Atsushi Ohnishi

Hokkaido University, Sapporo, Hokkaidō, Japan

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Publications (13)49.99 Total impact

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    ABSTRACT: Growth-blocking peptide (GBP) is a member of an insect cytokine family with diverse functions including growth and immunity controls. Members of this cytokine family have been reported in 15 species of Lepidoptera, and we have recently identified GBP-like peptides in Diptera such as Lucilia cuprina and Drosophila melanogaster, indicating that this peptide family is not specific to Lepidoptera. In order to extend our knowledge of this peptide family, we purified the same family peptide from one of the tenebrionids, Zophobas atratus,(1) isolated its cDNA, and sequenced it. The Z. atratus GBP sequence together with reported sequence data of peptides from the same family enabled us to perform BLAST searches against EST and genome databases of several insect species including Coleoptera, Diptera, Hymenoptera, and Hemiptera and identify homologous peptide genes. Here we report conserved structural features in these sequence data. They consist of 19-30 amino acid residues encoded at the C terminus of a 73-152 amino acid precursor and contain the motif C-x(2)-G-x(4,6)-G-x(1,2)-C-[KR], which shares a certain similarity with the motif in the mammalian EGF peptide family. These data indicate that these small cytokines belonging to one family are present in at least five insect orders.
    Insect biochemistry and molecular biology 03/2012; 42(6):446-54. DOI:10.1016/j.ibmb.2012.03.001 · 3.25 Impact Factor
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    ABSTRACT: Antimicrobial peptides (AMPs), major innate immune effectors, are induced to protect hosts against invading microorganisms. AMPs are also induced under non-infectious stress; however, the signaling pathways of non-infectious stress-induced AMP expression are yet unclear. We demonstrated that growth-blocking peptide (GBP) is a potent cytokine that regulates stressor-induced AMP expression in insects. GBP overexpression in Drosophila elevated expression of AMPs. GBP-induced AMP expression did not require Toll and immune deficiency (Imd) pathway-related genes, but imd and basket were essential, indicating that GBP signaling in Drosophila did not use the orthodox Toll or Imd pathway but used the JNK pathway after association with the adaptor protein Imd. The enhancement of AMP expression by non-infectious physical or environmental stressors was apparent in controls but not in GBP-knockdown larvae. These results indicate that the Drosophila GBP signaling pathway mediates acute innate immune reactions under various stresses, regardless of whether they are infectious or non-infectious.
    Scientific Reports 01/2012; 2:210. DOI:10.1038/srep00210 · 5.08 Impact Factor
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    ABSTRACT: Growth-blocking peptide (GBP) is an insect cytokine that stimulates a class of immune cells called plasmatocytes to adhere to one another and to foreign surfaces. Although extensive structure-activity studies have been performed on the GBP and its mutants in Lepidoptera Pseudaletia separata, the signaling pathway of GBP-dependent activation of plasmatocytes remains unknown. We identified an adaptor protein (P77) with a molecular mass of 77 kDa containing SH2/SH3 domain binding motifs and an immunoreceptor tyrosine-based activation motif (ITAM)-like domain in the cytoplasmic region of the C terminus. Although P77 showed no capacity for direct binding with GBP, its cytoplasmic tyrosine residues were specifically phosphorylated within seconds after GBP was added to a plasmatocyte suspension. Tyrosine phosphorylation of P77 also was observed when hemocytes were incubated with Enterobactor cloacae or Micrococcus luteus, but this phosphorylation was found to be induced by GBP released from hemocytes stimulated by the pathogens. Tyrosine phosphorylation of the integrin beta subunit also was detected in plasmatocytes stimulated by GBP. Double-stranded RNAs targeting P77 not only decreased GBP-dependent tyrosine phosphorylation of the integrin beta subunit, but also abolished GBP-induced spreading of plasmatocytes on foreign surfaces. P77 RNAi larvae also showed significantly higher mortality than control larvae after infection with Serratia marcescens, indicating that P77 is essential for GBP to mediate a normal innate cellular immunity in insects. These results demonstrate that GBP signaling in plasmatocytes requires the adaptor protein P77, and that active P77-assisted tyrosine phosphorylation of integrins is critical for the activation of plasmatocytes.
    Proceedings of the National Academy of Sciences 09/2010; 107(36):15862-7. DOI:10.1073/pnas.1003785107 · 9.81 Impact Factor
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    ABSTRACT: A small multifunctional cytokine, growth-blocking peptide (GBP), from the armyworm Pseudaletia separata larvae was expressed as a soluble and active recombinant peptide in the methylotrophic yeast Pichia pastoris. An expression vector for GBP secretion was constructed using vector pPIC9, and GBP was expressed under the control of the alcohol oxidase (AOX1) promoter. Although we first tried to cultivate GBP in shake flask cultures, the yield was low, probably due to proteolysis of the recombinant protein. To overcome this problem, we utilized a high-density fermentation method. The pH of the medium in the fermenter was kept at 3.0, and the medium was collected within 48h post methanol shift to minimize exposure of the target peptide to proteases. Recombinant GBP was purified through three reverse-phase HPLC columns. We characterized the 25 amino acid GBP by molecular mass spectrometry and amino acid sequencing. Plasmatocyte spreading, one of the activities of GBP, was similar between chemically synthesized GBP and purified recombinant GBP. Up to 50mg GBP was recovered per 1L of yeast culture supernatant.
    Protein Expression and Purification 09/2002; 25(3):416-25. DOI:10.1016/S1046-5928(02)00036-0 · 1.51 Impact Factor
  • A Ohnishi, Y Oda, Y Hayakawa
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    ABSTRACT: Insect cytokine, growth-blocking peptide (GBP), enhances cell proliferation of human keratinocyte cells with a potency almost equivalent to that of human epidermal growth factor (EGF). GBP consists of 25 amino acid residues containing a core region that shows a striking similarity to the C-terminal beta-loop domain of EGF and disordered N and C termini. The present study demonstrates that, although GBP lacks the N-terminal half-portion of EGF molecule, at least five amino acids of the disordered N-terminal six-amino acid region are indispensable for affecting the cell growth activity of GBP. Upon stimulating mitogenesis in keratinocyte cells, GBP directly binds and activates their EGF receptors. GBP also effects proliferative activity on insect Sf9 cells through the binding and activation of the specific receptor, which consists of a heterodimeric complex: a binding subunit (60 kDa) and a tyrosine phosphorylation subunit (58 kDa). These results indicate that GBP enhances cell proliferation of human keratinocyte and insect Sf9 cells through the activation of EGF and GBP receptors, respectively.
    Journal of Biological Chemistry 11/2001; 276(41):37974-9. DOI:10.1074/jbc.M104856200 · 4.60 Impact Factor
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    ABSTRACT: Growth-blocking peptide (GBP) is a 25-amino acid insect cytokine found in Lepidopteran insects that possesses diverse biological activities such as larval growth regulation, cell proliferation, and stimulation of immune cells (plasmatocytes). The tertiary structure of GBP consists of a structured core that contains a disulfide bridge and a short antiparallel beta-sheet (Tyr(11)-Arg(13) and Cys(19)-Pro(21)) and flexible N and C termini (Glu(1)-Gly(6) and Phe(23)-Gln(25)). In this study, deletion and point mutation analogs of GBP were synthesized to investigate the relationship between the structure of GBP and its mitogenic and plasmatocyte spreading activity. The results indicated that deletion of the N-terminal residue, Glu(1), eliminated all plasmatocyte spreading activity but did not reduce mitogenic activity. In contrast, deletion of Phe(23) along with the remainder of the C terminus destroyed all mitogenic activity but only slightly reduced plasmatocyte spreading activity. Therefore, the minimal structure of GBP containing mitogenic activity is 2-23 GBP, whereas that with plasmatocyte spreading activity is 1-22 GBP. NMR analysis indicated that these N- and C-terminal deletion mutants retained a similar core structure to wild-type GBP. Replacement of Asp(16) with either a Glu, Leu, or Asn residue similarly did not alter the core structure of GBP. However, these mutants had no mitogenic activity, although they retained about 50% of their plasmatocyte spreading activity. We conclude that specific residues in the unstructured and structured domains of GBP differentially affect the biological activities of GBP, which suggests the possibility that multifunctional properties of this peptide may be mediated by different forms of a GBP receptor.
    Journal of Biological Chemistry 09/2001; 276(34):31813-8. DOI:10.1074/jbc.M105251200 · 4.60 Impact Factor
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    ABSTRACT: Parasitization of the armyworm Pseudaletia separata by the endoparasitic wasp Cotesia kariyai inhibits larval growth and delays pupation, conditions necessary for proper maturation of the parasite larvae. Parasitization is correlated with an elevated level of a 25-amino-acid hormone-like peptide, growth-blocking peptide (GBP, ENFSGGCVAGYMRTPDGRCKPTFYQ). Injection of synthetic GBP into nonparasitized larvae dose dependently mimics the effects of parasitization by delaying the larval development. Here we studied the relationship between parasitization and both the production and distribution of GBP in central nervous tissues. We found that parasitization is correlated with an elevated expression of GBP mRNA, and increased concentrations of both proGBP and GBP in the host insect brain and subesophageal ganglion. The increase in proGBP precedes that of the mature GBP by about 12 h. In situ hybridization analysis using sections of parasitized and nonparasitized larval brains showed strong expression of GBP mRNA in perineural cells and/or class I neuroglia in the rind of both larval brains. The expression in parasitized larval brain-subesophageal ganglion is approximately two- to threefold higher than that in nonparasitized larvae. The presence of GBP in insect neural tissue, and its role in inhibiting growth, suggest an involvement in the regulation of neurosecretory cells.
    Cell and Tissue Research 07/2000; 300(3):459-64. DOI:10.1007/s004419900152 · 3.33 Impact Factor
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    Journal of Biosciences, New Dehli; 09/1999
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    ABSTRACT: Growth-blocking peptide (GBP) is an insect growth factor consisting of 25 amino acid residues that retards the development of lepidopteran larvae at high concentration while it stimulates larval growth at low concentration. In this study, we determined the solution structure of GBP by two-dimensional 1H NMR spectroscopy. The structure contains a short segment of double-stranded beta-sheet involving residues 11-13 and 19-21 and a type-II beta-turn in the loop region (residues 8-11), whereas the N and C termini are disordered. This is the first report of the three-dimensional structure of the peptiderigic insect growth factor, and the structure of the well defined region of GBP was found to share similarity with that of the C-terminal domain of the epidermal growth factor (EGF). Because GBP has been reported to stimulate DNA synthesis of not only insect cells but also human keratinocyte cells at the same level with EGF, the structural similarity between GBP and EGF may lead to the interaction of GBP to EGF receptor.
    Journal of Biological Chemistry 02/1999; 274(4):1887-90. DOI:10.1074/jbc.274.4.1887 · 4.60 Impact Factor
  • Yoichi Hayakawa, Atsushi Ohnishi
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    ABSTRACT: Growth-blocking peptide (GBP) is an insect biogenic peptide that retards the development of lepidopteran larvae. cDNAs encoding GBP were isolated from three lepidopteran species: Pseudaletia separata, Mamestra brassicae and Spodoptera litura. Comparison of these molecules revealed that the GBP coding regions were 70% homologous. In contrast, the upstream regions of the deduced propeptides were only 33% identical. Sequence analysis further suggested that GBP shares some structural similarities with human epidermal growth factors. Bioassay data revealed that several pmol/ml of GBP stimulated DNA synthesis of a human keratinocyte cell line and of SF-9 insect cells. However, several nmol/ml of GBP did not stimulate cell proliferation at all. In vivo studies similarly indicated that low concentrations of GBP stimulated larval growth while high concentrations of GBP retarded growth. These data suggest that GBP acts as a growth factor that regulates insect larval development.
    Biochemical and Biophysical Research Communications 10/1998; 250(2):194-9. DOI:10.1006/bbrc.1998.8959 · 2.28 Impact Factor
  • Yasuhisa Endo, Atsushi Ohnishi, Yoichi Hayakawa
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    ABSTRACT: Growth-blocking peptide (GBP) has been purified for the first time from the haemolymph of the host armyworm Pseudaletia separata whose growth is inhibited and shows developmental arrest in the last larval instar stage when parasitized by the parasitoid wasp Cotesia kariyai. GBP naturally occurs in the haemolymph of lepidopteran larvae but its concentration is very low during the last larval instar in comparison with that in the penultimate larval instar. However, by 24h after parasitization or polydnavirus (PdV)-infection on day 0 of the last larval instar, a four-fold increase in GBP level, compared with synchronous non-parasitized control larvae, is observed. Although Northern blot analysis indicates that GBP mRNA is transcribed in brain-nerve cord and fat body, plasma GBP is likely to be secreted mainly from fat body because the GBP mRNA level is approximately 100-fold higher in fat body than that in brain-nerve cord. RT-PCR analysis demonstrates the constant expression of GBP mRNA in both parasitized (or PdV-infected) and non-parasitized larval fat body, which suggests that parasitism does not influence transcriptional level, but might influence post-transcriptional level to elevate plasma GBP concentration. This interpretation was supported by estimating GBP precursor levels in fat body of PdV-infected and non-infected larvae. Virus infection appears to elevate the GBP precursor levels in fat body to about six times greater than that in non-infected last instar larvae by 6h after PdV-injection. The GBP processing enzyme activity that occurs in Golgi body-rich extract of the fat body is increased by about 90% after parasitization or PdV-injection.
    Journal of insect physiology 10/1998; 44(9):859-866. DOI:10.1016/S0022-1910(98)00017-1 · 2.24 Impact Factor
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    ABSTRACT: Growth-blocking peptide (GBP) has been isolated for the first time from the haemolymph of the host armyworm Pseudaletia separata whose development was halted in the last larval instar stage by parasitization with the parasitoid wasp Cotesia kariyai. Recent studies demonstrated that GBP not only exists in the plasma (haemolymph without cells) of parasitized last instar larvae, but also in the plasma of nonparasitized penultimate (5th) instar larvae. Monoclonal antibodies were prepared to measure the titers of GBP in nonparasitized and parasitized larval plasma. One of three monoclonal antibodies raised against GBP, which is the most specific for GBP, was used to quantify the concentration of plasma GBP. As this antibody recognized two plasma peptides other than GBP in crude plasma fractions, each plasma peptide fraction was separated by a reversed phase HPLC, and then plasma GBP level was measured by ELISA. The highest level of plasma GBP detected on Day 0 of the penultimate instar larvae was gradually decreased throughout the larval growth except for the temporary increase on Day 0 of last larval instar. After parasitization on Day 0 of last larval instar, two peaks of plasma GBP titer were detected during the last larval instar, one day and six days after parasitization. This characteristic increase and decrease in plasma GBP level was also observed by transferring last instar larvae of the armyworm from 25 to 10 degrees C, as a result of which larvae delayed pupation by more than 15 days. From these results, it is reasonable to propose that plasma GBP in lepidopteran larvae might control certain upstream steps in a cascade of events leading to pupation; thus, an elevated level of plasma GBP interferes with normal metamorphosis from larvae to pupae.
    Insect Biochemistry and Molecular Biology 01/1996; 25(10):1121-7. DOI:10.1016/0965-1748(95)00054-2 · 3.42 Impact Factor
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    ABSTRACT: Growth-blocking peptide (GBP) is an insect biogenic peptide that prevents the onset of metamorphosis from larva to pupa. A cDNA coding for GBP is described. Mixed oligonucleotides derived from a GBP peptide sequence were used to generate amplified DNA by the polymerase chain reaction (PCR). Based on the sequence of the amplified DNA, a 41 bases oligonucleotide was designed for screening a cDNA library which was constructed from the armyworm Pseudaletia separata larvae parasitized with the parasitic wasp Cotesia kariyai. The cloned cDNA for GBP was 809 base pairs in length. An open reading frame of 429 base pairs encodes a pre-pro-peptide of 143 amino acid residues in which GBP is localized at the C-terminal region, and other three peptides including a putative signal peptide and appropriate processing sites for endoproteolytic cleavage precede the GBP sequence. Northern blot analyses demonstrate the presence of a 800-base mRNA transcript in fat body and 2.5-kilobase transcript in brain and nerve cord, suggesting the possibility that the transcription of GBP gene is regulated in a tissue-dependent manner. This interpretation was supported by isolating a GBP cDNA fragment from cDNA pool of brain-nerve cords. GBP mRNA is constantly expressed in both parasitized and non-parasitized last instar larvae and there is no difference in the levels of the mRNA between both larvae, thus indicating that parasitism may effect on translational or posttranslational level to elevate plasma GBP concentration.
    FEBS Letters 01/1996; 376(3):185-9. DOI:10.1016/0014-5793(95)01273-7 · 3.34 Impact Factor

Publication Stats

221 Citations
49.99 Total Impact Points


  • 1996–2002
    • Hokkaido University
      • • Institute of Low Temperature Science
      • • Laboratory of Nutritional Biochemistry
      Sapporo, Hokkaidō, Japan
  • 1999
    • Toyama Medical and Pharmaceutical University
      Тояма, Toyama, Japan