WO2004035755A2 - Transporters and ion channels - Google Patents

Transporters and ion channels Download PDF

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Publication number
WO2004035755A2
WO2004035755A2 PCT/US2003/033087 US0333087W WO2004035755A2 WO 2004035755 A2 WO2004035755 A2 WO 2004035755A2 US 0333087 W US0333087 W US 0333087W WO 2004035755 A2 WO2004035755 A2 WO 2004035755A2
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WIPO (PCT)
Prior art keywords
seq
polynucleotide
polypeptide
amino acid
sequence
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PCT/US2003/033087
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French (fr)
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WO2004035755A3 (en
Inventor
April J.A. Hafalia
Reena Khare
Preeti G. Lal
Henry Yue
Mariah R. Baughn
Michael B. Thornton
Dyung Aina M. Lu
Craig H. Ison
Shanya D. Becha
Li Ding
Bridget A. Warren
Soo Yeun Lee
Anita Swarnakar
Vicki S. Elliott
Thomas W. Richardson
Joseph P. Marquis
Jayalaxmi Ramkumar
Jagi Murage
Brigitte E. Raumann
Monique G. Yao
Yan Lu
Kimberly J. Gietzen
Yonghong G. Yang
Hsin-Ru Chang
Narinder K. Chawla
Uyen K. Tran
Sally Lee
Junming Yang
Ameena R. Gandhi
Catherine M. Tribouley
Jennifer L. Policky
Huijun Z. Ring
Ernestine A. Lee
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Incyte Corporation
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Priority to AU2003279980A priority Critical patent/AU2003279980A1/en
Publication of WO2004035755A2 publication Critical patent/WO2004035755A2/en
Publication of WO2004035755A3 publication Critical patent/WO2004035755A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • 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 invention relates to novel nucleic acids, transporters and ion channels encoded by these nucleic acids, and to the use of these nucleic acids and proteins in the diagnosis, treatment, and prevention of transport, neurological, muscle, immunological and cell proliferative disorders.
  • the invention also relates to the assessment of the effects of exogenous compounds on the expression of nucleic acids and transporters and ion channels.
  • Eukaryotic cells are surrounded and subdivided into functionally distinct organelles by hydrophobic lipid bilayer membranes which are highly impermeable to most polar molecules.
  • Cells and organelles require transport proteins to import and export essential nutrients and metal ions including K + , NH 4 + , P réelle SO 4 2" , sugars, and vitamins, as well as various metabolic waste products.
  • Transport proteins also play roles in antibiotic resistance, toxin secretion, ion balance, synaptic neurotransmission, kidney function, intestinal absorption, tumor growth, and other diverse cell functions (Griffith, J. and C. Sansom (1998) The Transporter Facts Book. Academic Press, San Diego CA, pp. 3-29).
  • Transport can occur by a passive concentration-dependent mechanism, or can be linked to an energy source such as ATP hydrolysis or an ion gradient.
  • Proteins that function in transport include carrier proteins, which bind to a specific solute and undergo a conformational change that translocates the bound solute across the membrane, and channel proteins, which form hydrophilic pores that allow specific solutes to diffuse through the membrane down an electrochemical solute gradient.
  • Carrier proteins which transport a single solute from one side of the membrane to the other are called uniporters.
  • coupled transporters link the transfer of one solute with simultaneous or sequential transfer of a second solute, either in the same direction (symport) or in the opposite direction (antiport).
  • intestinal and kidney epithelium contains a variety of symporter systems driven by the sodium gradient that exists across the plasma membrane. Sodium moves into the cell down its electrochemical gradient and brings the solute into the cell with it. The sodium gradient that provides the driving force for solute uptake is maintained by the ubiquitous Na7K + ATPase system.
  • Sodium-coupled transporters include the mammalian glucose transporter (SGLT1), iodide transporter (NIS), and multivitamin transporter (SMVT). All three transporters have twelve putative transmembrane segments, extracellular glycosylation sites, and cytoplasmically- oriented N- and C-termini. NIS plays a crucial role in the evaluation, diagnosis, and treatment of various thyroid pathologies because it is the molecular basis for radioiodide thyroid-imaging techniques and for specific targeting of radioisotopes to the thyroid gland (Levy, O. et al. (1997) Proc. Natl. Acad. Sci. USA 94:5568-5573).
  • SMVT is expressed in the intestinal mucosa, kidney, and placenta, and is implicated in the transport of the water-soluble vitamins, e.g., biotin and pantothenate (Prasad, P.D. et al. (1998) J. Biol. Chem. 273:7501-7506).
  • MFS major facilitator superfamily
  • MFS transporters are single polypeptide carriers that transport small solutes in response to ion gradients.
  • MFS transporters found in all classes of living organisms, and include transporters for sugars, oligosaccharides, phosphates, nitrates, nucleosides, monocarboxylates, and drugs.
  • MFS transporters found in eukaryotes all have a structure comprising 12 transmembrane segments (Pao, S.S. et al. (1998) Microbiol. Molec. Biol. Rev. 62: 1-34).
  • the largest family of MFS transporters is the sugar transporter family, which includes the seven glucose transporters (GLUT1-GLUT7) found in humans that are required for the transport of glucose and other hexose sugars. These glucose transport proteins have unique tissue distributions and physiological functions.
  • GLUT1 provides many cell types with their basal glucose requirements and transports glucose across epithelial and endothelial barrier tissues; GLUT2 facilitates glucose uptake or efflux from the liver; GLUT3 regulates glucose supply to neurons; GLUT4 is responsible for insulin- regulated glucose disposal; and GLUT5 regulates fructose uptake into skeletal muscle. Defects in glucose transporters are involved in a recently identified neurological syndrome causing infantile seizures and developmental delay, as well as glycogen storage disease, Fanconi-Bickel syndrome, and non-insulin-dependent diabetes mellitus (Mueckler, M. (1994) Eur. J. Biochem. 219:713-725; Longo, N. and L.J. Elsas (1998) Adv. Pediatr. 45:293-313).
  • Monocarboxylate anion transporters are proton-coupled symporters with a broad substrate specificity that includes L-lactate, pyruvate, and the ketone bodies acetate, acetoacetate, and beta-hydroxybutyrate. At least seven isoforms have been identified to date. The isoforms are predicted to have twelve transmembrane (TM) helical domains with a large intracellular loop between TM6 and TM7, and play a critical role in maintaining intracellular pH by removing the protons that are produced stoichiometrically with lactate during glycolysis.
  • TM transmembrane
  • H + -monocarboxylate transporter is that of the erythrocyte membrane, which transports L-lactate and a wide range of other aliphatic monocarboxylates.
  • Other cells possess H + -linked monocarboxylate transporters with differing substrate and inhibitor selectivities.
  • cardiac muscle and tumor cells have transporters that differ in their K m values for certain substrates, including stereoselectivity for L- over D-lactate, and in their sensitivity to inhibitors.
  • Organic anion transporters are selective for hydrophobic, charged molecules with electron-attracting side groups.
  • Organic cation transporters such as the ammonium transporter, mediate the secretion of a variety of drugs and endogenous metabolites, and contribute to the maintenance of intercellular pH (Poole, R.C. and A.P. Halestrap (1993) Am. J. Physiol. 264:C761-C782; Price, N.T. et al. (1998) Biochem. J. 329:321-328; and Martinelle, K. and I. Haggstrom (1993) J. Biotechnol. 30:339-350).
  • ATP-binding cassette (ABC) transporters are members of a superfamily of membrane proteins that transport substances ranging from small molecules such as ions, sugars, amino acids, peptides, and phospholipids, to lipopeptides, large proteins, and complex hydrophobic drugs.
  • ABC transporters consist of four modules: two nucleotide-binding domains (NBD), which hydrolyze ATP to supply the energy required for transport, and two membrane-spanning domains (MSD), each containing six putative transmembrane segments.
  • NBD nucleotide-binding domains
  • MSD membrane-spanning domains
  • These four modules may be encoded by a single gene, as is the case for the cystic fibrosis transmembrane regulator (CFTR), or by separate genes. When encoded by separate genes, each gene product contains a single NBD and MSD.
  • CFTR cystic fibrosis
  • ALDP adrenoleukodystrophy protein
  • PMP70 peroxisomal membrane protein-70, PMP70
  • SUR hyperinsulinemic hypoglycemia
  • MDR multidrug resistance
  • a number of metal ions such as iron, zinc, copper, cobalt, manganese, molybdenum, selenium, nickel, and chromium are important as cofactors for a number of enzymes.
  • copper is involved in hemoglobin synthesis, connective tissue metabolism, and bone development, by acting as a cofactor in oxidoreductases such as superoxide dismutase, ferroxidase (ceruloplasmin), and lysyl oxidase.
  • Copper and other metal ions must be provided in the diet, and are absorbed by transporters in the gastrointestinal tract. Plasma proteins transport the metal ions to the liver and other target organs, where specific transporters move the ions into cells and cellular organelles as needed. Imbalances in metal ion metabolism have been associated with a number of disease states (Danks, D.M. (1986) J. Med. Genet. 23:99-106).
  • Fatty acid transport protein an integral membrane protein with four transmembrane segments, is expressed in tissues exhibiting high levels of plasma membrane fatty acid flux, such as muscle, heart, and adipose. Expression of FATP is upregulated in 3T3-L1 cells during adipose conversion, and expression in COS7 fibroblasts elevates uptake of long-chain fatty acids (Hui, T.Y. et al. (1998) J. Biol. Chem. 273:27420-27429).
  • the lipocalin superfamily constitutes a phylogenetically conserved group of more than forty proteins that function as extracellular ligand-binding proteins which bind and transport small hydrophobic molecules. Members of this family function as carriers of retinoids, odorants, chromophores, pheromones, allergens, and sterols, and in a variety of processes including nutrient transport, cell growth regulation, immune response, and prostaglandin synthesis. A subset of these proteins may be multifunctional, serving as either a biosynthetic enzyme or as a specific enzyme inhibitor. (Tanaka, T. et al. (1997) J. Biol. Chem. 272:15789-15795; and van't Hof, W. et al. (1997) J. Biol. Chem. 272:1837-1841.)
  • Lipocalins Members of the lipocalin family display unusually low levels of overall sequence conservation. Pairwise sequence identity often falls below 20%. Sequence similarity between family members is limited to conserved cysteines which form disulfide bonds and three motifs which form a juxtaposed cluster that functions as a target cell recognition site.
  • the lipocalins share an eight stranded, anti- parallel beta-sheet which folds back on itself to form a continuously hydrogen-bonded beta-barrel.
  • the pocket formed by the barrel functions as an internal ligand binding site. Seven loops (LI to L7) form short beta-hairpins, except loop LI which is a large omega loop that forms a lid to partially close the internal ligand-binding site (Flower (1996) Biochem. J. 318: 1-14).
  • Lipocalins are important transport molecules. Each lipocalin associates with a particular ligand and delivers that ligand to appropriate target sites within the organism.
  • Retinol-binding protein (RBP), one of the best characterized lipocalins, transports retinol from stores within the liver to target tissues.
  • Apolipoprotein D (apo D), a component of high density lipoproteins (HDLs) and low density lipoproteins (LDLs), functions in the targeted collection and delivery of cholesterol throughout the body. Lipocalins are also involved in cell regulatory processes.
  • Apo D which is identical to gross- cystic-disease-fluid protein (GCDFP)-24, is a progesterone/pregnenolone-binding protein expressed at high levels in breast cyst fluid. Secretion of apo D in certain human breast cancer cell lines is accompanied by reduced cell proliferation and progression of cells to a more differentiated phenotype. Similarly, apo D and another lipocalin, ⁇ ,-acid glycoprotein (AGP), are involved in nerve cell regeneration. AGP is also involved in anti-inflammatory and immunosuppressive activities. AGP is one of the positive acute-phase proteins (APP); circulating levels of AGP increase in response to stress and inflammatory stimulation.
  • APP positive acute-phase proteins
  • AGP accumulates at sites of inflammation where it inhibits platelet and neutrophil activation and inhibits phagocytosis.
  • the immunomodulatory properties of AGP are due to glycosylation.
  • AGP is 40% carbohydrate, making it unusually acidic and soluble.
  • the glycosylation pattern of AGP changes during acute-phase response, and deglycosylated AGP has no immunosuppressive activity (Flower (1994) FEBS Lett. 354:7-11; Flower (1996) supra).
  • the lipocalin superfamily also includes several animal allergens, including the mouse major urinary protein (mMUP), the rat ⁇ -2-microgloobulin (rA2U), the bovine ⁇ -lactoglobulin ( ⁇ lg), the cockroach allergen (Bla g4), bovine dander allergen (Bos d2), and the major horse allergen, designated Equus caballus allergen 1 (Equ cl).
  • Equ cl is a powerful allergen responsible for about 80% of anti- horse IgE antibody response in patients who are chronically exposed to horse allergens. It appears that lipocalins may contain a common structure that is able to induce the IgE response (Gregoire, C. et al., (1996) J. Biol. Chem. 271 :32951-32959).
  • Lipocalins are used as diagnostic and prognostic markers in a variety of disease states.
  • the plasma level of AGP is monitored during pregnancy and in diagnosis and prognosis of conditions including cancer chemotherapy, renal disfunction, myocardial infarction, arthritis, and multiple sclerosis.
  • RBP is used clinically as a marker of tubular reabsorption in the kidney
  • apo D is a marker in gross cystic breast disease (Flower (1996) supra).
  • the use of lipocalin animal allergens may help in the diagnosis of allergic reactions to horses (Gregoire supra), pigs, cockroaches, mice and rats.
  • Mitochondrial carrier proteins are transmembrane-spanning proteins which transport ions and charged metabolites between the cytosol and the mitochondrial matrix. Examples include the ADP, ATP carrier protein; the 2-oxoglutarate/malate carrier; the phosphate carrier protein; the pyruvate carrier; the dicarboxylate carrier which transports malate, succinate, fumarate, and phosphate; the tricarboxylate carrier which transports citrate and malate; and the Grave's disease carrier protein, a protein recognized by IgG in patients with active Grave's disease, an autoimmune disorder resulting in hyperthyroidism.
  • Proteins in this family consist of three tandem repeats of an approximately 100 amino acid domain, each of which contains two transmembrane regions (Stryer, L. (1995) Biochemistry, W.H. Freeman and Company, New York NY, p. 551; PROSITE PDOC00189 Mitochondrial energy transfer proteins signature; Online Mendelian Inheritance in Man (OMIM) *275000 Graves Disease).
  • This class of transporters also includes the mitochondrial uncoupling proteins, which create proton leaks across the inner mitochondrial membrane, thus uncoupling oxidative phosphorylation from ATP synthesis. The result is energy dissipation in the form of heat.
  • Mitochondrial uncoupling proteins