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WO1997035981A1 - Insect neuropeptides genes and peptides - Google Patents

Insect neuropeptides genes and peptides Download PDF

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Publication number
WO1997035981A1
WO1997035981A1 PCT/GB1997/000843 GB9700843W WO9735981A1 WO 1997035981 A1 WO1997035981 A1 WO 1997035981A1 GB 9700843 W GB9700843 W GB 9700843W WO 9735981 A1 WO9735981 A1 WO 9735981A1
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WIPO (PCT)
Prior art keywords
leu
gly
tyr
phe
peptide
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PCT/GB1997/000843
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French (fr)
Inventor
Alan Thorpe
Hanne Duve
Anders Holten Johnsen
Peter East
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Btg International Limited
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Application filed by Btg International Limited filed Critical Btg International Limited
Priority to JP09534145A priority Critical patent/JP2001501806A/en
Priority to EP97914442A priority patent/EP0923645A1/en
Priority to AU21688/97A priority patent/AU2168897A/en
Publication of WO1997035981A1 publication Critical patent/WO1997035981A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C07K14/43577—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies

Definitions

  • the present invention relates to novel DNA isolated from the blowfly C. vomi toria which encodes novel peptides of the callatostatin class of insect peptides.
  • the novel peptides have also been isolated and purified from C. vomi toria .
  • the DNA also encodes the other known Leu-callatostatin peptides of C. vomitoria.
  • the homologue of this gene has also been isolated from the Australian sheep blowfly Lucilia cuprina and peptides of this class have also been shown to exist in the codling moth, Cydia pomonella and the armyworm Helicoverpa armigera , both potential targets for insecticides based on these peptides. Variants of these peptides have been identified from the large willow sawfly Pteronida salici s .
  • the first group of callatostatins to be isolated from Calliphora vomi toria were shown to have some sequence homology to cockroach allatostatins (Duve et al in Proc . Na t ' l . Acad . Sci . USA 90: 2456-2460) and the peptides have the following sequences:
  • the first four peptides are known as Leu-callatostatins, now designated Leu-callatostatins 1, 2, 3 and 4.
  • the fifth peptide is known as Met-callatostatin or Met-cast (Duve et al Cell Ti ssue Res . 276 367-379 (1994)) .
  • Met-callatostatin or Met-cast Duve et al Cell Ti ssue Res . 276 367-379 (1994)
  • Met-callatostatin or Met-cast Duve et al Cell Ti ssue Res . 276 367-379 (1994)
  • Met-callatostatin or Met-cast Duve et al Cell Ti ssue Res . 276 367-379 (1994)
  • three further Met-callatostatins have been isolated (Duve et al J. Biol . Chem. 269 21059-21066 (1994) ; Duve et al Regul .
  • Met-callatostatinpeptides Two of the additional Met-callatostatinpeptides are post-translationally modified by hydroxylation at either the Pro 2 or Pro 3 amino acid residues of the parent Met-callatostatin.
  • the third Met- callatostatin is an N-terminally truncated des-Gly 1 -Pro 2 variant.
  • the peptides have the following sequences:
  • Pro-Tyr-Asp-Phe-Gly-Met-NH 2 (designated Met-callatostatin 3-8 or des-Gly-Pro-Met-callatostatin) .
  • the Leu-callatostatin sub-family of peptides appears to be characterised by the possession of the C-terminal sequence - Tyr-Xaa-Phe-Gly-Leu-NH 2 (where Xaa is Gly or Ser) .
  • the four Met-callatostatins differ from the Leu-callatostatins by having Asp in position Xaa and Met-NH 2 at the C-terminus.
  • This new class of insect peptides may be related to the allatostatin class of peptides isolated from other unrelated insect species.
  • the allatostatin class of insect neuropeptides in cockroaches have been shown capable of inhibiting the production of juvenile hormone (JH) by the corpus allaturn.
  • the corpus allatum (CA) is a classical endocrine gland of insects situated in close proximity to the brain with which it has nervous connection.
  • Members of the allatostatin class of neuropeptides have now been isolated and identified in five species (other than C. vomi toria) , from four different orders of insects (other than the order Diptera) .
  • Diploptera punctata Order: Blattodea
  • five allatostatins ranging in size from 8 to 18 amino acids have been characterised (Pratt et al in Proc . Nat ' l . Acad. Sci USA
  • the significance of the allatostatins lies in the fact that " in vi tro” they have been shown, with the exception of the allatostatin from the tobacco hornworm moth, to inhibit the production of juvenile hormone (JH) by the corpus allatum in cockroaches.
  • the allatostatin molecule isolated from the tobacco hornworm moth inhibits JH production in the species in which it is found i.e. Manduca sexta but not in cockroaches.
  • the Callatostatins are of interest because they are potent inhibitors of JH synthesis and release in cockroaches, but not in the blowfly, the species from they originate.
  • the callatostatins are also of interest because of their effect on normal gut physiology in insects.
  • Juvenile hormone plays a crucial role in insect development by controlling metamorphosis, adult sexual maturity and reproduction. Interference with juvenile hormone biosynthesis and release through exploitation of the allatostatins and callatostatins may lead to insect control strategies that do not damage the environment .
  • Use of the DNA encoding the callatostatin peptides and the novel callatostatin peptides of the present invention may also provide an additional insect control strategy which relies on interfering with the normal gut physiology in larvae and/or adults of major lepidopteran pest species, such as the codling moth Cydia pomonella and the armyworm Helicoverpa armigera .
  • the peptides may also have utility as insecticides against other insect species.
  • the present invention is based on the discovery and potential practical commercial application of the DNA encoding one or more of four novel peptides identified in the blowfly Calliphora vomi toria and the other known Leu-callatostatins 1 to 4 in C. vomitoria, and the isolation and purification of the novel peptides, designated callatostatins 5, 6, 7 and 8.
  • the present invention also extends to the homologous prohormone genes from the Australian sheep blowfly Lucilia cuprina and Helicoverpa armigera and to the peptides coded for by these genes. Variants of the peptides have also been isolated from Cydia pomonella and Pteronidia salicis .
  • a recombinant or isolated DNA sequence encoding the amino acid sequence shown in Figure 2 , in Figure 4 , in Figure 18, or an amino acid sequence which is substantially homologous thereto.
  • a third aspect of the present invention provides a recombinant or isolated DNA sequence comprising the protein coding region of the DNA sequence shown in Figure 2, in Figure 4 or in Figure 17.
  • FIG. 2 Particularly preferred coding sequences are shown in Figure 2 for the C. vomi toria Leu-callatostatin peptides gene, in Figures 4 and Figure 17 for the homologous L. cuprina and Helicoverpa armigera Leu-callatostatin peptides genes respectively, as will subsequently be described in the examples.
  • Figures 4 and Figure 17 Particularly preferred coding sequences are shown in Figure 2 for the C. vomi toria Leu-callatostatin peptides gene, in Figures 4 and Figure 17 for the homologous L. cuprina and Helicoverpa armigera Leu-callatostatin peptides genes respectively, as will subsequently be described in the examples.
  • Those skilled in the art will with the information given in this specification, be able to identify with sufficient precision the coding regions and to isolate and/or recombine DNA containing them.
  • the gene encoding the novel Leu- callatostatin peptides 5, 6, 7 and 8 and the known Leu ⁇ callatostatin peptides 1, 2, 3 and 4 of C. vomi toria , L . cuprina and H. armigera will be referred to as the Leu ⁇ callatostatin gene.
  • the Leu-callatostatin genes shown in Figure 2, Figure 4 and Figure 17 contain coding regions and the invention therefore also extends to the prohormone sequences coded for by the Leu ⁇ callatostatin genes.
  • the coding region contains a number of putative peptide sequences which would be excised after translation of the mRNA for the gene.
  • the putative coding sequences correspond with the peptides already identified and the novel peptides of the present invention.
  • amino acid sequences of the peptides according to this invention are as follows :-
  • Val-Glu-Arg-Tyr-Ala-Phe-Gly-Leu (designated Leu ⁇ callatostatin 7) , or
  • Leu-callatostatin 8 Leu-Pro-Val-Tyr-Asn-Phe-Gly-Leu.
  • the invention embraces Leu-callatostatins 5 to 8 defined above when isolated and/or when substantially purified and essentially free of other peptide material.
  • the present invention also extends to variants of the peptides of the present invention.
  • An example of a variant of the present invention is a Leu-callatostatin peptide as defined above, apart from the substitution of one or more amino acids with one or more other amino acids.
  • the skilled person is aware that various amino acids have similar properties. One or more such amino acids of a substance can often be substituted by one or more other such amino acids without eliminating a desired activity of that substance. Additionally, different amino acids as substituents may enhance the activity of a particular peptide.
  • amino acids glycine, alanine, valine, leucine and isoleucine can often be substituted for one another (amino acids having aliphatic side chains) .
  • amino acids having aliphatic side chains amino acids having aliphatic side chains
  • valine, methionine, leucine and isoleucine are used to substitute for one another (since they have larger aliphatic side chains which are hydrophobic) .
  • amino acids which can often be substituted for one another include: phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains) ; lysine, arginine and histidine (amino acids having basic side chains) ; aspartate and glutamate (amino acids having acidic side chains) ; asparagine and glutamine (amino acids having amide side chains) ; and cysteine and methionine (amino acids having sulphur containing side chains) . Substitutions of this nature are often referred to as “conservative" or “semi- conservative" amino acid substitutions.
  • Amino acid deletions or insertions may also be made relative to the amino acid sequences of the peptides of the present invention given above.
  • amino acids which do not have a substantial effect on the activity of the peptides of the present invention, or at least which do not eliminate such activity may be deleted.
  • Such deletions can be advantageous since the overall length and the molecular weight of a peptide can be reduced whilst still retaining activity. This can enable the amount of peptide required for a particular purpose to be reduced - for example, dosage levels can be reduced.
  • Amino acid insertions relative to the sequence of the peptides of the present invention can also be made. This may be done to alter the properties of a substance of the present invention, for example to assist in identification, purification or expression in relation to production of fusion proteins.
  • Amino acid changes relative to the sequences of the peptides of the present invention given above can be made using any suitable technique e.g. by using site-directed mutagenesis.
  • amino acid substitutions or insertions within the scope of the present invention can be made using naturally occurring or non-naturally occurring amino acids. Whether or not natural or synthetic amino acids are used, it is preferred that only L- amino acids are present. However, D- amino acid substitutions are known, for example D-phenylalanine in certain molluscan neuropeptides and the present invention is not limited in this regard.
  • preferred peptides of the present invention have at least 50% sequence identity with a peptide according to the present invention, more preferably the degree of sequence identity is at least 75%. Sequence identities of at least 90% or at least 95% are most preferred. The degree of amino acid sequence identity can be calculated using a program such as "bestfit” (Smith and Waterman, Advances in Appli ed Mathematics, 482-489 (1981) ) to find the best segment of similarity between any two sequences.
  • the alignment is based on maximising the score achieved using a matrix of ammo acid similarities, such as that described by Schwarz and Dayhof (Atlas of Protein Sequence and Structure, Dayhof, M.O., ed. 353-358 (1979)) . Where high degrees of sequence identity are present there will be relatively few differences in ammo acid sequence. Thus for example they may be less than 20, less than 10, or even less than 5 differences .
  • the present invention also extends to variants of the peptides of the present invention and these variants include the peptides isolated from Cydia pomonella, Helicoverpa armigera and Pteronidia salicis when isolated and/or when substantially purified and essentially free of other peptide material.
  • the peptides isolated from C. pomonella are as follows:
  • Lys-Met-Tyr-Asp-Phe-Gly-Leu (designated cydiastatin 7)
  • the peptides isolated from H. armigera are as follows:
  • Glu-Arg-Asp-Met-His-Arg-Phe-Ser-Phe-Gly-Leu (designated helicostatin 9) .
  • the peptides with the above sequences from C. vomitoria, L. cuprina, H. armigera, C. pomonella and P. salicis are amidated at the carboxy terminus.
  • the peptide Leu-callatostatin 5 isolated from C. vomitoria has the sequence ARPYSFGL-NH 2 and this sequence is shared by the peptides isolated from C. pomonella and H. armigera. In P. salicis the sequence is altered by the substitution of a leucine residue m place of the prolme at position 3, i.e. the peptide has the sequence ARLYSFGL-NH 2 .
  • C. pomonella and H. armigera share a peptide sequence SRPYSFGL-NH 2 which differs from Leu-callatostatm 5 by the substitution of a serine residue in place of the alanine residue at position 1.
  • Leu-callatostatin 6 from C. vomitoria has the peptide sequence RPYSFGL-NH 2 which represents a deletion of the N-terminal alanine residue.
  • the peptide Leu-callatostatin 7 from C. vomi toria has the sequence VERYAFGL-NH 2 and this sequence is shared in part by the peptide GGEDFGHRYAFGL-NH 2 from P. salicis .
  • the valine and glutamic acid residues at positions 1 and 2 in Leu ⁇ callatostatin 7 are replace by a glycine and a histidine residue respectively with the addition of prior pentapeptide sequence.
  • Leu-callatostatin 8 from C. vomi toria has the sequence LPVYNFGL-NH 2 and this sequence is shared by peptides isolated from C. pomonella and H. armigera .
  • C. pomonella peptides have also been isolated which have the valine residue at position 3 replaced by either an isoleucine or a leucine residue.
  • This sequence is also shared by peptides isolated from P. salicis .
  • Further changes include the replacement of the valine at position 3 by a methionine residue in H. armigera and the replacement of the leucine and proline residue at positions 1 and 2 by alanine and arginine respectively in P. salicis .
  • C. pomonella and H. armigera also share the peptide sequence AYSYVSEYKRLPVYNFGL-NH 2 which represents the sequence of Leu-callatostatin 8 with a prior decapeptide sequence.
  • the lepidopteran species C. pomonella and H. armigera also share some sequence homology in the newly isolated peptides which have aspartic acid as the post-tyrosyl residue.
  • the peptides ARGYDFGL-NH 2 and KMYDFGL-NH 2 in C. pomonella differ in that the residues 1 to 3 of the first peptide being alanine, arginine and glycine are replaced by lysine and methionine in the second peptide.
  • the peptide sequence is altered by the replacement of the glycine at position 3 in the first C. pomonella peptide by an alanine residue.
  • the invention also embraces insecticidal compositions which contain one or more of the above peptides described above, which may be a liposomal formulation, and methods of killing or controlling insects which involve applying to the insects or their environment such msecticidal compositions.
  • the insecticidal compositions may contain besides at least one msecticidally effective peptide described above, a suitable carrier, diluent or excipient therefor.
  • the formulations according to the present invention may be administered in the form of a spray.
  • Methods of making insecticidal compositions are also embraced by the present invention which comprises admixing one or more the said Leu-callatostatins, helicostatins, pteridiostat s or cydiastat s described above with a suitable carrier, diluent or excipient therefor.
  • DNA according to the present invention can be synthesised by standard techniques for the preparation of genetic material.
  • the process may comprise ligating together successive nucleic acid residues and/or oligonucleotides to produce DNA.
  • peptides described above can be synthesized by the skilled worker by routine peptide synthesis by selecting the appropriate ammo acids and reaction conditions.
  • Such peptides as an insecticide is also within the scope of the present invention which may comprise the inhibition of gut motility.
  • the use of such secticidal compositions may be specific for the codling moth Cydia pomonella or for the army worm Helicoverpa armigera .
  • the codling moth Cydia pomonella , is a major worldwide pest of apples and pears and causes the greatest economic damage of any lepidopteran pest the UK. Resistance is developing to chemicals currently being used for its control and there is a strong desire to reduce chemical inputs, particularly on unprocessed food crops.
  • the army worm, Helicoverpa armigera is reported to be the most important insect pest known to agricultur .
  • the invention also extends to vector systems comprising a DNA sequence which encodes a peptide as described above for expression m a host cell .
  • Suitable vectors are preferably specific for the target organism and may be baculovirus based.
  • Baculoviruses are currently being developed as pest control agents (Payne, C. C, in Biological Plant and Heal th Protection, ed. Franz, J.M. (Fort suitse der Zoologie 32) , G. Fischer Verlag, Stuttgart, New York (1986) ) and several commercial products based on these viruses are now available (W stanley et al in Exploitation of Microorganisms, ed. D. G. Jones, 105-136, Chapman & Hall, London (1993)) . Viruses are, however, slower to kill insects than chemical insecticides and feeding may continue for some time after application of the msecticidal agent. It has been possible to reduce this damage by using genetically-engineered viruses that express arachnid toxins (Stewart et al Nature 352 85-88
  • a more appropriate baculovirus would be Cydia pomonella granulosis virus (CpGV) .
  • CpGV Cydia pomonella granulosis virus
  • This virus has been isolated in the UK, is very effective against the codling moth C. pomonella , which is a ma or U.K. pest.
  • This baculovirus has a narrow host range which includes only a few closely related tortricid species, and recombinant strains can be produced.
  • CpGV is already produced commercially, improved strains, capable of faster killing, could also be used to reduce further the crop damage.
  • Expression of the callatostatin peptides at high levels by baculoviruses would have the highly desirable effect of inhibiting feeding in infected larvae.
  • the heterologous genes of the blowfly, Calliphora vomi toria , of the sheep blowfly, Lucillia cuprina , or of Helicoverap armigera may be used in such a baculovirus.
  • the present invention also comprehends the use of a synthetic gene based on identified peptide sequences from C. pomonella or the actual endogenous gene of C. pomonella .
  • a major advantage of the latter approach is that CpGV can be engineered to carry a gene that is found only in the host larvae, thus minimising any possible risk to the environment. Similar approaches may also suitable with respect to the development of insecticides against H. armigera .
  • the present invention has now demonstrated that similar peptides to the C.
  • vomitoria peptides are expressed in these pest species and in some cases the amino acid sequence is identical e.g. Leu-callatostatin 8 which has the structure LPVYNFGL-NH 2 and this peptide is also found in both C. pomonella and H. armigera .
  • CpGV was the most promising agent available, though the relatively slow rate of kill by the virus allows significant crop damage to be caused by the pest. Very recently this virus has gone into commercial production, for example Carpovirusine TM or GranupomTM.
  • the peptides described by the present invention are the third group of the callatostatin class of compounds to be isolated from this major order of insects, the Diptera.
  • the first and second groups were also from this species, the blowfly Calliphora vomi toria .
  • FIGURE 1 shows the design of an oligonucleotide callatostatin gene probe of low degeneracy for C. vomi toria library screening.
  • (c) shows the sequence of a 12-fold degenerate oligonucleotide probe PCRCastl based on the sequences of the four PCR amplicons.
  • I and N indicate that either inosine or all our nucleotides respectively were used at that position in the oligonucleotide.
  • FIGURE 2 shows the sequence of the C. vomi toria Dra I fragment showing two open reading frames.
  • Each of the putative peptides (boxed) is flanked by a pair of basic amino acid residues (bold underline) and has a glycine residue (italicised) at its C-terminal end.
  • Proposed intron splice sites are marked with a vertical bar and the region of the putative intron is indicate.
  • FIGURE 3a shows a dot matrix comparison of L. cuprina and C. vomi toria genomic DNA sequences.
  • the black bars indicate the position of the two open reading frames encoding callatostatin-like peptides in the C. vomi toria sequence.
  • FIGURE 3b shows a sequence comparison of the prohormone amino acid sequences identified in C. vomi toria and L. cuprina .
  • the symbol "•••” represents an identical amino acid residue and the symbol "-" represents an insertion of a residue relative to the C. vomi toria sequence.
  • FIGURE 4 shows L. cuprina cDNA and the deduced prohormone sequence. Putative peptides are boxed and numbered with Roman numerals. Pairs of basic residues proposed as sites of proteolytic cleavage are underlined in bold and the C-terminal glycine substrates for carboxylamidation are marked in italics. The translation termination codon is indicated by an asterisk (*) and vertical arrows mark the positions where introns occur in the genomic DNA. Locations of primers used for RT-PCR analysis are indicated.
  • FIGURE 5 shows a diagrammatic representation of the dipteran Leu-callatostatin prohormone deduced from L. cuprina genomic and cDNA and C. vomi toria genomic DNA clones.
  • the positions of the callatostatin peptides are marked with diagonal lines and numbered I-V according to the nomenclature of Table 1. Sites of endoproteolytic cleavage are indicated by solid black boxes and the proposed signal peptide domain is marked by vertical bars.
  • FIGURE 6 shows the organisation of the dipteran Leu ⁇ callatostatin prohormone gene showing sites of two introns. Numbers refer to nucleotide coordinates of the L cuprina cDNA. The positions of translation initiation (ATG) and termination (TAA) codons are indicated and the prohormone open reading frame is shaded.
  • FIGURE 7 shows nested reverse transcriptase PCR analysis of mRNA pools prepared from L. cuprina adult head and midgut .
  • Lane M contains molecular weight markers (lOObp ladder) .
  • the head and midgut (Gut) cDNA samples are indicated and "1", "2" are the first and second (nested) PCR reaction products respectively.
  • Lane "Gen” contains the amplification product from L. cuprina genomic DNA with the primer pair AST-2/AST-3.
  • FIGURE 8 shows a flow diagram of the purification of Leu-callatostatin 7 and Leu-callatostatin 8 from C. vomi toria .
  • M r * indicates the mass of the peptide after methylation.
  • FIGURE 9 shows a flow diagram of the purification of Leu-callatostatins 4, 5 and 6 from C. vomi toria [Leu- callatostatin 4 being a comparative example] . Chromatography being carried out according to conditions detailed in step 6. M r * indicates the mass after methylation.
  • FIGURE 10 shows dose responses for inhibition of spontaneous muscle contractions of the rectum (colon and rectal pouch) of C. vomi toria by Leu-callatostatins 5 and 8. Percentage inhibition calculated relative to basal spontaneous contraction movements. Each point represents the mean of 5 to 10 measurements from a number of different vitellogenic flies.
  • FIGURE 11 shows a chromatographic profile of an extract of 100 heads of adult blowflies of C. vomi toria assayed with five different callatostatin RIAs specific for five different types of peptides, shown in the figure, ending C-termmally as follows:
  • FIGURE 12 shows a chromatographic profile of an extract of 1000 Cydia pomonella larvae chromatographed and assayed in an identical manner to C. vomi toria as shown in Figure 11.
  • FIGURE 13 shows dissected C. pomonella 5th star larvae :
  • Figure 13A shows a normal larvae
  • Figure 13B shows a larvae fixed and stained with X-gal to show the presence of recombinant CpGV expressing ⁇ - galactosidase (blue-black coloration) .
  • This demonstrates the successful engineering of C. pomonella granulosis virus.
  • FIGURE 14 shows a drawing of the gut of C. pomonella to show the regions 1 to 5 from which Figures 15-1 to 15-5 are taken.
  • Figure 15-1 shows frontal ganglion containing 4 callatostatin-immunoreactive cells of which the two most anterior cells give rise to Leu-callatostatin- immunoreactive material in and around the valve between the foregut and the midgut (see Figure 15- 4) .
  • Figure 15-2 shows the dorsal part of the brain with Leu-callatostatin immunoreactivity in certain of the median neurosecretory cell groups.
  • Figure 15-3 shows callatostatin immunoreactive axons in the gastric nerve. These have their origins from cells of both the frontal ganglion and the brain.
  • Figure 15-4 shows accumulation of Leu-callatostatin immunoreactive material in the muscles of the valve separating the foregut from the midgut.
  • Figure 15-5 shows Leu-callatostatin immunoreactivity in posteriorly located midgut endocrine cells immediately anterior to the valve between the midgut and the hindgut .
  • FIGURE 16 shows dose-response for the inhibition of peristaltic contractions of the foregut of 5th instar larvae of C. pomonella by the blowfly callatostatin neuropeptide Leu-callatostatin 3 (Ala-Asn-Arg-Tyr-Gly- Phe-Gly-Leu-NH 2 ) .
  • the percentage inhibition is relative to basal spontaneous peristaltic contractions.
  • FIGURE 17 shows the cDNA sequence of the helicostatin gene of Helicoverpa armigera .
  • FIGURE 18 shows the deduced amino acid sequence of the open reading frame from the cDNA of the helicostatin gene of Helicoverpa armigera .
  • Insect rearing and tissue dissection C. vomitoria were obtained as pupae and allowed to eclose under laboratory conditions (25°C : 65% relative humidity : 12 h light/dark) .
  • the flies were fed sugar, beef heart and water for 1-3 weeks after which they were anaesthetized with C0 2 , frozen and stored at -20°C prior to use.
  • Larval L. cuprina were reared under a 12:12 hour light :dark cycle, on fresh minced sheep liver supplemented with meat meal, fish meal and cotton lint.
  • Adult L. cuprina were maintained on water and a protein biscuit comprising a solidified mixture of sugar, egg powder, milk powder and yeast.
  • Tissues for RNA extraction were dissected under sterile, ice-cold phosphate- buffered saline (139 mM NaCl, 10 mM sodium phosphate, pH 7.2) , transferred to a microfuge tube and snap frozen in liquid nitrogen. Tissues were stored frozen at -80°C until use.
  • Example 1 Preparation of a callatostatin gene probe
  • oligonucleotide primers based on the ammo- and carboxyl-terminal sequences of the hexadecapeptide Leu ⁇ callatostatin 1 were synthesised (Pharmacia Gene Assembler) and used to amplify a fragment of the callatostatin gene from C. vomi toria genomic DNA using the polymerase chain reaction (Pratt et al Proc . Nat ' l . Acad. Sci . USA 88 2412-2416 (1991)) .
  • the structures of the sense (ClSl) and antisense (ClAl) primers are shown m Figure 1.
  • PCR reactions consisted of 20 mM Tris-HCl, pH 8.4 , 50 mM KCI, 1.5 mM MgCl 2 , 0.2 mM each of dATP, dCTP, dGTP and dTTP, 50 pmoles of each primer, 1 ⁇ g of C. vomi toria genomic DNA, 2.5 units Tag DNA polymerase and H 2 0 to a final volume of 50 ⁇ l .
  • Example 2 Callatostatin gene cloning Approximately 1.5 x 10 5 recombinant bacteriophages from a C. vomi toria genomic D ⁇ A library were plated on the E. coli host strain KW251 and plaque D ⁇ A was transferred to nitrocellulose filters according to standard techniques (Sambrook et al Molecular Cloning: A Laboratory Manual 2nd edition. Cold Spring Harbour Laboratory Press, USA (1989)) .
  • the oligonucleotide PCRCastl was radiolabelled with ⁇ - 32 P-dATP using the Klenow fragment of E. coli D ⁇ A polymerase (Sambrook et al Molecular Cloning: A Labora tory Manual 2nd edition.
  • the L. cuprina callatostatin homologue was isolated from a recombinant genomic DNA library using a 936 bp Dra I restriction enzyme fragment containing the C. vomi toria prohormone gene as probe.
  • Hybridisation probes were prepared by random-primed synthesis using the NEBlot kit (New England Biolabs) and ⁇ - 32 P-dATP (NEN-DuPont) .
  • cuprina random-primed head cDNA library were screened using a lkb Hind III fragment containing the L. cuprina callatostatin gene as probe. Hybridisation and wash conditions were as described for the L. cuprina genomic library screen. The cDNA insert from hybridising phages was excised with Eco Rl and subcloned into pBluescript SK(+) for sequencing.
  • First strand cDNA was synthesised from 1 ⁇ g of poly(A) + RNA in a reaction consisting of: 50 mM Tris-HCL (pH 8.3) , 75 mM Kcl , 3 mM MgCl 2 , 1 mM DTT, 15 mM each of dATP, dCTP, dGTP and dTTP, 20 units RNasin (Promega Biotec) , 25 ng oligo(dT) primer, 1 ⁇ g mRNA, 200 units of Superscript II reverse transcriptase
  • lcap-1 5' -CTCAACTAGAGGATAAAAGC-3 '
  • lcap-2 5' -CGTTAGCCTTTTGATGTTGG-3'
  • lcap-3 5' -CGACGTCCTAAACCAAAGC-3 '
  • lcap-4 5' -GGAATTATTGGCTGGATAGTG-3'
  • the first PCR reaction contained; 20 mM Tris-HCl, pH 8.4 , 50 mM KCI, 1.5 mM MgCl 2 , 0.2 mM each of dATP, dCTP, dGTP and dTTP, 50 pmoles of the primer pair lcap-l/lcap-4, 10 ⁇ l of cDNA and H 2 0 to a final volume of 50 ⁇ l .
  • Amplification conditions were; 95°C for 5 min, 50°C for 1 min with addition of 2 ⁇ l Taq DNA polymerase, followed by 95°C for 1 min, 50°C for 1 min, 72°C for 1 min, for 30 cycles; then 1 cycle of 72°C for 5 min to end the reaction.
  • the nested PCR reaction conditions were identical to those above, except that the primer pair lcap-2/lcap-3 was used and the template was 1 ⁇ l of the first PCR reaction.
  • Double-stranded plasmid DNA for sequencing was prepared by a standard alkaline lysis mini-prep procedure (Sambrook et al
  • Example 5 Isolation of the C. vomi toria callatostatin gene Since the amino acid sequences of the four Leu-callatostatin peptides were not favourable for the design of oligonucleotides suitable for library screening, a PCR approach was used to amplify a short region of the callatostatin gene to provide a basis for probe design.
  • vomi toria genomic library were screened, from which a single positive ( ⁇ Cvastl)was obtained.
  • Restriction enzyme analysis of ⁇ Cvastl identified a 3.2 kb Bam El/Xba I fragment that hybridised to the oligonucleotide probe.
  • Partial sequencing of this fragment identified a 936 bp Dra I restriction enzyme fragment that contained the sequence of the oligonucleotide probe.
  • the Dra I fragment was subcloned and sequenced on both strands. Sequence analysis identified likely protein coding regions in two distinct open reading frames, that together encoded five separate putative Leu-callatostatin peptides, each of which was flanked by pairs of basic amino acids that constitute potential endoproteolytic cleavage sites, shown in Figure 2. Each of these peptides had a single glycine residue at the C-terminal end that provides a substrate for peptidyl glycine a-amidating monooxygenase, consistent with the observation that all previously identified callatostatin peptides are amidated at the C-terminus (Duve et al Proc. Nat'l. Acad. Sci. USA 90 2456-2460 (1993)) . The sequences of the five putative callatostatins are given in Table 1 in order of their appearance in the gene.
  • Peptide IV corresponds to Leu-callatostatin 4 of Duve et al ⁇ Proc. Nat'l. Acad. Sci. USA 902456-2460 (1993)) and resolves the uncertainties in the original sequence data for that peptide.
  • Peptide V corresponds to Leu-callatostatin 1.
  • the putative peptides I-III were not identified in the previous purification studies (Duve et al Proc. Nat'l. Acad. Sci. USA 902456-2460 (1993) , Duve et al J. Biol. Chem. 269 21059-21066
  • Peptide III is identical to one of the peptides encoded on the cockroach prohormone gene (Donly et al Proc. Nat'l. Acad. Sci. USA 90 8807-8811 (1993) ) .
  • the 936 bp Dra I fragment containing the callatostatin gene was radiolabelled and used to screen a L. cuprina genomic DNA library. Two hybridising clones were purified and found to contain a 4.3 kb Xba I restriction enzyme fragment that hybridised to the C. vomi toria probe. A 2.6 kb Cla I/Xba I fragment was subcloned from the Xba I fragment and partially sequenced to identify the region homologous to the callatostatin prohormone gene.
  • Figure 3 shows a dot matrix comparison of the L. cuprina sequence with that of the C. vomi toria Dra I fragment. The two sequences were 84% identical over this region and similarity was greatest m the two domains corresponding to the open reading frames identified in C. vomi toria indicating that gene structure was conserved between the two species.
  • a 1 kb Hind III fragment that encompassed the peptide coding region of the L. cuprina gene was used to probe a cDNA library prepared from adult head mRNA. A single positive was isolated from a sample of approximately 3 x 10 5 clones, suggesting that transcripts are relatively rare.
  • the cDNA clone was sequenced and found to contain a single open reading frame encoding a Lucilia Leu-callatostatin prohormone 179 amino acids m length, shown m Figure 4. Since the clone was isolated from a random-primed library it cannot represent a full length cDNA.
  • the open reading frame is flanked by 341 nucleotides of 5' and 271 nucleotides of 3' sequence, each of which contains multiple translation stop codons in all three reading frames.
  • the cDNA encodes all of the L. cuprina callatostatin pre-prohormone.
  • the first in-frame methionine residue, and proposed translation initiation site occurs at nucleotide 342 and the translation termination codon is at nucleotide 879.
  • the putative pre-prohormone begins with a hydrophobic domain of 19 amino acids that is a probable signal peptide. The most likely site of cleavage of this signal sequence is at residue 20 (von Heijne, G. Nucl . Acids Res . 14 4683-4690 (1986)) .
  • the deduced prohormone sequence contains five Leu-callatostatin- like peptides that are identical to those identified from the C. vomi toria genomic clone, shown in Figure 2 and in Table 1.
  • the proposed structure of the dipteran callatostatin prohormone is shown diagrammatically in Figure 5.
  • the first three peptides are tandemly arrayed, separated only by the residues required for proteolytic processing and carboxyamidation.
  • the other two peptides occur as a tandem pair at the carboxyl end of the prohormone. This structure is absolutely conserved in the two blowfly species.
  • L . cuprina Leu-callatostatin gene is composed of at least three exons, as shown in Figure 6. Introns are located between nucleotides 289/290 and 777/778 of the L . cuprina cDNA clone, as seen in Figure 5.
  • the first intron is located in the 5' untranslated region of the mRNA and although no attempt was made to identify exon 1 in genomic DNA, this intron must be at least 2 kb long, based on hybridisation of the cDNA to existing clones (not shown) .
  • the second intron is very short, 63 bp in length, and occurs within the prohormone open reading frame in the region between the two blocks of peptides. Direct comparison of the L. cuprina genomic and cDNA sequences confirmed the prohormone structure proposed from the C. vomi toria genomic analysis of Figure 2.
  • Example 8 Expression of the callatostatin gene Reverse transcriptase-PCR was used to investigate expression of the callatostatin prohormone gene in cDNA pools prepared from whole adult head, representing an enriched source of brain and sub-oesophageal ganglion tissue, and from isolated midgut. To provide maximum sensitivity a 'nested' PCR design was used (McPherson et al in M.J. McPherson, P. Quirke, P. and
  • the amplicon generated from genomic DNA was slightly larger than the corresponding product from the cDNA pools as expected, due to the 63 bp intron, shown in Figure 7.
  • This genomic PCR result demonstrated that the RT-PCR products were bona fide cDNA products and not artifacts produced from contaminating genomic DNA.
  • a genomic clone encoding the sequences of the peptides Leu- callatostatin 1 and Leu-callatostatin 4 was isolated from a C. vomi toria library. This clone contained two open reading frames, each of which contained Leu-callatostatin sequences.
  • Three related, but structurally distinct putative Leu ⁇ callatostatin peptides were encoded on the first open reading frame. These three putative peptides all possessed the C- terminal pentapeptide sequence -Tyr-Xaa-Phe-Gly-Leu characteristic of the callatostatin/ allatostatin peptide family.
  • the octapeptide Leu-callatostatin 4 contained two uncertain residue assignments from the Edman peptide sequencing. The first (N-terminal) residue was identified as either Asp or Asn and the fifth amino acid was tentatively identified as Ser. The prohormone gene sequence has allowed unequivocal assignment of these residues as Asn at position 1 and Ser at position 5.
  • Leu-callatostatins 1-3 were isolated, it was noted that the hexadecapeptide Leu-callatostatin 1 contained a pair of basic (Arg) residues at positions 7 and 8 which, if used as a proteolytic cleavage site during prohormone maturation, would give rise to the octapeptide Leu ⁇ callatostatin 3.
  • Leu-callatostatin 2 was identical to Leu-callatostatin 1 with the two amino- terminal residues, Asp-Pro, removed. At this time, it was not clear whether these three peptides were each encoded independently on the callatostatin prohormone, or if Leu- callatostatins 2 and 3 were derived by proteolytic processing of Leu-callatostatm 1.
  • the prohormone and deduced peptide sequences are identical in both blowfly species.
  • This absolute conservation of peptide structure might reflect a functional constraint on sequence evolution in the Leu-callatostatin peptide family, since at least one other multi-member neuropeptide family, the CalliFMRFamides, does not have the same degree of conservation in these two species (Duve et al in Perspectives in Comparative Endocrinology, 91-96, eds. K.G. Davey, R.E. Peter, and S.S. Tobe, National Research Council of Canada: Ottawa (1994) ) .
  • the allatostatin prohormones of the cockroaches D. puncta ta and P. ameri cana are also highly conserved (Stay et al Adv. Insect Physiol 25 269-337 (1994) ) . However, there is very little similarity in prohormone organisation between the cockroaches and flies.
  • the cockroach prohormone is approximately 370 amino acids long and encodes 13 and 14 allatostatin peptides in D . punctata (Donly et al Proc . Na t ' l . Acad . Sci . USA 90 8807-8811 (1993)) and P. americana (Stay et al Adv. Insect Physiol 25 269-337 (1994) ) respectively, with the peptides occurring in several distinct blocks separated by acidic spacer regions.
  • dipteran prohormone is approximately 180 residues in length and contains only five Leu-callatostatin peptides distributed in two blocks, separated by an acidic spacer.
  • the octapeptide Ala-Arg-Pro-Tyr-Ser-Phe-Gly-Leu-NH 2
  • the second putative peptide of the dipteran prohormone (peptide II) is which may be a structural homologue of the allatostatin ASB2 isolated by Pratt et al ( Proc . Na t ' l . Acad . Sci . USA 88 2412- 2416 (1991) ) , which is also the second peptide in the cockroach prohormone (Donly et al Proc . Nat ' l . Acad . Sci . USA 90 8807-8811 (1993)) .
  • Leu- callatostatin 1 is several orders of magnitude less potent than its C-terminal derivative Leu-callatostatin 3.
  • the allatostatins are also myoinhibitory in cockroaches (Lange et al Arch . Insect . Biochem . Physiol . 24 79-92 (1993) , Lange et al J. Insect Physiol . 41 581-588 (1995) , Duve et al Physiol . Ento ol . 20 33-44 (1995)) . All thirteen peptides encoded on the D .
  • Leu-callatostatin/allatostatin peptides appear to be quite old in evolutionary terms.
  • the presence of allatostatin immunoreactivity and biological activity of allatostatin peptides in a crab (Skiebe et al J. Exp . Biol . 194 195-208
  • Radioimmunoassay The two putative octapeptides deduced from the Leu- callatostatm gene sequence (East et al Regul . Pept . [m press] (1996)) , Ala-Arg-Pro-Tyr-Ser-Phe-Gly-Leu-NH 2 and Val- Glu-Arg-Tyr-Ala-Phe-Gly-Leu-NH 2 , together with the octapeptide Leu-Pro-Val-Tyr-Asn-Phe-Gly-Leu-NH 2 (assuming an enzymatic cleavage at the dibasic pair of residues the longer putativepeptideAla-Tyr-Thr-Tyr-Thr-Asn-Gly-Gly-Asn-Gly-Ile- Lys-Arg-Leu-Pro-Val-Tyr-Asn-Phe-Gly-Leu-NH 2 ) , were synthesized (by Affiniti Research
  • nb Results are expressed as a % of the reactivity shown by the peptide against which the antiserum was raised, in the same RIA
  • concentration of standards in RIAs used to monitor HPLC elution profiles ranged from 015nM to lOnM
  • Calliphora vomi toria were obtained as pupae and allowed to eclose under laboratory conditions (25°C : 65% relative humidity : 12 h light/dark) .
  • the flies were fed sugar, ox heart and water for 1-3 weeks after which they were anaesthetized with C0 2 , frozen and stored at -20°C prior to use. Approximately 40,000 heads were collected by shaking the frozen flies in a plastic bag containing solid C0 2 followed by sieving. They were ground in batches of 2000 in a mortar and pestle in solid C0 2 and homogenized further in a Waring blender m 400 ml methanol/acetic acid/water (87:5:8) at 4°C.
  • the extract was left overnight at 4°C after which it was centrifuged at 3000 x g for 30 min.
  • the pellet was re- extracted with 1 1 of extraction fluid.
  • the combined supernatants were concentrated to approximately 300 ml on a rotary evaporator at 35°C under vacuum.
  • the concentrate was allowed to stand overnight at 4°C before being filtered through Whatman No. 4 paper and re-centrifuged at 15000 x g for 10 min prior to purification by HPLC.
  • the HPLC system for Steps 1-4 comprised two Waters 6000M pumps and a Waters 741 detector.
  • a Waters 625 system was used at room temperature, and for some Step 6 procedures (61) and all of Step 7 a Hewlett-Packard model 1090 HPLC system at 50°C was used. Details of the reversed-phase HPLC columns, solvent systems, flow rates, gradients and their rates of change, used in the purification are as follows.
  • Step 2
  • Step 1 The same column and the same basic running conditions were used as in Step 1 except that 10 mM ammonium acetate (pH 6.5) replaced 0.1%TFA.
  • the pooled material from Step 1 was aliquotted to give 25 separate runs, each containing the equivalent of 1600 heads.
  • Radioimmunoassays of the profiles showed separation into five different areas: Gly/Leu together with Asp/Met material (i.e. those peptides previously identified and designated Leu- callatostatins 1-3 and the various forms of Met-callatostatin (Duve et al Proc . Na t ' l . Acad . Sci . USA 90 2456-2460 (1993) , Duve et al J. Biol . Chem . 269 21059-21066 (1994) , Duve et al Regul . Pept .
  • Step 3 Bio-Rad Hi-Pore C 4 column (250 x 4.6 mm, 5 ⁇ m, 300 A), linear gradient 0 - 80% CH 3 CN/10 mM ammonium acetate (pH 6.5) in 200 min, flow rate 1.5ml/min.
  • Step 4 Kromasil C 1B column (250 x 4.6 mm, 5 ⁇ m, 300 A) , gradient CH 3 CN/0.1% TFA 0 - 15% in 10 min; 15 - 50% in 140 min, flow rate 1 ml/min.
  • Step 5 Kromasil C IB column (250 x 3.2 mm, 5 ⁇ m, 300A) , gradient CH 3 CN/0.1% TFA 0 - 15% in 15 min; 15 - 50% in 175 min, flow rate 0.5 ml/min.
  • Vydac C 18 column (150 x 2.1 mm, 5 ⁇ m, 300 A) , gradient CH 3 CN/0.1% TFA (5 - 15% in 5 min; 15 - 25 % in 50 min, flow rate 0.2ml/min. [Ser/Leu Ilai and Ser/Leu Ilaii] Column temperature 50°C.
  • Step 7
  • Vydac C 18 column (150 x 2.1 mm, 5 ⁇ m, 300 A) gradient CH 3 CN/0.1% TFA (for Asn/Leu material 5 - 17% in 5 min; 17 - 27 in 50 min; for Ser/Leu I, Ser/Leu Ilbi and Ser/Leu Ilbii 5 - 11% in 5 min; 11 - 18% in 35 min, flow rate 0.2 ml/min.
  • the flow charts in Figure 8 and Figure 9 give the % acetonitrile at which immunoreactive material eluted, and details of the points in the procedures where M r determinations and amino acid sequencing were carried out (nb.
  • the abbreviations for the various types of peptide being isolated are those used in Table 2) .
  • the immunoreactive fractions at Step 6 or 6f were analyzed by matrix-assisted laser desorption mass spectrometry using a Biflex (Bruker- Franzen) instrument. For this, 0.5 ⁇ l was mixed with 0.5 ⁇ l of a solution of 33mM ⁇ -cyano-4-hydroxycinnamic acid in acetonitrile/ methanol (Hewlett-Packard) . After drying, the mixture was analyzed in the linear mode at 15 kV. The method has an accuracy of 0.1%. If a single molecular mass was recorded, the material was subjected to amino acid sequence analysis and definitive mass spectrometry. If more than one peptide was seen to be present in the sample, it was chromatographed further.
  • amino acid sequencing The amino acid sequences of the purified peptides (5 - 50 pmol) were determined with an automated protein sequencer (Applied Biosystems, Procise 494A) equipped with an on-line system for the detection of the amino acid phenylthiohydantoin derivatives. All reagents and solvents were from Applied Biosystems.
  • the third peptide with the sequence Asn-Arg-Pro-Tyr-Ser-Phe-Gly-Leu- NH 2 , previously identified as Asx-Arg-Pro-Tyr- (Ser) -Phe-Gly- NH 2 and designated Leu-callatostatin 4 (Duve et al Proc . Nat ' l . Acad . Sci . USA 90 2456-2460 (1993)) , proved impossible to separate from a lesser amount of Leu-callatostatin 6, shown in Figure 9 as Ser/Leu I and Ser/Leu Ilbi) . The major, unambiguous sequence, however, was that of Leu-callatostatin 4.
  • the M r values of the peptide before and after methylation were 952.8 and 953.7 respectively, thereby confirming carboxyamidation and the fact that the first residue was Asn and not Asp.
  • the final yields of the three peptides were Leu ⁇ callatostatin 4, 28 pmols ; Leu-callatostatin 5, 14 pmols; and Leu-callatostatin 6, 120 pmols.
  • Table 3 shows the sequences of the peptides isolated, together with their M r values, before and after methylation. Table 3: Amino acid sequences and M r values of Leu-callatostatin neuropeptides isol ated from C. vomitoria.
  • Leu-callatostatin I s D-P-L-N-E-E-R R-A-N-R-Y-G-F-G-L-NH 2 1906 1 19039 1949 1
  • Leu-callatostatin 3 s A-N-R-Y-G-F-G-L-NH 2 8960 8968 -
  • Leu-callatostatin 8 L-P-V-Y-N-F-G-L-NH 2 921 1 9214 9223
  • Leu-callatostatins 5 and 8 showed them consistently to have a potent, reversible, inhibitory effect on the spontaneous contractile activity of the rectum of C. vomi toria as shown in Figure 10.
  • Leu-callatostatin 5 effected complete inhibition at 10 "9 M, 90% inhibition at 10 "12 M and 50% inhibition at 10 ⁇ 14 M.
  • Leu-callatostatin 8 which showed a biphasic dose-response curve similar to that previously observed with members of the Met-callatostatin family (Duve et al J. Biol . Chem. 269 21059-21066 (1994) , Duve et al Regul . Pept .
  • Leu-callatostatin 1 which appears as a putative peptide on the gene, and is present in extracts together with small amounts
  • C. voinitoria possesses a peptide (ARPYSFGL-NH 2 ) identical to one of D. punctata (Leu-callatostatin 5 and dipstatin 6) .
  • vomitoria occurs as the C-terminal sequence preceded by a dibasic pair of residues Lys-Arg in the octadecapeptide designated dipstatin 2 in D. punctata.
  • Leu-callatostatin 3 the truncated octapeptide variant of Leu-callatostatin 1, which can now be seen to occur as a result of post-translational processing of the precursor by enzymatic cleavage at the dibasic pair of residues (Arg-Arg) , gives complete, reversible inhibition of the ileum at concentrations as low as 10 "15 M.
  • the 'parent' hexadecapeptide is also effective on this in vitro preparation, although with a reduced potency (maximum inhibition at 10 '10 M) .
  • Leu-callatostatin immunoreactivity in nerves on the surface of the rectum in the form of either Ser/Leu or Asn/Leu peptides, may provide the morphological basis for the potent effects of the Ser/Leu (Leu-callatostatin 5) and Asn/Leu (Leu-callatostatin 8) peptides seen. Although contractions of the ileum are inhibited by these two peptides, the effect appears to be irreversible, unlike that seen for Leu-callatostatins 1 and 3.
  • Examples 12 to 15 Improved control of the codling moth Cydia pomonella by baculoviruses expressing insect neuropeptides
  • Example 12 Insertion of the C. vomi toria callatostatin (cal) gene into CpGV
  • the cal gene may be cloned into the CpGV transfer vector, pCpDNl, which places the gene under the control of the CpGV granulin promoter.
  • This vector has already been used with the lacZ gene and demonstrated high levels of expression of ⁇ - galactosidase in larvae infected with the recombinant virus, as shown in Figure 13A and Figure 13B.
  • the vector pCpDNl is a granulin replacement vector which results in a non-occluded recombinant virus. Whilst this vector may be suitable for studying the peptides that are expressed, for detailed studies on larval gut paralysis and for measurements of LT 50 values, it will be preferable to produce an occluded recombinant virus for more extensive assays. For this, a second transfer vector could be constructed to allow insertion of the cal gene into an intergenic region without deleting the granulin gene.
  • Example 13 Purification of the callatostatin peptide homologues of C. pomonella
  • Example 15 Physiological and degradation studies of the callatostatin-like peptides in C. pomonella
  • a possible methodology for inserting the callatostatin gene or DNA encoding one or more of the peptides into a suitable delivery vehicle for use as an insecticide against a pest such as C. pomonella is as follows.
  • C. pomonella for the construction of a synthetic C. pomonella gene and, eventually, for producing oligonucleotide probes for the isolation of the endogenous callatostatin gene of C. pomonella .
  • the cal gene will be cloned into the CpGV transfer vector, pCpDNl, which places the gene under the control of the CpGV granulin promoter.
  • This is a granulin replacement vector which results in a non-occluded recombinant virus. Whilst this will be suitable for studying the peptides that are expressed and for detailed studies on larval gut paralysis and measurements of LT 50 values, it would also be desirable to produce an occluded recombinant virus for more extensive assays.
  • a second transfer vector will be constructed which will allow insertion of the cal gene into an intergenic region without deleting the granulin gene.
  • the non-occluded recombinant virus will be used to study the extent of paralysis in C. pomonella larvae and compare LT 50 values with wild-type virus. This will give a very good indication of how effective the cal gene is. Subsequently, accurate measurements of LD 50 values will be performed with an occluded recombinant CpGV using both C. pomonella and Cryptophlebia leucotreta (false codling moth) . In the laboratory, C. leucotreta can be infected with wild-type CpGV but only at a 1000-fold higher dose than is required for C. pomonella . Bioassays with both these species would indicate whether there is any change in infectivity or host range of CpGV due to insertion of the cal gene.
  • the cal gene will be cloned into pAcUW2B to give pAcUW- cal .
  • This transfer vector places the cal gene under the control of the high level plO promoter and also contains the polyhedrin gene.
  • Cotransfection of pAcUW-cal with linearized polyhedrin- negative AcNPV will give rise to polyhedrin-positive recombinants expressing callatostatins.
  • the production of normal occlusion bodies (polyhedra) by recombinant virus will allow accurate bioassays in both neonate and later instars and is the form of the virus which is used in formulations for insecticidal application.
  • Bioassays to determine the biological effect of insertion of the cal gene into the virus will be done in H. virescens using neonate and later instars. Precise dosing will be achieved by droplet feeding of the larvae and LD 50 and LT 50 values will be compared. AcNPV is able to infect a large number (>40) different moth species and less detailed assays with other species will also be carried out.
  • Cp-cal The DNA sequence of a synthetic C. pomonella callatostatin gene (Cp-cal) will be derived from the amino acid sequence (s) of the most active peptide (s) using codon preferences obtained from an analysis of eight CpGV genes previously characterized. A C-terminal Gly will be added for amidation and the gene will be preceded by a signal peptide sequence e.g. from chorion and adipokinetic hormone genes which have been used successfully with the baculovirus expression system. The gene will be flanked by appropriate restriction enzyme sites for cloning into the CpGV transfer vector and subsequent insertion into the CpGV genome. Studies on expression and pathogenicity will be performed as in (2) and (3) above.
  • Example 16 Identification and sequencing of helicostatin gene of Heli coverpa armicrera
  • the gene encoding the helicostatin peptides of Helicoverpa armigera was similarly identified and sequenced.
  • the cDNA sequence of the helicostatin gene of H. armigera is shown in Figure 17.
  • the cDNA is 838 nucleotides long and includes the entire protein open reading frame (nucleotides 100 to 781 inclusive) .
  • the deduced amino acid sequence of the open reading frame of the cDNA of the gene is shown in Figure 18.
  • the sequence is the putative peptide precursor sequence and contains 228 amino acid residues.
  • Example 17 Isolation of peptides from Cydia pomonella , Heli coverpa armigera and Pteronidia salici s
  • Table 6 shows the amino acid sequences and M r values of the Leu-callatostatin neuropeptides or cydiastatins isolated from C. pomonella .
  • the M r of those peptides lacking an acidic residue remains the same as the M r before methylation, thus showing that the carboxyl group at the C-terminus is amidated during processing.
  • M r shows an increase of approximately 30 following methylation. This indicates that (a) the Met residue has been oxidised during the methylation process, (b) that the Asp residue has been methylated, and that (c) that the C-terminal Leu residue is amidated.
  • Table 7 shows the amino acid sequences and M r values of the Leu-callatostatin neuropeptides or helicostatins isolated from H. armigera .
  • ° - M r shows an increase of approximately 16 following methylation. This result indicates that the Met residue has been oxidised during the methylation process and that the C-terminus is amidated.

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Abstract

The DNA encoding novel peptides isolated from the blowfly Calliphora vomitoria of the callatostatin class of insect neuropeptides has been isolated. The DNA which encodes these novel peptides also encodes the other known Leucallatostatin peptides that have already been isolated from C. vomitoria. The homologues of this gene have also been isolated from Helicoverpa armigera and the Australian sheep blowfly Lucilia cuprina. The peptides have inhibitory effects on gut motility in the blowfly C. vomitoria and may have allatostatic effects in cockroaches. The peptides have application as insecticides and the DNA encoding the peptides may be inserted into a baculovirus for use as an insecticide against the pest species Cydia pomonella or Helicoverpa armigera, or other target organisms. Variants of these peptides have also been isolated from Cydia pomonella, Helicoverpa armigera and Pteronida salicis.

Description

INSECT NEUROPEPTIDES GENES AND PEPTIDES
The present invention relates to novel DNA isolated from the blowfly C. vomi toria which encodes novel peptides of the callatostatin class of insect peptides. The novel peptides have also been isolated and purified from C. vomi toria . The DNA also encodes the other known Leu-callatostatin peptides of C. vomitoria. The homologue of this gene has also been isolated from the Australian sheep blowfly Lucilia cuprina and peptides of this class have also been shown to exist in the codling moth, Cydia pomonella and the armyworm Helicoverpa armigera , both potential targets for insecticides based on these peptides. Variants of these peptides have been identified from the large willow sawfly Pteronida salici s .
The first group of callatostatins to be isolated from Calliphora vomi toria were shown to have some sequence homology to cockroach allatostatins (Duve et al in Proc . Na t ' l . Acad . Sci . USA 90: 2456-2460) and the peptides have the following sequences:
Asp-Pro-Leu-Asn-Glu-Glu-Arg-Arg-Ala-Asn-Arg-Tyr-Gly- Phe-Gly-Leu-NH2,
Leu-Asn-Glu-Glu-Arg-Arg-Ala-Asn-Arg-Tyr-Gly-Phe-Gly-Leu-
NH2,
Ala-Asn-Arg-Tyr-Gly-Phe-Gly-Leu-NH2,
Asp or Asn-Arg-Pro-Tyr-Ser-Phe-Gly-Leu-NH2, and
Gly-Pro-Pro-Tyr-Asp-Phe-Gly-Met-NH2.
The first four peptides are known as Leu-callatostatins, now designated Leu-callatostatins 1, 2, 3 and 4. The fifth peptide is known as Met-callatostatin or Met-cast (Duve et al Cell Ti ssue Res . 276 367-379 (1994)) . Subsequently, three further Met-callatostatins have been isolated (Duve et al J. Biol . Chem. 269 21059-21066 (1994) ; Duve et al Regul . Pept . 57 237-245 (1995)) . Two of the additional Met-callatostatinpeptides are post-translationally modified by hydroxylation at either the Pro2 or Pro3 amino acid residues of the parent Met-callatostatin. The third Met- callatostatin is an N-terminally truncated des-Gly1-Pro2 variant. The peptides have the following sequences:
Gly-Hyp-Pro-Tyr-Asp-Phe-Gly-Met-NH2 (designated [Hyp2]Met-callatostatin or [Hyp2]Met-cast) ,
Gly-Pro-Hyp-Tyr-Asp-Phe-Gly-Met-NH2 (designated [Hyp3]Met-callatostatin or [Hyp3]Met-cast) , and
Pro-Tyr-Asp-Phe-Gly-Met-NH2 (designated Met-callatostatin 3-8 or des-Gly-Pro-Met-callatostatin) .
The Leu-callatostatin sub-family of peptides appears to be characterised by the possession of the C-terminal sequence - Tyr-Xaa-Phe-Gly-Leu-NH2 (where Xaa is Gly or Ser) . The four Met-callatostatins differ from the Leu-callatostatins by having Asp in position Xaa and Met-NH2 at the C-terminus.
This new class of insect peptides may be related to the allatostatin class of peptides isolated from other unrelated insect species.
The allatostatin class of insect neuropeptides in cockroaches have been shown capable of inhibiting the production of juvenile hormone (JH) by the corpus allaturn. The corpus allatum (CA) is a classical endocrine gland of insects situated in close proximity to the brain with which it has nervous connection. Members of the allatostatin class of neuropeptides have now been isolated and identified in five species (other than C. vomi toria) , from four different orders of insects (other than the order Diptera) . In the cockroach, Diploptera punctata (Order: Blattodea) five allatostatins ranging in size from 8 to 18 amino acids have been characterised (Pratt et al in Proc . Nat ' l . Acad. Sci USA
88, 2412-2416 (1991) and Woodhead et al in Proc . Nat ' l . Acad. Sci . USA 86, 5997-6001 (1989)) . Two other allatostatins have also been identified in the cockroach Periplaneta americana
(Order: Blattodea) (Weaver et al in C Comp . Physiol . Biochem .
107, 119-127 (1994)) and in the cockroach Bla tella germanica
(Belles et al Regul . Pept . 53 237-245 (1994)) . In the tobacco hornworm moth, Manduca sexta (Order: Lepidoptera) a further allatostatin has been identified, but this has been shown to be structurally completely different to the other allatostatins (Kramer et al in Proc . Nat ' l . Acad . Sci . USA 88, 9458-9462 (1991)) . Recently, allatostatin peptides have been reported to be present in the cricket Gryllus bimaculatus (Order: Orthoptera) (Lorenz et al Regul . Pept . 57 227-239 (1995) ) .
The significance of the allatostatins (and callatostatins) lies in the fact that " in vi tro" they have been shown, with the exception of the allatostatin from the tobacco hornworm moth, to inhibit the production of juvenile hormone (JH) by the corpus allatum in cockroaches. The allatostatin molecule isolated from the tobacco hornworm moth inhibits JH production in the species in which it is found i.e. Manduca sexta but not in cockroaches. The Callatostatins are of interest because they are potent inhibitors of JH synthesis and release in cockroaches, but not in the blowfly, the species from they originate. The callatostatins are also of interest because of their effect on normal gut physiology in insects.
Juvenile hormone plays a crucial role in insect development by controlling metamorphosis, adult sexual maturity and reproduction. Interference with juvenile hormone biosynthesis and release through exploitation of the allatostatins and callatostatins may lead to insect control strategies that do not damage the environment . Use of the DNA encoding the callatostatin peptides and the novel callatostatin peptides of the present invention may also provide an additional insect control strategy which relies on interfering with the normal gut physiology in larvae and/or adults of major lepidopteran pest species, such as the codling moth Cydia pomonella and the armyworm Helicoverpa armigera . The peptides may also have utility as insecticides against other insect species.
The present invention is based on the discovery and potential practical commercial application of the DNA encoding one or more of four novel peptides identified in the blowfly Calliphora vomi toria and the other known Leu-callatostatins 1 to 4 in C. vomitoria, and the isolation and purification of the novel peptides, designated callatostatins 5, 6, 7 and 8. The present invention also extends to the homologous prohormone genes from the Australian sheep blowfly Lucilia cuprina and Helicoverpa armigera and to the peptides coded for by these genes. Variants of the peptides have also been isolated from Cydia pomonella and Pteronidia salicis .
According to a first aspect of the present invention there is provided a recombinant or isolated DNA sequence encoding the amino acid sequence shown in Figure 2 , in Figure 4 , in Figure 18, or an amino acid sequence which is substantially homologous thereto.
In a second aspect of the present invention there is provided a recombinant or isolated DNA sequence encoding the amino acid sequence shown in Figure 2, in Figure 4 or in Figure 18.
A third aspect of the present invention provides a recombinant or isolated DNA sequence comprising the protein coding region of the DNA sequence shown in Figure 2, in Figure 4 or in Figure 17.
Particularly preferred coding sequences are shown in Figure 2 for the C. vomi toria Leu-callatostatin peptides gene, in Figures 4 and Figure 17 for the homologous L. cuprina and Helicoverpa armigera Leu-callatostatin peptides genes respectively, as will subsequently be described in the examples. Those skilled in the art, will with the information given in this specification, be able to identify with sufficient precision the coding regions and to isolate and/or recombine DNA containing them.
In this specification, the gene encoding the novel Leu- callatostatin peptides 5, 6, 7 and 8 and the known Leu¬ callatostatin peptides 1, 2, 3 and 4 of C. vomi toria , L . cuprina and H. armigera will be referred to as the Leu¬ callatostatin gene.
The Leu-callatostatin genes shown in Figure 2, Figure 4 and Figure 17 contain coding regions and the invention therefore also extends to the prohormone sequences coded for by the Leu¬ callatostatin genes. The coding region contains a number of putative peptide sequences which would be excised after translation of the mRNA for the gene. The putative coding sequences correspond with the peptides already identified and the novel peptides of the present invention.
The amino acid sequences of the peptides according to this invention are as follows :-
Ala-Arg-Pro-Tyr-Ser-Phe-Gly-Leu (designated Leu¬ callatostatin 5) ,
Arg-Pro-Tyr-Ser-Phe-Gly-Leu (designated Leu¬ callatostatin 6) ,
Val-Glu-Arg-Tyr-Ala-Phe-Gly-Leu (designated Leu¬ callatostatin 7) , or
Leu-Pro-Val-Tyr-Asn-Phe-Gly-Leu (designated Leu- callatostatin 8) . The invention embraces Leu-callatostatins 5 to 8 defined above when isolated and/or when substantially purified and essentially free of other peptide material.
The present invention also extends to variants of the peptides of the present invention. An example of a variant of the present invention is a Leu-callatostatin peptide as defined above, apart from the substitution of one or more amino acids with one or more other amino acids. The skilled person is aware that various amino acids have similar properties. One or more such amino acids of a substance can often be substituted by one or more other such amino acids without eliminating a desired activity of that substance. Additionally, different amino acids as substituents may enhance the activity of a particular peptide.
Thus the amino acids glycine, alanine, valine, leucine and isoleucine can often be substituted for one another (amino acids having aliphatic side chains) . Of these possible substitutions it is preferred that glycine and alanine are used to substitute for one another (since they have relatively short side chains) and that valine, methionine, leucine and isoleucine are used to substitute for one another (since they have larger aliphatic side chains which are hydrophobic) .
Other amino acids which can often be substituted for one another include: phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains) ; lysine, arginine and histidine (amino acids having basic side chains) ; aspartate and glutamate (amino acids having acidic side chains) ; asparagine and glutamine (amino acids having amide side chains) ; and cysteine and methionine (amino acids having sulphur containing side chains) . Substitutions of this nature are often referred to as "conservative" or "semi- conservative" amino acid substitutions.
Amino acid deletions or insertions may also be made relative to the amino acid sequences of the peptides of the present invention given above. Thus, for example, amino acids which do not have a substantial effect on the activity of the peptides of the present invention, or at least which do not eliminate such activity, may be deleted. Such deletions can be advantageous since the overall length and the molecular weight of a peptide can be reduced whilst still retaining activity. This can enable the amount of peptide required for a particular purpose to be reduced - for example, dosage levels can be reduced.
Amino acid insertions relative to the sequence of the peptides of the present invention can also be made. This may be done to alter the properties of a substance of the present invention, for example to assist in identification, purification or expression in relation to production of fusion proteins.
Amino acid changes relative to the sequences of the peptides of the present invention given above can be made using any suitable technique e.g. by using site-directed mutagenesis.
It should be appreciated that amino acid substitutions or insertions within the scope of the present invention can be made using naturally occurring or non-naturally occurring amino acids. Whether or not natural or synthetic amino acids are used, it is preferred that only L- amino acids are present. However, D- amino acid substitutions are known, for example D-phenylalanine in certain molluscan neuropeptides and the present invention is not limited in this regard.
Whatever amino acid changes are made (whether by means of substitution, insertion or deletion) , preferred peptides of the present invention have at least 50% sequence identity with a peptide according to the present invention, more preferably the degree of sequence identity is at least 75%. Sequence identities of at least 90% or at least 95% are most preferred. The degree of amino acid sequence identity can be calculated using a program such as "bestfit" (Smith and Waterman, Advances in Appli ed Mathematics, 482-489 (1981) ) to find the best segment of similarity between any two sequences. The alignment is based on maximising the score achieved using a matrix of ammo acid similarities, such as that described by Schwarz and Dayhof (Atlas of Protein Sequence and Structure, Dayhof, M.O., ed. 353-358 (1979)) . Where high degrees of sequence identity are present there will be relatively few differences in ammo acid sequence. Thus for example they may be less than 20, less than 10, or even less than 5 differences .
As described above, in addition to the preferred sequences of the peptides isolated from C. vomi toria and L. cuprina , the present invention also extends to variants of the peptides of the present invention and these variants include the peptides isolated from Cydia pomonella, Helicoverpa armigera and Pteronidia salicis when isolated and/or when substantially purified and essentially free of other peptide material.
The peptides isolated from C. pomonella are as follows:
Ser-Pro-His-Tyr-Asn-Phe-Gly-Leu (designated cydiastat 1) ,
Ala-Tyr-Ser-Tyr-Val-Ser-Glu-Tyr-Lys-Arg-Leu-Pro-Val-Tyr- Asn-Phe-Gly-Leu (designated cydiastatm 2) ,
Leu-Pro-Val-Tyr-Asn-Phe-Gly-Leu (designated cydiastatm 2(11-18) ) ,
Ser-Arg-Pro-Tyr-Ser-Phe-Gly-Leu (designated cydiastatm 3) ,
Ala-Arg-Pro-Tyr-Ser-Phe-Gly-Leu (designated cydiastatm 4) , Ala-Arg-Gly-Tyr-Asp-Phe-Gly-Leu (designated cydiastatin 5) ,
Leu-Pro-Leu-Tyr-Asn-Phe-Gly-Leu (designated cydiastatin 6) ,
Lys-Met-Tyr-Asp-Phe-Gly-Leu (designated cydiastatin 7) ,
Leu-Pro-lie-Ty -Asn-Phe-Gly-Leu, and
Ala-Arg-Leu-Tyr-Ser-Phe-Gly-Leu.
The peptides isolated from H. armigera are as follows:
Ser-Pro-His-Tyr-Asp-Phe-Gly-Leu (designatedhelicostatin 1) ,
Ala-Tyr-Ser-Tyr-Val-Ser-Glu-Tyr-Lys-Arg-Leu-Pro-Val-Tyr- Asn-Phe-Gly-Leu (designated helicostatin 2) ,
Leu-Pro-Val-Tyr-Asn-Phe-Gly-Leu (designatedhelicostatin 2 (11-18) ) ,
Ser-Arg-Pro-Tyr-Ser-Phe-Gly-Leu (designatedhelicostatin 3) ,
Ala-Arg-Pro-Tyr-Ser-Phe-Gly-Leu (designated helicostatin 4) ,
Ala-Arg-Ala-Tyr-Asp-Phe-Gly-Leu (designatedhelicostatin 5) ,
Leu-Pro-Met-Tyr-Asn-Phe-Gly-Leu (designatedhelicostatin 6) ,
Ala-Arg-Ser-Tyr-Asn-Phe-Gly-Leu (designatedhelicostatin 7) , Tyr-Ser-Lys-Phe-Asn-Phe-Gly-Leu (designated helicostatin 8) , and
Glu-Arg-Asp-Met-His-Arg-Phe-Ser-Phe-Gly-Leu (designated helicostatin 9) .
The peptides isolated from P. salicis are as follows:
Gly -Gly -Glu -Asp -Phe -Gly -His -Arg -Tyr -Ala -Phe -Gly -Leu (designated pteridostatm 1) ,
Ala-Arg-Pro-Tyr-Asn-Phe-Gly-Leu (designated pteridostatm 2) ,
Ala -Arg - Leu - Tyr - Ser - Phe -Gly- Leu (designated pteridostatm 3) ,
Leu-Pro-Ile-Tyr-Asn-Phe-Gly-Leu (designated pteπdostatin 4) , and
Leu- Pro -Leu- Tyr -Asn- Phe -Gly- Leu.
Preferably, the peptides with the above sequences from C. vomitoria, L. cuprina, H. armigera, C. pomonella and P. salicis are amidated at the carboxy terminus.
The peptide Leu-callatostatin 5 isolated from C. vomitoria, has the sequence ARPYSFGL-NH2 and this sequence is shared by the peptides isolated from C. pomonella and H. armigera. In P. salicis the sequence is altered by the substitution of a leucine residue m place of the prolme at position 3, i.e. the peptide has the sequence ARLYSFGL-NH2. C. pomonella and H. armigera share a peptide sequence SRPYSFGL-NH2 which differs from Leu-callatostatm 5 by the substitution of a serine residue in place of the alanine residue at position 1. Leu-callatostatin 6 from C. vomitoria has the peptide sequence RPYSFGL-NH2 which represents a deletion of the N-terminal alanine residue.
The peptide Leu-callatostatin 7 from C. vomi toria, has the sequence VERYAFGL-NH2 and this sequence is shared in part by the peptide GGEDFGHRYAFGL-NH2 from P. salicis . The valine and glutamic acid residues at positions 1 and 2 in Leu¬ callatostatin 7 are replace by a glycine and a histidine residue respectively with the addition of prior pentapeptide sequence.
Leu-callatostatin 8 from C. vomi toria has the sequence LPVYNFGL-NH2 and this sequence is shared by peptides isolated from C. pomonella and H. armigera . In C. pomonella peptides have also been isolated which have the valine residue at position 3 replaced by either an isoleucine or a leucine residue. This sequence is also shared by peptides isolated from P. salicis . Further changes include the replacement of the valine at position 3 by a methionine residue in H. armigera and the replacement of the leucine and proline residue at positions 1 and 2 by alanine and arginine respectively in P. salicis . C. pomonella and H. armigera also share the peptide sequence AYSYVSEYKRLPVYNFGL-NH2 which represents the sequence of Leu-callatostatin 8 with a prior decapeptide sequence.
The lepidopteran species C. pomonella and H. armigera also share some sequence homology in the newly isolated peptides which have aspartic acid as the post-tyrosyl residue. The peptides ARGYDFGL-NH2 and KMYDFGL-NH2 in C. pomonella differ in that the residues 1 to 3 of the first peptide being alanine, arginine and glycine are replaced by lysine and methionine in the second peptide. In H. armigera the peptide sequence is altered by the replacement of the glycine at position 3 in the first C. pomonella peptide by an alanine residue.
The invention also embraces insecticidal compositions which contain one or more of the above peptides described above, which may be a liposomal formulation, and methods of killing or controlling insects which involve applying to the insects or their environment such msecticidal compositions. The insecticidal compositions may contain besides at least one msecticidally effective peptide described above, a suitable carrier, diluent or excipient therefor. The formulations according to the present invention may be administered in the form of a spray.
Methods of making insecticidal compositions are also embraced by the present invention which comprises admixing one or more the said Leu-callatostatins, helicostatins, pteridiostat s or cydiastat s described above with a suitable carrier, diluent or excipient therefor.
DNA according to the present invention can be synthesised by standard techniques for the preparation of genetic material. The process may comprise ligating together successive nucleic acid residues and/or oligonucleotides to produce DNA.
The peptides described above can be synthesized by the skilled worker by routine peptide synthesis by selecting the appropriate ammo acids and reaction conditions.
The use of such peptides as an insecticide is also within the scope of the present invention which may comprise the inhibition of gut motility. The use of such secticidal compositions may be specific for the codling moth Cydia pomonella or for the army worm Helicoverpa armigera .
The codling moth, Cydia pomonella , is a major worldwide pest of apples and pears and causes the greatest economic damage of any lepidopteran pest the UK. Resistance is developing to chemicals currently being used for its control and there is a strong desire to reduce chemical inputs, particularly on unprocessed food crops. The army worm, Helicoverpa armigera , is reported to be the most important insect pest known to agricultur .
The invention also extends to vector systems comprising a DNA sequence which encodes a peptide as described above for expression m a host cell . Suitable vectors are preferably specific for the target organism and may be baculovirus based.
Baculoviruses are currently being developed as pest control agents (Payne, C. C, in Biological Plant and Heal th Protection, ed. Franz, J.M. (Fortschritte der Zoologie 32) , G. Fischer Verlag, Stuttgart, New York (1986) ) and several commercial products based on these viruses are now available (W stanley et al in Exploitation of Microorganisms, ed. D. G. Jones, 105-136, Chapman & Hall, London (1993)) . Viruses are, however, slower to kill insects than chemical insecticides and feeding may continue for some time after application of the msecticidal agent. It has been possible to reduce this damage by using genetically-engineered viruses that express arachnid toxins (Stewart et al Nature 352 85-88
(1991), Tomalski et al Nature 352 82-855 (1991) ) . This work has involved Autographa calif ornica nuclear polyhedrosis virus
(AcNPV) , which is widely used as a model system (King et al
The Baculovirus Expression System Chapman and Hall, London (1992) , O'Reilly et al Baculovirus Expression Vectors : A
Laboratory Manual Salt Lake City, UT, W. H. Freeman (1992)) .
However, its application with the UK has caused some concern
(Williamson, M. (1991) Na ture 353 394 (1991) , Tickell, O. Ne
Scientist 145 6 (1995) ) . Firstly, because it is not an endemic virus, secondly because it is not effective against any UK pest species, and thirdly because it has a broad host range which includes many non-pest lepidopterans.
A more appropriate baculovirus would be Cydia pomonella granulosis virus (CpGV) . This virus has been isolated in the UK, is very effective against the codling moth C. pomonella , which is a ma or U.K. pest. This baculovirus has a narrow host range which includes only a few closely related tortricid species, and recombinant strains can be produced. Although CpGV is already produced commercially, improved strains, capable of faster killing, could also be used to reduce further the crop damage. Expression of the callatostatin peptides at high levels by baculoviruses would have the highly desirable effect of inhibiting feeding in infected larvae.
The heterologous genes of the blowfly, Calliphora vomi toria , of the sheep blowfly, Lucillia cuprina , or of Helicoverap armigera , according to the present invention may be used in such a baculovirus. Alternatively, the present invention also comprehends the use of a synthetic gene based on identified peptide sequences from C. pomonella or the actual endogenous gene of C. pomonella . A major advantage of the latter approach is that CpGV can be engineered to carry a gene that is found only in the host larvae, thus minimising any possible risk to the environment. Similar approaches may also suitable with respect to the development of insecticides against H. armigera . The present invention has now demonstrated that similar peptides to the C. vomitoria peptides are expressed in these pest species and in some cases the amino acid sequence is identical e.g. Leu-callatostatin 8 which has the structure LPVYNFGL-NH2 and this peptide is also found in both C. pomonella and H. armigera .
Extensive trials have shown CpGV to be as effective as chemical control in reducing major damage (Ballard et al in Biotechnology and Crop Improvement and Protection 355-369, Monograph N. 34, ed. Day, P.R. BCPC, The British Crop Protection Council, Thornton Heath, U. K. , (1986)) . Additionally, in a recent review on biological control of codling moth (Falcon et al in World Crop Pests - Tortricid Pests and their Control 355-369, ed. L.P.S. van der Geest & H.H, Evenhuis, Elsevier, Amsterdam, (1991)) it was concluded that CpGV was the most promising agent available, though the relatively slow rate of kill by the virus allows significant crop damage to be caused by the pest. Very recently this virus has gone into commercial production, for example Carpovirusine ™ or Granupom™.
The person skilled in the art will be able to select other suitable vector systems, including baculoviruses, with reference to the target pest organism.
The peptides described by the present invention are the third group of the callatostatin class of compounds to be isolated from this major order of insects, the Diptera. The first and second groups were also from this species, the blowfly Calliphora vomi toria .
The invention will now be described by way of example with reference to the accompanying Examples and drawings which are provided for the purposes of illustration and neither of which are to be construed as being limiting on the present invention.
FIGURE 1 shows the design of an oligonucleotide callatostatin gene probe of low degeneracy for C. vomi toria library screening.
(a) shows the sequence of degenerate PCR primers
C1S1 and C1A1 based on amino acids at the N- and C- termini of Leu-callatostatin 1.
(b) shows the sequences of four independent amplicons derived from PCR amplification of
C. vomi toria genomic DNA with primers C1S1 and C1A1.
(c) shows the sequence of a 12-fold degenerate oligonucleotide probe PCRCastl based on the sequences of the four PCR amplicons. I and N indicate that either inosine or all our nucleotides respectively were used at that position in the oligonucleotide.
FIGURE 2 shows the sequence of the C. vomi toria Dra I fragment showing two open reading frames. Each of the putative peptides (boxed) is flanked by a pair of basic amino acid residues (bold underline) and has a glycine residue (italicised) at its C-terminal end. Proposed intron splice sites are marked with a vertical bar and the region of the putative intron is indicate.
FIGURE 3a shows a dot matrix comparison of L. cuprina and C. vomi toria genomic DNA sequences. The black bars indicate the position of the two open reading frames encoding callatostatin-like peptides in the C. vomi toria sequence.
FIGURE 3b shows a sequence comparison of the prohormone amino acid sequences identified in C. vomi toria and L. cuprina . C. vomi toria prohormone amino acid sequence from Figure 2 and L. cuprina prohormone amino acid sequence from Figure 4. The symbol "•••" represents an identical amino acid residue and the symbol "-" represents an insertion of a residue relative to the C. vomi toria sequence.
FIGURE 4 shows L. cuprina cDNA and the deduced prohormone sequence. Putative peptides are boxed and numbered with Roman numerals. Pairs of basic residues proposed as sites of proteolytic cleavage are underlined in bold and the C-terminal glycine substrates for carboxylamidation are marked in italics. The translation termination codon is indicated by an asterisk (*) and vertical arrows mark the positions where introns occur in the genomic DNA. Locations of primers used for RT-PCR analysis are indicated. FIGURE 5 shows a diagrammatic representation of the dipteran Leu-callatostatin prohormone deduced from L. cuprina genomic and cDNA and C. vomi toria genomic DNA clones. The positions of the callatostatin peptides are marked with diagonal lines and numbered I-V according to the nomenclature of Table 1. Sites of endoproteolytic cleavage are indicated by solid black boxes and the proposed signal peptide domain is marked by vertical bars.
FIGURE 6 shows the organisation of the dipteran Leu¬ callatostatin prohormone gene showing sites of two introns. Numbers refer to nucleotide coordinates of the L cuprina cDNA. The positions of translation initiation (ATG) and termination (TAA) codons are indicated and the prohormone open reading frame is shaded.
FIGURE 7 shows nested reverse transcriptase PCR analysis of mRNA pools prepared from L. cuprina adult head and midgut . Lane M contains molecular weight markers (lOObp ladder) . The head and midgut (Gut) cDNA samples are indicated and "1", "2" are the first and second (nested) PCR reaction products respectively. Lane "Gen" contains the amplification product from L. cuprina genomic DNA with the primer pair AST-2/AST-3.
FIGURE 8 shows a flow diagram of the purification of Leu-callatostatin 7 and Leu-callatostatin 8 from C. vomi toria . Mr * indicates the mass of the peptide after methylation.
FIGURE 9 shows a flow diagram of the purification of Leu-callatostatins 4, 5 and 6 from C. vomi toria [Leu- callatostatin 4 being a comparative example] . Chromatography being carried out according to conditions detailed in step 6. Mr * indicates the mass after methylation.
FIGURE 10 shows dose responses for inhibition of spontaneous muscle contractions of the rectum (colon and rectal pouch) of C. vomi toria by Leu-callatostatins 5 and 8. Percentage inhibition calculated relative to basal spontaneous contraction movements. Each point represents the mean of 5 to 10 measurements from a number of different vitellogenic flies.
FIGURE 11 shows a chromatographic profile of an extract of 100 heads of adult blowflies of C. vomi toria assayed with five different callatostatin RIAs specific for five different types of peptides, shown in the figure, ending C-termmally as follows:
-Tyr-Asp-Phe-Gly-Met-NH2
-Tyr-Asn-Phe-Gly-Met-NH2
-Tyr-Gly-Phe-Gly-Met-NH2
-Tyr-Ala-Phe-Gly-Met-NH2
-Tyr-Ser-Phe-Gly-Met-NH2
FIGURE 12 shows a chromatographic profile of an extract of 1000 Cydia pomonella larvae chromatographed and assayed in an identical manner to C. vomi toria as shown in Figure 11.
FIGURE 13 shows dissected C. pomonella 5th star larvae :
Figure 13A shows a normal larvae, and Figure 13B shows a larvae fixed and stained with X-gal to show the presence of recombinant CpGV expressing β- galactosidase (blue-black coloration) . This demonstrates the successful engineering of C. pomonella granulosis virus.
FIGURE 14 shows a drawing of the gut of C. pomonella to show the regions 1 to 5 from which Figures 15-1 to 15-5 are taken. Abbreviations in order from anterior (left) to posterior: br - brain, sog - suboesophageal ganglion, oe n - oesophageal nerve, gn - gastric nerve, cr - crop, gi - gizzard, mg - midgut, mt - Malphigian tubules, hg - hindgut, mnc -median neurosecretory cells, fg - frontal ganglion.
FIGURE 15 shows whole mounts of brain, frontal ganglion and gut tissues of C. pomonella 5th instar larvae with immunofluorescence technique using an antiserum raised against Leu-callatostatin 3. Left = anterior.
Figure 15-1 shows frontal ganglion containing 4 callatostatin-immunoreactive cells of which the two most anterior cells give rise to Leu-callatostatin- immunoreactive material in and around the valve between the foregut and the midgut (see Figure 15- 4) .
Figure 15-2 shows the dorsal part of the brain with Leu-callatostatin immunoreactivity in certain of the median neurosecretory cell groups.
Figure 15-3 shows callatostatin immunoreactive axons in the gastric nerve. These have their origins from cells of both the frontal ganglion and the brain.
Figure 15-4 shows accumulation of Leu-callatostatin immunoreactive material in the muscles of the valve separating the foregut from the midgut. Figure 15-5 shows Leu-callatostatin immunoreactivity in posteriorly located midgut endocrine cells immediately anterior to the valve between the midgut and the hindgut .
FIGURE 16 shows dose-response for the inhibition of peristaltic contractions of the foregut of 5th instar larvae of C. pomonella by the blowfly callatostatin neuropeptide Leu-callatostatin 3 (Ala-Asn-Arg-Tyr-Gly- Phe-Gly-Leu-NH2) . The percentage inhibition is relative to basal spontaneous peristaltic contractions.
FIGURE 17 shows the cDNA sequence of the helicostatin gene of Helicoverpa armigera .
FIGURE 18 shows the deduced amino acid sequence of the open reading frame from the cDNA of the helicostatin gene of Helicoverpa armigera .
Examples 1 to 8 Identification of the dipteran Leu¬ callatostatin peptide family : characterisation of the prohormone gene from Calliphora vomi toria and Lucilia cuprina
Insect rearing and tissue dissection C. vomitoria were obtained as pupae and allowed to eclose under laboratory conditions (25°C : 65% relative humidity : 12 h light/dark) . The flies were fed sugar, beef heart and water for 1-3 weeks after which they were anaesthetized with C02, frozen and stored at -20°C prior to use. Larval L. cuprina were reared under a 12:12 hour light :dark cycle, on fresh minced sheep liver supplemented with meat meal, fish meal and cotton lint. Adult L. cuprina were maintained on water and a protein biscuit comprising a solidified mixture of sugar, egg powder, milk powder and yeast. Tissues for RNA extraction were dissected under sterile, ice-cold phosphate- buffered saline (139 mM NaCl, 10 mM sodium phosphate, pH 7.2) , transferred to a microfuge tube and snap frozen in liquid nitrogen. Tissues were stored frozen at -80°C until use.
Example 1: Preparation of a callatostatin gene probe Degenerate oligonucleotide primers based on the ammo- and carboxyl-terminal sequences of the hexadecapeptide Leu¬ callatostatin 1 were synthesised (Pharmacia Gene Assembler) and used to amplify a fragment of the callatostatin gene from C. vomi toria genomic DNA using the polymerase chain reaction (Pratt et al Proc . Nat ' l . Acad. Sci . USA 88 2412-2416 (1991)) . The structures of the sense (ClSl) and antisense (ClAl) primers are shown m Figure 1. PCR reactions consisted of 20 mM Tris-HCl, pH 8.4 , 50 mM KCI, 1.5 mM MgCl2, 0.2 mM each of dATP, dCTP, dGTP and dTTP, 50 pmoles of each primer, 1 μg of C. vomi toria genomic DNA, 2.5 units Tag DNA polymerase and H20 to a final volume of 50 μl .
Reactions were performed m a thermocycler (Corbett Research, FTS-320) under the following conditions; 95°C for 1 mm, 50°C for 1 mm, 72°C for 1 mm, for 40 cycles; then 1 cycle of 72°C for 5 mm to end the reaction. The 44 bp amplicon generated in this PCR reaction was isolated and subcloned, using the method of Marchuk et al . (Nucl . Acids Res . 19 1154 (1991)) , into the T-tailed Eco RV site of pBluescπpt SK(+) for sequence analysis. Alignment of sequence from several independent clones was used to design a long oligonucleotide of low degeneracy (PCRCastl) for library screening as shown Figure 1.
Example 2: Callatostatin gene cloning Approximately 1.5 x 105 recombinant bacteriophages from a C. vomi toria genomic DΝA library were plated on the E. coli host strain KW251 and plaque DΝA was transferred to nitrocellulose filters according to standard techniques (Sambrook et al Molecular Cloning: A Laboratory Manual 2nd edition. Cold Spring Harbour Laboratory Press, USA (1989)) . The oligonucleotide PCRCastl was radiolabelled with γ-32P-dATP using the Klenow fragment of E. coli DΝA polymerase (Sambrook et al Molecular Cloning: A Labora tory Manual 2nd edition. Cold Spring Harbour Laboratory Press, USA (1989) ) and hybridised to the library filters overnight at 57°C in a solution containing 6X SSPE, 10X Denhardt' s solution, 0.1% w/v SDS, 0.1% w/v sodium pyrophosphate and lOOμg/ml denatured salmon sperm DNA. Filters were washed three times for 15 min each in 2X SSC, 0.1% w/v SDS at 57°C. Hybridising clones were plaque purified and DNA was extracted by a plate lysate method (Sambrook et al Molecular Cloning: A Laboratory Manual 2nd edition. Cold Spring Harbour Laboratory Press, USA (1989)) . Restriction enzyme fragments were subcloned into pBluescript SK(+) (Stratagene) .
The L. cuprina callatostatin homologue was isolated from a recombinant genomic DNA library using a 936 bp Dra I restriction enzyme fragment containing the C. vomi toria prohormone gene as probe. Hybridisation probes were prepared by random-primed synthesis using the NEBlot kit (New England Biolabs) and α-32P-dATP (NEN-DuPont) . Approximately three genome equivalents were screened at high stringency (65°C in 6X SSC, 5X Denhardt's solution, 1 mM EDTA, 0.1% w/v SDS, 200 μg/ml denatured salmon sperm DNA) and filters were washed three times for 30 min each in 0.5X SSC, 0.1% w/v SDS at 65°C. Positives were plaque purified, DNA was extracted, mapped by restriction enzyme digest and Southern blot analysis and fragments of interest were subcloned into pBluescript SK(+) as described above. Approximately 5 x 105 recombinant phages from a L. cuprina random-primed head cDNA library were screened using a lkb Hind III fragment containing the L. cuprina callatostatin gene as probe. Hybridisation and wash conditions were as described for the L. cuprina genomic library screen. The cDNA insert from hybridising phages was excised with Eco Rl and subcloned into pBluescript SK(+) for sequencing.
Example 3: Reverse transcriptase-PCR
Total RNA was extracted from 0.5g adult heads or 0.2g midguts dissected from adult female L. cuprina using Ultraspec II reagent (Biotecx) . Polyadenylated RNA was isolated by oligo(dT) cellulose chromatography using standard techniques
(Sambrook et al Molecular Cloning: A Laboratory Manual 2nd edition. Cold Spring Harbour Laboratory Press, USA (1989) ) . First strand cDNA was synthesised from 1 μg of poly(A) + RNA in a reaction consisting of: 50 mM Tris-HCL (pH 8.3) , 75 mM Kcl , 3 mM MgCl2, 1 mM DTT, 15 mM each of dATP, dCTP, dGTP and dTTP, 20 units RNasin (Promega Biotec) , 25 ng oligo(dT) primer, 1 μg mRNA, 200 units of Superscript II reverse transcriptase
(Gibco-BRL) and H20 to a final volume of 20 μl . The reaction was incubated at 37°C for 2 h, then diluted to 1 ml with TE buffer and stored at 4°C. Nested PCR primers were designed from the L . cuprina cDNA sequence as follows:
lcap-1 5' -CTCAACTAGAGGATAAAAGC-3 ' , lcap-2 5' -CGTTAGCCTTTTGATGTTGG-3' , lcap-3 5' -CGACGTCCTAAACCAAAGC-3 ' , lcap-4 5' -GGAATTATTGGCTGGATAGTG-3'
The first PCR reaction contained; 20 mM Tris-HCl, pH 8.4 , 50 mM KCI, 1.5 mM MgCl2, 0.2 mM each of dATP, dCTP, dGTP and dTTP, 50 pmoles of the primer pair lcap-l/lcap-4, 10 μl of cDNA and H20 to a final volume of 50 μl . Amplification conditions were; 95°C for 5 min, 50°C for 1 min with addition of 2 μl Taq DNA polymerase, followed by 95°C for 1 min, 50°C for 1 min, 72°C for 1 min, for 30 cycles; then 1 cycle of 72°C for 5 min to end the reaction. The nested PCR reaction conditions were identical to those above, except that the primer pair lcap-2/lcap-3 was used and the template was 1 μl of the first PCR reaction.
Example 4 : DNA sequencing and analysis
Double-stranded plasmid DNA for sequencing was prepared by a standard alkaline lysis mini-prep procedure (Sambrook et al
Mol ecular Cloning: A Laboratory Manual 2nd edition. Cold
Spring Harbour Laboratory Press, USA (1989) ) with the inclusion of an extended phenol extraction step. Templates were sequenced by di-deoxy chain termination thermal cycle sequencing, using dye-labelled primers (Applied Biosystems) . Sequencing reactions were run on the Applied Biosystems Model 370A DNA sequencer and sequence was analysed using the GCG software package (Genetics Computer Group Program manual for the GCG package, Version 8.0.1, Madison, Wisconsin (September 1994) .
Example 5: Isolation of the C. vomi toria callatostatin gene Since the amino acid sequences of the four Leu-callatostatin peptides were not favourable for the design of oligonucleotides suitable for library screening, a PCR approach was used to amplify a short region of the callatostatin gene to provide a basis for probe design.
Short, degenerate oligonucleotides based on N- and C-terminal sequences of the hexadecapeptide Leu-callatostatin 1, shown in Figure 1 (a) , were used to prime a PCR reaction with C. vomi toria genomic DNA as template. The 44 bp amplicon produced from this reaction was cloned and several independent clones were sequenced, shown in Figure 1 (b) . This sequence information allowed the design of a minimally degenerate oligonucleotide probe, shown in Figure 1(c) that was used for library screening. Approximately 1.5 x 105 independent recombinants from a C. vomi toria genomic library were screened, from which a single positive (λCvastl)was obtained. Restriction enzyme analysis of λCvastl identified a 3.2 kb Bam El/Xba I fragment that hybridised to the oligonucleotide probe. Partial sequencing of this fragment identified a 936 bp Dra I restriction enzyme fragment that contained the sequence of the oligonucleotide probe.
The Dra I fragment was subcloned and sequenced on both strands. Sequence analysis identified likely protein coding regions in two distinct open reading frames, that together encoded five separate putative Leu-callatostatin peptides, each of which was flanked by pairs of basic amino acids that constitute potential endoproteolytic cleavage sites, shown in Figure 2. Each of these peptides had a single glycine residue at the C-terminal end that provides a substrate for peptidyl glycine a-amidating monooxygenase, consistent with the observation that all previously identified callatostatin peptides are amidated at the C-terminus (Duve et al Proc. Nat'l. Acad. Sci. USA 90 2456-2460 (1993)) . The sequences of the five putative callatostatins are given in Table 1 in order of their appearance in the gene.
Table 1: Peptide sequences deduced from the callatostatin prohormone gene
Peptide Sequence
Cvast I VERYAFGL
Cvast II AYTYTNGGNGIKRLPVYNFGL
Cvast III ARPYSFGL
Cvast IV NRPYSFGL'
Cvast V DPLNEERRANRYGFGL'
' Leu-callatostatin 4 of Duve et al Proc. Nat'l Acad. Sci. USA 902456-2460 (1993) ' Leu-callatostatin 1 of Duve et al Proc Nat'l Acad Sci USA 902456-2460 (1993)
Peptide IV corresponds to Leu-callatostatin 4 of Duve et al {Proc. Nat'l. Acad. Sci. USA 902456-2460 (1993)) and resolves the uncertainties in the original sequence data for that peptide. Peptide V corresponds to Leu-callatostatin 1. The putative peptides I-III were not identified in the previous purification studies (Duve et al Proc. Nat'l. Acad. Sci. USA 902456-2460 (1993) , Duve et al J. Biol. Chem. 269 21059-21066
(1994) , Duve et al Regul. Pept. 57 237-245 (1995) ) but information on this matter has now been obtained (Duve et al
Regul. Pept. [in press] (1996)) . Peptide III is identical to one of the peptides encoded on the cockroach prohormone gene (Donly et al Proc. Nat'l. Acad. Sci. USA 90 8807-8811 (1993) ) .
A further 1.2 kb of DNA flanking the Dra I fragment was sequenced, but contained no open reading frames or additional callatostatin-like sequences. The occurrence of the peptides in two separate open reading frames suggested the presence of a short intron with the protein coding region of the gene. This was supported by the presence of a consensus donor splice sequence at the 3' end of the upstream open reading frame and a splice acceptor sequence at the 5' end of the downstream open reading frame, shown m Figure 2. To extend the understanding of the dipteran Leu-callatostatin gene and peptide family, a second species, the sheep blowfly L . cuprina, was examined.
Example 6: Genomic and cDNA analysis of the L. cuprina callatostatin gene
The 936 bp Dra I fragment containing the callatostatin gene was radiolabelled and used to screen a L. cuprina genomic DNA library. Two hybridising clones were purified and found to contain a 4.3 kb Xba I restriction enzyme fragment that hybridised to the C. vomi toria probe. A 2.6 kb Cla I/Xba I fragment was subcloned from the Xba I fragment and partially sequenced to identify the region homologous to the callatostatin prohormone gene. Figure 3 shows a dot matrix comparison of the L. cuprina sequence with that of the C. vomi toria Dra I fragment. The two sequences were 84% identical over this region and similarity was greatest m the two domains corresponding to the open reading frames identified in C. vomi toria indicating that gene structure was conserved between the two species.
A 1 kb Hind III fragment that encompassed the peptide coding region of the L. cuprina gene was used to probe a cDNA library prepared from adult head mRNA. A single positive was isolated from a sample of approximately 3 x 105 clones, suggesting that transcripts are relatively rare. The cDNA clone was sequenced and found to contain a single open reading frame encoding a Lucilia Leu-callatostatin prohormone 179 amino acids m length, shown m Figure 4. Since the clone was isolated from a random-primed library it cannot represent a full length cDNA. However, the open reading frame is flanked by 341 nucleotides of 5' and 271 nucleotides of 3' sequence, each of which contains multiple translation stop codons in all three reading frames. Thus, the cDNA encodes all of the L. cuprina callatostatin pre-prohormone.
The first in-frame methionine residue, and proposed translation initiation site occurs at nucleotide 342 and the translation termination codon is at nucleotide 879. The putative pre-prohormone begins with a hydrophobic domain of 19 amino acids that is a probable signal peptide. The most likely site of cleavage of this signal sequence is at residue 20 (von Heijne, G. Nucl . Acids Res . 14 4683-4690 (1986)) . The deduced prohormone sequence contains five Leu-callatostatin- like peptides that are identical to those identified from the C. vomi toria genomic clone, shown in Figure 2 and in Table 1.
The proposed structure of the dipteran callatostatin prohormone is shown diagrammatically in Figure 5. The callatostatin-like peptides occur in two blocks, separated by a region with a high percentage of acidic residues (theoretical pi = 4.31, including the two di-basic pairs of residues required for prohormone processing) . The first three peptides are tandemly arrayed, separated only by the residues required for proteolytic processing and carboxyamidation. The other two peptides occur as a tandem pair at the carboxyl end of the prohormone. This structure is absolutely conserved in the two blowfly species.
Example 7: Genomic organisation of the callatostatin prohormone gene
Comparison of the genomic and cDNA sequences (data not shown) , established that the L . cuprina Leu-callatostatin gene is composed of at least three exons, as shown in Figure 6. Introns are located between nucleotides 289/290 and 777/778 of the L . cuprina cDNA clone, as seen in Figure 5.
The first intron is located in the 5' untranslated region of the mRNA and although no attempt was made to identify exon 1 in genomic DNA, this intron must be at least 2 kb long, based on hybridisation of the cDNA to existing clones (not shown) . The second intron is very short, 63 bp in length, and occurs within the prohormone open reading frame in the region between the two blocks of peptides. Direct comparison of the L. cuprina genomic and cDNA sequences confirmed the prohormone structure proposed from the C. vomi toria genomic analysis of Figure 2.
Example 8: Expression of the callatostatin gene Reverse transcriptase-PCR was used to investigate expression of the callatostatin prohormone gene in cDNA pools prepared from whole adult head, representing an enriched source of brain and sub-oesophageal ganglion tissue, and from isolated midgut. To provide maximum sensitivity a 'nested' PCR design was used (McPherson et al in M.J. McPherson, P. Quirke, P. and
G.R. Taylor, (Eds.) , PCR : a practical approach 171-186
(1991)) , with primer pairs chosen to include the open reading frame of the prohormone and the small second intron. Since the callatostatin prohormone gene is interrupted by introns, a PCR reaction was performed on genomic DNA using only the nested primers lcap-2/lcap-3 which flank the small second intron. The positions of all four primers in the L . cuprina cDNA are indicated on Figure 4.
The two cDNA pools yielded identical-sized products with both primer pairs, shown in Figure 7, and sequencing of the amplicons established that the structure of the cDNA was the same in brain and midgut tissues. Since the primers include almost all of the prohormone open reading frame, and it has been established by in si tu hybridisation that the gene is transcribed in both the CNS and midgut endocrine cells of adult C. vomi toria and L. cuprina (East et al Cell Tissue Res . 280 355-364 (1995)), both of these major sites of callatostatin expression have the capacity to produce the full complement of Leu-callatostatin peptides. The amplicon generated from genomic DNA was slightly larger than the corresponding product from the cDNA pools as expected, due to the 63 bp intron, shown in Figure 7. This genomic PCR result demonstrated that the RT-PCR products were bona fide cDNA products and not artifacts produced from contaminating genomic DNA.
Discussion of examples 1 to 8
A genomic clone encoding the sequences of the peptides Leu- callatostatin 1 and Leu-callatostatin 4 was isolated from a C. vomi toria library. This clone contained two open reading frames, each of which contained Leu-callatostatin sequences. The Leu-callatostatin peptides identified by purification from neural tissue extracts (Duve et al Proc . Nat ' l . Acad . Sci . USA 90 2456-2460 (1993)) were encoded on the second open reading frame. Three related, but structurally distinct putative Leu¬ callatostatin peptides were encoded on the first open reading frame. These three putative peptides all possessed the C- terminal pentapeptide sequence -Tyr-Xaa-Phe-Gly-Leu characteristic of the callatostatin/ allatostatin peptide family.
The octapeptide Leu-callatostatin 4 contained two uncertain residue assignments from the Edman peptide sequencing. The first (N-terminal) residue was identified as either Asp or Asn and the fifth amino acid was tentatively identified as Ser. The prohormone gene sequence has allowed unequivocal assignment of these residues as Asn at position 1 and Ser at position 5. When the Leu-callatostatins 1-3 were isolated, it was noted that the hexadecapeptide Leu-callatostatin 1 contained a pair of basic (Arg) residues at positions 7 and 8 which, if used as a proteolytic cleavage site during prohormone maturation, would give rise to the octapeptide Leu¬ callatostatin 3. Similarly, the 14 residue Leu-callatostatin 2 was identical to Leu-callatostatin 1 with the two amino- terminal residues, Asp-Pro, removed. At this time, it was not clear whether these three peptides were each encoded independently on the callatostatin prohormone, or if Leu- callatostatins 2 and 3 were derived by proteolytic processing of Leu-callatostatm 1.
Since the C. vomi toria prohormone sequence contains a complete copy of the Leu-callatostatin 1 peptide flanked by appropriate post-translational processing signals, with no separate copies of the Leu-callatostatm 2 and 3 peptides, it appears that the shorter peptides must be derived from Leu-callatostatm 1. Since the octapeptide Leu-callatostatm 3 is considerably more potent than Leu-callatostatm 1 in bioassays on the lleal segment of the hindgut (Duve et al Cell Tissue Res . 216 367- 379 (1994) ) , it is possible that the longer peptide functions as a precursor for the transport and delivery of the more active, shorter form.
It is known that allatostatins are released into the hae olymph of cockroaches (Woodhead et al J. Insect Physiol . 39 1001-1005 (1993)) and the presence of Leu-callatostatm immunoreactivity m potential neurohaemal sites such as the corpus cardiacum and heart, and m gut endocrine cells (Duve et al Cell Tissue Res . 276 367-379 (1994)) suggests that this is likely to occur in blowflies also. If so, production of a precursor such as Leu-callatostatm 1 may provide a mechanism for protecting the biologically active form of the peptide from degradation by haemolymph am opeptidases.
The two open reading frames of the C. vomi toria Leu- callatostatm prohormone and an additional 1 kb of flanking sequence did not contain any copies of the Met- callatostatin peptide. In addition, an oligonucleotide probe based on the Met-callatostatm peptide sequence did not hybridise to the 16 kb genomic clone λCvastl which suggests that the Leu- and Met-callatostatms are not encoded on the same prohormone. To investigate this further, both genomic and cDNA clones of the Leu-callatostatm prohormone were isolated from a second blowfly species, L . cuprina . Sequence analysis of these clones confirmed the organisation deduced for the Calliphora gene and established that five putative Leu-callatostatin peptides are present in the blowfly prohormone.
The prohormone and deduced peptide sequences are identical in both blowfly species. This absolute conservation of peptide structure might reflect a functional constraint on sequence evolution in the Leu-callatostatin peptide family, since at least one other multi-member neuropeptide family, the CalliFMRFamides, does not have the same degree of conservation in these two species (Duve et al in Perspectives in Comparative Endocrinology, 91-96, eds. K.G. Davey, R.E. Peter, and S.S. Tobe, National Research Council of Canada: Ottawa (1994) ) .
The allatostatin prohormones of the cockroaches D. puncta ta and P. ameri cana are also highly conserved (Stay et al Adv. Insect Physiol 25 269-337 (1994) ) . However, there is very little similarity in prohormone organisation between the cockroaches and flies. The cockroach prohormone is approximately 370 amino acids long and encodes 13 and 14 allatostatin peptides in D . punctata (Donly et al Proc . Na t ' l . Acad . Sci . USA 90 8807-8811 (1993)) and P. americana (Stay et al Adv. Insect Physiol 25 269-337 (1994) ) respectively, with the peptides occurring in several distinct blocks separated by acidic spacer regions.
In contrast, the dipteran prohormone is approximately 180 residues in length and contains only five Leu-callatostatin peptides distributed in two blocks, separated by an acidic spacer. The octapeptide Ala-Arg-Pro-Tyr-Ser-Phe-Gly-Leu-NH2
(putative peptide III of the dipteran prohormone) is the only peptide that is identical in all four species. The second putative peptide of the dipteran prohormone (peptide II) is Which may be a structural homologue of the allatostatin ASB2 isolated by Pratt et al ( Proc . Na t ' l . Acad . Sci . USA 88 2412- 2416 (1991) ) , which is also the second peptide in the cockroach prohormone (Donly et al Proc . Nat ' l . Acad . Sci . USA 90 8807-8811 (1993)) . These peptides are both long, 21 residues in blowfly and 18 in cockroach, are identical for 10 amino acids at their C-terminus and are very similar at the tyrosine-rich N-terminus. Like Leu-callatostatin 1, this peptide has an internal pair of basic amino acids and recent work (Duve et al Regul . Pept . [ in press] (1996)) shows that proteolytic cleavage does indeed occur here. The remaining peptides are all quite different in sequence. Thus, there are some similarities and some marked differences, most notably in the total number of peptides, between the prohormone in these two insect orders.
The extent of the differences between flies and cockroaches raises some interesting questions concerning peptide function. It is already clear that the functions of callatostatin/ allatostatin peptides are not completely conserved. As their name indicates, the cockroach allatostatins were initially identified through their inhibitory activity on juvenile hormone synthesis by the corpus allatum (Woodhead et al Proc. Nat ' l . Acad . Sci . USA, 86 5997-6001 (1989)) . The callatostatin peptides, while being potent inhibitors of the cockroach corpus allatum, have no effect on juvenile hormone synthesis in the blowfly (Duve et al Proc . Nat ' l . Acad . Sci . USA 38 8807-8811 (1992) ) . Apart from their effect on juvenile hormone synthesis, the only other function so far ascribed to the callatostatin/allatostatin peptides is in regulation of visceral muscle contraction. In C. vomi toria Leu¬ callatostatin peptides are potent inhibitors of hindgut contraction (Duve et al Cell Tissue Res . 276 367-379 (1994)) . Moreover, this effect is highly specific, affecting only the ileum, the region of the hindgut between the point of entry of the Malpighian tubules and the rectal valve. There are significant differences in potency between peptides, with Leu- callatostatin 1 being several orders of magnitude less potent than its C-terminal derivative Leu-callatostatin 3. The allatostatins are also myoinhibitory in cockroaches (Lange et al Arch . Insect . Biochem . Physiol . 24 79-92 (1993) , Lange et al J. Insect Physiol . 41 581-588 (1995) , Duve et al Physiol . Ento ol . 20 33-44 (1995)) . All thirteen peptides encoded on the D . punctata allatostatin prohormone gene inhibit both spontaneous and proctolin-induced contractions of the isolated hindgut of that species, with considerable differences in potency between members of the family (Lange et al J. Insect Physiol . 41 581-588 (1995)) . The blowfly peptide Leu¬ callatostatin 3 was also active against this D. punctata hindgut preparation. Duve et al ( Physiol . Entomol . 20 33-44 (1995)) investigated the effects of several callatostatins on the gut of the cockroach, Leucophaea maderae and found strong inhibition of spontaneous contractions of the foregut by several peptides, including Leu-callatostatin 3, but no effect on hindgut contraction at concentrations up to 10 ~ η M for any of the peptides of this family. The difference in response for the two cockroach species is intriguing and warrants further investigation. It may reflect the diversity of allatostatin peptides in cockroaches, or a degree of peptide/receptor specialisation in different regions of the gut, that is revealed through the use of the heterologous blowfly peptides. Indeed, physiological studies with the newly described Leu-callatostatins provide direct evidence for differential effects of this peptide family on the blowfly hindgut (Duve et al Regul . Pept . [in press] 1996)) .
The Leu-callatostatin/allatostatin peptides appear to be quite old in evolutionary terms. The presence of allatostatin immunoreactivity and biological activity of allatostatin peptides in a crab (Skiebe et al J. Exp . Biol . 194 195-208
(1994)) suggests that this peptide family existed at least since the divergence of insects and crustaceans. In fact, a recent survey demonstrated the presence of allatostatin-like immunoreactivity in the nervous systems of most of the lower invertebrate phyla (Smart et al J. Comp . Neurol . 347 426-432 (1994)) . If the peptides responsible for this immunoreactivity prove to belong to the Leu-callatostatin/ allatostatin family, a widespread occurrence in nerves associated with the gut suggests that regulation of this organ may be one of their ancestral functions.
Although it is frequently difficult to establish clear homologies between specific neurones of different insects, immunocytochemical studies have shown that at least some of the peptides are expressed in interneurons within the CNS of both C. vomi toria (Duve et al Cell Tissue Res . 276 367-379 (1994)) and D. punctata (Stay et al Adv. Insect Physiol 25 269-337 (1994)) . The other clearly homologous site of expression shared between flies and cockroaches is in intrinsic endocrine cells of the gut. In blowflies the Leu¬ callatostatin prohormone gene (East et al Cell Tissue Res . 280 355-364 (1995)) and peptides (Duve et al Cell Tissue Res . 276 367-379 (1994) , East et al Cell Tissue Res . 280 355-364 (1995) ) are expressed in a population of endocrine cells at the posterior end of the midgut .
In D. punctata, allatostatin mRNA and peptide immunoreactivity have been identified in midgut endocrine cells (Reichwald et al Proc . Na t ' l . Acad . Sci . USA 91 11894- 11898 (1994)) although their spatial distribution was not reported. Although some allatostatins have been partially purified from cockroach midgut extracts, it is not known if the full gene complement of peptides is expressed in this tissue.
The existence of a peptide family with multiple functions raises several questions about mechanisms of receptor-mediated signal transduction and the specificity of response of individual cell types. One means for obtaining tissue specific responses is through differential expression of the peptides. This could be achieved transcriptionally, through alternative promoter utilisation or cell specific mRNA splicing, or post-translationally, at the level of prohormone processing or peptide maturation and delivery. The discovery that the callatostatin peptides are encoded on two separate exons raised the possibility that different cell types might generate different populations of peptides through a mechanism of alternative RNA splicing. To investigate this, a sensitive nested RT-PCR strategy was used to examine callatostatin gene expression in two different tissues, brain and midgut. Cloning and sequencing of the amplicons generated from these two tissue types established that mRNA encoding all members of the Leu-callatostatin family is expressed in both head and midgut. Thus, if the peptides are differentially expressed in these tissues, the effect must be mediated post- translationally.
Since the putative Leu-callatostatin peptides I-III identified from the gene sequence were not found in previous purifications of blowfly extracts, it is possible that they are not processed from the prohormone. However, the failure to recover these peptides might have resulted from the specificity of the antisera used for the radioimmunoassays to monitor peptide purification. Previous purification work used antisera directed against the C-terminus of Leu-callatostatin 1. Since the variable post-tyrosyl residue is Gly in this peptide, whereas the putative peptides encoded by sequences I-III have Ala, Asn and Ser in this position, they may have gone undetected in the radioimmunoassay. Further studies of blowfly tissue extracts with antisera raised against the putative Leu- callatostatin peptides encoded by the callatostatin gene sequences described here (peptides I-III) have now been carried out and the results provide a complete understanding of peptide expression (Duve et al Regul . Pept . [ in press] (1996) ) .
Examples 9 to 11 Identification of the dipteran Leu¬ callatostatin peptide family and precursor processing revealed by isolation studies in Calliyhora vomi toria
Radioimmunoassay The two putative octapeptides deduced from the Leu- callatostatm gene sequence (East et al Regul . Pept . [m press] (1996)) , Ala-Arg-Pro-Tyr-Ser-Phe-Gly-Leu-NH2 and Val- Glu-Arg-Tyr-Ala-Phe-Gly-Leu-NH2, together with the octapeptide Leu-Pro-Val-Tyr-Asn-Phe-Gly-Leu-NH2 (assuming an enzymatic cleavage at the dibasic pair of residues the longer putativepeptideAla-Tyr-Thr-Tyr-Thr-Asn-Gly-Gly-Asn-Gly-Ile- Lys-Arg-Leu-Pro-Val-Tyr-Asn-Phe-Gly-Leu-NH2) , were synthesized (by Affiniti Research Products Ltd. , Exeter, UK) with the N- terminal addition of Cys-jβAla-øAla and were conjugated through the N-termmus to keyhole limpet haemocyanm using m- maleimidobenzoic acid N-hydroxysucclnimide ester.
After the collection of pre-immune sera, three Dutch rabbits per peptide-camer protein-conjugate were immunized, initially w th antigen emulsified Freund's complete adjuvant, and subsequently at intervals of 2 weeks with antigen emulsified in Freund's incomplete adjuvant. After emulsification, 1 ml of immunogen (100 μg/conjugate) was injected subcutaneously at 4 sites on the flanks of each rabbit. Bleeding from the marginal ear vein was carried out 1 week after each immunization and tested in both immunocytochemistry and radioimmunoassay.
Immunocytochemistry of tissue sections and wholemounts revealed essentially the same populations of neurones and gut endocrine cells as previously reported (Duve, H. et al Cell Tissue Res . 276367-379 (1994)) . The radioimmunoassays showed a high degree of specificity for the Xaa residue occurring after Tyr. The data for cross-reactivity of the callatostatins known to occur in C. vomi toria from previous purifications (Duve et al Proc . Nat ' l . Acad . Sci . USA 90 2456- 2460 (1993) , Duve et al J. Biol . Chem. 26921059-21066 (1994) , Duve et al Regul . Pept . 57 237-245 (1995) ) and by deduction from the DNA sequence (East et al Regul . Pept . [ in press] (1996) ) against the complete range of antisera used in RIA in the present study are shown in Table 2. Table 2: Cross-reactivities in the radioimmunoassays used in the isolation and identification of Leu-callatostatins from the blowfly Calliphora vomi toria
ANTISERA PEPTIDES
Figure imgf000039_0001
nb Results are expressed as a % of the reactivity shown by the peptide against which the antiserum was raised, in the same RIA The concentration of standards in RIAs used to monitor HPLC elution profiles ranged from 015nM to lOnM
* Previously identified peptides (Duve et al Proc Nat ' l Acad Sci USA 902456-2460 (1993) , Duve et a l J Bwl Chem 26921059-21066 (1994), Duve et a l Regul Pept 57237-245 (1995))
** Peptides deduced from the callatostatin gene (East et al Regul Pept [ m press] (1996))
Animals and extracts
Calliphora vomi toria were obtained as pupae and allowed to eclose under laboratory conditions (25°C : 65% relative humidity : 12 h light/dark) . The flies were fed sugar, ox heart and water for 1-3 weeks after which they were anaesthetized with C02, frozen and stored at -20°C prior to use. Approximately 40,000 heads were collected by shaking the frozen flies in a plastic bag containing solid C02 followed by sieving. They were ground in batches of 2000 in a mortar and pestle in solid C02 and homogenized further in a Waring blender m 400 ml methanol/acetic acid/water (87:5:8) at 4°C. The extract was left overnight at 4°C after which it was centrifuged at 3000 x g for 30 min. The pellet was re- extracted with 1 1 of extraction fluid. The combined supernatants were concentrated to approximately 300 ml on a rotary evaporator at 35°C under vacuum. The concentrate was allowed to stand overnight at 4°C before being filtered through Whatman No. 4 paper and re-centrifuged at 15000 x g for 10 min prior to purification by HPLC.
HPLC procedures
The HPLC system for Steps 1-4 comprised two Waters 6000M pumps and a Waters 741 detector. For Step 5 and some of Step 6 a Waters 625 system was used at room temperature, and for some Step 6 procedures (61) and all of Step 7 a Hewlett-Packard model 1090 HPLC system at 50°C was used. Details of the reversed-phase HPLC columns, solvent systems, flow rates, gradients and their rates of change, used in the purification are as follows.
Step 1:
Waters μBondapak semi-preparative C1B column (300 x 7.8 mm, 10 μm, 125 A), linear gradient 0 tol00% CH3CN/0.1%TFA m 60 mm, flow rate 1.5 ml/min. Each run comprised 7.5 ml of concentrated extract, the equivalent of 1000 heads, diluted with 15 ml water/0.1% TFA, pumped onto the column. Profiles were analysed with the five RIAs detailed m Table 2. Immunoreactivity occurred predominantly in the fractions eluting between 17 and 30% acetonitrile. However, since at this stage it was difficult to separate the different forms of immunoreactivity (and hence the different peptides) , all fractions containing immunoreactivity were pooled for further processing.
Step 2 :
The same column and the same basic running conditions were used as in Step 1 except that 10 mM ammonium acetate (pH 6.5) replaced 0.1%TFA. The pooled material from Step 1 was aliquotted to give 25 separate runs, each containing the equivalent of 1600 heads.
Radioimmunoassays of the profiles showed separation into five different areas: Gly/Leu together with Asp/Met material (i.e. those peptides previously identified and designated Leu- callatostatins 1-3 and the various forms of Met-callatostatin (Duve et al Proc . Na t ' l . Acad . Sci . USA 90 2456-2460 (1993) , Duve et al J. Biol . Chem . 269 21059-21066 (1994) , Duve et al Regul . Pept . 57 237-245 (1995))) in fractions eluting between 14.4 and 24.8% CH3CN, Ala/Leu material 24.9 - 31.9%, Asn/Leu material 32 - 36%, and Ser/Leu 36.1 - 52%, including the peptide previously identified and designated Leu-callatostatin 4 (Duve et al Proc . Na t ' l . Acad . Sci . USA 90 2456-2460 (1993) ) .
In view of the previous identification of Leu-callatostatins 1-3 (Gly/Leu compounds) and also the Met-callatostatins (Duve et al Proc . Na t ' l . Acad . Sci . USA 90 2456-2460 (1993) , Duve et al J. Biol . Chem. 269 21059-21066 (1994) , Duve et al Regul . Pept . 57 237-245 (1995)) , purification of the pool of immunoreactive material between 14.4 and 24.8% CH3CN was discontinued at this point. For the Ala/Leu and Asn/Leu materials, further purification was straightforward and resulted in the identification of a single Ala/Leu peptide and a single Asn/Leu peptide. For the Ser/Leu material however, purification proved more difficult and resulted in the identification of three Ser/Leu peptides.
Step 3 : Bio-Rad Hi-Pore C4 column (250 x 4.6 mm, 5 μm, 300 A), linear gradient 0 - 80% CH3CN/10 mM ammonium acetate (pH 6.5) in 200 min, flow rate 1.5ml/min.
Step 4: Kromasil C1B column (250 x 4.6 mm, 5 μm, 300 A) , gradient CH3CN/0.1% TFA 0 - 15% in 10 min; 15 - 50% in 140 min, flow rate 1 ml/min.
Step 5: Kromasil CIB column (250 x 3.2 mm, 5μm, 300A) , gradient CH3CN/0.1% TFA 0 - 15% in 15 min; 15 - 50% in 175 min, flow rate 0.5 ml/min.
Step : Vydac C18 column (150 x 2.1 mm, 5 μm, 300 A) , gradient CH3CN/0.1% TFA (0 - 15% in 10 min; 15 - 50 in 175 min, flow rate 0.2 ml/min. [Ala/Leu; Asn/Leu; Ser/Leu I and Ser/Leu lib material] Column at room temperature (22°C) .
Step 61:
Vydac C18 column (150 x 2.1 mm, 5 μm, 300 A) , gradient CH3CN/0.1% TFA (5 - 15% in 5 min; 15 - 25 % in 50 min, flow rate 0.2ml/min. [Ser/Leu Ilai and Ser/Leu Ilaii] Column temperature 50°C.
Step 7 :
Vydac C18 column (150 x 2.1 mm, 5 μm, 300 A) gradient CH3CN/0.1% TFA (for Asn/Leu material 5 - 17% in 5 min; 17 - 27 in 50 min; for Ser/Leu I, Ser/Leu Ilbi and Ser/Leu Ilbii 5 - 11% in 5 min; 11 - 18% in 35 min, flow rate 0.2 ml/min. Column temperature 50°C. The flow charts in Figure 8 and Figure 9 give the % acetonitrile at which immunoreactive material eluted, and details of the points in the procedures where Mr determinations and amino acid sequencing were carried out (nb. The abbreviations for the various types of peptide being isolated are those used in Table 2) .
Mass spectro etry
To test the purity of the peptides, the immunoreactive fractions at Step 6 or 6f were analyzed by matrix-assisted laser desorption mass spectrometry using a Biflex (Bruker- Franzen) instrument. For this, 0.5 μl was mixed with 0.5 μl of a solution of 33mM α-cyano-4-hydroxycinnamic acid in acetonitrile/ methanol (Hewlett-Packard) . After drying, the mixture was analyzed in the linear mode at 15 kV. The method has an accuracy of 0.1%. If a single molecular mass was recorded, the material was subjected to amino acid sequence analysis and definitive mass spectrometry. If more than one peptide was seen to be present in the sample, it was chromatographed further.
For some of the initial Mr determinations, where direct application of the sample from HPLC gave a weak signal, and for the definitive mass spectrometry, a different procedure was used. Here, 50 μl of the samples were dried and redissolved in 5 μl of 0.1% TFA in 30% CH3CN, after which a 0.5 μl aliquot was analyzed as described above. The possible amidation of the COOH-terminus was assessed by determining the total number of free carboxyl groups by methylation of an aliquot and remeasurement of the molecular mass (Talbo et al
Eur. J. Biochem . 195 495-504 (1991)) . Each free carboxyl group results in an increase of 14 in Mr when methylated.
Amino acid sequencing The amino acid sequences of the purified peptides (5 - 50 pmol) were determined with an automated protein sequencer (Applied Biosystems, Procise 494A) equipped with an on-line system for the detection of the amino acid phenylthiohydantoin derivatives. All reagents and solvents were from Applied Biosystems.
Synthetic peptides
Synthesis of the Leu-callatostatins and the peptide conjugates for antibody production was carried out by Affiniti Research Products Ltd. (purity greater than 95%; Mr checked by fast atom bombardment or electron spray mass spectrometry.) For physiological studies, peptides were weighed out and dissolved in Ringer's solution (Duve et al Physiol . Entomol . 20 33-44 (1995)) on the day of use.
Immunocytochemistry The three types of antisera raised against the Ala/Leu, Asn/Leu and Ser/Leu peptides were tested in immunocytochemical procedures as described previously (Duve, H. et al Cell Tissue Res . 276 367-379 (1994)) .
Hindgut physiology
Experiments on the effects of peptides on the inhibition of spontaneous contractile activity of the hindgut of C. vomi toria were performed as previously described (Duve, H. et al Cell Ti ssue Res . 276 367-379 (1994) , Duve et al J. Biol . Chem . 269 21059-21066 (1994) ) .
Example 9: Identification of peptides Ala/Leu peptide:
Purification of the Ala/Leu immunoreactive material resulted in the isolation at Step 6, shown in Figure 8 of an octapeptide with the amino acid sequence Val-Glu-Arg-Tyr-Ala- Phe-Gly-Leu-NH2. The Mr value of this peptide, designated Leu¬ callatostatin 7, was 953.0 compared with the calculated value of 953.2. Following methylation, the recorded Mr value was 968.1, thus confirming C-terminal amidation (taking into account the accuracy of the method, an increase of 14.9 is in accord with methylation of only a single acidic residue [Glu2]) . The final yield was 80 pmol .
Asn/Leu peptide:
Purification of the Asn/Leu-immunoreactive material resulted in the isolation at Step 7, shown in Figure 8, of an octapeptide with the amino acid sequence Leu-Pro-Val-Tyr-Asn- Phe-Gly-Leu-NH2. The recorded Mr value of this peptide designated Leu-callatostatin 8, was 921.4 compared with the calculated value of 921.1. The Mr of the methylated peptide was 922.3, thus confirming carboxyamidation. The final yield was 160 pmol.
Ser/Leu peptides:
Purification of the Ser/Leu-immunoreactive material resulted in the identification of three peptides shown in Figure 9. Two of these, with the sequences Ala-Arg-Pro-Tyr-Ser-Phe-Gly-Leu- NH2 and Arg-Pro-Tyr-Ser-Phe-Gly-Leu-NH2, designated Leu- callatostatins 5 and 6, were isolated in pure form, shown in Figure 9 as Ser/Leu Ilbii and Ser/Leu Ilai. The Mr values were 908.8 and 837.9 respectively and since these were unchanged following methylation (910.1 and 839.1) , it was confirmed that they were both C-terminally amidated. The third peptide, with the sequence Asn-Arg-Pro-Tyr-Ser-Phe-Gly-Leu- NH2, previously identified as Asx-Arg-Pro-Tyr- (Ser) -Phe-Gly- NH2 and designated Leu-callatostatin 4 (Duve et al Proc . Nat ' l . Acad . Sci . USA 90 2456-2460 (1993)) , proved impossible to separate from a lesser amount of Leu-callatostatin 6, shown in Figure 9 as Ser/Leu I and Ser/Leu Ilbi) . The major, unambiguous sequence, however, was that of Leu-callatostatin 4. The Mr values of the peptide before and after methylation were 952.8 and 953.7 respectively, thereby confirming carboxyamidation and the fact that the first residue was Asn and not Asp. The final yields of the three peptides were Leu¬ callatostatin 4, 28 pmols ; Leu-callatostatin 5, 14 pmols; and Leu-callatostatin 6, 120 pmols. Table 3 shows the sequences of the peptides isolated, together with their Mr values, before and after methylation. Table 3: Amino acid sequences and Mr values of Leu-callatostatin neuropeptides isol ated from C. vomitoria.
Trivial name Amino acid sequence Mr value X* Mr value Y" Mr value Z***
Leu-callatostatin Is D-P-L-N-E-E-R R-A-N-R-Y-G-F-G-L-NH2 1906 1 19039 1949 1
1 pu-callatostahn Ϋ L-N-E-E-R-R-A-N-R-Y-G-F-G-L-NH2 16939 16945 -
Leu-callatostatin 3s A-N-R-Y-G-F-G-L-NH2 8960 8968 -
Leu-callatostatin 4§t N-R-P-Y-S-F-G-L-NH2 952.1 9528 9537
Leu-callatostatin 5 A-R-P-Y-S-F-G-L-NH2 9090 9088 910 1
Leu-callatostatin 6 R-P-Y-S-F-G-L-NH2 838 0 8379 839 1
Leu-callatostatin 7 V-E-R-Y-A-F-G-L-NH2 953 1 9532 968 1
Leu-callatostatin 8 L-P-V-Y-N-F-G-L-NH2 921 1 9214 9223
* X calculated Mr value
** Y Mr value of isolated peptide
*** Z Mr alue of isolated peptide after methylation
Data from earlier study (Duve et a l Proc Nat ' l Acad Sci USA 902456-2460 (1993))
Figure imgf000046_0001
Example 10: Immunocytochemistry
Immunocytochemical procedures were carried out on the neural and gut tissues of C. vomi toria with the newly-produced antisera raised against Leu-callatostatins 5, 7 and 8. Essentially, the same cells and axonal pathways that have been fully reported elsewhere (Duve, H. et al Cell Tissue Res . 276 367-379 (1994) , East et al Cell Tissue Res . 280 355-364 (1995)) were visualised. One exception is noteworthy. It has been reported earlier (East et al Cell Tissue Res . 280 355-364 (1995) ) that although in situ hybridisation studies showed Leu-callatostatin gene expression in a small 'satellite' cell accompanying a large (25 μm) Leu-callatostatin neurone in the lateral aspect of the dorsal protocerebrum close to the anterior optic tract, it showed immunoreactivity to the Leu- callatostatin antisera only rarely. However, in the many immunocytochemical preparations examined with the three new types of antisera, the satellite cells were always revealed with the Ala/Leu antisera, but never with either the Ser/Leu or the Asn/Leu antisera.
Example 11: Hindgut physiology
Experiments with Leu-callatostatins 5 and 8 showed them consistently to have a potent, reversible, inhibitory effect on the spontaneous contractile activity of the rectum of C. vomi toria as shown in Figure 10. Leu-callatostatin 5 effected complete inhibition at 10"9M, 90% inhibition at 10"12M and 50% inhibition at 10~14M. Leu-callatostatin 8, which showed a biphasic dose-response curve similar to that previously observed with members of the Met-callatostatin family (Duve et al J. Biol . Chem. 269 21059-21066 (1994) , Duve et al Regul . Pept . 57 237-245 (1995)) , had an even more potent inhibitory effect on the rectum, with 80% inhibition at 10"15M. Effects of Leu-callatostatins 5 and 8 on the ileu were inconsistent. In general a higher concentration was required to cause an inhibitory effect, after which the normal frequency of beating was often not restored. Discussion of results of examples 9 to 11
Four new members of the Leu-callatostatin neuropeptide family of the blowfly Calliphora vomi toria have been isolated and purified. However, the purification of the peptides presented particular problems.
The purification of the mixture of Leu-callatostatins 4, 5 and 6, with the post-tyrosyl Ser residue, proved to be especially difficult. Throughout the early stages of purification, the Ser/Leu- immunoreactivity occurred in broad bands and although there were some signs of definite peaks, these were always associated with extensive tailing not seen with either the Ala/Leu or Asn/Leu immunoreactivities. Such chromatographic profiles caused difficulties in selecting the most appropriate fractions for the next step in purification. The reasons for the difficulties have become clear in hindsight, since the extracts contained not only the two Ser/Leu octapeptides, Leu- callatostatins 4 and 5, which have a common heptapeptide C- terminus and either Ala or Asn as the first residue, and which were predictable from the DNA sequence, but also the N- terminally truncated heptapeptide form of these two peptides. Comparison of the yields of the three Ser/Leu peptides suggests that the truncated Leu-callatostatin 6 may be derived by aminopeptidase activity targeting both Leu-callatostatins 4 and 5.
There is a possibility that this enzymatic activity represents a step in the degradation of the two peptides. However, it is more likely that it represents a specific form of post- translational processing. Such N-terminal trimming has been reported in an analysis of peptides produced by the neuroendocrine light yellow cells (LYCs) of the gastropod mollusc Lymnaea stagnalis (Li et al J. Biol . Chem . 269 30288- 30292 (1994)) . Thus, examination of the cDNA encoding the precursor predicted a straightforward processing into three peptides (LYCP I, II and III) at conventional dibasic processing sites. However, mass spectrometry, confirmed by peptide isolation studies revealed N-terminally trimmed variant peptides derived from LYCP I and II (by removal of a single amino acid residue) . It is concluded that since the variants were much more abundant than the intact peptides, the indication was that LYCP I and II serve as intermediates in a peptide-processing sequence. In C. vomi toria , the fact that neither the Asn/Leu nor the Ala/Leu octapeptides show any evidence of N-terminal trimming suggests that, like the light yellow cell peptides of L. stagnalis, the occurrence of the Ser/Leu heptapeptide in extracts is not the result of enzymatic degradation, but rather that it is a product of specific cellular processing.
Examination of the Leu-callatostatin gene (East et al Regul . Pept. [ in press] (1996)) shows that the Ala/Leu octapeptide, Leu-callatostatin 7, appears to be processed in a predictable way. However, the Asn/Leu octapeptide, Leu-callatostatin 8, is clearly the product of post-translational processing by cleavage at the dibasic site in the putative heneicosapeptide AYTYTNGGNGIKRLPVYNFGL-NH2. The processing appears to go to completion since no other Asn/Leu immunoreactivity that could provide evidence of the heneicosapeptide was found in any of the profiles.
This is in contrast to the processing of the hexadecapeptide,
Leu-callatostatin 1 which appears as a putative peptide on the gene, and is present in extracts together with small amounts
(less than 10% of the parent compound) of the enzymatic cleavage products, the truncated tetradecapeptide and octapeptide, Leu-callatostatins 2 and 3 (Duve et al Proc . Nat ' l . Acad . Sci . USA 90 2456-2460 (1993)) . Since Leu¬ callatostatin 1 has an internal Arg-Arg site, the data suggest the presence of processing enzymes with preference for the Lys-Arg, seen N-terminally to all the other processed sites of the precursor, rather than the Arg-Arg combination. Such a phenomenon would be in line with mammalian dibasic processing enzymes which are known to show substrate specificity (Steiner et al J. Biol. Chem. 267 23435-23438 (1992) ) .
The present findings stress that it is impossible to predict peptide processing based solely on the DNA sequence. Knowledge of the finally expressed and post-translationally modified products, through independent biochemical analysis, is an absolute requirement if the true biologically-active molecules are to be identified.
Comparison of the Leu-callatostatins of C. vomitoria with the homologous allatostatins of the cockroaches Diploptera punctata ( oodhead et al Proc. Nat'l. Acad. Sci. USA 86 5997- 6001 (1989) , Woodhead et al Biochem. Mol. Biol. 24 257-263 (1994) , Pratt et al Biochem. Biophys. Res. Comm. 163 1243-1247 (1989) , Pratt et al Proc. Nat'l. Acad. Sci. USA 88 2412-2416 (1991) , Donly et al Proc. Nat'l. Acad. Sci. USA 90 8807-8811 (1993)) , Periplaneta americana (Weaver et al Co p. Biochem. Physiol. 107C 119-127 (1994)) and Bl at tell a germanica (Belles et al Regul Pept. 53 237-247 (1994)) and the cricket Gryllus bimaculatus (Lorenz et al Regul. Pept. 57 227-236 (1995)) shows that there has been a high degree of amino acid sequence conservation within this family of neuropeptides through the long period of insect evolution.
Not only has the C-terminal pentapeptide sequence Tyr-Xaa-Phe- Gly-Leu-NH2 been conserved (with the Xaa residue restricted to Gly, Ser, Ala or Asn, in those peptides so far identified) but also, in some instances, the N-terminal sequence too. Thus, C. voinitoria possesses a peptide (ARPYSFGL-NH2) identical to one of D. punctata (Leu-callatostatin 5 and dipstatin 6) . In addition, the amino acid sequence represented by the octapeptide Leu-callatostatin 8 in C. vomitoria, occurs as the C-terminal sequence preceded by a dibasic pair of residues Lys-Arg in the octadecapeptide designated dipstatin 2 in D. punctata. There is also a partial N-terminal sequence similarity if a comparison is made between the putative heneicosapeptide of C. vomi toria, from which Leu-callatostatin 8 is generated, and the isolated octadecapeptide of D. punctata . , shown in Table 4.
Table 4
C. vomi toria A-Y-T-Y-T-N-G-G-N-G- I -K-R-L- P-V-Y-N-F-G-L- H2
D. punctata A-Y-S-Y- V-S -E-Y - K-R-L- P-V-Y-N- F-G-L-NH2
A major difference between these two, however, is that whereas in C. vomi toria, extracts provide evidence only of the truncated octapeptide, with no sign of the extended heneicosapeptide, the results of studies on D. puncta ta show that it is the octadecapeptide and not the octapeptide that is present in tissue extracts (Pratt et al Proc . Na t ' l . Acad . Sci . USA 88 2412-2416 (1991)) .
It has been demonstrated previously that the Leu- callatostatins in which Xaa = Gly, are potent inhibitors of spontaneous contractile activity of the ileal segment of the hindgut of C. vomi toria (Duve, H. et al Cell Ti ssue Res . 276 367-379 (1994)) . Thus, Leu-callatostatin 3, the truncated octapeptide variant of Leu-callatostatin 1, which can now be seen to occur as a result of post-translational processing of the precursor by enzymatic cleavage at the dibasic pair of residues (Arg-Arg) , gives complete, reversible inhibition of the ileum at concentrations as low as 10"15M. The 'parent' hexadecapeptide is also effective on this in vitro preparation, although with a reduced potency (maximum inhibition at 10'10M) .
So far Leu-callatostatins 5 and 8 have been tested on the same type of preparation, and the interesting difference is that both have a reversible inhibitory effect on the contractions of the rectum, a complex part of the gut that is completely unaffected by Leu-callatostatins 1 to 3.
These results may be significant in providing an answer to the question why it is necessary for insects to possess such large numbers of closely related peptides. In this instance it appears that motility patterns in different (although immediately adjacent) regions of the gut, are regulated by different members of the Leu-callatostatin peptide family.
The results of these physiological studies may help to explain a problem presented by earlier immunocytochemical studies which showed extensive Leu-callatostatin innervation of the rectum compared with relatively modest innervation of the ileum (Duve, H. et al Cell Tissue Res . 276 367-379 (1994)) . At that time only antisera raised against Leu-callatostatin 3 was being used (Leu-callatostatin 3 being the same peptide that was being used for the physiological studies) and it was not easy to explain why the motility of the rectum was unaffected, whilst that of the ileum was strongly inhibited. The current immunocytochemical studies show, however, that antisera raised against Leu-callatostatins in which Xaa = Ser, Ala or Asn result in immunostaining patterns virtually identical to that realised by the Leu-callatostatin 3 antisera in which Xaa = Gly.
In situ hybridisation studies showed an almost direct correspondence between neurones expressing the Leu¬ callatostatin gene and the pattern of immunostaining represented by Leu-callatostatin 3 antisera (East et al Cell Tissue Res . 280 355-364 (1995)) and it would now appear from the combined results of the purification, genetic and immunocytochemical studies, that the processing of the precursor is carried out to give the major types of peptides in all those cells that express the gene.
Thus, visualisation of Leu-callatostatin immunoreactivity in nerves on the surface of the rectum, in the form of either Ser/Leu or Asn/Leu peptides, may provide the morphological basis for the potent effects of the Ser/Leu (Leu-callatostatin 5) and Asn/Leu (Leu-callatostatin 8) peptides seen. Although contractions of the ileum are inhibited by these two peptides, the effect appears to be irreversible, unlike that seen for Leu-callatostatins 1 and 3.
Since conservation of amino acid sequence is almost certainly associated with the conservation of a fundamental aspect of physiology, it is of interest that the particular function which instigated the search for these peptides, that of the inhibition of biosynthesis of juvenile hormone from the corpus allatum ( i.e. a truly allatostatic effect) , is not observed in flies (Duve et al Proc . Nat ' l . Acad . Sci . USA 90 2456-2460 (1993) ) . A possible ubiquitous role for the callatostatin type of peptides is in the regulation of gut motility. This function in flies, reported here and elsewhere (Duve, H. et al Cell Tissue Res . 276 367-379 (1994)) , is also observed in cockroaches (Duve et al Physiol . Entomol . 20 33-44 (1995) , Lange et al Arch . Insect . Biochem . Physiol . 24 79-92 (1993)) . However, the localized occurrence of the peptides in the brain and other parts of the central and peripheral nervous systems of insects, as well as their presence in midgut endocrine cells (Duve, H. et al Cell Tissue Res . 276 367-379 (1994) , East et al Cell Tissue Res . 280 355-364 (1995)) , suggests other possibilities for fundamental roles in neuromodulation, neurotransmission and endocrine regulation.
A comparison of the prohormone amino acid sequences identified in C. vomi toria and L. cuprina reveals the close sequence homology between the two prohormone sequences and as shown in Figure 3b.
Examples 12 to 15: Improved control of the codling moth Cydia pomonella by baculoviruses expressing insect neuropeptides
Example 12: Insertion of the C. vomi toria callatostatin (cal) gene into CpGV The cal gene may be cloned into the CpGV transfer vector, pCpDNl, which places the gene under the control of the CpGV granulin promoter. This vector has already been used with the lacZ gene and demonstrated high levels of expression of β- galactosidase in larvae infected with the recombinant virus, as shown in Figure 13A and Figure 13B.
The vector pCpDNl is a granulin replacement vector which results in a non-occluded recombinant virus. Whilst this vector may be suitable for studying the peptides that are expressed, for detailed studies on larval gut paralysis and for measurements of LT50 values, it will be preferable to produce an occluded recombinant virus for more extensive assays. For this, a second transfer vector could be constructed to allow insertion of the cal gene into an intergenic region without deleting the granulin gene.
Example 13: Purification of the callatostatin peptide homologues of C. pomonella
Results of preliminary analyses on an extract of 1000 C. pomonella larvae are shown in Figure 12. The purification procedure, chromatography and radioimmunoassay (RIA) are as described previously for C. vomi toria (Duve et al Proc . Nat ' l .
Acad. Sci . USA 90 2456-2460 (1993) , Duve et al J. Biol . Chem.
269 21059-21066 (1994) , Duve et al Regul . Pept . 57 237-245
(1995)) . The results of an equivalent assay on an extract of
1000 heads of adult blowflies of C. vomi toria are shown in Figure 11 for comparison. The considerable differences in amounts and types of peptides present in the two extracts is apparent .
These results show that partially purified extracts of C. pomonella contain callatostatin-immunoreactive peptides as the presence of a carboxyamidated C-terminus is an absolute requirement for the radioimmunoassay used. It would therefore appear that C. pomonella must be able to carry out all the post-translational processing required to produce functional callatostatin peptides. Example 14: Tissue distribution of the callatostatin homologues in Cγdia pomonella
Immunocytochemical studies provide important clues to the function of neuropeptides as exemplified by the identification of strong callatostatin innervation of the hindgut (Duve et al Cell Tissue Res . 276 367-379 (1994)) . The gut of the codling moth C. pomonella is shown diagrammatically in Figure 14. Recently, region-specific and residue-specific antisera have been developed that provide specificity to the variable residue at the -4 position from the COOH-terminus . Results of pilot studies on C. pomonella show intense innervation of the foregut valve, shown in Figure 15 (Figure 15-1 to Figure 15-5) .
Example 15: Physiological and degradation studies of the callatostatin-like peptides in C. pomonella
Preliminary experiments have shown that the spontaneous periodic contractions in the valve region between the foregut and midgut of C. pomonella are inhibited by the callatostatins. (IC50 =1 to 0.1 nM) , shown in Figure 16.
These experiments with codling moth larvae show that the callatostatins are be potent inhibitors of the contractions of the foregut of C. pomonella .
Use of the callatostatin gene as insecticide
A possible methodology for inserting the callatostatin gene or DNA encoding one or more of the peptides into a suitable delivery vehicle for use as an insecticide against a pest such as C. pomonella is as follows.
[A] To develop recombinant CpGV and AcNPV engineered with
(i) the C. vomi toria callatostatin gene and (ii) a synthetic C. pomonella callatostatin gene, and to test their efficacy as insecticides relative to wild-type viruses.
(1) Insertion of the C. vomi toria callatostatin gene into CpGV. (2) Characterization by RIA and ICC of the callatostatins expressed by recombinant CpGV in vi tro and in vivo .
(3) Bioassays of the recombinant and wild-type CpGV strains.
(4) Insertion of the C. vomi toria callatostatin gene into AcNPV.
(5) Characterization by RIA and ICC of the callatostatins expressed by the recombinant AcNPV in vi tro and in vivo .
(6) Bioassays of the recombinant and wild-type
AcNPV strains.
(7) Synthesis of a C. pomonella callatostatin gene (Cp- cal ) , insertion into CpGV. Studies on expression and pathogenicity as in (2) and (3) above .
[B] To characterize the callatostatin homologues of
C. pomonella for the construction of a synthetic C. pomonella gene and, eventually, for producing oligonucleotide probes for the isolation of the endogenous callatostatin gene of C. pomonella .
(8) Purification of the callatostatin peptide homologues of C. pomonella larvae.
(9) Biochemical characterization of C. pomonella callatostatins .
(10) Tissue localization of the callatostatins of
C. pomonella . (11) Physiological effects and degradation of the callatostatins in C. pomonella .
These proposed steps may be further explained as follows:
(1) Insertion of the C. vomi toria callatostatin (cal) gene into CpGV
The cal gene will be cloned into the CpGV transfer vector, pCpDNl, which places the gene under the control of the CpGV granulin promoter. This is a granulin replacement vector which results in a non-occluded recombinant virus. Whilst this will be suitable for studying the peptides that are expressed and for detailed studies on larval gut paralysis and measurements of LT50 values, it would also be desirable to produce an occluded recombinant virus for more extensive assays. For this a second transfer vector will be constructed which will allow insertion of the cal gene into an intergenic region without deleting the granulin gene.
(2) Study of the callatostatins expressed in C. pomonella cells and larvae by the recombinant CpGV
Expression of the callatostatins in cells will be followed using indirect immunofluorescence procedures with our range of different callatostatin antisera. In other experiments, larval or cell homogenates and perfusates of cell cultures will be analyzed by callatostatin RIAs following purification by analytical and narrow-bore HPLC.
(3) Pathogenicity of the recombinant CpGV compared to wild- type virus
The non-occluded recombinant virus will be used to study the extent of paralysis in C. pomonella larvae and compare LT50 values with wild-type virus. This will give a very good indication of how effective the cal gene is. Subsequently, accurate measurements of LD50 values will be performed with an occluded recombinant CpGV using both C. pomonella and Cryptophlebia leucotreta (false codling moth) . In the laboratory, C. leucotreta can be infected with wild-type CpGV but only at a 1000-fold higher dose than is required for C. pomonella . Bioassays with both these species would indicate whether there is any change in infectivity or host range of CpGV due to insertion of the cal gene.
(4) Insertion of the cal gene into AcNPV
The cal gene will be cloned into pAcUW2B to give pAcUW- cal . This transfer vector places the cal gene under the control of the high level plO promoter and also contains the polyhedrin gene. Cotransfection of pAcUW-cal with linearized polyhedrin- negative AcNPV will give rise to polyhedrin-positive recombinants expressing callatostatins. The production of normal occlusion bodies (polyhedra) by recombinant virus will allow accurate bioassays in both neonate and later instars and is the form of the virus which is used in formulations for insecticidal application.
(5) Study of the callatostatins expressed by the recombinant AcNPV
These studies will be carried out in vivo in Heliothis virescens and in vi tro using Spodoptera frugiperda cell lines using the techniques outlined in (Winstanley et al in " Exploi tation of Microorganisms" , ed. D. G. Jones. Chapman & Hall, London, 105-136 (1993)) for the recombinant CpGV. Based on previous studies with expression of proteins by AcNPV, the levels of callatostatin expression are estimated to provide a haemolymph concentration around 10"6M. This should provide large amounts of peptide sufficient for direct measurement and for purification and amino acid sequence determination.
(6) Pathogenicity of recombinant AcNPV compared to wild-type virus
Bioassays to determine the biological effect of insertion of the cal gene into the virus will be done in H. virescens using neonate and later instars. Precise dosing will be achieved by droplet feeding of the larvae and LD50 and LT50 values will be compared. AcNPV is able to infect a large number (>40) different moth species and less detailed assays with other species will also be carried out.
(7) Synthesis of a C. pomonella callatostatin gene (Cp-cal) and insertion into CpGV
The DNA sequence of a synthetic C. pomonella callatostatin gene (Cp-cal) will be derived from the amino acid sequence (s) of the most active peptide (s) using codon preferences obtained from an analysis of eight CpGV genes previously characterized. A C-terminal Gly will be added for amidation and the gene will be preceded by a signal peptide sequence e.g. from chorion and adipokinetic hormone genes which have been used successfully with the baculovirus expression system. The gene will be flanked by appropriate restriction enzyme sites for cloning into the CpGV transfer vector and subsequent insertion into the CpGV genome. Studies on expression and pathogenicity will be performed as in (2) and (3) above.
Example 16: Identification and sequencing of helicostatin gene of Heli coverpa armicrera
Using analogous techniques to those employed in characterising the genes of C. vomi toria and L. cuprina as described above, the gene encoding the helicostatin peptides of Helicoverpa armigera was similarly identified and sequenced. The cDNA sequence of the helicostatin gene of H. armigera is shown in Figure 17. The cDNA is 838 nucleotides long and includes the entire protein open reading frame (nucleotides 100 to 781 inclusive) . The deduced amino acid sequence of the open reading frame of the cDNA of the gene is shown in Figure 18. The sequence is the putative peptide precursor sequence and contains 228 amino acid residues.
Example 17: Isolation of peptides from Cydia pomonella , Heli coverpa armigera and Pteronidia salici s
The peptides from C. pomonella , H. armigera and P. salici s were isolated using the same techniques as described previously for C. vomi toria (Duve et al Proc . Na t ' l . Acad .
Sci . USA 90 2456-2460 (1993), Duve et al J. Biol . Chem . 269
21059-21066 (1994) , Duve et al Regul . Pept . 57 237-245
(1995)) . The peptides isolated are shown in Tables 6, 7 and 8.
Table 6 shows the amino acid sequences and Mr values of the Leu-callatostatin neuropeptides or cydiastatins isolated from C. pomonella .
The Mr of those peptides lacking an acidic residue remains the same as the Mr before methylation, thus showing that the carboxyl group at the C-terminus is amidated during processing.
Table 6
Peptide Armno acid sequence Mr values
X Y Z
D S-R-P-Y-S-F-G-L-NH, 9250 9265 9254
E A-R-P-Y-S-F-G-L-NH 909 0 909 9 909 2
I A-R-L-Y-S-F-G-L-NH, 9267 9267 9259
J A-Y-S-Y-V-S-E-Y-K-R-L P-V-Y-N-F-G-L-NH, 21685 21693 2184 T
F L-p.v-Y-N-F-G-L-NH, 921 1 921 9 921 9
G L-P-I-Y-N-F-G-L-Nrt, 935 1 936 4 935 8
H L-P-L-Y-N-F-G-L-NH, 935 1 9366 935 6
B S-P-H-Y-N-F-G-L-NH, 9330 9335 935 1
A A-R-G-Y-D-F-G-L-NR, 897 0 897 4 911 9"
C K-M-Y-D-F-G-L-NH, 8720 8722 9027"
Key to abbreviations used:
X - calculated Mr value
Y - Mr value of isolated peptide
Z - Mr value of isolated peptide after methylation
- Mr shows an increase of approximately 14 following methylation and indicates that the carboxyl group from the Glu or Asp residues has been methylated. The carboxyl group at the C-terminus of these peptides is amidated.
Mr shows an increase of approximately 30 following methylation. This indicates that (a) the Met residue has been oxidised during the methylation process, (b) that the Asp residue has been methylated, and that (c) that the C-terminal Leu residue is amidated.
Table 7 shows the amino acid sequences and Mr values of the Leu-callatostatin neuropeptides or helicostatins isolated from H. armigera .
Table 7
Peptide Amino acid sequence M, values
X Y 1
D S-R-P-Y-S-F-G-L-NH, 9250 9258 9258
E A-R-P-Y-S-F-G-L-NH, 9090 9096 9100
H A-Y-S-Y-V-S-E-Y-K-R-L-P-V-Y-N-F-G-L-NH, 21685 21699 21839"
F L-P-V-Y-N-F-G-L-NH, 9211 9223 9219
G L-P-M-Y-N-F-G-L-NH, 9531 9533 9705°
C (A)-R-S-Y-N-F-G-L(-NH,) 9260 9269 .
A A-R-A-Y-D-F-G-L-NH, 9116 9116 9263'
B (S)-P-H-Y-D-F-G-L-NH? 9340 9356 9497"
Key to abbreviations used:
X - calculated Mr value, Y - Mr value of isolated peptide Z - Mr value of isolated peptide after methylation
(A) and (S) - the first residues of peptides C and B from H. armigera were deduced by subtraction from the Mr value of the identified amino acid sequence of residues 2 to 8 from the Mr of the entire peptide. The complete sequences are in agreement with those deduced from the cDNA of H. armigera (Davey et al . , unpublished) .
(-NH2) - insufficient peptide C from H. armigera remained for methylation. However, it is assumed that this peptide, as with all the others, is amidated since the RIAs fail to recognise non-amidated peptides.
- Mr shows an increase of approximately 14 following methylation and indicates that the carboxyl group from the Glu or Asp residues has been methylated. The carboxyl group at the C-terminus of these peptides is amidated.
° - Mr shows an increase of approximately 16 following methylation. This result indicates that the Met residue has been oxidised during the methylation process and that the C-terminus is amidated.
A comparison of the amino acid sequences of Leu-callatostatins isolated from the blowfly C. vomitoria, the lepidopterans Cydia pomonella and Helicovera armigera, and the hymenopteran Pteronida sali cis is shown in Table 8.
Figure imgf000063_0001

Claims

1. A recombinant or isolated DNA sequence encoding an amino acid sequence as shown in Figure 18, in Figure 2, in Figure 4, or an amino acid sequence substantially homologous thereto.
2. A recombinant or isolated DNA sequence encoding an amino acid sequence as shown in Figure 18, in Figure 2 or in Figure 4.
3. A recombinant or isolated DNA sequence comprising the protein coding region of the DNA sequence shown in Figure 17, in Figure 2 or in Figure 4.
4. A peptide having one of the following sequences:
Ser-Pro-His-Tyr-Asp-Phe-Gly-Leu,
Ala-Tyr-Ser-Tyr-Val-Ser-Glu-Tyr-Lys-Arg-Leu-Pro-Val-
Tyr-Asn-Phe-Gly-Leu,
Leu-Pro-Val-Tyr-Asn-Phe-Gly- eu,
Ser-Arg-Pro-Tyr-Ser-Phe-Gly- eu,
Ala-Arg-Pro-Tyr-Ser-Phe-Gly-Leu,
Ala-Arg-Ala-Tyr-Asp-Phe-Gly-Leu,
Leu-Pro-Met-Tyr-Asn-Phe-Gly-Leu,
Ala-Arg-Ser-Tyr-Asn-Phe-Gly- eu,
Tyr-Ser-Lys-Phe-Asn-Phe-Gly-Leu, or Glu-Arg-Asp-Met-His-Arg-Phe-Ser-Phe-Gly-Leu
in an isolated or purified form.
5. A peptide having the following sequence: Ser-Pro-His-Tyr-Asp-Phe-Gly-Leu.
6. A peptide having the following sequence:
Ala-Tyr-Ser-Tyr-Val-Ser-Glu-Tyr-Lys-Arg-Leu-Pro-Val- Tyr-Asn-Phe-Gly-Leu.
7. A peptide having the following sequence: Leu-Pro-Val-Tyr-Asn-Phe-Gly-Leu.
8. A peptide having the following sequence: Ser-Arg-Pro-Tyr-Ser-Phe-Gly-Leu.
9. A peptide having the following sequence: Ala-Arg-Pro-Tyr-Ser-Phe-Gly-Leu.
10. A peptide having the following sequence: Ala-Arg-Ala-Tyr-Asp-Phe-Gly-Leu.
11. A peptide having the following sequence: Leu-Pro-Met-Tyr-Asn-Phe-Gly-Leu.
12. A peptide having the following sequence : Ala-Arg-Ser-Tyr-Asn-Phe-Gly-Leu.
13. A peptide having the following sequence: Tyr-Ser-Lys-Phe-Asn-Phe-Gly-Leu.
14. A peptide having the following sequence:
Glu-Arg-Asp-Met-His-Arg-Phe-Ser-Phe-Gly-Leu.
15. A peptide having the amino acid sequence as shown in Figure 18.
16. A peptide having one of the following sequences:
Ala-Arg-Pro-Tyr-Ser-Phe-Gly-Leu,
Arg-Pro-Tyr-Ser-Phe-Gly-Leu,
Val-Glu-Arg-Tyr-Ala-Phe-Gly-Leu, or
Leu-Pro-Val-Tyr-Asn-Phe-Gly-Leu
in an isolated or purified form.
17. A peptide having the following sequence: Arg-Pro-Tyr-Ser-Phe-Gly-Leu.
18. A peptide having the following sequence: Val-Glu-Arg-Tyr-Ala-Phe-Gly-Leu.
19. A peptide having the amino acid sequence as shown in Figure 2.
20. A peptide having one of the following sequences:
Ser-Pro-His-Tyr-Asn-Phe-Gly-Leu,
Ala-Tyr-Ser-Tyr-Val-Ser-Glu-Tyr-Lys-Arg-Leu-Pro-Val- Tyr-Asn-Phe-Gly-Leu,
Leu-Pro-Val-Tyr-Asn-Phe-Gly-Leu,
Ser-Arg-Pro-Tyr-Ser-Phe-Gly-Leu, Ala- Arg - Pro -Tyr- Ser- Phe -Gly -Leu ,
Ala-Arg-Gly-Tyr-Asp-Phe-Gly-Leu,
Leu-Pro-Leu-Tyr-Asn-Phe-Gly-Leu, or
Lys-Met-Tyr-Asp-Phe-Gly-Leu
in an isolated or purified form.
21. A peptide having the following sequence: Ser-Pro-His-Tyr-Asn-Phe-Gly-Leu.
22. A peptide having the following sequence: Ala-Arg-Gly-Tyr-Asp-Phe-Gly-Leu.
23. A peptide having the following sequence: Leu-Pro-Leu-Tyr-Asn-Phe-Gly-Leu.
24. A peptide having the following sequence: Lys-Met-Tyr-Asp-Phe-Gly-Leu.
25. A peptide having one of the following sequences:
Gly-Gly-Glu-Asp-Phe-Gly-His-Arg-Tyr-Ala-Phe-Gly-Leu,
Ala-Arg-Pro-Tyr-Asn-Phe-Gly-Leu,
Ala-Arg-Leu-Tyr-Ser-Phe-Gly-Leu, or
Leu-Pro-lie-Tyr-Asn-Phe-Gly-Leu
in an isolated or purified form.
26. A peptide having the following sequence: Gly-Gly-Glu-Asp- Phe -Gly-His -Arg -Tyr-Ala - Phe -Gly-Leu .
27. A peptide having the following sequence: Ala-Arg-Pro-Tyr-Asn-Phe-Gly-Leu,
28. A peptide having the following sequence: Ala-Arg-Leu-Tyr-Ser-Phe-Gly-Leu.
29. A peptide having the following sequence: Leu-Pro-lie-Tyr-Asn-Phe-Gly-Leu.
30. A peptide having the ammo acid sequence as shown in Figure 4.
31. A recombinant or isolated DNA sequence encoding a peptide having an ammo acid sequence as claimed m any one of claims 4 to 30.
32. A process for the preparation of a DNA sequence as claimed in any one of claims 1, 2, 3 or 31, the process comprising ligatmg together successive nucleotide residues and/or oligonucleotides.
33. A process for the preparation of a peptide as claimed in any one of claims 4 to 30, comprising the step of ligatmg together successive ammo acid residues and/or peptide fragments.
34. An secticidal composition comprising a peptide as claimed in any one of claims 4 to 30 and a suitable carrier, diluent or excipient therefor.
35. An secticidal formulation as claimed claim 34 which is a liposomal formulation.
36. A process for the preparation of an insecticidal composition, comprising admixing a peptide as claimed in any one of claims 4 to 30 with a suitable carrier, diluent or excipient therefor.
37. A method of killing insects comprising administering to the insects an effective amount of an insecticidal formulation as claimed in claim 34 or claim 35.
38. A method of controlling insects comprising administering to the insects an insecticidal formulation as claimed in claim 34 or claim 35.
39. A method as claimed in claim 37 or claim 38, in which the insecticidal formulation is administered in the form of a spray.
40. The use of a peptide as claimed in any one of claims 4 to 30 as an insecticide.
41. The use as claimed in claim 40, in which the peptide acts as an inhibitor of gut motility.
42. The use as claimed in claim 40 or claim 41, in which the insecticide is effective against a lepidopteran insect species.
43. The use as claimed in claim 40 or claim 41, in which the insecticide is effective against Helicoverpa armigera .
44. The use as claimed in claim 40 or claim 41, in which the insecticide is effective against Cydia pomonella .
45. A vector system comprising a DNA sequence as claimed in any one of claims 1 to 3 for expression in a host cell.
46. A vector system comprising a vector and a DNA sequence encoding a peptide or a protein including a peptide as claimed in any one of claims 4 to 30 for expression in a host cell.
47. A vector system as claimed in claim 45 or in claim 46, in which the vector is a baculovirus.
48. A host cell containing a vector system as claimed in any one of claims 45 to 47.
PCT/GB1997/000843 1996-03-26 1997-03-26 Insect neuropeptides genes and peptides WO1997035981A1 (en)

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WO2003014150A2 (en) 2001-08-08 2003-02-20 University Of Durham Fusion proteins for insect control
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US9804116B2 (en) 2014-12-26 2017-10-31 Kabushiki Kaisha Toshiba Method and device for detecting sample

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