Minoru Yamakawa

A genome-wide analysis of genes and gene families involved in innate immunity of Bombyx mori

A genome-wide analysis of innate immunity-related genes and gene families was conducted using the silkworm, Bombyx mori. We identified orthologs for a large number of genes involved in insect immunity that have been reported from Drosophila melanogaster (Diptera), Anopheles gambiae (Diptera), Apis mellifera (Hymenoptera) and Tribolium castaneum (Coleoptera). B. mori has a unique recognition gene and antimicrobial peptide genes that are not present in the Drosophila, Anopheles, Apis and Tribolium genomes, suggesting a lineage-specific gene evolution for lepidopteran insects. The comparative analysis of the insect immune repertoires indicated a dynamic and flexible gene expansion in recognition, modulation and effector mechanisms due to different selection pressures. Differential gene regulation by different bacterial species was found in PGRP and Serpin genes, suggesting that Bombyx has a highly selective gene regulation system depending on bacterial species.

A Lipase Isolated from the Silkworm Bombyx mori Shows Antiviral Activity against Nucleopolyhedrovirus

ABSTRACT A protein showing strong antiviral activity against Bombyx mori nucleopolyhedrovirus (BmNPV) was purified from the digestive juice of B. mori larvae. A homology search of the deduced amino acid sequence of the protein cDNA revealed 56% homology with Drosophila melanogaster lipase and 21% homology with human lipase. As lipase activity of the protein was confirmed in vitro, this protein was designated Bmlipase-1. Northern blot analysis showed that the Bmlipase-1 gene is expressed in the midgut but not in other tissues, nor is it activated by BmNPV infection. In addition, the Bmlipase-1 gene was shown not to be expressed in the molting and wandering stages, indicating that the gene is hormonally regulated. Our results suggest that an insect digestive enzyme has potential as a physiological barrier against BmNPV at the initial site of viral infection.

Transgenic expression of cecropin B, an antibacterial peptide from Bombyx mori, confers enhanced resistance to bacterial leaf blight in rice

The short persistence of cecropin B peptide in plants, due to post‐translational degradation, is a serious impediment in its effective utilization for developing bacterial resistance transgenic plants. Two DNA constructs encoding the full‐length precursor of cecropin B peptide and the mature sequence of cecropin B peptide preceded by a signal peptide derived from rice chitinase gene were transformed in rice. The differences in the transcriptional levels in independent transgenic lines showed moderate to high expression of cecropin B gene that correlated well with the differences in cecropin B accumulation observed by Western blot analysis. The development of lesions resulting from infection by Xanthomonas oryzae pv. oryzae was significantly confined in the infected leaflet of transgenic lines, when compared with the control plants.

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.

Efficient protein production using a Bombyx mori nuclear polyhedrosis virus lacking the cysteine proteinase gene

Infection by a baculovirus (Bombyx mori nuclear polyhedrosis virus, BmNPV) in silkworm (Bombyx mori) larvae is highly efficient as an expression system for the production of useful proteins. However, the amount of the protein of interest expressed tends to decrease in the later stages of infection presumably due, in part, to a proteinase produced in the larval haemolymph. The N-terminal amino acid sequence of a proteinase purified from the haemolymph of BmNPV-infected larvae was identical to the internal amino acid sequence of the viral cysteine proteinase gene of BmNPV, suggesting that the cysteine proteinase in the haemolymph originated from the BmNPV gene. We constructed a mutant virus (CPd) which had a deletion in the cysteine proteinase gene. No proteinase activity corresponding to this proteinase was detected in the haemolymph of silkworm larvae infected with CPd. The firefly luciferase and the human growth hormone genes were separately introduced into CPd under control of the polyhedrin promoter. These constructs produced these proteins very efficiently, because of a greatly reduced degree of degradation of these proteins. A BmNPV vector system using CPd enhances the stability of foreign expressed proteins, especially for those that are cysteine proteinase-sensitive.

Two isoforms of a member of the arthropod defensin family from the soft tick, Ornithodoros moubata (Acari: Argasidae).

We previously purified and determined the partial amino acid sequence of a 4 kDa peptide having high homology with scorpion defensin from the hemolymph of adult fed female soft ticks, Ornithodoros moubata. In this study, the full length sequences of two defensin isoforms were obtained. Deduced amino acid sequences reveal a precursor protein of 73 amino acid residues with a mature portion consisting of 37 amino acid residues. This mature peptide contains six cysteine residues conserved in the same location as other invertebrate defensins. Phylogenetic analysis reveals that Ornithodoros defensin is most closely related to scorpion defensin and other more ancient arthropods. Ornithodoros defensin mRNA is constitutively expressed and up-regulated by blood-feeding and bacterial injection. Ornithodoros defensin gene expression occurs mainly in the midgut. This is the first report of the cloning and gene expression of an antibacterial peptide from the Acari.

Molecular cloning, nucleotide sequence and gene expression of a cytochrome P450 (CYP6F1) from the pyrethroid-resistant mosquito, Culex quinquefasciatus Say

To analyze cytochrome P450s in the southern house mosquito, Culex quinquefasciatus, we quantified the content of P450s and b5 in larval microsomes of guts and carcasses. Results indicated that content was 30 times higher in guts than in carcasses. A conserved region in the alignment of insect P450 family 6 (CYP6) proteins served as a guide for the synthesis of degenerate oligonucleotide primers to clone P450 cDNAs. Primers were used in the reverse transcription-polymerase chain reaction (RT-PCR) of gut mRNA from 4th-instar larvae of the permethrin-susceptible or resistant C. quinquefasciatus. PCR products of ca. 250 base pairs (bp) were cloned, and nucleotide sequences of 35 clones from susceptible and 28 from resistant strains determined. Alignment of the deduced amino acid sequences from these clones showed them to be classifiable into six isoforms. We next screened a cDNA clone (CYP6F1) from a gut cDNA library and determined the nucleotide sequence. Northern blot analysis showed that the CYP6PF1 gene in the permethrin-resistant strain appeared to be expressed more strongly than in the susceptible strain. The deduced amino acid of CYP6F1 showed that it has conserved domains of a membrane-anchoring signal, reductase binding sites, a heme-binding site, ETLR motif and substrate recognition sites in P450s. Phylogenetic analysis showed that CYP6F1 is strongly related to CYP6D1 involved in pyrethroid detoxification.

Antibacterial peptide defensin is involved in midgut immunity of the soft tick, Ornithodoros moubata.

Two defensin genes A and B were previously demonstrated to be up‐regulated by blood feeding in the soft tick, Ornithodoros moubata[ Nakajima et al. (2001) Two isoforms of a member of the arthropod defensin family from the soft tick, Ornithodoros moubata (Acari: Argasidae). Insect Biochem Mol Biol 31: 747–751]. In this study, two defensin isoforms C and D similar to defensins A and B were newly cloned. A total of four defensins have been identified in O. moubata. All four Ornithodoros defensins are coded as prepro‐defensins. Ornithodoros defensin genes consist of four exons and three introns, an organization reported in mussel defensins but not insect defensins. Ornithodoros defensin C and D genes are predominantly expressed in the midgut and up‐regulated in response to blood feeding. The mature peptide of the previously cloned Ornithodoros defensin A was purified from the midgut lumen, indicating defensin is secreted into the midgut. These findings confirm the involvement of Ornithodoros defensin in midgut immunity.

Selective cancer cell cytotoxicity of enantiomeric 9-mer peptides derived from beetle defensins depends on negatively charged phosphatidylserine on the cell surface

Four enantiomeric 9-mer peptides named d-peptide A, B, C and D were designed and synthesized on the basis of 43-mer insect defensins from two beetles. The d-9-mer peptides maintained bacterial membrane disruptive activity similar to the original peptides and also showed various extents of growth inhibitory activity against different cancer cell lines. Of these peptides, d-peptide B exhibited the highest selective cancer cell cytotoxicity against the mouse myeloma cell line, P3-X63-Ag8.653. Flow cytometric and scanning electron microscopic analysis revealed d-peptide B disrupts mouse myeloma membrane construction, whereas no cytotoxic effect on normal leukocytes was observed. Moreover, a strong correlation between negatively charged phosphatidylserine (PS) density in cancer cell membrane surface and sensitivity to d-9-mer peptides were observed in various cancer cell lines. These results suggest that d-9-mer peptides have negative charge-dependent selective cancer cell cytotoxicity targeting PS in the cancer cell membrane. In addition, synergic growth inhibitory activity against mouse myeloma was observed in combinations of d-peptide B and dexamethasone. These results suggest d-9-mer peptides are promising candidates for novel anticancer drugs.

Immune proteins and their gene expression in the silkworm, Bombyx mori

Several self-defense proteins have been isolated from the silkworm, Bombyx mori and their amino acid sequences determined. These proteins include novel antibacterial proteins designated lebocin and moricin, and a novel lectin designated hemocytin, an insect homologue of mammalian von Willebrand factor. Antibacterial mechanisms of lebocin and moricin have been analyzed and their ability to form ion channels in bacterial membranes play an important role in defense against bacterial infection. cDNAs and genes encoding these proteins have been cloned to examine their induction mechanisms upon bacterial infection. Regulatory motifs such as the kappaB-like and GATA sequence have been identified in the B. mori antibacterial proteins. On the other hand, hemocytin gene expression was confirmed to occur upon bacterial infection and before pupation under naive conditions, suggesting that hemocytin plays an important role in both immunity and metamorphosis. Moreover, this review also describes the releasing mechanisms of a bacterial cell wall component, lipopolysaccharide (LPS), from intact bacteria, clearance of LPS from B. mori hemolymph and a possible signal transduction pathway for antibacterial protein gene expression.