Motoyuki Sumida

Cloning and expression of the gene of hemocytin, an insect humoral lectin which is homologous with the mammalian von Willebrand factor

Invertebrate lectins play an important role in a non-specific self-defense mechanism, as invertebrates do not synthesize specific antibodies. We report the cloning of several overlapping cDNAs encoding the entire silkworm (Bombyx mori) lectin, which we propose to call hemocytin. The sequence (10477 bp) encoded 3133 amino acids. The characteristics features of the carbohydrate-recognition domain of C-type animal lectin were revealed at C-terminal sequence of hemocytin. When cDNA encoding this region was introduced into baculovirus vector, hemagglutinating activities were detected in the culture fluid of a recombinant virus-infected cells. These activities were inhibited by D-mannose, N-acetyl-D-galactosamine, and D-maltose which are haptenic saccharides of authentic hemocytin. Analysis of dot and Northern blot hybridization revealed that hemocytin gene was transcribed in hemocytes of the silkworm at larval-pupal metamorphosis and/or after the injection of Escherichia coli and lipopolysaccharide. After silkworm larvae were injected with C-terminal portion of hemocytin, aggregation of hemocytes was observed in the hemolymph. Hemocytin has significant homology with mammalian von Willebrand factor which involves in platelet adhesion to subendothelium. Also, hemocytin has a homologous region with coagulation factor V and VIII. These results suggest that hemocytin molecule is an adhesive protein and relates to hemostasis or encapsulation of foreign substances for self-defense.

Foreign gene expression by a baculovirus vector with an expanded host range.

A nuclear polyhedrosis virus (NPV) (Baculoviridae)-based gene expression system was improved by DNA recombination. The BmN cell line established from Bombyx mori and the Sf21 cell line established from Spodoptera frugiperda are non-permissive for Autographa californica multicapsid NPV (AcMNPV) and B. mori NPV (BmNPV) replication, respectively. After cotransfection of AcMNPV DNA and BamHI-digested BmNPV DNA into Sf21 cells, progeny viruses were isolated by plaque purification on BmN cell monolayers and the host specificity of one viral isolate was analysed. The virus had a wider host range, and replicated and produced polyhedra in Sf21 cells, BmN cells and larvae of the silkworm, B. mori. DNA restriction endonuclease analysis showed that the isolate was a hybrid of AcMNPV and BmNPV. Using the AcMNPV transfer vector pAcYM1 a portion of the polyhedrin gene of the hybrid virus was replaced with the coding region of the firefly luciferase gene, producing a recombinant virus. The latter expressed firefly luciferase in both cell lines and in silkworm larvae under the control of the polyhedrin promoter.

Prothoracicotropic hormone is released five times in the 5th-larval instar of the silkworm, Bombyx mori

Abstract In order to elucidate the timing of the in vivo release of prothoracicotropic hormone (PTTH), the fluctuation of the PTTH titer in the hemolymph of the 5th-instar larva of Bombyx mori was determined using an in vitro system to monitor the activation of the prothoracic glands with PTTH. Five peaks in the PTTH titers were detected in the hemolymph. The first was observed in the early stage of the 5th-larval instar. The second and third peak were observed 3 days and 1 day before the onset of wandering, respectively. The fourth peak was detected in the pre-pupal stage and the last peak was observed just before the maximal level of the ecdysteroid titer in the hemolymph. The timing of these in vivo releases of PTTH was compatible with the in vitro release of PTTH from brain-corpus cardiacum-corpus allatum complex. Furthermore, a change in the ecdysone secretory activity of prothoracic gland in the 5th-larval instar was followed to clarify the physiological significance of these PTTH releases into the hemolymph. Consequently, this secretory activity was dependent on the PTTH released in vivo except for the PTTH released in the early stage of the 5th-larval instar. On the basis of the correlation between the timing of the in vivo releases of PTTH and the developmental events including the change in the secretory activity of prothoracic glands as well as wandering, the physiological significance of five releases of PTTH in the 5th-instar larva of Bombyx mori is discussed.

Expression of Bombyx mori cytoplasmic polyhedrosis virus polyhedrin in insect cells by using a baculovirus expression vector, and its assembly into polyhedra

A cDNA encoding the cytoplasmic polyhedrin of Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) strain H was introduced into an improved baculovirus expression vector which can be utilized to express foreign genes in the Spodoptera frugiperda cell line IPLB-SF-21AE (Sf21 cells) and the B. mori cell line BmN. A recombinant virus produced large, cubic inclusion body-like structures in infected Sf21 and BmN cells. Western blot analysis showed that these structures were BmCPV polyhedra. This result suggested that the supramolecular assembly of BmCPV polyhedrin is responsible for its properties.

cDNA and deduced amino acid sequences of apolipophorin‐IIIs from Bombyx mori and Bombyx mandarina

The cDNA sequence for apolipophorin-III from two strains of Bombyx mori (N4 and P50) and the Japanese and Chinese strains of Bombyx mandarina were determined. Both the cDNA and deduced amino acid sequences of the four apolipophorin-IIIs were highly similar (95-98%). The four Bombyx sequences also showed significant similarity to the sequence of apolipophorin-III from another lepidopteran, Manduca sexta (83-84%), particularly in the five amphipathic alpha-helices that are proposed to play a critical role in the binding of apolipophorin-III to lipophorin. In the coding region, the nucleotide sequences for the Chinese strain of B. mandarina and the P50 strain of B. mori were identical, supporting the suggestion that P50 is the current strain most closely related to the original domesticated strain. The N4 strain of B. mori is more closely related to these two strains than is the Japanese strain of B. mandarina, suggesting that Japanese strain of B. mandarina separated from the Chinese strain of B. mandarina before domestication of B. mori. Arch.

Expression of the Newcastle disease virus (NDV) fusion glycoprotein and vaccination against NDV challenge with a recombinant baculovirus.

The hybrid baculovirus constructed from Autographa california nuclear polyhedrosis virus (NPV) and Bombyx mori (silkworm) NPV was used for expression of fusion glycoprotein (F) of Newcastle disease virus (NDV) strain D26. The gene encoding F protein was introduced into the improved baculovirus expression vector derived from the host-range-expanded baculovirus. In Spodoptera frugiperda (fall armyworm) cells infected with a recombinant baculovirus, HyF121, the expressed F protein was properly located onto the cell surface. After silkworm pupae were infected with HyF121, a subunit vaccine against NDV was prepared from the HyF121-infected pupae. Chickens inoculated with the subunit vaccine were protected against virulent NDV challenge.

Structure and Chemical Composition of Silk Proteins in Relation to Silkworm Diet

We investigate the structure and chemical composition of silk proteins produced by silkworms reared entirely on an artificial (A) diet, and compared those with worms entirely on a mulberry leaf ( M ) diet. The artificial diet has tofu-cake as its main component. Two types of cocoons (A and M) are used as sample materials. The dissolution behavior of the A sericin in hot water is different from that of M sericin; the amount of A sericin dissolved (28%) is higher than that of M (24%), and the dissolution rate of A sericin is also higher. Both sericins are separated into four fractions according to dissolution rate in hot water, and the amino acid composition (AAC) of each fraction is determined. Although the AAC of unfractionated A sericin is almost the same as that of M sericin, the AACs of the fractionated sericin from the A sample are distinguished from those from the M sample. AACs of the A and M fibroins are also evaluated and found to be almost identical within experimental error. The x-ray diffraction pattern of the A fibroin is also nearly the same as that of M fibroin. The artificial diet used in this study affects the dissolution behavior and chemical com position of sericin proteins, but does not affect structure and chemical composition of fibroin proteins.

Changes in kinetic parameters and total activity of midgut sucrase in the silkworm, Bombyx mori during larval-pupal-adult development.

1. 1. Developmental changes in total activity of midgut sucrase in the silkworm, Bombyx mori were determined with two separate assay systems which were prepared based on kinetic parameters of midgut sucrase from larvae and pharate adults. 2. 2. pH Optima were at 6.0–6.6 for the fifth instar larvae and at pH 5.6 and 6.9 for the pharate adults. Km values toward sucrose at pH 5.6 were 10.0 mM for larval enzyme and 2.3 mM for pharate adult enzyme. For assay of the larval type of enzyme, a final concentration of 100 mM substrate at pH 6.0 was employed and for pharate adult type of enzyme, 15 mM substrate at pH 5.6. 3. 3. To determine the moment that midgut sucrase changes electrophoretically from larval to pharate adult type, electrophoresis of midgut tissue homogenates on native polyacrylamide gel at pH 8.9 was conducted during silkworm development and the change was observed on the day of larval-pupal ecdysis. 4. 4. Sucrase activity was low in the fourth instar. From the fifth instar it increased steeply to reach a maximum level at day 4–6 with Vm = 87.0 μmoles of glucose/min per individual midgut, which was approximately 9-fold greater than the level at day 0, fifth instar. Activity then decreased rapidly to a low level until larval-pupal ecdysis and the level was maintained until 1 day before adult emergence. 5. 5.In the fifth instar larvae, the membrane-bound fraction of midgut tissue homogenate exhibits low but significant sucrase activity, consisting of up to 10% of total activity. pH-Activity relationships of membrane-bound and soluble enzymes were similar in the pH range between pH 5.5 and 7.5 but the Km value for sucorse was 7.7 mM and 16.6 mM for membrane-bound and soluble enzyme, respectively. In high substrate concentration the membrane-bound enzyme showed activation by substrate whereas the soluble enzyme showed substrate inhibition.

Induction of a hemagglutinating activity in the hemolymph of the silkworm, Bombyx mori, infected with cytoplasmic polyhedrosis virus

Abstract The hemagglutinating activity increased significantly in the hemolymph of the fifth instar larvae of the silkworm, Bombyx mori, infected with cytoplasmic polyhedrosis virus (CPV). Analysis by immunoblotting clearly indicated the enhanced accumulation of a protein with hemagglutinating activity in the hemolymph of the silkworms infected with CPV, which is a lectin-like protein, possibly newly induced during infection with CPV. These results suggest the protein to be a candidate responsible in the silkworm for the immune defense against exogenous agents including CPV.

Purification and some properties of soluble β-fructofuranosidase from larval midgut of the silkworm, Bombyx mori

Abstract Soluble sucrase was purified from midgut of the silkworm, Bombyx mori , to near homogeneity by ammonium sulphate fractionation at 40–80% saturation, column chromatographies of ion-exchange cellulose, hydroxylapatite and Sephacryl S-100 HR. Specific activity of 384.7 U/mg protein of the purified soluble sucrase was the highest activity among the sucrases so far purified from insects. The purified soluble sucrase hydrolysed sucrose, raffinose and stachyose, but never hydrolysed any other di- and trisaccharides, and derivatives of glucoside. The results indicate that the purified soluble sucrase is a β-fructofuranosidase. Molecular weight under non-dissociating conditions of the soluble β-fructofuranosidase, was estimated to be 85,000, and the subunit molecular weight by SDS-PAGE was 58,000. It appears that the soluble β-fructofuranosidase is a homodimer. The pH optimum of the soluble β-fructofuranosidase was pH 6.2 and 6.0–7.0, when sucrose and raffinose were used as substrates, respectively. The K m values of the soluble β-fructofuranosidase were 6.5 and 8.6 mM, when sucrose and raffinose were used as substrates, respectively. The effect of metal ions on the soluble β-fructofuranosidase was inhibitory in 13 metal ions investigated. The notable ones were Hg 2+ , Cu 2+ , Pb 2+ , Fe 3+ and Cd 2+ , affecting activity to reduce 0–20% of the control activity. Tris and tricine were potent inhibitors for the soluble β-fructofuranosidase and inhibitor constant ( k i ) of Tris was 9.2 mM when sucrose was used as a substrate. After enzymatic reaction of the soluble β-fructofuranosidase with sucrose as a substrate, a compound corresponding in mobility to trisaccharides such as raffinose and melezitose was identified in the reaction mixture, which indicated a transferolytic activity of the soluble β-fructofuranosidase. Stability of the soluble β-fructofuranosidase against various pHs preserved for 48 hr at 37°C was investigated. Ninety percent, and more than 50% of the initial activity, was maintained at pH 6.5 and in the range pH 4.5–9.5, respectively, but almost no activity was detected at pH 4.0.