Sumiharu Nagaoka

Molecular Characterization of Bombyx mori Cytoplasmic Polyhedrosis Virus Genome Segment 4

ABSTRACT The complete nucleotide sequence of the genome segment 4 (S4) ofBombyx mori cytoplasmic polyhedrosis virus (BmCPV) was determined. The 3,259-nucleotide sequence contains a single long open reading frame which spans nucleotides 14 to 3187 and which is predicted to encode a protein with a molecular mass of about 130 kDa. Western blot analysis showed that S4 encodes BmCPV protein VP3, which is one of the outer components of the BmCPV virion. Sequence analysis of the deduced amino acid sequence of BmCPV VP3 revealed possible sequence homology with proteins from rice ragged stunt virus (RRSV) S2,Nilaparvata lugens reovirus S4, and Fiji disease fijivirus S4. This may suggest that plant reoviruses originated from insect viruses and that RRSV emerged more recently than other plant reoviruses. A chimeric protein consisting of BmCPV VP3 and green fluorescent protein (GFP) was constructed and expressed with BmCPV polyhedrin using a baculovirus expression vector. The VP3-GFP chimera was incorporated into BmCPV polyhedra and released under alkaline conditions. The results indicate that specific interactions occur between BmCPV polyhedrin and VP3 which might facilitate BmCPV virion occlusion into the polyhedra.

Biochemical properties of an omega-class glutathione S-transferase of the silkmoth, Bombyx mori

A cDNA encoding an omega-class glutathione S-transferase of the silkmoth, Bombyx mori (bmGSTO), was cloned by reverse transcriptase-polymerase chain reaction. The resulting clone was sequenced and deduced for amino acid sequence, which revealed 40, 40, and 39% identities to omega-class GSTs from human, pig, and mouse, respectively. A recombinant protein (rbmGSTO) was functionally overexpressed in Escherichia coli cells in a soluble form and purified to homogeneity. rbmGSTO was able to catalyze the biotranslation of glutathione with 1-chloro-2,4-dinitrobenzene, a model substrate for GST, as well as with 4-hydroxynonenal, a product of lipid peroxidation. This enzyme was shown to have high affinity for organophosphorus insecticide and was present abundantly in silkmoth strain exhibiting fenitrothion resistance. These results indicate that bmGSTO could be involved in the increase in level of insecticide resistance for lepidopteran insects.

Identification of the sperm-activating factor initiatorin, a prostatic endopeptidase of the silkworm, Bombyx mori.

Male Bombyx mori has a trypsin-type protease, called initiatorin, in the secretion from the posterior segment of the ejaculatory duct that is thought to be involved in the acquisition of sperm motility, although this inference remains to be demonstrated. Here, we revised the experimental procedures including that for purification and definitely identified the purified initiatorin protein as an activation factor of B. mori sperm by an in vitro study in which we treated isolated spermatozoa with this enzyme. Analysis of cDNA revealed that initiatorin consists of 281 amino acids with sequence similarity to bovine trypsin, and is highly homologous to the ejaculated accessory gland proteins not only of other Lepidoptera but also of Orthoptera. Recombinant initiatorin, expressed in Escherichia coli and purified, also showed proteolytic and sperm-activating activities. RT-PCR and Western blot analyses indicated that initiatorin is abundantly expressed in the glandula (g.) prostatica. It was also shown that pro-initiatorin is synthesized and stored in g. prostatica, and then converted to the mature form upon ejaculation. Fluorogenic peptides with a dibasic sequence were efficiently cleaved by initiatorin, and one such substrate, BOC-Gly-Arg-Arg-MCA, inhibited sperm activation by the extract of g. prostatica. These results delineate the idea that initiatorin has the most suitable protease property as an initiator of the protein degradation cascade in that it releases free arginines, which in turn become an energy resource for sperm motility.

Identification of two arginases generated by alternative splicing in the silkworm, Bombyx mori

Arginase (EC 3.5.3.1) catalyzes the hydrolysis of arginine to ornithine and urea. Here, we have cloned two arginase cDNAs from the silkworm, Bombyx mori. The analysis of exon/intron structures showed that the two mRNAs named bmarg-r and bmarg-f were generated from a single gene by alternative usage of exons. The bmarg-r and bmarg-f were predicted to encode almost the same amino acid sequences, except that the latter had additional ten N-terminal residues. Recombinant bmARG-r and bmARG-f in Escherichia coli cell lysates were roughly similar to each other in enzymatic characteristics, which did not show large difference from those of arginases assayed by using tissue extracts. Differential RT-PCR experiments and tissue distribution analyses of arginase activity indicated that the bmarg-r gene is expressed in the male reproductive organs, especially in the glandula lacteola and vesicular seminalis, from which it is secreted to the seminal fluid and transferred to the female during copulation, whereas the bmarg-f gene is expressed in the larval and adult nonreproductive organs including the fat body and muscle, where the produced arginase proteins are considered to stay in the cells. Thus, the two silkworm arginase isoforms may have a difference in whether or not the product is excreted out of the cells in which it is synthesized.

Cell wall sheath surrounding calcium oxalate crystals in mulberry idioblasts

Summary.The distribution and ultrastructural features of idioblasts containing calcium oxalate crystals were studied in leaf tissues of mulberry, Morus alba L. In addition to the calcium carbonate crystals formed in epidermal idioblasts, large calcium oxalate crystals were deposited in cells adjacent to the veins and surrounded by a cell wall sheath which had immunoreactivity with an antibody recognizing a xyloglucan epitope. The wall sheath formation indicates exclusion of the mature crystal from the protoplast.

Ultrastructural and immunochemical features of the cell wall sac formed in mulberry ( Morus alba ) idioblasts

A peculiar inward growth, named a “cell wall sac”, formed in mulberry (Morus alba) idioblasts, is a subcellular site for production of calcium carbonate crystals. On the basis of ultrastructural observations, a fully expanded cell wall sac could be divided into two parts—an amorphous complex consisting of multi-layered compartments with multiple fibers originating from the innermost cell wall layer, and a peripheral plain matrix with fiber aggregates. Immunofluorescent localization showed that low and highly esterified pectin epitopes were detected at the early stages of development of the cell wall sac, followed by complete disappearance from the both parts of fully enlarged mature sac. In contrast, the xyloglucan epitope remained in the compartment complex; this was supported by the observation that the xyloglucan epitope labeled with immuno-gold particles is found on fibers in the complex part.

The angiotensin converting enzyme (ACE) inhibitor, captopril disrupts the motility activation of sperm from the silkworm, Bombyx mori

Angiotensin I-converting enzyme (also known as peptidyl dicarboxypeptidase A, ACE, and EC 3.4.15.1), which is found in a wide range of organisms, cleaves C-terminal dipeptides from relatively short oligopeptides. Mammalian ACE plays an important role in the regulation of blood pressure. However, the precise physiological functions of insect ACE homologs have not been understood. As part of our effort to elucidate new physiological roles of insect ACE, we herein report a soluble ACE protein in male reproductive secretions from the silkmoth, Bombyx mori. Seminal vesicle sperm are quiescent in vitro, but vigorous motility is activated by treatment with either a glandula (g.) prostatica homogenate or trypsin in vitro. When seminal vesicle sperm were pre-incubated with captopril, a strong and specific inhibitor of mammalian ACE, and then stimulated to initiate motility by the addition of the g. prostatica homogenate or trypsin, the overall level of acquired motility was reduced in an inhibitor-concentration-dependent manner. In the course of this project, we detected ACE-related carboxypeptidase activity that was inhibited by captopril in both the vesicular (v.) seminalis of the noncopulative male reproductive tract and in the spermatophore that forms in the female bursa copulatrix at the time of mating, just as in an earlier report on the tomato moth, Lacanobia oleracea, which belongs to a different lepidopteran species (Ekbote et al., 2003a). Two distinct genes encoding ACE-like proteins were identified by analysis of B. mori cDNA, and were named BmAcer and BmAcer2, respectively [the former was previously reported by Quan et al. (2001) and the latter was first isolated in this paper]. RT-qPCR and Western blot analyses indicated that the BmAcer2 was predominantly produced in v. seminalis and transferred to the spermatophore during copulation, while the BmAcer was not detected in the adult male reproductive organs. A recombinant protein of BmAcer2 (devoid of a signal peptide) that was expressed in Escherichia coli cells exhibited captopril-sensitive carboxypeptidase activities. Our findings show that the BmAcre2 gene encodes a secreted ACE protein included in the Bombyx seminal plasma. In particular, the silkworm ACE protein in the seminal fluid might be involved in the signaling pathway that leads to the activation and regulation of sperm motility.