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Synthesis and biological activity of a photoaffinity-biotinylated pheromone-biosynthesis activating neuropeptide (PBAN) analog☆
Peptides 20 (1999) 787–794
Synthesis and biological activity of a photoaffinity-biotinylated pheromone-biosynthesis activating neuropeptide (PBAN) analog夞 Ada Rafaeli*, Carina Gileadi Department of Stored Products, Pheromone Research Laboratory, Volcani Center, Bet Dagan 50250, Israel Received 25 November 1998; accepted 22 February 1999
Abstract To study the mode of action of pheromone-biosynthesis activating neuropeptide (PBAN) at the receptor level and for receptor purification, we synthesized and tested the biologic properties of a photoaffinity biotinylated PBAN analog N-[N-(4-azido-tetrafluorobenzoyl)-biocytinyloxyl-succinimide (Atf-Bct-NHS-PBAN). The Atf-Bct-NHS-PBAN was separated from unreacted reagent and synthetic Hez-PBAN by high-performance liquid chromatography. Conjugated biotin was detected by using enzyme-linked assay as well as tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis. The biologic activity of purified Atf-Bct-NHS-PBAN was confirmed using both in vivo and in vitro pheromonotropic bioassays. These observations indicate that Atf-Bct-NHS-PBAN is a full agonist of PBAN action in pheromone glands and may be used to study PBAN receptors by employing avidin coupled to various reporter groups. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Pheromone biosynthesis; PBAN; Photoaffinity; Avidin-biotin interaction; Receptors
1. Introduction Female moths are attractive to males during specific periods when they emit their pheromone by extruding their ovipositor tips in a typical “calling” posture. The pheromonotropic activity of these females has been attributed to the regulation by a neurohormone that is produced in the brain-subesophageal ganglion (Br-SEG) complex [24]. This neurohormone, termed pheromone-biosynthesis activating neuropeptide (PBAN) is a 33 amino acid peptide that has been isolated and sequenced from three species of moths Helicoverpa zea [25], Bombyx mori (Bom-PBAN) [12] and Lymantria dispar (Lym-PBAN) [16]. Whether the action of PBAN is the sole mechanism creating pheromonotropic activity is controversial. On the one hand, there is evidence regarding its direct action in the induction of pheromone biosynthesis. PBAN has been
夞 This research was supported by the Israel Academy of Sciences & Humanities grant to A.R. and is a contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel, No. 440-98 series. * Corresponding author. Tel.: ⫹1-972-3-9683729; fax: ⫹1-972-39604428. E-mail address: [email protected] (A. Rafaeli)
shown to stimulate isolated ovipositor tips in vitro in several different species of moths [1,8,11,19,28]. Moreover, we have delineated the exact target tissue by demonstrating that PBAN stimulated isolated intersegmental membranes that are situated between the 8th and 9th abdominal segments of the ovipositor tips of H. armigera females to produce the main pheromone component, Z-11 hexadecenal [20,21]. In addition, research concerning the cellular events in the pheromone glandular tissue, that occur as a result of PBAN stimulation, have revealed the involvement of cAMP [28,29] and calcium [11] as second messengers. Further evidence for a direct hormonal action of PBAN on the pheromone glands has also become apparent in the identification of the circulating hormone and its bioactivity in the hemolymph during active reproductive periods [9,14,26]. On the other hand, it has been proposed that the innervation of the pheromone gland is crucial for pheromonotropic activity and that the biogenic amine, octopamine, may be involved in the stimulation of sex pheromone production in Lepidoptera [3–5]. Axons originating from the PBAN producing SEG cells were shown to travel to the neurohemal organs, the corpora cardiaca, as well as the entire length of the ventral nerve cord, indicating that some of these peptides may travel through these axons [15].
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Ventral nerve cord stimulation and injections of octopamine to mid-photophase females resulted in the stimulation of pheromone biosynthesis [4]. These workers also showed daily fluctuations in octopamine levels in the pheromone gland, parallel to the daily fluctuations of pheromone production, and suggested that octopamine was involved in the regulation of pheromone production [5]. They concluded that stimulation of pheromone biosynthesis occurs by both neural and hormonal mechanisms that depend on photoperiodic cues. Conclusive proof of the direct action of PBAN on the intersegmental membrane that is responsible for pheromone biosynthesis awaits the demonstration of surface receptors on the putative target tissue/s. The study of cell surface receptors including their surface distribution, internalization, intracellular compartmentation and recycling depends on the ability to visualize and localize them in a specific manner. Biotin-hormone conjugates are uniquely suited for this purpose as avidin conjugates can be readily coupled to the biotinylated hormone [31]. In addition, avidin affinity columns can be employed to purify receptors bound to the biotinylated ligands. Photoaffinity labeling offers an additional valuable technique to ensure strong cross-linking to receptor molecules. This method relies on a physiologically active analog of a natural hormone to bind to a specific binding site that is then covalently attached to the active site by selective irradiation of a chromophore with latent chemical reactivity [18]. The use of photogenerated species to study the interaction between ligands and biologic macromolecules, particularly proteins and nucleic acids, has become a widely used technique [2,6]. A number of biotinylated vertebrate peptide hormones have been prepared successfully [13,17,32]. However, to our knowledge, the preparation of a biotinylated-photoaffinity labeled analog by using an invertebrate peptide hormone has not been reported. The preparation of such an analog will allow us to accomplish some of the above-mentioned goals. In this report, we describe the synthesis and characterization of a labeled neuropeptide analog for Hez-PBAN that was shown, in preliminary studies [23], to bind through photocrosslinking to a protein in the intersegmental tissue of ovipositor tips.
2. Materials and methods 2.1. Insect culture The study was conducted on Helicoverpa armigera (Lepidoptera:Noctuidae). The larvae were raised on an artificial diet at a constant temperature of 26°C and 14:10 (light:dark) photoperiod as reported previously [28]. Pupae were sexed and males and females were allowed to emerge separately.
2.2. Materials Synthetic Hez-PBAN was purchased from Peninsula Lab. (Belmont, CA) and dissolved in 50% methanol containing 0.01 M HCl as a 100 pmol/l solution. This solution, kept frozen at ⫺20°C, maintained bioactivity for 6 months. For labeling purposes PBAN was dissolved in 50% methanol in the absence of HCl. N-[N-(4-azido-tetrafluorobenzoyl)-biocytinyloxyl-succinimide (Atf-Bct-NHS) was purchased from Boehringer Mannheim GmbH (Vienna, Austria). High-performance liquid chromatography (HPLC) grade acetonitrile was purchased from Carlo Erba Reagenti (Milan, Italy). 3,3⬘,5,5⬘-Tetramethyl benzidine (TMB) and ImmunoPure™ Avidin-HRP conjugate were purchased from Pierce (Rockford, IL, USA). Molecular weight standards were purchased from Bio-Rad (Hercules, CA, USA). Antiserum to Hez-PBAN was obtained as a gift from Dr Ashok Raina (USDA, Weslaco, TX, USA). All other chemicals were purchased from Sigma (St. Louis, MO, USA). Before use for bioassay, chemicals were diluted to the final concentration in Pipes buffered incubation medium, pH 6.6, modified from Jurenka et al. [11]. 2.3. Synthesis and purification of Atf-Bct-NHS-PBAN analog Synthetic Hez-PBAN was dissolved in 50% methanol to a concentration of 100 pmol/l. A sample (equivalent to 20.4 g) was dried down and dissolved in 15l dimethyl formamide (DMF) in a solution of borate buffer (1 M), pH 8.5. Atf-Bct-NHS was weighed in dimmed light and dissolved in DMF to a final solution of 1 mg/100 l. The Atf-Bct-NHS reagent (4 l) was added to the solution of Hez-PBAN and the reaction was allowed to proceed at 4°C overnight. A 2.5-l sample was removed and the reaction in this sample was stopped with 0.5 l of glycine (1 M) and 10 l of borate buffer (0.1 M), pH 8.5. The photoaffinitybiotinylated labeled PBAN was purified by HPLC with a Shimadzu LC-10AT gradient pump; Shimadzu GT-104 degasser; Tracor 970A UV/Vis detector and a Vydac RP-C4 column run at a shallow gradient from 20% acetonitrile in 0.1% trifluoroacetic acid to 30% acetonitrile in 0.1% trifluoroacetic acid at 0.1%/min. The flow rate was maintained at 0.5 ml/min. To ensure that separation of reacted PBAN from synthetic Hez-PBAN was successfully accomplished the sample was spiked with 5 g of Hez-PBAN. The eluant was monitored at absorbance of 220 nm for an elution profile and compared to a control run containing unreacted reagent, glycine and a Hez-PBAN spike. One milliliter fractions were collected every 2 min. Aliquots of the fractions were dried and used for biotin detection and for bioassay. The remainder of the reaction mixture was purified using the above conditions but omitting both ultraviolet detection and spiking with native peptide. The resultant fractions were therefore available for future testing.
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samples were run at the same voltage for another 60 min. The gels were transferred onto nitrocellulose membranes and the Western blots were analyzed by chemiluminescence by using Supersignal™ Substrate (Pierce) or by immunoblotting technique [10] by using PBAN antiserum (at 1:5 000 dilution). Molecular weight was estimated by using kaleidoscope polypeptide standards (range 4000 –39 800 Da).
Fig. 1. Enzyme-linked standard test for detection of Aft-Bct-NHS. Points were tested in triplicates, bars show the extent of SEM (in some treatments SEM is smaller than the point).
2.4. Detection of biotinylated reagent by using enzymelinked assay The Atf-Bct-NHS reagent was initially used by itself to test detection by using streptavidin-horseradish peroxidase conjugate. An example of such a standard assay is depicted in Fig. 1. Various concentrations of the reagent and aliquots of fractions from the HPLC purification were coated on micro-test plates, incubated in the dark for 3 h at room temperature by using a shaker. The samples were subsequently exposed to ultraviolet radiation (254 nm) (S-68 Mineralight™ Lamp, Ultra-Violet Products, San Gabriel, CA, USA) for 30 min. The contents were then discarded and the wells were washed in phosphate-buffered saline ⫹ 0.05% Tween 20. Blocking buffer was added by using phosphate-buffered saline ⫹ 1% bovine serum albumin. After washing, the plate was reacted with streptavidin-enzyme conjugate in phosphate-buffered saline ⫹ 0.1% bovine serum albumin for 1 h at room temperature. After further washing, TMB substrate was added. The reaction was stopped using 1 M H3PO4. The resultant product was quantified by using a plate-reader (Bio-Tek Instruments ELx800) at 450 nm. 2.5. Detection of Atf-Bct-NHS-PBAN analog by tricine sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) For the separation of Atf-Bct-NHS-PBAN by using tricine SDS-PAGE, the method of Schagger and Von Jagow [27] was modified for mini-gel electrophoresis by using a Bio-Rad 1000/500 Power supply. This technique was used successfully for PBAN-like peptides [10]. The method is based on a discontinuous SDS-PAGE system by using tricine as trailing ion for the separation of small proteins. The modified gel contained 4.5 cm of 16.5% acrylamide separating gel, 1 cm of 10% acrylamide spacer gel and 1 cm of 4% acrylamide stacking gel. Samples were loaded into wells onto the 4% gel at 30V for 20 min. The voltage was subsequently adjusted to 95V at which the samples reached the interface between 10 –16.5% after another 20 min. The
2.6. Pheromonotropic activity of Atf-Bct-NHS-PBAN analog by intersegmental membrane preparations (in vitro bioassay) We used the radiochemical bioassay as developed by us to monitor de novo pheromone production [28]. Ovipositor tips, consisting of the eighth and ninth abdominal segments with the attached intersegmental membrane, were removed during the 10 –12th h photophase from 2-day-old females. Clearing of internal tissues was performed by transecting the abdominal tips along the dorsal side and removing all internal tissues. The abdominal tips were further transected vertically between the intersegment and the 8th segment such that the intersegmental membrane tissue could be incubated separately. The tissues were washed twice in Pipes buffered incubation medium. After preincubation in normal saline for 30 min, the intersegments were dried lightly on tissue paper and then transferred individually to 10 l of incubation medium containing 0.25 Ci [1-14C]-acetate in the presence or absence of Hez-PBAN (0.05 pmol/female) or purified Atf-Bct-NHS-PBAN analog at various concentrations. All incubations for pheromone production were performed for 3 h at room temperature. To measure the incorporation of [1-14C]-acetate into lipids the glands were extracted in hexane and the amount of radioactivity was measured using a liquid scintillation counter (Packard TriCarb 4530) as reported previously [28,29]. Based on thinlayer chromatography and GC separations, we have previously shown that the total incorporation levels depict relative levels of incorporation into the pheromone component where the majority of the label was found to co-elute with the main pheromone component of H. armigera, (Z)-11 hexadecenal [20]. 2.7. Pheromonotropic activity of Atf-Bct-NHS-PBAN analog by intact and decapitated moths (in vivo bioassay) Female moths (2 day old) were injected between the 4th and 5th abdominal segments during the 11th h photophase, by using a Hamilton syringe, with 10 l of physiological saline containing Hez-PBAN (1 pmol/female) or purified Atf-Bct-NHS-PBAN analog (1 l eluant/female evaporated to near dryness). Similarly, moths that were decapitated for 24 h were injected as described above. After 1 h, the ovipositor tips were excised and extracted in hexane containing internal standard (tridecanyl acetate) for 15 min at room temperature. The extract was subsequently analyzed
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Fig. 2. Solubility of Hez-PBAN in DMF and the effect of DMF in the in vitro bioassay using isolated intersegmental membranes. PBAN (0.05 pmol/intersegment) was dissolved in DMF, dried down and reconstituted in physiological medium and the solution was subsequently tested for pheromonotropic activity (PBAN in DMF). No residual activity was found (vial rinse). DMF (1%) in physiological medium does not affect pheromonotropic activity (PBAN ⫹ DMF). Histograms represent means of nine replicates, bars show the extent of SEM.
on GC for (Z)-11 hexadecenal production under the conditions described previously [28].
3. Results 3.1. Solubility of PBAN in DMF and bioactivity in the in vitro bioassay Because the reagent is soluble in DMF we first tested the solubility of PBAN in DMF. An aliquot of stock PBAN (dissolved in 50% methanol and 0.01 M HCl) was dried down under a stream of N2. It was subsequently re-dissolved in DMF and an aliquot was dried down and dissolved in physiological medium for bioassay. The medium was aliquoted and the vial was rinsed in physiological medium. The rinse was used as a test for residual undissolved PBAN. Other treatments included the addition of 1% DMF into the physiological medium; a positive PBAN control and a control for basal levels of incorporation into pheromone (Fig. 2). PBAN was found to be soluble in DMF because no loss in bio-activity was observed when tested on intersegments (Fig. 2, PBAN in DMF) and the control rinsed vial did not contain any residual activity (Fig. 2, Vial rinse). In addition DMF, present at 1% in the incubation medium, did not affect the stimulatory response to PBAN (Fig. 2, PBAN ⫹ 1% DMF).
corresponding to Hez-PBAN (18.6 min) and another corresponding to residual reagent that was unreacted with glycine at 24.7 min (Fig. 3, lower profile). Analysis of the eluants for biotin levels also revealed a main peak of activity (Fig. 4, lower histogram) that corresponded to the retention time of Atf-Bct-NHS-PBAN observed under ultraviolet detection (33.5 min). Some biotin was detected in the void volume corresponding to glycinereacted reagent and in the residual reagent peak that appeared at 24.7 min. The eluants were also screened for bioactivity by using
3.2. HPLC Purification and identification of Atf-Bct-NHSPBAN analog HPLC purification of Atf-Bct-NHS-PBAN revealed the appearance of a single peak at retention time of 33.5 min (Fig. 3, upper profile). This peak was separated from HezPBAN that appeared at 18.6 min. A control run with a sample of reagent reacted with glycine alone and spiked with Hez-PBAN before injection revealed two peaks: one
Fig. 3. Typical elution profile of the separation of Atf-Bct-NHS-PBAN analog on a RP-Vydac C4 column at 20 –30% acetonitrile ⫹ 0.1% trifluoroacetic acid mobile phase, at a rate of 0.1%/min showing the appearance of a single peak at 33.5 min retention time corresponding to Atf-Bct-NHSPBAN analog (upper profile). Superimposed (lower profile) is an elution profile containing unreacted reagent and a spike of native Hez-PBAN).
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Fig. 4. Screening of bioactivity using the in vitro bioassay (above) and biotin levels (below) in 1-l and 10-l aliquots, respectively, of fractions from HPLC separation of the Atf-Bct-NHS-PBAN analog. Histograms represent means of triplicates for bioactivity and duplicates for biotin level determinations, bars show extent of SEM.
the in vitro bioassay for pheromone production by intersegmental membranes (Fig. 4, upper histogram). One major peak of activity (retention time 32–34 min) that corresponded both to the retention time of the biotinylated peak (retention time 32–34 min) and the peak observed under UV detection (33.5 min) was identified. Another activity peak corresponding to native Hez-PBAN (18.6 min) was also detected at retention time of 18 –20 min. A third activity peak at 40 – 42 min was also observed, however, it was not highly biotinylated. 3.3. Dose-response activity relationship of Atf-Bct-NHSPBAN analog Various doses of the HPLC-purified eluants containing the Atf-Bct-NHS-PBAN (at 32 min and 34 min) were compared to the dose-response relationship found by HezPBAN (Fig. 5) using the in vitro bioassay. It was found that the Atf-Bct-NHS-PBAN analog was highly active with dose-response saturation observed at 0.05 l eluant volume. From calculations, based on this dose-response activity re-
lationship, we estimate that a total of 14.7 g of Atf-BctNHS-PBAN analog was successfully biotinylated. 3.4. Identification of Atf-Bct-NHS-PBAN analog by tricine SDS-PAGE Samples of the active peak (corresponding to elution time 28 –36) and a control fraction at elution time of 20 min were subjected to tricine SDS-PAGE and Western blotting using chemiluminescence for detection. Some Western blots, containing a sample of native PBAN, were also detected by using antiserum to PBAN. Typical blots are depicted in Fig. 6A and B. It can be observed that fractions corresponding to elution time 30, 32, 34, and 36 contain biotin, fractions 32 and 34 corresponding to a strongly reactive biotinylated protein. This protein was not present in the fraction corresponding to native Hez-PBAN (20 min). The protein was separated in the 16.5% acrylamide gel section corresponding to a small protein in the range of 4 – 8.2 kDa. Reagent alone did not give a signal at this position (data not shown). The Western blot, reacted with PBAN antiserum (Fig. 6B) shows the position of native
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Fig. 5. Dose-response curves of Hez-PBAN, and active HPLC fractions by using the in vitro bioassay. Sample volume used is depicted as second x-axis. Points represent means of 7 replicates, bars show the extent of SEM.
3.5. Pheromonotropic activity of Atf-Bct-NHS-PBAN analog on intact female moths (in vivo bioassay)
intact female moths during the photophase at 1 l of eluant/ female significantly stimulated pheromone titers above saline control levels (P ⬍ 0.05) (Fig. 7). No significant difference was observed between the responses of intact and decapitated females to Atf-Bct-NHS-PBAN analog.
Both fractions 32 and 34, corresponding to Atf-BctNHS-PBAN analog, when injected into decapitated and
4. Discussion
Fig. 6. Western blot showing a separation of Atf-Bct-NHS-PBAN analog on a Tricine SDS-PAGE by using a discontinuous system: 16.5% acrylamide gel overlaid by a 10% spacer gel and 4% stacking gel, showing strong biotin label in fractions 32 and 34. (A) Transfer analyzed by chemiluminescence. (B) Transfer analyzed by chemiluminescence and immunoblotting (PBAN antiserum). PB ⫽ 5 pmol native Hez-PBAN. Molecular weights were marked using kaleidoscope polypeptide standards.
Much emphasis has been devoted to the study of the structure and function of peptide hormones in insects and other invertebrates however, there are very few reports directed at the characterization of peptide receptors in these organisms. The use of photoaffinity labeled analogs has been successful for the characterization of some soluble binding proteins [7,18], however, it has been of limited success in the quest for determining the character and structure of membrane-bound receptor proteins that may be much less abundant. Three stages are necessary in the synthesis of a photoaffinity-tagged analog with the aim at characterizing a receptor: 1) The design of a new chemical for the photoaffinity analog that will include a photochemically reactive functional group as well as a tag that may be detected by using a sensitive assay; 2) the demonstration that this analog is physiologically active both when tested in an in vitro and an in vivo system. Analogs, with activity that are not of physiological significance, are not useful because they may label proteins nonspecifically and thereby identify
PBAN that approximates in molecular size to Atf-Bct-NHSPBAN analog.
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Fig. 7. Stimulation of pheromone production in intact and decapitated moths (in vivo bioassay) by Atf-Bct-NHS-PBAN (1 l and 2 l eluant/female) as compared to the response to Hez-PBAN (1 pmol/female). Histograms represent the means of five replicates, bars indicate the extent of standard error. Different letters denote a significant difference (P ⬍ 0.05; analysis of variances followed by Fisher’s protected least squares difference test).
proteins that are not involved in the molecular action of the native hormone; and 3) The demonstration that this protein is ligand-specific, confirmed by competitive displacement in the presence of an excess of native ligand. In this study, we demonstrated in detail two of the essential stages in the synthesis of a photoaffinity analog (Atf-Bct-NHS-PBAN) that could be utilized in receptor characterization studies. A photochemically reactive functional group was successfully tagged onto Hez-PBAN. This was shown to be fully active in an in vitro bioassay as well as when injected to intact or decapitated females in vivo thereby demonstrating a physiological functional analog. Its molecular weight was confirmed to be in the region of 4 kDa, corresponding to the molecular weight expected of a PBAN analog. It should be noted that determination or estimation of the molecular weights of small peptides using tricine SDS-PAGE is not accurate due to an intrinsic charge and shape of small peptides that are relatively more important in determining their mobility on SDS gels than large proteins [30]. However, by comparing the mobility of the Atf-Bct-NHS-PBAN analog to native PBAN by using immunoblotting technique, a more accurate estimation could be made. Preliminary studies [23], using this Atf-Bct-NHS-PBAN analog, showed evidence for specific competitive labeling of a protein at the 50-kDa range in the intersegmental tissue of the pheromone glands. This protein was successfully displaced in the presence of a saturation level (⫻250) of synthetic Hez-PBAN, thereby demonstrating stage 3 of the requirements mentioned above. By incorporating avidin-biotin technology we have been successful in obtaining a highly biologically active analog that can amplify a relatively small abundance of receptors to a detectable level. This analog will be useful for the characterization and subsequent sequence determination of the receptor for PBAN. Moreover, by coupling of avidin-gold to this biotinylated hormone analog, it may also prove useful in
the study of the surface distribution, internalization, intracellular compartmentation and recycling of the PBAN receptor. Because moths constitute the major group of pest insects in agriculture and are therefore of economic importance, a great deal of effort has gone into the identification of their pheromones. Whereas the components of pheromone blends from a variety of moth species have been identified, pheromone analogs have had limited success in controlling pest populations. The biosynthesis and release of sex pheromone is necessary to attract a mate, therefore the interruption of this process is a potential strategy for manipulating adult moth behavior. Crucial to understanding the primary mode of action of any hormone is the identification and characterization of its receptor or receptors that constitute a key link in the signal transducing mechanism. Therefore the rationale in pursuing these studies not only lies in the advancement of basic scientific knowledge on comparative biochemistry but also specifically in the potential for finding ways of manipulating the reproductive behavior of pest moths. The development of specific and safe insect control strategies utilizing pheromone systems depends on a clear knowledge of the molecular mechanisms involved. The sequences of these receptors and subsequent study of their binding domains will provide future templates for the design of effective and specific antagonists. Furthermore, because PBAN belongs to a family of insect neuropeptides with more than one function in different life stages, this rationale can be extended to other physiological processes in different insects.
Acknowledgments We thank Mina Litvin for her dedication to maintaining a healthy insect culture.
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[16] Masler EP, Raina AK, Wagner RW, Kochansky JP. Isolation and identification of a pheromonotropic neuropeptide from the brainsuboesophageal ganglion complex of Lymantria dispar: a new member of the PBAN family. Insect Biochem Mol Biol 1994;24:829 –36. [17] Niendorf A, Dietel M, Arps H, Lloyd J, Childs GV. Visualization of binding sites for bovine parathyroid hormone (PTH 1– 84) on cultured kidney cells with a biotinyl-b-PTH (1– 84) antagonist. J Histochem Cytochem 1986;34:357– 61. [18] Prestwich GD. Photoaffinity labeling and biochemical characterization of binding proteins for pheromones, juvenile hormones and peptides. Insect Biochem 1991;21:27– 40. [19] Rafaeli A, Soroker V, Kamensky B, Raina AK. Action of PBAN on in vitro pheromone glands of Heliothis armigera females. J Insect Physiol 1990;36:641– 6. [20] Rafaeli A, Gileadi C. Modulation of the PBAN-induced pheromonotropic activity in Helicoverpa armigera. Insect Biochem Mol Biol 1995;25:827–34. [21] Rafaeli A, Gileadi C. Down regulation of pheromone biosynthesis: cellular mechanisms of pheromonostatic responses. Insect Biochem Mol Biol 1996;26:797– 807. [22] Rafaeli A, Soroker V. Cyclic-AMP mediation of the hormonal stimulation of 14C-acetate incorporation by Heliothis armigera pheromone glands in vitro. Mol Cell Endocrinol 1989;65:43– 8. [23] Rafaeli A, Gileadi C. Neuroendocrine control of pheromone production in moths. Invertebrate Neuroscience 1997;3:223–9. [24] Raina AK. Neuroendocrine control of sex pheromone biosynthesis in Lepidoptera. Ann Rev Entomol 1993;38:320 – 49. [25] Raina AK, Jaffe H, Kempe TG, Keim P, Blacher RW, Fales HM, Riley CT, Klun JA, Ridgway R, Hayes DK. Identification of a neuropeptide hormone that regulates sex pheromone production in female moths. Science 1989;244:796 – 8. [26] Ramaswamy SB, Jurenka RA, Linn CH Jr, Roelofs W. Evidence for the presence of a pheromonotropic factor in hemolymph and regulation of sex pheromone production. J Insect Physiol 1995;41:501– 8. [27] Schagger H, Von Jagow G. Tricine-SDS-PAGE for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 1987;166: 368 –79. [28] Soroker V, Rafaeli A. In vitro hormonal stimulation of acetate incorporation by Heliothis armigera pheromone glands. Insect Biochem 1989;67:1–5. [29] Soroker V, Rafaeli A. Multi-signal transduction of the pheromonotropic response by pheromone gland incubations of Helicoverpa armigera. Insect Biochem Mol Biol 1995;25:1–9. [30] Swank RT, Munkres KD. Molecular weight analysis of oligopeptides by electrophoresis in polyacrylamide gel with SDS. Anal Biochem 1971;39:462–77. [31] Wilchek M, Bayer ED. The avidin-biotin complex in bioanalytical applications. Anal Biochem 1988;171:1–32. [32] Westlund KN, Wynn PC, Chmielowiec S, Collins TJ, Childs GV. Characterization of a potent biotin-conjugated CRF analog and the response of anterior pituitary corticotropes. Peptides 1984;5:627–34.
Synthesis and biological activity of a photoaffinity-biotinylated pheromone-biosynthesis activating neuropeptide (PBAN) analog夞 Ada Rafaeli*, Carina Gileadi Department of Stored Products, Pheromone Research Laboratory, Volcani Center, Bet Dagan 50250, Israel Received 25 November 1998; accepted 22 February 1999
Abstract To study the mode of action of pheromone-biosynthesis activating neuropeptide (PBAN) at the receptor level and for receptor purification, we synthesized and tested the biologic properties of a photoaffinity biotinylated PBAN analog N-[N-(4-azido-tetrafluorobenzoyl)-biocytinyloxyl-succinimide (Atf-Bct-NHS-PBAN). The Atf-Bct-NHS-PBAN was separated from unreacted reagent and synthetic Hez-PBAN by high-performance liquid chromatography. Conjugated biotin was detected by using enzyme-linked assay as well as tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis. The biologic activity of purified Atf-Bct-NHS-PBAN was confirmed using both in vivo and in vitro pheromonotropic bioassays. These observations indicate that Atf-Bct-NHS-PBAN is a full agonist of PBAN action in pheromone glands and may be used to study PBAN receptors by employing avidin coupled to various reporter groups. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Pheromone biosynthesis; PBAN; Photoaffinity; Avidin-biotin interaction; Receptors
1. Introduction Female moths are attractive to males during specific periods when they emit their pheromone by extruding their ovipositor tips in a typical “calling” posture. The pheromonotropic activity of these females has been attributed to the regulation by a neurohormone that is produced in the brain-subesophageal ganglion (Br-SEG) complex [24]. This neurohormone, termed pheromone-biosynthesis activating neuropeptide (PBAN) is a 33 amino acid peptide that has been isolated and sequenced from three species of moths Helicoverpa zea [25], Bombyx mori (Bom-PBAN) [12] and Lymantria dispar (Lym-PBAN) [16]. Whether the action of PBAN is the sole mechanism creating pheromonotropic activity is controversial. On the one hand, there is evidence regarding its direct action in the induction of pheromone biosynthesis. PBAN has been
夞 This research was supported by the Israel Academy of Sciences & Humanities grant to A.R. and is a contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel, No. 440-98 series. * Corresponding author. Tel.: ⫹1-972-3-9683729; fax: ⫹1-972-39604428. E-mail address: [email protected] (A. Rafaeli)
shown to stimulate isolated ovipositor tips in vitro in several different species of moths [1,8,11,19,28]. Moreover, we have delineated the exact target tissue by demonstrating that PBAN stimulated isolated intersegmental membranes that are situated between the 8th and 9th abdominal segments of the ovipositor tips of H. armigera females to produce the main pheromone component, Z-11 hexadecenal [20,21]. In addition, research concerning the cellular events in the pheromone glandular tissue, that occur as a result of PBAN stimulation, have revealed the involvement of cAMP [28,29] and calcium [11] as second messengers. Further evidence for a direct hormonal action of PBAN on the pheromone glands has also become apparent in the identification of the circulating hormone and its bioactivity in the hemolymph during active reproductive periods [9,14,26]. On the other hand, it has been proposed that the innervation of the pheromone gland is crucial for pheromonotropic activity and that the biogenic amine, octopamine, may be involved in the stimulation of sex pheromone production in Lepidoptera [3–5]. Axons originating from the PBAN producing SEG cells were shown to travel to the neurohemal organs, the corpora cardiaca, as well as the entire length of the ventral nerve cord, indicating that some of these peptides may travel through these axons [15].
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Ventral nerve cord stimulation and injections of octopamine to mid-photophase females resulted in the stimulation of pheromone biosynthesis [4]. These workers also showed daily fluctuations in octopamine levels in the pheromone gland, parallel to the daily fluctuations of pheromone production, and suggested that octopamine was involved in the regulation of pheromone production [5]. They concluded that stimulation of pheromone biosynthesis occurs by both neural and hormonal mechanisms that depend on photoperiodic cues. Conclusive proof of the direct action of PBAN on the intersegmental membrane that is responsible for pheromone biosynthesis awaits the demonstration of surface receptors on the putative target tissue/s. The study of cell surface receptors including their surface distribution, internalization, intracellular compartmentation and recycling depends on the ability to visualize and localize them in a specific manner. Biotin-hormone conjugates are uniquely suited for this purpose as avidin conjugates can be readily coupled to the biotinylated hormone [31]. In addition, avidin affinity columns can be employed to purify receptors bound to the biotinylated ligands. Photoaffinity labeling offers an additional valuable technique to ensure strong cross-linking to receptor molecules. This method relies on a physiologically active analog of a natural hormone to bind to a specific binding site that is then covalently attached to the active site by selective irradiation of a chromophore with latent chemical reactivity [18]. The use of photogenerated species to study the interaction between ligands and biologic macromolecules, particularly proteins and nucleic acids, has become a widely used technique [2,6]. A number of biotinylated vertebrate peptide hormones have been prepared successfully [13,17,32]. However, to our knowledge, the preparation of a biotinylated-photoaffinity labeled analog by using an invertebrate peptide hormone has not been reported. The preparation of such an analog will allow us to accomplish some of the above-mentioned goals. In this report, we describe the synthesis and characterization of a labeled neuropeptide analog for Hez-PBAN that was shown, in preliminary studies [23], to bind through photocrosslinking to a protein in the intersegmental tissue of ovipositor tips.
2. Materials and methods 2.1. Insect culture The study was conducted on Helicoverpa armigera (Lepidoptera:Noctuidae). The larvae were raised on an artificial diet at a constant temperature of 26°C and 14:10 (light:dark) photoperiod as reported previously [28]. Pupae were sexed and males and females were allowed to emerge separately.
2.2. Materials Synthetic Hez-PBAN was purchased from Peninsula Lab. (Belmont, CA) and dissolved in 50% methanol containing 0.01 M HCl as a 100 pmol/l solution. This solution, kept frozen at ⫺20°C, maintained bioactivity for 6 months. For labeling purposes PBAN was dissolved in 50% methanol in the absence of HCl. N-[N-(4-azido-tetrafluorobenzoyl)-biocytinyloxyl-succinimide (Atf-Bct-NHS) was purchased from Boehringer Mannheim GmbH (Vienna, Austria). High-performance liquid chromatography (HPLC) grade acetonitrile was purchased from Carlo Erba Reagenti (Milan, Italy). 3,3⬘,5,5⬘-Tetramethyl benzidine (TMB) and ImmunoPure™ Avidin-HRP conjugate were purchased from Pierce (Rockford, IL, USA). Molecular weight standards were purchased from Bio-Rad (Hercules, CA, USA). Antiserum to Hez-PBAN was obtained as a gift from Dr Ashok Raina (USDA, Weslaco, TX, USA). All other chemicals were purchased from Sigma (St. Louis, MO, USA). Before use for bioassay, chemicals were diluted to the final concentration in Pipes buffered incubation medium, pH 6.6, modified from Jurenka et al. [11]. 2.3. Synthesis and purification of Atf-Bct-NHS-PBAN analog Synthetic Hez-PBAN was dissolved in 50% methanol to a concentration of 100 pmol/l. A sample (equivalent to 20.4 g) was dried down and dissolved in 15l dimethyl formamide (DMF) in a solution of borate buffer (1 M), pH 8.5. Atf-Bct-NHS was weighed in dimmed light and dissolved in DMF to a final solution of 1 mg/100 l. The Atf-Bct-NHS reagent (4 l) was added to the solution of Hez-PBAN and the reaction was allowed to proceed at 4°C overnight. A 2.5-l sample was removed and the reaction in this sample was stopped with 0.5 l of glycine (1 M) and 10 l of borate buffer (0.1 M), pH 8.5. The photoaffinitybiotinylated labeled PBAN was purified by HPLC with a Shimadzu LC-10AT gradient pump; Shimadzu GT-104 degasser; Tracor 970A UV/Vis detector and a Vydac RP-C4 column run at a shallow gradient from 20% acetonitrile in 0.1% trifluoroacetic acid to 30% acetonitrile in 0.1% trifluoroacetic acid at 0.1%/min. The flow rate was maintained at 0.5 ml/min. To ensure that separation of reacted PBAN from synthetic Hez-PBAN was successfully accomplished the sample was spiked with 5 g of Hez-PBAN. The eluant was monitored at absorbance of 220 nm for an elution profile and compared to a control run containing unreacted reagent, glycine and a Hez-PBAN spike. One milliliter fractions were collected every 2 min. Aliquots of the fractions were dried and used for biotin detection and for bioassay. The remainder of the reaction mixture was purified using the above conditions but omitting both ultraviolet detection and spiking with native peptide. The resultant fractions were therefore available for future testing.
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samples were run at the same voltage for another 60 min. The gels were transferred onto nitrocellulose membranes and the Western blots were analyzed by chemiluminescence by using Supersignal™ Substrate (Pierce) or by immunoblotting technique [10] by using PBAN antiserum (at 1:5 000 dilution). Molecular weight was estimated by using kaleidoscope polypeptide standards (range 4000 –39 800 Da).
Fig. 1. Enzyme-linked standard test for detection of Aft-Bct-NHS. Points were tested in triplicates, bars show the extent of SEM (in some treatments SEM is smaller than the point).
2.4. Detection of biotinylated reagent by using enzymelinked assay The Atf-Bct-NHS reagent was initially used by itself to test detection by using streptavidin-horseradish peroxidase conjugate. An example of such a standard assay is depicted in Fig. 1. Various concentrations of the reagent and aliquots of fractions from the HPLC purification were coated on micro-test plates, incubated in the dark for 3 h at room temperature by using a shaker. The samples were subsequently exposed to ultraviolet radiation (254 nm) (S-68 Mineralight™ Lamp, Ultra-Violet Products, San Gabriel, CA, USA) for 30 min. The contents were then discarded and the wells were washed in phosphate-buffered saline ⫹ 0.05% Tween 20. Blocking buffer was added by using phosphate-buffered saline ⫹ 1% bovine serum albumin. After washing, the plate was reacted with streptavidin-enzyme conjugate in phosphate-buffered saline ⫹ 0.1% bovine serum albumin for 1 h at room temperature. After further washing, TMB substrate was added. The reaction was stopped using 1 M H3PO4. The resultant product was quantified by using a plate-reader (Bio-Tek Instruments ELx800) at 450 nm. 2.5. Detection of Atf-Bct-NHS-PBAN analog by tricine sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) For the separation of Atf-Bct-NHS-PBAN by using tricine SDS-PAGE, the method of Schagger and Von Jagow [27] was modified for mini-gel electrophoresis by using a Bio-Rad 1000/500 Power supply. This technique was used successfully for PBAN-like peptides [10]. The method is based on a discontinuous SDS-PAGE system by using tricine as trailing ion for the separation of small proteins. The modified gel contained 4.5 cm of 16.5% acrylamide separating gel, 1 cm of 10% acrylamide spacer gel and 1 cm of 4% acrylamide stacking gel. Samples were loaded into wells onto the 4% gel at 30V for 20 min. The voltage was subsequently adjusted to 95V at which the samples reached the interface between 10 –16.5% after another 20 min. The
2.6. Pheromonotropic activity of Atf-Bct-NHS-PBAN analog by intersegmental membrane preparations (in vitro bioassay) We used the radiochemical bioassay as developed by us to monitor de novo pheromone production [28]. Ovipositor tips, consisting of the eighth and ninth abdominal segments with the attached intersegmental membrane, were removed during the 10 –12th h photophase from 2-day-old females. Clearing of internal tissues was performed by transecting the abdominal tips along the dorsal side and removing all internal tissues. The abdominal tips were further transected vertically between the intersegment and the 8th segment such that the intersegmental membrane tissue could be incubated separately. The tissues were washed twice in Pipes buffered incubation medium. After preincubation in normal saline for 30 min, the intersegments were dried lightly on tissue paper and then transferred individually to 10 l of incubation medium containing 0.25 Ci [1-14C]-acetate in the presence or absence of Hez-PBAN (0.05 pmol/female) or purified Atf-Bct-NHS-PBAN analog at various concentrations. All incubations for pheromone production were performed for 3 h at room temperature. To measure the incorporation of [1-14C]-acetate into lipids the glands were extracted in hexane and the amount of radioactivity was measured using a liquid scintillation counter (Packard TriCarb 4530) as reported previously [28,29]. Based on thinlayer chromatography and GC separations, we have previously shown that the total incorporation levels depict relative levels of incorporation into the pheromone component where the majority of the label was found to co-elute with the main pheromone component of H. armigera, (Z)-11 hexadecenal [20]. 2.7. Pheromonotropic activity of Atf-Bct-NHS-PBAN analog by intact and decapitated moths (in vivo bioassay) Female moths (2 day old) were injected between the 4th and 5th abdominal segments during the 11th h photophase, by using a Hamilton syringe, with 10 l of physiological saline containing Hez-PBAN (1 pmol/female) or purified Atf-Bct-NHS-PBAN analog (1 l eluant/female evaporated to near dryness). Similarly, moths that were decapitated for 24 h were injected as described above. After 1 h, the ovipositor tips were excised and extracted in hexane containing internal standard (tridecanyl acetate) for 15 min at room temperature. The extract was subsequently analyzed
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Fig. 2. Solubility of Hez-PBAN in DMF and the effect of DMF in the in vitro bioassay using isolated intersegmental membranes. PBAN (0.05 pmol/intersegment) was dissolved in DMF, dried down and reconstituted in physiological medium and the solution was subsequently tested for pheromonotropic activity (PBAN in DMF). No residual activity was found (vial rinse). DMF (1%) in physiological medium does not affect pheromonotropic activity (PBAN ⫹ DMF). Histograms represent means of nine replicates, bars show the extent of SEM.
on GC for (Z)-11 hexadecenal production under the conditions described previously [28].
3. Results 3.1. Solubility of PBAN in DMF and bioactivity in the in vitro bioassay Because the reagent is soluble in DMF we first tested the solubility of PBAN in DMF. An aliquot of stock PBAN (dissolved in 50% methanol and 0.01 M HCl) was dried down under a stream of N2. It was subsequently re-dissolved in DMF and an aliquot was dried down and dissolved in physiological medium for bioassay. The medium was aliquoted and the vial was rinsed in physiological medium. The rinse was used as a test for residual undissolved PBAN. Other treatments included the addition of 1% DMF into the physiological medium; a positive PBAN control and a control for basal levels of incorporation into pheromone (Fig. 2). PBAN was found to be soluble in DMF because no loss in bio-activity was observed when tested on intersegments (Fig. 2, PBAN in DMF) and the control rinsed vial did not contain any residual activity (Fig. 2, Vial rinse). In addition DMF, present at 1% in the incubation medium, did not affect the stimulatory response to PBAN (Fig. 2, PBAN ⫹ 1% DMF).
corresponding to Hez-PBAN (18.6 min) and another corresponding to residual reagent that was unreacted with glycine at 24.7 min (Fig. 3, lower profile). Analysis of the eluants for biotin levels also revealed a main peak of activity (Fig. 4, lower histogram) that corresponded to the retention time of Atf-Bct-NHS-PBAN observed under ultraviolet detection (33.5 min). Some biotin was detected in the void volume corresponding to glycinereacted reagent and in the residual reagent peak that appeared at 24.7 min. The eluants were also screened for bioactivity by using
3.2. HPLC Purification and identification of Atf-Bct-NHSPBAN analog HPLC purification of Atf-Bct-NHS-PBAN revealed the appearance of a single peak at retention time of 33.5 min (Fig. 3, upper profile). This peak was separated from HezPBAN that appeared at 18.6 min. A control run with a sample of reagent reacted with glycine alone and spiked with Hez-PBAN before injection revealed two peaks: one
Fig. 3. Typical elution profile of the separation of Atf-Bct-NHS-PBAN analog on a RP-Vydac C4 column at 20 –30% acetonitrile ⫹ 0.1% trifluoroacetic acid mobile phase, at a rate of 0.1%/min showing the appearance of a single peak at 33.5 min retention time corresponding to Atf-Bct-NHSPBAN analog (upper profile). Superimposed (lower profile) is an elution profile containing unreacted reagent and a spike of native Hez-PBAN).
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Fig. 4. Screening of bioactivity using the in vitro bioassay (above) and biotin levels (below) in 1-l and 10-l aliquots, respectively, of fractions from HPLC separation of the Atf-Bct-NHS-PBAN analog. Histograms represent means of triplicates for bioactivity and duplicates for biotin level determinations, bars show extent of SEM.
the in vitro bioassay for pheromone production by intersegmental membranes (Fig. 4, upper histogram). One major peak of activity (retention time 32–34 min) that corresponded both to the retention time of the biotinylated peak (retention time 32–34 min) and the peak observed under UV detection (33.5 min) was identified. Another activity peak corresponding to native Hez-PBAN (18.6 min) was also detected at retention time of 18 –20 min. A third activity peak at 40 – 42 min was also observed, however, it was not highly biotinylated. 3.3. Dose-response activity relationship of Atf-Bct-NHSPBAN analog Various doses of the HPLC-purified eluants containing the Atf-Bct-NHS-PBAN (at 32 min and 34 min) were compared to the dose-response relationship found by HezPBAN (Fig. 5) using the in vitro bioassay. It was found that the Atf-Bct-NHS-PBAN analog was highly active with dose-response saturation observed at 0.05 l eluant volume. From calculations, based on this dose-response activity re-
lationship, we estimate that a total of 14.7 g of Atf-BctNHS-PBAN analog was successfully biotinylated. 3.4. Identification of Atf-Bct-NHS-PBAN analog by tricine SDS-PAGE Samples of the active peak (corresponding to elution time 28 –36) and a control fraction at elution time of 20 min were subjected to tricine SDS-PAGE and Western blotting using chemiluminescence for detection. Some Western blots, containing a sample of native PBAN, were also detected by using antiserum to PBAN. Typical blots are depicted in Fig. 6A and B. It can be observed that fractions corresponding to elution time 30, 32, 34, and 36 contain biotin, fractions 32 and 34 corresponding to a strongly reactive biotinylated protein. This protein was not present in the fraction corresponding to native Hez-PBAN (20 min). The protein was separated in the 16.5% acrylamide gel section corresponding to a small protein in the range of 4 – 8.2 kDa. Reagent alone did not give a signal at this position (data not shown). The Western blot, reacted with PBAN antiserum (Fig. 6B) shows the position of native
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Fig. 5. Dose-response curves of Hez-PBAN, and active HPLC fractions by using the in vitro bioassay. Sample volume used is depicted as second x-axis. Points represent means of 7 replicates, bars show the extent of SEM.
3.5. Pheromonotropic activity of Atf-Bct-NHS-PBAN analog on intact female moths (in vivo bioassay)
intact female moths during the photophase at 1 l of eluant/ female significantly stimulated pheromone titers above saline control levels (P ⬍ 0.05) (Fig. 7). No significant difference was observed between the responses of intact and decapitated females to Atf-Bct-NHS-PBAN analog.
Both fractions 32 and 34, corresponding to Atf-BctNHS-PBAN analog, when injected into decapitated and
4. Discussion
Fig. 6. Western blot showing a separation of Atf-Bct-NHS-PBAN analog on a Tricine SDS-PAGE by using a discontinuous system: 16.5% acrylamide gel overlaid by a 10% spacer gel and 4% stacking gel, showing strong biotin label in fractions 32 and 34. (A) Transfer analyzed by chemiluminescence. (B) Transfer analyzed by chemiluminescence and immunoblotting (PBAN antiserum). PB ⫽ 5 pmol native Hez-PBAN. Molecular weights were marked using kaleidoscope polypeptide standards.
Much emphasis has been devoted to the study of the structure and function of peptide hormones in insects and other invertebrates however, there are very few reports directed at the characterization of peptide receptors in these organisms. The use of photoaffinity labeled analogs has been successful for the characterization of some soluble binding proteins [7,18], however, it has been of limited success in the quest for determining the character and structure of membrane-bound receptor proteins that may be much less abundant. Three stages are necessary in the synthesis of a photoaffinity-tagged analog with the aim at characterizing a receptor: 1) The design of a new chemical for the photoaffinity analog that will include a photochemically reactive functional group as well as a tag that may be detected by using a sensitive assay; 2) the demonstration that this analog is physiologically active both when tested in an in vitro and an in vivo system. Analogs, with activity that are not of physiological significance, are not useful because they may label proteins nonspecifically and thereby identify
PBAN that approximates in molecular size to Atf-Bct-NHSPBAN analog.
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Fig. 7. Stimulation of pheromone production in intact and decapitated moths (in vivo bioassay) by Atf-Bct-NHS-PBAN (1 l and 2 l eluant/female) as compared to the response to Hez-PBAN (1 pmol/female). Histograms represent the means of five replicates, bars indicate the extent of standard error. Different letters denote a significant difference (P ⬍ 0.05; analysis of variances followed by Fisher’s protected least squares difference test).
proteins that are not involved in the molecular action of the native hormone; and 3) The demonstration that this protein is ligand-specific, confirmed by competitive displacement in the presence of an excess of native ligand. In this study, we demonstrated in detail two of the essential stages in the synthesis of a photoaffinity analog (Atf-Bct-NHS-PBAN) that could be utilized in receptor characterization studies. A photochemically reactive functional group was successfully tagged onto Hez-PBAN. This was shown to be fully active in an in vitro bioassay as well as when injected to intact or decapitated females in vivo thereby demonstrating a physiological functional analog. Its molecular weight was confirmed to be in the region of 4 kDa, corresponding to the molecular weight expected of a PBAN analog. It should be noted that determination or estimation of the molecular weights of small peptides using tricine SDS-PAGE is not accurate due to an intrinsic charge and shape of small peptides that are relatively more important in determining their mobility on SDS gels than large proteins [30]. However, by comparing the mobility of the Atf-Bct-NHS-PBAN analog to native PBAN by using immunoblotting technique, a more accurate estimation could be made. Preliminary studies [23], using this Atf-Bct-NHS-PBAN analog, showed evidence for specific competitive labeling of a protein at the 50-kDa range in the intersegmental tissue of the pheromone glands. This protein was successfully displaced in the presence of a saturation level (⫻250) of synthetic Hez-PBAN, thereby demonstrating stage 3 of the requirements mentioned above. By incorporating avidin-biotin technology we have been successful in obtaining a highly biologically active analog that can amplify a relatively small abundance of receptors to a detectable level. This analog will be useful for the characterization and subsequent sequence determination of the receptor for PBAN. Moreover, by coupling of avidin-gold to this biotinylated hormone analog, it may also prove useful in
the study of the surface distribution, internalization, intracellular compartmentation and recycling of the PBAN receptor. Because moths constitute the major group of pest insects in agriculture and are therefore of economic importance, a great deal of effort has gone into the identification of their pheromones. Whereas the components of pheromone blends from a variety of moth species have been identified, pheromone analogs have had limited success in controlling pest populations. The biosynthesis and release of sex pheromone is necessary to attract a mate, therefore the interruption of this process is a potential strategy for manipulating adult moth behavior. Crucial to understanding the primary mode of action of any hormone is the identification and characterization of its receptor or receptors that constitute a key link in the signal transducing mechanism. Therefore the rationale in pursuing these studies not only lies in the advancement of basic scientific knowledge on comparative biochemistry but also specifically in the potential for finding ways of manipulating the reproductive behavior of pest moths. The development of specific and safe insect control strategies utilizing pheromone systems depends on a clear knowledge of the molecular mechanisms involved. The sequences of these receptors and subsequent study of their binding domains will provide future templates for the design of effective and specific antagonists. Furthermore, because PBAN belongs to a family of insect neuropeptides with more than one function in different life stages, this rationale can be extended to other physiological processes in different insects.
Acknowledgments We thank Mina Litvin for her dedication to maintaining a healthy insect culture.
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