Vol. 179, No. 2, 1991 September 16, 1991







Lehmberg lp4 , Ronald B. Ota2, Kenji Furuya2, David S. Kings, Shalom Applebaum’, Hans-J&g Ferenz’, and David A. Schooleyz * ‘Faculty

2Department 3Howard Hughes


of Agriculture, The Hebrew University Rehovot 76100, Israel

of Jerusalem,

of Biochemistry,

Reno, NV 89557


41nsect Physiology Received


of Nevada,

Institute, Dept. of Molecular & Cell Biology, of California, Berkeley, CA 94720

Group, University 3,


of Oldenburg,

D-2900 Oldenburg,


University &many


We have isolated a peptide from brains and corpora cardiaca of Locusta migratoria which is immunologically related to the diuretic hormone of Mancluca se&a. We determined its structure as a 46 amino acid linear peptide with 43-50% identity to the M. sexta hormone. Moreover, we showed that the new peptide functions as a diuretic hormone in L. migratoria, stimulating urine production by Malpighian Plx?SS,Inc.


and elevating

levels of CAMP in tubules.

o 1991Academic

A considerable literature exists on the physiology of water balance in insects and its regulation (1,2). Insect diuretic hormones are generally regarded as stimulants of urine secretion by the Malpighian tubules, but they might also act by decreasing fluid resorption by the rectum (2). Multiple factors may act on these tissues; three peptide factors affect Malpighian tubules of the mosquito Aedes aegypti (3), whilst two peptide factors in corpora cardiaca of Locusta migratoria affect Malpighian tubules of this species in different ways (4). A partial

sequence for one of the L. migratoria

factors, DP-1, has been reported


?I’he sequence reported in this paper has been deposited in the NBRF data base (accession number A23702). *To whom correspondence should be addressed. Abbreviations: Br, brains; CC, corpora cardiaca; DH, diuretic hormone; ELISA, enzyme-linked immunosorbent assay; FMOC, fluorenylmethoxycarbonyl; HFBA, heptafluorobutyric acid; HOBt, 1-hydroxybenzotriazole; LC, liquid chromatography; Lom DH, Locusta migratoria DH; Mas DH, Manduca sexta DH; TFA, trifluoroacetic acid. 0006-291x191 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.



179, No. 2, 1991




To date, the complete chemical structures of only two insect diuretic hormones are known: an antiparallel homodimer of [Ile2,Thr4]arginine vasotocin from ganglia of L. migratoria (the AVP-like DH) (51, and a 41 amino acid peptide (Mas DH) from “trimmed heads” of Manduca sexta (6). The latter has a high degree of identity to the corticotropin releasing factor/sauvagine/urotensin I family of peptides and stimulates in viva fluid secretion from two species of lepidoptera, but its exact mode of action is unreported. We screened an extract of brains (Br) and corpora cardiaca (CC) of L. migratoria with an ELISA assay developed (7) for Mas DH, and observed a strong cross reactivity. We herein report the isolation and identification of the immunoreactive factor, a 46 amino acid peptide with 4350% sequence identity (depending on alignment) to Mas DH. Moreover, this material functions as a diuretic hormone in L. migratoria, stimulating primary urine production by Malpighian tubules and causing a dose-dependent stimulation of CAMP levels in the tubules. MATERIALS


Insect cultu~? and tissue collection. Locusta migratoria migratorioides were kept under crowded conditions and constant illumination at a temperature of 28+2’ C, and fed flaked oats and fresh grass. Br/CC were excised from one animal at a time, the optic lobes removed, and the material frozen in liquid N2 and stored at -70 C. 4600 Br/CC from male and female locusts were collected. Ptrrillcation of peptides from the Br/CC. The tissue was homogenized in acetone at 0” C with a Polytron. Peptides were then extracted with an acidic buffer (1.0 M acetic acid with 20 mM H2SO4, 0.1 mM phenyhnethylsulfonyl fluoride, and 0.01 mM pepstatin A) at 0” C. The crude extract was adsorbed on 1 g of Vydac C4 packing. After washing with 0.1% TFA, the cartridge was eluted with 30 and 60% acetonitrile in 0.1% TFA/water. To each fraction 0.1% 2-(methylthio)ethanol was added as antioxidant. Purifications by LC were performed with a Perkin-Elmer Model 410 Bio pump, a Rheodyne loop injector, and a Perkin-Elmer Model 235 detector set at 220 and 280 nm. The pump was modified so that water-diluted fractions could be pumped into the column (8). The first purification was performed with a Vydac C4 column, 250 x 10 mm, using a O-60% CH&N/O.l% TFA gradient at 5 ml/min. The second step was done with a Vydac C4 column, 150 x 4.6 mm, with a O-30% 1-propanol/O.l % TFA gradient at 1.0 ml/min; the third step was done with the same column, but eluted with O-60% CH&N/O.l % HFBA at 1.0 mUmin. The fourth step was performed with a Vydac Crs column, 150 x 4.6 mm, using a O-60% CH$N/O.l % TFA gradient at 1.0 ml/mm. The fifth step was an ion exchange LC performed on TSK SP 5PW, eluted with a gradient of O-O.4 M NaCl containing 0.02 M HzPO4-, pH 6.25, and 10% CHsCN. The sixth step was a reversed-phase LC with Vydac C4 column, 150 x 4.6 mm, with a O-30% 1-propanol/O.l% HFBA gradient at 0.8 ml/mm. The final separation used a Vydac C4 column, 150 x 2.1 mm, eluted with O-60% CH&N/O.l% TFA at 0.2 ml/mm. Aliquots (always 1 Br/CC equivalent) of each fraction were assayed with a recently developed ELISA to determine materials immunoreactive with Mas DH (7). Structural analy&s of peptide Sequence analyses were performed with a Porton Model 2090 gas phase sequencer with integral PTH amino acid analyzer. Data 1037


179, No. 2, 1991




were collected with a Spectra Physics WINner data system. Enzymatic cleavages were conducted at 37” C for 24 hr with endoproteinase Asp-N (sequencing grade) or endoproteinase Glu-C, both from Boehringer Mannheim. For both the buffer was pH 8.0 Tris-HCl, 0.1 M for Glu-C and 0.05 M for Asp-N. Fragments were isolated as described for the final purification. Peptides were hydrolyzed in uucuo using 5.7 M HCl + 1% phenol at 110’ C for 20 hr, and the hydrolysate analyzed using a Hewlett Packard Amino Quant system with fluorescence detection. Peptiaesynthesis. The two hexapeptides corresponding to the C-terminal fragment Asp-Nl, Asp-Phe-Leu-Gln-Gln-Ile-NH2 and Asp-Phe-Leu-Gln-GlnIle-OH, were synthesized by Research Genetics, Huntsville, Alabama, USA. Lom DH was synthesized using solid phase methodology with an Applied Biosystems 431A automated synthesizer on FMOC-aminomethyl Rink resin utilizing HBTU (tetramethyluronium HOBt PFs3 (9) FMOC-amino acid activation in the presence of HOBT and DIEA with user-devised l-2 hr single coupling cycles with lo-15 fold molar excess of acylating species. Protecting groups were Arg(2,2,5,7,8-pentamethylchromansulfonyl), Asn(Trity1) and Gln(Trity1). Peptide resin cleavage and deprotection were accomplished in a single step using reagent K for 4 hr at 20° C (10) to afford crude peptide amide of -2O-30% purity. The sample was purified by a single pass on a Vydac C4 column, 250 x 10 mm, 30 to 45% CHsCN/O.l% TFA over 60 min, yielding material of -98% purity (-11% actual isolated yield) as judged by analytical LC and capillary zone electrophoresis. Structure and purity were confirmed by electrospray ionization mass spectrometry on a VG Bio-Q (calculated 5363.39, found 5363.41* 0.44). FtESULm AND DISCUSSION Pilot studies showed a very high level of Mas DH-like immunoreactivity in extracts of adult Br/CC. We isolated this factor using modifications of procedures used to isolate the DH and other hormones of M. se&a (8). After removing lipids from the tissue, the Mas-DH like factor was extracted and adsorbed on Vydac C4 bulk packing. The Mas DH-like factor was eluted with a 60% acetonitrile fraction, which was further purified by seven sequential LC separations. An apparent pair of immunoreactive peaks of about equal height, obtained in the fourth purification, appeared nearly homogenous in the following purification stages. Subsequent examination of the Vydac Crs column revealed a void in the packing, which probably caused artifactual appearance of a “doublet”. The final isolated sample used for microanalysis was also tested for biological affects. Isolated L. migrutoria Malpighian tubules were maintained in vitro (11) in a 25 ~1 droplet of minimal essential medium until secretion stopped. A group was stimulated with 0.5 Br/CC equivalent of purified peptide in 10 ul medium; secretion reached 0.45 ris0.16 nl/min (n= 14). We added 10 ul medium to a control group, which resumed secreting at 0.16 f 0.03 nl/min (n= 8). The level of CAMP induced by stimulation of tubules with the peptide was assayed by RIA (11) and a dose/response determined. Maximal stimulation was observed with 0.5 Br/CC equivalent (Fig. 1). Initial microsequence analysis of the pure peptide gave a sequence to 45 residues, but with ten of these undetermined (Fig. 2). Sequence analysis of the 1038


179, No. 2, 1991









0 18025





w. Measurement of CAMP levels in Malpighian tubules of L. migrutoria maintained in vitro with various levels of the pure immunoreactive peptide.

fragments Glu-Cl,


and Glu-C3

allowed the assignment of the blank residues. The most difficult were the Arg residues at positions 26,27,28, and 30, due to low recoveries of PTH-Ax-g observed. The assignment of these residues as Arg was confirmed by amino acid analysis of fragment Glu-C2 (Table 11, showing four Arg residues in this 11 amino acid fragment. The C-terminal Ile residue was not observed even on sequencing the Asp-N1 fragment due to “wash-off’ from the support. The presence of this residue was proven by amino acid analysis of Glu-C3, and of the crucial fragment Asp-Nl. Subsequently, the two possible hexapeptides corresponding to Asp-Nl, Asp-Phe-Leu-Gln-Gln-Ile-NH2 and Asp-Phe-Leu-Gln-Gln-Ile-OH were synthesized, and the fragment of native peptide was found to co-elute with the amidated form. Peptide with the sequence

? -&p-


? -u-Ile-Gln-Ala-

? -va1-Tyr-Lys





~-Lys-Val-His-Ala-LWI-~-Ala-~28 Az-Arg-ArgGlu-Cl Glu-CL



~-Bsn-Arg-Asn-~-LW1-Asn-ASp-~-NHZ 30 ~-~q-~sp-~-Glu-Glu-Gln-Ilr-Lys-~-BSn-Lys-Asp-~-~-Gln-Gln-~-NH~ --_-----------Glu~C3





a0 ----


Asp-N1 -






Fpure 2. Sequences of DP-l(4), M. s&u DH (Mr= 4,732) (61,and L. m@ztoriu DH (Mr= 5,364), summarizing the proof of sequence. Underlined amino acids are identical between the two DHs. Arrows underneath an amino acid residue show it was positively identified in the parent peptide (top line of arrows) or in various fragment peptides (lower arrows). 1039




179, No. 2, 1991





Table 1 acid composition of crucial fragment peptides of Lom DH

Asx (D/N) Ser (S) Glx (E/Q) Arg (R) Ala (A) Phe (F) Ile (I) Leu (L) LYS WI






i.29 (2)

ii.47 (2)

i 1.00(1) 1.02(1) 1.14(1) 0

3.92 2.37(4) (2) 0 1.06(1) 1.00(1) 0

2.11(2) 0.37 (0) 3.33 (3) 0 1.25(1) 1.00(1) 1.69(2) 0.98( 1) 2.46(2)

Residues per molecule of fragment peptide, normalized on Phe or Leu, compared to residues predicted from the sequence(Fig. 2). Amino acids not shown were not detected in hydrolysates.

assigned was synthesized using automated solid phase techniques (FMOC chemistry); the purified, synthetic peptide was found to have precisely coincident retention behavior with native Lom DH on coinjection on reversed-phase LC. We use the terminology Lom DH for this hormone in accord with a recent convention (12). The complete sequence shows a direct 43% sequence identity (not shown) with Mas DH. In addition to a two residue extension at the amino terminus, Lom DH has at least a three residue insertion between a conserved region ending at Arg27 and another conserved region beginning at Ala38. Sequence identity of 50% is observed by inserting a two residue gap in the Lom DH sequence and a five residue gap in the Mas DH sequence (Fig. 2). (With the more conventional alignment, the identity of Leu2’, Arg3’, and Ala32 is lost.) A partial sequence of eight residues was reported earlier for DP-1, a diuretic peptide which stimulates cAMP in locust Malpighian tubules (4). Lom DH and DP-1 both have Gly at residues 2 and 4; all other residues differ. The homology of Lom DH to the corticotropin releasing factor/sauvagine/urotensin I family of peptides is lower than for Mas DH, being only 22% identical with sauvagine (131, the most similar of the non-insect members. Lom DH has no similarity to the AVP-like DH previously reported (51, but multiple diuretic factors have been reported previously in single insect species (3) including the locust (4). Acknowledgments We are grateful to Dr. Steven J. Kramer for antibodies to Mas Dr. Jorge Li for synthetic Mas DH. This work was partly supported Crop Protection and the Nevada Agriculture Experiment Station (to the Mauerberger Foundation Fund (to SWA). EL was the recipient Award during her research in Rehovot. 1040

DH, and to by Sandoz DAS) and by of a Minerva

Vol. 179, No. 2, 1991




REFERENCES of Insects (R. G. H. Downer and H. 1. Phillips, J. E. (1983) in Endocrinology Laufer, eds.), pp. 411-425, A. R. Liss, New York. 2. Spring, J. H. (1990) J. Insect Physiol. 36,13-22. 3. Beyenbach, K. W., and Petzel, D. H. (1987) News Physiol. Sci. 2,171-175. 4. Morgan, P. J., Siege& K. J., and Mordue, W. (1987) Insect Biochem. 17, 383388. 5. 6. 7. 8.



Proux, J. P., Miller, C. A., Li, J. P., Carney, R. L., Girardie, A., Delaage, M., and Schooley, D. A. (1987) Biochem. Biophys. Res. Commun. 149,180-186. Kataoka, H., Troetschler, R. G., Li, J. P., Kramer, S. J., Carney, R. L., and Schooley, D. A. (1989) Proc. Natl. Acad. Sci. USA 86,2976-2980. Furuya, K., Ota, R. B., Reynolds, S. E., Schooley, D. A., Troetschler, R., and Kramer, S. J. (1991) Insect Biochem. submitted. Schooley, D. A., Carney, R. L., Kataoka, H., Kramer, S. J., Li, J. P., Toschi, A., and Troetschler, R. (1990) in Molecular Insect Science (H. H. Hagedorn, J. G. Hildebrand, M. G. Kidwell and J. H. Law, eds.), pp. 199-212, Plenum Press, New York. Knorr, A., Trzeciak, A., Bannwarth, W., and Gillessen, D. (1989) Tet. Lett. 30, 1927-1930. King, D. S., Fields, C., and Fields, G. (1990) Int. J. Pept. Protein Res. 36, 255266.

11. Rafaeli, A., Pines, M., Stern, P. S., and Applebaum, S. W. (1984) Gen. Comp. Endocrinol. 54,35-42. 12. Raina, A. K., and Gade, G. (1988) Insect Biochem. 18, 785-787. 13. Montecucchi, P. C., and Henschen, A. (1981) Int. J. Peptide Protein Res. 18, 113-120.


Identification of a diuretic hormone of Locusta migratoria.

We have isolated a peptide from brains and corpora cardiaca of Locusta migratoria which is immunologically related to the diuretic hormone of Manduca ...
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