Plasmatocyte sensitivity to plasmatocyte spreading peptide (PSP) fluctuates with the larval molting cycle

Plasmatocyte spreading peptide (PSP) is a cytokine from the moth Pseudoplusia includens that activates a class of hemocytes called plasmatocytes to bind and spread on foreign surfaces. Previous structure-function studies on PSP used plasmatocytes collected from P. includens larvae that were in the late stages of the last (fifth) instar. Here, we report that plasmatocyte sensitivity to PSP varied significantly during the fourth and fifth instar. PSP weakly activated plasmatocytes early in the instar when hemolymph juvenile hormone (JH) titers were relatively high and ecdysteroid titers were low, but strongly activated plasmatocytes late in the instar after JH titers declined and ecdysteroid titers rose. In contrast, plasmatocytes did not vary in their response to plasma, which contains other factors besides PSP that affect plasmatocyte function. In vitro assays indicated that 20-hydroxyecdysone (20E) dose-dependently synergized PSP activity, whereas the JH analog methoprene antagonized PSP activity. Methoprene had no effect on adhesion and spreading of granular cells, but plasmatocytes from larvae topically treated with methoprene exhibited a reduction in sensitivity to PSP. Collectively, these results indicate that plasmatocyte sensitivity to PSP fluctuates in relation to the molting cycle, and that PSP activity is affected by juvenoids and ecdysone.     

Plasmatocyte spreading peptide induces spreading of plasmatocytes but represses spreading of granulocytes.

In most Lepidoptera, plasmatocytes and granulocytes are the two hemocyte classes capable of adhering to foreign targets. Previously, we identified plasmatocyte spreading peptide (PSP1) from the moth Pseudoplusia includens and reported that it induced plasmatocytes to rapidly spread on foreign surfaces. Here we examine whether the response of plasmatocytes to PSP1 was influenced by cell density or culture conditions, and whether PSP1 affected the adhesive state of granulocytes. Plasmatocyte spreading rates were clearly affected by cell density in the absence of PSP1 but spreading was density independent in the presence of PSP1. PSP1 also induced plasmatocytes in agarose-coated culture wells to form homotypic aggregations rather than spread on the surface of culture wells. In contrast, granulocytes rapidly spread in a density independent manner in the absence of PSP1, but were dose-dependently inhibited from spreading by the addition of peptide. An anti-PSP1 polyclonal antibody neutralized the spreading activity of synthetic PSP1. This antibody also neutralized the plasmatocyte spreading activity of granulocyte-conditioned medium, and significantly delayed plasmatocyte spreading when cells were cultured at a high density in unconditioned medium. These results suggested that the spreading activity derived from granulocytes is due in part to PSP1. Pretreatment of plasmatocytes with trypsin had no effect on PSP1-induced aggregation but PSP1-induced aggregations were readily dissociated by trypsin. This suggested that PSP1 is not an adhesion factor but induces adhesion by stimulating a change in the cell surface of plasmatocytes. Synthetic PSP1 also induced aggregation of plasmatocytes from other Lepidoptera indicating that regulation of hemocyte activity by PSP1-related peptides may be widespread. Arch.     

Structure of a paralytic peptide from an insect, Manduca sexta.

Paralytic peptide 1 (PP1) from a moth, Manduca sexta, is a 23-residue peptide (Glu-Asn-Phe-Ala-Gly-Gly-Cys-Ala-Thr-Gly-Tyr-Leu-Arg-Thr-Ala-Asp-Gly-Arg -Cys-Lys-Pro-Thr-Phe) that was first found to have paralytic activity when injected into M. sexta larvae. Recent studies demonstrated that PP1 also stimulated the spreading and aggregation of a blood cell type called plasmatocytes and inhibited bleeding from wounds. We determined the solution structure of PP1 by two-dimensional 1H NMR spectroscopy to begin to understand structural-functional relationships of this peptide. PP1 has an ordered structure, which is composed of a short antiparallel beta-sheet at residues Tyr11-Thr14 and Arg18-Pro21, three beta turns at residues Phe3-Gly6, Ala8-Tyr11 and Thr14-Gly17, and a half turn at the carboxyl-terminus (residues Lys20-Phe23). The well-defined secondary and tertiary structure was stabilized by hydrogen bonding and side-chain hydrophobic interactions. In comparison with two related insect peptides, whose structures have been solved recently, the amino-terminal region of PP1 is substantially more ordered. The short antiparallel beta-sheet of PP1 has a folding pattern similar to the carboxyl-terminal subdomain of epidermal growth factor (EGF). Therefore, PP1 may interact with EGF receptor-like molecules to trigger its different biological activities.     

Biological activity of Manduca sexta paralytic and plasmatocyte spreading peptide and primary structure of its hemolymph precursor

A family of hemolymph peptides was previously identified in several lepidopteran insects, which exhibited multiple biological activities including rapid paralysis, blockage of growth and development, or stimulation of plasmatocyte spreading and aggregation. We synthesized Manduca sexta paralytic peptide 1 (PP1) and found that after it was injected into larvae, bleeding from wounds was dramatically reduced. PP1 also stimulated spreading and aggregation behavior of M. sexta plasmatocytes in vitro. Stimulation of plasmatocyte aggregation and adherence to the body wall may explain a decrease observed in the number of circulating plasmatocytes after injection of PP1. Such aggregates might rapidly form plugs in wounds to prevent bleeding. We cloned a cDNA for a Manduca paralytic peptide precursor, using polymerase chain reactions and cDNA library screening. The active 23-residue PP2 peptide encoded by this clone is at the carboxyl-terminal end of a precursor protein predicted to be 107 amino acid residues long after cleavage of a secretion signal peptide. Active PP2 was produced by processing of recombinant proPP2 by bovine factor X_a. A single proPP2 mRNA was present in fat body but not in hemocytes. The level of this mRNA was not affected by injection of bacteria into larvae. We produced recombinant proPP2 in Escherichia coli and used this protein to produce an antiserum. The antiserum detected proPP2 in plasma and was used to observe rapid proteolytic processing of proPP2 after hemolymph collection.     

PI3K和受体型酪氨酸激酶介导家蚕麻痹肽免疫诱导信号的传递

【目的】普遍存在于鳞翅目昆虫中的ENF肽具有非常相似的结构和生理功能, 对昆虫的发育、 免疫、 应激反应等方面都起着重要的调控作用。有研究报道, 作为ENF肽家族成员之一的家蚕Bombyx mori麻痹肽（paralytic peptide, PP）通过激活MAPK来调控家蚕的免疫应答, 但其完整的分子作用机制尚不明确。本研究旨在解析传递家蚕PP免疫诱导信号的分子通路。【方法】首先通过荧光定量PCR检测了多种昆虫细胞系在PP诱导下抗菌肽基因的转录水平, 并利用Western blot检测p38 MAPK的磷酸化水平, 然后利用不同信号传递分子的抑制剂处理BmE细胞筛选参与传递PP免疫诱导信号的分子。【结果】PI3K抑制剂LY294002和受体型酪酸激酶抑制剂Genistein抑制了PP对BmE细胞抗菌肽基因表达的诱导作用; 且BmE细胞和家蚕血细胞在PP的诱导下, Akt和大小约为70 kDa的细胞膜蛋白均出现磷酸化水平的动态变化。【结论】PI3K/Akt信号通路和Genistein敏感性的受体型酪氨酸激酶介导了PP免疫诱导信号的传递。     

Molecular cloning of silkworm paralytic peptide and its developmental regulation

The silkworm paralytic peptide (PP) is a member of the ENF peptide family that exerts multiple biological activities involved in defense reaction and growth regulation. We isolated its cDNA and examined mRNA expression profiles. cDNA encoded 131 amino acids from which the 23-residue PP sequence was found at the C-terminal portion. Immunoblot analysis and paralytic activity assay indicated that inactive pro-protein in larval hemolymph was processed into active peptide immediately after bleeding. In the last larval instar, 0.6-kb PP mRNA was expressed in various tissues, of which the fat body was predominant. Its expression in the fat body decreased during the feeding period and then increased during metamorphic process. Juvenile hormone and 20-hydroxyecdysone upregulated its expression. At the embryonic stage, 1.5-kb mRNA, in addition to 0.6-kb mRNA, was expressed from 1 day after oviposition to hatching. PP was thus expressed stage-specifically under hormonal control.     

Monoclonal antibodies bind distinct classes of hemocytes in the moth Pseudoplusia includens

Insect hemocytes have historically been identified on the basis of morphology, ultrastructure and hypothesized function. Among insects in the order Lepidoptera, five hemocyte classes are usually recognized: granular cells, plasmatocytes, spherule cells, oenocytoids and prohemocytes. We have generated a panel of monoclonal antibodies (mAbs) against hemocytes of the moth Pseudoplusia includens. In this study, hemocyte identification using 16 different mAbs was compared to identification methods using morphological characters. Three main categories of mAb binding activity were identified: (1) mAbs that specifically labeled only one morphological class of hemocytes, (2) mAbs that labeled granular cells and spherule cells, and (3) mAbs that labeled plasmatocytes and oenocytoids. With one exception, none of the antibodies bound to other tissues in P. includens. However, certain mAbs that specifically labeled granular cells and/or spherule cells in separated hemocyte populations also labeled plasmatocytes co-cultured with granular cells or cultured in granular cell conditioned medium. Overall, our results suggest that granular cells are antigenically related to spherule cells, and that plasmatocytes are antigenically related to oenocytoids. The use of mAbs as hemocyte markers are discussed.     

Parasitism by Cotesia plutellae alters the hemocyte population and immunological function of the diamondback moth, Plutella xylostella

Cotesia plutellae, a solitary endoparasitoid wasp, parasitizes the diamondback moth, Plutella xylostella, and induces host immunosuppression and lethality in the late larval stage. This study focused on changes of cellular immunity in the parasitized P. xylostella in terms of hemocyte composition and cellular functions. In third and fourth instar larvae of nonparasitized P. xylostella, granular cells represented the main hemocyte type (60-70%) and plasmatocytes were also present at around 15% among the total hemocytes. Following parasitization by C. plutellae, the relative proportions of these two major hemocytes changed very little, but the total hemocyte counts exhibited a significant reduction. Functionally, the granular cells played a significant role in phagocytosis based on a fluorescence assay using fluorecein isothiocyanate-labeled bacteria. The phagocytic activity of the granular cells occurred as early as 5 min after incubation with the bacteria, and increased during the first 40 min of incubation. The parasitism by C. plutellae significantly inhibited phagocytosis of the granular cells. Plasmatocytes also exhibited minor phagocytic activity. Moreover, plasmatocyte phagocytosis was not inhibited by parasitism. On the other hand, hemocyte-spreading behavior in response to pathogen infection was significant only for plasmatocytes, which exhibited a characteristic spindle shape upon infection. A significant spreading of the plasmatocytes was found as early as 5 min after pathogen incubation and their ratio increased during the first 40 min.An insect cytokine, plasmatocyte-spreading peptide 1 (PSP1) from Pseudoplusia includens, was highly active in inducing plasmatocyte-spreading behavior of P. xylostella in a dose-dependent manner. P. xylostella parasitized by C. plutella was significantly inhibited in plasmatocyte-spreading in response to an active dose of PSP1. An in vivo encapsulation assay showed that the parasitized P. xylostella could not effectively form the hemocyte capsules around injected agarose beads. This research demonstrates that the parasitism of C. plutellae adversely affects the total hemocyte populations in number and function, which would contribute to host immunosuppression.     

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 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.     

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.     

Morphological abnormalities and lethality in silkworm (Bombyx mori) larvae treated with high concentrations of insect growth-blocking peptide

Insect growth-blocking peptides (GBPs) exhibit growth-blocking and paralytic activity. Low concentrations of GBP stimulate larval growth, whereas high concentrations of GBP significantly retard larval growth. Here, we show that morphological abnormalities and lethality were induced in silkworm (Bombyx mori) larvae by high concentrations of GBP. Active B. mori GBP (BmGBP) was produced by treating recombinant proBmGBP (expressed in baculovirus-infected insect cells) with bovine factor Xa. When silkworm larvae on day 1 of the fifth-instar stage were injected between the seventh and eight abdominal segments with BmGBP (100 or 500 ng/larva), the larval–pupal and pupal–adult transformations of these silkworms were delayed in a dose-dependent manner. However, a high concentration (2000 ng/larva) of BmGBP or Spodoptera exigua GBP (SeGBP) acutely induced morphological abnormalities and death in silkworm larvae. In silkworm larvae treated with high concentrations of GBPs, the ingested food excessively accumulated in the foregut, which caused extreme swelling in both the thorax and the foregut and resulted in larval death. Therefore, these results not only provide insight into the effect of insect GBPs on gut physiology but also reveal a novel function of insect GBPs.     

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.     

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.     

Immunological, biochemical and physiological analyses of cardioacceleratory peptide 2 (CAP2) activity in the embryo of the tobacco hawkmoth Manduca sexta

The cells in the embryonic CNS of the tobacco hawkmoth, Manduca sexta, that synthesize a cardioacceleratory peptide 2 (CAP2)-like antigen were identified using immunohistochemical techniques. Two distinct neurosecretory cell types were present in the abdominal ventral nerve cord (VNC) that contain CAP2-like immunoreactivity during late embryogenesis: a pair of large (diameter range 15-20 microns) cells lying along the posterior, dorsal midline of abdominal ganglia A4-A8, and a bilateral set of four smaller (diameter range 6-11 microns) neurons which lie at the base of each ventral root in abdominal ganglia A2-A8. CAP2-like accumulation appeared to follow independent patterns in the two cell types. CAP2-like immunoreactivity began at 60% of embryo development (DT) in the medial cells, accumulated steadily throughout embryogenesis, and dropped markedly during hatching. Lateral cells synthesized the CAP2-like antigen later in development (70% DT) and showed a sharp drop in antigen levels between 75% and 80% of embryonic development. Extracts from developing M. sexta embryos were found to contain a cardioactive factor capable of accelerating the contraction frequency of the pharate adult moth heart in a fashion similar to CAP2. Immunoprecipitation with a monoclonal antibody that specifically recognizes the two endogenous Manduca cardioacceleratory peptides and purification using high pressure liquid chromatography identified this factor as cardioacceleratory peptide 2 (CAP2). Using an in vitro heart bioassay, the levels of this cardioactive neuropeptide were traced during the development of the M. sexta embryo. As with the immunohistochemical results, two periods during embryogenesis were identified in which the level of CAP2 dropped markedly: between 75% and 80% development, and at hatching. Embryo bioassays of CAP2 activity were used to identify possible target tissues for physiological activity during these two putative release times. CAP2 was found to accelerate contraction frequency in the embryonic heart and hindgut of Manduca in a dose-dependent fashion. Of these two possible targets, the hindgut proved to be more sensitive to CAP2, having a lower response threshold and a longer duration of response to a given concentration of the exogenously applied peptide. Based on these immunocytochemical, pharmacological and biochemical results, and on a previously published detailed analysis of Manduca embryogenesis, we conclude that CAP2 is probably released from a specific set of identified neurosecretory cells in the abdominal VNC to modulate embryonic gut activity at 75-80% of embryo development during ingestion of the extra-embryonic yolk.     

Analysis of epitope structure of PSP94 (prostate secretory protein of 94 amino acids): (I) immuno-dominant and immuno-recessive area

PSP94 is a potential biomarker for evaluating patients with prostate carcinoma. We have systematically studied the epitope structure of PSP94 by using a polyclonal antibody against human PSP94. Results of peptide mapping and ELISA tests of dose response to rabbit antiserum against human PSP94 protein showed that only the N-terminal peptides (N30 and M23) are immunoreactive while all the synthetic peptides (C28, C10) located closer to the C-terminus are completely devoid of antigenic activity with the polyclonal antibody. These results were confirmed by analysis of reciprocal competitive binding of PSP94 polyclonal antibody by the N-terminal peptides (N30 and M23) v. either recombinant GST-PSP94 fusion protein, purified recombinant PSP94, or natural PSP94 protein. To further delineate the antigenic activity of the N- and C-termini, we have also expressed N- and C-terminal half of the whole PSP94 (each 47 peptides) using the E. coli GST expression system. The recombinant N47/C47 peptides were released by thrombin cleavage from the GST fusion protein and characterized by Western blotting experiments. Dose response of the recombinant GST-PSP-N47 and -C47 peptides to PSP94 polyclonal antibody showed differential binding activities. Competitive binding of these recombinant N47/C47 proteins against the GST-PSP94 protein demonstrates that the polyclonal antibody has a higher affinity for the N47 peptide than the C47 peptide. Based on the immunological studies of both synthetic peptides and recombinant PSP94- N/C terminal proteins, we propose an epitope structure of human PSP94 with an immno-dominant N-terminus and an immuno-recessive C-terminus. J. Cell. Biochem. 65:172–185. © 1997 Wiley-Liss, Inc.     

Isolation and identification of paralytic peptides from hemolymph of the lepidopteran insects Manduca sexta, Spodoptera exigua, and Heliothis virescens

Seven paralytic peptides were isolated and identified from lepidopteran hemolymph. All of these peptides cause rapid, rigid paralysis when injected into Manduca sexta and some other lepidopteran larvae. Each peptide contains 23 amino acid residues including 2 cysteines and the carboxyl termini are acidic. Synthetic peptides in the disulfide or reduced forms, and as carboxyl-terminal acids or amides were equally paralytic. The most potent paralytic peptide, Mas PP I, has the following sequence: H-Glu-Asn-Phe-Ala-Gly-Gly-Cys-Ala-Thr-Gly-Tyr-Leu- Arg-Thr-Ala-Asp-Gly-Arg-Cys-Lys-Pro-Thr-Phe-OH. The two peptides from M. sexta hemolymph are remarkable in that they are autoparalytic (i.e. factors in collected hemolymph that are paralytic when injected into the same larvae).     

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.