Molecular characterization of iron binding proteins, transferrin and ferritin heavy chain subunit, from the bumblebee Bombus ignitus.

Transferrin and ferritin are iron-binding proteins involved in transport and storage of iron as part of iron metabolism. Here, we describe the cDNA cloning and characterization of transferrin (Bi-Tf) and the ferritin heavy chain subunit (Bi-FerHCH), from the bumblebee Bombus ignitus. Bi-Tf cDNA spans 2340 bp and encodes a protein of 706 amino acids and Bi-FerHCH cDNA spans 1393 bp and encodes a protein of 217 amino acids. Comparative analysis revealed that Bi-Tf appears to have residues comprising iron-binding sites in the N-terminal lobe, and Bi-FerHCH contains a 5'UTR iron-responsive element and seven conserved amino acid residues associated with a ferroxidase center. The Bi-Tf and Bi-FerHCH cDNAs were expressed as 79 kDa and 27 kDa polypeptides, respectively, in baculovirus-infected insect Sf9 cells. Northern blot analysis revealed that Bi-Tf exhibits fat body-specific expression and Bi-FerHCH shows ubiquitous expression. The expression profiles of the Bi-Tf and Bi-FerHCH in the fat body of B. ignitus worker bees revealed that Bi-Tf and Bi-FerHCH are differentially induced in a time-dependent manner in a single insect by wounding, bacterial challenge, and iron overload.

Catalase from the white-spotted flower chafer, Protaetia brevitarsis: cDNA sequence, expression, and functional characterization.

Catalase, which is one of the key enzymes of the cellular antioxidant defense system, prevents free hydroxyl radical formation by breaking down hydrogen peroxide into oxygen and water. Here, we show the cloning and characterization of a catalase gene in a coleopteran insect. This gene was isolated by searching the white-spotted flower chafer Protaetia brevitarsis cDNA library, and the gene itself encodes a protein of 505 amino acids in length, named PbCat. PbCat shows high similarities to the insect catalase genes known to date. The recombinant PbCat, which is expressed as a 56-kDa polypeptide in baculovirus-infected insect Sf9 cells, shows the highest activity at 30 degrees C and pH 7.0. Northern and Western blot analyses revealed the presence of PbCat in all tissues examined, showing its ubiquitous expression. P. brevitarsis larvae in which H(2)O(2) was overloaded, showed a marked up-regulation in PbCat expression. Moreover, P. brevitarsis larvae showed an apparent increase in PbCat expression even after a wounding through injection. These results indicate that PbCat is up-regulated after wounding and oxidative pressure induced by H(2)O(2), reflecting an important role of PbCat in H(2)O(2) scavenging.

Molecular cloning and characterization of a transferrin cDNA from the white-spotted flower chafer, Protaetia brevitarsis.

A full-length cDNA clone with high homology to insect transferrin genes was cloned by screening a Protaetia brevitarsis cDNA library. This gene (PbTf) had a total length of 2338 bp with an open reading frame (ORF) of 2163 bp, and encoded a predicted peptide of 721 amino acid residues. Like known cockroach, termite, and beetle transferrins, PbTf appears to have residues comprising iron-binding sites in both N- and C-terminal lobes. The deduced amino acid sequence of the PbTf cDNA was closest in structure to the beetle Apriona germari transferrin (68% protein sequence identity). Northern blot analysis revealed that PbTf exhibited fat body-specific expression and was upregulated by wounding, bacterial or fungal infection and iron overload, suggesting a functional role for PbTf in defense and stress responses.

Transferrin inhibits stress-induced apoptosis in a beetle.

Transferrin in insects is known as an iron transporter, an antibiotic agent, a vitellogenin, and a juvenile hormone-regulated protein. We show here a novel functional role for insect transferrin. Stresses, such as iron overload, bacterial or fungal challenge, cold or heat shock, wounding, and H2O2 or paraquat exposure, cause upregulation of the beetle Apriona germari transferrin (AgTf) gene in the fat body and epidermis, and they cause increased AgTf protein levels. RNA interference (RNAi)-mediated AgTf reduction results in rapid induction of apoptotic cell death in the fat body during exposure to heat stress. The observed effect of AgTf RNAi indicates that AgTf inhibits heat stress-induced apoptotic cell death, suggesting a functional role for AgTf in defense and stress responses in the beetle.

Effects of Paecilomyces tenuipes cultivated in egg yolk on lipid metabolism in rats on a high fat-cholesterol diet.

We investigated the effects of the fruiting bodies of cultivated Paecilomyces tenuipes grown on egg yolk (PTE) on lipid and antioxidant metabolisms. Forty 8-week-old male Sprague-Dawley rats were fed a high fat/high cholesterol diet (control) or a high fat/high cholesterol diet with 1%, 3%, or 5% PTE for 5 weeks. PTE was found to significantly lower plasma total lipid, total cholesterol, low-density lipoprotein cholesterol, and the atherogenic index, compared with the control. Hepatic total lipid and total cholesterol were also significantly lower than in the control group. The hypolipidemic activity of PTE was increased with increasing concentrations, and plasma lipid peroxidation was significantly lower in the 3% and 5% PTE groups than in the control or 1% PTE group. Plasma total radical trapping antioxidant potential, erythrocytic antioxidant enzyme, and leukocytic DNA damage were not significantly different among the groups. Our results indicate that P. tenuipes cultivated on egg yolk can improve lipid profiles and lipid peroxidation in rats fed a high fat/high cholesterol diet.

Bombus ignitus Cu,Zn superoxide dismutase (SOD1): cDNA cloning, gene structure, and up-regulation in response to paraquat, temperature stress, or lipopolysaccharide stimulation.

A Cu,Zn superoxide dismutase (SOD1) gene was cloned from the bumblebee, Bombus ignitus. The SOD1 gene of B. ignitus spans 1,317 bp and consists of three introns and four exons encoding 151 amino acid residues. The B. ignitus SOD1 (BiSOD1) possesses the typical metal-binding ligands of six histidines and one aspartic acid common to SOD1s. The deduced amino acid sequence of the BiSOD1 cDNA showed 82% identity to Apis mellifera SOD1 and 68-64% to SOD1 sequences from other insects. Northern blot analysis revealed the presence of BiSOD1 transcripts in all tissues examined. When paraquat (methyl viologen), a free radical-inducing agent, was injected into the body cavity of B. ignitus workers, BiSOD1 mRNA expression was up-regulated in the fat body. In addition, the expression levels of BiSOD1 mRNA in the fat body significantly increased when B. ignitus workers were exposed to low (4 degrees C) or high (37 degrees C) temperatures, or injected with lipopolysaccharide (LPS), which suggests that BiSOD1 possibly protects against oxidative stress caused by extreme temperatures and bacterial infection.

Molecular cloning and characterization of ATX1 cDNA from the mole cricket, Gryllotalpa orientalis.

To search for an insect homologue of antioxidant protein 1 (ATX1), a mole cricket, Gryllotalpa orientalis, cDNA library was screened and a cDNA clone, which encodes a 73 amino acid polypeptide with a predicted molecular mass of 8.0 kDa and pI of 5.68, was isolated. The G. orientalis ATX1 (GoATX1) cDNA features both a MTCXXC copper-binding site in the N-terminus and a KTGK lysine-rich region in the C-terminus. The deduced amino acid sequence of the GoATX1 cDNA showed 63% identity to Drosophila melanogaster ATX1 and 55% to Ixodes pacificus ATX1. Northern blot analysis revealed the presence of GoATX1 transcripts in midgut, fat body, and epidermis. When H2O2 was injected into the body cavity of G. orientalis adult, GoATX1 mRNA expression was up-regulated in the fat body tissue. Fat body expression level of GoATX1 mRNA in the fat body was increased following exposure to low (4 degrees C) and high (37 degrees C) temperatures, suggesting that GoATX1 plays a protective role against oxidative stress caused by temperature shock. This is the first report about a functional role of insect ATX1 in antioxidant defense.

Cloning and characterization of the Cu,Zn superoxide dismutase (SOD1) cDNA from the mole cricket, Gryllotalpa orientalis.

A Cu,Zn superoxide dismutase (SOD1) cDNA was cloned from the mole cricket, Gryllotalpa orientalis. The G. orientalis SOD1 (GoSOD1) cDNA contains an open reading frame of 462 bp encoding 154 amino acid polypeptide with a predicted molecular mass of 15.8 kDa and pI of 6.1, and possesses the typical metal binding ligands of six histidines and one aspartic acid common to SOD1s. The deduced amino acid sequence of the GoSOD1 cDNA showed 75% identity to Lasius niger SOD1, 73% to Apis mellifera SOD1, and 70-68% to SOD1 sequences from other insects. Northern blot analysis revealed the presence of GoSOD1 transcripts in all tissues examined. The expression level of GoATX1 mRNA in the fat body was induced when G. orientalis adult was exposed at low (4 degrees C) and high (37 degrees C) temperatures, suggesting that the GoSOD1 seems to play a protective role against oxidative stress caused by temperature shock.

Molecular cloning, expression and characterization of cDNAs encoding the ferritin subunits from the beetle, Apriona germari.

Insect secreted ferritins are composed of subunits, which resemble heavy and light chains of vertebrate cytosolic ferritins. We describe here the cloning, expression and characterization of cDNAs encoding the ferritin heavy-chain homologue (HCH) and light-chain homologue (LCH) from the mulberry longicorn beetle, Apriona germari (Coleoptera, Cerambycidae). The A. germari ferritin LCH and HCH cDNA sequences were comprised of 672 and 636 bp encoding 224 and 212 amino acid residues, respectively. The A. germari ferritin HCH subunit contained the conserved motifs for the ferroxidase center typical of vertebrate ferritin heavy chains and the iron-responsive element (IRE) sequence with a predicted stem-loop structure was present in the 5'-untranslated region (UTR) of ferritin HCH mRNA. However, the A. germari ferritin LCH subunit had no IRE at its 5'-UTR and ferroxidase center residues. Phylogenetic analysis confirmed the deduced protein sequences of A. germari ferritin HCH and LCH being divided into two types, G type (LCH) and S type (HCH). Southern blot analysis suggested the possible presence of each A. germari ferritin subunit gene as a single copy and Northern blot analysis confirmed a higher expression pattern in midgut than fat body. The cDNAs encoding the A. germari ferritin subunits were expressed as approximately 30 kDa (LCH) and 26 kDa (HCH) polypeptides in baculovirus-infected insect cells. Western blot analysis and iron staining assay confirmed that A. germari ferritin has a native molecular mass of approximately 680 kDa.

cDNA cloning, expression, and characterization of an arylphorin-like hexameric storage protein, AgeHex2, from the mulberry longicorn beetle, Apriona germari.

An arylphorin-like hexameric storage protein, AgeHex2, cDNA was cloned from the mulberry longicorn beetle, Apriona germari (Coleoptera, Cerambycidae), larval cDNA library. The complete cDNA sequence of AgeHex2 is comprised of 2,088 bp encoding 696 amino acid residues. The AgeHex2 had four potential N-glycosylation sites. The AgeHex2 contained the highly conserved two larval storage protein signature motifs. The deduced protein sequence of AgeHex2 showed high homology with A. germari hexamerin1 (51% amino acid identity), Tenebrio molitor hexamerin2 (49% amino acid identity), T. molitor early-staged encapsulation inducing protein (43% amino acid identity), and Leptinotarsa decemlineata diapause protein1 (43% amino acid identity). Phylogenetic analysis further confirmed the AgeHex2 is more closely related to coleopteran hexamerins than to the other insect storage proteins. Northern blot analysis confirmed that the AgeHex2 showed fat body-specific expression. The cDNA encoding AgeHex2 was expressed as a 75-kDa protein in the baculovirus-infected insect cells. Furthermore, N-glycosylation of the recombinant AgeHex2 was revealed by tunicamycin to the recombinant virus-infected Sf9 cells, demonstrating that the AgeHex2 is N-glycosylated. Western blot analysis using the polyclonal antiserum against recombinant AgeHex2 indicated that the AgeHex2 corresponds to a 75-kDa storage protein present in the A. germari larval hemolymph.