Proc. Nati. Acad. Sci. USAVol. 86, pp. 5997-6001, August 1989Neurobiology

Primary structure of four allatostatins: Neuropeptide inhibitors ofjuvenile hormone synthesis

(insect brain/corpora aliata/cockroach/Diploptera)

A. P. WOODHEAD*, B. STAY*t, S. L. SEIDEL*, M. A. KHANS, AND S. S. TOBEt*Department of Biology, University of Iowa, Iowa City, IA 52242; and tDepartment of Zoology, University of Toronto, Toronto, ON M5S lA1, Canada

Communicated by Berta Scharrer, May 12, 1989 (receivedfor review March 15, 1989)

ABSTRACT Four neuropeptides that inhibit juvenile hor-mone synthesis by the corpora allata have been isolated frombrains of the virgin female cockroach Diploptera punctata.These allatostatins are 8-13 amino acids long, are amidated,and show sequence similarity, including a 3-amino acid se-quence at the C-terminal end that is common to all fourpeptides. The peptide sequences are as follows: allatostatin 1,Ala-Pro-Ser-Gly-Ala-Gln-Arg-Leu-Tyr-Gly-Phe-Gly-Leu-NH2; allatostatin 2, Gly-Asp-Gly-Arg-Leu-Tyr-Ala-Phe-Gly-Leu-NH2; allatostatin 3, Gly-Gly-Ser-Leu-Tyr-Ser-Phe-Gly-Leu-NH2; and allatostatin 4, Asp-Arg-Leu-Tyr-Ser-Phe-Gly-Leu-NH2. An in vitro bioassay of the synthesized allatostatinsshowed >40% inhibition of juvenile hormone synthesis bycorpora allata of virgin females with 10-9M allatostatin 1, 10-8M allatostatins 2 and 4, and 7 x 10-7 M allatostatin 3.Inhibition by allatostatins 1-4 was reversible. In addition,allatostatin 1 inhibited juvenile hormone synthesis by corporaallata from mated females and last-instar larvae ofD. punctataand corpora allata of adult female Periplaneta americana.

The regulation of endocrine organs by neuropeptides fromthe brain is strikingly similar in organisms as different asvertebrates and insects (1). For example, in vertebrates,neuropeptides produced by the hypothalamus of the brainregulate a major endocrine gland, the anterior pituitary (2).Directly parallel to this in insects is the regulation ofthe majorendocrine organs, the corpora allata (CA) and the prothoracicglands, by neuropeptides produced by the pars intercerebra-lis and pars lateralis of the brain (3). Great advances havebeen made in isolating and characterizing neuropeptides thatregulate endocrine glands in vertebrates (4, 5). Far lessattention has been directed toward analysis of the analogousneuropeptides in insects (6), although insect systems becauseof their relative simplicity are advantageous for studying themechanisms of this basic neuroendocrine relationship (1).Recent progress has been made in establishing the struc-

tures of the prothoracicotropic hormones, neuropeptides thatstimulate the prothoracic gland to produce the growth hor-mones, ecdysteroids. In the lepidopterans Bombyx mori andManduca sexta, these appear to occur as large and smallpeptides and within these size groups in multiple forms (7-9).Although the primary structures of the two peptides in M.sexta are not yet known, both have prothoracicotropic ac-tivity in this species (7). In B. mori the primary structure ofthe small peptide, which resembles insulin, and part of thelarge peptide are known, but only the large peptide is activein this species (8, 9).We are concerned here with the brain-CA axis. The sole

known product of the endocrine cells of the CA is juvenilehormone (JH), in one or several forms depending on thespecies of insect (10). The modulated synthesis of JH is, in

most species, required not only for development in immaturestages but also for reproduction in the adult stage, especiallyfor vitellogenesis in the female (3, 11). Peptidergic neurose-cretory material from cells of the brain that project to the CAhave long been postulated to regulate the activity of the CA,either as stimulators, allatotropins (e.g., refs. 12-16), orinhibitors, allatostatins (e.g., refs. 17-21). Experimental ev-idence suggests that one of these effects may predominatedepending upon the species (10).A pioneering investigation on neurosecretion in the brain of

the cockroach Leucophaea maderae revealed that the CAwere released from inhibition when the nerve tract from thebrain to the CA was severed (22). Until recently littleprogress has been made toward isolating either allatotropinsor allatostatins. An allatotropin has been isolated from headsofM. sexta and its primary structure has been elucidated (23);as yet no primary structure for an allatostatin is known.However, brain extract has been shown for several species ofinsects to inhibit JH synthesis (19, 24). Extract of brains fromthe cockroach Diploptera punctata contains peptidergic ma-terial that inhibits JH synthesis by the CA in vitro (19, 25, 26).In this paper we describe the isolation and primary structureof allatostatins extracted from brains ofadult virgin female D.punctata. The activity of these native and synthetic allato-statins was tested on CA in vitro. In addition, the reversibilityand the stage and species specificity of synthetic allatostatinswere tested.

MATERIALS AND METHODSAnimals. The viviparous cockroach D. punctata was main-

tained and staged females were obtained as described (27,28). Adult Periplaneta americana females were removedfrom stock cultures on the day of emergence and maintainedat 270C.CA of virgin D. punctata females do not undergo cycles in

JH synthesis but rather are maintained at a relatively con-stant low rate of synthesis (29). Brains of virgin females werefound to be a good source of allatostatins and their CAresponded reproducibly to treatment with brain extract (19,25). Therefore, in this investigation we have continued to usethe brains of virgin females as a source of allatostatins andtheir CA to test for allatostatic activity.

Bioassay for Allatostatic Activity. The CA of D. punctataproduce only JH III (30). Rates ofJH synthesis by CA weredetermined by the in vitro radiochemical method of Feyer-eisen and Tobe (31) as modified by Tobe and Clarke (32). JHsynthesis was monitored by incorporation of L-[methyl-3H]methionine (200 mCi/mmol; 1 Ci = 37 GBq; New EnglandNuclear; in a final concentration of 50 ,uM). CA wereremoved from the incubation medium before extraction andquantification ofJH; thus the rates of synthesis represent the

Abbreviations: JH, juvenile hormone; CA, corpora allata; TFA,trifluoroacetic acid; ACN, acetonitrile.tTo whom reprint requests should be addressed.

5997

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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radiolabeled hormone released into the medium. CA wereincubated in 50 1.l of radiolabeled medium for 3 hr to establishthe normal rate of synthesis; then the glands were transferredto medium containing putative allatostatins or control solu-tions for a second 3-hr incubation. The percentage change inJH synthesis, [1 - (treated rate/normal rate)] x 100, wascalculated. Routinely, one treatment solution was tested onfour or five individual glands or pairs of glands; the means ofthese percentages were used to indicate whether the CA wereinhibited as a result of treatment. For screening high-pressureliquid chromatography (HPLC) fractions, any mean inhibi-tion >30% was considered to indicate allatostatic activitysince control glands sometimes showed up to 30% change.

Single CA from 2-day virgin females were used to testHPLC fractions for allatostatic activity, to establish dose-responses for inhibition of JH synthesis, and to test revers-ibility of inhibition of JH synthesis by synthetic peptides.Synthetic allatostatins were also tested on single CA of matedD. punctata and on pairs of CA from D. punctata larvae andP. americana adults.

Brain Extract. Brains were dissected from heads of virginfemales (adult age, 2-30 days). Optic lobes and adhering fatbody and tracheae were removed. Twenty to 30 brains werehomogenized in 0.9% NaCl (10 Al per brain) in a small glasshomogenizer. The homogenate was heated to 100'C for 5-10min and centrifuged at 4000 x g for 20 min, and the super-natant was stored (up to a month) at -80'C until sufficientextract was collected. Pilot separations were run on 50-450brain equivalents of extract. The final purification was ac-complished with extract from 1000-1200 brains.

Purification of Aliatostatins. 1. SEP-PAK preparation ofsample. Reverse-phase C18 cartridges (Waters) were pre-washed with 5 ml each of 0.1% bovine serum albumin(fraction V, Sigma) with 0.1% trifluoroacetic acid (TFA), 40%acetonitrile (ACN) with 0.1% TFA, and 0.1% aqueous TFA.Extract of 125 brains was loaded (about 3 ,tg of protein perbrain equivalent) and reapplied three times. The cartridgewas washed with 5 ml each of 0.1% TFA and 17% ACN with0.1% TFA, and material was eluted with 3 ml of 40% ACNwith 0.1% TFA. Protein was not detected in the eluate afterloading three times or in the washes. About 1 gg of proteinper brain equivalent was removed with the 40% ACN.Allatostatic activity was found in the 40% ACN eluate.

2. First HPLC separation. The 40% ACN eluate of 250brain equivalents from step 1 was reduced in volume withnitrogen to 200 ,l, diluted to 1 ml with 0.1% TFA, andinjected onto a 3.9 mm x 30 cm reverse-phase C18 column(10-,um particle size, ,uBondapak, Waters). HPLC was per-formed on a Waters system with an automated gradientcontroller and a model 490 detector. Solvent A was 0.1%TFA in water; solvent B was ACN with 0.1% TFA. A lineargradient from 0% to 50% ACN (1%/min) with a flow rate of1.5 ml/min was used. Compounds eluting from the columnwere detected by their absorbance at 220 and 280 nm.Fractions were collected at 1-min intervals. Allatostaticactivity was assayed in fractions 10-60 and was limited tofractions between 33 and 42, eluting with 22-31% ACN. Amixture of known peptides {0.5 ,ug each of thyrotropin-releasing hormone, [Lys8]vasopressin, luteinizing hormone-releasing hormone, somatostatin-14, bovine serum albumin(V), and glucagon; all from Sigma} was also separated by thisgradient system. Luteinizing hormone-releasing hormoneand somatostatin eluted in fractions 33 and 41 or 42, respec-tively, and therefore these peptides were subsequently usedfor standard runs to mark the retention time of fractions withallatostatic activity.

3. Second HPLC separation. Five hundred brain equiva-lents offraction 36 or 37 (the active fractions) from step 2 wasreduced in volume, diluted, and injected onto the C18 columnas before. A shallower linear gradient, 10-35% ACN over 50

min, with a flow rate of 1 ml/min was used. The regionsabsorbing at 220 nm were collected by hand into separatetubes and assayed for allatostatic activity.

4. Third HPLC separation. Purification to apparent ho-mogeneity was achieved by separation of active regions from1000 brain equivalents of the material from step 3. Thematerial was reduced in volume, diluted as before, andinjected onto a reverse-phase 4.5 mm x 15 cm C8 column(5-jim particle size, Jones Chromatography, Littleton, CO).A linear gradient from 11% to 44% ACN over 50 min with aflow rate of 1 ml/min was used. The UV absorbing peakswere again collected by hand into separate tubes. Each peakwas assayed for allatostatic activity.

Storage and Preparation of Eluates for Bioassay. Eluates inACN (with 0.1% TFA) from the chromatographic separationswere stored at -800C until assay or further analysis. Forassay aliquots were transferred to 1.5-ml tubes containing 10,ul of 0.1% bovine serum albumin and dried with nitrogen.The material was resuspended in 10 1.d of 1 M HCl, and theappropriate amount of incubation medium was added andthen neutralized with 1 M NaOH. Controls, eluates fromHPLC with no absorbance (at 0.1 absorbance unit full scale),were treated in the same way.Sequence Analysis. Four active peaks from the final HPLC

separation of 1000 brains were sequenced by Edman degra-dation using an Applied Biosystems gas-phase sequencer(model 470A) or pulsed liquid-phase sequencer (model 477A)with on-line HPLC. These determinations were made onabout 100 pmol of each peptide by the University of Wis-consin Biotechnology Center. Samples of three of the fouractive peaks (1, 3, and 4) isolated from a subsequent HPLCseparation of extract from 1200 brains were analyzed by theMassachusetts Institute of Technology Mass SpectrometryFacility. Mass determinations (tandem mass spectrometerJEOL HX110/HX110, Tokyo) were made to confirm themasses ofthe peptides and to assess amidation ofthe terminalamino acids. In addition, peptide 4 was subjected to collision-induced decomposition tandem mass spectrometry (33) todetermine whether tyrosine was modified, as Edman degra-dation analysis had tentatively indicated.

Peptide Synthesis. Allatostatins were synthesized on anApplied Biosystems model 430A automatic solid-phase pep-tide synthesizer (University of Wisconsin BiotechnologyCenter, allatostatins 1 and 2; University of Iowa ProteinStructure Facility, allatostatins 3 and 4). All four peptideswere purified by HPLC on a preparative C18 reverse-phasecolumn (Vydac), and amino acid analyses confirmed theircomposition.

Synthetic and Native Allatostatins. Elution times of thesynthetic allatostatins were compared with those of nativeallatostatins isolated from extract of 1000 brains. The foursynthetic peptides were run on HPLC using the final C8column step; this was followed by runs of native allatostatins1-3 and allatostatin 4.Computer Search for Similar Peptides. Two data bases,

Swiss-Prot§ and PIR, were searched for peptides withsequences similar to allatostatins 1-4 using the FASTA pro-gram (34) via BIONET to determine the degree of similaritywith other identified peptides.

RESULTSHPLC Separation of Allatostatins. Assays of material from

the first C18 column separation showed that material withallatostatic activity eluted between 22% and 30% ACN (frac-

§Swiss-Prot (1988) EMBL Nucleotide Sequence Data Library (Eur.Mol. Biol. Lab., Heidelberg), Release 9.0.Protein Identification Resource (1988) Protein Sequence Database(Nati. Biomed. Res. Found., Washington, DC), Release 18.0.

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0.04

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FIG. 1. Final separation of allatostatins 1-4 from extract of 1200brains of virgin female D. punctata by HPLC on a reverse-phase C8column with linear ACN gradient (dashed line). After a first sepa-ration on a C18 column, fractions 36 and 37 were rechromatographedon the C18 column with a shallower gradient of ACN. Material wasderived from fraction 36 (A) and fraction 37 (B).

tions 33-41). Assays of pooled fractions 33-35, 36-38, and39-41 using 4.5 brain equivalents per CA showed >50%inhibition ofJH synthesis in the three groups. However, onlythe material in pooled fraction 36-38 showed >50%o inhibitionwhen tested at 1 brain equivalent per CA. Subsequentseparations were limited to this group, specifically fractions36 and 37. Following the second C18 column separation,active fractions were isolated from both fractions 36 and 37(first fractionation). Chromatography on a C8 column (thirdfractionation) separated the active material to apparent ho-mogeneity. Four peaks showed allatostatic activity: one inmaterial derived from fraction 36 and three in materialderived from fraction 37 (Fig. 1 A and B). All but the first peakderived from fraction 37 (allatostatin 3) showed >50%o inhi-bition ofJH synthesis when tested at 5 brain equivalents per

1 Ala-Pro-Ser-Gly-Ala-GIn-Ar

2

3

4

CA. Allatostatin 3 showed 35-40% inhibition at 5 brainequivalents.Amino Acid Sequences. Extracts of 1000 and 1200 brains,

purified through three HPLC separations, each providedabout 100 pmol of material from each ofthe four active peaks.The amino acid sequences for the four peptides determinedby Edman degradation and mass spectral analysis are shownin Fig. 2. Mass spectral analysis showed that the C terminuswas amidated in allatostatins 1, 3, and 4 (allatostatin 2 was notanalyzed, but amidation is inferred because this form of thesynthetic peptide is bioactive). Values for mass determinedby spectrometry were: allatostatin 1, 1335.7; allatostatin 3,899.5; and allatostatin 4, 969.5. These values match preciselythe calculated molecular weights. The collision-induced de-composition tandem mass spectrum for allatostatin 4 wasconsistent with the sequence determined by Edman degra-dation (in addition, the collision spectrum showed that thetyrosine was unmodified).

Synthetic Allatostatins. All four allatostatins were synthe-sized with amidated C termini. In sequential HPLC runs, theelution time of the synthetic allatostatins was the same as thatof the native material. The dose-responses for inhibition ofJH synthesis for allatostatins 1-4 and the mean and range ofcontrol values are shown in Fig. 3. Inhibition ofJH synthesis>40o was achieved with 10-9 M allatostatin 1, 10-8 Mallatostatins 2 and 4, and 7 x 10-7 M allatostatin 3.

Reversibility of Inhibition. Allatostatins 1-4 were tested forreversibility of inhibition on CA of 2-day-old virgin females.After treatment with allatostatin, glands were transferred tomedium without allatostatin for a third 3-hr incubation.Results for allatostatins 1 and 2 are shown in Fig. 4. Data forallatostatins 3 and 4 are not shown. However, for all fourallatostatins rates of JH synthesis decreased 60-74% withallatostatin treatment. Rates of JH synthesis increased inthe third incubation period and were not significantly differ-ent from rates in the first incubation period (Mann-Whitneytest).

Inhibition of CA of Mated and Larval Females. The effectsof synthetic allatostatins on CA of physiological stages otherthan 2-day adult virgins were also determined. Allatostatin 1(10-8 M) was tested on CA from 6-day-old mated females,known on the basis of physiological age (the end of thevitellogenic cycle) to show declining rates of JH- synthesis.During the first 3-hr period (untreated), the mean rate of JHsynthesis (±SEM) was 10.3 ± 1.5 pmol hr-1 per gland; duringthe second 3-hr period (treated), the mean rate was 2.4 ± 0.3pmol hr-1 per gland: an inhibition of 77% (n = 13). The meanrate of JH synthesis of control glands (n = 4) decreased 10%during the corresponding time period.

Species Specificity. Allatostatin 1 (10-8 M) inhibited JHsynthesis by CA of adult female P. americana 2-5 days old.The mean untreated rate of JH synthesis (±SEM) was 2.5 ±0.5 pmol hr-1 per gland; the mean treated rate was 0.2 ± 0.1pmol hr-1 per gland: an inhibition of 92% (n = 9). The mean

g-Leu-Tyr -Gly-Phe-Gly-Leu-NH2

Gly-Asp-Gly-Arg- Leu-Tyr-Ala -Ph.-Gly-Leu-NH2

Gly -Gly-Ser-Leu-Tyr-Ser-Phe-Gly- Leu-NH2

Asp-Arg-Leu-Tyr-Ser-Phe-Gly-Leu-NH,2

FIG. 2. Primary structure of allatostatins 1-4. Identical amino acids (connected by solid lines) are found at the amidated C terminus.Allatostatins 1, 2, and 4 also contain arginine in the same position.

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FIG. 3. Dose-responses for inhibition of JH synthesis by syn-thetic allatostatins 1 (s), 2 (m), 3 (A), and 4 (o). Glands were incubatedfor 3 hr in normal medium and then transferred to medium withallatostatin for a second 3-hr incubation. Percentage inhibition wascalculated as [1 - (treated rate/normal rate)] x 100. Each point forallatostatin treatment is the mean of 5-37 measurements; verticalbars show standard error of the mean. The control value, C (®), isthe mean of the combined untreated controls for the first and second3-hr incubations from each day of assays; the range of these valuesis shown by the vertical bar.

rate of JH synthesis in control glands (n = 9) decreased 5%during the second incubation period.Computer Search for Similar Peptides. No peptides similar

to allatostatins 1-4 were found in either data base. Limitedsequence similarity was observed with internal sequences ofseveral animal proteins including cytochrome P-450 reduc-tase (60% similarity for 10 C-terminal amino acids of alla-tostatin 1) and thyroglobulin precursor (100% similarity for 6C-terminal amino acids of allatostatin 2).

DISCUSSIONThe four allatostatic neuropeptides isolated from the brain ofD. punctata inhibit the synthesis of JH by CA in vitro andthus are analogous to the somatostatic neuropeptide of ver-tebrates that inhibits secretion of somatotropin by the ante-rior pituitary gland (35). In fact, somatostatin has been shownto modulate JH synthesis in Blattella germanica, but indi-rectly, via the brain (36). The allatostatins are amidatedpeptides of 8-13 amino acids with considerable structuralsimilarity. The three C-terminal amino acids and the fifth andsixth amino acids from the C terminus are identical in the fourpeptides, and variability occurs at the N terminus. Thesequence differences in the peptides indicate that the smallerpeptides are not products of the largest one (Fig. 2). Thusthese allatostatins comprise a family of peptides. They arenot similar to any known peptides, but the similarity amongthe allatostatins at the C terminus is reminiscent of the familyof neuropeptides related to FMRFamide (Phe-Met-Arg-Phe-NH2) that has been described by gene isolation in Drosophila(37). Other families of neuropeptides in invertebrates are, forexample, in insects, adipokinetic hormones (38) and leuco-kinins (39, 40), and in molluscs, Aplysia egg-laying hormones

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. I3 6 9Time (hr)

FIG. 4. Reversibility of inhibition ofJH synthesis by allatostatins1 (Upper) and 2 (Lower). Right (e) and left (o) glands of 2-day-oldvirgin females were incubated for 3 hr in normal medium (Unt.); thenright glands were transferred to medium with allatostatin (All.)(allatostatin 1, 10-8 M; allatostatin 2, 10-7 M) and left glands weretransferred to normal medium (Unt.) for 3 hr. A final 3-hr incubationfor both right and left glands was in normal medium. Each point isthe mean of seven individual measurements. Vertical bars show thestandard error of the mean. Inhibition by allatostatins 3 and 4 wasalso reversible (see Results).

(41). It will be instructive to determine whether the otherfractions of brain extract, which have allatostatic activity butare as yet unidentified, contain additional members of thesame family or unrelated peptides.

Inhibition of JH synthesis was achieved with 10-9 Mallatostatin 1, 10-8 M allatostatins 2 and 4, and 7 x 10-7 Mallatostatin 3 (Fig. 3). These differences in activity may beaccounted for by the variability in structure at the N terminus(Fig. 2). Inhibition was completely reversible-i.e., JH rateswere restored to pretreatment levels after glands were trans-ferred to normal medium.

Although allatostatin 1 has been isolated from virgin femalebrains, its activity does not appear to be stage- or species-specific. Allatostatin 1 not only inhibits JH synthesis by CAof virgin females but also CA of mated females at the end ofvitellogenesis, larval CA, and CA of adult P. americanafemales. Since allatostatins 2-4 have not been tested on thesegroups, it is not known how general is their ability to inhibitJH synthesis. Nor is it known whether there is synergisticactivity of these forms.The allatotropin isolated from brains of the lepidopteran,

M. sexta, is a peptide of 13 amino acids; its stimulatory effecton CA in vitro appears to be stage-specific and limited tolepidopterans (23). Its amino acid sequence is not similar tothe four allatostatins that we have isolated.The regulation of the production of JH, one of the major

hormones regulating development and reproduction in in-sects, is a complex process (10). Identification of allatostatinsnow provides the molecules that can be used experimentallyto extend our understanding of the regulation ofJH synthesisat the cellular and organ level. In addition, the availability of

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allatostatins will facilitate studies on the regulatory mecha-nisms of their production and release.

We thank Mr. Sanjay Joshi for performing the bioassays and Mrs.Kuen Chan for preparing the figures. We acknowledge the contri-butions of the University of Wisconsin Biotechnology Center and theUniversity of Iowa Protein Structure Facility. We thank Dr. Cathe-rine Costello for mass spectrometric determinations. The Massachu-setts Institute of Technology Mass Spectrometry Facility receivessupport from National Institutes of Health Grant RR00317 to Dr.Klaus Biemann. BIONET is supported by National Institutes ofHealth Grant P41RR01685. We acknowledge support from NationalInstitutes of Health Grant A115320.

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