Neuropeptides (1990) l&107-109 @I Longman Group UK Ltd 1990

Structure of the Hypertrehalosemic Neuropeptide of the German Cockroach, Blattella germanica

J. A. VEENSTRA* and F. CAMPS

Department of Biological Organic Chemistry, Centro de Investigacibn y Desarrollo, C.S.I.C., Jorge Girona Salgado, 18-26, 08034 Barcelona, Spain. *Present address: Department of Entomology, College of Agriculture, The University of Arizona, 434 Forbes, Building No. 36, Tucson, AZ 85721, USA (Reprint requests to FCl

Abstract-The structure of the hypertrehalosemic neuropeptide of the German cockroach, Blattells germanica, was found to be identical to that of the cockroach species, Bfaberus

discoidalis and Nauphoera cinerea (Glp-Val-Asn-Phe-Ser-Pro-Gly-Trp-Gly-Thr-NH*.. Since Blatrella gerrnanica is not closely related to Blaberus discoidalis and Nauphoera cinerea, this supports the hypothesis that in this peptide family evolution of peptide structure may be related to evolution of peptide function.

Introduction

Crustacean red pigment concentrating hormone (RPCH) and locust adipokinetic hormone (AKH) were the first members to be fully characterized of an arthropod peptide family now known as the AKHIRPCH-family (1, 2). In crustaceans pep- tides of this family induce the concentration of red pigment, while in insects they increase haemo- lymph concentrations of lipid in locusts and moths (AKH) or trehalose in cockroaches (hypertreha- losemic hormone, HTH), and they may also increase the rate of heart beat. The structures of peptides of the AKI-URPCH family show consider- able variability between different insect species (3). unlike in crustaceans, where only a single RPCH has been found so far. Why the evolution of

Date received 24 July 1989 Date accepted 24 July 1989

the structures of these peptides has occurred at a higher rate in insects than in crustaceans is unclear.

Three members of this peptide family have been isolated from cockroaches. These are two octapep- tides from the American cockroach, Periplaneta americana, which have been called Periplanetins CC-l and CC-2, Ml and M2, or HTHl and HTH2 (4-6), while a decapeptide HTH has been isolated and sequenced from Blaberus discoidalis and Nau- phoeta cinerea (7, 8). We have recently described the isolation of the HTH of Blattelfagermanica (9). Since this cockroach species belongs to another family (Blattellidae) than either Blaberus and Nauphoeta (Blaberidae) or Periplaneta (Blatti- dae), it seemed of interest to determine the structure of Blattella HTH.

Materials and Methods

Isolation of the peptide. amino acid composition

107

108 NEUROPEPTIDES

analysis and enzymatic deblocking were per- formed as described before (9,lO). The deblocked HTH was sequenced by “Servicio de secuencia- ci6n de proteinas de la universidad de Barcelona” on a model 470A protein sequencer of Applied Biosystems Inc. (Foster City, CA, USA), accord- ing to the program supplied by the manufacturer; the generated phenylthiohydantoin amino acids were analyzed online by a model 120A of the same company.

Results and Discussion

The amino acid composition of the purified pep- tide yielded the presence of the following amino acid residues: Asx(l), Glx( l), Ser(l), Gly(2), Thr( 1)) Pro( 1)) Val( 1) and Phe( 1). This composi- tion is identical to that of the HTH of Bluberus and Nauphoeta, with the exception of tryptophan, which is destroyed during the hydrolysis, and thus suggested that Bluttellu HTH could be identical to Blaberus and Nauphoeta HTH. However, Scar- borough et al. have warned against relying only on amino acid compositional data and homology to deduce the structures of peptides of this family, since their initial guess for the structure of peri- planetin CC-2 based on such considerations pro- ved to be wrong (4).

Bluttellu I-ITH had the same retention time in HPLC when either heptafluorobutyric acid or trifluoroacetic acid (TFA) was used as the pairing ion. This indicates (11) that it did not have a free amino terminal, as was expected, since all peptides of the AKI-I/RPCH-family are N-terminally blocked by pyroglutamate. The peptide was there- fore digested with pyroglutamate amino pepti- dase. For the deblocked peptide the following sequence was obtained: Val-Asn-Phe-Ser-Pro- Gly-Trp-Gly-Thr. Together with the N-terminal pyroglutamate this leads to: Glp-Val-Asn-Phe- Ser-Pro-Gly-Trp-Gly-Thr, which accounts for all amino acid residues found in the compositional analysis. Since all members of the AKIWRPCH- family are C-terminally amidated, it was expected that Blatcella HTH would also have a C-terminal amide group. Peptides of this size with identical primary sequences, but which differ in the pres- ence or absence of a C-terminal amide have

different elution times on HPLC when a gradient between water and acetonitrile with TFA as the pairing ion is used (7, 11, 13). Since Bluttellu HTH eluted at the same time as the C-terminally amidated synthetic peptide (synthetic Nuuphoecu HTH from Peninsula Laboratories), Blattellu HTH must be amidated as well. The complete structure of Bluttellu HTH is thus: Glp-Val-Asn- Phe-Ser-Pro-Gly-Trp-Gly-Thr-amide.

The large structural variability of peptides of the AKWRPCH family is in marked contrast with peptides related to the leucosulfakinins, the only other insect peptide family for which the sequences are known from different species. These peptides, which are related to gastrin and cholecystokinin, have now been isolated and sequenced from the Madeira and American cockroach (10, 13, 14), and the sequence of their mRNA has been reported for Drosophila (15). This shows that there have been marked differ- ences in the rate of evolution of these two insect peptide families. One possible explanation for the faster rate of evolution in the AKWRPCH family might be that the receptors for these peptides do not distinguish very well between the different peptides, and that hence there has been no strong evolutionary pressure to conserve their structures. The almost equal potency of several members of the AKIWRPCH family in elevating trehalose concentrations in Periplunetu (16) and haemo- lymph lipid concentrations in the migratory locust Locusta migrutoria (17) seem to support this hypothesis. It is interesting to note, however, that these peptides mobilize quite different energy substrates in different insect groups, trehalose in cockroaches, lipid in moths and locusts, and proline and glucose in the Colorado potato beetle (18). This suggests that changes in structure may have coincided with changes in function. If that were the case, one would expect the structures of these peptides to have been conserved whenever their functions have been conserved. The presence of the same HTH in Blattella as in Nauphoeta and Blaberus, even though Blattella is not closely related to the other species, supports this second hypothesis on the evolution of the AKHRPCH family. This hypothesis can be further tested by determining the structures of peptides in this family in other insect species.

STRUCTURE OF THE HYPERTREHALOSEMIC NEUROPEPTIDE OF THE GERMAN COCKROACH 109

Acknowledgements

We thank Mr. A. Pons and Mrs. M.-T. Esteve for amino acid analysis, Mr. C. Buesa for sequencing, Mr. J. Lloria for some of the cockroaches used, and Dr. T. Marfnez for her interest. This work was supported by a postdoctoral fellowship to JAV from the Spanish Ministry of Education and Science and grants form C.A.I.C.Y.T. (84187) and C.S.I.C. (851263).

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