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Klaus Scheller - One of the best experts on this subject based on the ideXlab platform.
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Effect of 20-hydroxyecdysone on synthesis and uptake of arylphorin by the larval fat body of Calliphora vicina (Diptera: Calliphoridae)
European Journal of Entomology, 2013Co-Authors: Thorsten Burmester, U Matzner, Klaus SchellerAbstract:During the final larval instar of the blowfly Calliphora vicina the storage protein arylphorin is stage-specifically produced and re-absorbed by the fat body cells. In this study the hormonal regulation of arylphorin biosynthesis and endocytosis has been investigated in vitro and in vivo. Application of 10 to 50 ng 20-hydroxyecdysone (20E) in vivo or of 10-7 to 10-6 M 20E in vitro specifically inhibits arylphorin translation without altering the arylphorin mRNA level. After termination of arylphorin synthesis in vivo, it can be artificially re-induced by neck-ligation, an effect which can be reversed by application of 50 ng 20E. This suggests that the small rise in titre of 20E at the end of feeding stage is responsible for the inhibition of arylphorin-mRNA translation in vivo. 20E stage-specifically stimulates arylphorin uptake by the larval fat body in vitro, as well as in vivo (Burmester & Scheller, in prep.). We assume two defined but different post-translational effects of 20E on arylphorin metabolism.
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Functional dissection of the hexamerin receptor and its ligand arylphorin in the blowfly Calliphora vicina
Insect molecular biology, 2003Co-Authors: Immo A. Hansen, V. Gutsmann, Susanne R. Meyer, Klaus SchellerAbstract:The process of receptor-mediated uptake of hexamerin storage proteins from insect haemolymph by fat body cells is a unique feature of the class Insecta. We identified the binding domains of the hexamerin receptor and the hexamerin ligand arylphorin in the blowfly, by means of the yeast-two-hybrid-system. The receptor-binding domain of arylphorin was located within domain 3 of the arylphorin monomer. The ligand-binding domain of the hexamerin receptor was mapped to the extreme N-terminus of the receptor. The binding domains identified exhibit no similarity to any functional protein domains known to date. Additionally, we identified two previously unknown protein-interactors of the hexamerin receptor. The results of this study provide further insights regarding the mechanism of the receptor-mediated endocytosis of storage proteins in insects.
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Interaction of the anterior fat body protein with the hexamerin receptor in the blowfly Calliphora vicina.
European journal of biochemistry, 2002Co-Authors: Immo A. Hansen, Susanne R. Meyer, Ingo Schäfer, Klaus SchellerAbstract: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.
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Developmentally Controlled Cleavage of the Calliphora Arylphorin Receptor and Posttranslational Action of the Steroid Hormone 20-Hydroxyecysone
European journal of biochemistry, 1997Co-Authors: Thorsten Burmester, Klaus SchellerAbstract:In response to a rise in ecdysteroid titre, fat body cells of insect larvae take up storage proteins from the haemolymph by receptor-mediated endocytosis. Here we show that the receptor responsible for incorporation of the major haemolymph protein arylphorin of the blowfly, Calliphora vicina, is subject to an unusual posttranslational processing that involves three distinct cleavage steps. After the removal of a 17-amino-acid signal peptide, a receptor precursor of 141 kDa is released. Before reaching the cell surface, the precursor is cleaved a second time, giving rise to the active 92-kDa arylphorin receptor, plus a 48-kDa peptide. The function of this 48-kDa peptide may be the prevention of premature ligand-receptor interaction in the endoplasmic reticulum. 20-Hydroxyecdysone initiates a third cleavage step of the arylphorin receptor, which results in a 62-kDa arylphorin binding protein and a 30-kDa peptide. Contrary to the standard model of steroid hormone action, the process which give rise to receptor cleavage can be induced by 20-hydroxyecdysone in vivo and in vitro even in absence of protein biosynthesis.
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Conservation of Hexamerin Endocytosis in Diptera
European journal of biochemistry, 1997Co-Authors: Thorsten Burmester, Klaus SchellerAbstract:In cyclorrhaphan Diptera at least two different types of haemolymph proteins exist which belong to the class of hexamerins. In the last larval instar of Calliphora vicina, the highly aromatic hexamerin, arylphorin, and the second hexamerin, PII, make up about 90% of haemolymph proteins. Both of these proteins are selectively taken up by the fat body cells at the end of larval life and share a common membrane-bound receptor. In addition, hexamerins and possible hexamerin receptors of Calliphora vicina, Calliphora vomitoria, Drosophila melanogaster, Ceratitis capitata, Sarcophaga bullata, Musca domestica and Protophormia terraenovae were investigated. Uptake of arylphorin by the larval fat bodies of Calliphora vicina as well asarylphorin-receptor binding can be competed in vitro by haemolymph from other Diptera. Therefore, hexamerin-receptor binding must be conserved among related cyclorrhaphan Diptera and between different types of hexamerins within a species. As the degree of competition is in good agreement with the presumed phylogenetic distances between these species, the method described here provides a simple tool to estimate evolutionary distances.
Thorsten Burmester - One of the best experts on this subject based on the ideXlab platform.
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Expression and evolution of hexamerins from the tobacco hornworm, Manduca sexta, and other Lepidoptera.
Insect biochemistry and molecular biology, 2014Co-Authors: Thorsten BurmesterAbstract:Hexamerins are large hemolymph-proteins that accumulate during the late larval stages of insects. Hexamerins have emerged from hemocyanin, but have lost the ability to bind oxygen. Hexamerins are mainly considered as storage proteins for non-feeding stages, but may also have other functions, e.g. in cuticle formation, transport and immune response. The genome of the hornworm Manduca sexta harbors six hexamerin genes. Two of them code for Arylphorins (Msex2.01690, Msex2.15504) and two genes correspond to a methionine-rich hexamerin (Msex2.10735) and a moderately methionine-rich hexamerin (Msex2.01694), respectively. Two other genes do not correspond to any known hexamerin and distantly resemble the Arylphorins (Msex2.01691, Msex2.01693). Five of the six hexamerin genes are clustered within ∼45 kb on scaffold 00023, which shows conserved synteny in various lepidopteran genomes. The methionine-rich hexamerin gene is located at a distinct site. M. sexta and other Lepidoptera have lost the riboflavin-binding hexamerin. With the exception of Msex2.01691, which displays low mRNA levels throughout the life cycle, all hexamerins are most highly expressed during pre-wandering phase of the 5th larval instar of M. sexta, supporting their role as storage proteins. Notably, Msex2.01691 is most highly expressed in the brain, suggesting a divergent function. Phylogenetic analyses showed that hexamerin evolution basically follows insect systematics. Lepidoptera display an unparalleled diversity of hexamerins, which exceeds that of other hexapod orders. In contrast to previous analyses, the lepidopteran hexamerins were found monophyletic. Five distinct types of hexamerins have been identified in this order, which differ in terms of amino acid composition and evolutionary history: i. the Arylphorins, which are rich in aromatic amino acids (∼20% phenylalanine and tyrosine), ii. the distantly related arylphorin-like hexamerins, iii. the methionine-rich hexamerins, iv. the moderately methionine rich hexamerins, and v. the riboflavin-binding hexamerins.
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Effect of 20-hydroxyecdysone on synthesis and uptake of arylphorin by the larval fat body of Calliphora vicina (Diptera: Calliphoridae)
European Journal of Entomology, 2013Co-Authors: Thorsten Burmester, U Matzner, Klaus SchellerAbstract:During the final larval instar of the blowfly Calliphora vicina the storage protein arylphorin is stage-specifically produced and re-absorbed by the fat body cells. In this study the hormonal regulation of arylphorin biosynthesis and endocytosis has been investigated in vitro and in vivo. Application of 10 to 50 ng 20-hydroxyecdysone (20E) in vivo or of 10-7 to 10-6 M 20E in vitro specifically inhibits arylphorin translation without altering the arylphorin mRNA level. After termination of arylphorin synthesis in vivo, it can be artificially re-induced by neck-ligation, an effect which can be reversed by application of 50 ng 20E. This suggests that the small rise in titre of 20E at the end of feeding stage is responsible for the inhibition of arylphorin-mRNA translation in vivo. 20E stage-specifically stimulates arylphorin uptake by the larval fat body in vitro, as well as in vivo (Burmester & Scheller, in prep.). We assume two defined but different post-translational effects of 20E on arylphorin metabolism.
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diversity of stonefly hexamerins and implication for the evolution of insect storage proteins
Insect Biochemistry and Molecular Biology, 2007Co-Authors: Silke Hagnerholler, Christian Pick, Stefan Girgenrath, James H Marden, Thorsten BurmesterAbstract:Hexamerins are large storage proteins of insects in the 500 kDa range that evolved from the copper-containing hemocyanins. Hexamerins have been found at high concentration in the hemolymph of many insect taxa, but have remained unstudied in relatively basal taxa. To obtain more detailed insight about early hexamerin evolution, we have studied hexamerins in stoneflies (Plecoptera). Stoneflies are also the only insects for which a functional hemocyanin is known to co-occur with hexamerins in the hemolymph. Here, we identified hexamerins in five plecopteran species and obtained partial cDNA sequences from Perla marginata (Perlidae), Nemoura sp. (Nemouridae), Taeniopteryx burksi (Taeniopterygidae), Allocapnia vivipara (Capniidae), and Diamphipnopsis samali (Diamphipnoidae). At least four distinct hexamerins are present in P. marginata. The full-length cDNA of one hexamerin subunit was obtained (PmaHex1) that measures 2475 bp and translates into a native polypeptide of 702 amino acids. Phylogenetic analyses showed that the plecopteran hexamerins are monophyletic and positioned at the base of the insect hexamerin tree, probably diverging about 360 million years ago. Within the Plecoptera, distinct hexamerin types evolved before the divergence of the families. Mapping amino acid compositions onto the phylogenetic tree shows that the accumulation of aromatic amino acids (and thus the evolution of ‘‘Arylphorins’’) commenced soon after the hexamerins diverged from hemocyanins, but also indicates that hexamerins with distinct amino acid compositions reflect secondary losses of aromatic amino acids. r 2007 Elsevier Ltd. All rights reserved.
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Developmentally Controlled Cleavage of the Calliphora Arylphorin Receptor and Posttranslational Action of the Steroid Hormone 20-Hydroxyecysone
European journal of biochemistry, 1997Co-Authors: Thorsten Burmester, Klaus SchellerAbstract:In response to a rise in ecdysteroid titre, fat body cells of insect larvae take up storage proteins from the haemolymph by receptor-mediated endocytosis. Here we show that the receptor responsible for incorporation of the major haemolymph protein arylphorin of the blowfly, Calliphora vicina, is subject to an unusual posttranslational processing that involves three distinct cleavage steps. After the removal of a 17-amino-acid signal peptide, a receptor precursor of 141 kDa is released. Before reaching the cell surface, the precursor is cleaved a second time, giving rise to the active 92-kDa arylphorin receptor, plus a 48-kDa peptide. The function of this 48-kDa peptide may be the prevention of premature ligand-receptor interaction in the endoplasmic reticulum. 20-Hydroxyecdysone initiates a third cleavage step of the arylphorin receptor, which results in a 62-kDa arylphorin binding protein and a 30-kDa peptide. Contrary to the standard model of steroid hormone action, the process which give rise to receptor cleavage can be induced by 20-hydroxyecdysone in vivo and in vitro even in absence of protein biosynthesis.
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Conservation of Hexamerin Endocytosis in Diptera
European journal of biochemistry, 1997Co-Authors: Thorsten Burmester, Klaus SchellerAbstract:In cyclorrhaphan Diptera at least two different types of haemolymph proteins exist which belong to the class of hexamerins. In the last larval instar of Calliphora vicina, the highly aromatic hexamerin, arylphorin, and the second hexamerin, PII, make up about 90% of haemolymph proteins. Both of these proteins are selectively taken up by the fat body cells at the end of larval life and share a common membrane-bound receptor. In addition, hexamerins and possible hexamerin receptors of Calliphora vicina, Calliphora vomitoria, Drosophila melanogaster, Ceratitis capitata, Sarcophaga bullata, Musca domestica and Protophormia terraenovae were investigated. Uptake of arylphorin by the larval fat bodies of Calliphora vicina as well asarylphorin-receptor binding can be competed in vitro by haemolymph from other Diptera. Therefore, hexamerin-receptor binding must be conserved among related cyclorrhaphan Diptera and between different types of hexamerins within a species. As the degree of competition is in good agreement with the presumed phylogenetic distances between these species, the method described here provides a simple tool to estimate evolutionary distances.
Juan Luis Jurat-fuentes - One of the best experts on this subject based on the ideXlab platform.
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Alpha-arylphorin is a mitogen in the Heliothis virescens midgut cell secretome upon Cry1Ac intoxication.
PeerJ, 2017Co-Authors: Anaïs Castagnola, Jerreme Jackson, Omaththage P. Perera, Cris Oppert, Shigetoshi Eda, Juan Luis Jurat-fuentesAbstract:Insecticidal crystal (Cry) proteins produced by the bacterium Bacillus thuringiensis (Bt) target cells in the midgut epithelium of susceptible larvae. While the mode of action of Cry toxins has been extensively investigated, the midgut response to Cry intoxication and its regulation are not well characterized. In this work, we describe the secreted proteome (secretome) of primary mature midgut cell cultures from Heliothis virescens larvae after exposure to Cry1Ac toxin compared to control buffer treatment. The Cry1Ac-induced secretome caused higher proliferation and differentiation and an overall reduction in total cell mortality over time in primary H. virescens midgut stem cell cultures when compared to treatment with control buffer secretome. Differential proteomics identified four proteins with significant differences in abundance comparing Cry1Ac-treated and control secretomes. The most significant difference detected in the Cry1Ac secretome was an arylphorin subunit alpha protein not detected in the control secretome. Feeding of purified alpha-arylphorin to H. virescens larvae resulted in midgut hyperplasia and significantly reduced susceptibility to Cry1Ac toxin compared to controls. These data identify alpha-arylphorin as a protein with a new putative role in the midgut regeneration process in response to Cry1Ac intoxication and possibly pathogen/abiotic stress, identifying alpha-arylphorin as a potential gene to target with insecticidal gene silencing for pest control.
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Arylphorin is a mitogen in the Heliothis virescens midgut cell secretome upon Cry1Ac intoxication
2017Co-Authors: Anaïs Castagnola, Jerreme Jackson, Omaththage P. Perera, Cris Oppert, Shigetoshi Eda, Juan Luis Jurat-fuentesAbstract:Insecticidal crystal (Cry) proteins produced by the bacterium Bacillus thuringiensis (Bt) target cells in the midgut epithelium of susceptible larvae. While the mode of action of Cry toxins has been extensively investigated, the midgut response to Cry intoxication and its regulation are not well characterized. In this work, we report the secreted proteome (secretome) of primary mature midgut cell cultures from Heliothis virescens larvae after exposure to Cry1Ac toxin compared to control buffer treatment. Biological activity of the Cry1Ac-induced secretome was monitored as higher proliferation and differentiation and an overall reduction in total cell mortality over time in primary H. virescens midgut stem cell cultures when compared to treatment with control buffer secretome. Differential proteomics identified 4 proteins with significant differences in abundance comparing Cry1Ac-treated and control secretomes. The most significant difference detected in the Cry1Ac secretome was an arylphorin protein not detected in the control secretome. Feeding of purified arylphorin to H. virescens larvae resulted in midgut hyperplasia and significantly reduced susceptibility to Cry1Ac toxin compared to controls. These data identify arylphorin as a protein with a putative relevant role in the midgut regeneration process in response to Cry1Ac intoxication.
Shunji Natori - One of the best experts on this subject based on the ideXlab platform.
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Purification, Characterization, and cDNA Cloning of ABP-2 (Aryiphorin Gene-Specific Binding Protein-2) That Specifically Binds to the ABP-1-Binding Sequence in the Arylphorin Gene of Sarcophaga peregrina
Journal of biochemistry, 1996Co-Authors: Nobuaki Adachi, Takeo Kubo, Shunji NatoriAbstract:Previously, we demonstrated that ABP-1 (arylphorin gene-specific binding protein-1), which is suggested to be the transcriptional activator of the arylphorin gene of Sarcophaga peregrina, is present in NIH-Sape-4 cells, which do not express arylphorin. As well as ABP-1, these cells were found to contain another protein (ABP-2) that probably binds to the same sequence as that to which ABP-1 binds [Adachi, N., Kubo, T., and Natori, S. (1993) J. Biochem. 114, 55-60]. We purified ABP-2 from a nuclear extract of NIH-Sape-4 cells and compared its DNA-binding activity with that of ABP-1. Both ABP-1 and ABP-2 were found to bind to the same sequence in the arylphorin gene with the same affinity and stability, but an ABP-2-specific hypersensitive site was detected by DNase I footprinting analysis. Analyses of proteolytic fragments suggested that both ABP-1 and ABP-2 have Zn fingers showing high similarity with that of AEF-1, a transcriptional repressor of the Drosophila melanogaster alcohol dehydrogenase gene that binds to a sequence very similar to that binding ABP-1 and ABP-2. We isolated a candidate cDNA for ABP-2, and the protein it encoded contained nine Zn fingers and regions rich in alanine, glutamine, serine/threonine, glycine, histidine, and asparagine.
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Molecular Cloning and Sequencing of Arylphorin-binding Protein in Protein Granules of the Sarcophaga Fat Body IMPLICATION OF A POST-TRANSLATIONAL PROCESSING MECHANISM
The Journal of biological chemistry, 1995Co-Authors: Sung Ook Chung, Takeo Kubo, Shunji NatoriAbstract:Abstract Previously, we identified an arylphorin-binding protein of Sarcophagaperegrina (flesh fly) with a molecular mass of 120 kDa and suggested its participation in the selective uptake of arylphorin from the hemolymph into the pupal fat body at metamorphosis (Ueno, K., and Natori, S.(1984) J. Biol. Chem. 259, 12107-12111). This paper reports the isolation and sequencing of cDNA for the 120-kDa protein. This protein consists of 1146 amino acid residues. Immunoblotting and RNA blotting experiments revealed that this protein is present as two fragments of 76 kDa (695 residues) and 53 kDa (451 residues) in the larval fat body. When larvae pupate, the 120-kDa protein gene is further activated and the complete 120-kDa protein is synthesized without fragmentation. This suggests a novel mechanism for the production of the 120-kDa protein regulated by a proteinase depending upon the stage of development of Sarcophaga. All of these proteins were found to be localized in protein granules in the adipocytes.
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Purification and Characterization of the Arylphorin Gene Specific Binding Protein from an Embryonic Cell Line of Sarcophaga peregrina (Flesh Fly).
Journal of biochemistry, 1993Co-Authors: Nobuaki Adachi, Takeo Kubo, Shunji NatoriAbstract:Previously, we purified a DNA binding protein from a nuclear extract of the fat body of Sarcophaga peregrina larvae that binds to the ACCACAACA motif in the 5'-upstream region of the arylphorin gene, and suggested that this protein is a transcriptional activator of the arylphorin gene. In this study, we detected and purified the same protein (ABP-1) from an embryonic cell line of Sarcophaga that does not express the arylphorin gene. Unlike the fat body, which synthesizes arylphorin actively, the embryonic cells were found to contain an additional DNA binding protein (ABP-2) that bound to the same DNA probe as ABP-1, suggesting a novel mechanism of regulation of the arylphorin gene.
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Purification of a stage-specific and sequence-specific DNA-binding protein for the arylphorin gene of Sarcophaga peregrina.
Biochimica et biophysica acta, 1991Co-Authors: Jung Woo Kim, Hiroto Komano, Shunji NatoriAbstract:A protein that binds specifically to the nucleotide sequence ACCACAACA located at residues −247 to −255 upstream of the +1 site of the arylphorin gene of Sarcophaga peregrina was purified to homogeneity from fat body nuclei of third instar larvae. This DNA-binding protein consisted of two subunits with molecular masses of 40 kDa and 42 kDa, respectively. Accurate transcription initiation of a truncated arylphorin gene in a nuclear extract of NIH-Sape-4 cells, an embryonic cell line of Sarcophaga, was significantly enhanced in the presence of the purified DNA-binding protein.
Hartmut Kayser - One of the best experts on this subject based on the ideXlab platform.
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cDNA sequences of two arylphorin subunits of an insect biliprotein: phylogenetic differences and gene duplications during evolution of hexamerins-implications for hexamer formation.
Journal of experimental zoology. Part B Molecular and developmental evolution, 2016Co-Authors: Bernhard Lieb, Bettina Ebner, Hartmut KayserAbstract:Arylphorins represent a conserved class of hexameric ∼500 kDa insect hemolymph glycoproteins, rich in aromatic amino acids, which are produced in large quantities at the larval stage as reserves for metamorphosis and egg development. The recently isolated arylphorin from the moth Cerura vinula is unique in being complexed to a novel farnesylated bilin. Protein sequencing suggested the presence of two different ∼85 kDa subunits. Here, we report the complete coding sequences of two cDNAs encoding two Arylphorins subunits with 67% identity and calculated physicochemical characteristics in agreement with the isolated holoprotein. Our phylogenetic analyses of the hexamerins revealed monophyletic origins not only for each of the Arylphorins and methionine-rich proteins (H-type and M-type), the two major classes of hexamerins, but also for the minor groups of arylphorin-like and riboflavin-binding hexamerins. We named the latter proteins X-type (mixed type) hexamerins because they share sequence features with both major groups, and they show unique deletions and insertions at conserved sites located on the protein surface. We present a phylogenetic tree of lepidopteran hexamerins, which is in agreement with actual systematics. Overall, duplications of hexamerin genes occurred independently in several lepidopteran lineages. We also analyzed the hexamerin sequences for key parameters, which characterize each type of hexamerins. Based on the crystal structure of the homomeric arylphorin from Antheraea pernyi, we present a model for the heteromeric Cerura protein focusing on the role of N-glycan structures in stabilizing the hexamer structure.
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Very high-density lipoprotein and vitellin as carriers of novel biliverdins IXα with a farnesyl side-chain presumably derived from heme A in Spodoptera littoralis.
Insect biochemistry and molecular biology, 2015Co-Authors: Hartmut Kayser, Manfred Nimtz, Philippe Ringler, Shirley A. MüllerAbstract:Bilins in complex with specific proteins play key roles in many forms of life. Biliproteins have also been isolated from insects; however, structural details are rare and possible functions largely unknown. Recently, we identified a high-molecular weight biliprotein from a moth, Cerura vinula, as an arylphorin-type hexameric storage protein linked to a novel farnesyl biliverdin IXα; its unusual structure suggests formation by cleavage of mitochondrial heme A. In the present study of another moth, Spodoptera littoralis, we isolated two different biliproteins. These proteins were identified as a very high-density lipoprotein (VHDL) and as vitellin, respectively, by mass spectrometric sequencing. Both proteins are associated with three different farnesyl biliverdins IXα: the one bilin isolated from C. vinula and two new structurally closely related bilins, supposed to be intermediates of heme A degradation. The different bilin composition of the two biliproteins suggests that the presumed oxidations at the farnesyl side-chain take place mainly during egg development. The egg bilins are supposedly transferred from hemolymph VHDL to vitellin in the female. Both biliproteins show strong induced circular dichroism activity compatible with a predominance of the M-conformation of the bilins. This conformation is opposite to that of the arylphorin-type biliprotein from C. vinula. Electron microscopy of the VHDL-type biliprotein from S. littoralis provided a preliminary view of its structure as a homodimer and confirmed the biochemically determined molecular mass of ∼350 kDa. Further, images of S. littoralis hexamerins revealed a 2 × 3 construction identical to that known from the hexamerin from C. vinula.
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Isolation, characterisation and molecular imaging of a high-molecular-weight insect biliprotein, a member of the hexameric arylphorin protein family.
Journal of molecular biology, 2009Co-Authors: Hartmut Kayser, Karlheinz Mann, Gia Machaidze, Manfred Nimtz, Philippe Ringler, Shirley A. Müller, Ueli AebiAbstract:The abundant blue hemolymph protein of the last instar larvae of the moth Cerura vinula was purified and characterized by protein-analytical, spectroscopic and electron microscopic methods. Amino acid sequences obtained from a large number of cleavage peptides revealed a high level of similarity of the blue protein with Arylphorins from a number of other moth species. In particular, there is a high abundance of the aromatic amino acids tyrosine and phenylalanine amounting to about 19% of total amino acids and a low content of methionine (0.8%) in the Cerura protein. The mass of the native protein complex was studied by size-exclusion chromatography, analytical ultracentrifugation, dynamic light scattering and scanning transmission electron microscopy and found to be around 500 kDa. Denaturating gel electrophoresis and mass spectrometry suggested the presence of two proteins with masses of about 85 kDa. The native Cerura protein is, therefore, a hexameric complex of two different subunits of similar size, as is known for Arylphorins. The protein was further characterized as a weakly acidic (pI ∼ 5.5) glycoprotein containing mannose, glucose and N-acetylglucosamine in an approximate ratio of 10:1:1. The structure proposed for the most abundant oligosaccharide of the Cerura arylphorin was the same as already identified in Arylphorins from other moths. The intense blue colour of the Cerura protein is due to non-covalent association with a bilin of novel structure at an estimated protein subunit-to-ligand ratio of 3:1. Transmission electron microscopy of the biliprotein showed single particles of cylindrical shape measuring about 13 nm in diameter and 9 nm in height. A small fraction of particles of the same diameter but half the height was likely a trimeric arylphorin dissociation intermediate. Preliminary three-dimensional reconstruction based on averaged transmission electron microscopy projections of the individual particles revealed a double-trimeric structure for the hexameric Cerura biliprotein complex, suggesting it to be a dimer of trimers.
