INSECTICIDAL GENE AND PEPTIDE

Abstract: The present invention relates to FMRF amide type peptides and their non-amidated precursors, to recombinant nucleic acids encoding for such peptides, to the use of these nucleic acids, in forms substantially isolated from other nucleic acid or when incorporated into vectors, to transform organisms in order to render them capable of producing these peptides, to the use of such peptides, nucleic acid and vectors as pesticidal agents, eg. insecticidal, acaricidal and helminthecidal agents and to compositions comprising one or more of these. A peptide or salt thereof comprising an amino acid sequence Pro-Pro-(Xaa<1>)n-Phe-Xaa<2>-Arg-Xaa<3> or a conservatively substituted analogue thereof, wherein the N terminal of said peptide may be free amino or protected amino and the C-terminal of said peptide, which is the C-terminal -Xaa<3> of the above sequence, may consist of that residue with a free carboxylic acid group or with an amidated carboxyl of the formula CONR<1>R<2> wherein R<1> and R<2> are independently selected form hydrogen, alkyl, alkenyl, hydroxyalkyl, alkoxyalkyl and aminoalkyl or together with the nitrogen to which they are attached form a C3-8 heterocyclic ring; wherein Xaa<1> is any amino acid, which is independently selected at each repeat n, and n is an integer from 0 to 10 and Xaa<2> is Met, Val, Ile, Leu, Nle or Phe or a conservatively substituted analogue thereof and Xaa<3> is Phe, Asp or Glu, provided that when the peptide is Ile-Pro-Pro-Gln-Phe-Met-Arg-Phe it is not combined with skin of the frog Hylambates (Kassina maculata) is specifically claimed as are nucleic acids encoding therefor.

Description: INSECTICIDAL GENE AND PEPTIDE. The present invention relates to FMRFamide type peptides and their non-amidated precursors, to recombinant nucleic acids encoding for such peptides, to the use of these nucleic acids, in forms substantially isolated from other nucleic acid or when incorporated into vectors, to transform organisms in order to render them capable of producing these peptides, to the use of such peptides, nucleic acid and vectors as pesticidal agents, eg. insecticidal, acaricidal and helminthecidal agents and to compositions comprising one or more of these. The FMRFamide family of peptides are known to be implicated in invertebrate neurohormonal control and have been proposed for use as insect control agents. The cloning of Drosophila FMRF gene into baculovirus has been reported (Keeley et al., (1988) Biotechnol Biol. Pestle. ,99-106) while WO 9517515 (US Serial No. 08/172,653) describes a method for incapacitating insects by infecting them with an active recombinant baculovirus containing an FMRFamide like peptide precursor. The latter document describes the linking of a gene encoding for the FMRFamide precursor to a silkworm cytoplasmic actin promoter and enhancer from Bombyx morl. Vectors using the baculovirus 1E-1 gene are particularly favoured. The present inventors now report the isolation of the octapeptide, Ile-Pro-Pro-Gln- Phe-Met-Arg-Phe.amide (IPPQFMRFamide) from acidified ethanol extracts of the skin of the African rhacophorid frog, Hylambates (Kassina) maculata, commonly known as the red-legged pan frog. This peptide represents the first authentic member of the FMRFamide family of peptides isolated from a vertebrate source and the C-terminal tetrapeptide is identical to the original peptide isolated from the brain of the bivalve mollusc, Macrocallista nimbosa. This newly identified and isolated vertebrate FMRF amide-like peptide fulfills the criteria of activation of receptors on invertebrate tissues and the mode of action of this frog venom component is consistent with its role in protection from invertebrate predators. The identification and isolation of this peptide provides the first evidence that vertebrates produce anti-invertebrate peptides in their defensive secretions and that by extrapolation, the peptidergic regulatory system of these invertebrates is an appropriate and available target for peptidic insecticides/anthelmintics. Thus in a first aspect of the present invention there is provided a peptide or salt thereof comprising an amino acid sequence Pro-Pro-(Xaa 1 ) n -Phe-Xaa 2 -Arg-Xaa 3 or a conservatively substituted analogue thereof wherein the N terminal of said peptide may be free amino or protected amino and the C- terminal of said peptide, which is provided by the C-terminal Xaa 3 in the sequence above, may consist of that residue having a free carboxylic acid group or having an amidated carboxyl of formula CONR'R 2 wherein R 1 and R 2 are independently selected from hydrogen, alkyl, alkenyl, hydroxyalkyl, alkoxyalkyl and aminoalkyl or together with the nitrogen to which they are attached form a C 3 . 8 heterocylic ring; wherein Xaa 1 is any amino acid, which is independently selected at each repeat n and n is an integer from 0 to 10 ; Xaa 2 is Met, Val, He, Leu, Nle or Phe or a conservatively substituted analogue thereof and Xaa 3 is Phe, Asp or Glu. provided that when the peptide is Ile-Pro-Pro-Gln-Phe-Met-Arg-Phe it is in a form not associated with, eg. when not in the form of, skin of the frog Hylambates (Kassina) maculata. Where FMRFamide agonist activity is desired, Xaa 3 is preferably Phe. Where FMRFamide antagonist activity is desired, Xaa 3 is preferably Asp or Glu. Preferably n is from 1 to 3 and most preferably is 1. Preferably Xaa 1 is an alpha amino acid, particularly a naturally occurring amino acid but may be of D- or L- configuration, more preferably being glutamine or a conservative substitution thereof , most preferably being L-glutamine. Use of D-amino acids will be expected to give resistance to enzymolysis where used and may be beneficial. Preferably Xaa 2 is Met, lie, Leu or Nle. Preferably R 1 and R 2 are independently selected from hydrogen, C M n- or iso-alkyl or C 3.8 cycloalkyl, eg. cyclohexyl. Alternatively R 1 and R 2 together with the nitrogen to which they are attached may form a C 3 . 8 heterocyclic ring. Preferably the N-terminal, when protected, is protected with conventionally employed peptide chemistry N-terminal protecting goups, but more preferably such groups are pharmaceutically, agriculturally or horticulturally acceptable. Preferably the peptide is of 6 to 20 amino acids long, most preferably 7 to 10. For the purpose of production of the peptides of the invention it may be preferred to produce them as repeating polymers wherein the peptide sequence described above is repeated a number of times linked together by a specific enzyme lysable bond. Such principle is described in PCT/US93/0651 (WO 94/01451) where multicopy peptides are disclosed together with methods for producing amidated peptides. Such peptides may be provided by synthesising cDNA for the peptide and ligating numbers of copies of this together such that it encodes for eg. polypeptide [-Spacer-Pro-Pro-(Xaa') n -Phe-Xaa 2 -Arg- Xaa 3 ] p where p is an integer from 2 to several thousand, eg. 10,000, preferably 10 to 100. The cleavable spacer group may equally be on the C-terminal for this purpose. Suitable nucleic acid encoding for specifically cleavable spacer petides which can be cleaved without cleaving the peptide units of the invention will occur to those skilled in the art, particularly in the light of the aforesaid published PCT, and will offer more efficient expression targets for recombinant production of the present peptides. Preferably the amino acids are L-configuration amino acids such as those which are naturally occurring in biological systems. This is particularly preferred for the end four C- terminal amino acids, whereas the N-terminal may employ one or more D-configuration acids. When the peptide is in the form of a peptide salt, this may comprise the peptide together with any anion or cation conventionally used in pesticidal or pharmaceutical preparations as counterion in salt preparations. Thus the amino acid sequence above, when of the preferred 6 to 20 amino acid residues long, has up to 12 amino acid residues appended to its N-terminal. Most preferred peptides are those wherein n is 1 and/or where only 1 further amino acid residue is appended to the N-terminal. Preferred peptides of the first aspect of the present invention comprise an amino acid sequence Ile-Pro-Pro-Gln-Phe-Xaa-Arg-Phe or a conservatively substituted analogue thereof wherein Xaa represents Met, Val, He, Leu, Nle or Phe or a conservatively substituted analogue thereof. The sequence Phe-Xaa-Arg-Phe is that shared by the FMRF. amide family of invertebrate peptides while the sequence Ile-Pro-Pro-Gln is that determined as specific to the vertebrate peptide isolated as reported above. Thus a preferred such peptide of the invention is provided comprising an amino acid sequence Ile-Pro-Pro-Gln-Phe-Met-Arg- Phe or a conservatively substituted analogue thereof. Most preferred peptides of the invention are amide terminated at their C-terminal ends. Particular novel forms of the peptide, particularly of the native Ile-Pro-Pro-Gln-Phe- Met-Arg-Phe.amide and its precursor Ile-Pro-Pro-Gln-Phe-Met-Arg-Phe are characterised in that they are forms not encountered in nature, ie. substantially isolated or in novel or formulated form, eg. together with pharmaceutically, agriculturally or horticulturally acceptable excipients. Novel forms may include, inter alia, those in solid state, in substantially pure state, or in aqueous solution in a form not combined with other Hylambates (Kassina) maculata frog proteins or not combined with its skin. The preferred peptides of the invention have an amino acid sequence consisting of Ile-Pro-Pro-Gln-Phe-Met-Arg-Phe and particularly preferred is the wild type peptide.amide Ile-Pro-Pro-Gln-Phe-Met-Arg-Phe.amide. Also preferred are those peptides where the Met 5 in this sequence is replaced by an amino acid that is less susceptible to oxidation, whether in use or in preparation, eg. such as He. The present inventor has determined that the various amino acid sequences described above and their amidated forms will best retain the activity of the naturally occuring octapeptide with varying degrees of resistance to degredation by biological systems such as those including peptidase enzymes. It will be realised that the present peptides may include or be conjugated to a variety of other molecules such as labelling agents or other bioactives. Particularly advantageous will be fusion peptides incorporating a cytolytic peptide sequence which can aid penetration of the peptide of the invention through pest tissues. In a second aspect of the invention there is provided a nucleic acid encoding for a peptide as described above. For the preferred peptides of the invention this is characterised in that the nucleic acid is in recombinant form or a form free of skin of the frog Hylambates (Kassina) maculata. Preferred nucleic acids comprise a base sequence of SEQ ID No 1 AUH CCN CCN CAR UUY AUG MGN UUY wherein H is A, C or U; R is A or G; Y is C or U; M is A or C and N is A, C, G or U provided that when M is A, N is selected from A or G only and when the nucleic acid is an RNA, U is uracil and when it is a DNA U is thymine (T); or a sequence complementary thereto. The nucleic acid is most preferably in the form of recombinant nucleic acid, preferably in the form of a vector, but may be in the form of DNA suitable for direct incorporation into the genome of an organism using techniques such as electroporation or by use of agents such as lipofectin (see WO 9517515). Where the nucleic acid is in the form of a vector, that vector is preferably a virus or virus derived vector; particularly a baculovirus or baculovirus derived vector. Preferably the vector is one such as those described in WO 9517515 in so far as it is capable of early expression of the peptide once it has infected a pest, eg. insect, cell. Bacterial vectors suitable for plant protection or plant transformation will also occur to those skilled in the art and are provided by the present invention. A third aspect of the present invention provides the use of a peptide of the invention as defined as a pesticide to treat loci other than Hylambates (Kassina) maculata, eg. plants or animals, whether adminstered internally or externally. Preferred such use is of peptides of amino acid sequence Ile-Pro-Pro-Gln-Phe-Xaa 2 -Arg-Phe or a conservatively substituted analogue thereof. Again, it is preferred that the peptide has an amidated C-terminal, but this is not essential as the FMRFamide family is widespread in invertebrates and the necessary enzymes for specific or non-specific amidation of the FMRF C-terminal end will be present in many target pest tissues. Preferably Xaa is Met, Leu, He or Nle or a conservative substitution thereof. The preferred use of the pesticide is against invertebrates possessing FMRF or related tetrapeptide sequence receptors. Preferably the invertebrates are insects, acarids, helminthe-worms, eg. platyhelminthes, or nematodes. Most preferably the invertebrates are insects and helminthes. In a fourth aspect of the present invention there are provided compositions comprising a peptide or nucleic acid of the invention together with a carrier and/or excipients that are physiologically acceptable to one or more plants or vertebrates, ie. they are pharmaceutically, agriculturally or horticulturally acceptable. Preferably the carrier will be suitable for application to all commercially and aesthetically and ecologically desirable plants, but this will not be essential where the target area is not sensitive to such action. Carriers suitable for the application of peptide or vector agents will be well known to those skilled in the art. WO 9517515 discloses the use of suspensions of baculovirus occlusion bodies applied to leaves. Compositions suitable for use in plant protection will occur to those skilled in the art in the light of the present disclosure. Also provided are compositions for protecting or treating animals, eg. humans, against or for internal or external pests, eg. as topical compositions for application to skin, eg. as creams, lotions or aerosols, or for internal administration as is conventional in the pharmaceutical art. For treatment of gut parasites it may be preferred to use enterically coated compositions, with the advantage that the peptides of the invention will not be hydrolysed in the stomach. For treatment of blood borne parasites parenteral administration of peptide may be appropriate. All the compositions of the invention will be likely to benefit from inclusion of inhibitors of one or more enzymes, eg. amidases, capable of cleaving any of the peptide bonds present. These inhibitors may be non-peptide in nature but may be as simple as glycine amide or some similar amino acid or peptide based molecule. In a fifth aspect the present invention provides transgenic cells and organisms which express one or more peptides of the invention. Such organisms or cells may be invertebrate, eg. insect, in nature and be useful in production of the peptides of the invention for the purpose of their use as pesticides, or may be plant cells or plants which, besides this peptide production utility are capable of combatting their own infestation with pests by use of the expressed peptide as a defence mechanism. In a sixth aspect of the present invention there are provided antibodies and vaccines for their production comprising . The vaccines are characterised in that they comprise peptides of formula -Phe-Xaa 2 -Arg-Xaa 3 , preferably -Pro-Pro-(Xaa') n -Phe-Xaa 2 -Arg-Xaa 3 , that are antigenic in character. The present inventor has determined that by conjugating the -Phe-Xaa 2 -Arg-Xaa 3 residues to a suitable macromolecule, eg. ovalbumen or a similar hapten, eg by glutaraldehyde treatment, specific antibodies may be raised in animals. Eg. when Ile-Pro-Pro-Gln-Phe-Met-Arg-Phe.arnide is linked by glutaraldehyde crosslinking to ovalbumen and emulsified in adjuvant, particularly Freund's incomplete adjuvant, it induces a specific antibody response in rabbits. Such approach will readily lend itself to the production of protective antibodies in animals and allow production of monoclonal antibodies, using conventional techniques, which optionally may be engineered in known to fashion to produce humanised or otherwise optimised antibodies. A seventh aspect of the present invention provides the vaccines and antibodies of the invention for use in therapy, eg. of invertebrate parasitic conditions and for prophylaxis thereof. Methods of producing transgenic plant and animal cells will be well known to those skilled in the art. In addition to use of viral vectors and direct incorporation of nucleic acid into the genome of an organism using techniques such as electroporation and lipofectin as referred to above, bacterial vectors such as agrobacterium may be used to deliver the nucleic acid. For plants such incorporation may be into pollen or seeds. The choice of a particular nucleic acid sequence of the invention for transforming the cells of a particular organism will depend on that cells codon preferences. The use of degenerative substitutions is thus advantageous where the cell or vector expressing the protein is of such different type to the DNA source organism cell that it has different codon preferences for transcription/translation to that of the cDNA source cell. Such degenerative substitutions will thus be host specific. The expression 'conservative substitutions' as used with respect to amino acids relates to the substitution of a given amino acid by an amino acid having physicochemical characteristics in the same class. Thus where an amino acid in the SEQ ID No 2 has a hydrophobic characterising group, a conservative substitution replaces it by another amino acid also having a hydrophobic characterising group; other such classes are those where the characterising group is hydrophilic, cationic, anionic or contains a thiol or thioether. Such substitutions are well known to those of ordinary skill in the art, ie. see US 5380712. A still further aspect of the present invention provides methods for synthesis of the peptides of the present invention. Such methods include the production of such peptides by expression of the DNA of the invention in host cells such as will occur to those skilled in the art of recombinant DNA expression systems, see eg. WO 9517515, or may eg. comprise conventional solid or liquid phase peptide synthetic techniques, automated or otherwise. For synthesis and subsequent conversion of the peptides of the invention having free carboxylic acid C-terminals to their amidated forms standard peptide techniques will also occur to those skilled in the art, such as those employed JP 1050896 and JP 2257890 in FMRF. amide chemistry. The present invention will now be described further by way of illustration only by reference to the following non-limiting Examples and Sequence listing and Figures. Further embodiments falling within the scope of the invention will occur to those skilled in the art in the light of these. FIGURES Figure 1. Muscle tension recording showing the effect of 1 μM VF1 on an innervated muscle preparation of Ascaris suum. The addition of the peptide is indicated by the arrow head. Horizontal bar represents 2 min while vertical bar represents 0.25 g. Figure 2. Physiograph showing the effect of 10 μM VF1 on the contractility of insect hindgut. Peptide addition is marked by the arrow head while peptide removal is indicated by the inverted arrow head. Horizontal bar represents 1 min while vertical bar represents 10 mg. Figure 3. Physiograph displaying the effects of 10 μM VF1 on the contractility of the uterus of Ascaris suum. Peptide addition is indicated by an arrow head and peptide removal by an inverted arrow head. Horizontal bar represents 1 min and vertical bar represents 10 mg. SEQUENCE LISTING SEQ ID No 1 , that of hypothetical mRNA encoding for the naturally occurring peptide from frog skin. SEQ IDs No 2 to 14: those of peptides of the invention. EXAMPLES EXAMPLE 1 : Isolation of octapeptide The peptide Ile-Pro-Pro-Gln-Phe-Met-Arg-Phe. amide was confirmed to be present in the venom of Hylambates (Kassina) maculata species obtained from a number of living specimens thus proving that it is a secreted product from different individuals of the same species. The peptide was identified by radioimmunoassay of HPLC fractions of the extract using an antiserum raised against invertebrate FMRFamide. This detection system was used in conjunction with a series of HPLC fractionations, to isolate the peptide in pure form. The primary structure was established by a combination of microsequencing and mass spectroscopy. The molecular mass of the peptide was 1034 Da and the presence of a C-terminal amide was established by radioimmunoassay cross-reactivity and by methylation followed by mass spectroscopy. The peptide was synthesised to >95% purity using conventional peptide synthesis techniques and the identity of the synthetic replicate was confirmed using HPLC and microsequencing. EXAMPLE 2: Ascaris somatic muscle assay. The synthetic replicate was initially tested on a sensitive preparation for this family of peptide, the somatic muscle of the parasitic roundworm, Ascaris suum. At a concentration of 1 micromolar, the peptide caused rapid and prolonged relaxation of the test muscle strip (Figure 1). This established that the vertebrate peptide could in fact interact with endogenous invertebrate FMRFamide like peptide receptors. EXAMPLE 3: Insect hindgut assay. The presence of this peptide in a vertebrate defensive secretion was a mystery until we determined that one of the main groups of predators of this frog are giant carnivorous aquatic insects. The peptide was thus proposed to perform a defensive function against these insect predators, an action which would depend on its interaction with endogenous insect receptors for endogenous FMRFamide-related peptides. To test this hypothesis, the synthetic peptide was subjected to biological testing on an insect tissue, the hindgut. which is a standard insect bioassay for bioactive peptides including FMRFamide-related peptides. The results are shown in Figure 2. Application of the frog peptide to this preparation at a concentration of 10 micromolar caused a rapid increase in contraction frequency and magnitude. The effect was prolonged and only partially reversible with time. These data provided evidence that this vertebrate peptide was interactive with endogenous insect FaRP receptors and was highly stimulatory. EXAMPLE 4: Roundworm uterus assay. The peptide was applied to a novel physiological preparation, the roundworm uterus preparation as described in the legend to Fig 3. As shown in that Figure, the peptide was excitatory at a concentration of 10 micromolar. These data are consistent with activation of endogenous invertebrate FMRFamide-like peptide receptors. EXAMPLE 5: Antifeedant assay. Fifth instar larave of Schistocerca gregaria were injected into the hemolymph with saline or various amounts of peptide.amide of Example 1. Each test involved injection of ten controls and ten treated larvae. In table 1 below numbers represent the reduction grammes weight of a cole leaf during the test period, being the amount eaten plus evaporation. TABLE 1 SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: BRITISH TECHNOLOGY GROUP LIMITED (B) STREET: 10 FLEET PLACE (C) CITY: LONDON (E) COUNTRY: UNITED KINGDOM (GB) (F) POSTAL CODE (ZIP) : EC4M 7SB (A) NAME: SHAW CHRISTOPHER (B) STREET: 3 SWALLOW CLOSE (C) CITY: COMBER (E) COUNTRY: UNITED KINGDOM (GB) (F) POSTAL CODE (ZIP) : BT23 5UJ (n) TITLE OF INVENTION: INSECTICIDAL GENE AND PEPTIDE (in) NUMBER OF SEQUENCES: 14 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.30 (EPO) (vi) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: GB 9702189.3 (B) FILING DATE: 04-FEB-1997 (vi) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: GB 9725149.0 (B) FILING DATE: 27-NOV-1997 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 base pairs (B) TYPE : nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (li) MOLECULE TYPE: mRNA (in) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Hylambates (Kassina) maculata (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..24 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: AUHCCNCCNC ARUUYAUGMG NUUY 24 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 ammo acids (C) STRANDEDNESS: (D) TOPOLOGY: linear (n) MOLECULE TYPE: peptide (v) FRAGMENT TYPE : C-terminal (vi) ORIGINAL SOURCE: (A) ORGANISM: Hylambates (Kassina) maculata (xi) SEQUENCE DESCRIPTION : SEQ ID NO : 2 : Pro Pro Xaa Phe Xaa Arg Xaa 1 5 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids (B) TYPE : amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: C-terminal (vi) ORIGINAL SOURCE: (A) ORGANISM: Hylambates (Kassina) maculata (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: Pro Pro Xaa Phe Xaa Arg Phe 1 5 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino acids (B) TYPE : amino acid (C) STRANDEDNESS: (D) TOPOLOGY linear (ii) MOLECULE TYPE peptide (v) FRAGMENT TYPE C-terminal (vi) ORIGINAL SOURCE: (A) ORGANISM: Hylambates (Kassina) maculata (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Pro Pro Phe Xaa Arg Xaa 1 5 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE : amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: YES (v) FRAGMENT TYPE: C-terminal (vi) ORIGINAL SOURCE: (A) ORGANISM: Hylambates (Kassina) maculata (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION:!..24 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: Pro Pro Xaa Xaa Phe Xaa Arg Xaa 1 5 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: C-terminal (vi) ORIGINAL SOURCE: (A) ORGANISM: Hylambates (Kassina) maculata (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Pro Pro Xaa Xaa Xaa Phe Xaa Arg Xaa 1 5 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 amino acids (B) TYPE : amino acid (C) STRANDEDNESS: (D) TOPOLOGY linear (ii) MOLECULE TYPE peptide (v) FRAGMENT TYPE C-terminal (vi) ORIGINAL SOURCE: (A) ORGANISM: Hylambates (Kassina) maculata (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: Pro Pro Xaa Xaa Xaa Xaa Phe Xaa Arg Xaa 1 5 10 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 11 amino acids (B) TYPE : amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: C-terminal (vi) ORIGINAL SOURCE: (A) ORGANISM: Hylambates (Kassina) maculata (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Pro Pro Xaa Xaa Xaa Xaa Xaa Phe Xaa Arg Xaa 1 5 10 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 amino acids (B) TYPE : amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (11 ) MOLECULE TYPE : peptide (v) FRAGMENT TYPE : C-termmal (vi ) ORIGINAL SOURCE : (A) ORGANISM: Hylambates (Kassina) maculata (xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 9 : Pro Pro Xaa Xaa Xaa Xaa Xaa Xaa Phe Xaa Arg Xaa 1 5 10 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 amino acids (C) STRANDEDNESS: (D) TOPOLOGY linear (li) MOLECULE TYPE peptide (v) FRAGMENT TYPE C-terminal (vi) ORIGINAL SOURCE: (A) ORGANISM: Hylambates (Kassina) maculata (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: Pro Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Xaa Arg Xaa 1 5 10 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (li) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: C-terminal (vi) ORIGINAL SOURCE: (A) ORGANISM: Hylambates (Kassina) maculata (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: Pro Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Xaa Arg Xaa 1 5 10 (2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 ammo acids (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iv) ANTI-SENSE: NO (v) FRAGMENT TYPE: C-terminal (vi) ORIGINAL SOURCE: (A) ORGANISM: Hylambates (Kassina) maculata (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: Pro Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Xaa Arg Xaa 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) STRANDEDNESS : (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: C-terminal (vi) ORIGINAL SOURCE: (A) ORGANISM: Hylambates (Kassina) maculata (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: Pro Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Xaa Arg Xaa 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE : amino acid (C) STRANDEDNESS : (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: C-terminal vi) ORIGINAL SOURCE: (A) ORGANISM: Hylambates (Kassina) maculata (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: lie Pro Pro Gin Phe Met Arg Phe 1 5

Claims: CLAIMS. 1. A peptide or salt thereof comprising an amino acid sequence Pro-Pro-(Xaa ] ) n -Phe-Xaa 2 -Arg-Xaa 3 or a conservatively substituted analogue thereof wherein the N terminal of said peptide may be free amino or protected amino and the C- terminal of said peptide, which is the C-terminal -Xaa 3 of the above sequence, may consist of that residue with a free carboxylic acid group or with an amidated carboxyl of formula CONR'R 2 wherein R 1 and R 2 are independently selected from hydrogen, alkyl, alkenyl, hydroxyalkyl, alkoxyalkyl and aminoalkyl or together with the nitrogen to which they are attached form a C 3 . 8 heterocyclic ring; wherein Xaa 1 is any amino acid, which is independently selected at each repeat n, and n is an integer from 0 to 10 and Xaa 2 is Met, Val, He, Leu, Nle or Phe or a conservatively substituted analogue thereof and Xaa 3 is Phe, Asp or Glu, provided that when the peptide is Ile-Pro-Pro-Gln-Phe-Met-Arg-Phe it is not combined with skin of the frog Hylambates (Kassina) maculata 2. A peptide as claimed in claim 1 characterised in that n is from 1 to 3. 3. A peptide as claimed in claim 1 characterised in that n is 1. 4. A peptide as claimed in any one of the preceding claims characterised in that Xaa' is glutamine or a conservative substitution thereof . 5. A peptide as claimed in any one of the preceding claims characterised in that Xaa 2 is Met, He, Leu or Nle. 6. A peptide as claimed in any one of the preceding claims characterised in that Xaa 3 is Phe. 7. A peptide as claimed in any one of the preceding claims characterised in that it is of 6 to 20 amino acid residues long 8. A peptide as claimed in claim 7 characterised in that it is of 7 to 10 amino acid residues. 9. A peptide as claimed in claim 1 characterised in that it has an amino acid sequence consisting of Ile-Pro-Pro-Gln-Phe-Xaa 2 - Arg-Phe and that the C-terminal of said peptide consists of a free carboxylic acid group or an amidated carboxyl of formula CONR'R 2 wherein R 1 and R 2 are independently selected from hydrogen, alkyl, alkenyl, hydroxyalkyl, alkoxyalkyl and aminoalkyl. 10. A peptide as claimed in claim 1 comprising an amino acid sequence Ile-Pro-Pro- Gln-Phe-Xaa 2 -Arg-Phe or a conservatively substituted analogue thereof wherein Xaa 2 represents Met, Ala, He, Nle, Leu or Phe or a conservatively substituted analogue thereof provided that when the peptide is Ile-Pro-Pro-Gln-Phe-Met-Arg-Phe it is in a form not combined with skin of the frog Hylambates (Kassina) maculata. 11. A peptide as claimed in claim 10 characterised in that Xaa 2 is Met or a conservatively substituted analogue thereof. 12. A peptide as claimed in any one of the preceding claims characterised in that it is amide terminated at its C-terminal end. 13. A peptide as claimed in any one of the preceding claims characterised in that it is in substantially isolated, solid or formulated form. 14. A peptide as claimed in any one of the preceding claims characterised in that it is in aqueous solution in a form not combined with other Hylambates (Kassina) maculata frog proteins or peptides. 15 A peptide as claimed in any one of the preceding claims characterised in that its amino acid sequence consists of Ile-Pro-Pro-Gln-Phe-Xaa 2 -Arg-Phe. 16. A peptide as claimed in claim 15 being Ile-Pro-Pro-Gln-Phe-Met-Arg-Phe.amide. 17. A peptide as claimed in any one of the preceding claims characterised in that it comprises a cytolytic sequence. 18. A nucleic acid encoding for a peptide of amino acid sequence as claimed in any one of the preceding claims or a complementary sequence thereto, characterised in that the nucleic acid is in a form not combined with the skin of the frog Hylambates (Kassina) maculata. 19. Recombinant nucleic acid encoding for a peptide as claimed in any one of claims 1 to 17. 20. A nucleic acid as claimed in claim 18 or 19 characterised in that it comprises a base sequence of SEQ ID No 1 AUH CCN CCN CAR UUY AUG MGN UUY wherein H is A, C or T/U; R is A or G; Y is C or T/U; M is A or C andN is A, C, G or T/U provided that when M is A, N is selected from A or G only and when the nucleic acid is an RNA, U is uracil and when it is a DNA U is thymine; or a sequence complementary thereto. 21. A nucleic acid as claimed in claim 18, 19 or 20 being in the form of a vector. 22. A nucleic acid as claimed in claim 21 characterised in that the vector is a virus or virus derived vector. 23. A nucleic acid as claimed in claim 22 characterised in that the vector is a baculovirus or baculovirus derived vector. 24. Use of a peptide as claimed in any one of claims 1 to 17 as a pesticide for the treatment of loci other than Hylambates (Kassina) maculata. 25. Use as claimed in claim to 24 characterised in that the pesticide is used against invertebrates. 26. Use as claimed in claim 25 characterised in that the invertebrates are insects, acarids, flat- or ring-worms or nematodes. 27. A composition comprising a peptide as claimed in any one of claims 1 to 17 or a nucleic acid as claimed in any one of claims 17 to 23 together with a carrier physiologically acceptable to one or more plants or vertebrates. 28. A transgenic cell comprising a nucleic acid as claimed in any one of claims 17 to 22. 29. A method of producing a transgenic cell as claimed in claim 28 comprising incorporating a nucleic acid as claimed in any one of claims 17 to 22 into a cell to be transformed. 30. A transgenic organism comprising a transgenic cell as claimed in claim 28. 31. A method of producing a peptide as claimed in any one of claims 1 to 17 comprising ( a) expressing DNA as claimed in any one of claims 17 to 22 in an expression system to provide a peptide having a free carboxylic acid at its C-terminal and (b) reacting that if required with an amine. 32. A method of producing a peptide as claimed in any one of claims 1 to 17 comprising ( a) expressing peptide from cDNA as claimed in any one of claims 17 to 22, characterised in that the DNA C-terminal codon is deleted, in an expression system to provide a peptide having a free carboxylic acid at its C-terminal and (b) coupling the C-terminal amino acid Xaa 3 to that in amidated form. 33. A method of producing a peptide as claimed in any one of claims 1 to 17 or 31 and 32 comprising (a) synthesizing cDNA for the peptide (b) ligating copies of this cDNA together linked by spacer DNA, such that the ligated DNA encodes for expression of a polypeptide [spacer-Pro-Pro-(Xaa') n -Phe-Xaa 2 -Arg-Xaa 3 ] p or polypeptide [-Pro-Pro-(Xaa') n -Phe-Xaa 2 - Arg-Xaa 3 -spacer] p where p is an integer from 2 to several thousand and the spacer is an amino acid or peptide sequence (c) expressing the polypeptide and (d) cleaving the product of (c) with an amidase which cleaves within or to one side of the spacer but not within at least one of the sequences -Pro-Pro-(Xaa 1 ) n -Phe-Xaa 2 - Arg-Xaa 3 or -Pro-Pro-(Xaa 1 ) n -Phe-Xaa 2 -Arg-. 34. Use of a peptide comprising the C-terminal sequence Phe-Xaa 2 -Arg-Xaa 3 as a vaccine for prophylaxis or treatment of an animal or human insect, acarid or helminthe pest. 35. Use as claimed in claim 34 characterised in that the peptide is conjugated to protein. 36. A peptide as claimed in any one of the preceding claims in a form conjugated to a protein.

Inventors: SHAW CHRISTOPHER, SHAW, CHRISTOPHER
Applicants: BRITISH TECH GROUP, SHAW CHRISTOPHER, BRITISH TECHNOLOGY GROUP LIMITED, SHAW, CHRISTOPHER
Application year: 1998
Main classification: C12N15
IPC codes: A01N63, A61K39, C07K14, C12N15