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WO1998031797A1 - Zppar6, recepteur hormonal nucleaire sans queue (recepteur tlx) humain - Google Patents

Zppar6, recepteur hormonal nucleaire sans queue (recepteur tlx) humain Download PDF

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Publication number
WO1998031797A1
WO1998031797A1 PCT/US1998/000678 US9800678W WO9831797A1 WO 1998031797 A1 WO1998031797 A1 WO 1998031797A1 US 9800678 W US9800678 W US 9800678W WO 9831797 A1 WO9831797 A1 WO 9831797A1
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Prior art keywords
zppar6
human
nuclear hormone
amino acid
hormone receptor
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PCT/US1998/000678
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English (en)
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James L. Holloway
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Zymogenetics, Inc.
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Priority to AU62407/98A priority Critical patent/AU6240798A/en
Publication of WO1998031797A1 publication Critical patent/WO1998031797A1/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
    • C07K14/70567—Nuclear receptors, e.g. retinoic acid receptor [RAR], RXR, nuclear orphan receptors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Non-peptide hormones are involved in coordination of multiple events related to development, differentiation and physiological response to a wide range of stimuli. These hormones bind to intracellular nuclear hormone receptors that mediate the hormonal effect.
  • Nuclear hormone receptors have been identified as ligand-dependent transcription factors that initiate nuclear responses to steroids, retinoids, 1,25 dihydroxyvitamin D 3 and thyroid hormones. Peroxisome proliferator-activated receptors are also members of the NHR family. Based on shared characteristics, a superfamily of nuclear hormone receptors has been recognized. The superfamily includes structurally related proteins for which no ligand has been identified ("orphan nuclear hormone receptors") .
  • NHR superfamily share structural similarity. The majority exhibit three principal domains: (1) a variable, amino-terminal domain that often is related to transactivating activity; (2) a highly conserved DNA binding domain; ' and (3) a moderately conserved carboxy-terminal ligand-binding domain.
  • the DNA binding domain has two "zinc finger” motifs, and differences in the sequence of these motifs have been associated with differences in DNA binding or receptor dimerization or its absence. Further, the region immediately carboxy-terminal of the zinc fingers has been implicated in DNA recognition. This region contains two adjacent clusters of amino acids referred to as the "A- and T- boxes.” Differences in these regions correlate with the subdivision of the superfamily into four groups :
  • Nuclear receptors bind to DNA sequences, called “response elements", in the promoter region of target genes. These response elements have distinct sequence motifs, and the relative orientation and spacing of these sequence motifs are important for receptor binding specificity.
  • Drosophila gene "tailless" (TLL) Drosophila gene "tailless"
  • TLL is involved in establishing non- metameric domains at the termini of Drosophila embryos, and is required for differentiation of terminal structures such as brain, foregut , midgut and hindgut .
  • TLL is involved in development of the nervous system. Both cTLX and mTLX are involved in transcriptional control of undifferentiated neuroepithelial cells in anterior regions of the developing embryonic brain. Despite low homology in the ligand binding domain, cTLX can mimic the action of TLL in vivo, suggesting conservation between vertebrates and invertebrates within these early embryonic development pathways.
  • nuclear hormone receptors are likely to regulate such a large number of essential biological processes, identification of novel nuclear hormone receptors furthers an understanding of the complex transcriptional networks in which such receptors function. Identification and characterization of new receptors permits further dissection of the activities of related receptors .
  • a novel nuclear hormone receptor provides a means to find the natural ligand for that receptor. By identifying the corresponding ligand, the physiological role of the receptor can be determined.
  • the demonstrated in vivo activities of these nuclear hormone receptors illustrate the enormous clinical potential of, and need for, other nuclear hormone receptors, nuclear hormone receptor ligands and receptor agonists and antagonists.
  • the present invention addresses this need by providing a novel nuclear hormone receptor and related compositions and methods .
  • the present invention provides a novel human nuclear hormone receptor polypeptide and related compositions and methods
  • an isolated polynucleotide molecule encoding a human ZPPAR6 nuclear hormone receptor polypeptide comprising an amino acid sequence as shown in SEQ ID NO : 2 from amino acid residue 1 (Ala) to amino acid residue 139 (lie) .
  • an isolated polynucleotide molecule which encodes a full-length human ZPPAR6 nuclear hormone receptor polypeptide of about 380-390 amino acid residues.
  • an expression vector comprising a transcription promoter; a DNA segment encoding a human ZPPAR6 nuclear hormone receptor polypeptide comprising an amino acid sequence as shown in SEQ ID NO : 2 from amino acid residue 1 (Ala) to amino acid residue 139 (lie) ; and a transcription terminator.
  • a cultured cell into which has been introduced the expression vector which expresses the human ZPPAR6 nuclear hormone receptor polypeptide encoded by the DNA segment.
  • SEQ ID NO: 2 from amino acid residue 1 (Ala) to amino acid residue 139 (lie) .
  • the isolated human ZPPAR6 nuclear hormone receptor polypeptide has from about 380 to 390 amino acid residues.
  • a method for producing a human ZPPAR6 nuclear hormone receptor polypeptide comprising the steps of culturing a cell into which has been introduced an expression vector, whereby the cell expresses a human ZPPAR6 nuclear receptor polypeptide encoded by the DNA segment; and recovering the human ZPPAR6 nuclear hormone receptor polypeptide.
  • the invention further provides isolated antibodies and binding proteins that binds to an epitope of a human ZPPAR6 nuclear hormone receptor polypeptide as disclosed above.
  • the invention also provides a method for identifying a compound that modulates human ZPPAR6 nuclear hormone receptor-mediated transcription of a target gene in a cell, comprising the steps of incubating a test compound with eukaryotic cells that express a human ZPPAR6 nuclear hormone receptor polypeptide; and measuring the human ZPPAR6 -mediated transcription of the target gene in the presence and in the absence of the test compound, or measuring the effect of the test compound on target gene transcription in human ZPPAR6-overexpressing (+) cells and in human ZPPAR6 -deficient (-) cells, whereby a difference in target gene transcription in- the presence and absence of test compound, or in human ZPPAR6-overexpressing (+) cells and in human ZPPAR6 -deficient (-) cells, indicates a test compound that modulates human ZPPAR6 -mediated transcription of the target gene.
  • the invention further provides
  • Allelic variant Any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (i.e., no change in the encoded polypeptide) , or may encode polypeptides having altered amino acid sequence.
  • allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene.
  • Amino or N-terminal and carboxyl or C-terminal Used herein to denote positions within polypeptides. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain sequence positioned carboxyl-terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide.
  • Degenerate nucleotide sequence Denotes a sequence of nucleotides that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that encodes a polypeptide) .
  • Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (i.e., GAU and GAC triplets each encode Asp) .
  • Expression vector A DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription. Such additional segments may include promoter and terminator sequences, and optionally one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both.
  • Isolated When applied to a protein, the term “isolated” indicates that the protein is found in a condition other than its native environment, such as apart from blood and animal tissue. In a preferred form, the isolated protein is substantially free of other proteins, particularly other proteins of animal origin. It is preferred to provide the proteins in a highly purified form, i.e., greater than 95% pure, more preferably greater than 99% pure.
  • the term “isolated” indicates that the molecule is removed from its natural genetic milieu, and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems.
  • isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones.
  • Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, and may include naturally occurring 5 ' and 3 ' untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316:774-78, 1985) .
  • Nuclear hormone receptor denotes a ligand-dependent transcription factor that initiates nuclear responses to non-peptide hormones, such as steroids, retinoids, 1,25 dihydroxyvitamin D3 and thyroid hormones.
  • operably linked indicates that the segments are arranged so that they function in concert for their intended purposes, e.g., transcription initiates in the promoter and proceeds through the coding segment to the terminator.
  • Orphan nuclear hormone receptor Denotes a receptor for which a corresponding ligand has not been identified.
  • Ortholo (or "species homolocr") Denotes a polypeptide or protein obtained from one species that has homology to an analogous polypeptide or protein from a different species.
  • Polynucleotide Denotes a single- or double- stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5 1 to the 3' end.
  • Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vi tro, or prepared from a combination of natural and synthetic molecules.
  • Polypeptide A polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as "peptides" .
  • Receptor A cell-associated protein, or a polypeptide subunit of such protein, that binds to a bioactive molecule (the "ligand") and mediates the effect of the ligand on the cell. Binding of ligand to receptor results in a change in the receptor (and, in some cases, receptor multimerization, i.e., association of identical or different receptor subunits) that causes interactions between the effector domain (s) of the receptor and other molecule (s) in the cell. These interactions in turn lead to alterations in the metabolism of the cell.
  • ligand a bioactive molecule
  • Metabolic events that are linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, cell proliferation, increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids .
  • ZPPAR6 has the characteristics of a nuclear hormone receptor, as discussed in more detail below. All references cited herein are incorporated by reference in their entirety.
  • the present invention is based in part upon the discovery of a novel polynucleotide sequence (SEQ. ID. NO. 1) and corresponding polypeptide sequence (SEQ. ID. NO. 2) that have homology to the nuclear hormone receptor superfamily.
  • the polynucleotide sequence of the invention has a high degree of sequence identity with mouse (mTLL or mTLX) and chicken (cTlx) orthologs of the Drosophila gene "tailless" (Yu et al . , ibid. ;
  • the receptor the human ortholog of cTLX and mTLX, has been designated as ZPPAR6 receptor, also referred to herein as hZPPAR6, or ZPPAR6.
  • Novel ZPPAR6 receptor-encoding polynucleotides and polypeptides of the present invention were initially identified by querying an EST database for sequences homologous to individual members of the nuclear hormone receptor superfamily, as well as to conserved motifs within the family.
  • An EST from a human brain library was discovered and was determined to be a novel human protein having homology to cTLX and mTLX. From this information, a novel 419 bp human cDNA fragment (SEQ. ID. NO. 1) was derived from a clone containing the EST. This sequence contained the EST sequence and extended the sequence by more than 40 base pairs in the 5' and more than 15 base pairs in the 3' directions.
  • human ZPPAR6 ligand binding domain likely has an additional 60-70 amino acid residues in the N terminal region of the ligand binding domain, an 80-90 amino acid residue DNA binding domain, a 70-80 amino acid residue region joining these two domains and a 15-20 amino acid residue amino terminal sequence.
  • domain boundaries are approximate and are based on alignments with known proteins and predictions of protein folding.
  • the remainder of the ligand binding domain and the N-terminal portion of the receptor containing the amino terminal and DNA binding domains are derived by any one of numerous methods that are available to those skilled in the art.
  • An exemplary method is RACE, rapid amplification of cDNA ends (Marathon TM cDNA Amplification Kit, Clontech, Palo Alto, CA) , as discussed in the Example section below. Briefly, this long distance PCR method allows for generation of full length cDNA. 5' and 3' oligonucleotide probes are designed based on the sequence as represented in SEQ. ID. NO. 1. Using adaptor ligated cDNA as a template, both 3' and 5' RACE PCR reactions are performed to generate overlapping products that span the complete transcript. Based on comparisons with other members of the nuclear hormone receptor family, the human
  • ZPPAR6 receptor is a polypeptide of about 380-390 amino acid residues encoded by a polynucleotide of about 1,600 nucleotides.
  • SEQ ID NO : 1 shares 71% nucleotide identity with a Xenopus laevis orphan nuclear hormone receptor XTLL (XKU67886, Genbank Accession No. U67886) , 81% nucleotide identity with chicken TLX, 86% nucleotide identity with mouse Mtll and 94% nucleotide identity with a human tailless gene homologue (HSTAILLE, Genbank Accession No. Y13276) .
  • Chicken TLX and mouse TLX share 99.5% identity within the ligand binding domain, but only have 41% identity with the Drosophila TLL ligand binding domain.
  • Mouse TLX shares 42% identity within the ligand binding domain with human COUP-TF I and II, another brain expressed nuclear hormone receptor, and these receptors share regions of conserved amino acids independent from TLL.
  • Nuclear hormone receptors tend to be expressed in characteristic tissue-specific patterns that correlate with their primary physiological effects.
  • Northern blot analysis of various human tissue using a 27 bp probe to the 3' end of the published sequence for EST188758 revealed a 4.4 kb band in heart and liver; a 2.5 kb band in heart and pancreas; a 2.0 kb band in heart, skeletal muscle, brain and liver, and also in fetal lung, kidney, brain and liver.
  • a 1.5 kb band was also seen in small intestine and a 1.0 kb band in pancreas. Yu et al . ,
  • ZPPAR6 could play a role similar to that proposed for mTLX, cTLX or TLL, but it is likely that ZPPAR6 is also involved in tissue formation. If ZPPAR6 acts on primitive cells in early development and those cells remain in adult tissues, ZPPAR6 ligands would serve as therapeutics for tissue regeneration. The high degree of homology in the ligand binding domains of ZPPAR6, cTLX and mTLX suggests that they may be bound by the same or similar ligands. Differences in tissue distribution suggest that binding by a same or similar ligand could result in distinct and independent biological functions. As of this time, no ligand has been identified for any of Drosophila TLL, chicken TLX or mouse TLX receptors.
  • the present invention also provides polynucleotide molecules, including DNA and RNA molecules, that encode the ZPPAR6 polypeptides disclosed above.
  • SEQ ID NO : 6 is a degenerate DNA sequence that encompasses all DNAs that encode the ZPPAR6 polypeptide of
  • ZPPAR6 polypeptide-encoding polynucleotides comprising nucleotide 1 to nucleotide ??? of SEQ ID NO: 6 and their RNA equivalents are contemplated by the present invention.
  • Table 1 sets forth the one-letter codes used within SEQ ID NO: 6 to denote degenerate nucleotide positions. "Resolutions” are the nucleotides denoted by a code letter. "Complement” indicates the code for the complementary nucleotide (s) .
  • the code Y denotes either C or T
  • its complement R denotes A or G, A being complementary to T, and G being complementary to C.
  • degenerate codons used in SEQ ID NO : 6 encompassing all possible codons for a given amino acid, are set forth in Table 2.
  • any X NNN One of ordinary skill in the art will appreciate that some ambiguity is introduced in determining a degenerate codon, representative of all possible codons encoding each amino acid.
  • the degenerate codon for serine can, in some circumstances, encode arginine (AGR)
  • the degenerate codon for arginine (MGN) can, in some circumstances, encode serine (AGY) .
  • some polynucleotides encompassed by the degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequence of SEQ ID NO : 2. Variant sequences can be readily tested for functionality as described herein.
  • preferential codon usage or “preferential codons” is a term of art referring to protein translation codons that are most frequently used in cells of a certain species, thus favoring one or a few representatives of the possible codons encoding each amino acid (See Table 2) .
  • the amino acid Threonine (Thr) may be encoded by ACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonly used codon; in other species, for example, insect cells, yeast, viruses or bacteria, different Thr codons may be preferential.
  • Preferential codons for a particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art.
  • the degenerate codon sequence disclosed in SEQ ID NO : 6 serves as a template for optimizing expression of polynucleotides in various cell types and species commonly used in the art and disclosed herein. Sequences containing preferential codons can be tested and optimized for expression in various species, and tested for functionality as disclosed herein.
  • the isolated polynucleotides will hybridize to similar sized regions of SEQ ID NO:l, or a sequence complementary thereto, under stringent conditions.
  • stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • Typical stringent conditions are those in which the salt concentration is up to about 0.03 M at pH 7 and the temperature is at least about 60°C.
  • a human ZPPAR6 nuclear hormone receptor comprising the sequence disclosed in SEQ. ID. NO : 1 represents one allele of the human ZPPAR6 gene and that allelic variation and alternative splicing are expected to occur. Allelic variants of these sequences can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures. Allelic variants of the DNA sequence shown in SEQ ID NO : 1 including those containing silent mutations and those in which mutations result in amino acid sequence changes, are within the scope of the present invention, as are proteins which are allelic variants of SEQ ID NO : 2.
  • Splice variants of a human ZPPAR6 nuclear hormone receptor comprising the DNA sequence shown in SEQ ID NO:l include DNA sequences that result from mature RNA molecules created by known eukaryotic RNA splicing processes wherein intron sequence is removed and exon sequence is joined. Such DNA sequences encoding ZPPAR6 nuclear hormone receptor proteins which retain properties of the human ZPPAR6 protein comprising the sequence of SEQ ID NO: 2 are within the scope of the present invention. Allelic variants and splice variants of these sequences can be cloned by probing cDNA or genomic libraries from different individuals or tissues according to standard procedures known in the art . The present invention further provides counterpart receptors and polynucleotides from other species ("species orthologs").
  • ZPPAR6 receptors from other mammalian species, including porcine, ovine, bovine, canine, feline, equine, and other primate receptors .
  • a chicken and mouse ortholog of ZPPAR6 are known (Yu et al . ibid. ; Monaghan et al . , ibid. ) .
  • Species orthologs of the ZPPAR6 receptor can be cloned using information and compositions provided by the present invention in combination with conventional cloning techniques.
  • a cDNA can be cloned using mRNA obtained from a tissue or cell type that expresses the receptor. Suitable sources of mRNA can be identified by probing Northern blots with probes designed from the sequences disclosed herein.
  • a library is then prepared from mRNA of a positive tissue or cell line.
  • a receptor- encoding cDNA can then be isolated by a variety of methods, such as by probing with a complete or partial human cDNA or with one or more sets of degenerate probes based on the disclosed sequence.
  • a cDNA can also be cloned using the polymerase chain reaction (PCR) (Mullis, U.S. Patent No. 4,683,202), using primers designed from the sequences disclosed herein.
  • the cDNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to the receptor. Similar techniques can also be applied to the isolation of genomic clones.
  • polypeptides can be produced by engineering amino acid changes into the representative human polypeptide sequence shown in SEQ ID NO: 2 or an allelic variant or species ortholog thereof . It is preferred that these engineered variant polypeptides are at least 80% identical to the polypeptide of SEQ ID NO : 2. Such polypeptides will more preferably be at least 90% identical, and most preferably 95% or more identical to SEQ ID NO: 2.
  • isolated is meant a protein or polypeptide that is found in a condition other than its native environment, such as apart from blood and animal tissue. In a preferred form, the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin.
  • polypeptides in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure.
  • Percent sequence identity is determined by conventional methods. See, for example, Altschul et al . , Bull. Math. Bio. 48 : 603-616, 1986 and Henikoff and Henikoff, Proc . Natl. Acad. Sci. USA £9:10915-10919, 1992. Briefly, two amino acid sequences • are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff (ibid. ) as shown in Table 3 (amino acids are indicated by the standard one-letter codes). The percent identity is then calculated as:
  • Sequence identity of polynucleotide molecules is determined by similar methods using a ratio as disclosed above .
  • Engineered proteins and polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see Table 4) and other substitutions that do not significantly affect the folding or activity of the protein or polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or a small extension that facilitates purification (an affinity tag) , such as a poly-histidine tract, Glu-Glu tag (Grussenmeyer et al . , Proc . Natl. Acad. Sci .
  • an affinity tag such as a poly-histidine tract, Glu-Glu tag (Grussenmeyer et al . , Proc . Natl. Acad. Sci .
  • Aromatic phenylalanine tryptophan tyrosine
  • the proteins of the present invention can also comprise non-naturally occurring amino acid residues.
  • Non-naturally occurring amino acids include, without limitation, trans-3 -methylproline, 2 , -methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N- methylglycine, allo-threonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, tert- leucine, norvaline, 2-azaphenylalanine, 3- azaphenylalanine, 4-azaphenylalanine, and 4- fluorophenylalanine .
  • coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4- azaphenylalanine, or 4-fluorophenylalanine) .
  • the non-naturally occurring amino acid is incorporated into the protein in place of its natural counterpart. See, Koide et al . , Biochem. 3_3:7470-6, 1994.
  • Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2 : 395-403, 1993) .
  • Non-conservative amino acids amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for ZPRR6 amino acid residues.
  • "Unnatural amino acids” have been modified after protein synthesis, and/or have a chemical structure in their side chain (s) different from that of the standard amino acids.
  • Unnatural amino acids can be chemically synthesized, or preferably, are commercially available, and include pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, and 3,3- dimethylproline .
  • Essential amino acids in the receptor polypeptides of the present invention can be identified according to procedures known in the art, such as site- directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 24 : 1081-85, 1989; Bass et al., Proc. Natl. Acad. Sci. USA £8:4498-502, 1991).
  • site- directed mutagenesis or alanine-scanning mutagenesis
  • Single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity (e.g., ligand binding and signal transduction) to identify amino acid residues that are critical to the activity of the molecule.
  • Sites of ligand-receptor interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity; in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., Science 2J5J5: 306-12 , 1992; Smith et al . , J. Mol . Biol. 224 :899-904, 1992; Wlodaver et al . , FEBS Lett. 309:59-64, 1992.
  • the identities of essential amino acids can also be inferred from analysis of homologies with related receptors .
  • Mutagenesis methods as disclosed above can be combined with high-throughput screening methods to detect activity of cloned, mutagenized receptors in host cells.
  • Mutagenized DNA molecules that encode active receptors or portions thereof e.g., ligand-binding fragments
  • These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.
  • Variants of the disclosed ZPPAR6 DNA and polypeptide sequences can be generated through DNA shuffling as disclosed by Stemmer, Nature £70 . : 389-91, 1994 and Stemmer, Proc . Natl . Acad. Sci. USA £1:10747-51, 1994.
  • variant genes are generated by in vi tro homologous recombination by random fragmentation of a parent gene followed by reassembly using PCR, resulting in randomly introduced point mutations.
  • This technique can be modified by using a family of parent genes, such as allelic variants or genes from different species, to introduce additional variability into the process. Selection or screening for the desired activity, followed by additional iterations of mutagenesis and assay provides for rapid "evolution" of sequences by selecting for desirable mutations while simultaneously selecting against detrimental changes .
  • Mutagenesis methods as disclosed above can be combined with high-throughput , automated screening methods to detect activity of cloned, mutagenized polypeptides in host cells.
  • Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using modern equipment. These methods allow the rapid determination of the importance of individual amino acid residues • in a polypeptide of interest, and can be applied to polypeptides of unknown structure .
  • any ZPPAR6 polypeptide including variants and fusion proteins
  • one of ordinary skill in the art can readily generate a fully degenerate polynucleotide sequence encoding that variant using the information set forth in Tables 1 and 2 above .
  • the receptor polypeptides of the present invention can be produced in genetically engineered host cells according to conventional techniques.
  • Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryotic cells, particularly cultured cells of multicellular organisms, are preferred.
  • ZPPAR6 polypeptide is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator, within an expression vector.
  • the vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers. Cultured mammalian cells are preferred hosts within the present invention.
  • Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al . , Cell ⁇ :725, 1978; Corsaro and Pearson, Somatic Cell Genetics 1:603, 1981; Graham and Van der Eb, Virology £2:456, 1973), electroporation (Neumann et al . , EMBO J . 1:841-45, 1982), DEAE-dextran mediated transfection (Ausubel et al . , eds . , Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987), and liposome-mediated transfection (Hawley-Nelson et al .
  • Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL 1650) , COS-7 (ATCC No. CRL 1651), BHK (ATCC No.
  • CRL 1632 BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al . , J. Gen. Virol . £6:59-72, 1977) and Chinese hamster ovary (e.g., CH0-K1; ATCC No. CCL 61) cell lines. Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Rockville, Maryland. In general, strong transcription promoters are preferred, such as promoters from SV-40 or cytomegalovirus . See, e.g., U.S. Patent No. 4,956,288. Other suitable promoters include those from metallothionein genes (U.S. Patent Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.
  • Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as “transfectants” . Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as “stable transfectants.”
  • a preferred selectable marker is a gene encoding resistance to the antibiotic neomycin.
  • Selection is carried out in the presence of a neomycin- type drug, such as G-418 or the like. Selection systems may also be used to increase the expression level of the gene of interest, a process referred to as "amplification" .
  • Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes.
  • a preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate .
  • Other drug resistance genes e.g., hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
  • drug resistance genes e.g., hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
  • eukaryotic cells can also be used as hosts, including insect cells, plant cells and avian cells. Transformation of insect cells and production of foreign polypeptides therein is disclosed by Guarino et al., U.S. Patent No. 5,162,222; Bang et al . , U.S. Patent No. 4,775,624; and WIPO publication WO 94/06463.
  • the use of Agroba cteri um rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al . , J. Biosci. (Bangalore) £1:47-58, 1987.
  • Fungal cells including yeast cells, and particularly cells of the genus Saccharomyces , can also be used within the present invention, such as for producing receptor fragments or polypeptide fusions.
  • Methods for transforming yeast cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,311; Kawasaki et al., U.S. Patent No. 4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et al . , U.S. Patent No. 5,037,743; and Murray et al . , U.S. Patent No. -4,845,075.
  • Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine) .
  • a preferred vector system for use in yeast is the P0T1 vector system disclosed by Kawasaki et al . (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media.
  • Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Patent No. 4,599,311; Kingsman et al . , U.S. Patent No. 4,615,974; and Bitter, U.S. Patent No.
  • Transformation systems for other yeasts including Hansenula polymorpha , Schi zosaccharomyces pombe , Kluyveromyces lactis , Kluyveromyces fragilis , Ustilago maydis, Pichia pastoris , Pichia methanolica , Pichia guillermondii and Candida mal tosa are known in the art. See, for example, Gleeson et al . , J. Gen. Microbiol .
  • Prokaryotic host cells including strains of the bacteria Escherichia coli, Bacillus and other genera are also useful host cells within the present invention. Techniques for transforming these hosts and expressing foreign DNA sequences cloned therein are well known in the art (see, e.g., Sambrook et al . , ibid. ) .
  • the polypeptide When expressing a ZPPAR6 polypeptide in bacteria such as E. coli, the polypeptide may be retained in the cytoplasm, typically as insoluble granules, or may be directed to the periplasmic space by a bacterial secretion sequence.
  • the cells are lysed, and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea.
  • the denatured polypeptide can then be refolded and dimerized by diluting the denaturant , such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution.
  • the polypeptide can be recovered from the periplasmic space in a soluble and functional form by disrupting the cells (by, for example, sonication or osmotic shock) to release the contents of the periplasmic space and recovering the protein, thereby obviating the need for denaturation and refolding.
  • Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells.
  • suitable media including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media may also contain such components as growth factors or serum, as required.
  • the growth medium will generally select for cells containing the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or co- transfected into the host cell.
  • Chimeric receptors can be created, expressed by cultured host cells and used in an assays to screen for the natural ligand, as well as agonists and antagonists of the natural ligand. Briefly, the DNA binding domain of human ZPPAR6 is exchanged with the DNA binding domain of a known human nuclear hormone receptor. Such known nuclear hormone receptors as the estrogen and the glucocorticoid receptors have been used successfully to create chimeric receptors for ligand determination (Giguere et al . , Nature ££0:624-29, 1987; Green and Chambon, Nature 325 : 75-8, 1987; Petkovich et al .
  • the chimeric receptor is then transfected into a nuclear hormone receptor-deficient host cell line which has been engineered to express a reporter gene.
  • Suitable reporter genes include the bacterial chloramphenicol acetyl transferase (CAT) or a luciferase gene (de Wet et al . , Mol. Cell. Biol. £:725, 1987).
  • the reporter gene is linked to a hormone response element (native or synthetic) that is responsive to the DNA binding domain used in the chimeric receptor.
  • Ligand test samples are added to the chimeric receptor and if at least one ligand binds to the ligand binding domain of the chimeric receptor the assay detects activation of transcription of the reporter gene. For example, expression of the luciferase gene is detected by luminescence using methods known in the art (e.g., Baumgartner et al . , J. Biol. Chem. 269:29094-101, 1994; Schenborn and Goiffin, Promega Notes 41:11, 1993) . Luciferase activity assay kits are commercially available from, for example, Promega Corp., Madison, WI . Chimeric receptor containing cell lines of this type can be used to screen libraries of chemicals, cell-conditioned culture media, fungal broths, soil samples, water samples, and the like.
  • Expressed recombinant ZPPAR6 polypeptides can be purified using conventional purification methods, such as ammonium sulfate precipitation, acid or chaotrope extraction, and affinity chromatography, including ion exchange, hydrophobic interaction, hydroxyapatite, size exclusion, Q-Fast Flow Sepharose, MonoQ resin, phenyl Sepharose, Mono
  • Protein refolding (and optionally reoxidation) procedures may be advantageously used. It is preferred to purify the protein to >80% purity, more preferably to >90% purity, even more preferably >95%, and particularly preferred is a pharmaceutically pure state, that is greater than 99.9% pure with respect to contaminating macromolecules , particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents. Preferably, a purified protein is substantially free of other proteins, particularly other proteins of animal origin.
  • Human ZPPAR6 polypeptides or fragments thereof may also be prepared through chemical synthesis.
  • Human ZPPAR6 polypeptides may be monomers or multimers; glycosylated or non-glycosylated; pegylated or non- pegylated; and may or may not include an initial methionine amino acid residue.
  • ZPPAR6 polypeptide agonists and antagonists would be useful as therapeutic agents for modulating transcription of target genes.
  • the high degree of homology between ZPPAR6 , mTLL and cTlx suggests that they may share similar functions.
  • Agonists for such receptors could be used to influence transcription, cellular differentiation, and/or proliferation during development, in particular during embryogenesis .
  • Northern blot analysis revealed ZPPAR6 transcripts in pancreas which may indicate that one function of the receptor is to regulate pancreatic specific transcription of target genes.
  • Such genes could be involved in diabetes; for example in islet cell differentiation or in modulation of insulin expression.
  • Antagonists can be used to out-compete endogenous human ZPPAR6 ligand and exert control over the receptor.
  • Antagonists are also useful as research reagents for characterizing sites of ligand receptor interaction. Agonists and antagonists may also prove useful in the study of modulation of biological processes .
  • the receptor or receptor polypeptides may also act as antagonists to other nuclear hormone receptors, mediating transcriptional activity through repression of gene expression by competing with other transcriptional activators for binding sites (Luo et al . , Mol . Endocrinol . 9:1233-239,
  • transcription factors within a cell compete for transcriptional co-activators.
  • a nuclear receptor/transcription factor binds to its appropriate recognition sequence, and then interacts with proteins (“co-activators") that enable up-regulation of target gene expression.
  • Co-activators proteins that enable up-regulation of target gene expression.
  • Co-repressors that interact with nuclear receptors (and down-regulate gene expression) are also known. Co-activators are expressed at very low, limiting levels within a cell, thus transcription factors must compete for the limited amount of co-activators present.
  • a ZPPAR ⁇ -ligand combination may significantly out-compete other transcription factor/ligand combinations for the limited co-activator (s) .
  • the ZPPAR ⁇ -ligand combination would essentially "turn off" target genes regulated by the non-ZPPAR6-ligand combinations.
  • Human ZPPAR6 polypeptides may also be used within diagnostic systems for detection of circulating levels of ligand.
  • Antibodies or other agents that specifically bind to ZPPAR6 may be used to detect the presence of receptor in tissue samples. Detection methods could be used as diagnostic tools to monitor and quantify receptor or ligand levels. Elevated or depressed levels of ligand or receptor may be indicative of pathological conditions, including cancers.
  • an N- or C- terminal extension such as a poly-histidine tag, substance P, or a FlagTM peptide (Hopp et al., Bio/Technology _6: 1204-10, 1988; available from Eastman Kodak Co., New Haven, CT) , or another polypeptide or protein for which an antibody or other specific binding agent is available (such as maltose binding protein or immunoglobulin F c fragment) , can be fused to the receptor polypeptide.
  • Ligand-binding receptor polypeptide can be used for purification of ligand.
  • the receptor polypeptide is immobilized on a solid support, such as beads of agarose, cross-linked agarose, glass, cellulosic resins, silica- based resins, polystyrene, cross-linked polyacrylamide, or like materials that are stable under the conditions of use.
  • a solid support such as beads of agarose, cross-linked agarose, glass, cellulosic resins, silica- based resins, polystyrene, cross-linked polyacrylamide, or like materials that are stable under the conditions of use.
  • Methods for linking polypeptides to solid supports are known in the art, and include amine chemistry, cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, and hydrazide activation.
  • the resulting media will generally be configured in the form of a column, and fluids containing ligand are passed through the column one or more times to allow ligand to bind to the receptor polypeptide.
  • the ligand is then eluted using changes in salt concentration, chaotropic agents (MnCl2), or pH to disrupt ligand receptor binding.
  • An assay system that uses a ligand-binding receptor (or an antibody, one member of a complement/anti- complement pair) or a binding fragment thereof, and a commercially available biosensor instrument (BIAcoreTM, Pharmacia Biosensor, Piscataway, NJ) also may be advantageously employed.
  • Such receptor, antibody, member of a complement/anti-complement pair or fragment is immobilized onto the surface of a receptor chip.
  • a receptor, antibody, member or fragment is covalently attached, using amine or sulfhydryl chemistry, to dextran fibers that are attached to gold film within the flow cell .
  • a test sample is passed through the cell. If a ligand, epitope, or opposite member of the complement/anti-complement pair is present in the sample, it will bind to the immobilized receptor, antibody or member, respectively, causing a change in the refractive index of the medium, which is detected as a change in surface plasmon resonance of the gold film.
  • Ligand-binding receptor polypeptides can also be used within other assay systems known in the art. Such systems include Scatchard analysis for determination of binding affinity (see, Scatchard, Ann. NY Acad. Sci. 51 : 660-72, 1949) and calorimetric assays (Cunningham et al.,
  • viruses for this purpose include adenovirus, herpesvirus, vaccinia virus and adeno- associated virus (AAV) .
  • Adenovirus a double-stranded DNA virus, is currently the best studied gene transfer vector for delivery of heterologous nucleic acid (for a review, see T.C. Becker et al . , Meth. Cell Biol. 4£:161-89, 1994; and J.T. Douglas and D.T. Curiel, Science & Medicine 4.: 44- 53, 1997).
  • adenovirus can (i) accommodate relatively large DNA inserts; (ii) be grown to high-titer; (iii) infect a broad range of mammalian cell types; and (iv) be used with a large number of available vectors containing different promoters. Also, because adenoviruses are stable in the bloodstream, they can be administered by intravenous injection. Some disadvantages (especially for gene therapy) associated with adenovirus gene delivery include: (i) very low efficiency integration into the host genome; (ii) existence in primarily episomal form; and (iii) the host immune response to the administered virus, precluding readministration of the adenoviral vector.
  • inserts up to 7 kb of heterologous DNA can be accommodated. These inserts may be incorporated into the viral DNA by direct ligation or by homologous recombination with a co-transfected plasmid.
  • the essential El gene has been deleted from the viral vector, and the virus will not replicate unless the El gene is provided by the host cell (i.e., the human 293 cell line) .
  • the host cell i.e., the human 293 cell line
  • adenovirus When intravenously administered to intact animals, adenovirus primarily targets the liver. If the adenoviral delivery system has an El gene deletion, the virus cannot replicate in the host cells.
  • the host's tissue i.e., liver
  • the host's tissue will express and process (and, if a signal sequence is present, secrete) the heterologous protein.
  • Secreted proteins will enter the circulation in the highly vascularized liver, and effects on the infected animal can be determined.
  • the adenovirus system can also be used for protein production in vitro. By culturing adenovirus- infected non-293 cells under conditions where the cells are not rapidly dividing, the cells can produce proteins for extended periods of time. For instance, BHK cells are grown to confluence in cell factories, then exposed to the adenoviral vector encoding the secreted protein of interest. The cells are then grown under serum-free conditions, which allows infected cells to survive for several weeks without significant cell division.
  • adenovirus vector infected 293S cells can be grown in suspension culture at relatively high cell density to produce significant amounts of protein (see A. Gamier et al . , Cvtotechnol . 15:145-55, 1994). With either protocol, an expressed, secreted heterologous protein can be repeatedly isolated from the cell culture supernatant. Within the infected 293S cell production protocol, non-secreted proteins may also be effectively obtained.
  • the highly conserved amino acids in the DNA binding domain of nuclear hormone receptors can be used as a tool to identify new superfamily members.
  • reverse transcription-polymerase chain reaction RT-PCR
  • RT-PCR reverse transcription-polymerase chain reaction
  • highly degenerate primers designed from the zinc finger sequences are useful for this purpose.
  • Specific tissue can be chosen to based on desired characteristics such as high incidence of cancers or hyperplasia.
  • Low stringency hybridization has also been used to screen cDNA libraries using highly degenerate primers to conserved regions of nuclear hormone receptors such as the DNA binding ' domain.
  • Human ZPPAR6 polypeptides can also be used to prepare antibodies that specifically bind to respective human ZPPAR6 epitopes, peptides or polypeptides.
  • Methods for preparing polyclonal and monoclonal antibodies are well known in the art (see, for example, Sambrook et al . , Molecular Cloning: A Laboratory Manual , Second Edition, Cold Spring Harbor, NY, 1989; and Hurrell, J. G. R., Ed.,
  • polyclonal antibodies can be generated from a variety of warm-blooded animals, such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats.
  • the immunogenicity of a human ZPPAR6 polypeptide may be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant.
  • an adjuvant such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant.
  • Polypeptides useful for immunization also include fusion polypeptides, such as fusions of human ZPPAR6 or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein.
  • the polypeptide immunogen may be a full-length molecule or a portion thereof.
  • polypeptide portion is "hapten-like"
  • such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH) , bovine serum albumin (BSA) or tetanus toxoid) for immunization.
  • a macromolecular carrier such as keyhole limpet hemocyanin (KLH) , bovine serum albumin (BSA) or tetanus toxoid
  • antibodies includes polyclonal antibodies, affinity-purified polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments, such as F(ab')2 and Fab proteolytic fragments. Genetically engineered intact antibodies or fragments, such as chimeric antibodies, Fv fragments, single chain antibodies and the like, as well as synthetic antigen- binding peptides and polypeptides, are also included.
  • Non-human antibodies may be humanized by grafting only non-human CDRs onto human framework and constant regions, or by incorporating the entire non-human variable domains (optionally "cloaking" them with a human-like surface by replacement of exposed residues, wherein the result is a "veneered” antibody) .
  • humanized antibodies may retain non-human residues within the human variable region framework domains to enhance proper binding characteristics. Through humanizing antibodies, biological half-life may be increased, and the potential for adverse immune reactions upon administration to humans is reduced.
  • Alternative techniques for generating or selecting antibodies useful herein include in vi tro exposure of lymphocytes to human ZPPAR6 protein or peptide, and selection of antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled human ZPPAR6 protein or peptide) .
  • Antibodies are determined to be specifically binding if: 1) they exhibit a threshold level of binding activity, and/or 2) they do not significantly cross-react with related polypeptide molecules.
  • Antibodies herein specifically bind if they bind to a human ZPPAR ⁇ polypeptide, peptide or epitope with a binding affinity f. —1 • 7 —1
  • the binding affinity of an antibody can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis
  • Antibodies of the current invention do not significantly cross-react with related polypeptide molecules, for example, if they detect ZPPAR6 but not known related polypeptides using a standard Western blot analysis (Ausubel et al . , ibid. ) .
  • known related polypeptides are orthologs, a polypeptide or protein obtained from one species that has homology to an analogous polypeptide or protein from a different species, such chicken TLX, Drosophila TLL and mouse TLX, mutant
  • ZPPAR6 nuclear hormone receptor polypeptides and non- human ZPPAR6 nuclear hormone receptors.
  • antibodies may be "screened against" known related polypeptides to isolate a population that specifically binds to the inventive polypeptides. For example, antibodies raised to ZPPAR6 are adsorbed to related polypeptides adhered to insoluble matrix; antibodies specific to ZPPAR6 will flow through the matrix under the proper buffer conditions. Such screening allows isolation of polyclonal and monoclonal antibodies non-crossreactive to closely related polypeptides (Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988; Current Protocols in Immunology, Cooligan, et al .
  • assays known to those skilled in the art can be utilized to detect antibodies which specifically bind to human ZPPAR6 proteins or peptides. Exemplary assays are described in detail in Antibodies: A Laboratory Manual , Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include: concurrent immunoelectrophoresis , radioimmunoassay, radioimmuno-precipitation, enzyme-linked immunosorbent assay (ELISA) , dot blot or Western blot assay, inhibition or competition assay, and sandwich assay. In addition, antibodies can be screened for binding to wild-type versus mutant human ZPPAR6 protein or peptide .
  • Antibodies to human ZPPAR6 may be used for tagging cells that express human ZPPAR6; for isolating human ZPPAR6 by affinity purification; for diagnostic assays for determining circulating levels of human ZPPAR6 polypeptides; for detecting or quantitating soluble human ZPPAR6 as marker of underlying pathology or disease; in analytical methods employing FACS ; for screening expression libraries; for generating anti-idiotypic antibodies; and as neutralizing antibodies or as antagonists to block human ZPPAR6 in vi tro and in vivo .
  • Suitable direct tags or labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like; indirect tags or labels may feature use of biotin-avidin or other complement/anti-complement pairs as intermediates.
  • Antibodies herein may also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications.
  • Antibodies to ZPPAR6 may be used for tagging cells that express ZPPAR6 ; for isolating ZPPAR6 by affinity purification; for diagnostic assays for determining circulating levels of ZPPAR6 polypeptides; for detecting or quantitating soluble- ZPPAR6 as marker of underlying pathology or disease; in analytical methods employing FACS; for screening expression libraries; for generating anti-idiotypic antibodies; and as neutralizing antibodies or as antagonists to block ZPPAR6 in vi tro and in vivo .
  • Suitable direct tags or labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like; indirect tags or labels may feature use of biotin-avidin or other complement/anti- complement pairs as intermediates.
  • Antibodies herein may also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications.
  • ZPPAR6 polypeptide binding domains can be obtained by screening random peptide libraries displayed on phage
  • Nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as through random mutagenesis and random polynucleotide synthesis.
  • These random peptide display libraries can be used to screen for peptides which interact with a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances.
  • Techniques for creating and screening such random peptide display libraries are known in the art (Ladner et al . , US Patent NO. 5,223,409; Ladner et al . , US Patent NO. 4,946,778; Ladner et al . , US Patent NO.
  • Random peptide display libraries can be screened using the ZPPAR6 sequences disclosed herein to identify proteins which bind to ZPPAR6.
  • binding proteins which interact with ZPPAR6 polypeptides may be used for tagging cells; for isolating homolog polypeptides by affinity purification; they can be directly or indirectly conjugated to drugs, toxins, radionuclides and the like.
  • binding proteins can also be used in analytical methods such as for screening expression libraries and neutralizing activity.
  • the binding proteins can also be used for diagnostic assays for determining circulating levels of polypeptides; for detecting or quantitating soluble polypeptides as marker of underlying pathology or disease.
  • These binding proteins can also act as ZPPAR6 "antagonists" to block ZPPAR6 binding and signal transduction in vitro and in vivo. These anti-ZPPAR6 binding proteins would be useful for inhibiting receptor binding.
  • the ZPPAR6 polynucleotides and/or polypeptides disclosed herein may be useful as therapeutic targets, wherein agonists or antagonists could modulate one or more biological processes in cells, tissues and/or biological fluids.
  • the ZPPAR6 polypeptides can be used to screen test samples for the presence of natural ligand, or of agonists or antagonists of the natural ligand. Additionally, the corresponding response elements recognized and bound by ZPPAR6 can be analyzed.
  • the ZPPAR6 polynucleotide sequences can be used to obtain probes/oligonucleotides that can hybridize to counterpart sequences on individual chromosomes. Chromosomal identification and/or mapping of the ZPPAR6 gene will be a useful tool in determining disease association.
  • Polynucleotides encoding ZPPAR6 polypeptides are useful within gene therapy applications where it is desired to increase or inhibit ZPPAR6 activity. If a mammal has a mutated or absent ZPPAR6 gene, the ZPPAR6 gene can be introduced into the cells of the mammal. In one embodiment, a gene encoding a ZPPAR6 polypeptide is introduced in vivo in a viral vector.
  • viral vectors include an attenuated or defective DNA virus, such as, but not limited to, herpes simplex virus (HSV) , papillomavirus, Epstein Barr virus (EBV) , adenovirus, adeno-associated virus (AAV), and the like.
  • Defective viruses which entirely or almost entirely lack viral genes, are preferred.
  • a defective virus is not infective after introduction into a cell.
  • Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells.
  • Examples of particular vectors include, but are not limited to, a defective herpes simplex virus 1 (HSV1) vector (Kaplitt et al . , Molec . Cell. Neurosci . 2 . :320-30, 1991); an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al . , J. Clin. Invest.
  • HSV1 herpes simplex virus 1
  • a ZPPAR6 gene can be introduced in a retroviral vector, e.g., as described in Anderson et al . , U.S. Patent No. 5,399,346; Mann et al . Cell £:153, 1983; Temin et al . , U.S. Patent No. 4,650,764; Temin et al . , U.S. Patent No. 4,980,289; Markowitz et al . , J. Virol.£:1120, 1988; Temin et al . , U.S. Patent No. 5,124,263; International Patent Publication No. WO 95/07358, published March 16, 1995 by Dougherty et al .
  • the vector can be introduced by lipofection in vivo using liposomes.
  • Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Feigner et al . , Proc . Natl. Acad. Sci . USA £4:7413-7, 1987; Mackey et al . , Proc. Natl. Acad. Sci. USA £5:8027-31, 1988).
  • the use of lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages. Molecular targeting of liposomes to specific cells represents one area of benefit.
  • directing transfection to particular cells represents one area of benefit.
  • directing transfection to particular cell types would be particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney, and brain.
  • Lipids may be chemically coupled to other molecules for the purpose of targeting.
  • Targeted peptides e.g., hormones or neurotransmitters
  • proteins such as antibodies, or non-peptide molecules could be coupled to liposomes chemically. It is possible to remove the target cells from the body; to introduce the vector as a naked DNA plasmid; and then to re- implant the transformed cells into the body.
  • Naked DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun or use of a DNA vector transporter. See, e.g., Wu et al . , J. Biol. Chem. 267:963-7, 1992; Wu et al . , J. Biol. Chem. 263 :14621-4, 1988.
  • Antisense methodology can be used to inhibit ZPPAR6 gene transcription, such as to inhibit cell proliferation in vivo .
  • Polynucleotides that are complementary to a segment of a ZPPAR6-encoding protein e.g., a polynucleotide comprising a sequence as set froth in SEQ ID NO:l
  • Such antisense oligonucleotides are used to inhibit expression of ZPPAR6 polypeptide-encoding genes in cell culture or in a subject.
  • ZPPAR6 gene function referred to as "knockout mice”
  • mice may also be generated (Lowell et al . , Nature 366 :740-42. 1993). These mice may be employed to study the ZPPAR6 gene and the protein encoded thereby in an in vivo system.
  • Polynucleotides if the present invention can be used for chromosomal localization of ZPPAR ⁇ using techniques known to those skilled in the art.
  • Radiation hybrid mapping is a somatic cell genetic technique developed for constructing high-resolution, contiguous maps of mammalian chromosomes (Cox et al., Science 250:245-50, 1990). Partial or full knowledge of a gene's sequence allows one to design PCR primers suitable for use with chromosomal radiation hybrid mapping panels.
  • Commercially available radiation hybrid mapping panels which cover the entire human genome, such as the Stanford G3 RH Panel and the GeneBridge 4 RH Panel (Research Genetics, Inc., Huntsville, AL) , are available.
  • These panels enable rapid, PCR-based chromosomal localizations and ordering of genes, sequence-tagged sites (STSs), and other nonpolymorphic and polymorphic markers within a region of interest. This includes establishing directly proportional physical distances between newly discovered genes of interest and previously mapped markers.
  • the precise knowledge of a gene' s position can be useful for a number of purposes, including: 1) determining if a sequence is part of an existing contig and obtaining additional surrounding genetic sequences in various forms, such as YACs, BACs or cDNA clones; 2) providing a possible candidate gene for an inheritable disease which shows linkage to the same chromosomal region; and 3) cross-referencing model organisms, such as mouse, which may aid in determining what function a particular gene might have.
  • Sequence tagged sites can also be used independently for chromosomal localization.
  • An STS is a DNA sequence that is unique in the human genome and can be used as a reference point for a particular chromosome or region of a chromosome.
  • An STS is defined by a pair of oligonucleotide primers that are used in a polymerase chain reaction to specifically detect this site in the presence of all other genomic sequences. Since STSs are based solely on DNA sequence they can be completely described within an electronic database, for example,
  • the ZPPAR6 gene a probe comprising ZPPAR6 DNA or RNA or a subsequence thereof can be used to determine if the ZPPAR6 gene is present on that chromosome or if a mutation has occurred.
  • Detectable chromosomal aberrations at the ZPPAR6 gene locus include, but are not limited to, aneuploidy, gene copy number changes, insertions, deletions, restriction site changes and rearrangements.
  • Such aberrations can be detected using polynucleotides of the present invention by employing molecular genetic techniques, such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, and other genetic linkage analysis techniques known in the art (Sambrook et al . , ibid. ; Ausubel et . al . , ibid. ; Marian, Chest 108:255-65, 1995) .
  • molecular genetic techniques such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, and other genetic linkage analysis techniques known in the art (Sambrook et al . , ibid. ; Ausubel et . al . , ibid. ; Marian, Chest 108:255-65, 1995) .
  • RFLP restriction fragment length polymorphism
  • STR short tandem repeat
  • Example 1 Identification of the Human ZPPAR6 Receptor Polypeptides Analysis of the EST sequence derived from querying an EST database for sequences homologous to individual members of the nuclear receptor superfamily, as well as to conserved motifs within the family, resulted in an EST that corresponded to the ligand binding domain of a human nuclear hormone receptor.
  • a clone related to dbEST 188758 was obtained from I.M.A.G.E Consortium (Washington University, St. Louis, MO), and a 419 bp sequence (SEQ. ID.
  • SEQ. ID. NO. 2 There were six base pair substitutions and eight deleted base pairs within SEQ. ID. NO. 1, when compared to that of dbEST 188758. All unknown base pairs from dbEST 188758 were identified in the ZPPAR6 sequence. There was also an additional 45 bp 5' extension. Analysis of the deduced amino acid sequence (SEQ. ID. NO. 2) indicates that the ZPPAR6 fragment encoded by the sequence represented by SEQ. ID. NO. 1 includes a portion of the ligand binding domain and has homology to known nuclear hormone receptors cTLX and mTLX. To obtain a full length cDNA sequence of ZPPAR6 , 5' and 3' RACE (rapid amplification of cDNA ends) is used.
  • primers are designed, preferably from a sequence near the 3' end of the 419 bp fragment (SEQ. ID. NO. 1).
  • 3' RACE is then performed using "marathon ready" cDNA as a template.
  • Preferably fetal brain, lung, liver or kidney cDNA is used as a template.
  • Templates can be purchased (Clontech, Palo Alto, CA) or prepared using a Marathon- cDNA Amplification Kit (Clontech) according to the protocol provided by the manufacturer.
  • 3' RACE is then- carried out using a Marathon- cDNA Amplification Kit (Clontech) according to manufacturer's instructions.
  • PCR reaction product is isolated by low melt agarose gel electrophoresis and purified using a QIAquick column (Qiagen, Inc., Chatsworth, CA) according to the manufacturer's direction.
  • the PCR reaction product can be optionally used as a template for 3 ' nested RACE where 40 pmol of a second 3 ' primer (offset from the 3 ' sense primer by several base pairs) and marathon adapter primer AP-2 (Clontech) are used to generate a specific cDNA sequence.
  • the 3' nested RACE PCR reaction is performed for 1 min at 94°C followed by 30 cycles of (94°C for 30 sec; 68°C for 4 min).
  • the nested 3 ' RACE PCR reaction product is isolated by low melt agarose electrophoresis and purified using a QIAquick column (Qiagen, Inc.) according to the manufacturer's direction and submitted for sequence analysis. 5' RACE is carried out as described above.
  • Specific antisense 5' RACE and nested 5' RACE primers can be synthesized, preferably encoding sequence from near the 5' end of the 419 bp (SEQ. ID. NO. 1) fragment.
  • AP-2 (Clontech) can be used for both the sense and antisense primers.
  • a full length sequence can be constructed using the clonally derived 419 bp fragment (SEQ. ID. NO. 1) as well as the 5' and 3' RACE products as both templates and primers in a PCR reaction to fill any remaining sequence gaps.
  • Specific 5' and 3' sense and antisense primers are designed from the known RACE derived sequences to prime for amplification of a full length product.
  • MTN I Human Multiple Tissue Northern Blots
  • T4 polynucleotide kinase and forward reaction buffer (GIBCO BRL, Gaithersburg, MD) according to the manufacturer's specifications.
  • the probe was purified using a NUCTRAP push column (Stratagene Cloning Systems,
  • Northern blots Hybridization took place overnight at 42 C 6 using 4 x 10 cpm/ml of labeled probe, and the blots were then washed at 50 C in 6X SSC, 0.1% SDS. Five transcripts were seen. A 4.4 kb band was observed in liver and heart. A 2.5 kb band was seen in heart and pancreas . A 2.0 kb band was seen in heart, skeletal muscle, with faint bands in brain and liver, also in fetal lung, kidney, brain and liver. A 1.5 kb band was detected in small intestine and a 1.0 kb band was seen in pancreas. A more stringent wash at 62°C in 6X SSC, 0.1% SDS eliminated all bands except for that in fetal kidney.
  • AAA CTC CTG ⁇ G CTT TTG CCA GCT TTA CGT TCT ATT AGC CCA TCA ACT 335 Lys Leu Leu Leu Leu Leu Pro Ala Leu Arg Ser He Ser Pro Ser Thr 100 105 110
  • MOLECULE TYPE protein
  • FRAGMENT TYPE internal

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Abstract

Nouveaux polynucléotides et polypeptides récepteurs hormonaux nucléaires humains, et compositions et procédés associés. Ces polypeptides et polynucléotides présentent des caractéristiques de récepteurs d'hormone nucléaires ainsi qu'une homologie avec d'autre récepteurs d'hormone nucléaires de mammifère. Les polypeptides récepteurs peuvent être utilisés dans des procédés pour détecter le ligand humain naturel et les analogues du ligand. Les polypeptides récepteurs peuvent également être utilisés dans des procédés visant à influencer les fonctions biologiques et à maintenir l'homéostasie.
PCT/US1998/000678 1997-01-15 1998-01-15 Zppar6, recepteur hormonal nucleaire sans queue (recepteur tlx) humain WO1998031797A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU62407/98A AU6240798A (en) 1997-01-15 1998-01-15 Zppar6, human tailless nuclear hormone receptor (tlx receptor)

Applications Claiming Priority (2)

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US3376297P 1997-01-15 1997-01-15
US60/033,762 1997-01-15

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WO1998031797A1 true WO1998031797A1 (fr) 1998-07-23

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11136352B2 (en) 2008-10-01 2021-10-05 Immatics Biotechnologies Gmbh Immunotherapy against several tumors including neuronal and brain tumors

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0392691A2 (fr) * 1989-04-10 1990-10-17 Baylor College Of Medicine Dosages des interactions du facteur de transcription-coup
EP0441483A2 (fr) * 1990-01-16 1991-08-14 Baylor College Of Medicine Vecteurs d'expression pour la production de récepteurs stéroides, chimères de ces récepteurs, tests de dépistage pour ces récepteurs et tests cliniques utilisants les récepteurs synthétisés ainsi que leurs vecteurs

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0392691A2 (fr) * 1989-04-10 1990-10-17 Baylor College Of Medicine Dosages des interactions du facteur de transcription-coup
EP0441483A2 (fr) * 1990-01-16 1991-08-14 Baylor College Of Medicine Vecteurs d'expression pour la production de récepteurs stéroides, chimères de ces récepteurs, tests de dépistage pour ces récepteurs et tests cliniques utilisants les récepteurs synthétisés ainsi que leurs vecteurs

Non-Patent Citations (3)

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Title
DATABASE EMBL EBI, Hinxton, UK; 23 April 1995 (1995-04-23), HILLIER L. ET AL: "The WashU-Merck EST Project", XP002065918 *
MONAGHAN AP ET AL: "The mouse homolog of the orphan nuclear receptor tailless is expressed in the developing forebrain.", DEVELOPMENT, MAR 1995, 121 (3) P839-53, ENGLAND, XP002062071 *
YU RT ET AL: "Relationship between Drosophila gap gene tailless and a vertebrate nuclear receptor Tlx.", NATURE, AUG 4 1994, 370 (6488) P375-9, ENGLAND, XP002062021 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11136352B2 (en) 2008-10-01 2021-10-05 Immatics Biotechnologies Gmbh Immunotherapy against several tumors including neuronal and brain tumors
US11208434B2 (en) 2008-10-01 2021-12-28 Immatics Biotechnologies Gmbh Immunotherapy against several tumors including neuronal and brain tumors
US12221493B2 (en) 2008-10-01 2025-02-11 Immatics Biotechnologies Gmbh Immunotherapy against several tumors including neuronal and brain tumors
US12234298B2 (en) 2008-10-01 2025-02-25 Immatics Biotechnologies Gmbh Immunotherapy against several tumors including neuronal and brain tumors

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