WO2001019985A1 - Nouveaux recepteurs et genes codant pour ces memes recepteurs - Google Patents

Nouveaux recepteurs et genes codant pour ces memes recepteurs Download PDF

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Publication number
WO2001019985A1
WO2001019985A1 PCT/JP2000/006200 JP0006200W WO0119985A1 WO 2001019985 A1 WO2001019985 A1 WO 2001019985A1 JP 0006200 W JP0006200 W JP 0006200W WO 0119985 A1 WO0119985 A1 WO 0119985A1
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Prior art keywords
amino acid
acid sequence
protein
seq
bok
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PCT/JP2000/006200
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English (en)
Japanese (ja)
Inventor
Kazuhiko Kato
Kenji Asai
Hiroshi Nagaso
Takehiko Yokomizo
Takao Shimizu
Original Assignee
Meiji Seika Kaisha, Ltd.
Japan Health Sciences Foundation
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Application filed by Meiji Seika Kaisha, Ltd., Japan Health Sciences Foundation filed Critical Meiji Seika Kaisha, Ltd.
Priority to AU68782/00A priority Critical patent/AU6878200A/en
Publication of WO2001019985A1 publication Critical patent/WO2001019985A1/fr

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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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a novel protein (a novel receptor), a gene encoding the same, a plasmid or a transformant containing the gene, a method for producing the novel protein, a cell containing the novel protein or a cell membrane thereof.
  • the present invention relates to a method for treating or preventing a disease caused by deficiency or excess of raenoic acids, use of the agonist or antagonist, and an antibody against the novel protein or a fragment thereof.
  • Leukotriene day 4 is an unsaturated fatty acid that is biosynthesized from arachidonic acid by various enzymes, is the most potent leukocyte chemotactic substance among naturally occurring substances, and is a leukocyte activator. is there.
  • Leuco Toryen B 4 is made of white blood cells, seeds' s condition (e.g., infection, immunodeficiency, ulcerative colitis, Crohn's disease, necrotizing expansion after myocardial infarction reperfusion, psoriasis, atopic dermatitis, bronchial asthma, It is thought to be involved in the formation of acute respiratory distress syndrome, delayed neuronal death, vasospasm after subarachnoid hemorrhage, or perfusion injury after organ transplantation.
  • s condition e.g., infection, immunodeficiency, ulcerative colitis, Crohn's disease, necrotizing expansion after myocardial infarction reperfusion, psoriasis, atopic dermatitis, bronchial asthma. It is thought to be involved in the formation of acute respiratory distress syndrome, delayed neuronal death, vasospasm after subarachnoid hemorrhage, or perfusion injury after organ transplantation.
  • G protein-coupled receptor G- pr 0 te I ncoupledreceptor; GPCR
  • GPCR G protein-coupled receptor
  • HETE hydroxyeicosatetraenoic acid
  • HPETE Hydropel quiche Icosatetraenoic acid
  • Fig. 9 shows the structures and biosynthetic pathways of leukotrienes, HETEs and HPETEs.
  • Enzymes involved in the biosynthesis of HETEs and HPETEs have been isolated and analyzed from various animal species, and their primary structures and enzymatic properties are well preserved. It is presumed to play an important role, but there are still many unknowns, including its mechanism of action and the presence of receptors. In addition to suggesting the existence of specific cell membrane receptors, experimental facts that bind to the nuclear receptor PPAR (peroxisomeprolifera tor-activated receptors) and regulate the transcription of various genes are announced Have been.
  • nuclear receptor PPAR peroxisomeprolifera tor-activated receptors
  • 12-lipoxygenase leukocyte type
  • 12-lipoxygenase platelet type mice lacking these were created and analyzed. I have.
  • the phenotypes that have been identified so far include 12-lipoxygenase (leukocyte) -deficient mice (BI eich, D. et al., J. Clin. Invest., 103, 143). 1-13 36, 1999 and Sun, D. et al., J. Biol.
  • the present inventor has found and found a completely novel receptor, determined the structure thereof, and found that this is a low-affinity second receptor of leukotriene day 4. Was found.
  • the discovery of this second receptor is expected to lead to the development of new antagonists that are more potent (eg, inhibit both the first and second receptors), or to develop therapeutics using antibodies and the like. You.
  • the present inventors the present inventors the novel leuco which has found Toryen B 4 second receptor, as well as leuco Bok Lien B 4, a plurality of hydroxy Eiko Sate Bok Lae phosphate (HETE) acids and Hidoroperu It has been found that the receptor has a binding ability to human xyeicosatetraenoic acids (HPETEs) and an ability to transmit intracellular signals by these ligands. No receptor that binds to HETEs or HPETEs has been reported in the past, and the present inventors have found its existence for the first time.
  • HPETEs human xyeicosatetraenoic acids
  • the present invention is, (1) protein comprising the amino acid sequence represented by SEQ ID NO: 2 (hereinafter, sometimes referred to as "LT 8 4 receptor protein”>, (2) the functionally equivalent modified (Hereinafter sometimes simply referred to as “functionally equivalent variant”), or (3) a protein containing the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing.
  • LT 8 4 consisting of an amino acid sequence having 6 0% or more homologous sex respect to the amino acid sequence of the receptor protein) protein (hereinafter, Rukoto referred to as "homologous protein”) relates.
  • novel proteins i.e., to Ding 8 4 receptor protein, variation functionally equivalent, and the homologous protein
  • the novel proteins i.e., to Ding 8 4 receptor protein, variation functionally equivalent, and the homologous protein
  • the present invention relates to a gene encoding a.
  • the present invention also relates to a plasmid containing the gene.
  • the present invention also relates to a transformant containing the gene.
  • the present invention also relates to the method for producing a novel protein according to the present invention, which comprises culturing the transformant.
  • the present invention also relates to a cell containing the novel protein according to the present invention or a cell membrane fraction thereof.
  • the present invention also provides a novel protein according to the present invention, characterized by using the novel protein according to the present invention, a cell membrane fraction containing the same, or a cell containing the novel protein according to the present invention.
  • the present invention relates to a method for screening an agonist or an antagonist for a protein and a screening kit.
  • the present invention also relates to an agonist and an antagonist for the novel protein according to the present invention.
  • the present invention provides a method for treating or preventing a disease caused by a deficiency of leucotriene- 4 , hydroxyeicosatetraenoic acids, or hydroperoxyeicosatetraenoic acids by using the agonist according to the present invention. And administering to the subject in need thereof an effective amount.
  • the present invention is that the antagonistic Bok by the of the present invention, leuco Toryen beta 4, hydroxy Eiko satay Bok Raen acids, or Hidoroperu old Kishieikosate Bok Raen excessively subject in need of treatment or prevention of caused by diseases of acids,
  • the present invention relates to a method for treating or preventing the disease, which comprises administering an effective amount.
  • the present invention relates to the use of Agonisu Bok by the of the present invention, leuco Bok Lien beta 4, human
  • the present invention relates to a use for producing an agent for treating or preventing a disease caused by a deficiency of droxyeicosatetraenoic acids or hydroperoxyeicosatetraenoic acids.
  • the present invention provides a method for producing a therapeutic or prophylactic agent for a disease caused by an excess of leukotriene B 4 , hydroxyeicosatetraenoic acid, or hydropersylxieicosatetraenoic acid in the antagonist according to the present invention.
  • the present invention relates to an antibody or a fragment thereof, which specifically reacts with the novel protein according to the present invention.
  • Figure 1 is a graph showing [3 H] and the concentration of LTB 4, the relationship between [3 H] binding amount of LTB 4 to the cell membrane fraction.
  • FIG. 2 is a graph showing the results of a scout yard analysis performed based on the results shown in FIG.
  • BLT 1 cells is a graph showing the binding of [3 H] LTB 4 to the cell membrane fraction.
  • FIG. 4 is a graph showing the amount of [ 3 H] LTB 4 bound to a cell membrane fraction when CHO-HABLT 2 cells were used.
  • Figure 5 is based on the data shown in FIG. 3 and FIG. 4 is a graph showing the relative binding inhibition activity when formed into a 1 0 0% binding inhibition by LTB 4.
  • FIG. 6 is a graph showing the degree of increase in intracellular calcium ion (Ca 2+ ) concentration when CHO-FLAG BLT1 cells were used.
  • FIG. 7 is a graph showing the degree of increase in intracellular calcium ion ( Ca2 + ) concentration when CHO-HABLT2 cells were used.
  • Figure 8 is a repo one data one gene plus Mi de p CRE-L uc DNA and human Bok Roy co Bok Rie> B 4 second receptor expression plasmid (ph BLT 2) and Bok lance Fuekushiyon simultaneously with HEK 2 9 is a graph showing the expression level of luciferase in 93 cells.
  • FIG. 9 is an explanatory diagram showing the structures and biosynthetic pathways of leukotrienes, HETEs and HPETEs. BEST MODE FOR CARRYING OUT THE INVENTION
  • a novel protein according to the present invention (1) a protein comprising an amino acid sequence represented by SEQ ID NO: 2 (i.e., LT 8 4 receptor protein), (2) functionally equivalent modifications of their body (i.e., variation functionally equivalent), and (3) a protein comprising an amino acid sequence represented by SEQ ID NO: 2 (i.e., LTB 4 receptor protein) 6 0% or more homology with respect to the amino acid sequence of (Ie, a homologous protein) comprising an amino acid sequence having the formula:
  • the novel protein according to the invention proteins consisting Amino acid sequence shown in SEQ ID NO: 2 (i.e., human Bok leuco Bok Rie> B 4 second receptor), its functional equivalence variants,
  • protein comprising the amino acid sequence represented by SEQ ID NO: 2 i.e., human Bok 'leuco Toryen B 4 second receptor
  • has a function to 6 0% or more homology to the amino acid sequence of Proteins consisting of an amino acid sequence are preferred.
  • Human Bok leuco Bok Rie> B 4 second receptor which is one of the novel proteins of the present invention, 3 5 2 amino acids the amino acid sequence of a known human Bok leuco Toryen B 4 first receptor full length whereas consisting of residues, 3 5 consists of 8 amino acid residues, known prior Kihi Bok.
  • the Amino acid sequence of human Bok 'leuco Bok Rie> B 4 second receptor according to the invention so far plurality retain the amino acid common to a number
  • the reported G protein-coupled receptor (GPCR) Therefore, it is presumed that it is a GPCR.
  • human Bok 'leuco Bok Lien ⁇ 4 second receptor not only leuco Bok Lien B 4, a plurality of hydroxycarboxylic Eiko satay Bok Raen acid (HETE) acids and human mud pel O carboxymethyl Eiko satay The ability to bind to traenoic acid (HPETE) These receptors have intracellular signal transduction ability by these ligands.
  • HETE hydroxycarboxylic Eiko satay Bok Raen acid
  • HPETE traenoic acid
  • the HETEs and HPETEs are unsaturated fatty acids biosynthesized from arachidonic acid by various enzymes.
  • HETEs include, for example, 1 2 (R) 1 HETE, 1 2 (S) — HETE, 15 (S) — HETE, 5 (S) — HETE, or 8 (S) — HETE; Classes include, for example, 15 (S) -HPETE, 5 (S) -HPETE, 8 (S) -HPETE, 12 (S) -HPETE, or 15 (R) -HPETE.
  • HETE such multiple [e.g., 1 2 (R) -HETE, 1 2 (S) - HETE, or 1 5 (S) -HETE ]
  • HPETEs eg, 15 (S) — HPETE.
  • the term “functionally equivalent variant” means that the amino acid sequence has a deletion, substitution, or addition of one or more (particularly, one or several) amino acids in the amino acid sequence of the original protein. and an amino acid sequence, moreover, human Bok 'leuco tri E emissions B 4 second receptor substantially the same activity (e.g., binding ability to the leuco Bok Lien B 4, HETE acids, and Z or HPETE acids, And the resulting various cell stimulating activities).
  • the functional variant includes salts of the functional variant, and further includes both those having no sugar chain and those having a sugar chain.
  • human Bok 'leuco Bok Lien B 4 second receptor variants in human Bok e.g., human Bok' by alleles of the leuco Toryen B 4 second receptor gene co - proteins de
  • human Bok other organisms e.g ', baboon Bok mammal (e.g., mouse, rat Bok, hamster, or I j)] contained 4 second receptor derived from leuco Toryen B or its variants.
  • mutants derived from human Bok, or human leuco Bok Rie from organisms other than preparative> B 4 second receptor or a variant thereof) or human leuco Bok Lien B 4 second Second reception Proteins artificially modified by genetic engineering based on the condition are included.
  • variant refers to “var I at I 0 n”, that is, an individual difference observed in the same protein within the same species, or a difference observed in a homologous protein between several species. means.
  • primers one or probe For example, based on information of the human leuco Triester> B 4 nucleotide sequence of the second receptor gene to design appropriate primers one or probe, said primer one or probes, organisms and purpose [e.g., mammals Animals (eg, humans, mice, rats, hamsters, or dogs)] (eg, total RNA or mRNA fraction, cDNA library, or phage library).
  • the desired protein can be prepared by obtaining the gene of the target protein by performing the PCR method or the hybridization method, and expressing the gene using an appropriate expression system.
  • the above-mentioned protein which has been artificially modified by genetic engineering can be prepared by a conventional method, for example, by a site-directed mutagenesis method (site-specificmutagenews).
  • Examples of the protein containing the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing include, for example, a fusion protein of a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing and a fusion partner. it can.
  • the fusion partner can be bound to the N-terminal and Z- or C-terminal of the protein having the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing.
  • the fusion partner examples include a protein for purification [eg, 'all or a part of glutathione S-transferase (GST)], a protein for detection [eg, hemagglutinin or /?-Galactosidase peptide] (All or part of LacZa)] or an expression protein (eg, ', signal sequence).
  • GST glutathione S-transferase
  • a protein for detection eg, hemagglutinin or /?-Galactosidase peptide] (All or part of LacZa)
  • an expression protein eg, ', signal sequence
  • a proteolytic enzyme that undergoes limited degradation for example, thrombin or factor
  • An amino acid sequence that can be cleaved at X a) can be introduced as appropriate.
  • Homologous protein of the present invention as long as the amino acid sequence having 6 0% or more homology with respect to amino acid sequence of LT 8 4 receptor protein, is not particularly limited, the amino acid sequence of the LT beta receptor proteins With respect to amino acid sequences having a homology of preferably 70% or more, more preferably 80% or more, still more preferably 90% or more, still more preferably 95% or more, and particularly preferably 98% or more. Can be.
  • homologous proteins of the present invention LTB 4 receptor protein 6 0% or more with respect to the amino acid sequence of (preferably 70% or more, more preferably 80% or more, more preferably 90% or more, more preferably It is composed of an amino acid sequence having a homology of 95% or more, particularly preferably 98% or more, and has substantially the same activity as that of the human 4th receptor (for example, leuco). Toryen B 4, HETE compound, and or binding ability to HPETE acids, and is preferably thereby a protein with various cell-stimulating activity) occurring.
  • Novel proteins by these invention i.e., LTB 4 receptor protein, variation functionally equivalent, and the homologous protein
  • LTB 4 receptor protein i.e., LTB 4 receptor protein, variation functionally equivalent, and the homologous protein
  • novel proteins by these invention is possible to get by a variety of known methods, for example, by using a gene according to the invention known
  • It can be prepared by the genetic engineering technique described above. More specifically, a transformant according to the present invention described later (that is, a transformant containing a gene encoding a novel protein according to the present invention) is used under conditions that allow expression of the novel protein according to the present invention. It can be prepared by culturing under the following conditions, and separating and purifying the target protein from the culture by a method generally used for separating and purifying proteins.
  • the separation and purification methods include, for example, ammonium sulfate salting out, ion exchange column chromatography using ion exchange cellulose, molecular sieve column chromatography using molecular sieve gel, affinity column chromatography using protein A binding polysaccharide. Dialysis, lyophilization or the like.
  • the gene according to the invention is, in particular, so long as it encodes the novel protein according to the invention. It is not limited, and examples thereof include a gene consisting of the base sequence represented by SEQ ID NO: 1 in the sequence listing.
  • the term “gene” in the present specification includes:
  • Nucleotide sequence or Ranaru the gene represented by SEQ ID NO: 1 is human • leuco Bok Lien 8 4 second receptor co one de consisting of the amino acid sequence represented by SEQ ID NO: 2 in the Sequence Listing .
  • the plasmid according to the present invention is not particularly limited as long as it contains the gene according to the present invention.
  • the plasmid according to the present invention may be added to a known expression vector appropriately selected depending on the host cell used. Plasmids obtained by insertion can be mentioned.
  • the transformant of the present invention is not particularly limited as long as it contains the gene of the present invention.
  • the transformant of the present invention is a transformant in which the gene of the present invention has been integrated into a chromosome of a host cell.
  • it may be a transformant containing the gene according to the present invention in the form of a plasmid.
  • the transformant may be a transformant expressing the protein according to the present invention, or may be a transformant not expressing the protein according to the present invention.
  • the transformant of the present invention can be obtained, for example, by transforming a desired host cell with the plasmid of the present invention or with the gene itself of the present invention.
  • Examples of the host cell include, for example, commonly used known microorganisms, for example, Escherichia coli or yeast (Saccharomyces cerevisiae) or known cultured cells, for example, animal cells (for example, CHO cells, HEK- 293 cells or COS cells) or insect cells (eg, BmN4 cells)
  • Examples of the known expression vectors include, for example, p UC, p TV, pGEX, pKK, or pTrcHis; for yeast, pEMBL ⁇ or pYES2; for CH0 cells, pcDNA3 or pMAM neo; HEK— For 293 cells, pcDNA3; for COS cells, pcDNA3; for BmN4 cells, the polyhedrin promoter of silkworm nuclear polyhedrosis virus (BmNPV).
  • BmNPV silkworm nuclear polyhedrosis virus
  • Novel protein i.e., LT snake 4 receptor protein, variation functionally equivalent, and the homologous protein
  • the transformant according to the present invention that is, a cell transformed with a plasmid containing a gene encoding a novel protein according to the present invention
  • the transformant according to the present invention may be used in accordance with the present invention.
  • the cell membrane fraction according to the present invention containing the novel protein according to the present invention can be obtained, for example, by crushing the cells according to the present invention and separating a fraction containing a large amount of cell membranes.
  • the method for crushing cells include a method of crushing cells with a homogenizer (for example, a Potter-E levehjem type homogenizer), a method of crushing with a single ring blender or a polytron (Kinematica), and a method using ultrasonic waves. Crushing, or crushing by ejecting cells from a fine nozzle while applying pressure with a French press or the like can be mentioned.
  • the cell membrane fractionation method include, for example, a fractionation method using centrifugal force, for example, a fractionation centrifugation method or a density gradient centrifugation method.
  • a novel protein i.e., LT 8 4 receptor according to the invention body protein, variation functionally equivalent, and the homologous protein
  • cell membrane fraction according to the invention ie, a novel protein (i.e. according to the invention, LT 8 4 receptor protein, functionally equivalent variants, or homologous protein) cell membrane fraction containing, or cells according to the present invention [i.e., a novel protein (i.e. according to the present invention, containing 1_ Ding 8 4 receptor protein, variation functionally equivalent, or homologous protein) Cells].
  • a cell containing the novel protein of the present invention or a cell membrane fraction thereof when used, a cell containing no leukotrien day 4 first receptor or a cell membrane fraction thereof can be used.
  • Toryen B 4 HETE such, or cell stimulating activity caused by HPETE acids are attached, for example, an increase in intracellular calcium concentration, activity inhibition of adenyl two Le cyclase, promotion of cell migration activity, My Bokujiko It utilizes activation of kinase activated kinase, c-fos and c-jun inducing activity, AP-1 activating effect, vascular smooth muscle contraction activity, or tumor cell adhesion enhancing activity.
  • the novel protein according to the present invention or the cell membrane fraction according to the present invention or the cell according to the present invention is brought into contact with a test compound, and the novel protein according to the present invention or the cell membrane fraction according to the present invention is contacted. or a cell according to the invention, by a test compound to analyze whether binding, present invention Agonisu Bok or against the novel protein (particularly human Bok leuco Bok Rie> B 4 second receptor) by The screening can be performed without distinguishing the antagonist.
  • novel protein according to the present invention particularly a human 'Roikotoryen B 4 second receptor
  • Agonisu Bok or antagomir- varnish Bok against can be screened without distinction Agonisu Bok or antagomir- varnish Bok against.
  • test compound when the test compound is an agonist or an antagonist against the novel protein according to the present invention, a natural protein against the novel protein according to the present invention, the cell membrane fraction according to the present invention, or the cell according to the present invention in the absence of the test compound.
  • the specific binding amount in the presence of the test compound is lower than the specific binding amount of the ligand.
  • a labeled natural ligand can be used as the natural ligand.
  • a radioisotope for example, [ 3 H] or [′ 4 C] can be used.
  • a compound that binds to the novel protein according to the present invention, the cell membrane fraction according to the present invention, or the cell according to the present invention, and inhibits the binding of these to the natural ligand, (the Japanese human Bok 'leuco Bok Rie> B 4 second receptor) novel proteins by can be chosen indifferently as Agonisu preparative or Antagoni scan Bok against.
  • a cell according to the present invention and a labeled natural ligand are used in the absence and presence of a test compound. , Or HPETEs), comparing the specific binding amount of the natural ligand to the cells according to the present invention under the above conditions, and further measuring the specific cell stimulating activity of the natural ligand under the above conditions. by comparison, the new protein according to the invention (in particular human Bok 'leuco Bok Rie> B 4 second receptor) can be screened to distinguish Agonisu Bok or antagonistic Bok against.
  • a labeled natural ligand ie, leukotriene B or HETE
  • a compound having a cell stimulating activity via the receptor contained in the cells can be selected as an agonist.
  • the screening method of the present invention can be carried out by utilizing, for example, the inhibition of the activity of adenylate cyclase as the cell stimulating activity.
  • a novel protein according to the present invention expressed on the cell membrane preferably, by introducing human Bok leuco Bok Lien ⁇ 4 second receptor including expression base Kuta scratch excess And the cyclic AMP response
  • screening cells cells containing a luciferase gene (hereinafter referred to as screening cells) in which the system lj (CRE) is located 5 'upstream
  • novel proteins particularly, human leuco triae according to the present invention can be obtained.
  • > B 4 second receptor) to distinguish Agonisu Bok or ⁇ Ntago two be sampled for can be screened.
  • intracellular signal transduction caused by the natural ligand binds to the leuco Bok Lien B 4 second receptor, i.e., the active suppression of adenylate cyclase, which is one of the cell-stimulating activity of the leuco Toryen B 4 To use.
  • leuco Bok Lien B 4 natural ligand to the second receptor is bound, leuco Toryen B 4 second receptor which is one of the G protein family one that coupled to G, the family, Suppresses adenylate cyclase and reduces the amount of cyclic AMP (generated from ATP by adenylate cyclase) in cells, resulting in the reduction of CRE introduced into the screening cells. It utilizes the fact that the transcription of the luciferase gene in the promoter region is suppressed.
  • the CRE introduced into the screening cell is present in the transcriptional regulatory region of a group of genes (cyclic AMP inducible genes) whose expression is enhanced when the concentration of cyclic AMP in the cell is increased.
  • Base sequence Therefore, adding an activator of adenylate cyclase [eg, forskolin (FSK)] to screening cells increases the concentration of intracellular cyclic AMP, and consequently downstream of CRE.
  • the expression level of the luciferase gene located at the site is increased. The luciferase expression level can be easily measured by measuring the luminescence derived therefrom.
  • the test compound Simultaneous addition of the adenylate cyclase activator, the natural ligand of the novel protein according to the present invention (particularly the human / leukotrienyo 4 second receptor), and the test compound to the cells for screening
  • the test compound has an action as an antagonist
  • the same luciferase as when only the adenylate cyclase-active agent is administered alone (in the absence of the natural ligand and in the absence of the test compound).
  • -Expression level is observed [ie, it antagonizes the action of the natural ligand on the novel protein (receptor) of the present invention].
  • the luciferase was compared with the case where only the adenylate cyclase activator was administered alone (in the absence of the natural ligand and the absence of the test compound).
  • the expression level is reduced [that is, the novel protein (receptor) according to the present invention has the same action as a natural ligand].
  • the effect of the test compound is an effect of binding to the novel protein according to the present invention.
  • a screening cell that is, a cell expressing the novel protein of the present invention on the cell membrane and further containing a luciferase gene located 5 ′ upstream of the CRE
  • control cells for example, cells in which the CRE contains the luciferase gene located 5 ′ upstream but does not express the novel protein according to the present invention on the cell membrane.
  • the same phenomenon regarding the luciferase expression level is observed in the screening cells and the control cells, whereas the effect of the test compound is the effect of the binding to the novel protein according to the present invention.
  • different phenotypes of luciferase expression are observed between screening cells and control cells.
  • the luciferase used in the above embodiment is an enzyme used when fireflies and the like emit light, and mammals do not have this. Therefore, the use of mammalian cells is preferable because the background is low.
  • Luciferase is one of the reporter enzymes such as chloramphenicylase tranferase and /?-Galactosidase, but has excellent quantitative properties and high detection sensitivity. Because the measurement method is simple, it is widely used. Emission mechanisms are luciferin is a group substance (C, 3 H, 2 N 2 S 2 0 3) is oxidized by Lucifera Ichize in the presence ATP, luminescent substance due to be produced.
  • Luciferase has been applied to various Atsay methods (eg, promoter atsay, two hybrid assay, or screening for agonist or antagonist).
  • Test compounds that can be used in the screening method of the present invention are not particularly limited, and include, for example, natural or synthetic compounds (eg, lipids, proteins, or peptides), fermentation products, cell extraction Liquid, plant extract, or animal tissue extract.
  • novel protein according to the invention i.e., 1_ Chomi 4 receptor protein, variation functionally equivalent, and the homologous protein
  • Moshiku cell membrane fraction according to the present invention i.e., LT beta receptor protein, variation functionally equivalent, and cell membrane fractions containing homologous protein
  • cells according to the present invention i.e., L Ding 8 4 receptor protein, variation functionally equivalent, and containing homologous protein cells
  • the screening kit I of the present invention if desired, various reagents, for example ', labeled leuco Bok Rie> B 4 or HETE acids or HPETE acids were unlabeled leuco Toryen B 4 or HETE acids or HPETE acids, binding reaction And Z or a wash buffer.
  • the screening kit of the present invention comprises a novel protein according to the present invention.
  • labeled natural Li ligand i.e., labeled leuco Bok Lien B 4 or HETE acids or HPETE, in particular the [3 H] labeled leuco Bok Rie B 4 or HETEs or HPETEs
  • unlabeled natural ligand ie, unlabeled leukotriene day 4 or HETEs or HPETEs
  • binding reaction buffer optionally, labeled natural Li ligand (i.e., labeled leuco Bok Lien B 4 or HETE acids or HPETE, in particular the [3 H] labeled leuco Bok Rie B 4 or HETEs or HPETEs), unlabeled natural ligand (ie, unlabeled leukotriene day 4 or HETEs or HPETEs), and / or a binding reaction buffer.
  • labeled natural Li ligand i.e., labeled leuco Bok Lien B 4 or HETE acids or HPE
  • Another embodiment of the screening kit Bok of the present invention express a novel protein on the cell membrane according to the present invention (preferably, over-expression by introducing the expression vector one containing human Bok leuco Toryen B 4 second receptor) And a cell containing a luciferase gene having a cyclic AMP response element (CRE) located 5 ′ upstream, and optionally a luciferase substrate, an adenylate cyclase activator (eg, FSK) , A natural ligand, and / or a buffer for a binding reaction.
  • CRE cyclic AMP response element
  • a screening kit is a kit for expressing the novel protein of the present invention on a cell membrane (preferably, by introducing an expression vector containing a human / leukotriene day 4 second receptor). Overexpressed) and cyclic AMP response element
  • CRE contains the luciferase gene located at the 5 'upstream and the CRE contains the luciferase gene located at the 5' upstream, but expresses the novel protein of the present invention on the cell membrane.
  • a luciferase substrate, an adenylate cyclase activator (eg, FSK), and a buffer for Z or binding reaction can be included.
  • Agonisu Bok for new protein according to the present invention (particularly human Bok leuco Bok Rie> B 4 second receptors), for example, diseases caused by lack of leuco Toryen B 4, if example embodiment, such as infection or immunodeficiency It is useful as a therapeutic or prophylactic agent, and also as a therapeutic or prophylactic agent for diseases caused by deficiency of HETEs or HPETEs.
  • the agonist of the present invention is administered alone or, preferably, together with a usual pharmaceutically acceptable carrier or diluent to a subject in need of treatment or prevention of the above-mentioned diseases in an effective amount.
  • the agonist of the present invention can be used for producing an agent for treating or preventing the above-mentioned diseases.
  • the agonist of the present invention can be obtained, for example, by the agonist screening method of the present invention. it can.
  • antagonist to a novel protein for example, diseases you excessively due leuco Bok Lien B 4, for example, infectious diseases, immunodeficiency Ulcerative colitis, Crohn's disease, necrosis after myocardial infarction reperfusion, psoriasis, atopic dermatitis, bronchial asthma, acute respiratory distress syndrome, delayed neuronal cell death, vasospasm after subarachnoid hemorrhage, or organ
  • the antagonist of the present invention is administered alone or, preferably, together with a usual pharmaceutically acceptable carrier or diluent to a subject in need of treatment or prevention of the above-mentioned diseases in an effective amount. can do. Further, the antagonist of the present invention can be used for producing an agent for treating or preventing the above-mentioned disease. Furthermore, antagonists Bok of the present invention are also useful in inhibiting the binding of human Bok 'leuco Bok Rie> B 4 natural ligand against the second receptor. The antagonist of the present invention can be obtained, for example, by the antagonist screening method of the present invention.
  • Antibodies according to the present invention include monoclonal and polyclonal antibodies.
  • Monochromator port one monoclonal antibody according to the present invention, as a an antigen for immunization and subscription-learning for antigen, a novel protein (particularly human Bok 'leuco Bok Rie> B 4 second receptor) according to the invention or their partial fragment It can be obtained by a means known per se except for using.
  • a novel protein particularly human Bok 'leuco Bok Rie> B 4 second receptor
  • a mouse is immunized with the immunizing antigen, and spleen cells obtained from the mouse and mouse myeloma cells are isolated from each other by the method described in Nature, 256, 495 (1975).
  • Cell fusion method, or cell fusion using the electric cell fusion method described in Imm unol. Method, 100, 181-189 (19897), and the screening antigen is used.
  • E. coli a hybridoma producing the monoclonal antibody of the present invention can be obtained.
  • the medium for culturing the Hypri-doma may be any medium suitable for culturing Hypri-doma, and is preferably Dulbecco's modified Eagle's minimum essential medium (Du I beccos modified E eag I esminimumessent I a medium). ⁇ ⁇ A medium containing fetal serum, L-glutamine, L-pyruvic acid, and antibiotics (penicillin G and streptomycin) is used.
  • Culture of the High Priestess dormer when carried out in a medium can be carried out in under conditions of 5% C_ ⁇ 2 concentration and 3 7 ° C for about 3 days. When culturing in the abdominal cavity of a mouse, it can be performed in about 14 days.
  • the monoclonal antibody can be separated and purified from the thus obtained culture solution or mouse ascites by a method generally used for protein separation and purification.
  • Such methods include, for example, ammonium sulfate precipitation, ion exchange column chromatography using ion exchange cellulose, molecular sieve column chromatography using molecular sieve gel-1, affinity column chromatography using protein A-binding polysaccharides, dialysis, Or lyophilization and the like can be mentioned.
  • polyclonal antibody of the present invention also, as an antigen for immunization and antigen for screening (especially human Bok leuco Bok Rie> B 4 second receptor) novel protein according to the invention but using or their partial fragment It can be prepared by a method known per se, for example, the method shown below. That is, a physiological saline solution containing an antigen is mixed with an equal amount of Freund's complete adjuvant or incomplete adjuvant, or an equivalent thereof, for example, Hunter's Titer Max TM (Funakoshi; Cat. No. YT.
  • the antibody fragment according to the present invention is a partial fragment of the antibody of the present invention (including a monoclonal antibody and a polyclonal antibody) and has the same reaction characteristics as the original antibody. It is not particularly limited as long as it has isomerism.
  • Examples of the antibody fragment according to the present invention include Fab, Fab ', F (ab') 2 , or Fv.
  • the antibody fragment of the present invention can be obtained, for example, by digesting the polyclonal antibody or the monoclonal antibody of the present invention with a proteolytic enzyme by a conventional method, and subsequently by a conventional method of protein separation and purification. be able to.
  • the filter on which the phage DNA was immobilized was mixed with a hybridization buffer (6 XSSC, lOXD enhardt's solution, 0.5% SDS, and 100 gZm L heat-denatured salmon) containing the labeled probe. Hybridization was performed at 65 ° C in sperm DNA). After the filter was finally washed at 65 ° C in 0.1 XSSC and 0.1% SDS solution, the obtained radiogram was searched for a black that hybridized with the probe.
  • a hybridization buffer (6 XSSC, lOXD enhardt's solution, 0.5% SDS, and 100 gZm L heat-denatured salmon
  • the nucleotide sequence of the inserted DNA fragment was determined by a usual method, and a homology search was performed using the international gene database DDBJ / EMB LZG enbank database. it has been found that contains the new DNA having the human leuco Bok Lien B 4 first receptor homology. As shown in Example 4 described below, novel proteins encoded by the novel DNA is because it has a property of binding with leuco Bok Rie> B 4 were confirmed, "human Bok leuco Bok Rie> B 4 Second receptor ".
  • the primer one consisting synthesized, hereinafter, similarly, HA-human 'as a sense primer for leuco Bok Lien ⁇ 4 second receptor fusion protein, nucleotide sequence shown in SEQ ID NO: 4 in Sequence Listing:
  • a primer consisting of 5′-CGGGATCCCGCCATGTACCCCTACGACGTGCCCGACTACGCCTCGGTCTGCTACCGTCC-3 ′ was used as a dual-purpose antisense primer, and the nucleotide sequence represented by SEQ ID NO: 5 in the sequence listing:
  • the plasmid pNOK22 obtained in Example 1 was subjected to a PCR reaction with the ⁇ -type.
  • the amplified fragment was digested with restriction enzymes EcoRI and BamHI, electrophoresed in 1% agarose gel, cut out from agarose gel, and purified using glass beads.
  • CH 0-K 1 cells (5 ⁇ 10 5 cells) were placed in a Ham F F2 medium containing 10% fetal calf serum at 37 ° C. using a 10 cm diameter dish. and incubated for 24 hours at 95% air (air) / 5% C 0 2 conditions.
  • Example 4 Expression and binding of human leukotriene B4 second receptor in HEK-293 cells
  • HEX-293 cells were seeded on a 15 cm plate, and 37% and 95% air was added using a modified Dalbecco's Eagle's medium (DMEM medium) containing 10% fetal serum. No. The cells were cultured for 24 hours under 5% C% 2 conditions.
  • DMEM medium modified Dalbecco's Eagle's medium
  • This cell was subjected to Bok run Sufuekushiyon in Ripofuekushiyon method using the expression of Example human leuco Toryen B 4 obtained in 2 second receptor plasmid (ph BLT 2) 2 0 g . Three days after the transfection, the cells were collected, disrupted by sonication, and the cell membrane fraction (microsome fraction) was collected for binding experiments.
  • [3 H] binding experiments for leuco Toryen B 4 is as follows lines Natsuta. Binding assay buffer [5 0 mm ol ZL- T ris- HCI (p H 7. 4), 1 0 mm o I ZL- M g CI 2, 1 0 mmo IZL- N a C and and 0 - 0 5 % ⁇ shea serum albumin (BSA)] 0. in 1 m L, 2 0 ⁇ g equivalent with [3 H] LTB 4 at various concentrations (0.
  • BSA shea serum albumin
  • Figure 1 is a graph showing [3 H] and the concentration of LTB 4, the relationship between [3 H] binding amount of LTB 4 to the cell membrane fraction.
  • “open squares” represent the total amount of binding
  • “open circles” represent the amount of non-specific binding
  • “black circles” represent the amount of specific binding.
  • FIG. 2 is a graph showing the results of Scatchard analysis performed based on the results shown in FIG.
  • “8” means the amount of bound (B 0 und) [ 3 H] LTB 4
  • “BZF” means the amount of bound (B 0 und) [ 3 H] LTB 4
  • unbound (F ree) [3 H] refers to the ratio between the concentration of LTB 4.
  • Kd was 22.7 nmol ZL and Bma iS IS fmol Zmg protein.
  • Example 5 HA-human Bok leuco Bok Lien B 4 second receptor you express a fusion protein C HO- K 1 cell binding experiments using
  • [3 H] binding experiments for leuco Bok Rie> B 4 (LTB 4), except that was used in 5 nmo IL the [3 H] LTB 4, and the actual applied in accordance with the procedures described in Example 4.
  • the above procedure was repeated except that a 1000-fold concentration (ie, 5 / mo I / L) of various unlabeled eicosanoids (see FIGS. 3 to 5 described later) was added during the binding reaction.
  • the obtained value was compared with the value when only [ 3 H] LTB 4 was added.
  • these eicosanoids used were Cayman Chemical Noka ISA.
  • CHO-FL AG BLT1 cells stably Furaggupe peptide (F LAG) a F LAG- human Bok leuco added to the N-terminal side Toryen B 4 first receptor fusion protein
  • F LAG stably Furaggupe peptide
  • CHO-FL AG BLT1 cells specifically expressed CHO cells
  • FIG. 3 is a graph showing the amount of [ 3 H] LTB 4 bound to a cell membrane fraction when CHO-FLAG BLT1 cells were used.
  • FIG. 4 is a graph showing the amount of [ 3 H] LTB 4 bound to the cell membrane fraction when CHO—HA BLT 2 cells were used.
  • Figure 5 is based on the data shown in FIG. 3 and FIG. 4 is a graph showing the relative binding inhibition activity when formed into a 1 0 0% binding inhibition by LTB 4.
  • “black bars” show the results when CHO-FLAG BLT1 cells were used, and "open bars” show the results when CHO-HABLT2 cells were used.
  • ETE means eicosatetraenoic acid
  • X means lipoxin
  • HETE means hydroxyeicosatetraenoic acid
  • HPETE means hydroperic acid xyeicosatetraenoic acid, respectively.
  • N 0 ne as unlabeled Eikosanoi earth without addition means (ie, the case of adding only [3 H] LTB 4).
  • the CHO-HABLT 2 cells prepared in Example 3 were mixed with 3 mol mol of ZL-Fura-IIAM (acetoxymethyl ester of Fura-2; Doujin Chemical) and 0.01% cremophor.
  • HEPES—Tyrode-BSA buffer [25 mmol ZL— HEPES—NaOH (pH 7.4), 1 Ommo ⁇ / LNaCI, 2.7 mmol / L -CI, 1.0 mm o I / L — Ca , 12 mm o I / L Na HCO 3 , 5.6 mm o I / L— Dg I ucose, 0.37 mm ol / L- N a H; P 0 4 , 0. 4 9 mmo I / L- M g CI 2, 0. 1% (w / v) fatty acid free (fattyacid- free) BSA] at 37 ° C for 1 hour.
  • FIG. 6 is a graph showing the degree of increase in intracellular calcium ion (Ca 2+ ) concentration when CHO-FLAG BLT1 cells were used.
  • Figure 7 shows C HO—HAB
  • FL AG- human leuco Bok Lien B 4 first receptor fusion protein reacts only LTB 4, whereas the increased intracellular calcium, as shown in FIG. 7 to, HA-human Bok 'leuco Toryen B 4 second receptor fusion proteins not only LTB 4, 1 2 (S) - HETE or 1 5 (S) also reacts with one HETE, increased intracellular calcium I let it.
  • Example 7 Screening of agonist or antagonist using human 'leukotrien B4 second receptor
  • the plasmid p CRE-Luc DNA was a plasmid containing a luciferase gene having a CRE (cyclic AMP response element) 5 ′ upstream.
  • the CRE is a nucleotide sequence that is commonly present in the transcriptional regulatory region of a group of genes (cyclic AMP-inducible genes) whose expression increases when the concentration of intracellular cyclic AMP increases.
  • the transfection was carried out using a commercially available transfection kit (Fu GENE 6 Transfectionreagent; Boehringer Mannheim) according to the attached instructions. After said transformer Fuekushon After incubation day at 3 7 ° C, poly one L- lysine to co one Bok was 9 6 Uerupure Bok, were seeded cells to approximately 2 XI 0 4 cells Noueru. After further cultivation, 0.1% BSA, 0.1 mmo I / L—3-isobutyl-1- 1-methylxanthine (IBMX), 1 moi ZL fuirin ruskolin (forsko I i ⁇ ; FS) ), and 0.
  • DM EM medium containing LTB 4 1 mol / L leuco Bok Rie> B 4 (DM EM medium containing LTB 4) (hereinafter, in referred to as the incubation medium), the medium was exchanged such that 1 0 0 mu 1_ Noueru Incubation was performed at 37 ° C for 4 hours.
  • the FSK is an inducer that increases the expression level of a gene (ie, luciferase) located downstream of the CRE.
  • each of the cells was mixed with a luminescent substrate solution (LucLite; Packard) containing luciferin and ATP and a DMEM medium (excluding phenol red):
  • a luminescent substrate solution (LucLite; Packard) containing luciferin and ATP
  • a DMEM medium excluding phenol red
  • the negative controls i.e., the case of 4 absence FSK absence and LTB
  • the positive control ie, in the presence of FSK and in the absence of LTB 4
  • Fig. 8 shows the results.
  • the unit of the measurement value “RLU” in FIG. 8 is a relative luminescence unit (RelatativeLightUnits).
  • the novel protein of the present invention can be efficiently screened. According to the screening, for example, the development of powerful new antagonists are expected than known mouth I co Bok Lien B 4 receptor antagonist to target human Bok-Roikotoryen B 4 first receptor. Sequence listing free text
  • nucleotide sequence described in SEQ ID NO: 3 or 4 in the sequence listing is a sense primer
  • nucleotide sequence described in SEQ ID NO: 5 in the sequence listing is an antisense primer

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Abstract

L'invention concerne des nouvelles protéines; des nouveaux gènes codant pour ces protéines ainsi que des plasmides ou des transformants contenant ces gènes. L'invention concerne également un procédé permettant de fabriquer ces protéines, des cellules contenant ces protéines ou des fractions de membrane cellulaire de celles-ci. De plus, l'invention concerne, d'une part, un procédé et une trousse permettant de cribler des agonistes ou des antagonistes de ces nouvelles protéines, et, d'autre part, des agonistes ou antagonistes de ces protéines. Elle concerne également un procédé permettant de traiter ou de prévenir les maladies causées par l'insuffisance ou l'excès de leucotriène B4, d'acides hydroxyeicosatétranoïques ou d'acides hydroxypéroxyeicosatétranoïques; l'utilisation de ces agonistes ou antagonistes; et des anticorps contre ces nouvelles protéines ou des fragments de celles-ci. Les nouvelles protéines décrites dans la présente invention comprennent des nouveaux récepteurs du leucotriène B4, des acides hydroxyeicosatétranoïques et des acides hydroxypéroxyeicosatétranoïques chez l'homme. Ces nouvelles protéines présentent également des modifications fonctionnelles équivalentes.
PCT/JP2000/006200 1999-09-10 2000-09-11 Nouveaux recepteurs et genes codant pour ces memes recepteurs WO2001019985A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001040802A2 (fr) * 1999-12-02 2001-06-07 Glaxo Group Limited Nouvelle proteine
WO2001068844A2 (fr) * 2000-03-17 2001-09-20 Bayer Aktiengesellschaft Regulation du recepteur humain du leucotriene de type b4, couple a la proteine g
WO2001070815A1 (fr) * 2000-03-21 2001-09-27 Yamanouchi Pharmaceutical Co., Ltd. Nouveau recepteur du leucotriene b4
EP1270000A3 (fr) * 2001-06-28 2003-02-12 Pfizer Products Inc. Benzopyrannes substitués par l'acide benzoique pour le traitement de l'athérosclérose
WO2006137435A1 (fr) * 2005-06-22 2006-12-28 National University Corporation Gunma University Agoniste du recepteur g2a couple a la proteine g et methode de criblage du regulateur de l'activite g2a

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006312635A (ja) * 2005-04-08 2006-11-16 Hinode Sangyo Kk 鼻腔壁塗布組成物

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999042484A1 (fr) * 1998-02-20 1999-08-26 Smithkline Beecham Corporation Fishboy, un recepteur couple a la proteine g
WO2000022131A2 (fr) * 1998-10-13 2000-04-20 Arena Pharmaceuticals, Inc. Recepteurs non-endogenes de la proteine g humaine ayant une activite constitutive
WO2000029423A1 (fr) * 1998-11-12 2000-05-25 Merck & Co., Inc. Recepteur couple a la proteine g ressemblant au recepteur b4 de la leukotriene

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999042484A1 (fr) * 1998-02-20 1999-08-26 Smithkline Beecham Corporation Fishboy, un recepteur couple a la proteine g
WO2000022131A2 (fr) * 1998-10-13 2000-04-20 Arena Pharmaceuticals, Inc. Recepteurs non-endogenes de la proteine g humaine ayant une activite constitutive
WO2000029423A1 (fr) * 1998-11-12 2000-05-25 Merck & Co., Inc. Recepteur couple a la proteine g ressemblant au recepteur b4 de la leukotriene

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001040802A2 (fr) * 1999-12-02 2001-06-07 Glaxo Group Limited Nouvelle proteine
WO2001040802A3 (fr) * 1999-12-02 2002-02-21 Glaxo Group Ltd Nouvelle proteine
WO2001068844A2 (fr) * 2000-03-17 2001-09-20 Bayer Aktiengesellschaft Regulation du recepteur humain du leucotriene de type b4, couple a la proteine g
WO2001068844A3 (fr) * 2000-03-17 2002-01-24 Bayer Ag Regulation du recepteur humain du leucotriene de type b4, couple a la proteine g
WO2001070815A1 (fr) * 2000-03-21 2001-09-27 Yamanouchi Pharmaceutical Co., Ltd. Nouveau recepteur du leucotriene b4
EP1270000A3 (fr) * 2001-06-28 2003-02-12 Pfizer Products Inc. Benzopyrannes substitués par l'acide benzoique pour le traitement de l'athérosclérose
WO2006137435A1 (fr) * 2005-06-22 2006-12-28 National University Corporation Gunma University Agoniste du recepteur g2a couple a la proteine g et methode de criblage du regulateur de l'activite g2a

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