COMMUNICATION
Solution Structure of an Insect Growth Factor, Growth-blocking Peptide*

Tomoyasu AizawaDagger , Naoki FujitaniDagger , Yoichi Hayakawa§, Atsushi Ohnishi§, Tadayasu Ohkubo, Yasuhiro KumakiDagger , Keiichi Kawanoparallel **, Kunio HikichiDagger , and Katsutoshi NittaDagger

From the Dagger  Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan, the § Institute of Low Temperature Science, Hokkaido University, Sapporo 060-0819, Japan, the  Japan Advanced Institute of Science and Technology, Ishikawa 923-1292, Japan, and the parallel  Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Toyama 930-0194, Japan

    ABSTRACT
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Abstract
Introduction
References

Growth-blocking peptide (GBP) is an insect growth factor consisting of 25 amino acid residues that retards the development of lepidopteran larvae at high concentration while it stimulates larval growth at low concentration. In this study, we determined the solution structure of GBP by two-dimensional 1H NMR spectroscopy. The structure contains a short segment of double-stranded beta -sheet involving residues 11-13 and 19-21 and a type-II beta -turn in the loop region (residues 8-11), whereas the N and C termini are disordered. This is the first report of the three-dimensional structure of the peptiderigic insect growth factor, and the structure of the well defined region of GBP was found to share similarity with that of the C-terminal domain of the epidermal growth factor (EGF). Because GBP has been reported to stimulate DNA synthesis of not only insect cells but also human keratinocyte cells at the same level with EGF, the structural similarity between GBP and EGF may lead to the interaction of GBP to EGF receptor.

    INTRODUCTION
Top
Abstract
Introduction
References

Endoparasitic wasps can alter the development of their hosts from larvae to pupae. This developmental disturbance allows the wasps to complete their larval growth and to emerge while the host is still in the larval stage, otherwise the wasp larvae would be trapped in the sclerotized pupal cuticles. Growth-blocking peptide (GBP)1 was originally isolated from the larval hemolymph of the host armyworm Pseudaletia separata whose development is halted in the last larval instar stage from parasitization by the parasitoid wasp Cotesia kariyai (1-3). The fact that injection of 20 pmol of GBP into early last instar larvae of the armyworm retards larval growth and causes more than a few days delay in pupation clearly shows its growth blocking activity (2, 3). We have since determined that GBP is a host gene product and that the change of titers of this peptide correlate well with the growth rate of insect larvae (4, 5). Recently, injection studies of GBP indicated that low concentrations (lower than 1 pmol/larva) stimulated larval growth, whereas high concentrations (higher than 10 pmol/larva) retarded growth (6). Furthermore, bioassay data revealed that several pmol/ml of GBP stimulated DNA synthesis of SF-9 insect cells and human keratinocyte cells, although several nmol/ml of GBP did not stimulate cell proliferation at all. Therefore, it is thought that GBP can act as a growth factor controlling the proliferation of a broad range of cells and the growth rate of whole larvae.

To examine structural features of GBP, NMR was used in this study. The solution structure that we report here was obtained by two-dimensional 1H NMR spectroscopy with the aid of distance geometry and simulated annealing. The three-dimensional structure of GBP contains a short double-stranded beta -sheet and a beta -turn in the C-terminal region, while six residues in the N terminus and three residues in the C terminus are highly disordered.

    EXPERIMENTAL PROCEDURES

1H NMR Spectroscopy-- The 25-residue peptide GBP was prepared by the Peptide Institute (Osaka, Japan) using an Applied Biosystem Model 430A peptide synthesizer with tert-butyloxycarbonyl chemistry and was further purified by high performance liquid column chromatography as described previously (7). The two cysteines were oxidized to form an intramolecular disulfide bond.

The GBP sample was dissolved at a final concentration of 1.5-3.0 mM in 350 µl of either 99% D2O or 90% H2O/10% D2O at pH 4.4. The pH was adjusted to 4.4 by adding microliter increments of HCl and NaOH. The NMR experiments were performed on either a JEOL JNM-Alpha 600 or a Varian Unity-plus 750 spectrometer. The majority of NMR spectra were recorded at a temperature of 30 °C, and some experiments were also recorded at 10 °C to resolve ambiguities. DQF-COSY spectra (8) were collected at 30 and 10 °C. TOCSY spectra (9) were obtained at 30 and 10 °C with a mixing time ranging from 50 to 100 ms. NOESY spectra (10) were collected at 30 °C with a mixing time of 100-500 ms. The water signal was suppressed by the DANTE pulse (11). Each spectrum size was 1024-4096 complex points in the t2 dimension and 200-512 complex points in t1. The NMR data were zero filled in both dimensions, and a shifted sine-bell was applied as a window function for resolution enhancement. Amide protons protected from solvent exchange were identified on a sample that had been lyophilized and resuspended in D2O.

Structure Calculations-- A total of 117 distance restraints for the structure calculations were obtained from the NOESY spectrum acquired with a mixing time of 300 ms at 30 °C. The distance restraints can be subdivided into 37 intraresidue, 43 sequential, 20 medium range, and 17 long range. The cross-peak intensities were classified into three classes, strong (1.6-2.7 Å), medium (1.6-3.5 Å), and weak (1.6-5.0 Å). Seven dihedral angle restraints were derived from the 3JHN-Halpha coupling constants measured from the DQF-COSY spectrum. These coupling constants greater than 8 Hz were used to estimate torsion angles phi  to be constrained in the range from -160 ° to 80 °. The amide resonances that were still visible after more than 30 min in D2O were identified as hydrogen bond donors. Hydrogen bond acceptors could be unambiguously determined on the basis of the secondary structure and preliminary structure calculations. Three hydrogen bond restraints identified from a series of one-dimensional NMR spectra of hydrogen-deuterium exchange experiments were used as input for the calculations (1.7-2.7 Å (H-O) and 2.5-3.5 Å (N-O)). Three-dimensional structure calculations were carried out with the computer program X-PLOR 3.1 (12) using the simulated annealing protocol. Initial structures were generated with fully extended conformation. The constraints from backbone NOEs were used as input for the first distance geometry. Each structure was subjected to one round of simulated annealing with all distance and angle constraints. The simulated annealing consisted of 6000 cooling steps from 2000 K to a final temperature of 100 K. A total of 100 structures were calculated. All 100 structures were passed through the refinement, which consisted of 15000 cooling steps from 2000 to 100 K. The 20 structures with the lowest energy that contained no NOE restraint violations greater than 0.1 Å were selected. The RMSD values were calculated by using the programs X-PLOR and MOLMOL (13).

    RESULTS AND DISCUSSION

The GBP molecule was soluble in water and monomeric as determined by NMR spectroscopy. At various peptide concentrations (3, 0.3, and 0.06 mM), there were no concentration-dependent changes that indicate dimerization or aggregation in the chemical shifts or peak line widths of one-dimensional 1H NMR spectra of GBP. In addition, no NOE cross-peaks indicative of dimeric or multimeric structure were detected.

The proton NMR signals were identified according to the standard procedure of sequential assignment (14). The sequential assignment of the residues was achieved by first identifying spin systems by a combination of DQF-COSY spectra and TOCSY spectra acquired with different mixing times. This procedure was followed by assigning identified spin systems to particular residues in the peptide by the observation of sequential NH-NH (dNN), Calpha H-NH (dalpha N), and Cbeta H-NH (dbeta N) NOEs. A nearly complete assignment of the proton NMR signals of GBP was obtained. The NMR signals of the backbone protons for Glu1 and Asn2 were either not observed or not resolved. The fingerprint region of the NOESY is shown in Fig. 1.


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Fig. 1.   Fingerprint region from a NOESY spectrum of GBP recorded at pH 4.4 in H2O at 30 °C with a 300 ms mixing time. The sequential dalpha N connectivities are indicated. Residue 15 and 21 are prolines. The alpha proton of Met12 is not assigned. The cross-peak for Thr14 that is not visible in this spectrum is labeled because it was observed and confirmed in other spectra.

Fig. 2 shows the pattern of sequential and short range NOE cross-peaks, the 3JHN-Halpha coupling constants, and the slowly exchanging amide protons for GBP. Elements of the secondary structure were deduced from the pattern of sequential and medium range NOE connectivities. Additional information was obtained from the H-D exchange study and the values of 3JHN-Halpha coupling constants. Slowly exchanging amide protons due to the formation of hydrogen bonds were identified from a series of one-dimensional NMR spectra. In these spectra, three backbone amide proton signals remained intensely after more than 30 min and were classified as slowly exchanging amide protons. From the DQF-COSY spectrum, nine residues were determined to have 3JHN-Halpha greater than 8.0 Hz. The secondary structures that were identified from these data are summarized in Fig. 3. The secondary structure elements of GBP consist of a double-stranded antiparallel beta -sheet that involves Tyr11-Arg13 and Cys19-Pro21 and a beta -turn that involves Val8-Tyr11. The regions of the beta -sheet were further confirmed by the interstrand dalpha alpha NOEs between Tyr11 and Pro21 and between Arg13 and Cys19 (Fig. 4). The type-II beta -turn (Val8-Tyr11) is identified by patterns of NOE, a strong dalpha N NOE between Ala9 and Gly10, a strong dNN NOE between Gly10 and Tyr11, and a weak dalpha N NOE between Ala9 and Tyr11, which is held together by a hydrogen bond between Val8 and Tyr11. Position 3 of this beta -turn is Gly10, the preferred residue here in a type-II beta -turn.


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Fig. 2.   Diagram of NOE connectivities between protons of neighboring (dalpha N, dbeta N, and dNN), J-coupling constants (3JHN-Halpha ), and slowly exchanging amide protons in D2O. The strength of the observed NOE is represented by the thickness of the bars. Residues with 3JHN-Halpha of >8 Hz are indicated by arrows. The backbone amide protons, which exchange slowly with D2O, are indicated by filled circles. The dalpha N of Met12 is not identified because the alpha proton of Met12 is not assigned (*). Glu1 and Asn2 are not assigned (#).


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Fig. 3.   Identification of the antiparallel beta -sheet and the type II beta -turn. The NOE connectivities are indicated by arrows. The broken line indicates hydrogen bonds for which slowed amide-proton exchange was observed.


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Fig. 4.   alpha H-alpha H region from a NOESY spectrum of GBP recorded at pH 4.4 in H2O at 30 °C with a 300 ms mixing time. Cross-peaks between alpha protons are annotated.

A total of 130 NMR-derived distance and angle constraints were used as input for distance geometry and simulated annealing calculations using the program X-PLOR. A family of 20 accepted three-dimensional structures were used to represent the solution structure of GBP (Fig. 5 and Table I). The N-terminal and C-terminal residues, Glu1-Gly6 and Phe23-Gln25, are not well defined in the NMR structure because the NOE data are insufficient to ascertain a single conformation. The mean pairwise RMSD of the well defined region (Cys7-Thr22) is 0.89 Å for the backbone atoms and 1.72 Å for all heavy atoms. The core structure of GBP, the small antiparallel beta -sheet and the beta -turn were further confirmed by the conformation. The beta -sheet is twisted in a right-handed manner. The loop region, which is composed of five residues (Thr14-Arg18) with no secondary structure, is less converged than the beta -turn (Val8-Tyr11). The mean pairwise RMSDs of the loop region are 0.88 and 1.80 Å for the backbone and all heavy atoms, respectively. On the other hand, those values of the beta -turn are lower, 0.71 Å for the backbone and 1.01 Å for all heavy atoms. However, the disulfide bond between Cys7 and Cys19 appears to contribute significantly to the stability of the loop region, as well as to establish important conformational constraints for defining a single conformation. Side chains in the beta -turn were well defined, having RMSD values lower than 1.50 Å. The side chains of Cys7 and Cys19, and Pro21 also had low RMSD values. The other side chains were not well resolved. In particular, the charged side chains of Arg13, Arg18, Lys20, and Asp16 had high RMSD values. However, it is uncertain whether this result is due to conformational flexibility or a lack of distance restraints of these residues.


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Fig. 5.   Three-dimensional structure of GBP. A, stereo view of the ensemble of 20 NMR structures of GBP superimposed for best fit over the backbone atoms of residues 7-22 of the mean coordinate structure. B, ribbon drawing of the minimized average structure of GBP. These diagrams were generated using the program MOLMOL (13).

                              
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Table I
Structural statistics of 20 structures of GBP
Represents the structural statistics for the 20 energy minimized structures obtained through the refinement protocol in X-PLOR. Eimpr, EVDW, and ENOE are improper energy of torsion angles, van der Waals' repulsion energy, and the square well NOE potential energy, respectively. All energies and RMSD values were calculated using the programs X-PLOR and MOLMOL.

We recently reported that GBP and human EGF (epidermal growth factor) induce an almost equivalent increase in DNA synthesis of a human keratinocyte cell line (6). On the other hand, GBP induces a higher and more persistent increase in SF-9 insect cell proliferation than does EGF. These results suggest that GBP has an ability to stimulate cell proliferation more broadly than does EGF. Fig. 6 compares the overall structure of GBP with that of mouse EGF (15). The general folding of GBP, with the exception of the N-terminal disordered region, clearly conforms to the C-terminal domain of EGF with weak primary structural similarity (Fig. 7). The backbone RMSD between the average GBP structure and the average mouse EGF structure over the region of the beta -sheet and the beta -turn is 1.98 Å. In particular, the type II beta -turn exists in GBP and EGF, is composed of Val8-Tyr11 in GBP, and corresponds to Val34-Tyr37 in EGF with high amino acid sequence conservation. The three-dimensional structural similarity of backbone between GBP and EGF raises the possibility that these two growth factors may stimulate the same receptor. Moreover, some highly conserved amino acid residues in EGF family are conserved in GBP. It is known that these residues are very important in EGF receptor-ligand association. For example, substitution of the highly conserved Arg41 and Leu47 of EGF resulted in a marked reduction in the affinity of the EGF receptor, which suggests that these residues are essential for receptor affinity (16, 17). It is likely that Arg18 of GBP corresponds to Arg41 of EGF in light of their alignments and three-dimensional structures. On the other hand, GBP does not have a Leu residue that corresponds to Leu47 of EGF, although it is thought that the hydrophobicity of Leu47 is important for interaction with the EGF receptor. Thus, it is possible that Phe23 and/or Tyr24 of GBP may act instead of Leu47 of EGF. In fact, elimination of Phe23 leads to a reduction in the growth blocking activity of GBP (18), suggesting that Phe23 of GBP may play an important role just as Leu47 of EGF does. To our knowledge, such a small peptide as stimulates EGF receptor at high level has not been found. If further studies elucidate the interaction of GBP with EGF receptor clearly, the structure of GBP may offer clues to the interaction between EGF and EGF receptor, which is still not very clear.


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Fig. 6.   Comparison of the three-dimensional structures of mouse EGF (A) and GBP (B). The disulfide bonds, Cys33-Cys42 in EGF and Cys7-Cys19 in GBP, are shown with balls and sticks. The drawing of mouse EGF was made using atomic coordinates obtained from the Brookhaven Protein Data Bank (entry set 1EPG). This diagram was generated using the WebLab Viewer computer program (Molecular Simulations Inc.).


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Fig. 7.   Sequence alignment of human EGF, mouse EGF, and GBP. The amino acid sequence of GBP was aligned to the C-terminal region of EGF. The partial C-terminal amino acid sequence of human and mouse EGF shows 24-28% homology with the GBP. The disulfide connectivity is shown by a solid line.

GBP was initially identified as a hemolymphal peptide that delays the normal development of the host insect larvae parasitized by parasitic wasps (1-3). GBP cDNAs have been cloned from three lepidopteran species, P. separata, Mamestra brassicae, and Spodoptera litura, and the amino acid sequences of GBP in these three species are highly homologous (6, 19). In addition, the amino acid sequences of paralytic peptides and plasmatocyte-spreading peptide isolated from insect hemolymph have been shown to be highly homologous to those of GBP. Each of seven paralytic peptides found in hemolymph of Manduca sexta, Spodoptera exigua, and Heliothis virescens causes a temporary paralysis when injected into larvae (20). On the other hand, plasmatocyte-spreading peptide from the larvae of Pseudoplusia includens induces the adhesion and spreading of plasmatocytes to foreign surfaces (21). Although it has been reported that all these GBP and GBP-like peptides vary in function, they share at least 14 completely conserved amino acids. This is the first report on the three-dimensional structure of these insect peptides. In addition, there has been no report on the three-dimensional structure of a peptiderigic growth factor in insects. Knowledge of the structure of GBP is important not only for elucidating the mechanism of its activity but also for clarifying the physiological roles of these homologous peptides and growth factors in insects.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (code 1BQF) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.

** To whom correspondence should be addressed: Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan. Tel./Fax: 81-764-34-5061; E-mail: kawano{at}ms.toyama-mpu.ac.jp.

The abbreviations used are: GBP, growth-blocking peptide; EGF, epidermal growth factor; DQF-COSY, double quantum filtered correlation spectroscopy; NOESY, nuclear Overhauser effect spectroscopy; RMSD, root mean square difference; NOE, nuclear Overhauser effect; TOCSY, total correlation spectroscopy; DANTE, delays alternating with nutation for tailored excitation.
    REFERENCES
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Abstract
Introduction
References

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