Growth-blocking Peptide Research Articles

C-terminal Elongation of Growth-blocking Peptide Enhances Its Biological Activity and Micelle Binding Affinity

Growth-blocking peptide (GBP) is a hormone-like peptide that suppresses the growth of the host armyworm. Although the 23-amino acid GBP (1-23 GBP) is expressed in nonparasitized armyworm plasma, the parasitization by wasp produces the 28-amino acid GBP (1-28 GBP) through an elongation of the C-terminal amino acid sequence. In this study, we characterized the GBP variants, which consist of various lengths of the C-terminal region, by comparing their biological activities and three-dimensional structures. The results of an injection study indicate that 1-28 GBP most strongly suppresses larval growth. NMR analysis shows that these peptides have basically the same tertiary structures and that the extension of the C-terminal region is disordered. However, the C-terminal region of 1-28 GBP undergoes a conformational transition from a random coiled state to an alpha-helical state in the presence of dodecylphosphocholine micelles. This suggests that binding of the C-terminal region would affect larval growth activity.

Solution structure of 1-23 GBP (growth-blocking peptide)

solution structure of 1-23 GBP (growth-blocking peptide)

Analysis of ESTs generated from immune-stimulated hemocytes of larval Heliothis virescens

Heliothis virescens immunome components responding to baculoviral and bacterial infection were identified from expressed sequence tags (ESTs) generated from an immune-stimulated larval hemocyte cDNA library. A total of 5548 ESTs were generated comprising 448 contigs and 1114 singletons, totaling 1606 putative transcripts 1101 of which had BLAST scores, including many known orthologs from other insect species. Orthologs of known or putative immune function were identified among them melanization pathway components, proteases, antibacterial proteins, lectins, bacteria-binding proteins, ferritins, scavenger receptors, cell surface receptors, signaling pathway components, and stress response enzymes. Additionally, many enzymes of central metabolism, cytoskeletal, mitochondrial, and ribosomal components, as well as transcriptional and translational regulators were identified. The effect of bacterial and baculoviral infection upon transcript levels of three identified immunome targets from among the ESTs was quantitated using real time PCR. Scolexin-B, C-type lectin and growth-blocking peptide binding protein transcripts were significantly elevated by bacterial infection. Per os infection with the baculovirus Helicoverpa zea single nucleopolyhedrovirus however did not significantly alter transcript levels of these three genes. The ESTs reported here are the first large scale report of the H. virescens immunome responding to entomopathogens, and represent a first step to a more complete transcriptome for this pest moth.

A Novel Peptide Mediates Aggregation and Migration of Hemocytes from an Insect

Insect blood cells (hemocytes) comprise an essential arm of the immune system [1-7]. Several factors mediating recognition and phagocytosis of foreign intruders by hemocytes have been identified, but the mechanisms regulating hemocyte movement remain fragmentary. Embryonic hemocytes from Drosophila migrate along stereotypical routes in response to chemotactic signals from PVF ligands, members of the platelet-derived growth factor family [8-12]. Embryonic and larval hemocytes also accumulate at external wounds [11-13], but PVFs are not required for this response, suggesting involvement by other, unknown factors. Here we report the identification of hemocyte chemotactic peptide (HCP) from the moth Pseudaletia separata and present evidence that it stimulates aggregation and directed movement of phagocytic hemocytes. Spatiotemporal studies revealed that HCP is expressed in both epidermal cells and hemocytes, whereas structure-function studies identified post-translational modifications important for activity. HCP also shares similarities with another group of cytokines from moths called ENF peptides [14-17]. Taken together, our results identify HCP as a chemotactic cytokine that enhances clotting at wound sites in larvae.

Effects of the virus-like particles of a braconid endoparasitoid, Meteorus pulchricornis, on hemocytes and hematopoietic organs of its noctuid host, Pseudaletia separata

Previously, we reported that virus-like particles of Meteorus pulchricornis (MpVLPs) induced apoptosis in hemocytes of its host, Pseudaletia separata, and that MpVLPs seem to especially damage the granulocyte population in vivo. The present study was conducted to elucidate more clearly the distinct behavior of two hemocyte populations in response to MpVLPs or parasitization. First, we examined the differences between plasmatocytes and granulocytes in their sensitivity to MpVLP-induced apoptosis and also in changes of their numbers after parasitization. MpVLPs induced apoptosis in both hemocyte species in vitro. The number of plasmatocytes increased slightly after parasitization in vivo unlike that of granulocytes. The BrdU incorporation assay indicated that the parasitization or the injection of venom containing MpVLPs (V(+)VLP) enhanced the proliferation of plasmatocytes and suppressed that of granulocytes, accounting in part for the distinct responses shown by the respective hemocyte populations. Next, we examined the effects of V(+)VLP on hematopoietic organs (HPOs), major tissues for hematopoiesis of P. separata. A large number (approximately 70–80%) of the cells released from cultured HPOs were determined to be plasmatocytes by the morphology and response to growth-blocking peptide. The injection of V(+)VLP did not affect HPOs in terms of the hematopoietic activity and hemocyte species released. These results, together with our previous data, suggest that the plasmatocyte population was maintained by both supply from undamaged HPOs and the propagation of circulating cells during the initial phase of at least 24 h post-parasitization, while MpVLPs caused apoptosis irrespective of hemocyte species.

Insect cytokine growth‐blocking peptide signaling cascades regulate two separate groups of target genes

Growth-blocking peptide (GBP) is a 25 amino acid insect cytokine found in lepidopteran insects that has diverse biological activities, such as larval growth regulation, paralysis induction, cell proliferation, and stimulation of immune cells. GBP also enhances expression of the tyrosine hydroxylase (TH, EC 1.14.16.2) and 3,4-dihydroxy-l-phenylalanine (Dopa) decarboxylase (DDC, EC 4.1.1.26) genes, which elevate dopamine levels in insect epidermal cells. We used insect epidermis and cultured cells to define the role of the GBP signaling pathway in the enhancement of TH and DDC gene expression. It has been recently reported that robust expression of the DDC gene requires activation of extracellular signal-regulated kinase (ERK) in epidermal cells of wounded Drosophila embryos. This study confirmed that GBP activates ERK, but this activation is not directly linked to the enhancement of TH and DDC gene expression. One of the GBP pathway components is phospholipase C, whose activation is essential for the activation of ERK and elevation of expression of both enzyme genes. The downstream signaling pathways diverge to ERK activation through activated protein kinase C and expression of the enzyme genes through inositol triphosphate receptor-mediated Ca2+ influx from extracellular fluid. Our data indicate that the diverged GBP signaling pathways enable GBP to exert completely different biological functions, even in a single cell type.

Insect cytokine, growth‐blocking peptide, is a primary regulator of melanin‐synthesis enzymes in armyworm larval cuticle

The cuticles of most insect larvae have a variety of melanin patterns that function in the insects' interactions with their biotic and abiotic environments. Larvae of the armyworm Pseudaletia separata have black and white stripes running longitudinally along the body axis. This pattern is emphasized after the last larval molt by an increase in the contrast between the lines. We have previously shown that 3,4-dihydroxy-L-phenylalanine (Dopa) decarboxylase (DDC) is activated during the molt period by preferential enhancement of its transcription in the epidermal cells beneath the black stripes. This study demonstrated that tyrosine hydroxylase (TH) expression is activated synchronously with DDC. Furthermore, enhancement of DDC and TH transcription is due to an increase in cyotoplasmic Ca(2+), which is induced by the insect cytokine, growth-blocking peptide (GBP). Enhanced gene expression for both enzymes was induced by substitution of the calcium ionophore A23187, and completely blocked by EGTA. A GBP-induced increase in cytoplasmic Ca(2+) was seen in epidermal cells under the black stripes but not those beneath the white stripes, suggesting that a difference in Ca(2+) concentration in stripe cells leads to the specific expression of DDC and TH genes. Based on the fact that epidermal cells beneath the white stripes contain abundant granules composed mainly of uric acid, which can form a complex with Ca(2+) and hence decrease its free concentration, we discuss the possibility that uric acid, as well as GBP, contributes to the difference in cytoplasmic Ca(2+) within the epidermal cells.

Cloning and Characterization of Bombyx mori PP-BP a Gene Induced by Viral Infection

The ENF peptide family, so termed after the consensus sequence in their amino termini (Glu-Asn-Phe-), is assumed to play multiple important roles in defense reactions, growth regulation, and homeostasis of Lepidopteran insects. The paralytic peptide of Bombyx mori (BmPP) is one such peptide that is involved in the paralytic and plasmatocyte-spreading activities in the hemocyte immune reaction. The growth-blocking peptide of Pseudaletia separata (PsGBP), which is also a member of the ENF peptide family, has similar functions that can reportedly be attenuated by the growth-blocking peptide-binding protein (GBP-BP). Using the fluorescent differential display (FDD) technique, the differential expression pattern of genes in highly susceptible silkworm strain 306 were analyzed, following infection with B. mori nuclear polyhedrosis virus (BmNPV), and a differential band (G12 782) was obtained from the hemolymph RNA pools. Using 5′-RACE with a specially designed primer based on the FDD study, a 1 401 bp cDNA clone was obtained containing a 1 311 bp open reading frame (ORF, GenBank accession number DQ306881). The deduced protein was highly homologous in primary structure to GBP-BP and was termed B. mori paralytic peptide-binding protein (PP-BP). The B. mori PP-BP gene is organized into two exons and only one intron, using bioinformatics searches.Using RT-PCR analysis, it was found that the B. mori PP-BP gene was expressed almost exclusively in the hemolymph. Real-time quantitative PCR analysis indicated that the B. mori PP-BP mRNA level in B. mori strain 306 exposed to BmNPV was much higher than that in B. mori strain without the virus infection. This result implies that the B. mori PP-BP is related to the cellular immune response after BmNPV invades the hemolymph.

N-Terminal Mutational Analysis of the Interaction Between Growth- Blocking Peptide (GBP) and Receptor of Insect Immune Cells

GBP, a small insect cytokine isolated from lepidopterans, has a variety of functions. We constructed a series of mutants focusing on the unstructured N-terminal residues of GBP by acetylation, deletion, and elongation in order to investigate the interaction between GBP and its receptor in plasmatocytes. The 1H NMR spectra showed no significant changes in the tertiary structures of these peptides, which indicated that all the mutants maintained their core beta-sheet structures. The deletion and acetylated mutants, 2-25GBP, Ac2-25GBP, and AcGBP, lost their activity. 2-25GBP was the strongest antagonist, while Ac2-25GBP and AcGBP were moderate. In contrast, the elongated mutants, (-1R)GBP, (-1A)GBP, and (-2G,-1R)GBP maintained their plasmatocyte-spreading activity. These results demonstrate the importance of the GBP N-terminal charged amine and length of N-terminal GBP-peptide backbone for plasmatocyte-spreading activity. Next, we analyzed other mutant peptides, 1-25(N2A)GBP and 2-25(N2A)GBP, focusing on Asn2. Surprisingly, 2-25(N2A)GBP had slight plasmatocyte-spreading activity, whereas 2-25GBP lost its activity. Finally, substituted mutant, F3AGBP, had neither plasmatocyte-spreading activity nor antagonistic activity. These results demonstrate the function of each N-terminal residue in the interaction between GBP and its receptor in plasmatocytes.

Insect cytokine growth-blocking peptide (GBP) regulates insect development

Growth-blocking peptide (GBP) was first identified in the hemolymph of the host armyworm, Pseudaletia separata, whose growth is halted in the last instar larval stage by parasitization with the parasitoid wasp, Cotesia kariyai. Studies on the mechanism of growth retardation by GBP revealed that GBP titers in hemolymph fluctuate synchronously with dopamine levels: GBP and dopamine peaks coincide with each larval molt period, during which larvae temporarily cease moving and feeding. The fact that GBP induced the elevation of dopamine concentration in the hemolymph was demonstrated in armyworm larvae by GBP injection. Dopamine elevation was also observed in insects exposed to various stress conditions such as parasitization and chilling. These results, together with the fact that dopamine itself inhibits larval growth, indicate that GBP induces growth retardation via the elevation of dopamine levels. Further, we demonstrated that the diapause-inducing influence of short day length also elevates dopamine concentrations in hemolymphs and the brain-central nervous system (Br-CNS) of the cabbage armyworm, Mamestra brassicae. The elevation of dopamine levels contributes to the onset of pupal diapause. We therefore proposed that a GBP-dopamine system contributes to the control of growth rates in insects. Recent studies by ourselves and other laboratories have found more than 10 homologous peptides in various insect species. These peptides have diverse functions: larval growth retardation, paralysis induction, cell proliferation, immune cell stimulation and cardioacceleration. As demonstrated, GBP itself exerts most of these functions, so it is reasonable to conclude that GBP and GBP-like peptides widely present in insects should be regarded as insect cytokines with a variety of functions.

1P001 Structure-activity relationships and C-terminal elongation effects of growth-blocking peptide(1. Protein structure and dynamics (I),Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

1P001 Structure-activity relationships and C-terminal elongation effects of growth-blocking peptide(1. Protein structure and dynamics (I),Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

Regulation of growth-blocking peptide expression during embryogenesis of the cabbage armyworm

Growth-blocking peptide (GBP) is an insect cytokine with diverse biological functions. Northern blot analysis revealed high heterogeneity in the size distribution of GBP mRNAs as well as in the tissues where they are detected. The spatio-temporal transcription pattern is dynamic, especially during embryogenesis. Gel shift assays demonstrated that the cabbage armyworm embryo nuclear extract specifically binds to a 178-bp element, at position +234 to +411 from the transcription start site of the 1.3 kb GBP transcript, in which two Drosophila Deformed (Dfd) binding sites are repeated in tandem. The specific binding between this element and Dfd was demonstrated using recombinant cabbage armyworm Dfd protein. Silencing the Dfd expression in embryos by treating with Dfd double-stranded RNA did not reduce the expression level of GBP, but ectopic GBP expression was observed in the lateral region of the embryo, suggesting that Dfd could serve as a transcriptional repressor for the GBP gene.

The Gly-Gly Linker Region of the Insect Cytokine Growth-blocking Peptide Is Essential for Activity

Growth-blocking peptide (GBP) is a 25-amino acid cytokine isolated from the lepidopteran insect Pseudaletia separata. GBP exhibits various biological activities such as regulation of larval growth of insects, proliferation of a few kinds of cultured cells, and stimulation of a class of insect immune cells called plasmatocytes. The tertiary structure of GBP consists of a well structured core domain and disordered N and C termini. Our previous studies revealed that, in addition to the structured core, specific residues in the unstructured N-terminal region (Glu1 and Phe3) are also essential for the plasmatocyte-stimulating activity. In this study, a number of deletion, insertion, and site-directed mutants targeting the unstructured N-terminal residues of GBP were constructed to gain more detailed insight into the mode of interaction between the N-terminal region and GBP receptor. Alteration of the backbone length of the linker region between the core structure and N-terminal domain reduced plasmatocyte-stimulating activity. The substitutions of Gly5 or Gly6 in this linker region with more bulky residues, such as Phe and Pro, also remarkably reduced this activity. We conclude that the interaction of GBP with its receptor depends on the relative position of the N-terminal domain to the core structure, and therefore the backbone flexibility of Gly residues in the linker region is necessary for adoption of a proper conformation suited to receptor binding. Additionally, antagonistic experiments using deletion mutants confirmed that not only the core domain but also the N-terminal region of GBP are required for "receptor-binding," and furthermore Phe3 is a binding determinant of the N-terminal domain.

A cytokine secreted from the suboesophageal body is essential for morphogenesis of the insect head

The suboesophageal body of insects was identified over a century ago in the silkworm embryo, but its biological function is still unknown. We discovered that this tissue is differentiated in the earliest embryonic stages of the cabbage armyworm and secretes the insect cytokine, growth-blocking peptide (GBP), transiently from 24 to 60 h after oviposition when gastrulation is in progress. Over-expression of GBP, achieved by microinjection of the GBP gene driven by a cytomegalovirus (CMV) constitutive promoter, resulted in complex deformities of the procephalon (embryonic head). Severe abnormal phenotypes of the head structure were produced by silencing the GBP expression in the embryo by treating with GBP double-stranded RNA: the procephalon-containing optic lobes diminished and completely separated into bilateral halves. This indicates that GBP secreted from the suboesophageal body plays an essential role in the formation of the procephalic domain during early embryogenesis. The cytokine-induced fusion of bilateral procephalic lobes is thought to be evolutionarily conserved at least in insects, because of the widespread occurrence of the suboesophageal body in insect embryos.

Insect Cytokine Growth-blocking Peptide Triggers a Termination System of Cellular Immunity by Inducing Its Binding Protein

Growth-blocking peptide (GBP) is a 25-amino acid cytokine found in lepidopteran insects that possesses diverse biological activities such as stimulation of immune cells (plasmatocytes), cell proliferation, and larval growth regulation. We found another novel function of GBP that induces a hemolysis of another class of blood cells (oenocytoids). In the lysate of oenocytoids we identified a GBP-binding protein that shows a specific affinity for GBP. The characterization of purified GBP-binding protein and its cDNA demonstrated it as a 49.5-kDa novel protein with a C-terminal region displaying limited homology to several insect lipoproteins. Results of Northern and Western blotting indicated that the GBP-binding protein should be synthesized only in blood cells. Immunoelectron microscopic analyses confirmed that indirect immunoreactive signals were mostly localized in oenocytoids. Kinetic and biological analyses of interaction between GBP and the binding protein showed their strong binding was followed by clearance of GBP from hemolymph, thus indicating that this protein might function as an inhibitory factor against GBP. Based on these results, we propose that insect cytokine GBP shows multifunctions even in cellular immunity: it serves to stimulate immune cells and afterward silences its own action by inducing the binding protein through specific hemolysis.

Roles of Aromatic Residues in the Structure and Biological Activity of the Small Cytokine, Growth-blocking Peptide (GBP)

Growth-blocking peptide (GBP) is a small (25 amino acids) insect cytokine with a variety of functions: controlling the larval development of lepidopteran insects, acting as a mitogen for various types of cultured cells, and stimulating insect blood cells. The aromatic residues of GBP (Phe-3, Tyr-11, and Phe-23) are highly conserved in the ENF peptide family found in lepidopteran insects. We investigated the relationship between the biological activities and structural properties of a series of GBP mutants, in which each of the three aromatic residues is replaced by a different residue. The results of the hemocytes-stimulating assays of GBP mutants indicated that Phe-3 is the key residue in this activity: Ala or Tyr replacement resulted in significant loss of the activity, but Leu replacement did not. The replacements of other aromatic residues hardly affected the activity. On the other hand, NMR analysis of the mutants suggested that Tyr-11 is a key residue for maintaining the core structure of GBP. Surprisingly, the Y11A mutant maintained its biological activity, although its native-like secondary structure was disordered. Detailed analyses of the (15)N-labeled Y11A mutant by heteronuclear NMR spectroscopy showed that the native-like beta-sheet structure of Y11A was induced by the addition of 2,2,2-trifluoroethanol. The results suggest that Y11A has a tendency to form a native-like structure, and this property may give the Y11A mutant native-like activity.

Isolation and characterization of a dopa decarboxylase cDNA and the induction of its expression by an insect cytokine, growth-blocking peptide in Pseudaletia separata

Parasitization by the wasp, Cotesia kariyai, elevates the concentration of an insect cytokine, growth-blocking peptide (GBP), in hemolymph of last instar Pseudaletis separata larvae. The increase of epidermal and hemolymph dopamine level is associated with the GBP increase. Both GBP and dopamine disturb host development and metamorphosis ( Hayakawa, 1995). Dopa decarboxylase (DDC) converts Dopa to dopamine, and its cDNA was isolated from P. separata, and the deduced amino acid sequence showed that it was highly homologous to other lepidopteran DDCs, showing 96, 90 and 86% identity with those of Mamestra brassicae, Bombyx mori, and Manduca sexta, respectively. A 3.2 kb DDC mRNA transcript was constitutively expressed at low levels in the epidermis, brain-nerve cord and hemocytes, and the expression was enhanced by injection of GBP in these tissues. Detailed characterization of the DDC mRNA expression in the epidermis showed that its expression reached a plateau 3 hr after the injection. DDC activity and DDC protein (55 kDa) level mirrored the mRNA expression. Immunocytochemistry with anti-DDC antibody confirmed that the enhanced DDC expression was localized in the epidermal cells. Dopamine concentration in the epidermis gradually increased and reached maximum 6 hr after the injection. When the epidermis of Day 1 last instar larvae was cultured in vitro in the presence of GBP, DDC mRNA increased, indicating that GBP acted on the epidermal cells directly to induce expression of the DDC gene.

Solution structure of paralytic peptide of silkworm, Bombyx mori

Paralytic peptide of Bombyx mori (BmPP) is one of the multifunctional ENF-peptides; the name of “ENF” is the consensus N-terminal amino acid sequence of the family peptides. We revealed that BmPP significantly possesses growth-blocking activity and plasmatocyte-spreading activity and that its activity profiles are different from those of another ENF-family peptide, namely, the growth-blocking peptide of Pseudaletia separata (PsGBP). We also determined the NMR structures of BmPP and PsGBP under the same conditions, which revealed the structural differences of the first and second β-turn regions between the two peptides. On the basis of our results, it can be considered that the tertiary structural difference in these peptides may cause their different profiles of growth-blocking activity.

Expression and purification of a small cytokine growth-blocking peptide from armyworm Pseudaletia separata by an optimized fermentation method using the methylotrophic yeast Pichia pastoris

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 48 h 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 50 mg GBP was recovered per 1 L of yeast culture supernatant.

Expression of growth-blocking peptide in Mamestra brassicae (Lepidoptera: Noctuidae) cell lines.

Growth blocking peptide (GBP) is one of the growth regulators in lepidopteran insects. Using RT-PCR, we detected GBP expression in two insect cell lines from Mamestra brassicae, SES-MaBr-4 from the fat body and SES-MaBr-93 from hemocytes. The sizes of the RT-PCR products agreed with the sizes calculated from the GBP cDNA sequence. These findings suggest that GBP is a candidate for an essential growth factor in these insect cell lines.