Proc. Nati. Acad. Sci. USA Vol. 86, pp. 2976-2980, April 1989 Physiological Sciences Isolation and identification of a diuretic hormone from the tobacco hornworm, Manduca sexta (osmoregulation/amino acid sequence/corticotropin-releasing factor/sauvagine/urotensin I) HIROSHI KATAOKA*, RUTH G. TROETSCHLER, JORGE P. Li, STEVEN J. KRAMER, ROBERT L. CARNEY, AND DAVID A. SCHOOLEYt Zoecon Research Institute, Sandoz Crop Protection, P. 0. Box 10975, Palo Alto, CA 94303-1104 Communicated by Wendell L. Roelofs, January 9, 1989 (received for review October 24, 1988)

ABSTRACT A diuretic hormone (DH) has been isolated Several species of butterflies exhibit significant diuresis from pharate adult heads of Manduca sexta by a nine-step soon after adult eclosion. This phenomenon allowed devel- purification procedure. The primary structure of the amino- opment of a facile in vivo DH assay (16). Though the terminal 40 residues was determined by sequence analysis of well-studied tobacco hornworm moth, Manduca sexta, does intact DH. The structure of an amidated carboxyl-terminal not show diuresis at this stage, we were able to utilize the tryptic hexapeptide was characterized by sequence analysis of butterfly Pieris rapae in a similar bioassay and to purify a DH the peptide, and this hexapeptide was later compared by from trimmed head tissue of pharate adult M. sexta. We here reversed-phase liquid chromatography with two synthetic report the isolation and structure identification of a M. sexta hexapeptides with the free acid or amide at the carboxyl DH, which has homology with sauvagine, corticotropin- terminus. The complete structure of M. sexta DH was estab- releasing factor (CRF), and urotensin I. lished as a 41-residue peptide without disulfide bonds: H-Arg- Met-Pro-Ser-Leu-Ser-Ile-Asp-Leu-Pro-Met-Ser-Val-Leu-Arg- MATERIALS AND METHODS Gln-Lys-Leu-Ser-Leu-Glu-Lys-Glu-Arg-Lys-Val-His-Ala- Leu-Arg-Ala-Ala-Ala-Asn-Arg-Asn-Phe-Leu-Asn-Asp-Ile- Insects. M. sexta were reared on an artificial diet (17). NH2. M. sexta DH was synthesized and shown to have chro- Pharate adult M. sexta were beheaded 24-48 hr before adult matographic and biological properties identical with those of eclosion, and the heads were frozen. A posterior section of the native material. Synthetic DH stimulated fluid excretion in these frozen heads containing the brain and the CC/corpora vivo upon injection into larval M. sexta and newly emerged allata complex was punched out with a 5-mm-diameter cork adult Pieris rapae. M. sexta DH has borer and stored at -800C until extraction. considerable sequence Cabbageworms (P. rapae larvae) were reared on cabbage homology with corticotropin-releasing factor, urotensin I, and plants until about the third stadium, when they were trans- sauvagine. ferred to an artificial diet (17), incorporating fresh cabbage leaves as a phagostimulant. Leaves ofone immature cabbage Insects regulate the osmotic composition of their blood plant were heated (15 min, 1630C) before grinding in a blender within relatively narrow limits in spite of an unfavorable with each 3-liter batch of diet. P. rapae pupae were held at surface-to-volume ratio (1). The major organs responsible for 280C during the day, 270C during the night on a 16:8 light:dark fluid and ion secretion are the Malpighian tubules (Mt). cycle until the morning of emergence. Primary urine from the Mt moves into the gut and eventually In Vivo Bioassay. DH activity in fractions from each to the rectum, where selective resorption of essential metab- purification step was detected by using newly emerged adult olites and water typically occurs (for reviews see refs. 1-3). P. rapae, in an assay modified from one previously reported It is generally believed that regulation of fluid secretion in for Danaus plexippus (16). In addition to utilizing an increase insects is controlled by one or more peptidic diuretic hor- in temperature just after lights-on (16) to synchronize adult mones (DHs), while resorption may be regulated by antidi- eclosion, we placed pharate adults in a cardboard container uretic hormones (1, 2, 4, 5). By using in vitro Mt assays, seated on a warm water bath (s450C) to trigger almost substances with diuretic activity have been found in ganglia simultaneous emergence of large numbers of young adults. from the head, thorax, and abdomen, and in a glandular These young butterflies were anesthetized with CO2 within 1 tissue, the corpora cardiaca (CC) (4-8). Partial purifications min of eclosion. They were neck ligated within 15 min, then ofDH from several species indicate that there may be several beheaded, and the wound was sealed with melted wax. Each DHs which differ in size and possibly in modes of action (3, animal was suspended from the side of a 30-ml plastic cup 5, 8-12). until the wings expanded. Beheaded butterflies were weighed To date, isolation of two insect DHs, both from Locusta within 1 hr of the molt. Untreated controls were weighed migratoria, has been reported (13, 14). One of these was about 15 min after eclosion. isolated from subesophageal and thoracic ganglia, using its To reduce possible adsorption ofDH to containers, bovine immunological cross-reactivity with an antibody to [Arg8]- serum albumin (50 gg) was added to test fractions before vasopressin (AVP) as an assay (14). This DH was identified evaporating the solvent. Treated butterflies were injected in as an antiparallel dimer of [Leu2,Thr4,Arg8]vasotocin (15), the ventral thorax with the test fraction dissolved in 5 dul of and it occurs in tissue with the corresponding monomer, which has no diuretic activity (15). The other DH was isolated Abbreviations: DH, diuretic hormone; AVP, [Arg8lvasopressin; LC, from CC of this locust by monitoring in vitro stimulation of liquid chromatography; TFA, trifluoroacetic acid; Boc, t-butoxycar- cAMP in Mt during purification, but only a partial sequence bonyl; Mt, Malpighian tubule(s); CC, corpora cardiaca; CRF, cor- was determined (13). ticotropin-releasing factor; oCRF, ovine CRF; ACTH, adrenocorti- cotropic hormone. *Present address: Department of Agricultural Chemistry, University The publication costs of this article were defrayed in part by page charge of Tokyo, Tokyo 113, Japan. payment. This article must therefore be hereby marked "advertisement" tTo whom reprint requests should be addressed at: Biochemistry in accordance with 18 U.S.C. §1734 solely to indicate this fact. Department, University of Nevada, Reno, NV 89557. 2976 Downloaded by guest on September 28, 2021 Physiological Sciences: Kataoka et al. Proc. Natl. Acad. Sci. USA 86 (1989) 2977 "Manduca saline" (18) (4 mM NaCl/40 mM KCI/3 mM with 10-ml fractions collected. DH was recovered in the 48- CaCl2/18 mM MgCl2); beheaded controls received 5 1.L of to 52-min fractions. Manduca saline. The plastic cups were capped to reduce TSK SP-5PW LC (Step 7). After addition of 2 ml of 0.2 M evaporation. The butterflies were weighed again 3 hr after sodium phosphate buffer (pH 6.25), the active fractions from injection; weight loss during this period was considered to be step 6 were applied to a TSK SP-5PW ion-exchange column fluid excretion. (Beckman; 7.5 x 75 mm) equilibrated with 0.02 M sodium To determine net normal diuresis, the mean weight loss of phosphate buffer (pH 6.25) containing 10% CH3CN. The beheaded controls was subtracted from the mean weight loss column was eluted with two successive gradients at 1 of intact controls. The mean weight loss of a typical 114-mg ml/min: 10 min of0-0.1 M NaCl in 0.02 M sodium phosphate untreated intact control animal in the 3-hr assay period was buffer (pH 6.25) containing 10% CH3CN and 60 min of0.1-0.4 23 mg (20% of mean body weight), that of a typical 118 M NaCl in the same buffer. Two-milliliter fractions were mg-beheaded control animal injected with 5 pJ of saline was collected. DH was recovered in the 48- to 52-min fractions. 11 mg (9% of mean body weight), and that of a typical 118-mg Vydac C4 Analytical LC (Step 8). The active fractions from beheaded animal injected with 1 head equivalent of extract in step 7, containing buffer salts and =10% CH3CN, were 5 1Ld of saline was 24 mg (20% of mean body weight). The pooled and applied by means of a 5-ml loop injector to a weight loss of treated, beheaded animals was expressed as a Vydac C4 column (5-pum packing, 4.6 x 150 mm) equilibrated fraction of net normal diuresis. with 30% CH3CN in 0.1% heptafluorobutyric acid (HFBA). M. sexta in Vivo DH Bioassay. Although P. rapae retain The column was eluted with a 75-min linear gradient of 30- most of their fluids during the postfeeding prepupal period, 45% CH3CN in 0.1% HFBA at aflow rate of 1.5 ml/min, with M. sexta wandering fifth-stadium larvae lose =30% of their 3-ml fractions collected. DH was recovered only in the 36- to body weight of -9 g (19) during the 48 hr that precede 38-min fraction. pupation. We used prewandering postfeeding M. sexta just Vydac C4 Microbore LC (Step 9). Finally, the active after lights-out (12:12 photoperiod) to assay for increased fraction from step 8 was diluted with 6 ml ofwater and applied diuresis triggered by DH. These larvae were weighed, then to a Vydac C4 microbore column (5-,um packing, 2.1 x 250 injected with 25 ,u1 of Manduca saline or 25 ,ul of this saline mm) by using two injections with a 5-ml loop injector. The containing 0.1 ,ug of synthetic DH. After 4 hr, each larva was column was equilibrated with 20% CH3CN in 0.1% TFA. again weighed and dry weight of frass was subtracted to After elution with a 100-min linear gradient of 20-40% determine net weight of water lost. CH3CN/0. 1% TFA at a flow rate of0.3 ml/min, pure DH was Extraction and Preliminary Purification (Steps 1-4). Ten recovered in a peak at 69.0-71.5 min. thousand trimmed heads of M. sexta (fresh weight 420 g) Trypsin Digestion. Purified DH (-1.5 nmol) was dissolved were homogenized in 1500 ml of cold acetone and filtered. in 50 ,l of 0.1 M Tris HCI (pH 8.0)/0.01 M CaCl2 containing Residues were extracted with 1500 ml of 1 M HOAc/20 mM 1 ,g of trypsin (TPCK treated, Sigma). After incubation at HCI (containing 0.1 mM phenylmethylsulfonyl fluoride and 35°C for 2 hr, the reaction mixture was applied to a Vydac C18 0.01 mM pepstatin A, prepared freshly) and centrifuged at column (5-gm packing, 4.6 x 100 mm). The fragment pep- 10,000 X g for 20 min. After reextraction of the pellet with tides were eluted with an 80-min gradient of 0-40% CH3CN 1200 ml of the same solution, and recentrifugation, the in 0.1% TFA at a flow rate of 0.5 ml/min. combined supernatants were applied to an SP-Sephadex C-25 Sequence Analysis. Purified DH and tryptic peptides were column (Pharmacia; 25 mm inner diameter x 750 mm, sequenced by using an Applied Biosystems model 477A containing 300 ml of packing) equilibrated with 1 M HOAc. pulsed liquid-phase protein sequencer equipped with a model The column was eluted with 1000 ml each of 1 M HOAc, 0.05 120A on-line phenylthiohydantoin (PTH) amino acid ana- M NH4OAc (pH 4.0), and 0.05 M, 0.1 M, 0.4 M, and 0.8 M lyzer. NH4OAc (pH 7.0). The 0.4 M and 0.8 M NH4OAc fractions, Amino Acid Analyses. Purified DH and tryptic peptides which both had DH activity, were applied directly to 10 g of were hydrolyzed in vapor from 6 M HCI/1% phenol (110°C reversed-phase Vydac C4 packing material (20- to 30-,um for 20 hr). Hydrolysates were analyzed after conversion to packing contained in a 75-ml polypropylene syringe barrel phenylthiocarbamoyl amino acid derivatives by reversed- with polyethylene frit) equilibrated with 0.1% trifluoroacetic phase LC using an Ultrasphere ODS column (Beckman, acid (TFA). The cartridge was eluted with 100 ml each of 5-gm spheres, 4.6 x 150 mm) and buffers similar to those of 0.1% TFA and 20%, 35%, and 50% (vol/vol) CH3CN in 0.1% Ebert (20). TFA. DH was recovered in the 35% CH3CN/0.1% TFA Peptide Synthesis. The DH was synthesized by the solid- fraction. phase method (21) on a Biosearch 9600 peptide synthesizer, Vydac C4 Semipreparative Liquid Chromatography (LC) using a t-butoxycarbonyl (Boc) protocol and p-methylbenz- (Steps 5 and 6). This and subsequent purifications by LC were hydrylamine resin (0.35 meq/g, 0.50 g). Side chains of amino performed with a Perkin-Elmer Model 410 Bio pump, a acids were protected as follows: Boc-Arg(tosyl), Boc- Rheodyne loop injector, and a Kratos model 783 variable- Glu(benzyl), Boc-His(dinitrophenyl), Boc-Lys(o-chloro- wavelength detector, usually set at 220 nm. The pump was benzyloxycarbonyl), Boc-Ser(benzyl), and Boc-Asp(cyclo- modified with a Rheodyne model 5302 valve installed in the hexyl ester). Coupling was achieved by means ofsymmetrical "D" solvent tube before the solvent proportioning valve, so anhydrides, except asparagine and glutamine, which were that water-diluted fractions could be pumped into the col- introduced as preformed hydroxybenzotriazole active esters umn. The active fraction from step 4 was diluted with 200 ml (22). A yield of 2.06 g of the peptide-resin was obtained. The of water and pumped onto a Vydac C4 semipreparative dinitrophenyl protecting group on histidine was removed by column (10-,tm packing, 10 x 250 mm) previously equili- thiolysis (23) (thiophenol), giving 1.8 g of the peptide-resin. brated with 20% CH3CN in 0.1% TFA. The column was HF cleavage of 858 mg ofthe peptide-resin in the presence of eluted with an 80-min linear gradient of 20-40% CH3CN in 6% (vol/vol) anisole and 4% (vol/vol) ethyl methyl sulfide at 0.1% TFA at a flow rate of 5 ml/min; 10-ml fractions were 0°C for 1 hr afforded 437 mg of the crude peptide (with a collected. DH activity was found in fractions which eluted at carboxyl-terminal amide), which was purified by reversed- 34-38 min. These were combined and diluted with 40 ml of phase LC, using a 2.2 x 25 cm Vydac C4 column eluted with water, and again pumped onto the same column, but now 30% CH3CN/0. 1% TFA at a flow rate of9.9 ml/min. Pure DH equilibrated with 10% (vol/vol) 1-propanol/0.1% TFA. The was recovered at 13-14 min. The identity of the major peak retained materials were eluted with an 80-minlinear gradient was checked by sequence analysis and by amino acid of 10-30% 1-propanol in 0.1% TFA at a flow rate of 5ml/min, composition analysis. Downloaded by guest on September 28, 2021 2978 Physiological Sciences: Kataoka et al. Proc. Natl. Acad. Sci. USA 86 (1989)

0.2- 46840 0~~~~~~~~~~~~~ 0205 o ~~~~~~~~~~~~0 C4) 4)w h c 0~~~~~~~~~~~~ black0 bar indicates the DH-activ fraction.0 .0~~~ ~ ~ ~ ~ ~ ~ ~~~2.

.0~~~~~~~~~~~~~ a U 20 40 60 80 10 20 Retention Time (min) Retention Time (min) FIG. 1. Vydac C4 microbore, LC (step 9). Chromatographic FIG. 3. Comparative reversed-phase Vydac C4 LC of native T-5 conditions are described in Materials and Methods. The broken line and synthetic peptides. Upper trace, native T-5; lower trace, mixture shows the concentration of acetonitrile in 0.1% TFA in water. A of synthetic hexapeptides (standard one-letter symbols) with either black bar indicates the DH-active fraction. a free acid or an amidated carboxyl terminus. The carboxyl-terminal acid analogue of DH was synthe- Structural Analyses. Automated Edman degradation of sized in identical fashion, starting with Boc-L-Ile-O-resin intact DH (0.5 nmol) yielded a single amino acid sequence (0.74 meq/g, 0.3 g). After deprotection and HF cleavage, the with :80% initial yield and =z95% repetitive yield. Residues crude peptide was analyzed by reversed-phase LC, using a 1-40 were assigned after use of 10% of the isolated sample Vydac C4 0.46 x 15 cm column eluted with a linear gradient (Fig. 2). Trypsin digestion of DH (1.5 nmol) gave six of 20-40o CH3CN/0.1% TFA over 20 min at 1.5 ml/min. fragments (T-1 to T-6). Their amino acid composition and The peptide eluting at 14.6 min was analyzed by sequence sequences are shown in Table 1 and Fig. 2, These results analysis and by hydrolysis and amino acid analysis. suggest that T-5 is a carboxyl-terminal fragment of DH, as Hexapeptides with the sequence of the tryptic fragment only T-5 has neither arginine nor lysine at its carboxyl T-5, Asn-Phe-Leu-Asn-Asp-Ile, were synthesized as the terminus. Therefore, DH was regarded as a 41-residue carboxyl-terminal free acid and the amide by Biosearch under peptide. The nature of the carboxyl terminus was established contract. The crude peptides were purified by reversed-phase by comparison of reversed-phase LC retention times of T-5 LC essentially as described for tryptic digests. and the synthetic hexapeptide Asn-Phe-Leu-Asn-Asp-Ile prepared in the amidated and free acid forms. The data show clearly that the carboxyl terminus of T-5 is amidated (Fig. 3). RESULTS Hence, the complete structure of DH was established as Isolation Procedure. After lipids had been removed from shown in Fig. 2. This structure is also supported by amino the trimmed heads by extraction with acetone, DH was acid analysis of intact DH (Table 1). extracted by using an acidic solution containing two protease Synthesis and Biological Properties of M. sexta DH. We inhibitors. This acidic solution was applied directly to an synthesized M. sexta DH, as well as an analogue with an acid SP-Sephadex column, which was eluted stepwise with dif- at the carboxyl terminus, by using conventional automated ferent ammonium acetate buffers of increasing pH and/or solid-phase methods. The synthetic amidated and acidic ionic strength. The active fractions from SP-Sephadex were forms were compared to native DH by reversed-phase LC applied to a C4 reversed-phase "cartridge" similar to com- analysis, which again established the identity of native DH mercially available disposable cartridges, except containing with the synthetic amidated form. Synthetic DH and its acid 10 g of adsorbent. The cartridge was eluted stepwise with analogue were tested in the Pieris assay. The approximate CH3CN/0.1% aqueous TFA; DH-active material eluted in ED50 for the amide form was 0.1 ng per animal, that for the the 35% CH3CN fraction. The latter solution was diluted with acid was 0.1 Ag per animal. twice its volume ofwater and then loaded onto a semiprepar- We investigated the effects of synthetic DH on M. sexta ative reversed-phase LC column by using the pump. This larvae in vivo. When 0.1 ,ug of synthetic DH was injected into technique avoided losses of biological activity that occurred postfeeding, prewandering larvae, a mean of 226 mg of fluid when fractions were evaporated at each step. Two successive was lost over a 4-hr period from those injected with synthetic semipreparative reversed-phase purifications were per- DH, while controls excreted a mean of98 mg. This is a highly formed, using different organic modifiers. The active fraction significant difference (P < 0.001). from the second semipreparative reversed-phase LC was further purified by ion-exchange LC, analytical reversed- DISCUSSION phase LC, and finally microbore reversed-phase LC (Fig. 1). At the final step, DH activity coincided with a peak that Control of water balance is vital to the survival of insects, absorbed UV at 220 nm, but this material had no UV although the requirements for each species vary greatly. For absorbance at 280 nm (not shown). The overall yield was insects living in dry environments water retention is critical, about 5 nmol from 10,000 trimmed heads. while other species may consume their body weight in foliage H-Arg-Met-Pro-Ser-Leu-Ser-Ile-Asp-Leu-Pro-Met-Ser-Val-Leu-Arg-Gln-Lys-Leu-Ser-Leu- f T-6 t I T-4

Glu-Lys-Glu-Arg-Lys-Val-His-Ala-Leu-Arg-Ala-Ala-Ala-Asn-Arg-Asfn-Phe-Leu-Asn-Asp-Ile-NH2 T T-2 T-5 I T-l1 IT-3 FIG. 2. Amino acid sequence of M. sexta DH. T-1 to T-6 indicate sequences determined for tryptic peptides. Downloaded by guest on September 28, 2021 Physiological Sciences: Kataoka et al. Proc. Natl. Acad. Sci. USA 86 (1989) 2979 Table 1. Amino acid composition of DH and its tryptic peptides Residues per molecule Residue Intact DH T-1 T-2 T-3 T-4 T-5 T-6 Asx 4.92 (5) 0.73 (1) 3.14 (3) 0.79 (1) Glx 2.63 (3) 0.71 (1) Ser 3.69(4) 1.05 (1) 3.14 (3) Gly 0.24 (0) His 0.63 (1) 0.38 (1) 1.03 (1) Thr 0.41 (0) Ala 4.00 (4) 3.00 (3) 1.09 (1) 1.00 (1) Arg 5.43 (5) 1.20 (1) 1.04 (1) 1.09 (1) 1.14 (1) Pro 2.00 (2) 2.28 (2) Tyr 0.05 (0) Val 1.52 (2) 0.57 (1) 0.77 (1) 0.% (1) Met 1.56 (2) 1.59 (2) Ile 1.93 (2) 0.97 (1) 1.00 (1) Leu 6.72 (7) 1.00 (1) 1.04 (1) 2.00 (2) 1.06 (1) 3.00 (3) Phe 1.03 (1) 0.99 (1) Lys 3.38 (3) 0.89 (1) 0.95 (1) Position 1-41 31-35 26-30 25-30 18-22 36-41 2-15 Numbers in parentheses are from sequence analyses. Trp and Cys were not determined. each day, requiring elimination ofmost ofthe ingested water. DH, although the chromatographic conditions differ. About Diuresis is especially striking in blood-feeding insects which 5 nmol ofdiuretic hormone was isolated from 10,000 trimmed rapidly excrete the fluids obtained from a blood meal of 2-12 pharate adult heads. Proving that the carboxyl terminus was times the unfed body weight (1-3). Most aquatic insects must amidated required chemical synthesis ofthe tryptic fragment rid themselves of excess water acquired by osmosis (3, 6). T-5 in both the free acid and amidated forms. Certain insects excrete water during flight as a result of the In early experiments, we fractionated M. sexta DH by intense metabolism required (2). The most difficult osmoreg- using Sephadex G-50. The mobility of bioactive fractions ulatory problems may occur in insect larvae that dwell in salt suggested a molecular weight of 5000-6000. This is roughly water (3, 24). consistent with the value of 4732 calculated for the sequence Most attempts to characterize DH structurally have been shown in Fig. 2. based on difficult and tedious in vitro Mt bioassays (8). Other In an earlier attempt to isolate DH, we found that biological efforts have been based on an in vivo amaranth dye excretion activity was diminished or lost when we evaporated solvent test (9), and a semi-isolated Mt assay (10). Recently, more from either the reversed-phase cartridge step or the semi- indirect, but rapid in vitro bioassays have been adopted. preparative purifications, possibly due to association with Isolated Mt have been monitored for transepithelial voltage proteins, adsorption, or oxidation. Accordingly, we modified changes after DH stimulation (11). RIA techniques have been the pumping system so that water-diluted fractions contain- used to detect an increase in cAMP in DH-stimulated Mt (10, ing organic solvent could be loaded onto the column by the 12). pump. M. sexta DH has two methionine residues (positions Our isolation was guided by an in vivo bioassay on P. 2 and 11), perhaps rendering it quite sensitive to oxidation. rapae. Newly emerged adult butterflies of this species were We checked for sequence similarity of M. sexta DH with neck-ligated and beheaded, depriving the abdomen ofa pulse known peptides by using a computer program (Intelligenet- of DH, which ordinarily stimulates substantial loss of a clear ics). This located a substantial similarity with sauvagine (ref. fluid from the rectum. Neck-ligated animals excrete only a 26, Fig. 4). Sauvagine was isolated from skins of the frog dark meconium (waste products of pupal metabolism). In- Phyllomedusa sauvagei while using a bioassay based on its jection of DH-active fractions into ligated butterflies caused potent antidiuretic effect in rats (27). Ifallowance is made for a typical normal urine excretion. a one-amino acid insertion of histidine in M. sexta DH We isolated M. sexta DH in parallel with eclosion hormone (position 27), 40% ofthe residues are identical. An additional (EH), which we recently purified and sequenced (25). Both 16 amino acids in M. sexta DH could result from single-base hormones are extracted together, but they are separated by mutations in the sauvagine gene. The location of the muta- SP-Sephadex ion-exchange chromatography. The strategy of tions strengthens the supposition that histidine has been subsequent purification steps is nearly identical for EH and inserted in a well-conserved sequence. Sauvagine is but one

Carp Urotensin I N D D|P|P I|S I D LIT F H L L R NMI EMARNENQ R E Q A G L N RIK Y D E V NH2 Sucker Urotensin I N D D P P I SI D LIT F H LIL RIN MI E M A RI EN E R E Q A G L NR KY D E V NH2

Sauvagine Q G P P I S I D LIS L E LIL R K M I E I E K Q E K E K Q Q A A N NR L L DT I-NH2 Manduca sexta DH R M P S L S I D LI.P M S VIL R Q K L S L E K E R K V H A L R A A A N RIN F N D I-NH2

Caprine/ovine CRF S Q EIPI P I S L D L T F H LIL R E V L E M TKA D Q L A Q Q AH S N RI K LL D I NH2

Human/Rat CRF S E EIPIP I S L D L T F H LIL R EVLEMARAEQL AQQ A H S NR K L N E I I

FIG. 4. Comparison of amino acid sequence of Manduca DH with sequences of urotensin I (carp and sucker), sauvagine, and CRF (caprine, ovine, human, and rat). One-letter amino acid symbols are used. Residues identical with those in M. sexta DH are enclosed by solid lines. Downloaded by guest on September 28, 2021 2980 Physiological Sciences: Kataoka et al. Proc. Natl. Acad Sci. USA 86 (1989) member of an important peptide family including CRF and of another peptide, which would act as a DH on Mt. These urotensin I. CRF, originally isolated and identified from data do suggest that the concept of a unique "post-eclosion sheep hypothalami (oCRF, ref. 28), has been sequenced DH" (16) may be in error. subsequently from the rat (rCRF, ref. 29), goat (cCRF, ref. In summary, we have isolated a diuretic factor from M. 30), and cow (bCRF, ref. 31). The structure of human CRF sexta that exhibits diuretic effects in vivo, although we cannot was deduced by sequencing its gene isolated from a genomic say at this time whether it acts directly on the Mt or triggers library (hCRF, ref. 32). The sequence identity ofM. sexta DH release of another peptide from a tissue outside the head. The with oCRF/cCRF is 32%; an additional 41% similarity would latter result is suggested by the sequence similarity to a family result from single-base changes in the gene. Urotensin I, of releasing factors and by our inability to observe direct peptides biologically and chemically homologous to sau- effects of M. sexta "DH" on the presumed target tissue. vagine and CRF, were isolated and identified from fish urophysis (analogous to the hypothalamus). M. sexta DH We thank the staff of the insect culture group for providing shares 27% sequence identity with both white sucker (33) and animals, Marilyn Bennett for technical assistance, Blake Cesarin for carp (34) urotensin I; an additional 44% identity with sucker determining amino acid compositions, Dr. Alan Roter for a computer urotensin I could come from single-base changes in the gene. search of peptide homology, and Dr. Herbert Roller for encourage- Thus, M. sexta DH is the newest member ofthe sauvagine/ ment. CRF/urotensin I family of peptides, now represented in the 1. Phillips, J. E. (1984) Am. J. Physiol. 241, R241-R257. The most 2. Maddrell, S. (1986) in Insect Neurochemistry and Neurophysiology: classes Insecta/Amphibia/Mammalia/Pisces. 1986, eds. Borkovec, A. B. & Gelman, D. B. (Humana, Clifton, NJ), pp. studied physiological/pharmacological effects of this peptide 79-90. family appear to be the stimulation of adrenocorticotropic 3. Bradley, T. J. (1987) Annu. Rev. Entomol. 32, 439-462. hormone (ACTH) release and hypotensive/vasodilatory 4. Gee, J. D. (1977) in Transport ofIons and Water in Animals, eds. Gupta, properties; the antidiuretic effect of sauvagine has been B. L., Moreton, R. B., Oschman, J. L. & Wall, B. L. (Academic, New York), pp. 265-281. attributed to its potent hypotensive action (26). In addition, 5. Herault, J. E. & Proux, J. P. (1987) J. Insect Physiol. 33, 487-491. stimulation of f-endorphin release accompanies ACTH re- 6. Nicholls, S. P. (1985) J. Exp. Biol. 116, 53-67. lease induced by oCRF (28) or sauvagine (26). The potency 7. Nicolson, S. W. (1980) J. Insect Physiol. 26, 841-846. and character ofthe effects elicited by each peptide vary with 8. Aston, R. J. & Hughes, L. (1980) in Neurohormonal Techniques in Insects, ed. Miller, R. A. (Springer, New York), pp. 91-115. the species in which it is tested. For example, both types of 9. Proux, J., Rougon, G. & Cupo, A. (1982) Gen. Comp. Endocrinol. 47, urotensin I and sauvagine elicit a strong and selective 448-457. hypotensive/vasodilatory effect in mammals, actually more 10. Mordue, W. & Morgan, P. J. (1985) in Neurosecretion and the Biology than that caused by the mammalian form oCRF (34). ofNeuropeptides, eds. Kobayashi, H., Bern, H. A. & Urano, A. (Japan potent Sci. Soc. Press, Tokyo and Springer, New York), pp. 418-424. In contrast, injection of 500 pmol of urotensin I, sauvagine, 11. Petzel, D. H., Hagedorn, H. H. & Beyenbach, K. W. (1985) Am. J. and oCRF did not stimulate fluid excretion in the P. rapae Physiol. 249, R379-R386. assay (R.G.T., unpublished data). By comparison, the ED50 12. Rafaeli, A. (1984) in Insect Neurochemistry and Neurophysiology, eds. M. sexta DH is :0.02 Borkovec, A. B. & Kelly, T. J. (Plenum, New York), pp. 463-466. of pmol. 13. Morgan, P. J., Siegert, K. J. & Mordue, W. (1987) Insect Biochem. 17, Proux et al. (15) recently identified an antiparallel ho- 383-388. modimer of [Leu2,Thr4,Arg8]vasotocin as a DH of L. migra- 14. Schooley, D. A., Miller, C. A. & Proux, J. P. (1987) Arch. Insect toria. This result seems difficult to reconcile with our Biochem. Physiol. 5, 157-166. a member of a structurally divergent peptide 15. Proux, J. P., Miller, C. A., Li, J. P., Carney, R. L., Girardie, A., identifying Delaage, M. & Schooley, D. A. (1987) Biochem. Biophys. Res. Commun. family as a DH of M. sexta. However, the AVP-like locust 149, 180-186. DH has no activity in the P. rapae assay (R.G.T., unpub- 16. Dores, R. M., Dallmann, S. H. & Herman, W. S. (1979) J. Insect lished data), and conversely, immunological studies have Physiol. 25, 895-901. shown there is no AVP-like factor in M. sexta (S.J.K. and A. 17. Troetschler, R. G., Malone, C. M., Bucago, E. R. & Johnston, M. R. (1985) J. Econ. Entomol. 78, 1521-1523. Toschi, unpublished data). It is interesting that AVP (while 18. Cherbas, P. (1973) Ph.D. Thesis (Harvard Univ., Cambridge, MA). less potent than CRF) also triggers release of ACTH from 19. Baker, F. C., Tsai, L. W., Reuter, C. C. & Schooley, D. A. (1987) Insect mammalian pituitary (28). It is also intriguing that urotensin Biochem. 17, 989-996. been of a role in osmoregulation (35). 20. Ebert, R. F. (1986) Anal. Biochem. 154, 431-435. I has suspected playing 21. Barany, G. & Merrifield, R. B. (1980) in The Peptides, eds. Gross, E. & Thus, there may be some overlap in biological roles of these Meienhofer, J. (Academic, New York), Vol. 2, pp. 1-284. two distinct peptide families. 22. Konig, W. & Geiger, R. (1970) Chem. Ber. 103, 788-798. ACTH has been implicated in water balance in vertebrates 23. Shaltiel, S. & Fridkin, M. (1970) Biochemistry 9, 5122-5127. to its on aldosterone Accordingly, 24. Garrett, M. S. & Bradley, T. J. (1987) J. Exp. Biol. 129, 231-238. due control production. 25. Kataoka, H., Troetschler, R. G., Kramer, S. J., Cesarin, B. J. & Rafaeli et al. (36) recently investigated the possible existence Schooley, D. A. (1987) Biochem. Biophys. Res. Commun. 146, 746-750. of an ACTH-like material in locust CC. They found that the 26. Montecucchi, P. C. & Henschen, A. (1981) Int. J. Pept. Protein Res. 18, CC contain an ACTH-like material, and that ACTH at 10 ,AM 113-120. and increases cAMP 27. Montecucchi, P. C., Anastasi, A., de Castiglione, R. & Erspamer, V. stimulates liquid excretion from, in, (1980) Int. J. Pept. Protein Res. 16, 191-199. locust Mt. Extracts of CC produce similar effects in the 28. Vale, W., Spiess, J., Rivier, C. E. & Rivier, J. (1981) Science 213, 1394- traditional (8) in vitro Mt assay (36). Morgan et al. (13), 1397. assaying the increase in cAMP in Mt induced by tissue 29. Rivier, J., Spiess, J. & Vale, W. (1983) Proc. Natl. Acad. Sci. USA 80, a for a diuretic 4851-4855. fractions, reported partial sequence peptide 30. Ling, N., Esch, F., Bohlen, P., Baird, A. & Guillemin, R. (1984) also isolated from locust CC. This partial sequence has no Biochem. Biophys. Res. Commun. 122, 1218-1224. similarity to M. sexta DH or to the AVP-like locust DH (15). 31. Esch, F., Ling, N., B6hlen, P., Baird, A., Benoit, R. & Guillemin, R. Curiously, synthetic M. sexta DH does not increase cAMP in (1984) Biochem. Biophys. Res. Commun. 122, 899-905. 32. Shibahara, S., Morimoto, Y., Furutani, Y., Notake, M., Takahashi, H., Mt of M. sexta larvae (R.G.T., unpublished data). Shimizu, S., Horikawa, S. & Numa, S. (1983) EMBO J. 2, 775-779. In ourP. rapae assay, injection ofsynthetic DH and its acid 33. Lederis, K., Letter, A., McMaster, D., Moore, G. & Schlesinger, D. analogue revealed that the acid form was about 1/1000th as (1982) Science 218, 162-164. bioactive as the amide form. Injection of DH into M. sexta 34. Ichikawa, T., McMaster, D., Lederis, K. & Kobayashi, H. (1982) a loss of not via the but Peptides 3, 859-867. larvae caused profound fluid, only gut 35. Marshall, W. S. & Bern, H. A. (1981) Gen. Comp. Endocrinol. 43, 484- through the epidermis as well. Our results do not rule out the 491. possibility, suggested by the work of Rafaeli et al. (36), that 36. Rafaeli, A., Moskitzky, P. & Applebaum, S. W. (1987) Gen. Comp. the CRF-like M. sexta "DH" could actually stimulate release Endocrinol. 67, 1-6. Downloaded by guest on September 28, 2021