubata were raised in a rabbit and used to monitor the purification of a small amount of ferritins from both tick species. The authenticity of ferritin purified from O. moubata was confirmed by mass-fingerprinting analysis. In the native state, the tick ferritins are apparently larger (~500 kDa) than horse spleen ferritin (440 kDa). On SDS-PAGE tick ferritins migrate as a single band of about 21 kDa. These results suggest that tick ferritins are homo-oligomers of 24 identical subunits of heavy-chain type. The Northern blot analysis revealed that O. moubata ferritin mRNA level is likely not up-regulated after ingestion of a blood meal.
[Show abstract][Hide abstract] ABSTRACT: Comparison of the genomes and proteomes of the two diptera Anopheles gambiae and Drosophila melanogaster, which diverged about 250 million years ago, reveals considerable similarities. However, numerous differences are also observed; some of these must reflect the selection and subsequent adaptation associated with different ecologies and life strategies. Almost half of the genes in both genomes are interpreted as orthologs and show an average sequence identity of about 56%, which is slightly lower than that observed between the orthologs of the pufferfish and human (diverged about 450 million years ago). This indicates that these two insects diverged considerably faster than vertebrates. Aligned sequences reveal that orthologous genes have retained only half of their intron/exon structure, indicating that intron gains or losses have occurred at a rate of about one per gene per 125 million years. Chromosomal arms exhibit significant remnants of homology between the two species, although only 34% of the genes colocalize in small "microsyntenic" clusters, and major interarm transfers as well as intra-arm shuffling of gene order are detected.
[Show abstract][Hide abstract] ABSTRACT: Konrad Bloch developed an interest in insects because they are unable to make sterols, and in yeast because these cells need oxygen to make sterols and unsaturated fatty acids. Insects, like all other organisms, must deal with the toxic effects of oxygen in the presence of iron, which itself is a vital nutrient. They do so by making proteins with high affinity for ferric or ferrous ions. Two such proteins are transferrins and ferritins. Insects produce both of these proteins, but use them in different ways from most other organisms. Insect transferrins appear to be involved in innate immunity, perhaps by sequestering ferric ions to prevent pathogens and parasites from utilizing them. Insect ferritins, unlike those of any other group of organisms, are exported into the extracellular space (hemolymph). They may be involved in iron transport and/or protection against iron overload in the diet.
Biochemical and Biophysical Research Communications 05/2002; 292(5):1191-5. · 2.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Drosophila melanogaster secreted ferritin like the cytosolic ferritins of other organisms is composed of two subunits, a heavy chain homologue (HCH) and a light chain homologue (LCH). We report the cloning of a cDNA encoding the ferritin LCH of this insect. As predicted from the gene sequence, it contains no iron responsive element (IRE). Northern blot analysis reveals two mRNAs that differ in length due to the choice of polyadenylation signals. Message levels vary through the life cycle of the fly and are markedly increased by high levels of dietary iron. The gut is the main site of increased message synthesis and iron preferentially increases the amount of shorter messages. Western blotting reveals that LCH is the predominant ferritin subunit in all life stages. The amount of LCH protein corresponds well with the message levels in control animals, while in iron-fed animals LCH does not increase proportionally with the message levels. In contrast, the amount of HCH is less than that would be predicted from message levels in control animals, but corresponds well in iron-fed animals. Ferritin is abundant in gut and hemolymph of larvae and adults and in ovaries of adult flies. At pupariation, ferritin becomes more abundant in hemolymph than in other tissues.
[Show abstract][Hide abstract] ABSTRACT: Secreted ferritin in the mosquito, Aedes aegypti, has several subunits that are the products of at least two genes, one encoding a homologue of the vertebrate heavy chain (HCH) and the other the light chain homologue (LCH). Here we report the developmental and organ specific pattern of expression of the ferritin HCH messages and of both subunit types in control sugar-fed mosquitoes, in those exposed to high levels of dietary iron, and after blood feeding. When Northern blots were probed with a HCH cDNA, two bands were observed, representing at least two messages of different sizes that result from the choice of two different polyadenylation sites. Either raising mosquito larvae in an iron-enriched medium, or blood feeding adult female mosquitoes resulted in a marked increase in the HCH message level, particularly of the shorter message. Changes in the amount and length of messages and amount of ferritin subunits were studied over the life span of the mosquito and in different organs of female mosquitoes after blood feeding. The midgut of blood-fed insects is the main site of increased ferritin message synthesis. Ferritin protein levels also increase in midgut, fat body and hemolymph after blood feeding. Ferritin messages and subunits are synthesized in the ovaries and ferritin is found in the eggs. These observations are discussed in terms of translational and transcriptional control of ferritin synthesis and are compared to similar events in the regulation of Drosophila melanogaster ferritin.
Journal of Insect Science 02/2002; 2:7. · 0.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Like other organisms, insects must balance two properties of ionic iron, that of an essential nutrient and a potent toxin. Iron must be acquired to provide catalysis for oxidative metabolism, but it must be controlled to avoid destructive oxidative reactions. Insects have evolved distinctive forms of the serum iron transport protein, transferrin, and the storage protein, ferritin. These proteins may serve different functions in insects than they do in other organisms. A form of translational control of protein synthesis by iron in insects is similar to that of vertebrates. The Drosophila melanogaster genome contains many genes that may encode other proteins involved in iron metabolism.
Annual Review of Entomology 02/2002; 47:535-59. · 13.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Secreted ferritin in the mosquito, Aedes aegypti, has several subunits that are the products of at least two genes, one encoding a homologue of the vertebrate heavy chain (HCH) and the other the light chain homologue (LCH). Here we report the developmental and organ specific pattern of expression of the ferritin HCH messages and of both subunit types in control sugar-fed mosquitoes, in those exposed to high levels of dietary iron, and after blood feeding.
[Show abstract][Hide abstract] ABSTRACT: In yellow fever mosquito cells (Aag2 clone), iron treatment induces a threefold increase in ferritin message (fer mRNA) and protein (ferritin) by 16 h. These data contrast with work in mammalian hepatocytes and fibroblasts in which fer mRNA levels do not change with iron stimulation, but ferritin levels increase 50-fold. Pretreatment of the Aag2 cells with actinomycin D blocks induction of fer mRNA and reduces the ferritin subunit synthesis, suggesting that iron induction of ferritin subunit synthesis is subjected to transcriptional control. A putative iron-regulatory protein has also been identified in cytoplasmic extracts from Aag2 cells.
European Journal of Biochemistry 12/1999; 266(1):236-40. · 3.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A new type of insect lipoprotein was isolated from the hemolymph of the female cochineal insect Dactylopius confusus. The lipoprotein from the cochineal insect hemolymph was found to have a relative molecular mass of 450 000. It contains 48% lipid, mostly diacylglycerol, phospholipids and hydrocarbons. The protein moiety of the lipoprotein consists of two apoproteins of approximately 25 and 22 kDa, both of which are glycosylated. Both apolipoproteins are also found free in the hemolymph, unassociated with any lipid. Purified cochineal apolipoproteins can combine with Manduca sexta lipophorin, if injected together with adipokinetic hormone into M. sexta. This could indicate that the cochineal lipoprotein can function as a lipid shuttle similar to lipophorins of other insects, and that the cochineal insect apolipoproteins have an overall structure similar to insect apolipophorin-III.
European Journal of Biochemistry 05/1999; 261(1):285-90. · 3.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Drosophila melanogaster transferrin cDNA was cloned from an ovarian cDNA library by using a PCR fragment amplified by two primers designed from other dipteran transferrin sequences. The clone (2035 bp) encodes a protein of 641 amino acids containing a signal peptide of 29 amino acids. Like other insect transferrins, Drosophila transferrin appears to have a functional iron-binding site only in the N-terminal lobe. The C-terminal lobe lacks iron-binding residues found in other transferrins, and has large deletions which make it much smaller than functional C-terminal lobes in other transferrins. In-situ hybridization using a digoxigenin labeled transferrin cDNA probe revealed that the gene is located at position 17B1-2 on the X chromosome. Northern blot analysis showed that transferrin mRNA was present in the larval, pupal and adult stages, but was not detectable in the embryo. Iron supplementation of the diet resulted in lower levels of transferrin mRNA. When adult flies were inoculated with bacteria (Escherichia coli), transferrin mRNA synthesis was markedly increased relative to controls.
European Journal of Biochemistry 04/1999; 260(2):414-20. · 3.58 Impact Factor