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PubMed2000

cDNA cloning of acyl-CoA desaturase homologs in the silkworm, Bombyx mori.

We have isolated two acyl-CoA desaturase clones from a pheromone gland cDNA library by using the EST (expressed sequence tag) database of Bombyx mori. The putative acyl-CoA desaturases encoded by the clones desat 1 (2029bp) and desat 2 (2341bp) have 98% identity, and both proteins show 61% identities to Trichoplusia ni acyl-CoA Delta(11) desaturase. The deduced amino acid sequences conserve well the histidine clusters that are catalytically essential for acyl-CoA desaturase activity. Northern blot and RT-PCR analyses revealed that both transcripts of desat 1 and desat 2 were expressed predominantly in the pheromone gland. Both transcripts detected 3days before adult eclosion dramatically increased a day before adult eclosion, keeping the mRNA levels high even after eclosion. These results, combined with the fact that Delta(11) and Delta(10, 12) desaturation of palmitate is a key step to synthesize pheromone in B. mori, suggest that the desaturases encoded by desat 1 and desat 2 are involved in either or both of the desaturation steps in the pheromone biosynthetic pathway of B. mori. The mRNA levels of desat 1 and desat 2 were not affected by decapitation or injection of the pheromone biosynthesis activating neuropeptide (PBAN) into the adult female moth, suggesting that the transcription of desat 1 and desat 2 is not regulated by PBAN. In addition to the clones in the pheromone gland, eight other clones encoding the same Delta(9) desaturase homolog were found in an embryonic cDNA library by searching from the EST database of B. mori. The deduced amino acid sequence from one of the clones (desat 3) shows 79% identity to T. ni Delta(9) desaturase but only 52% identity to the desaturases in the pheromone gland of B. mori. Northern blot analysis showed that the mRNA corresponding to the desat 3 was detected in the ovary and fat body, but not in the pheromone gland. Abundance of the Delta(9) desaturase clones (eight out of the 762 randomly sequenced clones) in the library prepared from diapause-destined embryos (40h after oviposition) suggests that the Delta(9) desaturase encoded by desat 3 plays an important role in embryonic development in B. mori.

Amino Acid Sequence↗
PubMed2002

Molecular cloning and expression pattern of a Cubitus interruptus homologue from the mulberry silkworm Bombyx mori.

A homologue of the segment polarity gene Cubitus interruptus from Bombyx mori, (BmCi) has been cloned and characterized. This region harbouring Zn(2+) finger motif is highly conserved across species. In B. mori, BmCi RNA expression was first detected at stage 6 of embryogenesis, which reached maximum levels at stage 21C and was maintained until larval hatching. The segmentally reiterated striped pattern of transcript distribution in stage 21C embryos was in conformity with its predicted segment polarity nature. BmCi was expressed in the fore- and hind-wing discs, ovaries, testes and gut during fifth larval intermolt, reminiscent of its expression domains in Drosophila. Besides, BmCi expression was seen in the anterior part of the middle silkglands in late embryonic stages, and this pattern was maintained during larval development. The transition from third to fourth and fifth larval intermolts was accompanied by an increase in the transcript levels in the middle silkglands. Our results demonstrate the presence of a novel expression domain for Ci in Bombyx.

Amino Acid Sequence↗
PubMed2000

Synthesis and resorption of a humoral chymotrypsin inhibitor, CI-8, by fat body of the silkworm, Bombyx mori.

The Bombyx mori hemolymph contains up to 16 chymotrypsin inhibitors (CIs). The present in vitro culture of tissues in Grace's medium indicated that CI-8, which belongs to the largest molecular-size group of CIs with sugar moiety, is synthesized in the fat body and secreted from it during the feeding period. When the fat body from other strain which synthesizes an allelic component (CI-7) instead of CI-8 was incubated in vitro in hemolymph from the strain which has CI-8, the fat body was found to receive CI-8. Thus it was concluded that CI-8, once secreted into the hemolymph, was again sequestered into the fat body after the onset of spinning. Protein granules isolated from the pupal fat body were shown to contain CI-8, indicating that the sequestered CI-8 is present in the protein granules.

Animals↗
PubMed2001

Characterization of acyl-CoA-binding protein (ACBP) in the pheromone gland of the silkworm, Bombyx mori.

Various fatty acyl-CoAs are involved as intermediates or precursors of sex pheromone components in the biosynthetic pathway of the pheromones in many lepidopteran insects. We have purified a 10-kDa protein from the cytosolic fraction of Bombyx mori pheromone glands by using affinity chromatography with a palmitoyl-CoA-agarose column and reversed-phase HPLC. Amino acid sequence analysis of the fragment peptides obtained from the purified protein, and a homology search, revealed that this protein was a member of acyl-CoA-binding proteins (ACBPs). MALDI-TOF mass spectral analysis of the purified protein and cloning of the gene from a pheromone gland cDNA library confirmed B. mori ACBP to be a 90 amino acid protein with 78.9% identity to that of Manduca sexta ACBP. The secondary structure of the recombinant B. mori ACBP was determined by NMR spectroscopy. Northern blot analysis demonstrated that B. mori ACBP was predominantly expressed in the pheromone gland and the corresponding transcript was expressed from the day before adult eclosion. Present results suggest that ACBP plays a significant role in the production of sex pheromones regulated by the neurohormone, pheromone biosynthesis activating neuropeptide (PBAN).

Acyl Coenzyme A↗
PubMed2001

Multiple transport pathways for dibasic amino acids in the larval midgut of the silkworm Bombyx mori.

The transport pathways for dibasic amino acids were investigated in brush border membrane vesicles (BBMV) from the anterior-middle (AM) and posterior (P) regions of Bombyx mori midgut. In the absence of K(+), a low-affinity saturable transport of arginine in both AM- and P-BBMV (K(m) 1.01 mM, V(max) 4.07 nmol/7s/mg protein and K(m) 1.38 mM, V(max) 2.26 nmol/7s/mg protein, respectively) was detected. Arginine influx was dependent on the membrane electrical potential (Deltapsi) and increased raising the alkalinity of the external medium from pH 7.2 to 10.6. Competition experiments indicated the following order of substrate affinity: arginine, homoarginine, N(G)-monomethylarginine, N(G)-nitroarginine>lysine>>ornithine>cysteine>methionine. Leucine, valine and BCH (2-amino-2-norbornanecarboxylic acid) did not inhibit arginine influx. In the presence of external K(+), the influx of arginine as a function of arginine concentration fitted to a complex saturation kinetics compatible with both a low-affinity and a high-affinity component. The latter (K(m) 0.035 mM, V(max) 2.54 nmol/7s/mg protein) was fully characterized. The influx rate had an optimum at pH 8.8, was strongly affected by Deltapsi and was homogeneous along the midgut. The substrate affinity rank was: homoarginine>arginine, N(G)-monomethylarginine>>cysteine, lysine>>N(G)-nitroarginine>ornithine>methionine. Leucine and amino acids with a hydrophobic side chain were not accepted. This system is also operative in the absence of potassium, with the same order of specificity but a very low activity. Lysine influx is mediated by two more transport systems, the leucine uniport and the K(+)/leucine symport specific for amino acids with a hydrophobic side chain that recognizes lysine at extravesicular pH values (pH(out)) exceeding 9. Both the uniport and the symport differ from the cationic transport systems so far identified in mammals because they are unaffected by N-ethylmaleimide, have no significant affinity for neutral amino acids in the presence of the cation and show a striking difference in their optimum pH.

Alkalies↗
PubMed2001

Adenylate cyclase in prothoracic glands during the last larval instar of the silkworm, Bombyx mori.

We have previously reported that the absence of prothoracicotropic hormone (PTTH) signal transduction during the early last larval instar of Bombyx mori plays a role in leading to very low ecdysteroid levels in the hemolymph, inactivation of the corpora allata, as well as larval-pupal transformation. In the present study, adenylate cyclase was characterized in crude preparations of prothoracic gland cell membranes in an effort to localize the cause of refractoriness to PTTH. It was found that cyclase activity of the prothoracic glands from the day 6 last instar showed activation responses to fluoride, a guanine nucleotide analogue, as well as calmodulin (CaM) in dose-dependent fashions. The additive effects of day 5 prothoracic gland adenylate cyclase stimulation by fluoride and CaM imply that there may exist Gs protein-dependent and CaM-dependent forms of adenylate cyclase. For day 1 last instar prothoracic glands, which showed no response to stimulation by PTTH in either cAMP generation or ecdysteroidogenesis, adenylate cyclase activity exhibited far less responsiveness to Ca(2+)/CaM than did that from day 5 glands. These findings suggest that day 1 prothoracic glands may possess some lesions in the receptor-Ca(2+) influx-adenylate cyclase signal transduction pathway and these impairments in PTTH signal transduction may be, at least in part, responsible for decreased ecdysteroidogenesis.

Adenylyl Cyclases↗
PubMed2002

Ecdysteroid-dependent expression of a novel cuticle protein gene BMCPG1 in the silkworm, Bombyx mori.

When insects molt, the exoskeleton is renewed under the controls of insect hormones via the biosynthesis and degradation of cuticle proteins. To understand the hormonal control of cuticle formation, we used the differential display method to look for stage-specific cuticle genes, and identified a novel cDNA named Bombyx mori Cuticle Protein GlyGlyTyr-repeat 1 (BMCPG1). Expression of BMCPG1 mRNA peaked sharply immediately after a pulse of ecdysteroid during the fourth molt and pre-pupal stages, concurrent with the expression of genes for FTZF1 and dopa decarboxylase. BMCPG1 was expressed only in the epidermis, but not in any other tissue. We cultured the larval epidermis and found that BMCPG1 expression is not induced by the continuous presence of ecdysteroid. Removal of ecdysteroid from the medium, which constitutes a pulse treatment, is required for the induction of BMCPG1 transcription. These results explain well the stage-specific expression of BMCPG1 by ecdysteroid in vivo. Based on its expression patterns and unique structure, we propose that BMCPG1 may be a novel component of epicuticle of B. mori, and is probably involved in cross-linking of proteins via its GGY repeats.

Amino Acid Sequence↗
PubMed2002

3xP3-EGFP marker facilitates screening for transgenic silkworm Bombyx mori L. from the embryonic stage onwards.

Transgenesis was recently achieved in Bombyx mori L., but it has proved difficult and time-consuming to screen the numerous progeny to identify the transgenic individuals. As the 3xP3-EGFP marker has been shown to be a suitable universal marker for transgenic insects (Nature 402 (1999) 370), we evaluated its use for embryonic-stage screening for B. mori L. germline transformation. Using the piggyBac-derived vector pBac[3xP3-EGFPaf], we were able to isolate four transgenic individuals from about 120,000 embryos (560 broods). The screening was straightforward due to EGFP production in the G1 embryonic stemmata, which was visible through the translucent egg chorion. EGFP was produced in the stemmata and central and peripheral nervous systems from the fifth day of embryonic development. It persisted at high levels in the stemmata throughout the larval stage, and was also present in the compound eyes and nervous tissues of the pupae and the compound eyes of the moths.

Animals↗
PubMed2002

Protein purification, cDNA cloning and gene expression of attacin, an antibacterial protein, from eri-silkworm, Samia cynthia ricini.

Attacin was isolated from immunized larval hemolymph of the wild silkmoth, Samia cynthia ricini. The antibacterial effect of the attacin was limited to some species of Gram-negative bacteria. Two cDNA clones encoding attacin A and B, respectively, were isolated by screening the cDNA library from immunized fat body. The two cDNAs encoded the same length of precursor protein with 233 amino acid residues. The 46-residue prepropeptides of the two attacins were identical to each other, but 4 out of 187 residues of the mature proteins were different in each other. The two attacins show 98% identity at the amino acid level, while 92% identity at the nucleotide level. Both of the mature proteins were highly homologous to cecropia basic attacin with identity of 96%. The attacin transcripts were detected at significant level in fat body, hemocytes and Malpighian tube after injection with peptidoglycan, but not in the midgut and the silkgland. The induction of attacin gene expression was elicited most effectively by peptidoglycan and UV-killed bacteria in the fat body.

Amino Acid Sequence↗
PubMed2003

Protein purification, cDNA cloning and characterization of a protease inhibitor from the Indian tasar silkworm, Antheraea mylitta.

An inhibitor of Aspergillus oryzae fungal protease was purified to homogeneity from the hemolymph of fifth instar larvae of Antheraea mylitta by ammonium sulfate precipitation, anion exchange and gel filtration (FPLC) chromatography, and termed as AmFPI-1. The extent of purification was checked by two-dimensional gel electrophoresis, and the molecular weight of purified inhibitor was determined by SDS-PAGE as 10.4 kDa. Fifteen N-terminal amino acid sequences of this protein were determined, and degenerate oligonucleotides were synthesized on the basis of these sequences. A cDNA library of A. mylitta integument was constructed, and protease inhibitor cDNA was partially amplified by PCR using degenerate oligonucleotides and CDS primers. A full-length inhibitor cDNA clone obtained by screening the library with PCR amplified DNA as probe was sequenced. The cDNA consists of 543 nucleotides with an ORF of 315 bp and encodes a protein of 105 amino acids. The sequence exhibits similarity to several Bombyx mori ESTs, and in particular to N-terminal amino acid sequence of an inducible serine protease inhibitor (ISPI-1) from Galleria mellonella indicating its relatedness to ISPI-1 of G. mellonella. The presence of this protease inhibitor in the hemolymph may play an important role as a natural defense system against invading microorganisms.

Amino Acid Sequence↗
PubMed1998

Molecular cloning of chitinase cDNAs from the silkworm, Bombyx mori and the fall webworm, Hyphantria cunea.

cDNAs encoding chitinases were cloned and characterized from Bombyx mori and Hyphantria cunea, and their gene expression during the metamorphosis was also studied. The chitinase cDNA from B. mori encodes a protein of 565 amino acids with a calculated molecular mass of 63.4 kDa and the H. cunea chitinase cDNA encodes a protein of 553 amino acids with a calculated molecular mass of 62.0 kDa. Amino acid alignment of the two chitinases revealed 75% homology and 77-80% with M. sexta chitinase. The putative cleavage site of the signal peptide was between amino acid residues 20 and 21 for both chitinases. There were three potential N-glycosylation sites in the chitinase of B. mori at the amino acid residues 86-89, NFTS 304-307, NATG, 398-401, NYTV, whereas two potential N-glycosylation sites were present at the amino acid residues 86-89, NFTA and 304-307, NATG, in that of H. cunea. Southern blot analysis of total genomic DNA suggested that the B. mori genome has only one chitinase gene detectable by the cDNA probe and the H. cunea genome has one or two chitinase gene copies. Northern analysis indicated that gene expression was up-regulated during the molting process, larval-pupal transformation and pupal-adult transformation, when enzymatic degradation of cuticle was occurring.

Amino Acid Sequence↗
PubMed1999

Minimum structure of peptidoglycan required for induction of antibacterial protein synthesis in the silkworm, Bombyx mori.

Various peptidoglycan fragments, different in mode of cross-linking and molecular size, were isolated, and the elicitor activity was tested for induction of antibacterial protein synthesis in larvae of Bombyx mori. Linear uncross-linked peptidoglycans from Bacillus licheniformis and Micrococcus luteus were effective elicitors, similar to the directly cross-linked peptidoglycan from B. licheniformis cell wall. The fragments of uncross-linked peptidoglycan with a sugar chain length of four or more were active elicitors, but the disaccharide unit had no elicitor activity. The minimum structure of peptidoglycan required for induction of antibacterial protein synthesis was determined to be two repeating N-acetylglucosamine-N-acetylmuramic acid units with peptide side chains.

Animals↗
PubMed2002

Determination of a sugar chain and its linkage site on a glycoprotein TIME-EA4 from silkworm diapause eggs by means of LC-ESI-Q-TOF-MS and MS/MS.

The electrospray ionization (ESI)-tandem quadrupole/orthogonal-acceleration time-of-flight (Q-TOF) mass spectrometer combined with the nano-HPLC system was utilized to determine the glycosylation site and the glycan structure in glycoprotein TIME-EA4 (EA4) from Bombyx diapause eggs. LC-MS analysis of EA4 and deglycosylated EA4 indicated that the carbohydrate moiety of EA4 has the mass of 730.58 Da. Then, EA4 was digested with trypsin and chymotrypsin to identify the glycosylated peptide. The peptide fragment from G1y21 to Phe25 was found to carry the carbohydrate moiety. LC-MS/MS analysis of this peptide fragment revealed the sequence of the attached oligosaccharide and the glycosylation site at the same time. The present methodology utilizing the combination of the nano-HPLC system and a highly sensitive Q-TOF mass spectrometer is demonstrated to be quite effective for analyses of glycoproteins of relatively low purity and limited availability from natural sources.

Amidohydrolases↗
PubMed1998

Structural study on silkworm eclosion hormone fragment (1-34) in solution by proton nuclear magnetic resonance spectroscopy.

Eclosion hormone (EH) is a neuropeptide hormone which controls the ecdysis behavior in insect. The three dimensional structure of the N-terminal fragment (1-34) of the eclosion hormone which was predicted to contain a compact region crucial for the EH activity was studied in 50% d3-trifluoroethanol(TFE)/50% H2O at pH 3 and 298 K by 1H NMR spectroscopy with the combined use of distance geometry and molecular dynamics calculations. NMR results indicated that the fragment actually assumes an alpha-helix between Ala10 and Gln20, but no rigid structure is present from Cys21 through the C-terminus and for the N-terminal region (Ser1-Asp9). The elucidated structure was compared with the predicted structure of the native EH for the further development of the design of the insecticide.

Amino Acid Sequence↗
PubMed2003

Sexual attraction in the silkworm moth. Nature of binding of bombykol in pheromone binding protein--an ab initio study.

An analysis of the crystal structure of [BmPBP...bombykol] complex identified nine amino acid residues involved in a variety of intermolecular interactions binding the ligand. Using simple model fragments as the representatives of the residues, the interaction energies of their complexes with bombykol were calculated using high-level ab initio methods. The results were discussed in terms of the method and basis set dependence and were further corrected to account for their pair nonadditivities. This enabled us to describe quantitatively the nature and origin of the binding forces in terms of contribution of the individual amino acids and individual types of interaction to the overall stability. All of these interactions are well defined and cannot be considered as nonspecific hydrophobic interactions, one of the major conclusions of this work.

Amino Acids↗
PubMed2001

Protein purification, cDNA cloning and gene expression of lysozyme from eri-silkworm, Samia cynthia ricini.

Lysozyme was isolated from immunized hemolymph of Samia cynthia ricini larvae by heat treatment, cation exchange and reverse-phase chromatography. A cDNA encoding lysozyme was cloned by screening the cDNA library from immunized fat body using, as a probe, a DNA fragment obtained by PCR-based differential display method. The deduced amino acid sequence showed high homology with other chicken-type lysozymes. The calculated molecular mass of the mature peptide was 13785, which agreed precisely with that obtained by MALDI-TOF mass spectrometry of the isolated protein. The lysozyme transcripts were detected at a significant level in naïve fat body, and the level increased 5-10-fold upon injection of the larvae with UV-killed bacteria or peptidoglycan.

Animals↗
PubMed2006

Characterization by Raman microspectroscopy of the strain-induced conformational transition in fibroin fibers from the silkworm Samia cynthia ricini.

Raman microspectroscopy has been used to quantitatively study the effect of a mechanical deformation on the conformation and orientation of Samia cynthia ricini (S. c. ricini) silk fibroin. Samples were obtained from the aqueous solution stored in the silk gland and stretched at draw ratios (lambda) ranging from 0 to 11. Using an appropriate band decomposition procedure, polarized and orientation-insensitive spectra have been analyzed to determine order parameters and the content of secondary structures, respectively. The data unambiguously show that, in response to mechanical deformation, S. c. ricini fibroin undergoes a cooperative alpha-helix to beta-sheet conformational transition above a critical draw ratio of 4. The alpha-helix content decreases from 33 to 13% when lambda increases from 0 to 11, while the amount of beta-sheets increases from 15 to 37%. In comparison, cocoon silk is devoid of alpha-helical structure and always contains a larger amount of beta-sheets. Although the presence of isosbestic points in different spectral regions reveals that the conformational change induced by mechanical deformation is a two-state process, our results suggest that part of the glycine residues might be incorporated into beta-poly(alanine) structures. The beta-sheets are initially isotropically distributed and orient along the fiber axis as lambda increases, but do not reach the high level of orientation found in the cocoon fiber. The increase in the orientation level of the beta-sheets is found to be concomitant with the alpha --> beta conformational conversion, whereas alpha-helices do not orient under the applied strain but are rather readily converted into beta-sheets. The components assigned to turns exhibit a small orientation perpendicular to the fiber axis in stretched samples, showing that, overall, the polypeptide chains are aligned along the stretching direction. Our results suggest that, in nature, factors other than stretching contribute to the optimization of the amount of beta-sheets and the high degree of orientation found in natural cocoon silk.

Algorithms↗