Neuropeptides (1989) 14,145-149 @) Longman Group UK Ltd 1989

0143-4179/89/0014-0145/$10.00

Isolation and Structure of Two Gastrin/CCK-Like Neuropeptides From the American Cockroach Homologous to The Leucosulfakinins

J. A. VEENSTRA

Department of Biological Organic Chemistry, Centro de Investigacidn y Desarrollo, C.S.I.C., Jorge Girona Salgado 18-26, 08034 Barcelona, Spain. Correspondence to: Dept.of Entomology, 434 Forbes Building, The University of Arizona, Tucson, AZ 85721, USA

Abstract-Perisulfakinin, a peptide with sequence similarity to gastrin and cholecystokinin, was isolated from the corpora cardiaca of the American cockroach. Its sequence was determined to be Glu-Gln-Phe H-Asp-Asp-Tyr(SOaH)-Gly-His-Met-Arg-Phe-amide. The peptide induced hindgut contractions in the same species at concentrations as low as 250pM. A related non-sulfated peptide was also isolated and sequenced; it was found to be identical with non-sulfated leucosulfakinin II (pGlu-Ser-Asp-Asp-Tyr-Gly-His-Met-Arg-Phe-amide). This peptide did not stimulate hindgut contractions. The structures of the cockroach peptides of the leucosulfakinin family are thus much more conserved than the cockroach hypertrehalosemic hormones.

Introduction

In recent years a large number of vertebrate peptides have been demonstrated by immunocyto- chemistry and/or radioimmunoassay in inverte- brates. Although the immunological reaction of an invertebrate peptide with an antiserum to a verte- brate peptide may indicate some resemblance between the immunoreactive invertebrate com- pound and the vertebrate peptide, this does not necessarily indicate an evolutionary relationship between the two (1). Invertebrate peptide charac- terization studies are therefore essential to a full

understanding of the evolution of a peptide family. Several interesting homologies or even complete identity in structure between vertebrate and invertebrate neuropeptides have been reported (2-6), which include the leucosulfakinins, two gastrin/cholecystokinin related peptides from the Madeira cockroach (7, 8). Here the isolation and structure of two related peptides from the Ameri- can cockroach is reported.

Materials and Methods

Experimental animals

Date received 17 April 1989 American cockroaches, Periplaneta americana, Date accepted 18 April 1989 were derived from a laboratory culture kept at 27

145

146 NEUROPEPTIDES

+- 2°C. They were fed dog chow and had access to water.

Preliminary extraction

For peptide extraction 2- to 6-month-old animals were used. They were decapitated and the corpora cardiaca (CC) dissected under 0.9% NaCl and stored in a glass homogenizer on dry ice. Batches of up to 600 pair of corpora cardiaca were homo- genized in lml of Bennett’s mixture (1% NaCl, 5% formic acid,,l% trifluoroacetic acid (TFA) and 1M HCI in water (9)) to which 12~1 thiodiglycol had been added. The extract was centrifuged and the pellet reextracted in OSml of Bennett’s mix- ture. The combined supernatants were loaded on a previously activated and equilibrated C-18 Sep- Pak (Waters Associates, MA, USA). The Sep-Pak was washed with 5ml water and 5ml of 13% acetonitrile, and the peptides were eluted with 5 ml 32% acetonitrile (all eluants contained 0.1% TFA). This fraction was concentrated by nitrogen evaporation and purified by high performance liquid chromatography (HPLC).

Peptide HPLC

The HPLC apparatus consisted of two Waters model 510 pumps, a Waters model M730 data module, a model 680 gradient controller and a model 481 spectrophotometer. Separations were performed on a 15 x 0.46cm column and a 5 x 0.46cm precolumn (Tecnocroma, San Cugat des Valles, Spain) filled with 5 pm particles of Spher- isorb ODS-II. Solvents were water (A) and 65% acetonitrile (B), which contained either TFA (0.1% and 0.11%) or heptafluorobutyric acetic acid (HFBA, 0.13% and 0.145%) as pairing ions. In both cases the column was eluted isocratically for 30 min with 21% B, followed by a linear gradient from 21% to 51% B over 60 min at flow rate of lml/min. Absorption was monitored at 210nm.

Amino acid composition

The amino acid composition was determined using a Pica-Tag system (Waters Associated, MA, USA), after hydrolysis in HCl vapour for 1 hr at 150°C (10).

Enzymatic deblocking

Peptide was solubilized in 10 ~1 0.1 N HCl; 100 ~1 of Zalut’s buffer (11) containing 2.5 units of pyroglutamate amino peptidase (Sigma) was then added and the mixture incubated for 15 min at 37°C under nitrogen. The reaction was terminated by addition of 2501~1 of 0.1% TFA in water. The deblocked peptide was isolated by HPLC using TFA as pairing ion at a flow rate of 0.8mYmin. After 20 min isocratic elution of 21% B a linear gradient to 81% B over 30 min was used.

Sequence analysis

Sequencing of the peptides was performed by “Servicio de sequenciacion de Proteinas de la Universidad de Barcelona”, using a model 470A protein sequencer from Applied Biosystems Inc. (Foster City, CA, USA), according to the program supplied by the manufacturer. Phenylthiohydan- toin (PTH) amino acids were analyzed online by a model 120A detector made by the same company.

C-terminal analysis

Dry peptide (ca 600pmol perisulfakinin or 4OOp- mol non-sulfated leucosulfakinin-II) was solu- bilized in 20 (~1 0.1 M ammonium bicarbonate; 30 ~1 of the same buffer containing 1 kg TPCK- treated trypsin was then added and the mixture incubated under nitrogen for lhr at 37°C. The reaction was stopped by freezing and after lyophil- ization the mixture was derivatized with PITC as described (10) and analyzed by HPLC as follows: column (25 x 0.4cm) filled with 10krn particles Spherisorb ODS-II (Tecnocroma, San Cugat des Valles, Spain); solvent A: 25mM sodium acetate, 0.05% triethylamine, pH 6.35; solvent B: 60% acetonitrile; column temperature 52°C; gradient: linear from 20% to 44% B over 40 min; absor- bance measured at 254nm; flow rate O.Bml/min.

Tyrosine-sulfate determination

Tyrosine-O-sulfate was synthetized and purified as described (12, 13). Purified peptide was hydro- lyzed at 105°C for 18hr in 0.2M barium hydroxide. After cooling the solution was carefully neutral- ized with concentrated sulfuric acid and the barium sulfate removed by centrifugation. The supernatants were analyzed by HPLC using the

ISOLATION AND STRUCTURE OF TWO GASTRINICCK LIKE NEUROPEPTIDES 147

HPLC fractionation of Sep-Pak prepurified peptides pairs of corpora cardiaca using HPEBA as pairing ion.

The peak indicated by the closed arrow contains non-sulfated leucosulfakinin II, the one indicated by the open arrow perisulfakinin. Peaks 1 and 2 contain periplanetins CC1 and CC2 respectively.

same conditions as described for the analysis of the C-terminal groups. The only differences were that solvent A contained 200mM sodium acetate, and the gradient was linear from 10% to 52% B over 20 min. Under these conditions standard phenylthio- carbamyl (PTC) amino aids were well resolved, but the separations of the PITC derivatized pep- tide hydrolysates were not perfect, although the individual PTC amino acid peaks could be identi- fied. The absence or presence of PTC tyrosine sulfate was therefore confirmed by collecting the peaks which should contain these amino acids and reanalyzing them using 25mM sodium acetate in solvent A. Under these conditions the elution time of all PTC amino acids, but especially of PTC tyrosine sulfate is different. This procedure led to well resolved peaks and the presence or absence of tyrosine sulfate was confirmed.

Bioamays The assay on the hindgut was performed as described (14), using young adult males as hindgut donors.

Results and Discussion

Perisulfakinin was accidentally found while look- ing for a hypertrehalosemic peptide in extracts of

the corpora cardiaca from the American cock- roach. When fractionating a Sep-Pak prepurified extract by HPLC using the TFA system, hyper- trehalosemic activity was found to coelute with a peak with a retention time in between the two octapeptides periplanetin CC1 and CC2 from this species (15). In a pilot experiment this peak was found to elute later using HFBA as pairing ion. We therefore tried to purify the hypertrehalosemic activity, thought to be associated with this peak, by two successive HPLC steps, the first using HFBA as pairing ion (Fig. l), and the second using TFA (Fig. 2). Amino acid analysis showed that the peptide was pure, but it did not have hypertre- halosemic activity. The following amino acids were found: Asx(2), Glx(2), Gly( l), His(l), Arg(l), Tyr(l), Met(l) and Phe(2), and a content of 2Spmol of peptide per pair of CC. This amino acid composition suggested the presence of a peptide related to the leucosulfakinins. Auto- mated gas phase sequencing yielded the following sequence: Glu-Gln-Phe-Asp-Asp-Tyr-Gly-His- Met-Arg-Phe, while no amino acid residue was found in the next degradation cycle. After trypsin digestion of the peptide Phe-amide was found and hydrolysis in barium hydroxide revealed the presence of tyrosine sulfate. Thus the complete structure of this peptide, which was called peri- sulfakinin, is: Glu-Gin-Phe-Asp-Asp-Tyr(S03H)- Gly-His-Met-Arg-Phe-amide. Later a second pep- tide with a similar amino acid composition was

50 55 60 65 70 75 80 a5 ml”

hne

Fig. 2 Final puritication of perisulfakinin (open arrow) using TFA as pairing ion. The small arrows labelled 1 and 2 indicate the elution position of periplanetins CC1 and CC2 in this system.

148 NEUROPEPTIDES

r,me

Fig. 3 Final purification of non-sulfated leucosulfakinin II (closed arrow) using TFA as pairing ion.

found. This peptide eluted earlier in the HFBA (Fig. 1) and TFA systems (Fig. 3). Amino acid analysis of this peak showed the presence of a single peptide with the following composition: Asx(2), Glx(l), Ser(l), Gly(l), His(l), Arg(l), Tyr(l), Met(l), Phe(l) and the presence of 1.1 pmol peptide per CC pair. The N-terminus of this peptide appeared to be blocked, and the peptide was therefore incubated with pyrogluta- mate aminopeptidase, which digested it and yielded a new peptide, the sequence of which was found to be Ser-Asp-Asp-Tyr-Gly-His-Met-Arg- Phe, while no amino acid residue was detected in the next degradation cycle. Digestion of the intact peptide with trypsin yielded Phe-amide, while alkaline hydrolysis showed the absence of tyrosine sulfate. Combined these data led to the following structure: Glp-Ser-Asp-Asp-Tyr-Gly-His-Met-Arg- Phe-amide, or non-sulfated leucosulfakinin II. Perisulfakinin stimulated hindgut contractions at a concentration of 0.1 CC pair equivalents per ml (ca

Fig. 4 Effects of perisulfakinin (closed bars) and non-sulfated leucosulfakinin II (open bar) on the hingut contractions of the American cockroach. Numbers indicate the concentrations of the peptides in the organ bath in pair equivalents of corpora cardiaca per ml.

250pM) in five preparations tested, 0.05 CC equiv- alents per ml was in all cases without effect (Fig 4). Non-sulfated leucosulfakinin II, however, had no effect in four preparations tested (maximum con- centration tested was 5.OCC pair equivalents per ml). This was expected as non-sulfated leuco- sulfakinins are also unable to stimulate hindgut contraction in the Madeira cockroach (7,8).

It is of interest to note that leucosulfakinin I and perisulfakinin are almost identical in structure, only the fourth amino acid residue differs, Asp in perisulfakinin and Glu in leucosulfakinin I, which is a conservative change. Analysis of mRNA coding for two related peptides in Drosophila also showed Asp residues in this position (16). The finding of non-sulfated leucosulfakinin II in Peri- planeta suggests that sulfated leucosulfakinin II may be present in this species’ as well. The American and Madeira cockroach belong to the same insect order but this in itself does not explain the close structural similarity of these peptides, since they belong to different superfamilies which have separated long enough to allow evolution of substantial differences in neuropeptides, as is shown by the hypertrehalosemic peptides of these species. Whereas the American cockroach has two octapeptides (15), only one peptide has been found in the Madeira cockroach (17), which is most likely identical to the decapeptide isolated from other species of the same superfamily (17, 19). This raises interesting questions about the causes of such disparate rates of evolution for different insect peptides.

Acknowledgements

I thank Dr. T. Martinez for her excellent assistance with the bioassays, Mr. N. Kregar for several gifts of cockroaches from which my laboratory culture was started, Mr. A. Pons and Mrs. Ma.-T. Esteve for amino acid composition analysis, Mr. C. Buesa for sequencing the peptides, Drs. F. Camps and T. Martinez for their interest, support and critical reading of the manuscript, and Drs E. Giralt and F. X. Aviles for helpful discussions. This work was supported by a postdoctoral fellow- ship from the Spanish Ministry of Education and Science and grants from C.A.I.C.Y.T. (84187) and C.S.I.C. (851263).

References

1. Veenstra, J. A. (1988). Immunocytochemical demon- stration of vertebrate peptides in invertebrates: The homology concept. Neuropeptides 12: 49-54.

ISOLATION AND STRUCIURE OF TWO GASTRIN/CCK LIKE NEUROPEF’TIDES 149

2. Bodenmiiller. H. and Schaller, H. C. (1981). Conserved amino acid sequence of a neuropeptide, the head activator, from coelenterates to humans. Nature 293: 579-580.

3. Leung, M.K. and Stefano, G.B. (1984). Isolation and identification of enkephalins in pedal ganglia of Myrilus edulis (mollusca). Proceedings of the National Academy of Sciences USA 84: 955-958.

4. Nagasawa, H., Kataoka. H., Isogai, A., Tamura. S., Suzuki. A., Mizoguchi, A., Fujiwara, Y., Suzuki. A., Takahashi, S. T. and ishizaki, H. (1986). Amino acid sequence of a prothoracicotropic hormone of the silkworm Bombyx mori. Proceedings of the National Academy of Sciences USA 83: 5840-5843.

5. Matsumoto. S., Isogai, A. and Suzuki, A. (1985). N-termi- nal amino acid sequence of an insect *neurohormone. melanization and reddish coloration hormone (MRCH): heterogeneity and sequence homology with human insulin- like growth factor II. FEBS Letters 189: 115-118.

6. Proux, J. P.. Miller, C. A., Li, J. P., Carney, R. L., Girardie, A., DeLaage. M. and Schooley, D. A. (1987). Identification of an arginine vasopressin-like diuretic hormone from tocusru migmoria. Biochemical and Bio- physical Research Communications 149: 180-186.

7. Nachman, R. J., Holman, G. M., Haddon. W. F. and Ling, N. (1986). Leucosulfakinin, a sulfated insected neuro- peptide with homology to gastrin and cholecystokinin. Science 234: 71-73.

8. Nachman, R. J., Holman, G. M., Cook, B. J., Haddon, W, F. and Ling, N. (1986). Leucosulfakinin II, a blocked sulfated insect neuropeptide with homology to cholecysto- kinin and gastrin. Biochemical and Biophysical Research Communications 140: 357-364.

9. Bennett, H. P. J., Brown, C. A. and Solomon, S. (1981). Purification of the two major forms of rat pituitary corticotropin using only reversed-phase liquid chroma- tography. Biochemistry 20: 4530-4538.

10. Bidlingmeyer, B. A., Cohen, S. A. and Tarvin. T. L. (1984). Rapid analysis of amino acids using pre-column derivatization. Journal of Chromatography 336: 93-104.

11. Zalut, C., Henzel, W. J. and Harris, H. W.. Jr, (1980). Micro quantitative Edman manual sequencing. Journal of Biochemical and Biophysical Methods 3: 11-30.

12. Reitz, H. C., Ferrel, R. E., Fraenkel-Conrat, H. and Olcott, H. S. (1946). Action of sulfating agents on proteins and model substances. I. Concentrated sulfuric acid. Journal of American Chemical Society 68: 1024-1031.

13. Hutter, W. B. (1984). Determination and occurrence of tyrosine-O-sulfate in proteins. Methods in Enzymology 107: 200-223.

14. Starratt. A. N. and Steele, R. W. (1980). Proctolin: Bioassay, Isolation and Structure. In: Miller, T. A. (ed.), Neurohormonal Techniques in Insects. Springer-Verlag. New York. p. l-30.

15. Scarborough, R. M., Jamieson, G. C., Kalish, F.. Kramer, S. J.. McEnroe, G. A., Miller, C. A. and Schooley, D. A. (1984). Isolation and primary structure of two peptides with cardioacceleratory and hyperglycaemic activity from the corpora cardiaca of Periplaneta americana. Proceed- ings of the National Academy of Sciences USA 81: 5575-5579.

16. Nichols, R.. Schneuwly, S. A. and Dixon, J. E. (1988). Identification and characterization of a Drosophila homo- logue to the vertebrate neuropeptide cholecystokinin. Journal of Biological Chemistry 263: 12167-12170.

17. Gade. G. and Scheid, M. (1986). A comparative study on the isolation of adipokinetic and hypertrehalosemic factors from insect corpora cardiaca. Physiological Entomology 11: 145-157.

18. Hayes, T. K.. Keeley, L. L. and Knight, D. W. (1986). Insect hypertrehalosemic hormone: Isolation and primary structure from Balberus discoidalis cockroaches. Bio- chemical and Biophysicai Research Communications 140: 674-678.

19. Glde, G. and Rinehart, K. L.. Jr.,(1986). Amino acid sequence of a hypertrehalosaemic neuropeptide from the corpus cardiacum of the cockroach, Nauphoera cinerea. Biochemical and Biophysical Research Communications 141: 774-781.