Receptor-mediated endocytosis of storage proteins by the fat body of Helicoverpa zea

The selective uptake of storage proteins by the fat body of the corn earworm Helicoverpa zea is mediated by a membrane-bound receptor protein. In this study, the major storage proteins of this insect species, arylphorin and very high density lipoprotein, were directly labeled with colloidal gold-particles of different size. After the fat body had been incubated with the labeled storage proteins, the distribution of these proteins was examined by electron microscopy. Both storage proteins were found at the extracellular side of coated pits and within coated vesicles. Moreover, fusion products of several coated vesicles such as endosomes and multivesicular bodies contained both proteins in their lumen. Ultimately, the proteins accumulated in electron-dense storage granules. Equal numbers of either storage protein were present in each organelle, supporting the notion that a single receptor mediates the uptake of both proteins. In contrast, only small numbers of gold-labeled immunoglobulin G molecules were found in the organelles, indicating that the protein uptake is specific for storage proteins. The results show that storage protein uptake in this lepidoteran species occurs in a process of receptor-mediated endocytosis that is similar to the well-established uptake of specific proteins into mammalian tissues.     

Expression and localization of storage protein 1 (SP1) in differentiated fat body tissues of red hairy caterpillar, Amsacta albistriga Walker

The accumulation and utilization of storage proteins are prominent events linked to the metamorphosis of holometabolous insects. The female-specific storage protein 1 (SP1) is the major storage protein found in the hemolymph and fat body of female larvae of the groundnut pest, Amsacta albistriga. Here we show SP1 expression and localization in differentiated fat body tissues using biochemical and immunohistochemistry scrutiny. Comparison of A. albistriga SP1 with that of other species with respect to amino acid composition and N-terminal sequences show that SP1 is a methonine-rich protein and its identity was confirmed by means of immunoblot analysis. Northern blot studies revealed that the SP1 gene demonstrates stage- and tissue-specific expression in the peripheral fat body cells during the mid-larval period of fifth instar of A. albistriga. During the larval pupal transformation, SP1 are sequestered mainly by the perivisceral fat body tissues, until they serve the purpose of supplying amino acids for the production of egg yolk proteins. Further, electron microscopic studies using immunogold tracer techniques confirmed the localization of crystalline SP1 reserves, stored in the perivisceral fat body tissues. Hence, the peripheral fat body is responsible for biosynthesis of storage proteins, whereas the perivisceral fat body is a specialized storage organ.     

The fate and possible role of arylphorin during the metamorphosis of Mamestra brassicae.

1. Arylphorin, one of the storage proteins has been isolated from the hemolymph of Mamestra brassicae. 2. It has been established that Mamestra arylphorin is the most similar to manducin from among the known storage proteins of other species. 3. A rabbit polyclonal antibody has been developed against arylphorin, and its concentration changes have been determined quantitatively by ELISA in the hemolymph and fat body from the 1st day of the last larval instar to the 3rd day of the imago stage. 4. Histological sections were made on each day during the investigated period and it was shown by immunohistochemical methods that the main quantity of arylphorin was accumulated in the storage protein granules of the fat body and it could be detected even in the imaginal fat body. 5. The uptake of arylphorin by the fat body is induced by 20-hydroxyecdysone. 6. During differentiation of the imaginal cuticle arylphorin is incorporated first in the epidermal cells and it is built in the endocuticular layer of the integument thereafter.     

丽蝇蛹集金小蜂寄生对寄主蛹可溶性蛋白与芳基蛋白组成与含量的影响

为了探讨蛹期寄生蜂对寄主蛋白代谢的寄生生理效应,利用Bradford蛋白含量测定法、Western免疫印迹法及酶联免疫吸附检测法研究了棕尾别麻蝇Boettcherisca peregrina蛹被丽蝇蛹集金小蜂Nasonia vitripennis寄生后其脂肪体和血淋巴中可溶性蛋白及芳基蛋白组成与含量的变化。结果表明：寄生蛹脂肪体和血淋巴中可溶性蛋白的组成与未寄生相比基本无明显差异; 不论寄生与否寄主蛹脂肪体和血淋巴中芳基蛋白亚基分子量均为80 kDa,该亚基在脂肪体中未出现降解现象,而在血淋巴中仅于寄生后12 h的寄主蛹中呈现2条分子量相近的Western免疫印迹带,说明其降解可能先于未寄生对照。就含量而言,寄生蛹脂肪体中可溶性蛋白含量除寄生后24 h外均显著低于未寄生对照,芳基蛋白含量除寄生后48 h外也均显著低于未寄生对照,其中寄生后12 h的含量仅为未寄生的32.0%。寄生蛹血淋巴中可溶性蛋白含量多低于未寄生蛹,且寄生后2,12,24 h的差异达显著水平;芳基蛋白的含量均有低于未寄生的趋势,其中寄生后12 h的含量为未寄生的17.0%。综合认为,丽蝇蛹集金小蜂的寄生可导致寄主脂肪体和血淋巴中可溶性蛋白及芳基蛋白含量下降。     

Binding of insecticides to lipophorin and arylphorin,two hemolymph proteins of Heliothis zea

The binding of different insecticides to hemolymph proteins of the corn earworm, Heliothis zea, was studied by an analytical isoelectric focusing technique. Two proteins capable of binding hydrophobic substrates were discovered and identified as lipophorin and arylphorin. Lipophorin, the major lipoprotein of Heliothis zea, demonstrated rather unspecific binding of xenobiotics of different hydrophobicities. Arylphorin, a putative storage protein, revealed strong affinity for compounds of medium polarity, binding only weakly to insecticides of higher or lower polarity.     

Protein release from the larval fat body of the southwestern corn borer, Diatraea grandiosella: An in vitro study

The release of protein from the perivisceral fat body of non-diapausing, pre-diapausing and diapausing larvae of the southwestern corn borer, Diatraea grandiosella, was examined in vitro. Time course studies showed a selective release of proteins into macromolecule-free Grace's medium. The rate of release of individual proteins differed. The release of some proteins was partially inhibited by the incorporation of potassium cyanide (10^?2 M) and ouabain (5 × 10^?3 M) into the medium. During a 5 min incubation a single major high molecular weight protein fraction was released at a high rate from the fat body of both non-diapausing and diapausing larvae. A low molecular weight protein (the diapause-associated protein) was also released readily from the fat body of diapausing larvae. Although most proteins released from the fat body in vitro appeared to be present in the haemolymph in vivo, one notable exception was the absence of the diapause-associated protein from the haemolymph. The method holds promise for facilitating further studies of protein release from insect fat body.     

Interaction of the anterior fat body protein with the hexamerin receptor in the blowfly Calliphora vicina.

In late larvae of the blowfly, Calliphora vicina, arylphorin and LSP-2 proteins, which belong to the class of hexamerins, are selectively taken up by the fat body from the haemolymph. Hexamerin endocytosis is mediated by a specific membrane-bound receptor, the arylphorin-binding protein (ABP). Using the two-hybrid technique, we found that the anterior fat body protein (AFP) interacts with the hexamerin receptor. AFP, a homologue of the mammalian calcium-binding liver protein regucalcin (senescence marker protein-30), exhibits a strong binding affinity for a naturally occurring C-terminal cleavage fragment of the hexamerin receptor precursor (the P30 peptide) and other receptor cleavage products that contain P30. Expression of AFP mRNA and protein is restricted to the anterior part of the fat body tissue and to haemocytes in last-instar larvae. AFP mRNA occurs in all postembryonic developmental stages. Our results suggest that AFP plays a role in the regulation of hexamerin uptake by fat body cells along the anterior-posterior axis.     

Juvenile hormone binding protein traffic — Interaction with ATP synthase and lipid transfer proteins

Juvenile hormone (JH) controls insect development, metamorphosis and reproduction. In insect hemolymph a significant proportion of JH is bound to juvenile hormone binding protein (JHBP), which serves as a carrier supplying the hormone to the target tissues. To shed some light on JHBP passage within insect tissues, the interaction of this carrier with other proteins from Galleria mellonella (Lepidoptera) was investigated. Our studies revealed the presence of JHBP within the tracheal epithelium and fat body cells in both the membrane and cytoplasmic sections. We found that the interaction between JHBP and membrane proteins occurs with saturation kinetics and is specific and reversible. ATP synthase was indicated as a JHBP membrane binding protein based upon SPR-BIA and MS analysis. It was found that in G. mellonella fat body, this enzyme is present in mitochondrial fraction, plasma membranes and cytosol as well. In the model system containing bovine F₁ ATP synthase and JHBP, the interaction between these two components occurs with K_d = 0.86 nM. In hemolymph we detected JHBP binding to apolipophorin, arylphorin and hexamerin. These results provide the first demonstration of the physical interaction of JHBP with membrane and hemolymph proteins which can be involved in JHBP molecule traffic.     

In vitro synthesis, release and uptake of storage proteins by the fat body of Manduca sexta: putative hormonal control

1. Two major proteins (P1 and P2) are synthesized by the fifth instar larval fat body of Manduca sexta and then released into the hemolymph. 2. These proteins are later sequestered by the pre-pupal fat body. 20-Hydroxyecdysone does not appear to affect the synthesis of either protein. 3. When day 2 fifth instar larvae are neck-ligated there is an excessive synthesis (supersynthesis) of P2 (arylphorin). 4. Juvenile hormone I (JH I) applications to ligated animals had no effect, but brain homogenate injections resulted in the inhibition of P2 synthesis. 5. Neck ligations of larvae between days 5 and 6 revealed a head critical period between day 5 + 12 hr and day 5 + 18 hr, after which the head is unnecessary for the sequestration of either protein by the fat body. 6. JH I and JH III applications to ligated larvae before the head critical period do not restore the ability of the fat body to sequester the storage proteins. 7. P1 and P2 appear to be synthesized differentially and P2 is sequestered by the fat body to a much lesser extent than P1. 8. P2 is the hemolymph storage protein of both larval and pupal stages, whereas P1 appears to be the storage protein of the pupal fat body. 9. The data indicate that the synthesis of arylphorin and the resorption of both proteins are controlled by a putative head factor(s).     

Stimulation of midgut stem cell proliferation by Manduca sexta alpha-arylphorin

Extracts of the green-colored perivisceral fat body of newly ecdysed Manduca sexta pupae stimulate mitosis in midgut stem cells of Heliothis virescens cultured in vitro. Using a combination of cation- and anion-exchange chromatography, we have isolated a protein from these fat body extracts that accounts for the observed stem cell proliferation. SDS-PAGE analysis of the protein results in a single band of 77 kDa. Sequences of tryptic peptides from this protein are identical to internal sequences of the storage hexamer alpha-arylphorin. The alpha-arylphorin isolated by our procedure represents a small fraction of the total arylphorin present in the fat body extract. However, it alone seems responsible for the stimulation of mitotic activity in H. virescens midgut stem cells.     

Hormonal regulation of phase polymorphism and storage-protein fluctuation in the common cutworm, Spodoptera litura

Three storage proteins are synthesised by Spodoptera litura last-instar larvae as detected by an antiserum against pupal fat body proteins. The putative pupal storage proteins 1 and 2, appear in the haemolymph of the last-instar larvae 36 h after ecdysis under crowded rearing conditions: they appear 1 day later in isolated conditions. The appearance of these proteins in the haemolymph is prevented by juvenile hormone treatment and enhanced by allatectomy. Injection of 20-hydroxyecdysone into ligatured larvae does not induce appearance of these 2 proteins. Accumulation of protein 3 that reacts with Bombyx mori arylphorin antiserum is not blocked by juvenile hormone and is similar in both phases. It also accumulates to a small extent in the haemolymph during the moult to the final-larval instar and then disappears at ecdysis. One-hundred ng/ml ecdysteroid caused the sequestration of these proteins by the fat body, but a higher concentration of ecdysteroid (200 ng/ml) produced pupal cuticle in the isolated abdomens, suggesting that different ecdysteroid concentrations are necessary for these two events.     

Juvenile hormone binding components of locust fat body

Juvenile hormone (JH) binding components from the fat body of the African migratory locust were analyzed in a search for a potential nuclear JH receptor. Biosynthetically prepared 10R[³H]JH III gave a high proportion of specific binding to isolated nuclei and extracted proteins; data obtained with the JH analogs, [³H]methoprene and [³H]pyriproxyfen, on the other hand, were obscured by abundant non-specific binding. The vast majority of the high affinity JH III binding activity present in cytosolic and nuclear extracts was due to a high molecular weight JH binding protein (JHBP) which has previously been identified in locust hemolymph. This protein has several chromatographic forms which interfered in the search for a nuclear JH receptor. When specific antiserum was used to remove JHBP from nuclear extracts, a novel JH binding activity (NBP) was detected. NBP could be separated from JHBP by precipitation with ammonium sulfate. NBP displayed a high affinity for JH III (Kd = 0.25 nM) and JH I and JH II competed strongly for JH III binding, whereas methoprene and pyriproxyfen showed apparent competition when present in 1,000-fold excess. NBP was present in nuclear extracts at approximately 25,000 sites per cell; levels were similar in male and female locusts and were not greatly affected by the presence or absence of JH. The characteristics of NPB make it a strong candidate for a nuclear JH receptor. © 1995 Wiley-Liss, Inc.     

Protein degradation in silkworm peritracheal athrocytes and its physiological role in metamorphosis

The major functions of silkworm peritracheal athrocytes (nephrocytes) include endocytosis. Although athrocytes are also believed to function in protein degradation, there is limited experimental evidence for this. In this study, we detected the uptake and degradation of foreign proteins in peritracheal athrocytes by immunohistochemical, Western blot, and ex vivo analyses. IgG-FITC was detected in the athrocytes of silkworm larvae following injection, and LysoTracker analysis showed endosomal and lysosomal colocalizations. Athrocytes from larvae injected with IgG were incubated in Grace's medium for 2 days before being analyzed for the degradation of IgG by Western blotting. The level of incorporated IgG decreased and degradation products appeared following ex vivo culture. The highest level of IgG incorporation and degradation in the athrocytes was observed at the early pupal stage. The athrocytes also incorporated arylphorin, a major larval haemolymph protein and storage protein in silkworms. At the early pupal stage, arylphorin was actively degraded in the athrocytes. These results indicate that, in cooperation with the fat body, peritracheal athrocytes may function in the digestion of arylphorin during silkworm metamorphosis.     

Solubilization and Characterization of Striatal Dopamine Receptors

Abstract: Dopamine receptor binding proteins were sol-ubilized with the detergent 3–(3–cholamidopropyl) dimethylammonio - 2 - hydroxy - 1– propanesulfonate (CHAPSO) from bovine and rat striatal membranes. The binding of the dopamine antagonist [³H]spiroperidol ([³H]Spi) to the solubilized dopamine receptors was determined by the polyethyleneglycol method. The CHAPSO-solubilized dopamine receptor binding proteins remain in the supernatant fraction following centrifuga-tion at 100,000 ×g for 2 h. The CHAPSO-solubilized dopamine receptor proteins, as well as the prelabeled [³H]Spi-receptor protein complex, bind specifically to wheat germ agglutinin (WGA)-agarose columns, which is consistent with an identification as glycoproteins. HPLC analysis of the CHAPSO-solubilized, prelabeled [³H]Spi-receptor protein complex (CHAPSO preparation) reveals association with a high molecular weight form, indicating the formation of aggregates and/or micelles. Treatment of the WGA-agarose-bound [³H]Spi-receptor protein complex with digitonin (CHAPSO-digitonin preparation) results in dissociation of the high molecular weight form into lower molecular weight forms. The HPLC profile of the prelabeled [³H]Spi-receptor complex in the CHAPSO-digitonin preparation reveals two radioactive peaks. The major peak had a retention time of 16 min, corresponding to an apparent MW of 175,000, whereas the minor peak had a retention time of 21 min, corresponding to an apparent MW of 49,000. The CHAPSO-solubilized dopamine receptor binding proteins are sensitive to modulation by GTP, indicating that the association with the GTP binding component is preserved in the “soluble” state. The potencies of dopamine antagonists and agonists for inhibiting the binding of [³H]Spi to CHAPSO-solubilized dopamine receptor proteins are similar to those for membrane-bound proteins. Chronic treatment with haloperidol increases the B_max, and does not change the K_D for [³H]Spi in the CHAPSO-solubilized and in the membrane-bound preparations. Thus, the CHAPSO-solubilized dopamine receptor proteins retain the binding characteristics of the supersensitive membrane-bound dopamine receptors.     

Common origin of arthropod tyrosinase,arthropod hemocyanin,insect hexamerin,and dipteran arylphorin receptor

Dipteran arylphorin receptors, insect hexamerins, cheliceratan and crustacean hemocyanins, and crustacean and insect tyrosinases display significant sequence similarities. We have undertaken a systematic comparison of primary and secondary structures of these proteins. On the basis of multiple sequence alignments the phylogeny of these proteins was investigated. Hexamerin subunits, hemocyanin subunits, and tyrosinases share extensive similarities throughout the entire amino acid sequence. Our studies suggest the origin of arthropod hemocyanins from ancient tyrosinase-like proteins. Insect hexamerins likely evolved from hemocyanins of ancient crustaceans, supporting the proposed sister-group position of these subphyla. Arylphorin receptors, responsible for incorporation of hexamerins into the larval fat body of diptera, are related to hexamerins, hemocyanins, and tyrosinase. The receptor sequences display extensive similarities to the first and third domains of hemocyanins and hexamerins. In the middle region only limited amino acid conservation was observed. Elements important for hexamer formation are deleted in the receptors. Phylogenetic analysis indicated that dipteran arylphorin receptors diverged from ancient hexamerins, probably early in insect evolution. Correspondence to: T. Burmester     

Purification and characterization of three major hemolymph proteins of an insect,Calpodes ethlius (lepidoptera,hesperiidae)

Like many other Lepidoptera, fifth-stage Calpodes larvae have three major hemolymph proteins. Their molecular weights were estimated by 3-15% nondenaturing polyacrylamide gel electrophoresis (N-PAGE) as 470,000 (arylphorin; Ar), 580,000 (storage protein 2; SP2) and 720,000 (storage protein 1; SP1). Carbohydrate is associated with all three, but only Ar has lipid. The three proteins have been purified by preparative N-PAGE and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. On 3-15% SDS gels, Ar dissociated into 82,000 M_r subunits, SP2 into 86,000 M_r subunits, and SP1 into both 86,000 and 90,000 M_r subunits. The 470,000 M_r protein is identified as Ar because it is rich in aromatic amino acids. The 580,000 and 720,000 M_r proteins are rich in glycine and are called storage proteins. Electron microscopy of negatively stained preparations shows that each polymer has a different geometrical arrangement of subunits. SP1 is a cube made from eight subunits. SP2 is a hexamer in the form of a pentahedral prism. Ar is probably an octahedron made from six subunits. All three geometrical arrangements could permit the presence of a central carrying space.