Classifications

• • 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
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide

Definitions

• the present invention concerns insect hormone receptor proteins, genes encoding said proteins and uses of said receptor proteins and genes in the identification of insect control agents and in the regulation of gene expression.
• the hormone receptor is a diuretic hormone (DH) receptor.
• DH diuretic hormone
• Mt Malpighian tubule
• Mas-DH Manduca sexta diuretic hormone
• Mas-DH-R Manduca sexta diuretic hormone receptor
• Lorn DH Locusta migratoria diuretic hormone
• Acd DH Acheta domesticus diuretic hormone
• BSA bovine serum albumin
• TFA trifluoroacetic acid
• BOC t-butoxycarbonyl
• SF9 Spodoptera frugiperda.
• the control of fluid secretion in insects is modulated at least in part by diuretic hormones and antidiuretic hormones.
• the primary target site for diuretic hormones is the Malpighian tubules (Mt) which are the main organs for fluid and ion secretion in insects.
• diuretic hormones may act by decreasing fluid resorption by the rectum.
• Mt Malpighian tubules
• Achete domesticus and Periplenata americana have been identified and sequenced and these peptides share between 34% and 50% sequence identity with the 41 amino acid peptide isolated from Manduca sexta.
• the 41 amino acid diuretic hormone isolated from Manduca sexta is an amidated peptide which has been shown to stimulate fluid secretion in a number of insects such as Manduca sexta, Pieiris rapae and Achete domesticus.
• the high degree of sequence homology with the other diuretic hormones referred to above suggests that these hormones belong to a single family of corticotropin releasing factor (CFR)-related peptides.
• CFR corticotropin releasing factor
• the present invention therefore provides a polypeptide comprising an insect diuretic hormone receptor or analog or fragment thereof which is substantially free from associated insect polypeptides and exhibits biological activity characteristic of a native insect diuretic hormone receptor.
• the invention concerns an insect diuretic hormone receptor isolated from Manduca sexta having the amino acid sequence shown in SEQ ID NO. 2 and analogs or fragments thereof.
• the receptor consists of 395 amino acids and contains seven putative membrane spanning regions.
• Analogs and fragments of an insect diuretic hormone receptor as used herein refers to modified amino acid sequences resulting from truncation of, deletion of, substitution of, addition of or extension by one or more amino acids which retain the biological activity characteristic of a native insect diuretic hormone receptor.
• Preferred such analogs and fragments are preferably at least 70% homologous, more preferably 80% homologous even more preferably 85% homologous with a native insect diuretic hormone receptor.
• the invention also provides DNA sequences encoding an insect diuretic hormone receptor and analogs or fragments thereof. These DNA sequences are preferably combined with heterologous DNA sequences such as promoter or other operator and regulator sequences to form systems for the expression, production and/or delivery of polypeptides having the biological activity characteristic of a native insect receptor hormone receptor. Such systems may also contain other DNA sequences encoding other functional polypeptides whereby the DNA sequence encoding an insect diuretic hormone receptor or an analog or fragment thereof can be used to regulate expression.
• SEQ ID No. 1 shows a DNA sequence encoding a native insect diuretic hormone receptor isolated from Manduca sexta. It will be appreciated that this sequence is but one of a variety which may be constructed to encode an insect diuretic hormone receptor or an analog or fragment thereof. Different codons encoding the same amino acids may be readily substituted as allowed by the genetic code and any DNA sequence encoding a particular amino acid sequence according to the invention is included within the invention.
• Preferred DNA sequences according to the invention are those which will hybridize under stringent conditions to the DNA sequence shown in SEQ ID No. 1.
• Stringent hybridization conditions as contemplated herein are those in which hybridization is effected in a standard manner at 60°C in 2.5 X saline citrate buffer (a.k.a. SSC buffer) followed by merely washing at 37°C at a reduced buffer concentration, which will not affect true hybrids which have formed.
• the invention also includes procaryotes and eukaryotes transformed with DNA according to the invention.
• examples include viruses and bacterial, insect, yeast, mammalian and plant cells.
• Transformation can be effected in conventional manner with naked DNA or with the DNA cloned into suitable vectors such as plasmids such as described e.g. in Chu, G,; Hayakawa, H.; Berg, P. Electroporation for the efficient transfection of mammalian cells with DNA. Nuc. Acid. Res..15, 1311-1326 (1987); Trevors, J.T. Electrotransformation of Bacteria Met. in Mol. and Cell. Biol. 2:247-253 (1991). Transformed cells can themselves be employed to transform other cells of the same or different type e.g. plant cells by breeding or insect cells by transformed baculoviruses.
• Other aspects of the invention include: rDNA comprising a regulating element which is responsive to a ligand which binds to an insect diuretic hormone receptor or analog or fragment thereof according to the invention and cells transformed therewith;
• Antibodies both poly- and mono-clonal and binding fragments thereof with binding specificity for an epitope characteristic for an insect diuretic hormone receptor or analog or fragment thereof according to the invention
• Chimeric polypeptides comprising a diuretic hormone binding domain of an insect diuretic hormone receptor or analog or fragment thereof according to the invention and at least one further polypeptide and DNA encoding same;
• Methods of producing a polypeptide by introducing into a cell which is insensitive to ligands which bind to an insect diuretic hormone receptor or analog or fragment thereof according to the invention a receptor for that ligand and gene encoding the polypeptide which is operably linked to a regulating element which is responsive to said ligand and exposing the cell thus transformed to said ligand.
• Detergents useful in solubilization include Triton ® X 100, CHAPS digitonin and deoxycholate, Haga et. al. in Receptor Biochemistry: A Practical Approach Hulme E.C. ed. pp 1-50 OUP, 1990 describes other techniques and the contents thereof are incorporated herein by reference. Colonies of Manduca sexta are raised on an artificial diet (Troetschler etal. J. Econ. Entomol. 78, 1521-1523 (1985)). Peptides are prepared by solid phase synthesis on a Biosearch 9600 peptide synthesizer using a t-butoxycarbonyl protocol and p-methylbenzylhydrylamine resin (Kataoka et. al. Proc. Nat. Acad. Sci. 86, pp 2976-80 (1989)).
• Figure 1 shows a saturation isotherm of N ⁇ -[2,3- 3 H-propanoyl]Mas-DH binding to CHAP solubilized Mt membrane from 5th stage larvae Manduca sexta;
• Figure 2 shows a time course of N ⁇ -[2,3- 3 H-propanoyl]Mas-DH binding to CHAP solubilized Mt membrane from 5th stage larvae Manduca sexta:
• Figure 3 shows displacement of N ⁇ -[2,3- 3 H-propanoyl]Mas-DH binding by Mas-DH and biotinylated Mas-DH;
• Figure 4 shows ligand bound solubilized Mas-DH receptor
• Figure 5 shows the peak corresponding to the receptor-hormone complex when CHAPS solubilized Mt membranes are treated with N ⁇ -[2,3- 3 H-propanoyl]Mas-DH binding by Mas-DH and separated;
• Figure 6 shows the affinity of the receptor for Mas-DH depicted as saturation kinetics
• Figure 7 shows the affinity of the receptor for Mas-DH depicted as the reciprocal of Figure 6 kinetics
• Figure 8 shows the affinity of DH structurally related proteins for the recombinant Mas- DH-R
• Figure 9 shows the plasmid pKSl
• Figure 10 shows the expression of Mas-DH-R in recombinant baculovirus infected Sf9 cells
• Figure 11 shows the stimulation of cAMP synthesis by Mas-DH and N-terminal truncated analogs.
• SEQ ID No. 1 shows the cDNA sequence of the Mas-DH-R
• SEQ ID No. 2 shows a 395 amino acid sequence having 7 putative membrane spanning regions and corresponding to the cDNA open reading frame in SEQ ID No. 1;
• SEQ ID No. 3 shows the nucleotide sequences of pKSl
• SEQ ID Nos. 4 and 5 show respectively forward and reverse PCR primers used in the modification of the 5' and 3' non-coding regions of the cDNA encoding the Mas-DH-R.
• Malpighian tubules are isolated from 60 pre-wandering fifth instar Manduca sexta larvae weighing between 7 and 11 g. The Mt are washed in modified Manduca saline (4 mM NaCl, 40 mM KCl, 18 mM CaCl 2 , 3 mM CaCl 2 , 5 mM HEPES, pH 6.6) homogenized on ice, and centrifuged at 1200 x g for 10 minutes at 4°C. The supernatant is removed and the pellet washed further with Manduca saline.
• modified Manduca saline (4 mM NaCl, 40 mM KCl, 18 mM CaCl 2 , 3 mM CaCl 2 , 5 mM HEPES, pH 6.6) homogenized on ice, and centrifuged at 1200 x g for 10 minutes at 4°C. The supernatant is removed and the pellet washed further with Manduca
• the pooled supernatants are ultracentrifuged in a Beckman SW28 rotor at 112,700 x g f or 1 hr at 4°C and the resulting pellet resuspended in 6 mL of 45% sucrose/25 mM HEPES, pH 6.6 in an SW28 polyallomer centrifuge tube.
• Ten percent sucrose/25 mM HEPES pH 6.6 is carefully layered upon the 45% sucrose solution and the resulting step gradient is centrifuged as above.
• the membranes located at the interface of the sucrose gradient are removed, diluted with 25 mM HEPES pH 6.6 and pelleted at 112,700 x g.
• the pellet is resuspended in 1 mL of 25 mM HEPES pH 6.6 and stored at -70°C. Protein is determined using a BCA protein assay reagent (Pierce, Rockford, IL). Mt membranes are solubilized at a protein concentration of 2.25 mg/ml in 0.6% CHAPS, 25 mM HEPES, pH 7.0 for 2 h at 4°C. The membranes are then centrifuged in a Beckman, TL-100 ultracentrifuge at 125,000 x g for 45 minutes at 4°C. The supernatant is removed and may be used for solubilized receptor studies. Protein concentrations are determined by use of a BioRad protein assay kit (Bio-Rad, Richmond, CA).
• Biotin is incorporated into the ⁇ -amino group of Mas-DH (cf Kataoka et.al. ibid) via an amidation reaction using sulfosuccinimidyl 2-(biotinamido) ethyl- 1,3-dithiopropionate (NHS- SS-Biotin, Pierce, Rockford, IL).
• Fully protected Mas-DH is assembled on MBHA resin by the Boc/DIC method on a Biosearch/Millgen 9600 Peptide Synthesizer as described previously (Kataoka et. al. ibid).
• the Ncc-Boc group of the peptidyl resin (1.64 g, 0.157 mmol) is deprotected with 50% TFA in DCM and the DNP group on His 27 is removed by treating with 17.5% thiophenol in DMF in the usual manner.
• the selectively deprotected peptidyl resin is treated two times with NHS-SS-biotin reagent (100 mg, 0.165 mmol) and diisopropyl ethylamine (DIEA) 55 ⁇ l, 0.314 mmol) in 10 ml DMF for 45 min, with DMF wash between biotinylation reactions.
• biotinylated peptide (crude yield 0.82 g) is completely deprotected and cleaved from the solid support by treating 1.81 g biotinylated peptidyl resin with 15 ml HF in the presence of 1.5 ml anisole and 1 mL ethyl sulf ⁇ de at 0°C for 1 hr.
• the crude peptide is purified by reversed-phase liquid chromatography (RPLC) using a Vydac C 18 column, 0.46 x 15 cm, particle size 5 ⁇ m, flow rate at 1 ml/min linear gradient elution form 28% to 31.5% acetonitrile in 0.1% trifluoroacetic acid (TFA) over 21 min, monitoring at 220nm and or 280nm.
• RPLC reversed-phase liquid chromatography
• the purified peptide is a white powder and has the correct amino acid composition with a 43% peptide content.
• Solubilized membranes are dissolved in 250 ⁇ l of ice cold binding buffer (0.2% CHAPS, 25 mM HEPES, pH 7.0, 100 mM NaCl, 10 mg/ml BSA, 2.5 mg/ml bacitracin, 1 mg/ml amastatin) and various concentrations of N ⁇ -[2,3- 3 H-propanoyl] Mas-DH are added (N ⁇ -[2,3- 3 H- propanoyl] Mas-DH is dissolved in 20% propanol 0.1% TFA). To tubes used to determine ' nonspecific binding, 0.1 ⁇ g of unlabelled Mas-DH is added. The tubes are incubated on ice for 2 hr.
• the samples are filtered through a GF/B filter (pretreated for 1 hour with 0.1% PEI).
• the filters are washed with ice cold 25 mM HEPES, pH 7.0 containing 14% isopropanol and dried under a heat lamp.
• the dried filters are placed in 5 ml of Ready Protein scintillation cocktail (Beckman, Palo Alto, CA) and counted in a Beckman LS5000 scintillation counter- Data are analyzed by the computer program LIGAND (Munson et.al. 1980).
• Figure 1 shows a saturation isotherm of N ⁇ -[2,3- 3 H-propanoyl] Mas-DH binding to CHAPS solubilized Mt membranes from 5th stadium Manduca sexta larvae.
• the binding is saturable and specific.
• Solubilized Mt membranes are incubated with N ⁇ -[2,3- 3 H-propanoyl] Mas-DH (unlabelled Mas-DH is included for determination of nonspecific binding) for 2 hours on ice and then layered upon a 5% to 20% linear sucrose gradient and centrifuged in a VTi65.1 rotor (Beckman, Palo Alto, CA) at 269,000xg for 1.5 hours using a slow acceleration and no brake profile. Fractions (1 ml) are collected and the radioactivity in each sample is determined by liquid scintillation counting as described above.
• solubilized Mt membranes are incubated with N ⁇ -[2,3- 3 H-propanoyl] Mas-DH (unlabelled Mas-DH is included for determination of nonspecific binding) for 2 hours on ice.
• the solubilized membranes are then loaded on a 26 cm x 1 cm Sepharose CL-6B column and eluted a buffer containing 0.2% CHAPS, 25 mM HEPES, pH 7.0, 100 mM NaCl at a flow rate of 0.5 ml/min. Fractions (1 ml) are collected and the radioactivity in each sample is determined as above.
• Mas-DH-R is isolated via an expression cloning procedure developed by Aruffo, A. Seed, B. Proc. (1987) Molecular Cloning of CD28 cDNA by a High Efficiency COS Cell Expression System. Proc. Natl. Acad. Sci 84, 8573-8577.
• a directional cDNA library is prepared from the Malpighian tubules of 5th instar M. sexta and cloned into the EcoRl/Xhol site of the vector pKSl (obtainable from Dr. Peter Mclntyre, Sandoz Institute for Medical Research, London, U.K.). The library is divided into pools of 2200 individuals.
• Plasmid DNA is prepared from each pool, transfected into Cos7 cells and after three days the cells are screened for Mas-DHR expression using a 125-1 labelled Mas-DH followed by emulsion autoradiography. Positive pools are subdivided until single clones are obtained which is then sequenced.
• the cDNA sequence of the complete Mas-DH-R is shown in SEQ ID No. 1.
• the cDNA contains an open reading frame which codes for a protein 395 amino acids in length and has 7 putative membrane spanning domains (SEQ ID No. 2).
• the recombinant receptor has a high affinity for Mas-DH as shown in Figures 6 and 7.
• EXAMPLE G Expression of Mas-DH-R in recombinant Baculovirus Infected Insect Cells (a).
• Cell culture and virus infection Spodoptera frugiperda (Sf9) cells are grown as monolayers in TMN-FH medium (Sigma, St. Louis, MO) supplemented with 10% heat inactivated fetal calf serum, gentamicin and fungizone (2.5 ⁇ g/ml). Sf9 cells are treated with either wild type virus or recombinant virus at the indicated multiplicity of infection (MOI) in cell medium at room temperature. After one hour, the medium is replaced with fresh medium and the cells are incubated at 27°C.
• MOI multiplicity of infection
• the PCR product is also ligated into the Sbal and Smal sites of pBluescript (Stratagene, San Diego, CA) and sequenced by the dideoxy nucleotide chain termination method using Sequenase version 2.0 (U.S. Biochemical Corp., Cleveland, OH) to ensure that no mutations are introduced by Taq polymerase.
• pVL 1393/Mas-DH-R and linearized AcRP23.1acZ baculovirus DNA are co-transfected into Sf9 cells by CaPO 4 precipitation and the recombinant baculovirus (BN/Mas-DH-R) is plaque purified using standard procedures.
• a high titer stock (4.5 x 10 7 pfu/ml) is prepared by infecting Sf9 cells at a MOI of 0.1 and collecting the supernatant 4 days after infection.
• FIG. 10 shows a time course of Mas-DH-R expression in Sf9 cells.
• the Kd value is in close agreement with the Kd values of Mt-membranes and COS-7 cells transfected with the Mas-DH-R (Table 1).
• the level of Mas-DH-R expression is considerably higher.
• cAMP assay Approximately 1 x 10 6 Sf9 cells are infected with BV/Mas-DH-R at a MOI of 1 in 6 well tissue culture plates. Three days after infection, cells are incubated in fresh cell media containing 1 mM isobutylmethyxanthine and various concentrations of peptide hormones for 20 min at 27°C. The reaction is terminated by aspiration of the cell media and addition of 70% ice-cold ethanol. cAMP is measured by radioimmunoassay (Amersham, Arlington Heights, IL).
• Figure 11 depicts the stimulation of cAMP synthesis by Mas-DH and N-terminal truncated analogs-
• the N-terminal truncated analog [3-41] Mas- DH and [13-41] Mas-DH displays high affinity for Mas-DH-R-
• the [13-41] Mas-DH high affinity for the receptor, it is unable to stimulate cAMP synthesis.
• ACAGGAAGGC AAAATGCCGC AAAAAAGGGA ATAAGGGCGA CACGGAAATG TTGAATACTC 1800
• ATACTCTTCC TTTTTCAATC GCGTTGACAT TGATTATTGA CTAGTTATTA ATAGTAATCA 1860
• CTACCGCCAG CACAATGGAT CTCGAGGGAT CTTCCATACC TACCAGTTCT GCGCCTGCAG 2940
• CAGCACCTCC ATACCCCCTT T ATAAGCAG TTTGGGAACG GGTGCGGGTC TTACTCCGCC 4500
• CATCCCGCCC CTAACTCCGC CCAGTTCCGC CCATTCTCCG CCCCATGGCT GACTAATTTT 4560

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