Growth-blocking Peptide Research Articles

Characterization of Receptors of Insect Cytokine, Growth-blocking Peptide, in Human Keratinocyte and Insect Sf9 Cells

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.

Structure and Activity of the Insect Cytokine Growth-blocking Peptide: ESSENTIAL REGIONS FOR MITOGENIC AND HEMOCYTE-STIMULATING ACTIVITIES ARE SEPARATE

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.

2P025サイトカインgrowth-blocking peptide(GBP)のN末端領域と活性

2P025サイトカインgrowth-blocking peptide(GBP)のN末端領域と活性

Distribution of growth-blocking peptide in the insect central nervous tissue.

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.

Plasmatocyte spreading peptide (PSP1) and growth blocking peptide (GBP) are multifunctional homologs

Recently, we identified Plasmatocyte spreading peptide (PSP1) from the moth Pseudoplusia includens and reported that it mediates adhesion of hemocytes to foreign surfaces. PSP1 is structurally very similar to three classes of peptides identified earlier from other species of Lepidoptera: growth blocking peptide (GBP) originally identified in Pseudaletia separata, and a series of related peptides from other species designated as paralytic (PP) or cardioactive (CAP) peptides. In this study, we conducted parallel experiments in P. includens and P. separata to determine whether PSP1 and GBP have distinct or multiple biological activities. Both peptides affected the adhesive state of hemocytes from each moth very similarly. PSP1 and GBP exhibited significant growth blocking and paralytic activity in P. separata. Both peptides also had growth blocking activity in P. includens although larvae had to be injected with higher doses of each peptide to reduce weight gain than was observed for P. separata. However, GBP and PSP1 had little paralytic activity in P. includens. Collectively, our results indicate that GBP and PSP1 are multifunctional, but that some interspecific variation also exists in their growth blocking and paralytic activities. We suggest that all PSP1, GBP, PP and CAP family members are homologs that likely have multiple biological activities. Based upon the unique consensus sequence of their N termini, we propose that these molecules be henceforth referred to as members of the “ENF” peptide family.

3G1500 ファージ提示法によるGBP結合ペプチドのスクリーニングおよびその解析

3G1500 ファージ提示法によるGBP結合ペプチドのスクリーニングおよびその解析

1D1515 昆虫成長因子GBPの全体構造とC末端残基の活性への影響

1D1515 昆虫成長因子GBPの全体構造とC末端残基の活性への影響

Solution Structure of an Insect Growth Factor, Growth-blocking Peptide

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.

Mechanism of parasitism-induced elevation of haemolymph growth-blocking peptide levels in host insect larvae ( Pseudaletia separata)

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 24 h 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 6 h 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.

Cell Growth Activity of Growth-Blocking Peptide

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 brassicaeandSpodoptera 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 vivostudies 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.

Growth-blocking peptide expressed in the insect nervous system--cloning and functional characterization.

Growth-blocking peptide (GBP) is an insect biogenic peptide that retards larval growth and, consequently, delays the onset of pupation of lepidopteran larvae. Transcription of the GBP gene has been proven to be regulated in a tissue-dependent manner by an approximately 0.8-kb mRNA transcript in fat body and 2.5-kb transcript in the brain central nervous system (CNS) [Hayakawa, Y., Ohnishi, A., Yamanaka, A., Izumi, S. & Tomino, S. (1995) FEBS Lett. 367, 185-189]. The GBP cDNA in the brain CNS was isolated, sequenced and found to be almost identical with that in the fat body, except that it encodes a long 5'-untranslated region (approximately 1.1 kb), thus indicating that the brain CNS is synthesized as a polyprotein precursor and released through posttranslational processing in the same manner as that predicted in the fat body. Sequence analyses of the genomic GBP gene fragments demonstrated that the 5'-untranslated region contains an intron encoding a sequence element, TGATAA, which has been reported to be important for expression in the insect larval fat body, suggesting that this element contributes to a tissue-specific expression of the shorter GBP mRNA in fat body. Ribonuclease protection assays showed that parasitization by a parasitic wasp elevated the expression of GBP mRNA in brain CNS. The parasitization-induced increase of GBP precursor and GBP concentrations was also demonstrated in brain CNS. Brain CNS dopamine was increased eightfold by incubating the tissue with GBP for 8 h through the elevation of dopa decarboxylase activity by a transcriptional enhancement perhaps due to GBP-induced increase of cytoplasmic Ca2+ concentration. Dopamine elevation reduced in vitro incorporation of methionine into proteins synthesized in brain CNS.

Mechanism of parasitism-induced elevation of dopamine levels in host insect larvae

Parasitization with the parasitoid wasp, Cotesia kariyai, causes elevation of dopamine (DA) levels in the haemolymph and nerve cord of the last larval instar of the armyworm, Pseudaletia separata. Injection of DA or DA aganist [(−)-Quinpirole] during the early stages of the last larval instar retards normal growth and delays pupation. This suggests the possibility that DA plays an important role in the retardation of larval development by parasitism. The present study demonstrated that the DA level in the integument was also increased through the elevation of dopa decarboxylase (DDC) activity during parasitism. The total DA content in the integument was calculated to be approximately 50 times greater than in the haemolymph of parasitized Day 1 last instar larvae, thereby suggesting that parasitism-induced elevation of haemolymphal DA could be from DA in the integument. This hypothesis was partly confirmed by evidence that radiolabelled DA in the integument was secreted into in vitro incubation medium. Furthermore, DA elevation due to an increase in DDC activity in the integument was also observed in nonparasitized larvae in their last instar after injection of an insect biogenic peptide, growth-blocking peptide (GBP), whose concentration is elevated by parasitism. Therefore it is proposed that the plasma GBP fiter elevated by parasitism causes the rise in integumental DDC activity and this induces an increase in the DA level in the integument. This integument secretes DA into the haemolymph, raising DA levels in the haemolymph of parasitized larvae.

Molecular cloning and characterization of cDNA for insect biogenic peptide, growth-blocking peptide

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.

Growth-blocking peptide titer during larval development of parasitized and cold-stressed armyworm

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°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.

Elevation of dopamine levels in parasitized insect larvae

Last instar larvae of the armyworm, Pseudaletia separata, parasitized with the parasitoid wasp, Cotesia kariyai, suffer from retarded growth and do not initiate metamorphosis. Dopamine levels in hemolymph and nerve cord of host last instar larvae were increased by parasitization with parasitoid wasps. The elevation of dopamine levels was reproduced by injection with polydnavirus or growth-blocking peptide (GBP). Injection of dopamine or the dopamine agonist, (-)-Quinpirole, disturbs normal development of the armyworm larvae. These results suggest that GBP elevated by polydnavirus infection causes an increase of dopamine in hemolymph and nervous system, which is closely related to a disturbance of normal larval development.

Inclusion of a beta-aspartyl peptide bond causes the loss of inherent physiological activities.

Growth-blocking peptide (GBP) is a peptide which was initially isolated from the plasma of last instar larvae of the insect armyworm, Pseudaletia separata, parasitized by the parasitoid wasp, Cotesia kariyai. A standard GBP acid, a free carboxyl terminal GBP, was synthesized by solid phase methods, using 7-fluorenylmethoxycarbonyl (Fmoc) and tert-butyloxycarbonyl (Boc) amino acids, respectively. The peptide synthesized using Boc amino acids gave the expected structure of GBP, while approximately 90% of the peptide synthesized using Fmoc amino acids was a peptide with a beta-aspartyl (Asp) bond at position 16. The peptide containing the beta-Asp bond does not exhibit any physiological effect on armyworm larvae.

Growth-blocking peptide or polydnavirus effects on the last instar larvae of some insect species

The parasitoid wasp Apanteles kariyai perturbs the endocrinological process of the host that controls normal metamorphosis from larva to pupa. The effect is particularly evident when parasitization occurs during the late stages of larval development of the host Pseudaletia separata. This developmental disturbance allows the parasitoid wasp to complete its larval growth and emerge while the host is still in a larval stage, otherwise the parasitoid larvae would be trapped in the sclerotized pupal cuticle. A peptidergic factor, growth-blocking peptide (GBP), has recently been purified from the parasitized larval hemolymph. Injection of GBP into unparasitized last instar larvae of the armyworm clearly retards larval growth and, consequently, delays the onset of pupation of the larvar. Suppression of larval development also occurs when unparasitized last instar larvae are injected with a mixture of symbiotic polydnavirus and venom fluid from the reproductive tract of the Apanteles female wasps. The effects of injecting GBP or a mixture of polydnavirus and venom fluid into the last instar larvae was investigated in three species of Lepidoptera, armyworm ( Pseudaletia separata), common cutworm ( Spodoptera litura) and silkworm ( Bombyx mori), in order to compare the species-specificity of both factors. Injection of GBP causes suppression of larval development in all tested insects. In contrast, developmental suppression by the injection of polydnavirus plus venom fluid occured only in the armyworm larvae. Common cutworm larvae injected with virus plus venom were slightly suppressed but the larval development of the silkworm was not affected by virus-venom injection. Furthermore, GBP in the armyworm larval plasma was induced by virus-venom injection, but the detectable amount of GBP was not observed in the plasma of the common cutworm and silkworm larvae by the injection.

A putative new juvenile peptide hormone in lepidopteran insects

A growth-blocking peptide (GBP) with repressive activity against juvenile hormone (JH) esterase has been isolated from the last (6th) instar larval plasma of the armyworm Pseudaletia separata (Lepidoptera: Noctuidae) parasitized by the parasitoid wasp Apanteles kariyai (Hymenoptera: Braconidae) (1,2). This study demonstrates that GBP not only exists in the plasma of parasitized last instar larvae, but also in the plasma of unparasitized penultimate (5th) instar larvae, while the plasma of last instar larvae does not contain any detectable amount of GBP. The detection of GBP in unparasitized penultimate instar larvae, before the final larval molt, demonstrates that this factor is naturally occurring in the insect larva before the last larval instar and is seemingly coordinating, along with JH, the regulation of juvenile characteristics. This finding suggests the existence of a new type of juvenile peptide hormone in lepidopteran insects.

Structure of a growth-blocking peptide present in parasitized insect hemolymph

Last instar larvae of the insect armyworm, Pseudaletia separata, parasitized with the parasitoid wasp, Apanteles kariyai, do not initiate metamorphosis and, ultimately, the wasp larvae emerge from the host larvae about 10 days after parasitization (Tanaka, T., Agui, N., and Hiruma, K. (1987) Gen. Comp. Endocrinol. 67, 364-374). It is necessary for the parasitoid wasp to perturb the armyworm's endocrinological processes that control normal metamorphosis from larvae to pupae. This endocrinological perturbation allows the parasitoid to complete its larval growth before emerging from the host larvae. It is obligatory for the parasitoid larvae to emerge while the host is still in a larval stage because the sclerotized pupal cuticle is impenetrable for the parasitoid larvae. A growth-blocking peptide with repressive activity against juvenile hormone esterase has been proven to exist in the parasitized host larval plasma (Hayakawa, Y. (1990) J. Biol. Chem. 265, 10813-10816). Here, I describe the detailed structure of this peptide and also the corresponding synthetic peptide to confirm this structure.