Molecular cloning and characterization of the α-glucosidase II from Bombyx mori and Spodoptera frugiperda

The α-glucosidase II (GII) is a heterodimer of α- and β-subunits and important for N-glycosylation processing and quality control of nascent glycoproteins. Although high concentration of α-glucosidase inhibitors from mulberry leaves accumulate in silkworms (Bombyx mori) by feeding, silkworm does not show any toxic symptom against these inhibitors and N-glycosylation of recombinant proteins is not affected. We, therefore, hypothesized that silkworm GII is not sensitive to the α-glucosidase inhibitors from mulberry leaves. However, the genes for B. mori GII subunits have not yet been identified, and the protein has not been characterized. Therefore, we isolated the B. mori GII α- and β-subunit genes and the GII α-subunit gene of Spodoptera frugiperda, which does not feed on mulberry leaves. We used a baculovirus expression system to produce the recombinant GII subunits and identified their enzyme characteristics. The recombinant GII α-subunits of B. mori and S. frugiperda hydrolyzed p-nitrophenyl α-d-glucopyranoside (pNP-αGlc) but were inactive toward N-glycan. Although the B. mori GII β-subunit was not required for the hydrolysis of pNP-αGlc, a B. mori GII complex of the α- and β-subunits was required for N-glycan cleavage. As hypothesized, the B. mori GII α-subunit protein was less sensitive to α-glucosidase inhibitors than was the S. frugiperda GII α-subunit protein. Our observations suggest that the low sensitivity of GII contributes to the ability of B. mori to evade the toxic effect of α-glucosidase inhibitors from mulberry leaves.     

Purification of ecdysone oxidase and 3-dehydroecdysone 3α-reductase from the cotton leafworm,Spodoptera littoralis

The 3-epimerization of ecdysteroids (insect moulting hormones) is an inactivation pathway of the hormones that has been reported to occur in midgut cytosol of Lepidoptera. The pathway involves ecdysone oxidase-catalysed conversion of ecdysone into 3-dehydroecdysone, which is then irreversibly reduced to 3-epiecdysone by 3DE 3α-reductase. In this study, ecdysone oxidase and 3DE 3α-reductase from the cotton leafworm, S. littoralis, have been purified by extensive chromatography together with electrophoresis on native gels. Gel filtration suggested that the native ecdysone oxidase might be a trimer with apparent molecular mass of approximately 190 kDa, since the apparent molecular mass of the oxidase subunit was determined to be 64 kDa by SDS-PAGE. Two forms of 3DE 3α-reductase were observed during the purification, the 26 kDa form reductase has been purified to homogeneity and the second form of the reductase identified as a 51 kDa protein. The former reductase may be a trimer with apparent molecular mass of 76 kDa, whilst the latter was suggested to be a monomer by gel filtration. Chromatographic behaviour suggested that the 26 kDa form of the reductase has a lower pI value and a higher degree of hydrophobicity than that of the 51 kDa reductase. Substrate specificity and the tissue distribution of these enzymes are discussed.     

Genetics of insect hemolymph β-N-acetylglucosaminidase in the silkworm,Bombyx mori

The distribution of insect hemolymph -N-acetylglucosaminidase was investigated in the silkworm, Bombyx mori. Activity in 115 varieties was 6.92±3.22 units/ml, ranging from 1.4 to 17.0 units/ml. No enzyme-deficient individuals were observed. By selecting individuals showing either high or low enzyme activities, homozygotes were separated with activities varying 10-fold between isolates. No differences in activity of -mannosidase and -galactosidase were observed. Thus, it appears that high- or low-enzyme silkworms (High or Low lines) shared the same genetic background except for the gene concerning the activity of -N-acetylglucosaminidase. Studies on the heredity of the enzyme indicated that the synthesis of the enzyme protein was controlled by an autosomal allele. Examination by immunotitration and CM₅₂-cellulose column chromatography revealed that the difference in activity between High and Low lines was due to the amount of the active enzyme, but not to an endogeneous activator or inhibitor. Furthermore, there was no isozymic difference in -N-acetylglucosaminidase. Slab gel electrophoresis on polyacrylamide showed a species of enzyme (A) that stained more intensely in the High line. For the second species of enzyme (B), the converse was true. This evidence suggests that enzyme levels in hemolymph are under the control of a gene affording association of enzyme subunits to the active enzyme molecule.     

Genetics of insect hemolymph α-mannosidase in the silkworm,Bombyx mori

The genetics of hemolymph alpha-mannosidase was investigated in the silkworm, Bombyx mori. By selecting individuals showing either high or low enzyme activities, homozygotes were separated, with activities varying about five-fold. No differences in the activities of beta-galactosidase and beta-N-acetylglucosaminidase were observed. Thus, it seems that high- and low-enzyme silkworms (High and Low Lines) share the same genetic background except for the gene concerning the activity of alpha-mannosidase. The synthesis of the enzyme is controlled by an autosomal allele. Furthermore, expression of the gene varies from tissue to tissue, and there is no correlation between enzyme activity and growth rate. The difference in activity between High and Low lines is due to the amount of active enzyme, not to an endogeneous activator or inhibitor. There was no isozymic difference in alpha-mannosidase.     

Functional and pharmacological characterization of a β-adrenergic-like octopamine receptor from the silkworm Bombyx mori

A cDNA encoding a seven-transmembrane receptor was cloned from the nervous tissues of silkworm (Bombyx mori) larvae. Sequence analysis indicated that the gene is an ortholog of CG6989, which encodes a Drosophila β-adrenergic-like octopamine (OA) receptor (DmOctβ2R). As very little information is available regarding this class of receptors, we generated a cell line that stably expressed the gene in HEK-293 cells and we then performed functional and pharmacological studies of this receptor. [³H]OA-binding assays using membrane preparations of this cell line showed that the receptor possesses a higher affinity for OA than for tyramine (TA) or dopamine (DA). The cell line elicited a bell-shaped, OA concentration-dependent increase in intracellular cAMP levels, with a maximum at 100 nM. (R)-OA was more potent than (S)-OA. TA and DA had weak or marginal effects on cAMP production. The OA receptor agonist demethylchlordimeform elicited a similar biphasic response, although the maximum response was attained at a concentration as low as 1 nM. The rank order of potency of other agonists was as follows: naphazoline > tolazoline, clonidine. Among the antagonists tested, only chlorpromazine significantly attenuated the OA-induced increase in cAMP levels. No increase in intracellular Ca²⁺ levels was observed with OA at concentrations up to 100 μM. These findings indicate that the cloned receptor is a β-adrenergic-like OA receptor with unique functional and pharmacological properties.     

Identification and characterization of genes abnormally expressed in wing-deficient mutant (flügellos) of the silkworm,Bombyx mori

The wing-deficient mutant, flügellos (fl), of the silkworm lacks four wings in the pupa and the adult, due to aberrant wing morphogenesis during metamorphosis. To elucidate the mechanisms of wing-specific deficiencies in the fl mutant, we used mRNA differential display and identified five genes abnormally expressed in the fl wing discs. Northern blot and RT-PCR analyses revealed that four genes were overexpressed, but the fifth one was not transcribed in the fl wing discs. The expression level of ribosome-associated protein p40 in the fl wing discs was elevated approximately 10 times compared to the wild-type (WT) discs. Another overexpressed gene CB10 encodes a novel wing-specific protein with a putative zinc-finger motif. Overexpression of two components of extracellular matrix, cuticle protein 18 (BMCP18) and a fibrillin-like protein AD10, may result in the abnormal wing morphogenesis in the fl mutant. In contrast, a novel member of multifunctional Ca2+-binding protein annexins, designated as annexin b13 (Anx b13), was expressed dominantly in the wing discs of WT but completely repressed in the fl tissues. Strong expression of Anx b13 in wing discs during the fourth and fifth instar indicates that ANX B13 plays an important role in wing morphogenesis.     

Cell junctions in the cyst envelope in the silkworm testis,Bombyx mori linné

Summary The cell junctions of the cyst envelope in the testes of Bombyx mori were examined by electron microscopy utilizing a thin-sectioning technique following conventional fixation, tannic acid fixation and lanthanum tracer study, and also using a freeze-fracture technique. There are three kinds of junctions; septate junctions, gap junctions and tight junctions. Septate junctions are of the pleated type. Gap junctions are characterized by four electron-dense lines and three electronlucent lines in the reduced intercellular spaces seen by thin-sectioning. They are of the E type, having clusters of intramembraneous particles on the E-fracture face. The most striking finding is the frequent presence of tight junctions on the fracture planes, while focally fused outer leaflets of the junctional unit membranes are rarely detected on thin-sectioned preparations. Tight junctions are characterized by branching zigzag ridges on the P-fracture face and complementary grooves on the E-fracture face. It is proposed that tight junctions are new morphological evidence of blood-germ cell barrier in an insect. Acknowledgements: For helpful assistance the authors are indebted to their colleagues Miss N. Minemoto, Miss H. Kiyotake and Mr. Y. Goto     

Immunocytochemical localization of β-1,3-glucan recognition protein in the silkworm,Bombyx mori

Summary A monospecific antibody against -1,3-glucan recognition protein (a 62 kDa protein) of the larval silkworm prophenoloxidase activating system was used to study the localization of the protein. Among tissues from 5th instar larvae, only hemocytes and plasma were shown to contain a 62 kDa polypeptide immunoreactive with the antibody. Ultra-thin sections of the hemocytes were stained by an indirect immunogold staining method. Labelling occurred in the granules and cytoplasm of granulocytes and in the spherules and cytoplasm of spherulocytes. It was most conspicuous in granules of granulocytes and uniformly labelled spherules of spherulocyte, whereas no labelling was evident in prohemocytes, plasmatocytes and oenocytoids. The results are discussed in relation to the mode of recognition of fungi as non-self in insect hemocoel.     

Construction and analysis of the protein–protein interaction network for the olfactory system of the silkworm Bombyx mori

Olfaction plays an essential role in feeding and information exchange in insects. Previous studies on the olfaction of silkworms have provided a wealth of information about genes and proteins, yet, most studies have only focused on a single gene or protein related to the insect's olfaction. The aim of the current study is to determine key proteins in the olfactory system of the silkworm, and further understand protein–protein interactions (PPIs) in the olfactory system of Lepidoptera. To achieve this goal, we integrated Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and network analyses. Furthermore, we selected 585 olfactory-related proteins and constructed a (PPI) network for the olfactory system of the silkworm. Network analysis led to the identification of several key proteins, including GSTz1, LOC733095, BGIBMGA002169-TA, BGIBMGA010939-TA, GSTs2, GSTd2, Or-2, and BGIBMGA013255-TA. A comprehensive evaluation of the proteins showed that glutathione S-transferases (GSTs) had the highest ranking. GSTs also had the highest enrichment levels in GO and KEGG. In conclusion, our analysis showed that key nodes in the biological network had a significant impact on the network, and the key proteins identified via network analysis could serve as new research targets to determine their functions in olfaction.     

Differences in nutrient uptake between the fat body and embryonic primary cultures of silkworm （Bombyx mori）

Nutrition utilization and by-product formation in cultured insect cells has been investigated in several insect cells and has been of great interest to cell culturists and physiologists. In this research the biochemical changes in embryonic and fat body primary cultures of silkworm, Bombyx mori, have been compared. TC-100 medium supplemented with 10% and 20% FBS was used in embryonic and fat body primary cultures, respectively. Medium was renewed every week and the amount of glucose, uric acid, urea, total protein and alkaline phosphatase were measured in the samples from medium of primary cultures using spectrophotometeric methods. All biochemical macromolecules except uric acid showed significant changes. Glucose decreased in embryonic tissues, while in fat body culture its amount increased. Urea accumulation in embryonic culture was higher than in the fat body cultures. Since urea is a by-product, this accumulation could be due to higher utilization of amino acids. Total protein showed considerable changes and was consumed by embryonic culture more than the fat body＇ s. Alkaline phosphatase showed stronger activity in embryonic cells.     

Molecular characterization of heat shock proteins 90 (HSP83?) and 70 in tropical strains of Bombyx mori

Following the concept of whole organism, we have extracted total protein from the Bombyx mori for the identification and analysis of HSPs. Expression of 90 kDa HSP in first, second and third instars, 84 kDa in fourth instar and 90‐, 84‐, 62‐, 60‐, 52‐ and 33‐kDa HSPs in fifth instar larvae of tropical polyvoltine and bivoltine silkworm strains were obvious. Further, we have combined single and 2‐DE with MALDI‐TOF for analysis of BmHSPs. Ninety kilodalton band excised from 1‐DE gel was identified as HSP83 by MALDI‐TOF‐MS. The immunoblot analysis confirmed the expression of HSP90 in all the instars larvae of B. mori. Heat shock‐induced protein spots were excised from 2‐DE gels for MALDI‐TOF‐MS analysis. The Mascot search results are for HSP68, HSC70‐1 and HSP70Ba in Pure Mysore, and major HSP70Bbb, HSP68, HSC‐3 and HSP83 in NB₄D₂. Multiple sequence alignment explicit the variations in amino acid sequence between Pure Mysore and NB₄D₂. Notably, the PMF of spot 2 matched the coding sequence of B. mori and its gene annotation was determined on chromosome 9. With this novel approach, expression of BmHSP90 was confirmed in all the instars and uncovered isoforms of BmHSP70, which provided unequivocal insight to analyze and understand the biological significance in B. mori.     

The biochemical effects of potassium chloride on the silkworm, （ Bombyx mori L.）

The supplementation with 50, 100 and 150μg/mL potassium chloride to the fifth instar larvae of the silkworm Bombyx mori on fat body glycogen, protein, total lipids and haemolymph protein and trehalose were analyzed. The fat body glycogen and protein and haemolymph protein were increased significantly in all the treated groups; whereas fat body total lipids increased only in 100 and 150μg/mL and haemolymph trehalose increased only in 150μg/mL potassium chloride-treated groups when compared with those of the corresponding parameters of the carrier controls.     

Molecular cloning,expression and characterization of Pekin duck interferon-λ

Interferons (IFNs) are the first line of defense against viral infections in vertebrates. Type III interferon (IFN-λ) is recognized for its key role in innate immunity of tissues of epithelial origin. Here we describe the identification of the Pekin duck IFN-λ ortholog (duIFN-λ). The predicted duIFN-λ protein has an amino acid identity of 63%, 38%, 37% and 33% with chicken IFN-λ and human IFN-λ3, IFN-λ2 and IFN-λ1, respectively. The duck genome contains a single IFN-λ gene that is comprised of five exons and four introns. Recombinant duIFN-λ up-regulated OASL and Mx-1 mRNA in primary duck hepatocytes. Our observations suggest evolutionary conservation of genomic organization and structural features implicated in receptor binding and antiviral activity. The identification and expression of duIFN-λ will facilitate further study of the role of type III IFN in antiviral defense and inflammatory responses of the Pekin duck, a non-mammalian vertebrate and pathogen host with relevance for human and animal health.     

Molecular cloning and characterization of the β-catenin gene from fine-wool sheep

β-Catenin is an evolutionarily conserved molecule that functions as a crucial effector in both cell-to-cell adhesion and Wnt signaling. To gain a better understanding of its role in the development of hair follicles, we cloned the cDNA sequence of the β-catenin gene from the skin of Aohan fine-wool sheep and performed a variety of bioinformatics analyses. We obtained the full-length sequence, which was 4573-bp long and contained a 2346-bp open reading frame encoding a protein of 781 amino acids. The protein had a predicted molecular weight of 85.4 kDa and a theoretical isoelectric point of 5.57. Domain architecture analysis of the β-catenin protein revealed an armadillo repeat region, which is a common feature of β-catenin in other species. The ovine β-catenin gene shares 97.91%, 94.25%, 94.59%, 83.89%, and 89.39% sequence identity with its homologs in Bos taurus, Homo sapiens, Sus scrofa, Gallus gallus, and Mus musculus, respectively, while the amino acid sequence is more than 99% identical with each of these species. The expression of β-catenin mRNA was detected in the heart, liver, spleen, lung, kidney, skin, muscle, and adipose tissue. Expression levels were maximal in the lung and minimal in the muscle, and the difference in expression in these tissues was significant (P < 0.01). Western blot analysis revealed the presence of the β-catenin protein in all tissues examined; expression was lowest in the skin and adipose tissues.