WO2001083523A2 - Stmst protein and nucleic acid molecules and uses therefor - Google Patents

Stmst protein and nucleic acid molecules and uses therefor Download PDF

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Publication number
WO2001083523A2
WO2001083523A2 PCT/US2001/013795 US0113795W WO0183523A2 WO 2001083523 A2 WO2001083523 A2 WO 2001083523A2 US 0113795 W US0113795 W US 0113795W WO 0183523 A2 WO0183523 A2 WO 0183523A2
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stmst
seq
nucleic acid
polypeptide
protein
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PCT/US2001/013795
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French (fr)
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WO2001083523A3 (en
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Sean A. Mccarthy
Gu Wei
David White
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Millennium Pharmaceuticals, Inc.
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Priority to AU2001259240A priority Critical patent/AU2001259240A1/en
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Publication of WO2001083523A3 publication Critical patent/WO2001083523A3/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
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • GPCRs form one of the largest protein superfamilies found in nature, and it is estimated that greater than 1000 different such receptors exist in mammals. Upon binding of extracellular ligands, GPCRs interact with a specific subset of heterotrimeric G-proteins that can then, in their activated forms, inhibit or activate various effector enzymes and/or ion channels.
  • the ligands for many of these receptors are known although there exists an ever-increasing number of GPCRs which have been identified in the sequencing of the human genome which for have no ligands have yet been identified. This latter subfamily of GPCRs is called the ophan family of GPCRs.
  • GPCR-like molecules which share significant homology as well as many of the structural properties of the GPCR superfamily.
  • GPCR-like proteins which arises from three alternatively-spliced forms of a gene occurring between the CD4 and triosephosphate isomerase genes at human chromosome 12pl3, has been recently identified (including protein A-l, A-2, and A-3).
  • Ansari-Lari et al. (1996) Genome Res. 6(4):314-326. Comparative sequence analysis of the syntenic region in mouse chromosome 6 has further revealed a murine homologue of at least the A-2 splice product.
  • Ansari-Lari et al. (1998) Genome Res. 8(l):29-40.
  • the present invention is based, at least in part, on the discovery of nucleic acid molecules which encode a novel family of G protein-coupled receptor-like proteins, referred to herein as the Seven Transmembrane Signal Transducer ("STMST" family or "STMST proteins").
  • STMST Seven Transmembrane Signal Transducer
  • the STMST molecules of the present invention as well as STMST ligands and/or STMST modulators, are useful in regulating a variety of cellular processes.
  • this invention provides isolated nucleic acid molecules encoding STMST proteins or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of STMST-encoding nucleic acids.
  • an STMST nucleic acid molecule is 75% homologous to the nucleotide sequence shown in SEQ ID NO:l, or complement thereof. In another embodiment, an STMST nucleic acid molecule is 80% homologous to the nucleotide sequence shown in SEQ ID NO:4, or a complement thereof. In a preferred embodiment, an isolated STMST nucleic acid molecule has the nucleotide sequence shown SEQ ID NO:3, or a complement thereof. In another embodiment, an STMST nucleic acid molecule further comprises nucleotides 1-403 of SEQ ID NO:l. In another embodiment, an STMST nucleic acid molecule further comprises nucleotides 1295-2915 of SEQ ID NO: 1. In another preferred embodiment, an isolated STMST nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 1.
  • an isolated STMST nucleic acid molecule has the nucleotide sequence shown SEQ ID NO: 6, or a complement thereof. In another embodiment, an STMST nucleic acid molecule further comprises nucleotides 1-333 of SEQ ID NO:4. In another embodiment, an STMST nucleic acid molecule further comprises nucleotides 2161-4166 of SEQ ID NO:4. In another preferred embodiment, an isolated STMST nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:4.
  • an STMST nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence sufficiently homologous to the amino acid sequence of SEQ ID NO:2, or the amino acid sqeuence of SEQ ID NO:5.
  • an STMST nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence at least 75% homologous to the amino acid sequence of SEQ ID NO:2.
  • an STMST nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence at least 60% homologous to the amino acid sequence of SEQ ID NO:5.
  • an isolated nucleic acid molecule of the present invention encodes an STMST protein which includes at least one transmembrane domain. In another embodiment, an isolated nucleic acid molecule of the present invention encodes a protein which includes a 7 transmembrane receptor profile. In another embodiment, an isolated nucleic acid molecule of the present invention encodes a protein which includes a spectrin ⁇ -chain motif. In yet another embodiment, an STMST nucleic acid molecule encodes an STMST protein and is a naturally occurring nucleotide sequence.
  • Another embodiment of the invention features STMST nucleic acid molecules which specifically detect STMST nucleic acid molecules relative to nucleic acid molecules encoding non-STMST proteins.
  • STMST nucleic acid molecules which specifically detect STMST nucleic acid molecules relative to nucleic acid molecules encoding non-STMST proteins.
  • an STMST nucleic acid molecules which specifically detect STMST nucleic acid molecules relative to nucleic acid molecules encoding non-STMST proteins.
  • STMST nucleic acid molecule is at least 350 nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO:l, SEQ ID NO:4.
  • Another embodiment of the invention provides an isolated nucleic acid molecule which is antisense to the coding strand of an STMST nucleic acid.
  • Another aspect of the invention provides a vector comprising an STMST nucleic acid molecule.
  • the vector is a recombinant expression vector.
  • the invention provides a host cell containing a vector of the invention.
  • the invention also provides a method for producing an STMST protein by culturing in a suitable medium, a host cell of the invention containing a recombinant expression vector such that an STMST protein is produced.
  • an isolated STMST protein includes at least one transmembrane domain.
  • an isolated STMST protein includes at least six transmembrane domains.
  • an isolated STMST protein includes seven transmembrane domains.
  • an isolated STMST protein includes a 7 transmembrane receptor profile.
  • an isolated STMST protein includes a spectrin -chain profile.
  • an isolated STMST protein has an amino acid sequence sufficiently homologous to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:5.
  • an STMST protein has an amino acid sequence at least about 75% homologous to the amino acid sequence of SEQ ID NO:2. In another preferred embodiment, an STMST protein has an amino acid sequence at least about 60% homologous to the amino acid sequence of SEQ ID NO:5. In another embodiment, an STMST protein has the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:5.
  • Another embodiment of the invention features an isolated STMST protein which is encoded by a nucleic acid molecule having a nucleotide sequence at least about 75% homologous to a nucleotide sequence of SEQ ID NO: 1 , or a complement thereof.
  • Another embodiment of the invention features an isolated STMST protein which is encoded by a nucleic acid molecule having a nucleotide sequence at least about 80% homologous to a nucleotide sequence of SEQ ID NO:4, or a complement thereof.
  • This invention further features an isolated STMST protein which is encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l, SEQ ID NO:4, or a complement thereof.
  • the STMST proteins of the present invention can be operatively linked to a non-STMST polypeptide to form STMST fusion proteins.
  • the invention further features antibodies that specifically bind STMST proteins, such as monoclonal or polyclonal antibodies.
  • the STMST proteins or biologically active portions thereof can be incorporated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers.
  • the present invention provides a method for detecting STMST expression in a biological sample by contacting the biological sample with an agent capable of detecting an STMST nucleic acid molecule, protein or polypeptide such that the presence of an STMST nucleic acid molecule, protein or polypeptide is detected in the biological sample.
  • the present invention provides a method for detecting the presence of STMST activity in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of STMST activity such that the presence of STMST activity is detected in the biological sample.
  • the invention provides a method for modulating STMST activity comprising contacting the cell with an agent that modulates STMST activity such that STMST activity in the cell is modulated.
  • the agent inhibits STMST activity.
  • the agent stimulates STMST activity.
  • the agent is an antibody that specifically binds to an STMST protein.
  • the agent modulates expression of STMST by modulating transcription of an STMST gene or translation of an STMST mRNA.
  • the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of an STMST mRNA or an STMST gene.
  • the methods of the present invention are used to treat a subject having a disorder characterized by aberrant STMST protein or nucleic acid expression or activity by administering an agent which is an STMST modulator to the subject.
  • the STMST modulator is an STMST protein.
  • the STMST modulator is an STMST nucleic acid molecule.
  • the STMST modulator is an STMST ligand (e.g., a peptide ligand or neurotransmitter).
  • the STMST modulator is a peptide, peptidomimetic, or other small molecule.
  • the disorder characterized by aberrant STMST protein or nucleic acid expression is a developmental, differentiative, proliferative disorder, an inflammatory disorder, a respiratory disorder (e.g., asthma), or cell death.
  • the present invention also provides a diagnostic assay for identifying the presence or absence of a genetic alteration characterized by at least one of (i) aberrant modification or mutation of a gene encoding an STMST protein; (ii) mis-regulation of said gene; and (iii) aberrant post-translational modification of an STMST protein, wherein a wild-type form of said gene encodes an protein with an STMST activity.
  • the invention provides a method for identifying a compound that binds to or modulates the activity of an STMST protein.
  • the invention provides a method for identifying a compound which binds to an STMST protein which involves contacting the STMST protein, or a cell expressing the STMST protein with a test compound and determining whether the STMST protein binds to the test compound.
  • the invention provides a method for identifying a compound which modulates the activity of an STMST protein which involves contacting an STMST protein with a test compound, and determining the effect of the test compound on the activity of the polypeptide to thereby identify a compound which modulates the activity of the STMST protein.
  • Figure 1 depicts the cDNA sequence and predicted amino acid sequence of human STMST- 1.
  • the nucleotide sequence corresponds to nucleic acids 1 to 2915 of SEQ ID NO: 1.
  • the amino acid sequence corresponds to amino acids 1 to297 of SEQ ID NO:2.
  • Figure 2 depicts the cDNA sequence and predicted amino acid sequence of human STMST-2.
  • the nucleotide sequence corresponds to nucleic acids 1 to 4166 of SEQ ID NO:4.
  • the amino acid sequence corresponds to amino acids 1 to 609 of SEQ ID NO:5.
  • Figure 3 depicts an alignment of the amino acid sequences of human STMST- 1
  • SEQ ID NO:2 human STMST-2 (SEQ ID NO:5), human protein A-2 (Accession No. U47928, SEQ ID NO:9), and human protein A-3 (Accession No. U47929, SEQ ID NO: 10).
  • the 7 transmembrane receptor profile is indicated in italics.
  • the transmembrane domains are underlined.
  • the spectrin ⁇ -chain profile is indicated in bold.
  • Figure4A-B is a graphic representation of relative STMST expresion levels as determined by TaqManTM RT-PCR of mRNA samples from various cells including osteoblast cells lines and primary osteoblasts treated as indicated.
  • Figure 4C is a graphic representation of STMST expression levels as determined by transcription profiling analysis using a cDNA array.
  • the present invention is based on the discovery of novel molecules, referred to herein as STMST protein and nucleic acid molecules, which comprise a family of molecules having certain conserved structural and functional features.
  • family when referring to the protein and nucleic acid molecules of the invention is intended to mean two or more proteins or nucleic acid molecules having a common structural domain and having sufficient amino acid or nucleotide sequence homology as defined herein.
  • family members can be naturally occurring and can be from either the same or different species.
  • a family can contain a first protein of human origin, as well as other, distinct proteins of human origin or alternatively, can contain homologues of non-human origin.
  • Members of a family may also have common functional characteristics.
  • GPCRs G protein-coupled receptors
  • STMST proteins of the present invention comprise an N- terminal extracellular domain, seven transmembrane domains (also referred to as membrane-spanning domains), three extracellular domains (also referred to as extracellular loops), three cytoplasmic domains (also referred to as cytoplasmic loops), and a C-terminal cytoplasmic domain (also referred to as a cytoplasmic tail).
  • GPCRs G protein-coupled receptors
  • Members of the GPCR family also share certain conserved amino acid residues, some of which have been determined to be critical to receptor function and/or G protein signaling.
  • GPCRs contain the following features: a conserved asparagine residue in the first transmembrane domain; a cysteine residue in the first extracellular loop which is believed to form a disulfide bond with a conserved cysteine residue in the second extracellular loop; a conserved leucine and aspartate residue in the second transmembrane domain; an aspartate-arginine-tyrosine motif (DRY motif) at the interface of the third transmembrane domain and the second cytoplasmic loop of which the arginine residue is almost invariant (members of the rhodopsin subfamily of GPCRs comprise a histidine-arginine-methionine motif (HRM motif) as compared to a DRY motif); a conserved tryptophan and proline residue in the fourth transmembrane domain; a conserved phenylalanine residue which is commonly found as part of the motif FXXCXXP; and a conserved leucine residue in the seventh
  • Table I depicts an alignment of the transmembrane domain of 5 GPCRs. The conserved residues described herein are indicated by asterices. An alignment of the transmembrane domains of 44 representative GPCRs can be found at http://mgdkkl.nidll.nih.gov.8000/extended.html.
  • thrombin accesion No. P25116
  • rhodopsin accesion No. P08100
  • ml ACh accesion No. P08482
  • IL-8A accesion No.
  • GPCR-like proteins such as the STMST proteins of the present invention contain a siginificant number of structural characteristics of the GPCR family.
  • the STMSTs of the present invention contain conserved cysteines found in the first 2 extracellular loops (prior to the third and fifth transmembrane domains) of most GPCRs (cys 83 and cys 161 of SEQ ID NO:2 or SEQ ID NO:5).
  • a highly conserved asparagine residue in the first transmembrane domain is present (asn25 in SEQ ID NO:2 or SEQ ID NO:5).
  • Transmembrane domain two of the STMST proteins contains a highly conserved leucine (leu49 of SEQ ID NO:2 or SEQ ID NO:5). The two cysteine residues are believed to form a disulfide bond that stabilizes the functional protein structure.
  • a highly conserved tryptophan and proline in the fourth transmembrane domain of the STMST proteins is present (trpl35 and pro 145 of SEQ ID NO:2 or SEQ ID NO: 5).
  • the third cytoplasmic loop contains 49 amino acid residues and is thus the longest cytoplasmic loop of the three, characteristic of G protein coupled receptors. Moreover, a highly conserved proline in the sixth transmembrane domain is present (pro260 of SEQ ID NO:2 and SEQ ID NO:5). The proline residues in the fourth, fifth, sixth, and seventh transmembrane domains are thought to introduce kinks in the alpha- helices and may be important in the formation of the ligand binding pocket.
  • the conserved (in the second cytoplasmic loop) HRM motif found in almost all Rhodopsin family GPCRs is present in the STMST proteins of the instant invention (hisl07, argl08, metl09 of SEQ ID NO:2 or SEQ ID NO:5).
  • the arginine of the HRM sequence is thought to be the most important amino acid in GPCRs and is invariant).
  • an almost invariant proline is present in the seventh transmembrane domain of STMST-2 (pro294 of SEQ ID NO:5).
  • the STMST proteins of the present invention are proteins having an amino acid sequence of about 150-450, preferably about 200-400, more preferably about 225-375, more preferably about 250-350, or about 275-325 amino acids in length. In another embodiment, the STMST proteins of the present invention are proteins having an amino acid sequence of about 450-750, preferably about 500-700, more preferably about 525-675, even more preferably about 550-650, and even more preferably about 575-625 amino acid residues in length. In one embodiment, the STMST proteins of the present invention contain at least one transmembrane domain.
  • transmembrane domain includes an amino acid sequence having at least about 10, preferably about 13, preferably about 16, more preferably about 19, 21, 23, 25, 30, 35 or 40 amino acid residues, of which at least about 50-60%, 60- 70%, preferably about 70-80% more preferably about 80-90%, or about 90-95% of the amino acid residues contain non-polar side chains, for example, alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, and methionine.
  • a transmembrane domain is lipophillic in nature. For example, a transmembrane domain can be found at about amino acids 11-34 of SEQ ID NO:2 or SEQ ID NO:5.
  • an STMST protein of the present invention has more than one transmembrane domain, preferably 2, 3, 4, 5, 6, or 7 transmembrane domains.
  • transmembrane domains can be found at about amino acids 11-34, 44-67, 85-106, 127-149, 172-196, and 244-262 of SEQ ID NO:2 as well as at 11-34, 44-67, 85-106, 127-149, 172-196, 245-269, and 277-300 of SEQ ID NO:5.
  • an STMST protein of the present invention has 7 transmembrane domains.
  • an STMST family member is identified based on the presence of at least one cytoplasmic loop, also referred to herein as a cytoplasmic domain.
  • an STMST family member is identified based on the presence of at least one extracellular loop.
  • the term "loop" includes an amino acid sequence having a length of at least about 4, preferably about 5-10, preferably about 10-20, and more preferably about 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, or 100-150 amino acid residues, and has an amino acid sequence that connects two transmembrane domains within a protein or polypeptide.
  • the N-terminal amino acid of a loop is adjacent to a C-terminal amino acid of a transmembrane domain in a naturally-occurring GPCR or GPCR-like molecule
  • the C-terminal amino acid of a loop is adj acent to an N-terminal amino acid of a transmembrane domain in a naturally-occurring GPCR or GPCR-like molecule.
  • a "cytoplasmic loop” includes an amino acid sequence located within a cell or within the cytoplasm of a cell.
  • a cytoplasmic loop is found at about amino acids 35-43, 107-126, and 197-243 of SEQ ID NO:2, or alternatively, at about amino acid residues 35-43, 107-126, and 197-244 of SEQ ID NO:5.
  • an "extracellular loop” includes an amino acid sequence located outside of a cell, or extracellularly.
  • an extracellular loop can be found at about amino acid residues 68-84 and 150-171 of SEQ ID NO:2, or alternatively, at about amino acid residues 86-84, 150-171, or 270-276 of SEQ ID NO:5.
  • an STMST family member is identified based on the presence of a "C-terminal cytoplasmic domain", also referred to herein as a C-terminal cytoplasmic tail, in the sequence of the protein.
  • a "C- terminal cytoplasmic domain” includes an amino acid sequence having a length of at least about 10, preferably about 10-25, more preferably about 25-50, more preferably about 50-75, even more preferably about 75-100, 100-150, 150-200, 200-250, 250-300, 300-400, 400-500, or 500-600 amino acid resudues and is located within a cell or within the cytoplasm of a cell.
  • the N-terminal amino acid residue of a "C- terminal cytoplasmic domain” is adjacent to a C-terminal amino acid residue of a transmembrane domain in a naturally-occurring GPCR or GPCR-like protein.
  • a C-terminal cytoplasmic domain is found at about amino acid residues 301- 609 of SEQ ID NO:5.
  • an STMST family member is identified based on the presence of an "N-terminal extracellular domain", also referred to herein as an N- terminal extracellular loop in the amino acid sequence of the protein.
  • an "N-terminal extracellular domain” includes an amino acid sequence having about 1-500, preferably about 1-400, more preferably about 1-300, more preferably about 1-200, even more preferably about 1-100, and even more preferably about 1-50, 1-25, or 1-10 amino acid residues in length and is located outside of a cell or extracellularly.
  • the C-terminal amino acid residue of a "N-terminal extracellular domain” is adjacent to an N-terminal amino acid residue of a transmembrane domain in a naturally-occurring GPCR or GPCR-like protein.
  • an N-terminal cytoplasmic domain is found at about amino acid residues 1-10 of SEQ ID NO:2 or SEQ ID NO:5.
  • an STMST family member includes at least one, preferably 6 or 7, transmembrane domains and and/or at least one cytoplasmic loop, and/or at least one extracellular loop.
  • the STMST family member further includes an N-terminal extracellular domain and/or a C- terminal cytoplasmic domain.
  • the STMST family member can include six transmembrane domains, three cytoplasmic loops, and two extracellular loops, or can include six transmembrane domains, three extracellular loops, and 2 cytoplasmic loops.
  • the former embodiment can further include an N-terminal extracellular domain.
  • the latter embodiment can further include a C-terminal cytoplasmic domain.
  • the STMST family member can include seven transmembrane domains, three cytoplasmic loops, and three extracellular loops and can further include an N-terminal extracellular domain or a C-terminal cytoplasmic domain.
  • an STMST family member is identified based on the presence of at least one "7 transmembrane receptor profile", also referred to as a "Rhodopsin family sequence profile", in the protein or corresponding nucleic acid molecule.
  • the term "7 transmembrane receptor profile" includes an amino acid sequence having at least about 100-400, preferably about 150-350, more preferably about 200-300 amino acid residues, or at least about 250-275 amino acids in length and having a bit score for the alignment of the sequence to the 7tm_l family Hidden Markov Model (HMM) of at least 20, preferably 20-30, more preferably 30-40, more preferably 40-50, 50-75, 75-100, 100-200 or greater.
  • HMM Hidden Markov Model
  • the 7tm_l family HMM has been assigned the PFAM Accession PF00001 (http://genome.wustl.edu/Pfam/WWWdata 7tm_l .html).
  • the amino acid sequence of the protein family member is searched against a database of HMMs (e.g., the Pfam database, release 2.1) using the default parameters (http://www.sanger.ac.uk/Software/Pfam/HMM_search).
  • HMMs e.g., the Pfam database, release 2.1
  • the default parameters http://www.sanger.ac.uk/Software/Pfam/HMM_search.
  • the hmmsf program which is available as part of the HMMER package of search programs, is a family specific default program for PFOOOOl and score of 15 is the default threshold score for determining a hit.
  • a search using the amino acid sequence of SEQ ID NO:2 was performed against the HMM database resulting in the identification of a 7 TM receptor profile in the amino acid sequence of SEQ ID NO:2. The results of the search are set forth below.
  • an STMST protein is a human STMST- 1 or a human STMST-2 protein having a 7 transmembrane receptor profile at about amino acids 24-191 of SEQ ID NO:2 or SEQ ID NO:5, respectively.
  • Such a 7 transmembrane receptor profile has the amino acid sequence: ANAWGILSVGAKQKKWKPLEFLLCTLAATHMLNVAVPIATYSVVQLRRQR PDFE NEGLCKVFVSTFYTLTLATCFSVTSLSYHRMWMVC PVNYRLSNA KKQAVHTVMGIWMVSFILSALPAVGWHDTSERFYTHGCRFIVAEIGLGFG VCFLLLVGGSVAMGVICT. (SEQIDNO:9)
  • STMST family members having at least 50-60% homology, preferably about 60-70%, more preferably about 70-80%, or about 80-90% homology with the 7 transmembrane receptor profile of human STMST- 1 or STMST-2 (e.g., SEQ ID NO: 9) are within the scope of the invention.
  • an STMST family member is identified based on the presence of a "spectrin ⁇ -chain profile" in the protein or corresponding nucleic acid molecule.
  • the term "spectrin ⁇ -chain profile” includes a protein domain having an amino acid sequence of about 50-250, preferably about 75-225, more preferably about 100-200 amino acid residues, or about 125-175 amino acids and having a bit score for the alignment of the sequence to the spectrin family (HMM) of at least 7, preferably 8-10, more preferably 10-30, more preferably 30-50, even more preferably 50-75, 75-100, 100-200 or greater.
  • the spectrin family HMM has been assigned the PFAM Accession PF00435 (http://genome.wustl.edu/Pfam/WWWdata/spectrin.html).
  • the amino acid sequence of the protein is searched against a database of HMMs (e.g., the Pfam database, release 2.1) using the default parameters
  • the hrnmsf program which is available as part of the HMMER package of search programs, is a family specific default program for PF00435 and a score of 15 is the default threshold score for determining a hit.
  • the threshold score for determining a hit can be lowered (e.g. , to 8 bits).
  • an STMST protein is human STMST-2 protein which includes a spectrm ⁇ -chain profile at about amino acids 266-372 of SEQ ID NO:5.
  • a spectrin ⁇ -chain profile has the amino acid sequence:
  • STMST family members having at least 50-60% homology, preferably about 60-70%, more preferably about 70-80%, or about 80-90% homology with a spectrin ⁇ -chain profile of human STMST-2 (e.g., SEQ ID NO: 10) are within the scope of the invention.
  • an STMST protein includes at least a spectrin ⁇ -chain profile.
  • an STMST protein includes a spectrin ⁇ -chain profile and a 7 transmembrane receptor profile.
  • an STMST protein is human STMST-2 which includes a spectrin ⁇ -chain profile having about amino acids 266-372 of SEQ ID NO:5.
  • an STMST protein is human STMST-2 which includes a 7 transmembrane receptor profile having about amino acids 24-191 of SEQ ID NO: 5 and a spectrin ⁇ -chain profile having about amino acids 266-372 of SEQ ID NO:5.
  • Preferred STMST molecules of the present invention have an amino acid sequence sufficiently homologous to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:5.
  • the term "sufficiently homologous” refers to a first amino acid or nucleotide sequence which contains a sufficient or minimum number of identical or equivalent (e.g. , an amino acid residue which has a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences share common structural domains and/or a common functional activity.
  • amino acid or nucleotide sequences which share common structural domains have at least about 50% homology, preferably 60% homology, more preferably 70%-80%, and even more preferably 90-95% homology across the amino acid sequences of the domains and contain at least one and preferably two structural domains, are defined herein as sufficiently homologous.
  • amino acid or nucleotide sequences which share at least 50%, preferably 60%, more preferably 70-80, or 90-95% homology and share a common functional activity are defined herein as sufficiently homologous.
  • an "STMST activity”, “biological activity of STMST” or “functional activity of STMST”, refers to an activity exerted by an STMST protein, polypeptide or nucleic acid molecule on an STMST responsive cell as determined in vivo, or in vitro, according to standard techniques.
  • an STMST activity is a direct activity, such as an association with an STMST-traget molecule.
  • a "target molecule” or “binding partner” is a molecule with which an STMST protein binds or interacts in nature, such that STMST-mediated function is acheived.
  • An STMST target molecule can be a non-STMST molecule or an STMST protein or polypeptide of the present invention.
  • an STMST target molecule is an STMST ligand (e.g., a peptide ligand or neurotransmitter).
  • an STMST activity is an indirect activity, such as a cellular signaling activity (e.g., neuronal or glial cell signaling) mediated by interaction of the STMST protein with an STMST ligand.
  • an STMST activity is at least one or more of the following activities: (i) interaction of an STMST protein with soluble STMST ligand; (ii) interaction of an STMST protein with a membrane-bound non-STMST protein; (iii) interaction of an STMST protein with an intracellular protein (e.g., an intracellular enzyme or signal transduction molecule); and (iv) indirect interaction of an STMST protein with an intracellular protein (e.g., a downstream signal transduction molecule).
  • STMST is a GPCR-like protein having significant homology to at least the A-l family of GPCR-like proteins.
  • STMST has been found to be expressed in many tissues, including but not limited to heart, brain (including fetal brain), placenta, lung, liver, skeletal muscle and kidney (fetal). Within the brain, STMST expression has been further localized to the hypothalamus, a major center controlling food intake and body weight. In particular, STMST expression has been localized to the arcuate nucleus ("AC") and the ventormedial hypothalamic nucleus (VMN), two brain areas having specialized roles in energy homeostasis.
  • AC arcuate nucleus
  • VNN ventormedial hypothalamic nucleus
  • an STMST activity for example, a hypothalamic activity
  • a hypothalamic activity is at least one or more of the following activities: (1) modulation (e.g., repression or stimulation) of brain anabolic circuits or pathways; (2) modulation (e.g., repression or stimulation of brain catabolic pathways; (3) modulation of food intake and/or feeding behavior (e.g., stimulation of or inhibition/supression of food intake and/or feeding behavior); (4) modulation of energy expenditure (e.g., supression or stimulation of energy expenditure); (5) regulation of energy homeostasis; and (6) regulation of body fat mass.
  • modulation e.g., repression or stimulation
  • energy expenditure e.g., supression or stimulation of energy expenditure
  • regulation of energy homeostasis e.g., supression or stimulation of energy expenditure
  • an STMST activity is at least one or more of the following activities: (1) modulation of cellular signal transduction, either in vitro or in vivo; (2) regulation of gene transcription in a cell expressing an STMST protein; (3) regulation of gene transcription in a cell expressing an STMST protein, wherein said cell is involved inflammation; (4) regulation of cellular proliferation; (5) regulation of cellular differentiation; (6) regulation of development; (7) regulation of cell death; (8) regulation of inflammation; (9) regulation of respiratory cell function (e.g. , asthma); (10) regulation of actin binding; (11) regulation of cytoskeletal attachment; and (12) regulation of chemotaxis, trafficking and/or migration.
  • modulation of cellular signal transduction either in vitro or in vivo
  • (2) regulation of gene transcription in a cell expressing an STMST protein regulation of gene transcription in a cell expressing an STMST protein, wherein said cell is involved inflammation
  • (4) regulation of cellular proliferation (5) regulation of cellular differentiation; (6) regulation of development; (7) regulation of cell death;
  • an STMST activity is at least one or more of the following: (1) regulation of body temperature; (2) regulation of the sleep-wake cycle; (3) regulation of memory and/or behavior; (4) control of thirst; and (5) regulation of autonomic nervous system function.
  • STMST is highly expressed in osteoblasts (e.g., osteoblastic cell lines as well as primary osteoblasts).
  • Expression in primary osteobasts is further inducible by treatment of cells with parathyroid hormone (PTH) suggesting that STMST and/or STMST agonism may mimic PTH anabolic effects on bone.
  • PTH parathyroid hormone
  • Expression is also inducible by dexamethasone treatment which stimulates primary osteoblasts to differentiate in vitro.
  • Northern blot analysis confirms expression of STMST in primary osteoblasts.
  • STMST has further been determined by in situ analysis to be expressed in osteoblasts of human fetal bone.
  • an STMST modulator is useful for (i) modulating osteogenic cell function (e.g., osteoblast function); (ii) modulating bone homeostasis; (iii) modulation of bone resorption; and (iv) modulation of bone formation (e.g., stimulation of bone mass and/or inhibition of bone loss).
  • osteogenic cell function e.g., osteoblast function
  • bone homeostasis e.g., bone homeostasis
  • modulation of bone resorption e.g., stimulation of bone mass and/or inhibition of bone loss
  • an STMST modulator is useful for (1) regulating, preventing and/or treating bone- related disorders including, but not limited to osteoporosis, Paget's disease, osteoarthritis, degenerative arthritis, osteogenesis imperfecta, fibrous displasia, hypophosphatasia, bone sarcoma, myeloma bone disorder (e.g., osteolytic bone lesions) and hypercalcemia; (2) managment of bone fragility (e.g., decrease bone fragility); and (3) prevention and/or treatment of bone pain, bone deformaties, and/or bone fractures.
  • bone- related disorders including, but not limited to osteoporosis, Paget's disease, osteoarthritis, degenerative arthritis, osteogenesis imperfecta, fibrous displasia, hypophosphatasia, bone sarcoma, myeloma bone disorder (e.g., osteolytic bone lesions) and hypercalcemia; (2) managment of bone fragility (e.g., decrease bone fragility); and (3) prevention and/or treatment of bone pain, bone
  • an STMST activity is at least one or more of the following activities: (1) modulation of cellular signal transduction, either in vitro or in vivo; (2) regulation of gene transcription in a cell expressing an STMST protein; (3) regulation of gene transcription in a cell expressing an STMST protein, wherein said cell is involved inflammation; (4) regulation of cellular proliferation; (5) regulation of cellular differentiation; (6) regulation of develpoment; (7) regulation of cell death; (8) regulation of regulation of inflammation; (9) regulation of respiratory cell function (e.g., asthma); (10) regulation of actin binding; (11) regulation of cytoskeletal attachment; and (12) regulation of chemotaxis, trafficking and/or migration.
  • STMST proteins and polypeptides having an STMST activity are isolated STMST proteins and polypeptides having an STMST activity.
  • Preferred STMST proteins have at least one transmembrane domain and an STMST activity.
  • an STMST protein has a 7 transmembrane receptor profile and an STMST activity.
  • an STMST protein has a spectrin ⁇ -chain profile and an STMST activity.
  • an STMST protein has a 7 transmembrane receptor profile, a spectrin ⁇ -chain profile, and STMST activity.
  • an STMST protein has a 7 transmembrane receptor profile, a spectrin ⁇ -chain profile, an STMST activity, and an amino acid sequence sufficiently homologous to an amino acid sequence of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, or SEQ ID NO: 11.
  • the human STMST- 1 cDNA which is approximately 2915 nucleotides in length, encodes a protein which is approximately 297 amino acid residues in length.
  • the human STMST- 1 protein contains 6 transmembrane domains at about amino acids 11-34, 44-67, 85-106, 127-149, 172-196, and 244-262 of SEQ ID NO:2
  • the human STMST- 1 protein further contains a 7 transmembrane receptor profile.
  • the 7 transmembrane receptor profile can be found at least, for example, from about amino acids 24-191 of SEQ ID NO:2.
  • the human STMST-2 cDNA which is approximately 4166 nucleotides in length, encodes approximately 609 amino acid residues of the human STMST- 1 protein.
  • the human STMST-2 protein contains 7 transmembrane domains at about amino acids 11-34, 44-67, 85-106, 127-149, 172-196, 245-269, and 277-300 of SEQ ID NO:5.
  • the human STMST-2 protein further contains a 7 transmembrane receptor profile.
  • the 7 transmembrane receptor profile can be found at least, for example, from about amino acids 24-191 of SEQ ID NO:5.
  • the human STMST protein contains a spectrin ⁇ -chain profile from about amino acids 266-372 of SEQ ID NO:5.
  • nucleic acid molecules that encode STMST proteins or biologically active portions thereof, as well as nucleic acid fragments sufficient for use as hybridization probes to identify STMST-encoding nucleic acids (e.g., STMST mRNA) and fragments for use as PCR primers for the amplification or mutation of STMST nucleic acid molecules.
  • nucleic acid molecule is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs.
  • the nucleic acid molecule can be single-stranded or double- stranded, but preferably is double-stranded DNA.
  • an “isolated” nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid.
  • an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • the isolated STMST nucleic acid molecule can contain less than about 5 kb, 4kb, 3kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
  • an "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • a nucleic acid molecule of the present invention e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l, the nucleotide sequence of SEQ ID NO:4, or a portion thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or portion of the nucleic acid sequence of SEQ ID NO: 1 , or the nucleotide sequence of SEQ ID NO:4, as a hybridization probe, STMST nucleic acid molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
  • nucleic acid molecule encompassing all or a portion of SEQ ID NO:l or SEQ ID NO:4 can be isolated by the polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based upon the sequence of SEQ ID NO:l, SEQ ID NO:4.
  • PCR polymerase chain reaction
  • a nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
  • the nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • oligonucleotides corresponding to STMST nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
  • an isolated nucleic acid molecule of the invention comprises the nucleotide sequence shown in SEQ ID NO: 1.
  • the sequence of SEQ ID NO:l corresponds to the human STMST- 1 cDNA.
  • This cDNA comprises sequences encoding the human STMST- 1 protein (i.e., "the coding region", from nucleotides 404- 1294), as well as 5' untranslated sequences (nucleotides 1-403) and 3' untranslated sequences (nucleotides 1295-2915).
  • the nucleic acid molecule can comprise only the coding region of SEQ ID NO:l (e.g., nucleotides 404-1294, corresponding to SEQ ID NO:3).
  • an isolated nucleic acid molecule of the invention comprises the nucleotide sequence shown in SEQ ID NO:4.
  • the sequence of SEQ ID NO:l corresponds to the human STMST-2 cDNA.
  • This cDNA comprises sequences encoding the human STMST-2 protein (i.e., "the coding region", from nucleotides 334-2160), as well as 5' untranslated sequences (nucleotides 1-333) and 3' untranslated sequences (nucleotides 2161-4166).
  • the nucleic acid molecule can comprise only the coding region of SEQ ID NO: 1 (e.g. , nucleotides 334- 2160, corresponding to SEQ ID NO:6).
  • an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement of the nucleotide sequence shown in SEQ ID NO:l, SEQ ID NO:4, or a portion of any of these nucleotide sequences.
  • a nucleic acid molecule which is complementary to the nucleotide sequence shown in SEQ ID NO:l or SEQ ID NO:4 is one which is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO:l or SEQ ID NO: 4 thereby forming a stable duplex.
  • an isolated nucleic acid molecule of the present invention comprises a nucleotide sequence which is at least about 60-65%, preferably at least about 70-75%, more preferable at least about 80-85%, and even more preferably at least about 90-95% or more homologous to the nucleotide sequences shown in SEQ ID NO:l or SEQ ID NO:4, or a portion of any of these nucleotide sequences.
  • the nucleic acid molecule of the invention can comprise only a portion of the nucleic acid sequence of SEQ ID NO:l or SEQ ID NO:4, for example a fragment which can be used as a probe or primer or a fragment encoding a biologically active portion of an STMST protein.
  • the nucleotide sequence determined from the cloning of the STMST- 1 genes allows for the generation of probes and primers designed for use in identifying and/or cloning other STMST family members, as well as STMST homologues from other species.
  • the probe/primer typically comprises substantially purified oligonucleotide.
  • the oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, preferably about 25, more preferably about 40, 50 or 75 consecutive nucleotides of a sense sequence of SEQ ID NO:l or SEQ ID NO:4, of an anti-sense sequence of SEQ ID NO:l or SEQ ID NO:4, or of a naturally occurring mutant of SEQ ID NO: 1 or SEQ ID NO:4.
  • a nucleic acid molecule of the present invention comprises a nucleotide sequence which is greater that 350, 351-450, 451-550, 551-650, 651-750, or 751-850, 851-950, 951-1050, 1051-1150, or 1151-1250 nucleotides in length and hybridizes under stringent hybridization conditions to a nucleic acid molecule of SEQ ID NO: l or SEQ ID NO:4. Probes based on the STMST nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins.
  • the probe further comprises a label group attached thereto, e.g., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
  • the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
  • Such probes can be used as a part of a diagnostic test kit for identifying cells or tissue which misexpress an STMST protein, such as by measuring a level of an STMST protein
  • STMST-encoding nucleic acid in a sample of cells from a subject e.g., detecting STMST mRNA levels or determining whether a genomic STMST gene has been mutated or deleted.
  • a nucleic acid fragment encoding a "biologically active portion of an STMST protein” can be prepared by isolating a portion of the nucleotide sequence of SEQ ID NO:l or SEQ ID NO:4 which encodes a polypeptide having an STMST biological activity (the biological activities of the STMST proteins have previously been described), expressing the encoded portion of the STMST protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of the STMST protein.
  • the invention further encompasses nucleic acid molecules that differ from the nucleotide sequence shown in SEQ ID NO:l or SEQ ID NO:4 due to degeneracy of the genetic code and thus encode the same STMST proteins as those encoded by the nucleotide sequence shown in SEQ ID NO:l or SEQ ID NO:4.
  • an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NO:2 or SE ID NO:5.
  • STMST nucleotide sequences shown in SEQ ID NO:l or SEQ ID NO:4 DNA sequence polymorphisms that lead to changes in the amino acid sequences of the STMST proteins may exist within a population (e.g. , the human population). Such genetic polymorphism in the STMST genes may exist among individuals within a population due to natural allelic variation.
  • the terms "gene” and "recombinant gene” refer to nucleic acid molecules comprising an open reading frame encoding an STMST protein, preferably a mammalian STMST protein.
  • Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of an STMST gene. Any and all such nucleotide variations and resulting amino acid polymorphisms in STMST genes that are the result of natural allelic variation and that do not alter the functional activity of an STMST protein are intended to be within the scope of the invention.
  • nucleic acid molecules encoding other STMST family members and thus which have a nucleotide sequence which differs from the STMST- 1 sequences of SEQ ID NO:l or SEQ ID NO:4 are intended to be within the scope of the invention.
  • an STMST-3 cDNA can be identified based on the nucleotide sequence of human STMST-1 or STMST-2.
  • nucleic acid molecules encoding STMST proteins from different species, and thus which have a nucleotide sequence which differs from the STMST sequences of SEQ ID NO:l or SEQ ID NO:4 are intended to be within the scope of the invention.
  • an mouse STMST cDNA can be identified based on the nucleotide sequence of a human STMST.
  • Nucleic acid molecules corresponding to natural allelic variants and homologues of the STMST cDNAs of the invention can be isolated based on their homology to the STMST nucleic acids disclosed herein using the cDNAs disclosed herein, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
  • an isolated nucleic acid molecule of the invention is at least 15 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l or SEQ ID NO:4.
  • the nucleic acid is at least 30, 50, 100, 250 or 500 nucleotides in length.
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other.
  • the conditions are such that sequences at least about 70%, more preferably at least about 80%, even more preferably at least about 85% or 90% homologous to each other typically remain hybridized to each other.
  • stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • a preferred, non- limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45C, followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50-65°C.
  • an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO:l or SEQ ID NO:4 or corresponds to a naturally-occurring nucleic acid molecule.
  • a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
  • allelic variants of the STMST sequences that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences of SEQ ID NO:l or SEQ ID NO:4, thereby leading to changes in the amino acid sequence of the encoded STMST proteins, without altering the functional ability of the STMST proteins.
  • nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence of SEQ ID NO: 1 or SEQ ID NO:4.
  • non-essential amino acid residue is a residue that can be altered from the wild-type sequence of STMST (e.g., the sequence of SEQ ID NO:2) without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity.
  • amino acid residues that are conserved among the STMST proteins of the present invention are predicted to be particularly unamenable to alteration.
  • amino acid residues that are defined by the 7 transmembrane signature profile and the spectrin ⁇ -chain, repeated domain signature profile are particularly unamenable to alteration.
  • amino acid residues that are conserved between the STMST proteins of the present invention and members of the G protein coupled receptor protein family are not likely to be amenable to alteration (e.g. , the conserved asn residue within the first TM domain,. asn25 of SEQ ID NO:2 or SEQ ID NO:5; the conserved cys in the first extracellular loop, cys83 of SEQ ID NO:2 or SEQ ID NO:5; the conserved arg at the interface of the third TM domain and the first cytoplasmic loop, arg 108 of SEQ Dl NO:2 or SEQ ID NO:5; the conserved trp and pro in the fourth TM domain, trpl35 and ⁇ rol45 pf SEQ ID NO:2 or SEQ ID NO:5; the conserved cys residue in the second extracellular domain, cysl ⁇ l of SEQ ID NO:2 or SEQ ID NO: 5; the conserved phe residue in the fifth TM domain, p
  • nucleic acid molecules encoding STMST proteins that contain changes in amino acid residues that are not essential for activity.
  • STMST proteins differ in amino acid sequence from SEQ ID NO:2 and SEQ ID NO:5 yet retain biological activity.
  • the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 75% homologous to the amino acid sequence of SEQ ID NO:2.
  • the protein encoded by the nucleic acid molecule is at least about 75-80% homologous to SEQ ID NO:2, more preferably at least about 80-85% homologous to SEQ ID NO:2, even more preferably at least about 85-90% homologous to SEQ ID NO:2, and even more preferably at least about 90-95% homologous to SEQ ID NO:2.
  • the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 65% homologous to the amino acid sequence of SEQ ID NO: 5.
  • the protein encoded by the nucleic acid is at least about 65-70% homologous to SEQ ID NO:2, more preferably at least about 70- 75% homologous to SEQ ID NO:5, even more preferably at least about 75-80%, and even more preferably at least about 80-85%, 85-90%, or 90-95% homologous to SEQ ID NO:5.
  • An isolated nucleic acid molecule encoding an STMST protein homologous to the protein of SEQ ID NO:2 or SEQ ID NO:5 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO:l, SEQ ID NO: 4, or such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into SEQ ID NO:l or SEQ ID NO:4 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues.
  • a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta- branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • a predicted nonessential amino acid residue in an STMST protein is preferably replaced with another amino acid residue from the same side chain family.
  • mutations can be introduced randomly along all or part of an STMST coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for STMST biological activity to identify mutants that retain activity. Following mutagenesis of SEQ ID NO:l, SEQ ID NO:4, or the encoded protein can be expressed recombinantly and the activity of the protein can be determined.
  • a mutant STMST protein can be assayed for the ability to (1) modulate cellular signal transduction, either in vitro or in vivo; (2) regulate gene transcription in a cell expressing an STMST protein; (3) regulate gene transcription in a cell expressing an STMST protein, wherein said cell is involved inflammation; (4) regulate cellular proliferation; (5) regulate cellular differentiation; (6) regulate development; (7) regulate cell death; (8) regulate inflammation; and (9) regulate respiratory cell function; and (10) regulate osteogenic cell function.
  • an antisense nucleic acid comprises a nucleotide sequence which is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid.
  • the antisense nucleic acid can be complementary to an entire STMST coding strand, or to only a portion thereof.
  • an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding STMST.
  • the term "coding region” refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues (e.g., the coding region of human STMST-1 corresponds to
  • the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding STMST.
  • noncoding region refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).
  • antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing.
  • the antisense nucleic acid molecule can be complementary to the entire coding region of STMST mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of STMST mRNA.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of STMST mRNA.
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
  • An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5- chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7- methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta- D-mannosylqueosine, 5'-methoxycarbox
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
  • the antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding an STMST protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation.
  • the hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix.
  • An example of a route of administration of antisense nucleic acid molecules of the invention include direct injection at a tissue site.
  • antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.
  • antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens.
  • the antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
  • the antisense nucleic acid molecule of the invention is an ⁇ -anomeric nucleic acid molecule.
  • An ⁇ -anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641).
  • the antisense nucleic acid molecule can also comprise a 2'-o- methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).
  • an antisense nucleic acid of the invention is a ribozyme.
  • Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region.
  • ribozymes e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave STMST mRNA transcripts to thereby inhibit translation of STMST mRNA.
  • a ribozyme having specificity for an STMST-encoding nucleic acid can be designed based upon the nucleotide sequence of an STMST- 1 or STMST-2 cDNA disclosed herein (i.e., SEQ ID NO:l or SEQ ID NO:4).
  • SEQ ID NO:l or SEQ ID NO:4 a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in an STMST- encoding mRNA. See, e.g., Cech et al. U.S. Patent No. 4,987,071; and Cech et al. U.S. Patent No. 5,116,742.
  • STMST mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J.W. (1993) Science 261:1411-1418.
  • STMST gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the STMST (e.g. , the STMST promoter and/or enhancers) to form triple helical structures that prevent transcription of the STMST gene in target cells.
  • nucleotide sequences complementary to the regulatory region of the STMST e.g. , the STMST promoter and/or enhancers
  • the STMST gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the STMST (e.g. , the STMST promoter and/or enhancers) to form triple helical structures that prevent transcription of the STMST gene in target cells.
  • the STMST nucleic acid molecules of the present invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule.
  • the deoxyribose phosphate backbone of the nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup B. et al. (1996) Bioorganic & Medicinal Chemistry 4 (1): 5-23).
  • peptide nucleic acids refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
  • the neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.
  • the synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup B. et al. (1996) supra; Perry-O'Keefe et al. PNAS 93: 14670-675.
  • PNAs of STMST nucleic acid molecules can be used in therapeutic and diagnostic applications.
  • PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, for example, inducing transcription or translation arrest or inhibiting replication.
  • PNAs of STMST nucleic acid molecules can also be used in the analysis of single base pair mutations in a gene, (e.g. , by PNA-directed PCR clamping); as 'artificial restriction enzymes' when used in combination with other enzymes, (e.g., SI nucleases (Hyrup B. (1996) supra)); or as probes or primers for DNA sequencing or hybridization (Hyrup B. et al.
  • PNAs of STMST can be modified, (e.g., to enhance their stability or cellular uptake), by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.
  • PNA-DNA chimeras of STMST nucleic acid molecules can be generated which may combine the advantageous properties of PNA and DNA.
  • DNA recognition enzymes e.g. , RNAse H and DNA polymerases
  • PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup B. (1996) supra).
  • the synthesis of PNA-DNA chimeras can be performed as described in Hyrup B. (1996) supra and Finn P.J. et al. (1996) Nucleic Acids Res. 24 (17): 3357-63.
  • a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry and modified nucleoside analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can be used as a between the PNA and the 5' end of DNA (Mag, M. et al. (1989) Nucleic Acid Res. 17: 5973-88). PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn P.J. et al. (1996) supra).
  • modified nucleoside analogs e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite
  • chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment (Peterser, K.H. et al. (1975) Bioorganic Med. Chem. Lett. 5: 1119-11124).
  • the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. US. 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci.
  • oligonucleotides can be modified with hybridization-triggered cleavage agents (See, e.g., Krol et al. (1988) BioTechniques 6:958-976) or intercalating agents. (See, e.g, Zon (1988) Pharm. Res. 5:539-549).
  • the oligonucleotide may be conjugated to another molecule, (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization-triggered cleavage agent).
  • STMST molecules of the present invention are also within the scope of the present invention: isolated nucleic acids which encode and STMST ligands or STMST modulators, probes and/or primers useful for identifying STMST ligands or STMST modulators based on the sequences of nucleic acids which encode and STMST ligands or STMST modulators, isolated nucleic acid molecules which are complementary or antisense to the sequences of nucleic acids which encode and STMST ligands or STMST modulators, isolated nucleic acid molecules which are at least about 60-65%, preferably at least about 70-75%, more preferable at least about 80-85%, and even more preferably at least about 90-95% or more homologous to the sequences of nucleic acids which encode and STMST ligands or STM
  • One aspect of the invention pertains to isolated STMST proteins, and biologically active portions thereof, as well as polypeptide fragments suitable for use as immunogens to raise anti-STMST antibodies.
  • native STMST proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques.
  • STMST proteins are produced by recombinant DNA techniques.
  • Alternative to recombinant expression an STMST protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
  • an “isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the STMST protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • the language “substantially free of cellular material” includes preparations of STMST protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinanfly produced.
  • the language "substantially free of cellular material” includes preparations of STMST protein having less than about 30% (by dry weight) of non-STMST protein (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-STMST protein, still more preferably less than about 10% of non-STMST protein, and most preferably less than about 5% non-STMST protein.
  • non-STMST protein also referred to herein as a "contaminating protein”
  • the STMST protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of STMST protein in which the protein is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of STMST protein having less than about 30%) (by dry weight) of chemical precursors or non-STMST chemicals, more preferably less than about 20% chemical precursors or non-STMST chemicals, still more preferably less than about 10% chemical precursors or non-STMST chemicals, and most preferably less than about 5% chemical precursors or non-STMST chemicals.
  • Biologically active portions of an STMST protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence of the STMST protein, e.g., the amino acid sequence shown in SEQ ID NO:2 or the amino acid sequence shown in SEQ ID NO:5, which include less amino acids than the full length STMST proteins, and exhibit at least one activity of an STMST protein.
  • biologically active portions comprise a domain or motif with at least one activity of the STMST protein.
  • a biologically active portion of an STMST protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length.
  • a biologically active portion of an STMST protein comprises at least a transmembrane domain.
  • a biologically active portion of an STMST protein comprises at least one 7 transmembrane receptor profile. In another embodiment, a biologically active portion of an STMST protein comprises at least a spectrin a-chain, repeated domain profile. In another embodiment a biologically active portion of an STMST protein comprises at least a 7 transmembrane receptor profile and a spectrin ⁇ -chain profile.
  • a preferred biologically active portion of an STMST protein of the present invention may contain at least one of the above-identified structural domains and/or profiles.
  • a more preferred biologically active portion of an STMST protein may contain at least two of the above-identified structural domains and/or profiles.
  • other biologically active portions in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native STMST protein.
  • the STMST protein has an amino acid sequence shown in SEQ ID NO:2.
  • the STMST protein is substantially homologous to SEQ ID NO:2, and retains the functional activity of the protein of SEQ ID NO:2, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail in subsection I above.
  • the STMST protein is a protein which comprises an amino acid sequence at least about 75% homologous to the amino acid sequence of SEQ ID NO:2 and retains the functional activity of the STMST proteins of SEQ ID NO:2, respectively.
  • the protein is at least about 75-80% homologous to SEQ ID NO:2, more preferably at least about 80-85% homologous to SEQ ID NO:2, even more preferably at least about 85-90% homologous to SEQ ID NO:2, and most preferably at least about 90- 95% or more homologous to SEQ ID NO:2.
  • the STMST protein has an amino acid sequence shown in SEQ ID NO:5.
  • the STMST protein is substantially homologous to SEQ ID NO:5, and retains the functional activity of the protein of SEQ ID NO:5, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail in subsection I above.
  • the STMST protein is a protein which comprises an amino acid sequence at least about 65% homologous to the amino acid sequence of SEQ ID NO:2 and retains the functional activity of the STMST proteins of SEQ ID NO:2, respectively.
  • the protein is at least about 65-70%% homologous to SEQ ID NO:2, more preferably at least about 70-75% homologous to SEQ ID NO:2, even more preferably at least about 75-80% homologous to SEQ ID NO:2, and most preferably at least about 80- 85%,85-90%, or 90-95% homologous to SEQ ID NO:2.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence and non-homologous sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, or 90% of the length of the reference sequence (e.g., when aligning a second sequence to the STMST amino acid sequence of SEQ ID NO:2 having 293 amino acid residues, at least 88, preferably at least 117, more preferably at least 147, even more preferably at least 176, and even more preferably at least 205, 234 or 264 amino acid residues are aligned).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • amino acid or nucleic acid "homology” is equivalent to amino acid or nucleic acid "identity”).
  • the comparison of sequences and determination of percent homology between two sequences can be accomplished using a mathematical algorithim.
  • a preferred, non- limiting example of a mathematical algorithim utilized for the comparison of sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.
  • Gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic Acids Research 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.
  • an STMST “chimeric protein” or “fusion protein” comprises an STMST polypeptide operatively linked to a non-STMST polypeptide.
  • a “STMST polypeptide” refers to a polypeptide having an amino acid sequence corresponding to STMST
  • a non- STMST polypeptide refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the STMST protein, e.g., a protein which is different from the STMST protein and which is derived from the same or a different organism.
  • an STMST fusion protein the STMST polypeptide can correspond to all or a portion of an STMST protein.
  • an STMST fusion protein comprises at least one biologically active portion of an STMST protein.
  • an STMST fusion protein comprises at least two biologically active portions of an STMST protein.
  • the term "operatively linked" is intended to indicate that the STMST polypeptide and the non-STMST polypeptide are fused in-frame to each other.
  • the non-STMST polypeptide can be fused to the N-terminus or C-terminus of the STMST polypeptide.
  • the fusion protein is a GST-STMST fusion protein in which the STMST sequences are fused to the C-terminus of the GST sequences.
  • Such fusion proteins can facilitate the purification of recombinant STMST.
  • the fusion protein is an STMST protein containing a heterologous signal sequence at its N-terminus.
  • a native STMST signal sequence can be removed and replaced with a signal sequence from another protein.
  • expression and/or secretion of STMST can be increased through use of a heterologous signal sequence.
  • the STMST fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject in vivo.
  • the STMST fusion proteins can be used to affect the bioavailability of an STMST substrate.
  • Use of STMST fusion proteins may be useful therapeutically for the treatment of respiratory disorders (e.g., asthma).
  • the STMST-fusion proteins of the invention can be used as immunogens to produce anti-STMST antibodies in a subject, to purify STMST ligands and in screening assays to identify molecules which inhibit the interaction of STMST with an STMST ligand.
  • an STMST chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques.
  • DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).
  • anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence
  • many expression vectors are commercially available that already encode a fusion moiety (e.g. , a GST polypeptide).
  • An STMST- encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the STMST protein.
  • the present invention also pertains to variants of the STMST proteins which function as either STMST agonists (mimetics) or as STMST antagonists.
  • Variants of the STMST proteins can be generated by mutagenesis, e.g. , discrete point mutation or truncation of an STMST protein.
  • An agonist of the STMST proteins can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of an STMST protein.
  • An antagonist of an STMST protein can inhibit one or more of the activities of the naturally occurring form of the STMST protein by, for example, competitively inhibiting the protease activity of an STMST protein.
  • specific biological effects can be elicited by treatment with a variant of limited function.
  • treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the STMST protein.
  • variants of an STMST protein which function as either
  • STMST agonists or as STMST antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of an STMST protein for STMST protein agonist or antagonist activity.
  • a variegated library of STMST variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library.
  • a variegated library of STMST variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential STMST sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of STMST sequences therein.
  • a degenerate set of potential STMST sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of STMST sequences therein.
  • STMST variants from a degenerate oligonucleotide sequence Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential STMST sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang, S.A. (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477.
  • libraries of fragments of an STMST protein coding sequence can be used to generate a variegated population of STMST fragments for screening and subsequent selection of variants of an STMST protein.
  • a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of an STMST coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S 1 nuclease, and ligating the resulting fragment library into an expression vector.
  • an expression library can be derived which encodes N-terminal, C-terminal and internal fragments of various sizes of the STMST protein.
  • Recrusive ensemble mutagenesis (REM), a new technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify STMST variants (Arkin and Yourvan (1992) PNAS S9:7811 -7815; Delgrave et al. (1993) Protein Engineering 6(3)327-331).
  • cell based assays can be exploited to analyze a variegated STMST library.
  • a library of expression vectors can be transfected into a cell line which ordinarily synthesizes STMST.
  • the transfected cells are then cultured such that a particular mutant STMST is expressed and the effect of expression of the mutant on STMST activity in the cell can be detected, e.g. , by any of a number of activity assays for native STMST protein.
  • Plasmid DNA can then be recovered from the cells which score for modulated STMST activity, and the individual clones further characterized.
  • An isolated STMST protein, or a portion or fragment thereof, can be used as an immunogen to generate antibodies that bind STMST using standard techniques for polyclonal and monoclonal antibody preparation.
  • a full-length STMST protein can be used or, alternatively, the invention provides antigenic peptide fragments of STMST for use as immunogens.
  • the antigenic peptide of STMST comprises at least 8 amino acid residues of the amino acid sequence shown in SEQ ID NO:2 and encompasses an epitope of STMST such that an antibody raised against the peptide forms a specific immune complex with STMST.
  • the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.
  • Preferred epitopes encompassed by the antigenic peptide are regions of STMST that are located on the surface of the protein, e.g. , hydrophilic regions.
  • An STMST immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal) with the immunogen.
  • An appropriate immunogenic preparation can contain, for example, recombinantly expressed STMST protein or a chemically synthesized STMST polypeptide.
  • the preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent. Immunization of a suitable subject with an immunogenic STMST preparation induces a polyclonal anti-STMST antibody response.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen, such as STMST.
  • immunologically active portions of immunoglobulin molecules include F(ab) and F(ab')2 fragments which can be generated by treating the antibody with an enzyme such as pepsin.
  • the invention provides polyclonal and monoclonal antibodies that bind STMST.
  • polyclonal antibody refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of STMST.
  • a monoclonal antibody composition thus typically displays a single binding affinity for a particular STMST protein with which it immunoreacts.
  • Polyclonal anti-STMST antibodies can be prepared as described above by immunizing a suitable subject with an STMST immunogen. The anti-STMST antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized STMST.
  • ELISA enzyme linked immunosorbent assay
  • the antibody molecules directed against STMST can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction.
  • antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256:495-497) (see also, Brown et al. (1981) J. Immunol. 127:539-46; Brown et al. (1980) J. Biol.
  • an immortal cell line typically a myeloma
  • lymphocytes typically splenocytes
  • the immortal cell line e.g., a myeloma cell line
  • the immortal cell line is derived from the same mammalian species as the lymphocytes.
  • murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the present invention with an immortalized mouse cell line.
  • Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium").
  • HAT medium culture medium containing hypoxanthine, aminopterin and thymidine
  • Any of a number of myeloma cell lines can be used as a fusion partner according to standard techniques, e.g, the P3-NSl/l-Ag4-l, P3-x63-Ag8.653 or Sp2/O-Agl4 myeloma lines. These myeloma lines are available from ATCC.
  • HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol ("PEG").
  • PEG polyethylene glycol
  • Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed).
  • Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind STMST, e.g., using a standard ELISA assay.
  • a monoclonal anti-STMST antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with STMST to thereby isolate immunoglobulin library members that bind STMST.
  • Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27- 9400-01 ; and the Stratagene SurfZAPTM Phage Display Kit, Catalog No. 240612).
  • examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, Ladner et al.
  • recombinant anti-STMST antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention.
  • Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in Robinson et al. International Application No. PCT/US 86/02269; Akira, et al. European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al. European Patent Application 173,494; Neuberger et al. PCT International Publication No.
  • An anti-STMST antibody (e.g., monoclonal antibody) can be used to isolate STMST by standard techniques, such as affinity chromatography or immunoprecipitation.
  • An anti-STMST antibody can facilitate the purification of natural STMST from cells and of recombinantly produced STMST expressed in host cells.
  • an anti-STMST antibody can be used to detect STMST protein (e.g. , in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the STMST protein.
  • Anti-STMST antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, -galactosidase, or acetylcholinesterase;
  • suitable prosthetic 5 group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable
  • radioactive material include 125 1, 131 1, 35 S or 3 H.
  • STMST molecules of the present invention are in the screening for STMST ligands (e.g., surrogate ligands) and/or STMST modulators, it is intended that the following are also within the scope of the present invention: "isolated” or “purified” STMST ligands or STMST modulators,
  • vectors preferably expression vectors, containing a nucleic acid encoding an STMST protein (or a portion thereof).
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. , bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non-episomal mammalian vectors
  • Other vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • Such vectors are referred to herein as "expression vectors".
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and vector can be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g. , replication defective retro viruses, adenoviruses and adeno- associated viruses), which serve equivalent functions.
  • the recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed.
  • "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
  • the expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g. , STMST proteins, mutant forms of STMST proteins, fusion proteins, etc.).
  • the recombinant expression vectors of the invention can be designed for expression of STMST proteins in prokaryotic or eukaryotic cells.
  • STMST proteins can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase. Expression of proteins in prokaryotes is most often carried out in E.
  • Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein.
  • Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D.B. and Johnson, K.S.
  • an STMST fusion protein expressed in a retroviral expression vector of the present invention can be utilized to infect bone marrow cells which are subsequently transplanted into irradiated recipients.
  • E. coli expression vectors include pTrc (Amann et al, (1988) Gene 69:301-315) andpET l id (Studier et al, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89).
  • Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter.
  • Target gene expression from the pET l id vector relies on transcription from a T7 gnlO-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gnl).
  • This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident prophage harboring a T7 gnl gene under the transcriptional control of the lacUV 5 promoter.
  • One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128).
  • Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al, (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
  • the STMST expression vector is a yeast expression vector.
  • yeast expression vectors for expression in yeast S. cerivisae include pYepSecl (Baldari, et al, (1987) Embo J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al, (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, CA), and picZ (InVitrogen Corp, San Diego, CA).
  • STMST proteins can be expressed in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).
  • a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include ⁇ CDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195).
  • the expression vector's control functions are often provided by viral regulatory elements.
  • promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
  • suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
  • the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g. , tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific regulatory elements are known in the art.
  • suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBOJ.
  • promoters are also encompassed, for example the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the ⁇ -fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).
  • the invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to STMST mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA.
  • the antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced.
  • a high efficiency regulatory region the activity of which can be determined by the cell type into which the vector is introduced.
  • host cell and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • an STMST protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g. , DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, D ⁇ A ⁇ -dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A
  • a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
  • selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate.
  • Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding an STMST protein or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
  • a host cell of the invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i. e. , express) an STMST protein.
  • the invention further provides methods for producing an STMST protein using the host cells of the invention.
  • the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding an STMST protein has been introduced) in a suitable medium such that an STMST protein is produced.
  • the method further comprises isolating an STMST protein from the medium or the host cell.
  • the host cells of the invention can also be used to produce nonhuman transgenic animals.
  • a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which STMST-coding sequences have been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous STMST sequences have been introduced into their genome or homologous recombinant animals in which endogenous STMST sequences have been altered. Such animals are useful for studying the function and/or activity of an STMST and for identifying and/or evaluating modulators of STMST activity.
  • a "transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene.
  • transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc.
  • a transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal.
  • a "homologous recombinant animal” is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous STMST gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
  • a transgenic animal of the invention can be created by introducing an STMST- encoding nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinj ection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal.
  • the STMST-1 cDNA sequence e.g., that of SEQ ID NO: 1 or SEQ ID NO:4
  • a nonhuman homologue of a human STMST gene such as a mouse or rat STMST gene, can be used as a transgene.
  • an STMST gene homologue such as an STMST-3 gene can be isolated based on hybridization to the STMST cDNA sequences of SEQ ID NO:l or SEQ ID NO:4 (described further in subsection I above) and used as a transgene.
  • Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene.
  • a tissue-specific regulatory sequence(s) can be operably linked to an STMST transgene to direct expression of an STMST protein to particular cells.
  • transgenic founder animal can be identified based upon the presence of an STMST transgene in its genome and/or expression of STMST mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding an STMST protein can further be bred to other transgenic animals carrying other transgenes.
  • a vector which contains at least a portion of an STMST gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the STMST gene.
  • the STMST gene can be a human gene (e.g. , the cDNA of SEQ ID NO: 1 or SEQ ID NO:4), but more preferably, is a non-human homologue of a human STMST gene (e.g., a cDNA isolated by stringent hybridization with the nucleotide sequence of SEQ ID NO:l or SEQ ID NO:4).
  • a mouse STMST gene can be used to construct a homologous recombination vector suitable for altering an endogenous STMST gene in the mouse genome.
  • the vector is designed such that, upon homologous recombination, the endogenous STMST gene is functionally disrupted (i.e., , no longer encodes a functional protein; also referred to as a "knock out" vector).
  • the vector can be designed such that, upon homologous recombination, the endogenous STMST gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous STMST protein).
  • the altered portion of the STMST gene is flanked at its 5' and 3' ends by additional nucleic acid sequence of the STMST gene to allow for homologous recombination to occur between the exogenous STMST gene carried by the vector and an endogenous STMST gene in an embryonic stem cell.
  • the additional flanking STMST nucleic acid sequence is of sufficient length for successful homologous recombination with the endogenous gene.
  • flanking DNA both at the 5' and 3' ends
  • are included in the vector see e.g., Thomas, K.R. and Capecchi, M. R.
  • the vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced STMST gene has homologously recombined with the endogenous STMST gene are selected (see e.g., Li, E. et al. (1992) Cell 69:915).
  • the selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see e.g., Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, EJ. Robertson, ed.
  • a chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term.
  • Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene.
  • Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley, A. (1991) Current Opinion in Biotechnology 2:823-829 and in PCT International Publication Nos.: WO 90/11354 by Le Mouellec et al.
  • transgenic non-humans animals can be produced which contain selected systems which allow for regulated expression of the transgene.
  • One example of such a system is the cre/loxP recombinase system of bacteriophage PI .
  • cre/loxP recombinase system see, e.g., Lakso et al. (1992) PNAS 89:6232-6236.
  • a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al. ( 1991 ) Science 251:1351-1355. If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
  • Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, I. et al. (1997) Nature 385:810- 813.
  • a cell e.g., a somatic cell
  • the quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated.
  • the recontructed oocyte is then cultured such that it develops to morula or blastocyte and then transferred to pseudopregnant female foster animal.
  • the offspring borne of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.
  • compositions suitable for administration can be incorporated into pharmaceutical compositions suitable for administration.
  • Such compositions typically comprise the nucleic acid molecule, protein, antibody, or modulatory compound and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound (e.g., an STMST protein or anti-STMST antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets.
  • the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules.
  • Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.
  • Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the .
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • a sweetening agent such as sucrose or saccharin
  • a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • suppositories e.g., with conventional suppository bases such as cocoa butter and other glycerides
  • retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polygly colic acid, collagen, polyorthoesters, and polylactic acid.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50%) of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio
  • LD50/ED50 Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
  • the nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Patent 5,328,470) or by stereotactic injection (see e.g. , Chen et al (1994) PNAS 91 :3054-3057).
  • the pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
  • the pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • nucleic acid molecules, proteins, protein homologues, and antibodies described herein can be used in one or more of the following methods: a) screening assays; b) predictive medicine (e.g., diagnostic assays, prognostic assays, monitoring clinical trials, and pharmacogenetics); and c) methods of treatment (e.g., therapeutic and prophylactic).
  • an STMST protein of the invention has one or more of the following activities: (i) interaction of an STMST protein with soluble STMST ligand; (ii) interaction of an STMST protein with a membrane-bound non-STMST protein; (iii) interaction of an STMST protein with an intracellular protein (e.g., an intracellular enzyme or signal transduction molecule); and (iv) indirect interaction of an STMST protein with an intracellular protein (e.g., a downstream signal transduction molecule, and can thus be used, for example, in at least one of the following.
  • an intracellular protein e.g., an intracellular enzyme or signal transduction molecule
  • indirect interaction of an STMST protein with an intracellular protein e.g., a downstream signal transduction molecule
  • an STMST activity for example, a hypothalamic activity, is at least one or more of the following activities: (1) modulation (e.g., repression or stimulation) of brain anabolic circuits or pathways; (2) modulation (e.g., repression or stimulation of brain catabolic pathways; (3) modulation of food intake and/or feeding behavior (e.g., stimulation of or inhibition/suppression of food intake and/or feeding behavior); (4) modulation of energy expenditure (e.g., suppression or stimulation of energy expenditure); (5) regulation of energy homeostasis; (6) regulation of body fat mass; (7) regulation of body temperature; (8) regulation of the sleep-wake cycle; (9) regulation of memory and/or behavior; (10) control of thirst; and (11) regulation of autonomic nervous system function; (12) modulation of cellular signal transduction, either in vitro or in vivo; (13) regulation of gene transcription in a cell expressing an STMST protein; (14) regulation of gene transcription in a cell expressing an STMST
  • hypothalamic dysfunction includes a mis-regulated or aberrantly regulated function or activity attributed to the hypothalamus in an animal (e.g. , in a human), for example, a mis-regulated or aberrantly regulated hypothalamic activity, as described herein.
  • hypothalamic disorder includes a disease or disorder characterized by at least one phenotypic manifestation (e.g., a clinically detectable manifestation or symptom) of a hypothalamic dysfunction, as defined herein.
  • Modulation of an STMST activity as described above may be included as part of a multi-drug regime that targets multiple sites within the weight regulatory system, temperature regulatory system, sleep-wake cycle control system, memory and/or behavior regulatory systems, thirst regulatory system and/or autonomic nervous system.
  • an STMST modulator is useful for (1) modulating bone homeostasis (e.g., stimulation of bone homeostasis) and/or modulation of bone formation (e.g.
  • bone-related and/or bone resorption disorders including, but not limited to osteoporosis, Paget's disease, osteoarthritis, degenerative arthritis, osteogenesis imperfecta, fibrous displasia, hypophosphatasia, bone sarcoma, myeloma bone disorder (e.g., osteolytic bone lesions) and hypercalcemia; (3) managment of bone fragility (e.g., decrease bone fragility); and (4) prevention and/or treatment of bone pain, bone deformaties, and/or bone fractures.
  • bone-related and/or bone resorption disorders including, but not limited to osteoporosis, Paget's disease, osteoarthritis, degenerative arthritis, osteogenesis imperfecta, fibrous displasia, hypophosphatasia, bone sarcoma, myeloma bone disorder (e.g., osteolytic bone lesions) and hypercalcemia
  • managment of bone fragility e.g., decrease bone fragility
  • the isolated nucleic acid molecules of the invention can be used, for example, to express STMST protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect STMST mRNA (e.g., in a biological sample) or a genetic alteration in an STMST gene, and to modulate STMST activity, as described further below.
  • STMST proteins can be used to treat disorders characterized by insufficient or excessive production of an STMST protein and/or STMST ligand.
  • the STMST proteins can be used to screen drugs or compounds which modulate the STMST activity as well as to treat disorders characterized by insufficient or excessive production of STMST protein or production of STMST protein forms which have decreased or aberrant activity compared to STMST wild type protein.
  • the anti-STMST antibodies of the invention can be used to detect and isolate STMST proteins, regulate the bioavailability of STMST proteins, and modulate STMST activity.
  • the invention provides a method (also referred to herein as a "screening assay") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) which bind to STMST proteins, or have a stimulatory or inhibitory effect on, for example, STMST expression or STMST activity.
  • modulators i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) which bind to STMST proteins, or have a stimulatory or inhibitory effect on, for example, STMST expression or STMST activity.
  • the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of an STMST protein or polypeptide or biologically active portion thereof.
  • test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one-compound' library method; and synthetic library methods using affinity chromatography selection.
  • biological libraries include biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one-compound' library method; and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K.S. (1997) Anticancer Drug Des. 12:145).
  • an assay is a cell-based assay in which a cell which expresses an STMST protein on the cell surface is contacted with a test compound and the ability of the test compound to bind to the STMST protein determined.
  • the cell for example, can be of mammalian origin or a yeast cell. Determining the ability of the test compound to bind to an STMST protein can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the STMST protein can be determined by detecting the labeled compound in a complex.
  • test compounds can be labeled with ⁇ 1, 35s, l ⁇ C, or ⁇ H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting.
  • test compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
  • a microphysiometer can be used to detect the interaction of a test compound with an STMST protein without the labeling of either the test compound or the receptor. McConnell, H. M. et al (1992) Science 257:1906-1912.
  • a "microphysiometer” e.g., CytosensorTM
  • LAPS light-addressable potentiometric sensor
  • the assay comprises contacting a cell which expresses an STMST protein or biologically active portion thereof, on the cell surface with an STMST ligand (e.g., a peptide ligand or neurotransmitter), to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the STMST protein or biologically active portion thereof, wherein determining the ability of the test compound to interact with the
  • STMST protein or biologically active portion thereof comprises determining the ability of the test compound to preferentially bind to the STMST protein or biologically active portion thereof, as compared to the ability of the STMST ligand to bind to the STMST protein or biologically active portion thereof.
  • Determining the ability of the STMST ligand or STMST modulator to bind to or interact with an STMST protein or biologically active portion thereof can be accomplished by one of the methods described above for determining direct binding.
  • determining the ability of the STMST ligand or modulator to bind to or interact with an STMST protein or biologically active portion thereof can be accomplished by determining the activity of an STMST protein or of a downstream STMST target molecule.
  • the target molecule can be a cellular second messenger, and the activity of the target molecule can be determined by detecting induction of of the target (i.e.
  • a reporter gene comprising an STMST-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase
  • a cellular response for example, a proliferative response, an inflammatory response or a hypothalamic response.
  • the present invention involves a method of identifying a compound which modulates the activity of an STMST protein, comprising contacting a cell which expresses an STMST protein with a test compound, determining the ability of the test compound to modulate the activity the STMST protein, and identifying the compound as a modulator of STMST activity.
  • the present invention involves a method of identifying a compound which modulates the activity of an STMST protein, comprising contacting a cell which expresses an STMST protein with a test compound, determining the ability of the test compound to modulate the activity of a downstream STMST target molecule, and identifying the compound as a modulator of STMST activity.
  • an assay of the present invention is a cell-free assay in which an STMST protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to bind to the STMST protein or biologically active portion thereof is determined. Binding of the test compound to the STMST protein can be determined either directly or indirectly as described above.
  • Binding of the test compound to the STMST protein can also be accomplished using a technology such as real-time Biomolecular Interaction Analysis (BIA). Sjolander, S. and Urbaniczky, C. (1991) Anal Chem. 63:2338-2345 and Szabo et al (1995) Curr. Opin. Struct. Biol. 5:699-705.
  • BIOA Biomolecular Interaction Analysis
  • the assay includes contacting the STMST protein or biologically active portion thereof with a known ligand (e.g., a peptide ligand or neurotransmitter) which binds STMST to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with an STMST protein, wherein determining the ability of the test compound to interact with an STMST protein comprises determining the ability of the test compound to preferentially bind to STMST or biologically active portion thereof as compared to the known ligand.
  • a known ligand e.g., a peptide ligand or neurotransmitter
  • the assay is a cell-free assay in which an STMST protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the STMST protein or biologically active portion thereof is determined.
  • Determining the ability of the test compound to modulate the activity of an STMST protein can be accomplished, for example, by determining the ability of the STMST protein to modulate the activity of a downstream STMST target molecule by one of the methods described above for cell- based assays. For example, the catalytic/enzymatic activity of the target molecule on an appropriate substrate can be determined as previously described.
  • the cell-free assay involves contacting an STMST protein or biologically active portion thereof with a known ligand (e.g., a peptide ligand or neurotransmitter) which binds the STMST protein to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the STMST protein, wherein determining the ability of the test compound to interact with the STMST protein comprises determining the ability of the test compound to preferentially bind to or modulate the activity of an STMST target molecule, as compared to the known ligand.
  • a known ligand e.g., a peptide ligand or neurotransmitter
  • the cell-free assays of the present invention are amenable to use of both soluble and/or membrane-bound forms of isolated proteins (e.g. STMST proteins or biologically active portions thereof or STMST proteins).
  • isolated proteins e.g. STMST proteins or biologically active portions thereof or STMST proteins.
  • a membrane-bound form an isolated protein e.g., an STMST protein
  • non-ionic detergents such as n-o
  • binding of a test compound to an STMST protein, or interaction of an STMST protein with a target molecule in the presence and absence of a candidate compound can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro-centrifuge tubes.
  • a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix.
  • glutathione-S -transferase/ STMST fusion proteins or glutathione-S-transferase/target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtitre plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or STMST protein, and the mixture incubated under conditions conducive to complex formation (e.g. , at physiological conditions for salt and pH). Following incubation, the beads or microtitre plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of STMST binding or activity determined using standard techniques.
  • an STMST protein or an STMST target molecule can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated STMST protein or target molecules can be prepared from biotin-NHS (N- hydroxy-succinimide) using techniques well known in the art (e.g. , biotinylation kit,
  • modulators of STMST expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of STMST mRNA or protein in the cell is determined.
  • the level of expression of STMST mRNA or protein in the presence of the candidate compound is compared to the level of expression of STMST mRNA or protein in the absence of the candidate compound.
  • the candidate compound can then be identified as a modulator of STMST expression based on this comparison. For example, when expression of STMST mRNA or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of STMST mRNA or protein expression.
  • the candidate compound when expression of STMST mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of STMST mRNA or protein expression.
  • the level of STMST mRNA or protein expression in the cells can be determined by methods described herein for detecting STMST mRNA or protein.
  • the STMST proteins can be used as "bait proteins" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al.
  • STMST-binding proteins proteins which bind to or interact with STMST
  • STMST-binding proteins proteins which bind to or interact with STMST
  • STMST-binding proteins are also likely to be involved in the propagation of signals by the STMST proteins as, for example, downstream elements of an STMST-mediated signaling pathway.
  • STMST-binding proteins are likely to be cell-surface molecules associated with non- STMST expressing cells, wherein such STMST-binding proteins are involved in chemoattraction.
  • the two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains.
  • the assay utilizes two different DNA constructs.
  • the gene that codes for an STMST protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4).
  • a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey" or "sample”) is fused to a gene that codes for the activation domain of the known transcription factor.
  • the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the STMST protein.
  • a reporter gene e.g., LacZ
  • Secondary screening assays can also be used to confirm the identification of an STMST modulator, for example, a compound identified according to one of the above- described screening assays (e.g., high throughput screening assays).
  • candidate compounds of interest can be assayed in vivo for a variety of serum biomarkers and/or selected histomorphometric parameters of bone formation and resorption including, but not limited to, levels of serum osteocalcin, percentage of osteoblast surface, percentage of osteoclast surface, percentage of osteoid surface, percentage of bone volume, trabecular thickness and bone formation.
  • the present invention further features assays (e.g., secondary screening assays or validation assays) designed to confirm the activity of a test compound, for example, as an STMST modulator.
  • the invention features screening assays (e.g., secondary screening assays or validation assays) which include administering a test compound, for example, a test compound that demonstrates binding to an STMST protein or modulation of an STMST activity in at least one of the above-described cell- based or cell-free assays, to an animal and determining the ability of the test compound to modulate STMST activity in vivo.
  • Determining the ability of a compound to modulate activity in vivo can include, for example, determining the ability of the compound to modulate hypothalamic activity.
  • Exemplary animals for determining STMST modulatory activity include normal animals as well as animal models of hypothalamic dysfunction. It is also within the scope of this invention to use an agent or compound as described herein (e.g., an STMST modulating agent, an antisense STMST nucleic acid molecule, an STMST-specific antibody, or an STMST-binding partner) in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent.
  • This invention further pertains to novel agents identified by the above- described screening assays. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein. Moreover, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model.
  • an agent identified as described herein e.g., an STMST modulating agent, an antisense STMST nucleic acid molecule, an STMST-specific antibody, or an STMST-binding partner
  • an agent identified as described herein can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent.
  • an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent.
  • cDNA sequences identified herein can be used in numerous ways as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome; and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. These applications are described in the subsections below.
  • this sequence can be used to map the location of the gene on a chromosome. This process is called chromosome mapping. Accordingly, portions or fragments of the STMST nucleotide sequences, described herein, can be used to map the location of the STMST genes on a chromosome. The mapping of the STMST sequences to chromosomes is an important first step in correlating these sequences with genes associated with disease.
  • STMST genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the STMST nucleotide sequences. Computer analysis of the STMST sequences can be used to predict primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the STMST sequences will yield an amplified fragment. Somatic cell hybrids are prepared by fusing somatic cells from different mammals (e.g., human and mouse cells).
  • PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the STMST nucleotide sequences to design oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes. Other mapping strategies which can similarly be used to map a 9o, lp, or 1 v sequence to its chromosome include in situ hybridization (described in Fan, Y. et al. (1990) PNAS, 87:6223-27), pre-screening with labeled flow- sorted chromosomes, and pre-selection by hybridization to chromosome specific cDNA libraries.
  • Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step.
  • Chromosome spreads can be made using cells whose division has been blocked in metaphase by a chemical such as colcemid that disrupts the mitotic spindle.
  • the chromosomes can be treated briefly with trypsin, and then stained with Giemsa. A pattern of light and dark bands develops on each chromosome, so that the chromosomes can be identified individually.
  • the FISH technique can be used with a DNA sequence as short as 500 or 600 bases.
  • clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection.
  • 1,000 bases, and more preferably 2,000 bases will suffice to get good results at a reasonable amount of time.
  • Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.
  • differences in the DNA sequences between individuals affected and unaffected with a disease associated with the STMST gene can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.
  • the STMST sequences of the present invention can also be used to identify individuals from minute biological samples.
  • the United States military for example, is considering the use of restriction fragment length polymo ⁇ hism (RFLP) for identification of its personnel.
  • RFLP restriction fragment length polymo ⁇ hism
  • an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification.
  • This method does not suffer from the current limitations of "Dog Tags" which can be lost, switched, or stolen, making positive identification difficult.
  • the sequences of the present invention are useful as additional DNA markers for RFLP (described in U.S. Patent 5,272,057).
  • sequences of the present invention can be used to provide an alternative technique which determines the actual base-by-base DNA sequence of selected portions of an individual's genome.
  • the STMST nucleotide sequences described herein can be used to prepare two PCR primers from the 5' and 3' ends of the sequences. These primers can then be used to amplify an individual's DNA and . subsequently sequence it.
  • Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences.
  • the sequences of the present invention can be used to obtain such identification sequences from individuals and from tissue.
  • the STMST nucleotide sequences of the invention uniquely represent portions of the human genome. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases.
  • Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes.
  • the noncoding sequences of SEQ ID NO: 1 can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers which each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEQ ID NO:3 are used, a more appropriate number of primers for positive individual identification would be 500-2,000.
  • a panel of reagents from STMST nucleotide sequences described herein is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual.
  • Using the unique identification database positive identification of the individual, living or dead, can be made from extremely small tissue samples.
  • Forensic biology is a scientific field employing genetic typing of biological evidence found at a crime scene as a means for positively identifying, for example, a perpetrator of a crime.
  • PCR technology can be used to amplify DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, or semen found at a crime scene.
  • the amplified sequence can then be compared to a standard, thereby allowing identification of the origin of the biological sample.
  • sequences of the present invention can be used to provide polynucleotide reagents, e.g., PCR primers, targeted to specific loci in the human genome, which can enhance the reliability of DNA-based forensic identifications by, for example, providing another "identification marker" (i.e. another DNA sequence that is unique to a particular individual).
  • an "identification marker” i.e. another DNA sequence that is unique to a particular individual.
  • actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments.
  • Sequences targeted to noncoding regions of SEQ ID NO:l or SEQ ID NO:4 are particularly appropriate for this use as greater numbers of polymorphisms occur in the noncoding regions, making it easier to differentiate individuals using this technique.
  • polynucleotide reagents include the STMST nucleotide sequences or portions thereof, e.g., fragments derived from the noncoding regions of SEQ ID NO:l or SEQ ID NO:4, having a length of at least 20 bases, preferably at least 30 bases.
  • the STMST nucleotide sequences described herein can further be used to provide polynucleotide reagents, e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue, e.g., brain tissue. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such STMST probes can be used to identify tissue by species and/or by organ type. In a similar fashion, these reagents, e.g., STMST primers or probes can be used to screen tissue culture for contamination (i.e. screen for the presence of a mixture of different types of cells in a culture).
  • polynucleotide reagents e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue, e.g., brain tissue. This can be very useful in cases where a
  • the present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trails are used for prognostic (predictive) purposes to thereby treat an individual prophylactically.
  • diagnostic assays for determining STMST protein and/or nucleic acid expression as well as STMST activity, in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant STMST expression or activity.
  • a biological sample e.g., blood, serum, cells, tissue
  • the invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with STMST protein, nucleic acid expression or activity. For example, mutations in an STMST gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive purpose to thereby phophylactically treat an individual prior to the onset of a disorder characterized by or associated with STMST protein, nucleic acid expression or activity. Another aspect of the invention pertains to monitoring the influence of agents
  • An exemplary method for detecting the presence or absence of STMST protein or nucleic acid in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting STMST protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes STMST protein such that the presence of STMST protein or nucleic acid is detected in the biological sample.
  • a compound or an agent capable of detecting STMST protein or nucleic acid e.g., mRNA, genomic DNA
  • a preferred agent for detecting STMST mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to STMST mRNA or genomic DNA.
  • the nucleic acid probe can be, for example, a full-length STMST nucleic acid, such as the nucleic acid of SEQ ID NO: 1 or SEQ ID NO:4, or a fragment or portion of an STMST nucleic acid such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to STMST mRNA or genomic DNA.
  • Other suitable probes for use in the diagnostic assays of the invention are described herein.
  • a preferred agent for detecting STMST protein is an antibody capable of binding to STMST protein, preferably an antibody with a detectable label.
  • Antibodies can be polyclonal, or more preferably, monoclonal.
  • an intact antibody, or a fragment thereof can be used.
  • labeled with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled.
  • indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.
  • biological sample is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect STMST mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo.
  • in vitro techniques for detection of STMST mRNA include Northern hybridizations and in situ hybridizations.
  • in vitro techniques for detection of STMST protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence.
  • In vitro techniques for detection of STMST genomic DNA include Southern hybridizations.
  • in vivo techniques for detection of STMST protein include introducing into a subject a labeled anti-STMST antibody.
  • the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
  • the biological sample contains protein molecules from the test subject.
  • the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject.
  • a preferred biological sample is a serum sample isolated by conventional means from a subject.
  • the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting STMST protein, mRNA, or genomic DNA, such that the presence of STMST protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of STMST protein, mRNA or genomic DNA in the control sample with the presence of STMST protein, mRNA or genomic DNA in the test sample.
  • kits for detecting the presence of STMST in a biological sample can comprise a labeled compound or agent capable of detecting STMST protein or mRNA in a biological sample; means for determining the amount of STMST in the sample; and means for comparing the amount of STMST in the sample with a standard.
  • the compound or agent can be packaged in a suitable container.
  • the kit can further comprise instructions for using the kit to detect STMST protein or nucleic acid.
  • the diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a disease or disorder associated with aberrant STMST expression or activity.
  • the assays described herein such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing a disorder associated with STMST protein, nucleic acid expression or activity such as an inflammatory disorder or hypothalamic disorder.
  • the prognostic assays can be utilized to identify a subject having or at risk for developing an inflammatory disorder or hypothalamic disorder.
  • the present invention provides a method for identifying a disease or disorder associated with aberrant STMST expression or activity in which a test sample is obtained from a subject and STMST protein or nucleic acid (e.g., mRNA, genomic DNA) is detected, wherein the presence of STMST protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant STMST expression or activity.
  • a test sample refers to a biological sample obtained from a subject of interest.
  • a test sample can be a biological fluid (e.g., serum), cell sample, or tissue.
  • the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant STMST expression or activity.
  • an agent e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
  • agents e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
  • agents e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
  • such methods can be used to determine whether a subject can be effectively treated with an agent for a disorder, such as an inflammatory disorder or hypothalamic disorder.
  • such methods can be used to determine whether
  • the present invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant STMST expression or activity in which a test sample is obtained and STMST protein or nucleic acid expression or activity is detected (e.g., wherein the abundance of STMST protein or nucleic acid expression or activity is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant STMST expression or activity.)
  • the methods of the invention can also be used to detect genetic alterations in an STMST gene, thereby determining if a subject with the altered gene is at risk for a disorder.
  • the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic alteration characterized by at least one of an alteration affecting the integrity of a gene encoding an STMST-protein, or the mis-expression of the STMST gene.
  • such genetic alterations can be detected by ascertaining the existence of at least one of 1) a deletion of one or more nucleotides from an STMST gene; 2) an addition of one or more nucleotides to an STMST gene; 3) a substitution of one or more nucleotides of an STMST gene, 4) a chromosomal rearrangement of an STMST gene; 5) an alteration in the level of a messenger RNA transcript of an STMST gene, 6) aberrant modification of an STMST gene, such as of the methylation pattern of the genomic DNA, 7) the presence of a non- wild type splicing pattern of a messenger RNA transcript of an STMST gene, 8) a non- wild type level of an STMST-protein, 9) allelic loss of an STMST gene, and 10) inappropriate post-translational modification of an STMST-protein.
  • a preferred biological sample is a tissue or serum sample isolated by conventional means from a subject (e.g., a brain, heart, lung or kidney tissue or tissue section from the hypothalamus).
  • detection of the alteration involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) PNAS 91 -.360-364), the latter of which can be particularly useful for detecting point mutations in the STMST-gene (see Abravaya et al (1995) Nucleic Acids Res .23:675-682).
  • PCR polymerase chain reaction
  • LCR ligation chain reaction
  • This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g. , genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to an STMST gene under conditions such that hybridization and amplification of the STMST-gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.
  • nucleic acid e.g. , genomic, mRNA or both
  • Alternative amplification methods include: self sustained sequence replication (Guatelli, J.C. et al, 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh, D.Y. et al, 1989, Proc. Natl. Acad. Sci. USA 86:1173- 1177), Q-Beta Replicase (Lizardi, P.M. et all, 1988, Bio/Technology 6:1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.
  • mutations in an STMST gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns.
  • sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA.
  • sequence specific ribozymes see, for example, U.S. Patent No. 5,498,531 can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.
  • genetic mutations in STMST can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high density arrays containing hundreds or thousands of oligonucleotides probes (Cronin, M.T. et al (1996) Human Mutation 7: 244-255; Kozal, MJ. et al. (1996) Nature Medicine 2: 753- 759).
  • a sample and control nucleic acids e.g., DNA or RNA
  • high density arrays containing hundreds or thousands of oligonucleotides probes e.g., DNA or RNA
  • genetic mutations in STMST can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin, M.T. et al. supra.
  • a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential ovelapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.
  • any of a variety of sequencing reactions known in the art can be used to directly sequence the STMST gene and detect mutations by comparing the sequence of the sample STMST with the corresponding wild-type (control) sequence.
  • Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert ((1977) PNAS 74:560) or Sanger ((1977) PNAS 74:5463). It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays ((1995) Biotechniques 19:448), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen et al (1996) Adv. Chromatogr.
  • RNA/RNA or RNA/DNA heteroduplexes methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes.
  • Myers et al. (1985) Science 230:1242 methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes.
  • the art technique of "mismatch cleavage" starts by providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA containing the wild-type STMST sequence with potentially mutant RNA or DNA obtained from a tissue sample.
  • RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with SI nuclease to enzymatically digesting the mismatched regions.
  • either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, for example, Cotton et al. (1988) Proc. Natl Acad Sci USA 85:4397; Saleeba et al. (1992) Methods Enzymol 217:286-295.
  • the control DNA or RNA can be labeled for detection.
  • the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in STMST cDNAs obtained from samples of cells.
  • DNA mismatch repair enzymes
  • the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al (1994) Carcinogenesis 15:1657-1662).
  • a probe based on an STMST sequence e.g.
  • a wild-type STMST sequence is hybridized to a cDNA or other DNA product from a test cell(s).
  • the duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, for example, U.S. Patent No. 5,459,039.
  • alterations in electrophoretic mobility will be used to identify mutations in STMST genes.
  • single strand conformation polymorphism SSCP
  • SSCP single strand conformation polymorphism
  • Single-stranded DNA fragments of sample and control STMST nucleic acids will be denatured and allowed to renature.
  • the secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change.
  • the DNA fragments may be labeled or detected with labeled probes.
  • the sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence.
  • the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).
  • the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495).
  • DGGE denaturing gradient gel electrophoresis
  • DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR.
  • a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:12753).
  • oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al. (1986) Nature 324:163); Saiki et al (1989) Proc. Natl Acad. Sci USA 86:6230).
  • Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.
  • allele specific amplification technology which depends on selective PCR amplification may be used in conjunction with the instant invention.
  • Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11 :238).
  • amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3' end of the 5' sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
  • the methods described herein may be performed, for example, by utilizing prepackaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving an STMST gene.
  • any cell type or tissue in which STMST is expressed may be utilized in the prognostic assays described herein.
  • an STMST protein e.g., modulation of an inflammatory response, modulation of energy homeostasis, modulation of hypothalamic dysfunction, and modulation of bone homeostasis
  • agents e.g. , drugs, compounds
  • an STMST protein e.g., modulation of an inflammatory response, modulation of energy homeostasis, modulation of hypothalamic dysfunction, and modulation of bone homeostasis
  • an agent determined by a screening assay as described herein to increase STMST gene expression, protein levels, or upregulate STMST activity can be monitored in clinical trails of subjects exhibiting decreased STMST gene expression, protein levels, or downregulated STMST activity.
  • the effectiveness of an agent determined by a screening assay to decrease STMST gene expression, protein levels, or downregulate STMST activity can be monitored in clinical trails of subjects exhibiting increased STMST gene expression, protein levels, or upregulated STMST activity.
  • the expression or activity of an STMST gene, and preferably, other genes that have been implicated in, for example, an inflammatory disorder, hypothalamic disorder or bone-related disorder can be used as a "read out" or markers of the phenotype of a particular cell.
  • genes, including STMST, that are modulated in cells by treatment with an agent e.g., compound, drug or small molecule
  • an agent e.g., compound, drug or small molecule
  • STMST activity e.g., identified in a screening assay as described herein
  • cells can be isolated and RNA prepared and analyzed for the levels of expression of STMST and other genes implicated in the disorder.
  • the levels of gene expression can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of STMST or other genes.
  • the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent.
  • the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of an STMST protein, mRNA, or genomic DNA in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the STMST protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the STMST protein, mRNA, or genomic DNA in the pre-administration sample with the STMST protein, mRNA, or genomic DNA in the post
  • increased administration of the agent may be desirable to increase the expression or activity of STMST to higher levels than detected, i.e., to increase the effectiveness of the agent.
  • decreased administration of the agent may be desirable to decrease expression or activity of STMST to lower levels than detected, i.e. to decrease the effectiveness of the agent.
  • STMST expression or activity may be used as an indicator of the effectiveness of an agent, even in the absence of an observable phenotypic response.
  • the present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant STMST expression or activity.
  • treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics.
  • “Pharmacogenomics”, as used herein, refers to the application of genomics technologies such as gene sequencing, statistical genetics, and gene expression analysis to drugs in clinical development and on the market.
  • the term refers the study of how a patient's genes determine his or her response to a drug (e.g., a patient's "drug response phenotype", or "drug response genotype”.)
  • a drug e.g., a patient's "drug response phenotype", or "drug response genotype”.
  • another aspect of the invention provides methods for tailoring an individual's prophylactic or therapeutic treatment with either the STMST molecules of the present invention or STMST modulators according to that.individual's drug response genotype.
  • Pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drug-related side effects.
  • the invention provides a method for preventing in a subject, a disease or condition associated with an aberrant STMST expression or activity, by administering to the subject an STMST or an agent which modulates STMST expression or at least one STMST activity.
  • Subjects at risk for a disease which is caused or contributed to by aberrant STMST expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein.
  • Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the STMST aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression.
  • an STMST, STMST agonist or STMST antagonist agent can be used for treating the subject.
  • the appropriate agent can be determined based on screening assays described herein. The prophylactic methods of the present invention are further discussed in the following subsections.
  • the modulatory method of the invention involves contacting a cell with a * STMST molecule of the present invention such that the activity of an STMST is modulated.
  • the modulatory method of the invention involves contacting a ⁇ cell with an agent that modulates one or more of the activities of STMST protein activity associated with the cell.
  • An agent that modulates STMST protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring target molecule of an STMST protein (e.g., a carbohydrate), an STMST antibody, an STMST agonist or antagonist, a peptidomimetic of an STMST agonist or antagonist, or other small molecule.
  • the agent stimulates one or more STMST activites. Examples of such stimulatory agents include active STMST protein and a nucleic acid molecule encoding STMST that has been introduced into the cell.
  • the agent inhibits one or more STMST activites.
  • inhibitory agents include antisense STMST nucleic acid molecules and anti-STMST antibodies. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g, by administering the agent to a subject).
  • the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant expression or activity of an STMST protein or nucleic acid molecule.
  • the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., upregulates or downregulates) STMST expression or activity.
  • the method involves administering an STMST protein or nucleic acid molecule as therapy to compensate for reduced or aberrant STMST expression or activity.
  • Stimulation of STMST activity is desirable in situations in which STMST is abnormally downregulated and/or in which increased STMST activity is likely to have a beneficial effect.
  • inhibition of STMST activity is desirable in situations in which STMST is abnormally upregulated and/or in which decreased STMST activity is likely to have a beneficial effect.
  • STMST molecules of the present invention as well as agents, or modulators which have a stimulatory or inhibitory effect on STMST activity (e.g., STMST gene expression) as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) disorders (e.g., inflammatory disorders, hypothalamic disorders or bone-related disorders) associated with aberrant STMST activity.
  • disorders e.g., inflammatory disorders, hypothalamic disorders or bone-related disorders
  • pharmacogenomics i.e., the study , of the relationship between an individual's genotype and that individual's response to a . foreign compound or drug
  • Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug.
  • a physician or clinician may consider applying knowledge obtained in relevant pharmacogenomics studies in determining whether to administer an STMST molecule or STMST modulator as well as tailoring the dosage and/or therapeutic regimen of treatment with an STMST molecule or STMST modulator.
  • Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See e.g., Eichelbaum, M., Clin Exp Pharmacol Physiol, 1996, 23(10-11) :983- 985 and Linder, M.W., Clin Chem, 1997, 43(2):254-266.
  • two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmacogenetic conditions can occur either as rare genetic defects or as naturally-occurring polymorphisms.
  • G6PD glucose-6-phosphate dehydrogenase deficiency
  • oxidant drugs anti-malarials, sulfonamides, analgesics, nitrofurans
  • a genome-wide association relies primarily on a high-resolution map of the human genome consisting of already known gene-related markers (e.g., a "bi- allelic” gene marker map which consists of 60,000-100,000 polymorphic or variable sites on the human genome, each of which has two variants.)
  • gene-related markers e.g., a "bi- allelic” gene marker map which consists of 60,000-100,000 polymorphic or variable sites on the human genome, each of which has two variants.
  • Such a high-resolution genetic map can be compared to a map of the genome of each of a statistically significant number of patients taking part in a Phase 11/111 drug trial to identify markers associated with a particular observed drug response or side effect.
  • such a high resolution map can be generated from a combination of some ten-million known single nucleotide polymorphisms (SNPs) in the human genome.
  • SNP single nucleotide polymorphisms
  • a "SNP" is a common alteration that occurs in a single nucleotide base in a stretch of DNA. For example, a SNP may occur once per every 1000 bases of DNA.
  • a SNP may be involved in a disease process, however, the vast majority may not be disease- associated.
  • individuals Given a genetic map based on the occurrence of such SNPs, individuals can be grouped into genetic categories depending on a particular pattern of SNPs in their individual genome. In such a manner, treatment regimens can be tailored to groups of genetically similar individuals, taking into account traits that may be common among such genetically similar individuals.
  • a method termed the "candidate gene approach” can be utilized to identify genes that predict drug response.
  • a gene that encodes a drugs target e.g., an STMST protein or STMST protein of the present invention
  • all common variants of that gene can be fairly easily identified in the population and it can be determined if having one version of the gene versus another is associated with a particular drug response.
  • the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action.
  • drug metabolizing enzymes e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19
  • NAT 2 N-acetyltransferase 2
  • CYP2D6 and CYP2C19 cytochrome P450 enzymes
  • CYP2D6 and CYP2C19 cytochrome P450 enzymes
  • These polymorphisms are expressed in two phenotypes in the population, the extensive metabolizer (EM) and poor metabolizer (PM). The prevalence of PM is different among different populations.
  • the gene coding for CYP2D6 is highly polymorphic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite morphine. The other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to C YP2D6 gene amplification.
  • a method termed the "gene expression profiling" can be utilized to identify genes that predict drug response.
  • a drug e.g., an STMST molecule or STMST modulator of the present invention
  • the gene expression of an animal dosed with a drug can give an indication whether gene pathways related to toxicity have been turned on.
  • Information generated from more than one of the above pharmacogenomics approaches can be used to determine appropriate dosage and treatment regimens for prophylactic or therapeutic treatment an individual. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with an STMST molecule or STMST modulator, such as a modulator identified by one of the exemplary screening assays described herein.
  • a program termed 'signal sequence trapping' was utilized to analyse the sequences of several cDNAs of a cDNA library derived from bronchial epithelial cells which had been stimulated with the cytokine, TNF ⁇ .
  • This analysis identified a partial human clone having an insert of approximately 231 kb containing a protein-encoding sequence of approximately 225 nucleotides capable of encoding approximately 75 amino acids of STMST (e.g. , the start met through residue 74 of, for example, SEQ ID NO:2).
  • This cDNA was used to re-screen the library. Two full-length cDNA clones were isolated. Sequencing of these clones revealed the nucleotide sequences of human STMST-1 and STMST-2.
  • the nucleotide sequence encoding the human STMST-1 protein is shown in Figure 1 and is set forth as SEQ ID NO: 1.
  • the full length protein encoded by this nucleic acid is comprised of about 297 amino acids and has the amino acid sequence shown in Figure 1 and set forth as SEQ ID NO:2.
  • the coding portion (open reading frame) of SEQ ID NO:l is set forth as SEQ ID NO:3.
  • the nucleotide sequence encoding the human STMST-2 protein is shown in Figure 2 and is set forth as SEQ ID NO: 4.
  • the full length protein encoded by this nucleic acid is comprised of about 609 amino acids and has the amino acid sequence shown in Figure 2 and set forth as SEQ ID NO:5.
  • the coding portion (open reading frame) of SEQ ID NO:4 is set forth as SEQ ID NO:6.
  • a BLAST search (Altschul et al. (1990) J. Mol. Biol. 215:403) of the nucleotide sequence of human STMST-2 has revealed that STMST-2 is significantly similar to a protein identified as protein A-2 (human A-2, Accession No. U47928; murine A-2, Acession No. AC002393) which were sequenced as part of the sequencing of human chromosome 12pl3 and mouse chromosome 6, respectively.
  • the human A-2 protein appears to be one of a family of alternatively-spliced gene products which further includes protein A-l (Acession No. U47925) as well as A-3 (Acession No. U47929).
  • the A-2 proteins like the STMST proteins of the present invention, include many features indicative of the G protein-coupled receptor family of proteins.
  • the STMSTs of the present invention contain conserved cysteines found in the first 2 extracellular loops (prior to the third and fifth transmembrane domains) of most GPCRs (cys 83 and cys 161 of SEQ ID NO:2 or SEQ ID NO:5).
  • a highly conserved asparagine residue in the first transmembrane domain is present (asn25 in SEQ ID NO:2 or SEQ ID NO:5).
  • Transmembrane domain two of the STMST proteins contains a highly conserved leucine (leu49 of SEQ ID NO:2 or SEQ ID NO:5). The two cysteine residues are believed to form a disulfide bond that stabilizes the functional protein structure.
  • a highly conserved tryptophan and proline in the fourth transmembrane domain of the STMST proteins is present (trpl 35 and pro 145 of SEQ ID NO:2 or SEQ ID NO:5).
  • the third cytoplasmic loop contains 49 amino acid residues and is thus the longest cytoplasmic loop of the three, characteristic of G protein coupled receptors.
  • a highly conserved proline in the sixth transmembrane domain is, present (pro260 of SEQ ID NO:2 and SEQ ID NO:5).
  • the proline residues in the fourth, fifth, sixth, and seventh transmembrane domains are thought to introduce kinks in the alpha-helices and may be important in the formation of the ligand binding pocket.
  • the conserved (in the second cytoplasmic loop) HRM motif found in almost all Rhodopsin family GPCRs is present in the STMST proteins of the instant invention (hisl07, argl08, metl09 of SEQ ID NO:2 or SEQ ID NO:5).
  • the arginine of the HRM sequence is thought to be the most important amino acid in GPCRs and is invariant).
  • an almost invariant proline is present in the seventh transmembrane domain of STMST-2 (pro294 of SEQ ID NO:5).
  • the STMST family of proteins like the A-2 family of proteins, are refered to herein as G protein-coupled receptor-like proteins.
  • STMST-1 is also predicted to contain the following sites: cAMP and cGMP-dependent protein kinase phosphorylation site at aa 225-228 (KRRS); Protein kinase C phosphorylation sites at aa 153-155 (SER) and at aa 290-292 (SSR); Casein kinase II phosphorylation sites at aa 228-231 (SSID) and at aa 291-294 (SRQD); N- myristoylation sites at aa 9-14 (GSAVGW), aa 169-174 (GLGFGV), aa 181-186 (GGSVAM), aa 187-192 (GVICTA), aa 232-237 (GSEPAK), and at aa 244-249 (GLVTTI); Amidation site at aa 223-226 (QGKR).
  • KRRS Protein kinase C phosphorylation sites at aa 153-155
  • SSR Casein kinase II
  • STMST is predicted to contain the following sites: cAMP- and cGMP-dependent protein kinase phosphorylation sites at aa 225-228 (KRRS), aa 393-396 (RRFS), aa 436-439 (RRAS), and at aa 453-456 (RRRS); Protein kinase C phosphorylation sites at aa 253-255 (SER), aa 268-270 (SLR), aa 392-394 (TRR), aa 462-464 (SLR), aa 482-484 (SPR), and at aa 560-562 (SLR); Casein kinase II phosphorylation sites at aa 228-231 (SSID), aa 324-327 (SDDE), aa 328-331 (TSLE), aa 364-367 (SALE), aa 396-399 (SHDD), aa 417-420 (SGED), aa 466-469 (SALD), aa 506-509
  • GSAVGW aa 169-174 (GLGFGV), aa 181-186 (GGSVAM), aa 187-192 (GVICTA), aa 232-237 (GSEPAK), aa 244-249 (GLVTTI), aa 531-536 (GADPGE), aa 564-569 (GLSASW), aa 573-578 (GGLRAA), and at aa 579-584 (GGGGST); Amidation site at aa 223-226 (QGKR).
  • QGKR Amidation site at aa 223-226
  • This Example describes the tissue distribution of STMST mRNA, as determined by Northern blot hybridization.
  • Northern blot hybridizations with the various RNA samples were performed (Clontech Multi-tissue Northern I and human fetal tissue northern) under standard conditions and washed under stringent conditions.
  • a 4.5 Kb mRNA transcript was detected in heart, brain, placenta, lung, liver, skeletal muscle, fetal brain, fetal lung, and fetal kidney. Expression was highest in fetal brain.
  • STMST expression levels were measured in a variety of tissue and cell samples using the TaqmanTM procedure.
  • the TaqmanTM procedure is a quantitative, real-time PCR-based approach to detecting mRNA.
  • the RT-PCR reaction exploits the 5' nuclease activity of AmpliTaq GoldTM DNA Polymerase to cleave a TaqManTM probe during PCR.
  • cDNA is generated from the samples of interest and serves as the starting materials for PCR amplification.
  • a gene-specific oligonucleotide probe is included in the reaction (i.e., the TaqmanTM probe).
  • the TaqManTM probe incldes the oligonucleotide with a fluorescent reporter dye covalently linked to the 5' end of the probe (such as FAM (6-carboxyfluorescein), TET (6-carboxy- 4,7,2',7'-tetrachlorofluorescein), JOE (6-carboxy-4,5-dichloro-2,7- dimethoxyfluorescein), or VIC) and a quencher dye (TAMRA (6-carboxy-N,N,N',N'- tetramethylrhodamine) at the 3' end of the probe.
  • a fluorescent reporter dye covalently linked to the 5' end of the probe
  • TAM 6-carboxyfluorescein
  • TET 6-carboxy- 4,7,2',7'-tetrachlorofluorescein
  • JOE 6-carboxy-4,5-dichloro-2,7- dimethoxyfluorescein
  • VIC a quencher dye
  • cleavage of the probe separates the reporter dye and the quencher dye, resulting in increased fluorescence of the reporter. Accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the reporter dye. When the probe is intact, the proximity of the reporter dye to the quencher dye results in suppression of the reporter fluorescence.
  • the probe specifically anneals between the forward and reverse primer sites. The 5 '-3' nucleolytic activity of the AmpliTaqTM Gold DNA Polymerase cleaves the probe between the reporter and the quencher only if the probe hybridizes to the target. The probe fragments are then displaced from the target, and polymerization of the strand continues. The 3' end of the probe is blocked to prevent extension of the probe during PCR. This process occurs in every cycle and does not interfere with the exponential accumulation of product. Table II sets forth the relative expression of STMST mRNA in a variety of tissues.
  • FIG. 4B depict relative STMST expression levels in primary osteoblasts treated for 0, 1, 6 or 24 hours with either parathyroid hormone (PTH), interleukin- 1 (IL- 1 ) or dexamethasone (DEX).
  • PTH parathyroid hormone
  • IL- 1 interleukin- 1
  • DEX dexamethasone
  • FIG. 4C Transcriptional profiling analysis of a cDNA array
  • Northern blot hybridization of poly A+ from the following samples was performed under standard hybridization and wash conditions: human bone (total mRNA), human bone (poly A+ RNA), HuBCOB ⁇ (primary osteoblasts), HuBCOBl 1 (primary osteoblasts), huBCOB12 (primary osteoblasts), U2OS (osteoblast cell line), human spleen control (total mRNA) and human skeletal muscle (total mRNA). STMST transcript was also detected in human spleen mRNA.

Abstract

Novel STMST polypeptides, proteins, and nucleic acid molecules are disclosed. In addition to isolated, full-length STMST proteins, the invention further provides isolated STMST fusion proteins, antigenic peptides and anti-STMST antibodies. The invention also provides STMST nucleic acid molecules, recombinant expression vectors containing a nucleic acid molecule of the invention, host cells into which the expression vectors have been introduced and non-human transgenic animals in which an STMST gene has been introduced or disrupted. Diagnostic, screening and therapeutic methods utilizing compositions of the invention are also provided.

Description

NOVEL STMST PROTEIN AND NUCLEIC ACID MOLECULES
AND USES THEREFOR
Background of the Invention Molecular cloning studies have shown that G protein-coupled receptors
("GPCRs") form one of the largest protein superfamilies found in nature, and it is estimated that greater than 1000 different such receptors exist in mammals. Upon binding of extracellular ligands, GPCRs interact with a specific subset of heterotrimeric G-proteins that can then, in their activated forms, inhibit or activate various effector enzymes and/or ion channels. The ligands for many of these receptors are known although there exists an ever-increasing number of GPCRs which have been identified in the sequencing of the human genome which for have no ligands have yet been identified. This latter subfamily of GPCRs is called the ophan family of GPCRs. In addition to both GPCRs with known ligands, as well as orphan GPCRs, there exist a family of GPCR-like molecules which share significant homology as well as many of the structural properties of the GPCR superfamily. For example, a family of GPCR-like proteins which arises from three alternatively-spliced forms of a gene occurring between the CD4 and triosephosphate isomerase genes at human chromosome 12pl3, has been recently identified (including protein A-l, A-2, and A-3). Ansari-Lari et al. (1996) Genome Res. 6(4):314-326. Comparative sequence analysis of the syntenic region in mouse chromosome 6 has further revealed a murine homologue of at least the A-2 splice product. Ansari-Lari et al. (1998) Genome Res. 8(l):29-40.
The fundamental knowledge that GPCRs play a role in regulating that activity of virtually every cell in the human body has fostered an extensive search for modulators of such receptors for use as human therapeutics. In fact, the superfamily of GPCRs has proven to be among the most successful drug targets. Consequently, it has been recognized that the newly isolated orphan GPCRs, as well as the GPCR-like proteins, have great potential for drug discovery.
With the identification of each new GPCR, orphan GPCR5j and GPCR-like protein, there exists a need for identifying the surrogate ligands for such molecules as well as for modulators of such molecules for use in regulating a variety of cellular responses.
Summary of the Invention The present invention is based, at least in part, on the discovery of nucleic acid molecules which encode a novel family of G protein-coupled receptor-like proteins, referred to herein as the Seven Transmembrane Signal Transducer ("STMST" family or "STMST proteins"). The STMST molecules of the present invention as well as STMST ligands and/or STMST modulators, are useful in regulating a variety of cellular processes. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding STMST proteins or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of STMST-encoding nucleic acids.
In one embodiment, an STMST nucleic acid molecule is 75% homologous to the nucleotide sequence shown in SEQ ID NO:l, or complement thereof. In another embodiment, an STMST nucleic acid molecule is 80% homologous to the nucleotide sequence shown in SEQ ID NO:4, or a complement thereof. In a preferred embodiment, an isolated STMST nucleic acid molecule has the nucleotide sequence shown SEQ ID NO:3, or a complement thereof. In another embodiment, an STMST nucleic acid molecule further comprises nucleotides 1-403 of SEQ ID NO:l. In another embodiment, an STMST nucleic acid molecule further comprises nucleotides 1295-2915 of SEQ ID NO: 1. In another preferred embodiment, an isolated STMST nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 1.
In another preferred embodiment, an isolated STMST nucleic acid molecule has the nucleotide sequence shown SEQ ID NO: 6, or a complement thereof. In another embodiment, an STMST nucleic acid molecule further comprises nucleotides 1-333 of SEQ ID NO:4. In another embodiment, an STMST nucleic acid molecule further comprises nucleotides 2161-4166 of SEQ ID NO:4. In another preferred embodiment, an isolated STMST nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:4.
In another embodiment, an STMST nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence sufficiently homologous to the amino acid sequence of SEQ ID NO:2, or the amino acid sqeuence of SEQ ID NO:5. In another preferred embodiment, an STMST nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence at least 75% homologous to the amino acid sequence of SEQ ID NO:2. In yet another preferred embodiment, an STMST nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence at least 60% homologous to the amino acid sequence of SEQ ID NO:5.
In another embodiment, an isolated nucleic acid molecule of the present invention encodes an STMST protein which includes at least one transmembrane domain. In another embodiment, an isolated nucleic acid molecule of the present invention encodes a protein which includes a 7 transmembrane receptor profile. In another embodiment, an isolated nucleic acid molecule of the present invention encodes a protein which includes a spectrin α-chain motif. In yet another embodiment, an STMST nucleic acid molecule encodes an STMST protein and is a naturally occurring nucleotide sequence.
Another embodiment of the invention features STMST nucleic acid molecules which specifically detect STMST nucleic acid molecules relative to nucleic acid molecules encoding non-STMST proteins. For example, in one embodiment, an
STMST nucleic acid molecule is at least 350 nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO:l, SEQ ID NO:4. Another embodiment of the invention provides an isolated nucleic acid molecule which is antisense to the coding strand of an STMST nucleic acid.
Another aspect of the invention provides a vector comprising an STMST nucleic acid molecule. In certain embodiments, the vector is a recombinant expression vector. In another embodiment, the invention provides a host cell containing a vector of the invention. The invention also provides a method for producing an STMST protein by culturing in a suitable medium, a host cell of the invention containing a recombinant expression vector such that an STMST protein is produced.
Another aspect of this invention features isolated or recombinant STMST proteins and polypeptides. In one embodiment, an isolated STMST protein includes at least one transmembrane domain. In another embodiment, an isolated STMST protein includes at least six transmembrane domains. In another embodiment, an isolated STMST protein includes seven transmembrane domains. In another embodiment, an isolated STMST protein includes a 7 transmembrane receptor profile. In another embodiment, an isolated STMST protein includes a spectrin -chain profile. In another embodiment, an isolated STMST protein has an amino acid sequence sufficiently homologous to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:5. In a preferred embodiment, an STMST protein has an amino acid sequence at least about 75% homologous to the amino acid sequence of SEQ ID NO:2. In another preferred embodiment, an STMST protein has an amino acid sequence at least about 60% homologous to the amino acid sequence of SEQ ID NO:5. In another embodiment, an STMST protein has the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:5.
Another embodiment of the invention features an isolated STMST protein which is encoded by a nucleic acid molecule having a nucleotide sequence at least about 75% homologous to a nucleotide sequence of SEQ ID NO: 1 , or a complement thereof. Another embodiment of the invention features an isolated STMST protein which is encoded by a nucleic acid molecule having a nucleotide sequence at least about 80% homologous to a nucleotide sequence of SEQ ID NO:4, or a complement thereof. This invention further features an isolated STMST protein which is encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l, SEQ ID NO:4, or a complement thereof.
The STMST proteins of the present invention, or biologically active portions thereof, can be operatively linked to a non-STMST polypeptide to form STMST fusion proteins. The invention further features antibodies that specifically bind STMST proteins, such as monoclonal or polyclonal antibodies. In addition, the STMST proteins or biologically active portions thereof can be incorporated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers. In another aspect, the present invention provides a method for detecting STMST expression in a biological sample by contacting the biological sample with an agent capable of detecting an STMST nucleic acid molecule, protein or polypeptide such that the presence of an STMST nucleic acid molecule, protein or polypeptide is detected in the biological sample. In another aspect, the present invention provides a method for detecting the presence of STMST activity in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of STMST activity such that the presence of STMST activity is detected in the biological sample.
In another aspect, the invention provides a method for modulating STMST activity comprising contacting the cell with an agent that modulates STMST activity such that STMST activity in the cell is modulated. In one embodiment, the agent inhibits STMST activity. In another embodiment, the agent stimulates STMST activity. In one embodiment, the agent is an antibody that specifically binds to an STMST protein. In another embodiment, the agent modulates expression of STMST by modulating transcription of an STMST gene or translation of an STMST mRNA. In yet another embodiment, the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of an STMST mRNA or an STMST gene.
In one embodiment, the methods of the present invention are used to treat a subject having a disorder characterized by aberrant STMST protein or nucleic acid expression or activity by administering an agent which is an STMST modulator to the subject. In one embodiment, the STMST modulator is an STMST protein. In another embodiment, the STMST modulator is an STMST nucleic acid molecule. In another embodiment, the STMST modulator is an STMST ligand (e.g., a peptide ligand or neurotransmitter). In yet another embodiment, the STMST modulator is a peptide, peptidomimetic, or other small molecule. In a preferred embodiment, the disorder characterized by aberrant STMST protein or nucleic acid expression is a developmental, differentiative, proliferative disorder, an inflammatory disorder, a respiratory disorder (e.g., asthma), or cell death.
The present invention also provides a diagnostic assay for identifying the presence or absence of a genetic alteration characterized by at least one of (i) aberrant modification or mutation of a gene encoding an STMST protein; (ii) mis-regulation of said gene; and (iii) aberrant post-translational modification of an STMST protein, wherein a wild-type form of said gene encodes an protein with an STMST activity.
In another aspect the invention provides a method for identifying a compound that binds to or modulates the activity of an STMST protein. In one embodiment, the invention provides a method for identifying a compound which binds to an STMST protein which involves contacting the STMST protein, or a cell expressing the STMST protein with a test compound and determining whether the STMST protein binds to the test compound. In another embodiment, the invention provides a method for identifying a compound which modulates the activity of an STMST protein which involves contacting an STMST protein with a test compound, and determining the effect of the test compound on the activity of the polypeptide to thereby identify a compound which modulates the activity of the STMST protein.
Other features and advantages of the invention will be apparent from the following detailed description and claims.
Brief Description of the Drawings
Figure 1 depicts the cDNA sequence and predicted amino acid sequence of human STMST- 1. The nucleotide sequence corresponds to nucleic acids 1 to 2915 of SEQ ID NO: 1. The amino acid sequence corresponds to amino acids 1 to297 of SEQ ID NO:2.
Figure 2 depicts the cDNA sequence and predicted amino acid sequence of human STMST-2. The nucleotide sequence corresponds to nucleic acids 1 to 4166 of SEQ ID NO:4. The amino acid sequence corresponds to amino acids 1 to 609 of SEQ ID NO:5. Figure 3 depicts an alignment of the amino acid sequences of human STMST- 1
(SEQ ID NO:2), human STMST-2 (SEQ ID NO:5), human protein A-2 (Accession No. U47928, SEQ ID NO:9), and human protein A-3 (Accession No. U47929, SEQ ID NO: 10). The 7 transmembrane receptor profile is indicated in italics. The transmembrane domains are underlined. The spectrin α-chain profile is indicated in bold.
Figure4A-B is a graphic representation of relative STMST expresion levels as determined by TaqMan™ RT-PCR of mRNA samples from various cells including osteoblast cells lines and primary osteoblasts treated as indicated. Figure 4C is a graphic representation of STMST expression levels as determined by transcription profiling analysis using a cDNA array.
Detailed Description of the Invention
The present invention is based on the discovery of novel molecules, referred to herein as STMST protein and nucleic acid molecules, which comprise a family of molecules having certain conserved structural and functional features. The term "family" when referring to the protein and nucleic acid molecules of the invention is intended to mean two or more proteins or nucleic acid molecules having a common structural domain and having sufficient amino acid or nucleotide sequence homology as defined herein. Such family members can be naturally occurring and can be from either the same or different species. For example, a family can contain a first protein of human origin, as well as other, distinct proteins of human origin or alternatively, can contain homologues of non-human origin. Members of a family may also have common functional characteristics.
For example, the family of G protein-coupled receptors (GPCRs), to which the STMST proteins of the present invention bear significant homology, comprise an N- terminal extracellular domain, seven transmembrane domains (also referred to as membrane-spanning domains), three extracellular domains (also referred to as extracellular loops), three cytoplasmic domains (also referred to as cytoplasmic loops), and a C-terminal cytoplasmic domain (also referred to as a cytoplasmic tail). Members of the GPCR family also share certain conserved amino acid residues, some of which have been determined to be critical to receptor function and/or G protein signaling. For example, GPCRs contain the following features: a conserved asparagine residue in the first transmembrane domain; a cysteine residue in the first extracellular loop which is believed to form a disulfide bond with a conserved cysteine residue in the second extracellular loop; a conserved leucine and aspartate residue in the second transmembrane domain; an aspartate-arginine-tyrosine motif (DRY motif) at the interface of the third transmembrane domain and the second cytoplasmic loop of which the arginine residue is almost invariant (members of the rhodopsin subfamily of GPCRs comprise a histidine-arginine-methionine motif (HRM motif) as compared to a DRY motif); a conserved tryptophan and proline residue in the fourth transmembrane domain; a conserved phenylalanine residue which is commonly found as part of the motif FXXCXXP; and a conserved leucine residue in the seventh transmembrane domain which is commonly found as part of the motif DPXXY or NPXXY. Table I depicts an alignment of the transmembrane domain of 5 GPCRs. The conserved residues described herein are indicated by asterices. An alignment of the transmembrane domains of 44 representative GPCRs can be found at http://mgdkkl.nidll.nih.gov.8000/extended.html.
TABLE I ALIGNMENT OF : thrombin (6.) human P25116 rhodopsin (19.) human P08100 mlACh (21.) rat P08482
I -8A (30.) human P25024 octopamine (40.) Drosophila melanogaster P22270
TMl
6. 102 TLFVPSVYTGVFWSLPLNIMAIWFILKMK 132 19. 37 FSMLAAYMFLLIVLGFPINFLTLYVTVQHKK 67 21. 25 VAFIGITTGLLSLATVTGNLLVLISFKVNTE 55 30. 39 KYWIIAYALVFLLSLLGNSLVMLVILYSRV 69 40. 109 ALLTALVLSVIIVLTIIGNILVILSVFTYKP 139
I 1111111111111111111111111111111 3333333344444444445555555555666 2345678901234567890123456789012 T 2
6. 138 WYMLHLATADVLFVSVLPFKISYYFSG 165
19. 73 NYILLNLAVADLFMVLGGFTSTLYTSLH 100
21. 61 NYFLLSLACADLIIGTFSMNLYTTYLLM 88
30. 75 DVYLLNLALADLLFALTLPIWAASKVNG 102
40. 145 NFFIVSLAVADLTVALLVLPFNVAYSIL 172
2222222222222222222222222222 4444444444555555555566666666 0123456789012345678901234567
TM3
6. 176 RFVTAAFYCNMYASILLMTVISIDR 200 19. 111 NLEGFFATLGGEIALWSLWLAIER 135 21. 99 DLWLALDYVASNASV NLLLISFDR 123 30. 111 KWSLLKEVNFYSGILLLACISVDR 135 40. 183 KLWLTCDVLCCTSSILNLCAIALDR 207
I 3333333333333333333333333
2222333333333344444444445
6789012345678901234567890 TM4
6. 215 TLGRASFTCLAIWALAIAGWPLVLKE 241 19. 149 GENHAIMGVAFTWVMALACAAPPLAGW 175 21. 138 TPRRAAL IGLA LVSFVL APAILF 164 30. 149 RHLVKFVCLGC GLS NLSLPFFLFR 175
40. 222 TVGRVLLLISGV LLSLLISSPPLIG 248
I 444444444444444444444444444 334444444444555555555566666
890123456789012345678901234
TM5 6. 268 AYYFSAFSAVFFFVPLIISTVCYVSIIRC 296
19. 201 ESFVIYMFWHFTIPMIIIFFCYGQLVFT 229
21. 186 PIITFGTAMAAFYLPVTVMCTLY RIYRE 214
30. 200 MVLRILPHTFGFIVPLFVMLFCYGFTLRT 228
40. 267 RGYVIYSSLGSFFIPLAIMTIVYIEIFVA 295
55555555555555555555555555555 33334444444444555555555566666 67890123456789012345678901234 TM6
6. 313 FLSAAVFCIFIICFGPTNVLLIAHYSFL 340
19. 252 RMVIIMVIAFLIC VPYASVAFYIFTHQ 279
21. 365 RTLSAILLAFILT TPYNIMVLVSTFCK 397 30. 242 RVIFAW IFLLC LPYNLVLLADTLMR 269
40. 529 RTLGIIMGVFVIC LPFFLMYVILPFCQ 556
6666666666666666666666666666
3333344444444445555555556666 5678901234567890123456789012
TM7
** *
6. 347 EAAYFAYLLCVCVSS1SSCIDPLIYYYASSECQ 379 19. 282 NFGPIFMTIPAFFA SAAIYNPVIYIMMNKQFR 314
21. 394 CVPETL ELGYWLCYVNSTVNPMCYALCNKAFR 426
30. 281 NNIGRALDATEILGFLHSCLNPIIYAFIGQNFR 313
40. 559 CPTNKFKNFITWLGYINSGLNPVIYTIFNLDYR 591 777777777777777777777777777777777
233333333334444444444555555555566 901234567890123456789012345678901
The amino acid sequences of thrombin (Accession No. P25116), rhodopsin (Accession No. P08100), ml ACh (Accession No. P08482), IL-8A (Accession No.
P25024), octopamine (Accession No. P22270), can be found as SEQ ID NO:l 1, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively. Accordingly, GPCR-like proteins such as the STMST proteins of the present invention contain a siginificant number of structural characteristics of the GPCR family. For instance, the STMSTs of the present invention contain conserved cysteines found in the first 2 extracellular loops (prior to the third and fifth transmembrane domains) of most GPCRs (cys 83 and cys 161 of SEQ ID NO:2 or SEQ ID NO:5). A highly conserved asparagine residue in the first transmembrane domain is present (asn25 in SEQ ID NO:2 or SEQ ID NO:5). Transmembrane domain two of the STMST proteins contains a highly conserved leucine (leu49 of SEQ ID NO:2 or SEQ ID NO:5). The two cysteine residues are believed to form a disulfide bond that stabilizes the functional protein structure. A highly conserved tryptophan and proline in the fourth transmembrane domain of the STMST proteins is present (trpl35 and pro 145 of SEQ ID NO:2 or SEQ ID NO: 5). The third cytoplasmic loop contains 49 amino acid residues and is thus the longest cytoplasmic loop of the three, characteristic of G protein coupled receptors. Moreover, a highly conserved proline in the sixth transmembrane domain is present (pro260 of SEQ ID NO:2 and SEQ ID NO:5). The proline residues in the fourth, fifth, sixth, and seventh transmembrane domains are thought to introduce kinks in the alpha- helices and may be important in the formation of the ligand binding pocket. Furthermore, the conserved (in the second cytoplasmic loop) HRM motif found in almost all Rhodopsin family GPCRs is present in the STMST proteins of the instant invention (hisl07, argl08, metl09 of SEQ ID NO:2 or SEQ ID NO:5). (The arginine of the HRM sequence is thought to be the most important amino acid in GPCRs and is invariant). Moreover, an almost invariant proline is present in the seventh transmembrane domain of STMST-2 (pro294 of SEQ ID NO:5). In one embodiment, the STMST proteins of the present invention are proteins having an amino acid sequence of about 150-450, preferably about 200-400, more preferably about 225-375, more preferably about 250-350, or about 275-325 amino acids in length. In another embodiment, the STMST proteins of the present invention are proteins having an amino acid sequence of about 450-750, preferably about 500-700, more preferably about 525-675, even more preferably about 550-650, and even more preferably about 575-625 amino acid residues in length. In one embodiment, the STMST proteins of the present invention contain at least one transmembrane domain. As used herein, the term "transmembrane domain" includes an amino acid sequence having at least about 10, preferably about 13, preferably about 16, more preferably about 19, 21, 23, 25, 30, 35 or 40 amino acid residues, of which at least about 50-60%, 60- 70%, preferably about 70-80% more preferably about 80-90%, or about 90-95% of the amino acid residues contain non-polar side chains, for example, alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, and methionine. A transmembrane domain is lipophillic in nature. For example, a transmembrane domain can be found at about amino acids 11-34 of SEQ ID NO:2 or SEQ ID NO:5. In a preferred embodiment, an STMST protein of the present invention has more than one transmembrane domain, preferably 2, 3, 4, 5, 6, or 7 transmembrane domains. For example, transmembrane domains can be found at about amino acids 11-34, 44-67, 85-106, 127-149, 172-196, and 244-262 of SEQ ID NO:2 as well as at 11-34, 44-67, 85-106, 127-149, 172-196, 245-269, and 277-300 of SEQ ID NO:5. In a particularly preferred embodiment, an STMST protein of the present invention has 7 transmembrane domains. In another embodiment, an STMST family member is identified based on the presence of at least one cytoplasmic loop, also referred to herein as a cytoplasmic domain. In another embodiment, an STMST family member is identified based on the presence of at least one extracellular loop. As defined herein, the term "loop" includes an amino acid sequence having a length of at least about 4, preferably about 5-10, preferably about 10-20, and more preferably about 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, or 100-150 amino acid residues, and has an amino acid sequence that connects two transmembrane domains within a protein or polypeptide. Accordingly, the N-terminal amino acid of a loop is adjacent to a C-terminal amino acid of a transmembrane domain in a naturally-occurring GPCR or GPCR-like molecule, and the C-terminal amino acid of a loop is adj acent to an N-terminal amino acid of a transmembrane domain in a naturally-occurring GPCR or GPCR-like molecule.
As used herein, a "cytoplasmic loop" includes an amino acid sequence located within a cell or within the cytoplasm of a cell. For example, a cytoplasmic loop is found at about amino acids 35-43, 107-126, and 197-243 of SEQ ID NO:2, or alternatively, at about amino acid residues 35-43, 107-126, and 197-244 of SEQ ID NO:5. Also as used herein, an "extracellular loop" includes an amino acid sequence located outside of a cell, or extracellularly. For example, an extracellular loop can be found at about amino acid residues 68-84 and 150-171 of SEQ ID NO:2, or alternatively, at about amino acid residues 86-84, 150-171, or 270-276 of SEQ ID NO:5. In another embodiment of the invention, an STMST family member is identified based on the presence of a "C-terminal cytoplasmic domain", also referred to herein as a C-terminal cytoplasmic tail, in the sequence of the protein. As used herein, a "C- terminal cytoplasmic domain" includes an amino acid sequence having a length of at least about 10, preferably about 10-25, more preferably about 25-50, more preferably about 50-75, even more preferably about 75-100, 100-150, 150-200, 200-250, 250-300, 300-400, 400-500, or 500-600 amino acid resudues and is located within a cell or within the cytoplasm of a cell. Accordingly, the N-terminal amino acid residue of a "C- terminal cytoplasmic domain" is adjacent to a C-terminal amino acid residue of a transmembrane domain in a naturally-occurring GPCR or GPCR-like protein. For example, a C-terminal cytoplasmic domain is found at about amino acid residues 301- 609 of SEQ ID NO:5. In another embodiment, an STMST family member is identified based on the presence of an "N-terminal extracellular domain", also referred to herein as an N- terminal extracellular loop in the amino acid sequence of the protein. As used herein, an "N-terminal extracellular domain" includes an amino acid sequence having about 1-500, preferably about 1-400, more preferably about 1-300, more preferably about 1-200, even more preferably about 1-100, and even more preferably about 1-50, 1-25, or 1-10 amino acid residues in length and is located outside of a cell or extracellularly. The C-terminal amino acid residue of a "N-terminal extracellular domain" is adjacent to an N-terminal amino acid residue of a transmembrane domain in a naturally-occurring GPCR or GPCR-like protein. For example, an N-terminal cytoplasmic domain is found at about amino acid residues 1-10 of SEQ ID NO:2 or SEQ ID NO:5.
Accordingly in one embodiment of the invention, an STMST family member includes at least one, preferably 6 or 7, transmembrane domains and and/or at least one cytoplasmic loop, and/or at least one extracellular loop. In another embodiment, the STMST family member further includes an N-terminal extracellular domain and/or a C- terminal cytoplasmic domain. In another embodiment, the STMST family member can include six transmembrane domains, three cytoplasmic loops, and two extracellular loops, or can include six transmembrane domains, three extracellular loops, and 2 cytoplasmic loops. The former embodiment can further include an N-terminal extracellular domain. The latter embodiment can further include a C-terminal cytoplasmic domain. In another embodiment, the STMST family member can include seven transmembrane domains, three cytoplasmic loops, and three extracellular loops and can further include an N-terminal extracellular domain or a C-terminal cytoplasmic domain. In another embodiment, an STMST family member is identified based on the presence of at least one "7 transmembrane receptor profile", also referred to as a "Rhodopsin family sequence profile", in the protein or corresponding nucleic acid molecule. As used herein, the term "7 transmembrane receptor profile" includes an amino acid sequence having at least about 100-400, preferably about 150-350, more preferably about 200-300 amino acid residues, or at least about 250-275 amino acids in length and having a bit score for the alignment of the sequence to the 7tm_l family Hidden Markov Model (HMM) of at least 20, preferably 20-30, more preferably 30-40, more preferably 40-50, 50-75, 75-100, 100-200 or greater. The 7tm_l family HMM has been assigned the PFAM Accession PF00001 (http://genome.wustl.edu/Pfam/WWWdata 7tm_l .html).
To identify the presence of a 7 transmembrane receptor profile in an STMST family member, the amino acid sequence of the protein family member is searched against a database of HMMs (e.g., the Pfam database, release 2.1) using the default parameters (http://www.sanger.ac.uk/Software/Pfam/HMM_search). For example, the hmmsf program, which is available as part of the HMMER package of search programs, is a family specific default program for PFOOOOl and score of 15 is the default threshold score for determining a hit. For example, a search using the amino acid sequence of SEQ ID NO:2 was performed against the HMM database resulting in the identification of a 7 TM receptor profile in the amino acid sequence of SEQ ID NO:2. The results of the search are set forth below.
Score:44.14 SEQ ID NO:2: aa24-191 HMM: aal-174
GNiLVIWvIcRyRRMRTPMNYFIvNLAvADLLFslft.MPFWMvYyvMq 24 ANAWGILSVGAKQKK KPLEFLLCTLAATHMLN-VAVPIATYSVVQLRR
gRWpFGdfMCrlWmYFDYMNMYASIFfLTcISIDRYLWAICHPMrYmR 72 QRPDFE NEGLCKVFVSTFYTLTLATCFSVTSLSYHRM MVCWPVNYRL
WMTpRHRAWvMIiilWvMSFlISMPPFLMFr styrDEne NmTWCrαlyD 121 SNAKK-QAVHTVMGI MVSFILSALPA-VG- -HDTSERFYTHG-CRFIV
PewMWr YvILmtii gFYIPMilMlF 166 AEIGLGFGVCFLLLV-GGSVA-MGVICT
Likewise, a search using the amino acid sequence of SEQ ID NO: 5 results in an identical hit with a score of 44.14 against the 7tm_l family HMM Accession PF00001. Accordingly, in one embodiment of the invention, an STMST protein is a human STMST- 1 or a human STMST-2 protein having a 7 transmembrane receptor profile at about amino acids 24-191 of SEQ ID NO:2 or SEQ ID NO:5, respectively. Such a 7 transmembrane receptor profile has the amino acid sequence: ANAWGILSVGAKQKKWKPLEFLLCTLAATHMLNVAVPIATYSVVQLRRQR PDFE NEGLCKVFVSTFYTLTLATCFSVTSLSYHRMWMVC PVNYRLSNA KKQAVHTVMGIWMVSFILSALPAVGWHDTSERFYTHGCRFIVAEIGLGFG VCFLLLVGGSVAMGVICT. (SEQIDNO:9)
Accordingly, STMST family members having at least 50-60% homology, preferably about 60-70%, more preferably about 70-80%, or about 80-90% homology with the 7 transmembrane receptor profile of human STMST- 1 or STMST-2 (e.g., SEQ ID NO: 9) are within the scope of the invention.
In another embodiment, an STMST family member is identified based on the presence of a "spectrin α-chain profile " in the protein or corresponding nucleic acid molecule. As used herein, the term "spectrin α-chain profile" includes a protein domain having an amino acid sequence of about 50-250, preferably about 75-225, more preferably about 100-200 amino acid residues, or about 125-175 amino acids and having a bit score for the alignment of the sequence to the spectrin family (HMM) of at least 7, preferably 8-10, more preferably 10-30, more preferably 30-50, even more preferably 50-75, 75-100, 100-200 or greater. The spectrin family HMM has been assigned the PFAM Accession PF00435 (http://genome.wustl.edu/Pfam/WWWdata/spectrin.html).
To identify the presence of a spectrin alpha chain profile in a STMST family member, make the determination that a protein of interest has a particular profile, the amino acid sequence of the protein is searched against a database of HMMs (e.g., the Pfam database, release 2.1) using the default parameters
(http://www.sanger.ac.uk/Software/Pfam/HMM_search). For example, the hrnmsf program, which is available as part of the HMMER package of search programs, is a family specific default program for PF00435 and a score of 15 is the default threshold score for determining a hit. Alternatively, the threshold score for determining a hit can be lowered (e.g. , to 8 bits). A description of the Pfam database can be found in
Sonhammer et al. (1997) Proteins 28(3)405-420 and a detailed description of HMMs can be found, for example, in Gribskov et α/.(1990) Meth. Enzymol. 183:146-159; Gribskov et /.(1987) Proc. Natl. Acad. Sci. USA 84:4355-4358; Krogh et al.(1994) J. Mol. Biol. 235:1501-1531; and Stultz et α/.(1993) Rrotew S . 2:305-314, the contents of which are incorporated herein by reference. A search was performed against the HMM database resulting in the identification of a spectrin alpha chain profile in the amino acid sequence of SEQ ID NO:2. The results of the search are set forth below.
Score:8.78 SEQ ID NO:2: aa26β-372 HMM: aal-106
IqeRMnElndR erLkelMeqRRQMLedSmrlQQFfRDmDEeEsWInEK 266 FSSLRADASAPWMALCVL CSVAQALLLPVFLWACDRYRADLKAVREKC
EqilnSDDYGkDLtsVQnLlkKHQaFEaDIaAHE . dRIqalnefaqqLIq 315 MALMANDEESDDETSLEGGISPDLVLERSLDYGYGGDFVA DRMAKYEIS
enHYasEe 365 ALEGGLPQ
All amino acids are described using universal single letter abbreviations according to these motifs. Accordingly, in one embodiment, an STMST protein is human STMST-2 protein which includes a spectrm α-chain profile at about amino acids 266-372 of SEQ ID NO:5. Such a spectrin α-chain profile has the amino acid sequence:
FSS RADASAPWMA CVLWCSVAQALL PVF WACDRYRADL AV REKCMALMANDEESDDETSLEGGISPDLVLERSLDYGYGGDFVAL DRMAKYEISALEGGLPQ (SEQ ID O:10).
Accordingly, STMST family members having at least 50-60% homology, preferably about 60-70%, more preferably about 70-80%, or about 80-90% homology with a spectrin α-chain profile of human STMST-2 (e.g., SEQ ID NO: 10) are within the scope of the invention. In another embodiment, an STMST protein includes at least a spectrin α-chain profile. In another embodiment, an STMST protein includes a spectrin α-chain profile and a 7 transmembrane receptor profile. In another embodiment, an STMST protein is human STMST-2 which includes a spectrin α-chain profile having about amino acids 266-372 of SEQ ID NO:5. In yet another embodiment, an STMST protein is human STMST-2 which includes a 7 transmembrane receptor profile having about amino acids 24-191 of SEQ ID NO: 5 and a spectrin α-chain profile having about amino acids 266-372 of SEQ ID NO:5.
Preferred STMST molecules of the present invention have an amino acid sequence sufficiently homologous to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:5. As used herein, the term "sufficiently homologous" refers to a first amino acid or nucleotide sequence which contains a sufficient or minimum number of identical or equivalent (e.g. , an amino acid residue which has a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences share common structural domains and/or a common functional activity. For example, amino acid or nucleotide sequences which share common structural domains have at least about 50% homology, preferably 60% homology, more preferably 70%-80%, and even more preferably 90-95% homology across the amino acid sequences of the domains and contain at least one and preferably two structural domains, are defined herein as sufficiently homologous. Furthermore, amino acid or nucleotide sequences which share at least 50%, preferably 60%, more preferably 70-80, or 90-95% homology and share a common functional activity are defined herein as sufficiently homologous. As used interchangeably herein, an "STMST activity", "biological activity of STMST" or "functional activity of STMST", refers to an activity exerted by an STMST protein, polypeptide or nucleic acid molecule on an STMST responsive cell as determined in vivo, or in vitro, according to standard techniques. In one embodiment, an STMST activity is a direct activity, such as an association with an STMST-traget molecule. As used herein, a "target molecule" or "binding partner" is a molecule with which an STMST protein binds or interacts in nature, such that STMST-mediated function is acheived. An STMST target molecule can be a non-STMST molecule or an STMST protein or polypeptide of the present invention. In an exemplary embodiment, an STMST target molecule is an STMST ligand (e.g., a peptide ligand or neurotransmitter). Alternatively, an STMST activity is an indirect activity, such as a cellular signaling activity (e.g., neuronal or glial cell signaling) mediated by interaction of the STMST protein with an STMST ligand.
In a preferred embodiment, an STMST activity is at least one or more of the following activities: (i) interaction of an STMST protein with soluble STMST ligand; (ii) interaction of an STMST protein with a membrane-bound non-STMST protein; (iii) interaction of an STMST protein with an intracellular protein (e.g., an intracellular enzyme or signal transduction molecule); and (iv) indirect interaction of an STMST protein with an intracellular protein (e.g., a downstream signal transduction molecule). STMST is a GPCR-like protein having significant homology to at least the A-l family of GPCR-like proteins. STMST has been found to be expressed in many tissues, including but not limited to heart, brain (including fetal brain), placenta, lung, liver, skeletal muscle and kidney (fetal). Within the brain, STMST expression has been further localized to the hypothalamus, a major center controlling food intake and body weight. In particular, STMST expression has been localized to the arcuate nucleus ("AC") and the ventormedial hypothalamic nucleus (VMN), two brain areas having specialized roles in energy homeostasis.
Accordingly, in one embodiment, an STMST activity, for example, a hypothalamic activity, is at least one or more of the following activities: (1) modulation (e.g., repression or stimulation) of brain anabolic circuits or pathways; (2) modulation (e.g., repression or stimulation of brain catabolic pathways; (3) modulation of food intake and/or feeding behavior (e.g., stimulation of or inhibition/supression of food intake and/or feeding behavior); (4) modulation of energy expenditure (e.g., supression or stimulation of energy expenditure); (5) regulation of energy homeostasis; and (6) regulation of body fat mass. In another embodiment, an STMST activity (e.g., a hypothalamic activity or activity in another tissue in which STMST is expressed) is at least one or more of the following activities: (1) modulation of cellular signal transduction, either in vitro or in vivo; (2) regulation of gene transcription in a cell expressing an STMST protein; (3) regulation of gene transcription in a cell expressing an STMST protein, wherein said cell is involved inflammation; (4) regulation of cellular proliferation; (5) regulation of cellular differentiation; (6) regulation of development; (7) regulation of cell death; (8) regulation of inflammation; (9) regulation of respiratory cell function (e.g. , asthma); (10) regulation of actin binding; (11) regulation of cytoskeletal attachment; and (12) regulation of chemotaxis, trafficking and/or migration.
In another embodiment, an STMST activity is at least one or more of the following: (1) regulation of body temperature; (2) regulation of the sleep-wake cycle; (3) regulation of memory and/or behavior; (4) control of thirst; and (5) regulation of autonomic nervous system function.
Moreover, detailed analysis of expression by TaqMan™ RT-PCR analysis indicates that STMST is highly expressed in osteoblasts (e.g., osteoblastic cell lines as well as primary osteoblasts). Expression in primary osteobasts is further inducible by treatment of cells with parathyroid hormone (PTH) suggesting that STMST and/or STMST agonism may mimic PTH anabolic effects on bone. Expression is also inducible by dexamethasone treatment which stimulates primary osteoblasts to differentiate in vitro. Northern blot analysis confirms expression of STMST in primary osteoblasts. STMST has further been determined by in situ analysis to be expressed in osteoblasts of human fetal bone.
Accordingly, in one embodiment, an STMST modulator, is useful for (i) modulating osteogenic cell function (e.g., osteoblast function); (ii) modulating bone homeostasis; (iii) modulation of bone resorption; and (iv) modulation of bone formation (e.g., stimulation of bone mass and/or inhibition of bone loss). In another embodiment, an STMST modulator is useful for (1) regulating, preventing and/or treating bone- related disorders including, but not limited to osteoporosis, Paget's disease, osteoarthritis, degenerative arthritis, osteogenesis imperfecta, fibrous displasia, hypophosphatasia, bone sarcoma, myeloma bone disorder (e.g., osteolytic bone lesions) and hypercalcemia; (2) managment of bone fragility (e.g., decrease bone fragility); and (3) prevention and/or treatment of bone pain, bone deformaties, and/or bone fractures. In another embodiment, an STMST activity is at least one or more of the following activities: (1) modulation of cellular signal transduction, either in vitro or in vivo; (2) regulation of gene transcription in a cell expressing an STMST protein; (3) regulation of gene transcription in a cell expressing an STMST protein, wherein said cell is involved inflammation; (4) regulation of cellular proliferation; (5) regulation of cellular differentiation; (6) regulation of develpoment; (7) regulation of cell death; (8) regulation of regulation of inflammation; (9) regulation of respiratory cell function (e.g., asthma); (10) regulation of actin binding; (11) regulation of cytoskeletal attachment; and (12) regulation of chemotaxis, trafficking and/or migration.
Yet another embodiment of the invention features isolated STMST proteins and polypeptides having an STMST activity. Preferred STMST proteins have at least one transmembrane domain and an STMST activity. In a preferred embodiment, an STMST protein has a 7 transmembrane receptor profile and an STMST activity. In another preferred embodiment, an STMST protein has a spectrin α-chain profile and an STMST activity. In still another preferred embodiment, an STMST protein has a 7 transmembrane receptor profile, a spectrin α-chain profile, and STMST activity. In still another preferred embodiment, an STMST protein has a 7 transmembrane receptor profile, a spectrin α-chain profile, an STMST activity, and an amino acid sequence sufficiently homologous to an amino acid sequence of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, or SEQ ID NO: 11.
The human STMST- 1 cDNA, which is approximately 2915 nucleotides in length, encodes a protein which is approximately 297 amino acid residues in length. The human STMST- 1 protein contains 6 transmembrane domains at about amino acids 11-34, 44-67, 85-106, 127-149, 172-196, and 244-262 of SEQ ID NO:2 The human STMST- 1 protein further contains a 7 transmembrane receptor profile. The 7 transmembrane receptor profile can be found at least, for example, from about amino acids 24-191 of SEQ ID NO:2.
The human STMST-2 cDNA, which is approximately 4166 nucleotides in length, encodes approximately 609 amino acid residues of the human STMST- 1 protein. The human STMST-2 protein contains 7 transmembrane domains at about amino acids 11-34, 44-67, 85-106, 127-149, 172-196, 245-269, and 277-300 of SEQ ID NO:5. The human STMST-2 protein further contains a 7 transmembrane receptor profile. The 7 transmembrane receptor profile can be found at least, for example, from about amino acids 24-191 of SEQ ID NO:5. Moreover, the human STMST protein contains a spectrin α-chain profile from about amino acids 266-372 of SEQ ID NO:5.
Various aspects of the invention are described in further detail in the following subsections:
I. Isolated Nucleic Acid Molecules
One aspect of the invention pertains to isolated nucleic acid molecules that encode STMST proteins or biologically active portions thereof, as well as nucleic acid fragments sufficient for use as hybridization probes to identify STMST-encoding nucleic acids (e.g., STMST mRNA) and fragments for use as PCR primers for the amplification or mutation of STMST nucleic acid molecules. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double- stranded, but preferably is double-stranded DNA.
An "isolated" nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. Preferably, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated STMST nucleic acid molecule can contain less than about 5 kb, 4kb, 3kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
A nucleic acid molecule of the present invention, e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l, the nucleotide sequence of SEQ ID NO:4, or a portion thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or portion of the nucleic acid sequence of SEQ ID NO: 1 , or the nucleotide sequence of SEQ ID NO:4, as a hybridization probe, STMST nucleic acid molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
Moreover, a nucleic acid molecule encompassing all or a portion of SEQ ID NO:l or SEQ ID NO:4 can be isolated by the polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based upon the sequence of SEQ ID NO:l, SEQ ID NO:4.
A nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to STMST nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer. In a preferred embodiment, an isolated nucleic acid molecule of the invention comprises the nucleotide sequence shown in SEQ ID NO: 1. The sequence of SEQ ID NO:l corresponds to the human STMST- 1 cDNA. This cDNA comprises sequences encoding the human STMST- 1 protein (i.e., "the coding region", from nucleotides 404- 1294), as well as 5' untranslated sequences (nucleotides 1-403) and 3' untranslated sequences (nucleotides 1295-2915). Alternatively, the nucleic acid molecule can comprise only the coding region of SEQ ID NO:l (e.g., nucleotides 404-1294, corresponding to SEQ ID NO:3).
In another preferred embodiment, an isolated nucleic acid molecule of the invention comprises the nucleotide sequence shown in SEQ ID NO:4. The sequence of SEQ ID NO:l corresponds to the human STMST-2 cDNA. This cDNA comprises sequences encoding the human STMST-2 protein (i.e., "the coding region", from nucleotides 334-2160), as well as 5' untranslated sequences (nucleotides 1-333) and 3' untranslated sequences (nucleotides 2161-4166). Alternatively, the nucleic acid molecule can comprise only the coding region of SEQ ID NO: 1 (e.g. , nucleotides 334- 2160, corresponding to SEQ ID NO:6).
In another preferred embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement of the nucleotide sequence shown in SEQ ID NO:l, SEQ ID NO:4, or a portion of any of these nucleotide sequences. A nucleic acid molecule which is complementary to the nucleotide sequence shown in SEQ ID NO:l or SEQ ID NO:4 is one which is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO:l or SEQ ID NO: 4 thereby forming a stable duplex.
In still another preferred embodiment, an isolated nucleic acid molecule of the present invention comprises a nucleotide sequence which is at least about 60-65%, preferably at least about 70-75%, more preferable at least about 80-85%, and even more preferably at least about 90-95% or more homologous to the nucleotide sequences shown in SEQ ID NO:l or SEQ ID NO:4, or a portion of any of these nucleotide sequences. Moreover, the nucleic acid molecule of the invention can comprise only a portion of the nucleic acid sequence of SEQ ID NO:l or SEQ ID NO:4, for example a fragment which can be used as a probe or primer or a fragment encoding a biologically active portion of an STMST protein. The nucleotide sequence determined from the cloning of the STMST- 1 genes allows for the generation of probes and primers designed for use in identifying and/or cloning other STMST family members, as well as STMST homologues from other species. The probe/primer typically comprises substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, preferably about 25, more preferably about 40, 50 or 75 consecutive nucleotides of a sense sequence of SEQ ID NO:l or SEQ ID NO:4, of an anti-sense sequence of SEQ ID NO:l or SEQ ID NO:4, or of a naturally occurring mutant of SEQ ID NO: 1 or SEQ ID NO:4. In an exemplary embodiment, a nucleic acid molecule of the present invention comprises a nucleotide sequence which is greater that 350, 351-450, 451-550, 551-650, 651-750, or 751-850, 851-950, 951-1050, 1051-1150, or 1151-1250 nucleotides in length and hybridizes under stringent hybridization conditions to a nucleic acid molecule of SEQ ID NO: l or SEQ ID NO:4. Probes based on the STMST nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In preferred embodiments, the probe further comprises a label group attached thereto, e.g., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a diagnostic test kit for identifying cells or tissue which misexpress an STMST protein, such as by measuring a level of an
STMST-encoding nucleic acid in a sample of cells from a subject e.g., detecting STMST mRNA levels or determining whether a genomic STMST gene has been mutated or deleted.
A nucleic acid fragment encoding a "biologically active portion of an STMST protein" can be prepared by isolating a portion of the nucleotide sequence of SEQ ID NO:l or SEQ ID NO:4 which encodes a polypeptide having an STMST biological activity (the biological activities of the STMST proteins have previously been described), expressing the encoded portion of the STMST protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of the STMST protein.
The invention further encompasses nucleic acid molecules that differ from the nucleotide sequence shown in SEQ ID NO:l or SEQ ID NO:4 due to degeneracy of the genetic code and thus encode the same STMST proteins as those encoded by the nucleotide sequence shown in SEQ ID NO:l or SEQ ID NO:4. In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NO:2 or SE ID NO:5.
In addition to the STMST nucleotide sequences shown in SEQ ID NO:l or SEQ ID NO:4, it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of the STMST proteins may exist within a population (e.g. , the human population). Such genetic polymorphism in the STMST genes may exist among individuals within a population due to natural allelic variation. As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame encoding an STMST protein, preferably a mammalian STMST protein. Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of an STMST gene. Any and all such nucleotide variations and resulting amino acid polymorphisms in STMST genes that are the result of natural allelic variation and that do not alter the functional activity of an STMST protein are intended to be within the scope of the invention.
Moreover, nucleic acid molecules encoding other STMST family members and thus which have a nucleotide sequence which differs from the STMST- 1 sequences of SEQ ID NO:l or SEQ ID NO:4 are intended to be within the scope of the invention. For example, an STMST-3 cDNA can be identified based on the nucleotide sequence of human STMST-1 or STMST-2. Moreover, nucleic acid molecules encoding STMST proteins from different species, and thus which have a nucleotide sequence which differs from the STMST sequences of SEQ ID NO:l or SEQ ID NO:4 are intended to be within the scope of the invention. For example, an mouse STMST cDNA can be identified based on the nucleotide sequence of a human STMST.
Nucleic acid molecules corresponding to natural allelic variants and homologues of the STMST cDNAs of the invention can be isolated based on their homology to the STMST nucleic acids disclosed herein using the cDNAs disclosed herein, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is at least 15 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l or SEQ ID NO:4. In another embodiment, the nucleic acid is at least 30, 50, 100, 250 or 500 nucleotides in length. As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other. Preferably, the conditions are such that sequences at least about 70%, more preferably at least about 80%, even more preferably at least about 85% or 90% homologous to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred, non- limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45C, followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50-65°C. Preferably, an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO:l or SEQ ID NO:4 or corresponds to a naturally-occurring nucleic acid molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
In addition to naturally-occurring allelic variants of the STMST sequences that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences of SEQ ID NO:l or SEQ ID NO:4, thereby leading to changes in the amino acid sequence of the encoded STMST proteins, without altering the functional ability of the STMST proteins. For example, nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence of SEQ ID NO: 1 or SEQ ID NO:4. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of STMST (e.g., the sequence of SEQ ID NO:2) without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity. For example, amino acid residues that are conserved among the STMST proteins of the present invention, are predicted to be particularly unamenable to alteration. Moreover, amino acid residues that are defined by the 7 transmembrane signature profile and the spectrin α-chain, repeated domain signature profile are particularly unamenable to alteration. Furthermore, additional amino acid residues that are conserved between the STMST proteins of the present invention and members of the G protein coupled receptor protein family are not likely to be amenable to alteration (e.g. , the conserved asn residue within the first TM domain,. asn25 of SEQ ID NO:2 or SEQ ID NO:5; the conserved cys in the first extracellular loop, cys83 of SEQ ID NO:2 or SEQ ID NO:5; the conserved arg at the interface of the third TM domain and the first cytoplasmic loop, arg 108 of SEQ Dl NO:2 or SEQ ID NO:5; the conserved trp and pro in the fourth TM domain, trpl35 and ρrol45 pf SEQ ID NO:2 or SEQ ID NO:5; the conserved cys residue in the second extracellular domain, cyslδl of SEQ ID NO:2 or SEQ ID NO: 5; the conserved phe residue in the fifth TM domain, phe251 of SEQ ID NO:2 or SEQ ID NO:5; or the conserved pro in the seventh TM domain, pro294 of SEQ ID NO:5).
Accordingly, another aspect of the invention pertains to nucleic acid molecules encoding STMST proteins that contain changes in amino acid residues that are not essential for activity. Such STMST proteins differ in amino acid sequence from SEQ ID NO:2 and SEQ ID NO:5 yet retain biological activity. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 75% homologous to the amino acid sequence of SEQ ID NO:2. Preferably, the protein encoded by the nucleic acid molecule is at least about 75-80% homologous to SEQ ID NO:2, more preferably at least about 80-85% homologous to SEQ ID NO:2, even more preferably at least about 85-90% homologous to SEQ ID NO:2, and even more preferably at least about 90-95% homologous to SEQ ID NO:2. In another embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 65% homologous to the amino acid sequence of SEQ ID NO: 5. Preferably, the protein encoded by the nucleic acid is at least about 65-70% homologous to SEQ ID NO:2, more preferably at least about 70- 75% homologous to SEQ ID NO:5, even more preferably at least about 75-80%, and even more preferably at least about 80-85%, 85-90%, or 90-95% homologous to SEQ ID NO:5. An isolated nucleic acid molecule encoding an STMST protein homologous to the protein of SEQ ID NO:2 or SEQ ID NO:5 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO:l, SEQ ID NO: 4, or such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into SEQ ID NO:l or SEQ ID NO:4 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta- branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in an STMST protein is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of an STMST coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for STMST biological activity to identify mutants that retain activity. Following mutagenesis of SEQ ID NO:l, SEQ ID NO:4, or the encoded protein can be expressed recombinantly and the activity of the protein can be determined.
In a preferred embodiment, a mutant STMST protein can be assayed for the ability to (1) modulate cellular signal transduction, either in vitro or in vivo; (2) regulate gene transcription in a cell expressing an STMST protein; (3) regulate gene transcription in a cell expressing an STMST protein, wherein said cell is involved inflammation; (4) regulate cellular proliferation; (5) regulate cellular differentiation; (6) regulate development; (7) regulate cell death; (8) regulate inflammation; and (9) regulate respiratory cell function; and (10) regulate osteogenic cell function.
In addition to the nucleic acid molecules encoding STMST proteins described above, another aspect of the invention pertains to isolated nucleic acid molecules which are antisense thereto. An "antisense" nucleic acid comprises a nucleotide sequence which is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid. The antisense nucleic acid can be complementary to an entire STMST coding strand, or to only a portion thereof. In one embodiment, an antisense nucleic acid molecule is antisense to a "coding region" of the coding strand of a nucleotide sequence encoding STMST. The term "coding region" refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues (e.g., the coding region of human STMST-1 corresponds to
SEQ ID NO:3). In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding STMST. The term "noncoding region" refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).
Given the coding strand sequences encoding STMST disclosed herein (e.g., SEQ ID NO:3 and SEQ ID NO:6), antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of STMST mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of STMST mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of STMST mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5- chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7- methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta- D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio- N6-isopentenyladenine, uracil-5 -oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5- methyluracil, uracil-5- oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl- 2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding an STMST protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of administration of antisense nucleic acid molecules of the invention include direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
In yet another embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2'-o- methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).
In still another embodiment, an antisense nucleic acid of the invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave STMST mRNA transcripts to thereby inhibit translation of STMST mRNA. A ribozyme having specificity for an STMST-encoding nucleic acid can be designed based upon the nucleotide sequence of an STMST- 1 or STMST-2 cDNA disclosed herein (i.e., SEQ ID NO:l or SEQ ID NO:4). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in an STMST- encoding mRNA. See, e.g., Cech et al. U.S. Patent No. 4,987,071; and Cech et al. U.S. Patent No. 5,116,742. Alternatively, STMST mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J.W. (1993) Science 261:1411-1418.
Alternatively, STMST gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the STMST (e.g. , the STMST promoter and/or enhancers) to form triple helical structures that prevent transcription of the STMST gene in target cells. See generally, Helene, C. (1991) Anticancer Drug Des. 6(6):569-84; Helene, C. et al. (1992) Ann. N. Y. Acad. Sci. 660:27-36; and Maher, L.J. (1992) Bioassays 14(12):807-15. In yet another embodiment, the STMST nucleic acid molecules of the present invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup B. et al. (1996) Bioorganic & Medicinal Chemistry 4 (1): 5-23). As used herein, the terms "peptide nucleic acids" or "PNAs" refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup B. et al. (1996) supra; Perry-O'Keefe et al. PNAS 93: 14670-675. PNAs of STMST nucleic acid molecules can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, for example, inducing transcription or translation arrest or inhibiting replication. PNAs of STMST nucleic acid molecules can also be used in the analysis of single base pair mutations in a gene, (e.g. , by PNA-directed PCR clamping); as 'artificial restriction enzymes' when used in combination with other enzymes, (e.g., SI nucleases (Hyrup B. (1996) supra)); or as probes or primers for DNA sequencing or hybridization (Hyrup B. et al. (1996) supra; Perry-O'Keefe supra). In another embodiment, PNAs of STMST can be modified, (e.g., to enhance their stability or cellular uptake), by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras of STMST nucleic acid molecules can be generated which may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes, (e.g. , RNAse H and DNA polymerases), to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup B. (1996) supra). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup B. (1996) supra and Finn P.J. et al. (1996) Nucleic Acids Res. 24 (17): 3357-63. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry and modified nucleoside analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can be used as a between the PNA and the 5' end of DNA (Mag, M. et al. (1989) Nucleic Acid Res. 17: 5973-88). PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn P.J. et al. (1996) supra). Alternatively, chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment (Peterser, K.H. et al. (1975) Bioorganic Med. Chem. Lett. 5: 1119-11124). In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. US. 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCT Publication No. W088/09810, published December 15, 1988) or the blood-brain barrier (see, e.g. , PCT Publication No. W089/10134, published April 25, 1988). In addition, oligonucleotides can be modified with hybridization-triggered cleavage agents (See, e.g., Krol et al. (1988) BioTechniques 6:958-976) or intercalating agents. (See, e.g, Zon (1988) Pharm. Res. 5:539-549). To this end, the oligonucleotide may be conjugated to another molecule, (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization-triggered cleavage agent).
Furthermore, given the fact that an important use for the STMST molecules of the present invention is in the screening for STMST ligands (e.g., surrogate ligands) and/or STMST modulators, it is intended that the following are also within the scope of the present invention: isolated nucleic acids which encode and STMST ligands or STMST modulators, probes and/or primers useful for identifying STMST ligands or STMST modulators based on the sequences of nucleic acids which encode and STMST ligands or STMST modulators, isolated nucleic acid molecules which are complementary or antisense to the sequences of nucleic acids which encode and STMST ligands or STMST modulators, isolated nucleic acid molecules which are at least about 60-65%, preferably at least about 70-75%, more preferable at least about 80-85%, and even more preferably at least about 90-95% or more homologous to the sequences of nucleic acids which encode and STMST ligands or STMST modulators, portions of nucleic acids which encode and STMST ligands or STMST modulators (e.g., biologically-active portions), naturally-occurring allelic variants of nucleic acids which encode and STMST ligands or STMST modulators, nucleic acid molecules which hybridize under stringent hybridization conditions to nucleic acids which encode and STMST ligands or STMST modulators, functionally-active mutants of nucleic acids which encode and STMST ligands or STMST modulators, PNAs of nucleic acids which encode and STMST ligands or STMST modulators, as well as vectors containing a nucleic acid encoding an STMST ligand or STMST modulator, described herein, host cells into which an expression vector encoding an STMST ligand or STMST modulator has been introduced, and homologous recombinant animal which express STMST ligands or STMST modulators.
II. Isolated STMST Proteins and Anti-STMST Antibodies
One aspect of the invention pertains to isolated STMST proteins, and biologically active portions thereof, as well as polypeptide fragments suitable for use as immunogens to raise anti-STMST antibodies. In one embodiment, native STMST proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, STMST proteins are produced by recombinant DNA techniques. Alternative to recombinant expression, an STMST protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques. An "isolated" or "purified" protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the STMST protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of STMST protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinanfly produced. In one embodiment, the language "substantially free of cellular material" includes preparations of STMST protein having less than about 30% (by dry weight) of non-STMST protein (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-STMST protein, still more preferably less than about 10% of non-STMST protein, and most preferably less than about 5% non-STMST protein. When the STMST protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.
The language "substantially free of chemical precursors or other chemicals" includes preparations of STMST protein in which the protein is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. In one embodiment, the language "substantially free of chemical precursors or other chemicals" includes preparations of STMST protein having less than about 30%) (by dry weight) of chemical precursors or non-STMST chemicals, more preferably less than about 20% chemical precursors or non-STMST chemicals, still more preferably less than about 10% chemical precursors or non-STMST chemicals, and most preferably less than about 5% chemical precursors or non-STMST chemicals.
Biologically active portions of an STMST protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence of the STMST protein, e.g., the amino acid sequence shown in SEQ ID NO:2 or the amino acid sequence shown in SEQ ID NO:5, which include less amino acids than the full length STMST proteins, and exhibit at least one activity of an STMST protein. Typically, biologically active portions comprise a domain or motif with at least one activity of the STMST protein. A biologically active portion of an STMST protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length. In one embodiment, a biologically active portion of an STMST protein comprises at least a transmembrane domain. In another embodiment, a biologically active portion of an STMST protein comprises at least one 7 transmembrane receptor profile. In another embodiment, a biologically active portion of an STMST protein comprises at least a spectrin a-chain, repeated domain profile. In another embodiment a biologically active portion of an STMST protein comprises at least a 7 transmembrane receptor profile and a spectrin α-chain profile.
It is to be understood that a preferred biologically active portion of an STMST protein of the present invention may contain at least one of the above-identified structural domains and/or profiles. A more preferred biologically active portion of an STMST protein may contain at least two of the above-identified structural domains and/or profiles. Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native STMST protein.
In a preferred embodiment, the STMST protein has an amino acid sequence shown in SEQ ID NO:2. In other embodiments, the STMST protein is substantially homologous to SEQ ID NO:2, and retains the functional activity of the protein of SEQ ID NO:2, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail in subsection I above. Accordingly, in another embodiment, the STMST protein is a protein which comprises an amino acid sequence at least about 75% homologous to the amino acid sequence of SEQ ID NO:2 and retains the functional activity of the STMST proteins of SEQ ID NO:2, respectively. Preferably, the protein is at least about 75-80% homologous to SEQ ID NO:2, more preferably at least about 80-85% homologous to SEQ ID NO:2, even more preferably at least about 85-90% homologous to SEQ ID NO:2, and most preferably at least about 90- 95% or more homologous to SEQ ID NO:2.
In a preferred embodiment, the STMST protein has an amino acid sequence shown in SEQ ID NO:5. In other embodiments, the STMST protein is substantially homologous to SEQ ID NO:5, and retains the functional activity of the protein of SEQ ID NO:5, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail in subsection I above. Accordingly, in another embodiment, the STMST protein is a protein which comprises an amino acid sequence at least about 65% homologous to the amino acid sequence of SEQ ID NO:2 and retains the functional activity of the STMST proteins of SEQ ID NO:2, respectively.
Preferably, the protein is at least about 65-70%% homologous to SEQ ID NO:2, more preferably at least about 70-75% homologous to SEQ ID NO:2, even more preferably at least about 75-80% homologous to SEQ ID NO:2, and most preferably at least about 80- 85%,85-90%, or 90-95% homologous to SEQ ID NO:2. To determine the percent homology of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, or 90% of the length of the reference sequence (e.g., when aligning a second sequence to the STMST amino acid sequence of SEQ ID NO:2 having 293 amino acid residues, at least 88, preferably at least 117, more preferably at least 147, even more preferably at least 176, and even more preferably at least 205, 234 or 264 amino acid residues are aligned). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are homologous at that position (i.e., as used herein amino acid or nucleic acid "homology" is equivalent to amino acid or nucleic acid "identity"). The percent homology between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = # of identical positions/total # of positions x 100).
The comparison of sequences and determination of percent homology between two sequences can be accomplished using a mathematical algorithim. A preferred, non- limiting example of a mathematical algorithim utilized for the comparison of sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to STMST nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to STMST protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al, (1997) Nucleic Acids Research 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov. Another preferred, non-limiting example of a mathematical algorithim utilized for the comparison of sequences is the algorithm of Myers and Miller (1988) ComputAppl Biosci. 4:11-17. Such an algorithm is incorporated into the ALIGN program available, for example, at the GENESTREAM network server, IGH Montpellier, FRANCE (http://vega.igh.cnrs.fr) or at the ISREC server (http://www.ch.embnet.org). When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
The invention also provides STMST chimeric or fusion proteins. As used herein, an STMST "chimeric protein" or "fusion protein" comprises an STMST polypeptide operatively linked to a non-STMST polypeptide. A "STMST polypeptide" refers to a polypeptide having an amino acid sequence corresponding to STMST, whereas a "non- STMST polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the STMST protein, e.g., a protein which is different from the STMST protein and which is derived from the same or a different organism. Within an STMST fusion protein the STMST polypeptide can correspond to all or a portion of an STMST protein. In a preferred embodiment, an STMST fusion protein comprises at least one biologically active portion of an STMST protein. In another preferred embodiment, an STMST fusion protein comprises at least two biologically active portions of an STMST protein. Within the fusion protein, the term "operatively linked" is intended to indicate that the STMST polypeptide and the non-STMST polypeptide are fused in-frame to each other. The non-STMST polypeptide can be fused to the N-terminus or C-terminus of the STMST polypeptide.
For example, in one embodiment, the fusion protein is a GST-STMST fusion protein in which the STMST sequences are fused to the C-terminus of the GST sequences. Such fusion proteins can facilitate the purification of recombinant STMST. In another embodiment, the fusion protein is an STMST protein containing a heterologous signal sequence at its N-terminus. For example, a native STMST signal sequence can be removed and replaced with a signal sequence from another protein. In certain host cells (e.g., mammalian host cells), expression and/or secretion of STMST can be increased through use of a heterologous signal sequence.
The STMST fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject in vivo. The STMST fusion proteins can be used to affect the bioavailability of an STMST substrate. Use of STMST fusion proteins may be useful therapeutically for the treatment of respiratory disorders (e.g., asthma). Moreover, the STMST-fusion proteins of the invention can be used as immunogens to produce anti-STMST antibodies in a subject, to purify STMST ligands and in screening assays to identify molecules which inhibit the interaction of STMST with an STMST ligand.
Preferably, an STMST chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g. , a GST polypeptide). An STMST- encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the STMST protein.
The present invention also pertains to variants of the STMST proteins which function as either STMST agonists (mimetics) or as STMST antagonists. Variants of the STMST proteins can be generated by mutagenesis, e.g. , discrete point mutation or truncation of an STMST protein. An agonist of the STMST proteins can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of an STMST protein. An antagonist of an STMST protein can inhibit one or more of the activities of the naturally occurring form of the STMST protein by, for example, competitively inhibiting the protease activity of an STMST protein. Thus, specific biological effects can be elicited by treatment with a variant of limited function. In one embodiment, treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the STMST protein. In one embodiment, variants of an STMST protein which function as either
STMST agonists (mimetics) or as STMST antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of an STMST protein for STMST protein agonist or antagonist activity. In one embodiment, a variegated library of STMST variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of STMST variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential STMST sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of STMST sequences therein. There are a variety of methods which can be used to produce libraries of potential
STMST variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential STMST sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang, S.A. (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477.
In addition, libraries of fragments of an STMST protein coding sequence can be used to generate a variegated population of STMST fragments for screening and subsequent selection of variants of an STMST protein. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of an STMST coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S 1 nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes N-terminal, C-terminal and internal fragments of various sizes of the STMST protein.
Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of STMST proteins. The most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recrusive ensemble mutagenesis (REM), a new technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify STMST variants (Arkin and Yourvan (1992) PNAS S9:7811 -7815; Delgrave et al. (1993) Protein Engineering 6(3)327-331).
In one embodiment, cell based assays can be exploited to analyze a variegated STMST library. For example, a library of expression vectors can be transfected into a cell line which ordinarily synthesizes STMST. The transfected cells are then cultured such that a particular mutant STMST is expressed and the effect of expression of the mutant on STMST activity in the cell can be detected, e.g. , by any of a number of activity assays for native STMST protein. Plasmid DNA can then be recovered from the cells which score for modulated STMST activity, and the individual clones further characterized.
An isolated STMST protein, or a portion or fragment thereof, can be used as an immunogen to generate antibodies that bind STMST using standard techniques for polyclonal and monoclonal antibody preparation. A full-length STMST protein can be used or, alternatively, the invention provides antigenic peptide fragments of STMST for use as immunogens. The antigenic peptide of STMST comprises at least 8 amino acid residues of the amino acid sequence shown in SEQ ID NO:2 and encompasses an epitope of STMST such that an antibody raised against the peptide forms a specific immune complex with STMST. Preferably, the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.
Preferred epitopes encompassed by the antigenic peptide are regions of STMST that are located on the surface of the protein, e.g. , hydrophilic regions.
An STMST immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal) with the immunogen. An appropriate immunogenic preparation can contain, for example, recombinantly expressed STMST protein or a chemically synthesized STMST polypeptide. The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent. Immunization of a suitable subject with an immunogenic STMST preparation induces a polyclonal anti-STMST antibody response.
Accordingly, another aspect of the invention pertains to anti-STMST antibodies. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen, such as STMST. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab')2 fragments which can be generated by treating the antibody with an enzyme such as pepsin. The invention provides polyclonal and monoclonal antibodies that bind STMST. The term "monoclonal antibody" or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of STMST. A monoclonal antibody composition thus typically displays a single binding affinity for a particular STMST protein with which it immunoreacts. Polyclonal anti-STMST antibodies can be prepared as described above by immunizing a suitable subject with an STMST immunogen. The anti-STMST antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized STMST. If desired, the antibody molecules directed against STMST can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction. At an appropriate time after immunization, e.g., when the anti-STMST antibody titers are highest, antibody- producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256:495-497) (see also, Brown et al. (1981) J. Immunol. 127:539-46; Brown et al. (1980) J. Biol. Chem .255:4980-83; Yeh et al. (1976) PNAS 76:2927-31; and Yeh et al. (1982) Int. J. Cancer 29:269-75), the more recent human B cell hybridoma technique (Kozbor et al. (1983) Immunol Today 4:72), the EBV-hybridoma technique (Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques. The technology for producing monoclonal antibody hybridomas is well known (see generally R. H. Kenneth, in Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, New York (1980); E. A. Lerner (1981) Yale J. Biol. Med., 54:387-402; M. L. Gefter et al. (1977) Somatic Cell Genet. 3 :231 -36). Briefly, an immortal cell line (typically a myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with an STMST immunogen as described above, and the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds STMST. Any of the many well known protocols used for fusing lymphocytes and immortalized cell lines can be applied for the purpose of generating an anti-STMST monoclonal antibody (see, e.g., G. Galfre et al. (1977) Nature 266:55052; Gefter et al. Somatic Cell Genet., cited supra; Lerner, Yale J. Biol. Med., cited supra; Kenneth, Monoclonal Antibodies, cited supra). Moreover, the ordinarily skilled worker will appreciate that there are many variations of such methods which also would be useful. Typically, the immortal cell line (e.g., a myeloma cell line) is derived from the same mammalian species as the lymphocytes. For example, murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the present invention with an immortalized mouse cell line. Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium"). Any of a number of myeloma cell lines can be used as a fusion partner according to standard techniques, e.g, the P3-NSl/l-Ag4-l, P3-x63-Ag8.653 or Sp2/O-Agl4 myeloma lines. These myeloma lines are available from ATCC. Typically, HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol ("PEG"). Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed). Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind STMST, e.g., using a standard ELISA assay.
Alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal anti-STMST antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with STMST to thereby isolate immunoglobulin library members that bind STMST. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27- 9400-01 ; and the Stratagene SurfZAP™ Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, Ladner et al. U.S. Patent No. 5,223,409; Kang et al. PCT International Publication No. WO 92/18619; Dower et al. PCT International Publication No. WO 91/17271; Winter et al. PCT International Publication WO 92/20791 ; Markland et al. PCT International Publication No. WO 92/15679; Breitling et al. PCT International Publication WO 93/01288; McCafferty et al. PCT International Publication No. WO 92/01047; Garrard et al. PCT International Publication No. WO 92/09690; Ladner et al. PCT International Publication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275- 1281; Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J. Mol. Biol. 226:889-896; Clarkson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc. Acid Res. 19:4133-4137; Barbas et al. (1991) PNAS 88:7978-7982; and McCafferty et al. Nature (1990) 348:552-554.
Additionally, recombinant anti-STMST antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in Robinson et al. International Application No. PCT/US 86/02269; Akira, et al. European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al. European Patent Application 173,494; Neuberger et al. PCT International Publication No. WO 86/01533; Cabilly et al. U.S. Patent No. 4,816,567; Cabilly et al. European Patent Application 125,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987) PNAS 84:3439-3443; Liu et al. (1987) J. Lmmunol. 5 139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimura et al. (1987) Cane. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et al. (1986) BioTechniques 4:214; Winter U.S. Patent 5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988) J.
10 Immunol. 141:4053-4060.
An anti-STMST antibody (e.g., monoclonal antibody) can be used to isolate STMST by standard techniques, such as affinity chromatography or immunoprecipitation. An anti-STMST antibody can facilitate the purification of natural STMST from cells and of recombinantly produced STMST expressed in host cells.
1.5 Moreover, an anti-STMST antibody can be used to detect STMST protein (e.g. , in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the STMST protein. Anti-STMST antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be
20 facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, -galactosidase, or acetylcholinesterase; examples of suitable prosthetic 5 group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable
30 radioactive material include 1251, 1311, 35S or 3H.
Furthermore, given the fact that an important use for the STMST molecules of the present invention is in the screening for STMST ligands (e.g., surrogate ligands) and/or STMST modulators, it is intended that the following are also within the scope of the present invention: "isolated" or "purified" STMST ligands or STMST modulators,
35 biologically-active portions of STMST ligands or STMST modulators, chimeric or fusion proteins comprising all or a portion of an STMST ligand or STMST modulator, and antibodies comprising all or a portion of an STMST ligand or STMST modulator. III. Recombinant Expression Vectors and Host Cells
Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding an STMST protein (or a portion thereof). As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. , bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g. , replication defective retro viruses, adenoviruses and adeno- associated viruses), which serve equivalent functions.
The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription translation system or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g. , STMST proteins, mutant forms of STMST proteins, fusion proteins, etc.).
The recombinant expression vectors of the invention can be designed for expression of STMST proteins in prokaryotic or eukaryotic cells. For example, STMST proteins can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase. Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D.B. and Johnson, K.S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. Purified fusion proteins can be utilized in STMST activity assays, (e.g. , direct assays or competitive assays described in detail below), or to generate antibodies specific for STMST proteins, for example. In a preferred embodiment, an STMST fusion protein expressed in a retroviral expression vector of the present invention can be utilized to infect bone marrow cells which are subsequently transplanted into irradiated recipients. The pathology of the subject recipient is then examined after sufficient time has passed (e.g six (6) weeks). Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al, (1988) Gene 69:301-315) andpET l id (Studier et al, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89). Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter. Target gene expression from the pET l id vector relies on transcription from a T7 gnlO-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident prophage harboring a T7 gnl gene under the transcriptional control of the lacUV 5 promoter.
One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al, (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques. In another embodiment, the STMST expression vector is a yeast expression vector. Examples of vectors for expression in yeast S. cerivisae include pYepSecl (Baldari, et al, (1987) Embo J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al, (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, CA), and picZ (InVitrogen Corp, San Diego, CA). Alternatively, STMST proteins can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf 9 cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39). In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include ρCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g. , tissue-specific regulatory elements are used to express the nucleic acid). Tissue- specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBOJ. 8:729-733) and immunoglobulins (Banerji etal. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) RN4S 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Patent No. 4,873,316 and European Application Publication No. 264, 166). Developmentally-regulated promoters are also encompassed, for example the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).
The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to STMST mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see Weintraub, H. et al. , Antisense RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986.
Another aspect of the invention pertains to host cells into which a recombinant expression vector of the invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. A host cell can be any prokaryotic or eukaryotic cell. For example, an STMST protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g. , DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DΕAΕ-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A
Laboratory Manual. 2nd, ed, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), and other laboratory manuals.
For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding an STMST protein or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i. e. , express) an STMST protein. Accordingly, the invention further provides methods for producing an STMST protein using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding an STMST protein has been introduced) in a suitable medium such that an STMST protein is produced. In another embodiment, the method further comprises isolating an STMST protein from the medium or the host cell. The host cells of the invention can also be used to produce nonhuman transgenic animals. For example, in one embodiment, a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which STMST-coding sequences have been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous STMST sequences have been introduced into their genome or homologous recombinant animals in which endogenous STMST sequences have been altered. Such animals are useful for studying the function and/or activity of an STMST and for identifying and/or evaluating modulators of STMST activity. As used herein, a "transgenic animal" is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc. A transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. As used herein, a "homologous recombinant animal" is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous STMST gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
A transgenic animal of the invention can be created by introducing an STMST- encoding nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinj ection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. The STMST-1 cDNA sequence, e.g., that of SEQ ID NO: 1 or SEQ ID NO:4, can be introduced as a transgene into the genome of a non- human animal. Alternatively, a nonhuman homologue of a human STMST gene, such as a mouse or rat STMST gene, can be used as a transgene. Alternatively, an STMST gene homologue, such as an STMST-3 gene can be isolated based on hybridization to the STMST cDNA sequences of SEQ ID NO:l or SEQ ID NO:4 (described further in subsection I above) and used as a transgene. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequence(s) can be operably linked to an STMST transgene to direct expression of an STMST protein to particular cells. Methods for generating transgenic animals via embryo manipulation and microinj ection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Patent No. 4,873,191 by Wagner et al. and in Hogan, B., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of an STMST transgene in its genome and/or expression of STMST mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding an STMST protein can further be bred to other transgenic animals carrying other transgenes.
To create a homologous recombinant animal, a vector is prepared which contains at least a portion of an STMST gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the STMST gene. The STMST gene can be a human gene (e.g. , the cDNA of SEQ ID NO: 1 or SEQ ID NO:4), but more preferably, is a non-human homologue of a human STMST gene (e.g., a cDNA isolated by stringent hybridization with the nucleotide sequence of SEQ ID NO:l or SEQ ID NO:4). For example, a mouse STMST gene can be used to construct a homologous recombination vector suitable for altering an endogenous STMST gene in the mouse genome. In a preferred embodiment, the vector is designed such that, upon homologous recombination, the endogenous STMST gene is functionally disrupted (i.e., , no longer encodes a functional protein; also referred to as a "knock out" vector). Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous STMST gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous STMST protein). In the homologous recombination vector, the altered portion of the STMST gene is flanked at its 5' and 3' ends by additional nucleic acid sequence of the STMST gene to allow for homologous recombination to occur between the exogenous STMST gene carried by the vector and an endogenous STMST gene in an embryonic stem cell. The additional flanking STMST nucleic acid sequence is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both at the 5' and 3' ends) are included in the vector (see e.g., Thomas, K.R. and Capecchi, M. R. (1987) Cell 51 :503 for a description of homologous recombination vectors). The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced STMST gene has homologously recombined with the endogenous STMST gene are selected (see e.g., Li, E. et al. (1992) Cell 69:915). The selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see e.g., Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, EJ. Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene. Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley, A. (1991) Current Opinion in Biotechnology 2:823-829 and in PCT International Publication Nos.: WO 90/11354 by Le Mouellec et al. ; WO 91/01140 by Smithies et al; WO 92/0968 by Zijlstra et al; and WO 93/04169 by Berns et al. In another embodiment, transgenic non-humans animals can be produced which contain selected systems which allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage PI . For a description of the cre/loxP recombinase system, see, e.g., Lakso et al. (1992) PNAS 89:6232-6236. Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al. ( 1991 ) Science 251:1351-1355. If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, I. et al. (1997) Nature 385:810- 813. In brief, a cell, e.g., a somatic cell, from the transgenic animal can be isolated and induced to exit the growth cycle and enter G0 phase. The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The recontructed oocyte is then cultured such that it develops to morula or blastocyte and then transferred to pseudopregnant female foster animal. The offspring borne of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.
IV. Pharmaceutical Compositions
The STMST nucleic acid molecules, STMST proteins, anti-STMST antibodies, STMST ligands, and STMST modulators (also referred to herein as "active compounds") of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the nucleic acid molecule, protein, antibody, or modulatory compound and a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound (e.g., an STMST protein or anti-STMST antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the . following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polygly colic acid, collagen, polyorthoesters, and polylactic acid.
Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50%) of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio
LD50/ED50. Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects. The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography. The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Patent 5,328,470) or by stereotactic injection (see e.g. , Chen et al (1994) PNAS 91 :3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system. The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
V. Uses and Methods of the Invention
The nucleic acid molecules, proteins, protein homologues, and antibodies described herein can be used in one or more of the following methods: a) screening assays; b) predictive medicine (e.g., diagnostic assays, prognostic assays, monitoring clinical trials, and pharmacogenetics); and c) methods of treatment (e.g., therapeutic and prophylactic). As described herein, an STMST protein of the invention has one or more of the following activities: (i) interaction of an STMST protein with soluble STMST ligand; (ii) interaction of an STMST protein with a membrane-bound non-STMST protein; (iii) interaction of an STMST protein with an intracellular protein (e.g., an intracellular enzyme or signal transduction molecule); and (iv) indirect interaction of an STMST protein with an intracellular protein (e.g., a downstream signal transduction molecule, and can thus be used, for example, in at least one of the following. Accordingly, in one embodiment, an STMST activity, for example, a hypothalamic activity, is at least one or more of the following activities: (1) modulation (e.g., repression or stimulation) of brain anabolic circuits or pathways; (2) modulation (e.g., repression or stimulation of brain catabolic pathways; (3) modulation of food intake and/or feeding behavior (e.g., stimulation of or inhibition/suppression of food intake and/or feeding behavior); (4) modulation of energy expenditure (e.g., suppression or stimulation of energy expenditure); (5) regulation of energy homeostasis; (6) regulation of body fat mass; (7) regulation of body temperature; (8) regulation of the sleep-wake cycle; (9) regulation of memory and/or behavior; (10) control of thirst; and (11) regulation of autonomic nervous system function; (12) modulation of cellular signal transduction, either in vitro or in vivo; (13) regulation of gene transcription in a cell expressing an STMST protein; (14) regulation of gene transcription in a cell expressing an STMST protein, wherein said cell is involved inflammation; (15) regulation of cellular proliferation; (16) regulation of cellular differentiation; (17) regulation of development; (18) regulation of cell death; (19) regulation of inflammation; (20) and regulation of respiratory cell function.
Moreover, modulation of STMST activity has particular applicability in treating, for example, (1) hypothalamic dysfunction and/or disorders (2) body weight disorders (e.g., anorexia, obesity and or hyperphagia); (3) eating disorders (e.g., anorexia nervosa and/or bulimia nervosa); (4) cachexia; (5) AIDS-related wasting; and (6) cancer-related wasting. As used herein, the term "hypothalamic dysfunction" includes a mis-regulated or aberrantly regulated function or activity attributed to the hypothalamus in an animal (e.g. , in a human), for example, a mis-regulated or aberrantly regulated hypothalamic activity, as described herein. As used herein, the term "hypothalamic disorder" includes a disease or disorder characterized by at least one phenotypic manifestation (e.g., a clinically detectable manifestation or symptom) of a hypothalamic dysfunction, as defined herein. Modulation of an STMST activity as described above may be included as part of a multi-drug regime that targets multiple sites within the weight regulatory system, temperature regulatory system, sleep-wake cycle control system, memory and/or behavior regulatory systems, thirst regulatory system and/or autonomic nervous system. In another embodiment, an STMST modulator is useful for (1) modulating bone homeostasis (e.g., stimulation of bone homeostasis) and/or modulation of bone formation (e.g. , stimulation of bone mass and/or inhibition of bone loss); (2) regulating, preventing and/or treating bone-related and/or bone resorption disorders including, but not limited to osteoporosis, Paget's disease, osteoarthritis, degenerative arthritis, osteogenesis imperfecta, fibrous displasia, hypophosphatasia, bone sarcoma, myeloma bone disorder (e.g., osteolytic bone lesions) and hypercalcemia; (3) managment of bone fragility (e.g., decrease bone fragility); and (4) prevention and/or treatment of bone pain, bone deformaties, and/or bone fractures. The isolated nucleic acid molecules of the invention can be used, for example, to express STMST protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect STMST mRNA (e.g., in a biological sample) or a genetic alteration in an STMST gene, and to modulate STMST activity, as described further below. The STMST proteins can be used to treat disorders characterized by insufficient or excessive production of an STMST protein and/or STMST ligand. In addition, the STMST proteins can be used to screen drugs or compounds which modulate the STMST activity as well as to treat disorders characterized by insufficient or excessive production of STMST protein or production of STMST protein forms which have decreased or aberrant activity compared to STMST wild type protein. Moreover, the anti-STMST antibodies of the invention can be used to detect and isolate STMST proteins, regulate the bioavailability of STMST proteins, and modulate STMST activity.
A. Screening Assays: The invention provides a method (also referred to herein as a "screening assay") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) which bind to STMST proteins, or have a stimulatory or inhibitory effect on, for example, STMST expression or STMST activity. In one embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of an STMST protein or polypeptide or biologically active portion thereof. The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one-compound' library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K.S. (1997) Anticancer Drug Des. 12:145).
Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al (1993) Proc. Natl Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303; Carrell etal (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al (1994) Angew. Chem. Int. Ed. Engl 33:2061; and in Gallop et al. (1994) J. Med. Chem. 37:1233. Libraries of compounds may be presented in solution (e.g., Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner USP 5,223,409), spores (Ladner USP *409), plasmids (Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science 249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladner supra.).
In one embodiment, an assay is a cell-based assay in which a cell which expresses an STMST protein on the cell surface is contacted with a test compound and the ability of the test compound to bind to the STMST protein determined. The cell, for example, can be of mammalian origin or a yeast cell. Determining the ability of the test compound to bind to an STMST protein can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the STMST protein can be determined by detecting the labeled compound in a complex. For example, test compounds can be labeled with ^^1, 35s, l^C, or ^H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting. Alternatively, test compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
It is also within the scope of this invention to determine the ability of a test compound to interact with an STMST protein without the labeling of any of the interactants. For example, a microphysiometer can be used to detect the interaction of a test compound with an STMST protein without the labeling of either the test compound or the receptor. McConnell, H. M. et al (1992) Science 257:1906-1912. As used herein, a "microphysiometer" (e.g., Cytosensor™) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between ligand and receptor. In a preferred embodiment, the assay comprises contacting a cell which expresses an STMST protein or biologically active portion thereof, on the cell surface with an STMST ligand (e.g., a peptide ligand or neurotransmitter), to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the STMST protein or biologically active portion thereof, wherein determining the ability of the test compound to interact with the
STMST protein or biologically active portion thereof, comprises determining the ability of the test compound to preferentially bind to the STMST protein or biologically active portion thereof, as compared to the ability of the STMST ligand to bind to the STMST protein or biologically active portion thereof.
Determining the ability of the STMST ligand or STMST modulator to bind to or interact with an STMST protein or biologically active portion thereof, can be accomplished by one of the methods described above for determining direct binding. In a preferred embodiment, determining the ability of the STMST ligand or modulator to bind to or interact with an STMST protein or biologically active portion thereof, can be accomplished by determining the activity of an STMST protein or of a downstream STMST target molecule. For example, the target molecule can be a cellular second messenger, and the activity of the target molecule can be determined by detecting induction of of the target (i.e. intracellular Ca2+, diacylglycerol, IP3, etc.), detecting catalytic/enzymatic activity of the target on an appropriate substrate, detecting the induction of a reporter gene (comprising an STMST-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a cellular response, for example, a proliferative response, an inflammatory response or a hypothalamic response. Accordingly, in one embodiment the present invention involves a method of identifying a compound which modulates the activity of an STMST protein, comprising contacting a cell which expresses an STMST protein with a test compound, determining the ability of the test compound to modulate the activity the STMST protein, and identifying the compound as a modulator of STMST activity. In another embodiment, the present invention involves a method of identifying a compound which modulates the activity of an STMST protein, comprising contacting a cell which expresses an STMST protein with a test compound, determining the ability of the test compound to modulate the activity of a downstream STMST target molecule, and identifying the compound as a modulator of STMST activity.
In yet another embodiment, an assay of the present invention is a cell-free assay in which an STMST protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to bind to the STMST protein or biologically active portion thereof is determined. Binding of the test compound to the STMST protein can be determined either directly or indirectly as described above.
Binding of the test compound to the STMST protein can also be accomplished using a technology such as real-time Biomolecular Interaction Analysis (BIA). Sjolander, S. and Urbaniczky, C. (1991) Anal Chem. 63:2338-2345 and Szabo et al (1995) Curr. Opin. Struct. Biol. 5:699-705. As used herein, "BIA" is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g.,
BIAcore™). Changes in the optical phenomenon of surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules. In a preferred embodiment, the assay includes contacting the STMST protein or biologically active portion thereof with a known ligand (e.g., a peptide ligand or neurotransmitter) which binds STMST to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with an STMST protein, wherein determining the ability of the test compound to interact with an STMST protein comprises determining the ability of the test compound to preferentially bind to STMST or biologically active portion thereof as compared to the known ligand.
In another embodiment, the assay is a cell-free assay in which an STMST protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the STMST protein or biologically active portion thereof is determined. Determining the ability of the test compound to modulate the activity of an STMST protein can be accomplished, for example, by determining the ability of the STMST protein to modulate the activity of a downstream STMST target molecule by one of the methods described above for cell- based assays. For example, the catalytic/enzymatic activity of the target molecule on an appropriate substrate can be determined as previously described.
In yet another embodiment, the cell-free assay involves contacting an STMST protein or biologically active portion thereof with a known ligand (e.g., a peptide ligand or neurotransmitter) which binds the STMST protein to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the STMST protein, wherein determining the ability of the test compound to interact with the STMST protein comprises determining the ability of the test compound to preferentially bind to or modulate the activity of an STMST target molecule, as compared to the known ligand.
The cell-free assays of the present invention are amenable to use of both soluble and/or membrane-bound forms of isolated proteins (e.g. STMST proteins or biologically active portions thereof or STMST proteins). In the case of cell-free assays in which a membrane-bound form an isolated protein is used (e.g., an STMST protein) it may be desirable to utilize a solubilizing agent such that the membrane-bound form of the isolated protein is maintained in solution. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100, Triton® X-l 14, Thesit®, Isotridecypoly (ethylene glycol ether)n, 3-[(3- cholamidopropyl)dimethylamminio]-l -propane sulfonate (CHAPS), 3-[(3- cholamidopropyl)dimethylammimo]-2-hydroxy-l -propane sulfonate (CHAPSO), orN- dodecyl=N,N-dimethyl-3-ammonio-l -propane sulfonate. In more than one embodiment of the above assay methods of the present invention, it may be desirable to immobilize either STMST or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to an STMST protein, or interaction of an STMST protein with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S -transferase/ STMST fusion proteins or glutathione-S-transferase/target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtitre plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or STMST protein, and the mixture incubated under conditions conducive to complex formation (e.g. , at physiological conditions for salt and pH). Following incubation, the beads or microtitre plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of STMST binding or activity determined using standard techniques.
Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either an STMST protein or an STMST target molecule can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated STMST protein or target molecules can be prepared from biotin-NHS (N- hydroxy-succinimide) using techniques well known in the art (e.g. , biotinylation kit,
Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with STMST protein or target molecules but which do not interfere with binding of the STMST protein to its target molecule can be derivatized to the wells of the plate, and unbound target or STMST protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the STMST protein or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the STMST protein or target molecule. In another embodiment, modulators of STMST expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of STMST mRNA or protein in the cell is determined. The level of expression of STMST mRNA or protein in the presence of the candidate compound is compared to the level of expression of STMST mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified as a modulator of STMST expression based on this comparison. For example, when expression of STMST mRNA or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of STMST mRNA or protein expression. Alternatively, when expression of STMST mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of STMST mRNA or protein expression. The level of STMST mRNA or protein expression in the cells can be determined by methods described herein for detecting STMST mRNA or protein.
In yet another aspect of the invention, the STMST proteins can be used as "bait proteins" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identify other proteins, which bind to or interact with STMST ("STMST-binding proteins" or "STMST-bp") and are involved in STMST activity. Such STMST-binding proteins are also likely to be involved in the propagation of signals by the STMST proteins as, for example, downstream elements of an STMST-mediated signaling pathway. Alternatively, such STMST-binding proteins are likely to be cell-surface molecules associated with non- STMST expressing cells, wherein such STMST-binding proteins are involved in chemoattraction. The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for an STMST protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein ("prey" or "sample") is fused to a gene that codes for the activation domain of the known transcription factor. If the "bait" and the "prey" proteins are able to interact, in vivo, forming an STMST- dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the STMST protein.
Secondary screening assays can also be used to confirm the identification of an STMST modulator, for example, a compound identified according to one of the above- described screening assays (e.g., high throughput screening assays). For example, candidate compounds of interest can be assayed in vivo for a variety of serum biomarkers and/or selected histomorphometric parameters of bone formation and resorption including, but not limited to, levels of serum osteocalcin, percentage of osteoblast surface, percentage of osteoclast surface, percentage of osteoid surface, percentage of bone volume, trabecular thickness and bone formation.
The present invention further features assays (e.g., secondary screening assays or validation assays) designed to confirm the activity of a test compound, for example, as an STMST modulator. In one embodiment, the invention features screening assays (e.g., secondary screening assays or validation assays) which include administering a test compound, for example, a test compound that demonstrates binding to an STMST protein or modulation of an STMST activity in at least one of the above-described cell- based or cell-free assays, to an animal and determining the ability of the test compound to modulate STMST activity in vivo. Determining the ability of a compound to modulate activity in vivo can include, for example, determining the ability of the compound to modulate hypothalamic activity. Exemplary animals for determining STMST modulatory activity include normal animals as well as animal models of hypothalamic dysfunction. It is also within the scope of this invention to use an agent or compound as described herein (e.g., an STMST modulating agent, an antisense STMST nucleic acid molecule, an STMST-specific antibody, or an STMST-binding partner) in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent.
This invention further pertains to novel agents identified by the above- described screening assays. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein. Moreover, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g., an STMST modulating agent, an antisense STMST nucleic acid molecule, an STMST-specific antibody, or an STMST-binding partner) can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent. B. Detection Assays
Portions or fragments of the cDNA sequences identified herein (and the corresponding complete gene sequences) can be used in numerous ways as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome; and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. These applications are described in the subsections below.
1. Chromosome Mapping
Once the sequence (or a portion of the sequence) of a gene has been isolated, this sequence can be used to map the location of the gene on a chromosome. This process is called chromosome mapping. Accordingly, portions or fragments of the STMST nucleotide sequences, described herein, can be used to map the location of the STMST genes on a chromosome. The mapping of the STMST sequences to chromosomes is an important first step in correlating these sequences with genes associated with disease.
Briefly, STMST genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the STMST nucleotide sequences. Computer analysis of the STMST sequences can be used to predict primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the STMST sequences will yield an amplified fragment. Somatic cell hybrids are prepared by fusing somatic cells from different mammals (e.g., human and mouse cells). As hybrids of human and mouse cells grow and divide, they gradually lose human chromosomes in random order, but retain the mouse chromosomes. By using media in which mouse cells cannot grow, because they lack a particular enzyme, but human cells can, the one human chromosome that contains the gene encoding the needed enzyme, will be retained. By using various media, panels of hybrid cell lines can be established. Each cell line in a panel contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes, allowing easy mapping of individual genes to specific human chromosomes. (D'Eustachio P. et al (1983) Science 220:919-924). Somatic cell hybrids containing only fragments of human chromosomes can also be produced by using human chromosomes with translocations and deletions.
PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the STMST nucleotide sequences to design oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes. Other mapping strategies which can similarly be used to map a 9o, lp, or 1 v sequence to its chromosome include in situ hybridization (described in Fan, Y. et al. (1990) PNAS, 87:6223-27), pre-screening with labeled flow- sorted chromosomes, and pre-selection by hybridization to chromosome specific cDNA libraries.
Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step. Chromosome spreads can be made using cells whose division has been blocked in metaphase by a chemical such as colcemid that disrupts the mitotic spindle. The chromosomes can be treated briefly with trypsin, and then stained with Giemsa. A pattern of light and dark bands develops on each chromosome, so that the chromosomes can be identified individually. The FISH technique can be used with a DNA sequence as short as 500 or 600 bases. However, clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. Preferably 1,000 bases, and more preferably 2,000 bases will suffice to get good results at a reasonable amount of time. For a review of this technique, see Verma et al, Human Chromosomes: A Manual of Basic Techniques (Pergamon Press, New York 1988).
Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.
Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. (Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man, available on-line through Johns Hopkins University Welch Medical Library). The relationship between a gene and a disease, mapped to the same chromosomal region, can then be identified through linkage analysis (co-inheritance of physically adjacent genes), described in, for example, Egeland, J. et al. (1987) Nature, 325:783-787.
Moreover, differences in the DNA sequences between individuals affected and unaffected with a disease associated with the STMST gene, can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.
2. Tissue Typing
The STMST sequences of the present invention can also be used to identify individuals from minute biological samples. The United States military, for example, is considering the use of restriction fragment length polymoφhism (RFLP) for identification of its personnel. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification. This method does not suffer from the current limitations of "Dog Tags" which can be lost, switched, or stolen, making positive identification difficult. The sequences of the present invention are useful as additional DNA markers for RFLP (described in U.S. Patent 5,272,057).
Furthermore, the sequences of the present invention can be used to provide an alternative technique which determines the actual base-by-base DNA sequence of selected portions of an individual's genome. Thus, the STMST nucleotide sequences described herein can be used to prepare two PCR primers from the 5' and 3' ends of the sequences. These primers can then be used to amplify an individual's DNA and . subsequently sequence it.
Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences. The sequences of the present invention can be used to obtain such identification sequences from individuals and from tissue. The STMST nucleotide sequences of the invention uniquely represent portions of the human genome. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases. Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequences of SEQ ID NO: 1 , can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers which each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEQ ID NO:3 are used, a more appropriate number of primers for positive individual identification would be 500-2,000.
If a panel of reagents from STMST nucleotide sequences described herein is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual. Using the unique identification database, positive identification of the individual, living or dead, can be made from extremely small tissue samples.
3. Use of Partial STMST Sequences in Forensic Biology DNA-based identification techniques can also be used in forensic biology.
Forensic biology is a scientific field employing genetic typing of biological evidence found at a crime scene as a means for positively identifying, for example, a perpetrator of a crime. To make such an identification, PCR technology can be used to amplify DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, or semen found at a crime scene. The amplified sequence can then be compared to a standard, thereby allowing identification of the origin of the biological sample.
The sequences of the present invention can be used to provide polynucleotide reagents, e.g., PCR primers, targeted to specific loci in the human genome, which can enhance the reliability of DNA-based forensic identifications by, for example, providing another "identification marker" (i.e. another DNA sequence that is unique to a particular individual). As mentioned above, actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments. Sequences targeted to noncoding regions of SEQ ID NO:l or SEQ ID NO:4 are particularly appropriate for this use as greater numbers of polymorphisms occur in the noncoding regions, making it easier to differentiate individuals using this technique. Examples of polynucleotide reagents include the STMST nucleotide sequences or portions thereof, e.g., fragments derived from the noncoding regions of SEQ ID NO:l or SEQ ID NO:4, having a length of at least 20 bases, preferably at least 30 bases.
The STMST nucleotide sequences described herein can further be used to provide polynucleotide reagents, e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue, e.g., brain tissue. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such STMST probes can be used to identify tissue by species and/or by organ type. In a similar fashion, these reagents, e.g., STMST primers or probes can be used to screen tissue culture for contamination (i.e. screen for the presence of a mixture of different types of cells in a culture).
C. Predictive Medicine:
The present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trails are used for prognostic (predictive) purposes to thereby treat an individual prophylactically. Accordingly, one aspect of the present invention relates to diagnostic assays for determining STMST protein and/or nucleic acid expression as well as STMST activity, in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant STMST expression or activity. The invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with STMST protein, nucleic acid expression or activity. For example, mutations in an STMST gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive purpose to thereby phophylactically treat an individual prior to the onset of a disorder characterized by or associated with STMST protein, nucleic acid expression or activity. Another aspect of the invention pertains to monitoring the influence of agents
(e.g. , drugs, compounds) on the expression or activity of STMST in clinical trials.
These and other agents are described in further detail in the following sections.
1. Diagnostic Assays An exemplary method for detecting the presence or absence of STMST protein or nucleic acid in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting STMST protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes STMST protein such that the presence of STMST protein or nucleic acid is detected in the biological sample. A preferred agent for detecting STMST mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to STMST mRNA or genomic DNA. The nucleic acid probe can be, for example, a full-length STMST nucleic acid, such as the nucleic acid of SEQ ID NO: 1 or SEQ ID NO:4, or a fragment or portion of an STMST nucleic acid such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to STMST mRNA or genomic DNA. Other suitable probes for use in the diagnostic assays of the invention are described herein. A preferred agent for detecting STMST protein is an antibody capable of binding to STMST protein, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g. , Fab or F(ab')2) can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin. The term "biological sample" is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect STMST mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of STMST mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of STMST protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence. In vitro techniques for detection of STMST genomic DNA include Southern hybridizations. Furthermore, in vivo techniques for detection of STMST protein include introducing into a subject a labeled anti-STMST antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
In one embodiment, the biological sample contains protein molecules from the test subject. Alternatively, the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject. A preferred biological sample is a serum sample isolated by conventional means from a subject.
In another embodiment, the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting STMST protein, mRNA, or genomic DNA, such that the presence of STMST protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of STMST protein, mRNA or genomic DNA in the control sample with the presence of STMST protein, mRNA or genomic DNA in the test sample.
The invention also encompasses kits for detecting the presence of STMST in a biological sample. For example, the kit can comprise a labeled compound or agent capable of detecting STMST protein or mRNA in a biological sample; means for determining the amount of STMST in the sample; and means for comparing the amount of STMST in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect STMST protein or nucleic acid.
2. Prognostic Assays
The diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a disease or disorder associated with aberrant STMST expression or activity. For example, the assays described herein, such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing a disorder associated with STMST protein, nucleic acid expression or activity such as an inflammatory disorder or hypothalamic disorder. Alternatively, the prognostic assays can be utilized to identify a subject having or at risk for developing an inflammatory disorder or hypothalamic disorder. Thus, the present invention provides a method for identifying a disease or disorder associated with aberrant STMST expression or activity in which a test sample is obtained from a subject and STMST protein or nucleic acid (e.g., mRNA, genomic DNA) is detected, wherein the presence of STMST protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant STMST expression or activity. As used herein, a "test sample" refers to a biological sample obtained from a subject of interest. For example, a test sample can be a biological fluid (e.g., serum), cell sample, or tissue.
Furthermore, the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant STMST expression or activity. For example, such methods can be used to determine whether a subject can be effectively treated with an agent for a disorder, such as an inflammatory disorder or hypothalamic disorder. Alternatively, such methods can be used to determine whether a subject can be effectively treated with an agent for an inflammatory disease or hypothalamic disorder. Thus, the present invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant STMST expression or activity in which a test sample is obtained and STMST protein or nucleic acid expression or activity is detected (e.g., wherein the abundance of STMST protein or nucleic acid expression or activity is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant STMST expression or activity.)
The methods of the invention can also be used to detect genetic alterations in an STMST gene, thereby determining if a subject with the altered gene is at risk for a disorder. In preferred embodiments, the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic alteration characterized by at least one of an alteration affecting the integrity of a gene encoding an STMST-protein, or the mis-expression of the STMST gene. For example, such genetic alterations can be detected by ascertaining the existence of at least one of 1) a deletion of one or more nucleotides from an STMST gene; 2) an addition of one or more nucleotides to an STMST gene; 3) a substitution of one or more nucleotides of an STMST gene, 4) a chromosomal rearrangement of an STMST gene; 5) an alteration in the level of a messenger RNA transcript of an STMST gene, 6) aberrant modification of an STMST gene, such as of the methylation pattern of the genomic DNA, 7) the presence of a non- wild type splicing pattern of a messenger RNA transcript of an STMST gene, 8) a non- wild type level of an STMST-protein, 9) allelic loss of an STMST gene, and 10) inappropriate post-translational modification of an STMST-protein. As described herein, there are a large number of assay techniques known in the art which can be used for detecting alterations in an STMST gene. A preferred biological sample is a tissue or serum sample isolated by conventional means from a subject (e.g., a brain, heart, lung or kidney tissue or tissue section from the hypothalamus).
In certain embodiments, detection of the alteration involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) PNAS 91 -.360-364), the latter of which can be particularly useful for detecting point mutations in the STMST-gene (see Abravaya et al (1995) Nucleic Acids Res .23:675-682). This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g. , genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to an STMST gene under conditions such that hybridization and amplification of the STMST-gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein. Alternative amplification methods include: self sustained sequence replication (Guatelli, J.C. et al, 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh, D.Y. et al, 1989, Proc. Natl. Acad. Sci. USA 86:1173- 1177), Q-Beta Replicase (Lizardi, P.M. et all, 1988, Bio/Technology 6:1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.
In an alternative embodiment, mutations in an STMST gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA. Moreover, the use of sequence specific ribozymes (see, for example, U.S. Patent No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.
In other embodiments, genetic mutations in STMST can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high density arrays containing hundreds or thousands of oligonucleotides probes (Cronin, M.T. et al (1996) Human Mutation 7: 244-255; Kozal, MJ. et al. (1996) Nature Medicine 2: 753- 759). For example, genetic mutations in STMST can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin, M.T. et al. supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential ovelapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.
In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the STMST gene and detect mutations by comparing the sequence of the sample STMST with the corresponding wild-type (control) sequence. Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert ((1977) PNAS 74:560) or Sanger ((1977) PNAS 74:5463). It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays ((1995) Biotechniques 19:448), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen et al (1996) Adv. Chromatogr. 36:127-162; and Griffin et al. (1993) Appl. Biochem. Biotechnol. 38:147-159). Other methods for detecting mutations in the STMST gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science 230:1242). In general, the art technique of "mismatch cleavage" starts by providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA containing the wild-type STMST sequence with potentially mutant RNA or DNA obtained from a tissue sample. The double-stranded duplexes are treated with an agent which cleaves single-stranded regions of the duplex such as which will exist due to basepair mismatches between the control and sample strands. For instance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with SI nuclease to enzymatically digesting the mismatched regions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, for example, Cotton et al. (1988) Proc. Natl Acad Sci USA 85:4397; Saleeba et al. (1992) Methods Enzymol 217:286-295. In a preferred embodiment, the control DNA or RNA can be labeled for detection.
In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in STMST cDNAs obtained from samples of cells. For example, the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al (1994) Carcinogenesis 15:1657-1662). According to an exemplary embodiment, a probe based on an STMST sequence, e.g. , a wild-type STMST sequence, is hybridized to a cDNA or other DNA product from a test cell(s). The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, for example, U.S. Patent No. 5,459,039.
In other embodiments, alterations in electrophoretic mobility will be used to identify mutations in STMST genes. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al. (1989) Proc Natl Acad. Sci USA: 86:2766, see also Cotton (1993) MutatRes 285:125-144; and Hayashi (1992) Genet Anal Tech Appl 9:73-79). Single-stranded DNA fragments of sample and control STMST nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In a preferred embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).
In yet another embodiment the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495). When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:12753). Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al. (1986) Nature 324:163); Saiki et al (1989) Proc. Natl Acad. Sci USA 86:6230). Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.
Alternatively, allele specific amplification technology which depends on selective PCR amplification may be used in conjunction with the instant invention.
Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11 :238). In addition it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection (Gasparini et al. (1992) Mol Cell Probes 6: 1). It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3' end of the 5' sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification. The methods described herein may be performed, for example, by utilizing prepackaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving an STMST gene.
Furthermore, any cell type or tissue in which STMST is expressed may be utilized in the prognostic assays described herein.
3. Monitoring of Effects During Clinical Trials Monitoring the influence of agents (e.g. , drugs, compounds) on the expression or activity of an STMST protein (e.g., modulation of an inflammatory response, modulation of energy homeostasis, modulation of hypothalamic dysfunction, and modulation of bone homeostasis) an be applied not only in basic drug screening, but also in clinical trials. For example, the effectiveness of an agent determined by a screening assay as described herein to increase STMST gene expression, protein levels, or upregulate STMST activity, can be monitored in clinical trails of subjects exhibiting decreased STMST gene expression, protein levels, or downregulated STMST activity. Alternatively, the effectiveness of an agent determined by a screening assay to decrease STMST gene expression, protein levels, or downregulate STMST activity, can be monitored in clinical trails of subjects exhibiting increased STMST gene expression, protein levels, or upregulated STMST activity. In such clinical trials, the expression or activity of an STMST gene, and preferably, other genes that have been implicated in, for example, an inflammatory disorder, hypothalamic disorder or bone-related disorder, can be used as a "read out" or markers of the phenotype of a particular cell. For example, and not by way of limitation, genes, including STMST, that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) which modulates STMST activity (e.g., identified in a screening assay as described herein) can be identified. Thus, to study the effect of agents on certain disorders, for example, in a clinical trial, cells can be isolated and RNA prepared and analyzed for the levels of expression of STMST and other genes implicated in the disorder. The levels of gene expression (i.e., a gene expression pattern) can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of STMST or other genes. In this way, the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent.
Accordingly, this response state may be determined before, and at various points during treatment of the individual with the agent. In a preferred embodiment, the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of an STMST protein, mRNA, or genomic DNA in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the STMST protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the STMST protein, mRNA, or genomic DNA in the pre-administration sample with the STMST protein, mRNA, or genomic DNA in the post administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly. For example, increased administration of the agent may be desirable to increase the expression or activity of STMST to higher levels than detected, i.e., to increase the effectiveness of the agent. Alternatively, decreased administration of the agent may be desirable to decrease expression or activity of STMST to lower levels than detected, i.e. to decrease the effectiveness of the agent. According to such an embodiment, STMST expression or activity may be used as an indicator of the effectiveness of an agent, even in the absence of an observable phenotypic response.
Methods of Treatment:
The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant STMST expression or activity. With regards to both prophylactic and therapeutic methods of treatment, such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics. "Pharmacogenomics", as used herein, refers to the application of genomics technologies such as gene sequencing, statistical genetics, and gene expression analysis to drugs in clinical development and on the market. More specifically, the term refers the study of how a patient's genes determine his or her response to a drug (e.g., a patient's "drug response phenotype", or "drug response genotype".) Thus, another aspect of the invention provides methods for tailoring an individual's prophylactic or therapeutic treatment with either the STMST molecules of the present invention or STMST modulators according to that.individual's drug response genotype. Pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drug-related side effects. 1. Prophylactic Methods
In one aspect, the invention provides a method for preventing in a subject, a disease or condition associated with an aberrant STMST expression or activity, by administering to the subject an STMST or an agent which modulates STMST expression or at least one STMST activity. Subjects at risk for a disease which is caused or contributed to by aberrant STMST expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the STMST aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending on the type of STMST aberrancy, for example, an STMST, STMST agonist or STMST antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein. The prophylactic methods of the present invention are further discussed in the following subsections.
2. Therapeutic Methods
Another aspect of the invention pertains to methods of modulating STMST expression or activity for therapeutic purposes. Accordingly, in an exemplary embodiment, the modulatory method of the invention involves contacting a cell with a * STMST molecule of the present invention such that the activity of an STMST is modulated. Alternatively, the modulatory method of the invention involves contacting a ■ cell with an agent that modulates one or more of the activities of STMST protein activity associated with the cell. An agent that modulates STMST protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring target molecule of an STMST protein (e.g., a carbohydrate), an STMST antibody, an STMST agonist or antagonist, a peptidomimetic of an STMST agonist or antagonist, or other small molecule. In one embodiment, the agent stimulates one or more STMST activites. Examples of such stimulatory agents include active STMST protein and a nucleic acid molecule encoding STMST that has been introduced into the cell. In another embodiment, the agent inhibits one or more STMST activites. Examples of such inhibitory agents include antisense STMST nucleic acid molecules and anti-STMST antibodies. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g, by administering the agent to a subject). As such, the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant expression or activity of an STMST protein or nucleic acid molecule. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., upregulates or downregulates) STMST expression or activity. In another embodiment, the method involves administering an STMST protein or nucleic acid molecule as therapy to compensate for reduced or aberrant STMST expression or activity.
Stimulation of STMST activity is desirable in situations in which STMST is abnormally downregulated and/or in which increased STMST activity is likely to have a beneficial effect. Likewise, inhibition of STMST activity is desirable in situations in which STMST is abnormally upregulated and/or in which decreased STMST activity is likely to have a beneficial effect.
3. Pharmacogenomics
The STMST molecules of the present invention, as well as agents, or modulators which have a stimulatory or inhibitory effect on STMST activity (e.g., STMST gene expression) as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) disorders (e.g., inflammatory disorders, hypothalamic disorders or bone-related disorders) associated with aberrant STMST activity. In conjunction with such treatment, pharmacogenomics (i.e., the study , of the relationship between an individual's genotype and that individual's response to a . foreign compound or drug) may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, a physician or clinician may consider applying knowledge obtained in relevant pharmacogenomics studies in determining whether to administer an STMST molecule or STMST modulator as well as tailoring the dosage and/or therapeutic regimen of treatment with an STMST molecule or STMST modulator.
Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See e.g., Eichelbaum, M., Clin Exp Pharmacol Physiol, 1996, 23(10-11) :983- 985 and Linder, M.W., Clin Chem, 1997, 43(2):254-266. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmacogenetic conditions can occur either as rare genetic defects or as naturally-occurring polymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency (G6PD) is a common inherited enzymopathy in which the main clinical complication is haemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.
One pharmacogenomics approach to identifying genes that predict drug response, known as "a genome-wide association", relies primarily on a high-resolution map of the human genome consisting of already known gene-related markers (e.g., a "bi- allelic" gene marker map which consists of 60,000-100,000 polymorphic or variable sites on the human genome, each of which has two variants.) Such a high-resolution genetic map can be compared to a map of the genome of each of a statistically significant number of patients taking part in a Phase 11/111 drug trial to identify markers associated with a particular observed drug response or side effect. Alternatively, such a high resolution map can be generated from a combination of some ten-million known single nucleotide polymorphisms (SNPs) in the human genome. As used herein, a "SNP" is a common alteration that occurs in a single nucleotide base in a stretch of DNA. For example, a SNP may occur once per every 1000 bases of DNA. A SNP may be involved in a disease process, however, the vast majority may not be disease- associated. Given a genetic map based on the occurrence of such SNPs, individuals can be grouped into genetic categories depending on a particular pattern of SNPs in their individual genome. In such a manner, treatment regimens can be tailored to groups of genetically similar individuals, taking into account traits that may be common among such genetically similar individuals.
Alternatively, a method termed the "candidate gene approach", can be utilized to identify genes that predict drug response. According to this method, if a gene that encodes a drugs target is known (e.g., an STMST protein or STMST protein of the present invention), all common variants of that gene can be fairly easily identified in the population and it can be determined if having one version of the gene versus another is associated with a particular drug response.
As an illustrative embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action. The discovery of genetic polymorphisms of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an explanation as to why some patients do not obtain the expected drug effects or show exaggerated drug response and serious toxicity after taking the standard and safe dose of a drug. These polymorphisms are expressed in two phenotypes in the population, the extensive metabolizer (EM) and poor metabolizer (PM). The prevalence of PM is different among different populations. For example, the gene coding for CYP2D6 is highly polymorphic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite morphine. The other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to C YP2D6 gene amplification.
Alternatively, a method termed the "gene expression profiling", can be utilized to identify genes that predict drug response. For example, the gene expression of an animal dosed with a drug (e.g., an STMST molecule or STMST modulator of the present invention) can give an indication whether gene pathways related to toxicity have been turned on.
Information generated from more than one of the above pharmacogenomics approaches can be used to determine appropriate dosage and treatment regimens for prophylactic or therapeutic treatment an individual. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with an STMST molecule or STMST modulator, such as a modulator identified by one of the exemplary screening assays described herein.
This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application are hereby incorporated by reference. References throughout the instant specification to websites maintained as part of the World Wide Web are referred to herein by the prefix http.7/. The information contained in such websites is publically-avialable and can be accessed elctronically by contacting the cited address. The information referred to herein is also provided in paper format as Appendix A to the instant specification.
EXAMPLES
Example 1: Identification And Characterization of STMST-1 cDNAs
In this example, the identification and characterization of the genes encoding human STMST-1 and STMST-2 (also referred to as "TANGO123a" and "TANGO 123c", respectively) is described.
Isolation of the human STMST cDNAs
In order to identify novel secreted and/or membrane-bound proteins, a program termed 'signal sequence trapping' was utilized to analyse the sequences of several cDNAs of a cDNA library derived from bronchial epithelial cells which had been stimulated with the cytokine, TNFα. This analysis identified a partial human clone having an insert of approximately 231 kb containing a protein-encoding sequence of approximately 225 nucleotides capable of encoding approximately 75 amino acids of STMST (e.g. , the start met through residue 74 of, for example, SEQ ID NO:2). This cDNA was used to re-screen the library. Two full-length cDNA clones were isolated. Sequencing of these clones revealed the nucleotide sequences of human STMST-1 and STMST-2.
The nucleotide sequence encoding the human STMST-1 protein is shown in Figure 1 and is set forth as SEQ ID NO: 1. The full length protein encoded by this nucleic acid is comprised of about 297 amino acids and has the amino acid sequence shown in Figure 1 and set forth as SEQ ID NO:2. The coding portion (open reading frame) of SEQ ID NO:l is set forth as SEQ ID NO:3.
The nucleotide sequence encoding the human STMST-2 protein is shown in Figure 2 and is set forth as SEQ ID NO: 4. The full length protein encoded by this nucleic acid is comprised of about 609 amino acids and has the amino acid sequence shown in Figure 2 and set forth as SEQ ID NO:5. The coding portion (open reading frame) of SEQ ID NO:4 is set forth as SEQ ID NO:6.
Analysis of Human STMST-1 and STMST-2
Examination of the cDNA sequences depicted in Figures 1 and 2 showed that they were likely encoded by alternatively spliced mRNAs derived from the same gene. Thus, the amino acid sequence of STMST-1 diverges from that of STMST-2 at about amino acid residue 263 of SEQ ID NO:2 or SEQ ID NO:5. The amino acid sequence of STMST-1 lacks the extensive cytoplasmic domain of STMST-2.
A BLAST search (Altschul et al. (1990) J. Mol. Biol. 215:403) of the nucleotide sequence of human STMST-2 has revealed that STMST-2 is significantly similar to a protein identified as protein A-2 (human A-2, Accession No. U47928; murine A-2, Acession No. AC002393) which were sequenced as part of the sequencing of human chromosome 12pl3 and mouse chromosome 6, respectively. The human A-2 protein appears to be one of a family of alternatively-spliced gene products which further includes protein A-l (Acession No. U47925) as well as A-3 (Acession No. U47929). The A-2 proteins, like the STMST proteins of the present invention, include many features indicative of the G protein-coupled receptor family of proteins.
For instance, the STMSTs of the present invention contain conserved cysteines found in the first 2 extracellular loops (prior to the third and fifth transmembrane domains) of most GPCRs (cys 83 and cys 161 of SEQ ID NO:2 or SEQ ID NO:5). A highly conserved asparagine residue in the first transmembrane domain is present (asn25 in SEQ ID NO:2 or SEQ ID NO:5). Transmembrane domain two of the STMST proteins contains a highly conserved leucine (leu49 of SEQ ID NO:2 or SEQ ID NO:5). The two cysteine residues are believed to form a disulfide bond that stabilizes the functional protein structure. A highly conserved tryptophan and proline in the fourth transmembrane domain of the STMST proteins is present (trpl 35 and pro 145 of SEQ ID NO:2 or SEQ ID NO:5). The third cytoplasmic loop contains 49 amino acid residues and is thus the longest cytoplasmic loop of the three, characteristic of G protein coupled receptors. Moreover, a highly conserved proline in the sixth transmembrane domain is, present (pro260 of SEQ ID NO:2 and SEQ ID NO:5). The proline residues in the fourth, fifth, sixth, and seventh transmembrane domains are thought to introduce kinks in the alpha-helices and may be important in the formation of the ligand binding pocket. Furthermore, the conserved (in the second cytoplasmic loop) HRM motif found in almost all Rhodopsin family GPCRs is present in the STMST proteins of the instant invention (hisl07, argl08, metl09 of SEQ ID NO:2 or SEQ ID NO:5). (The arginine of the HRM sequence is thought to be the most important amino acid in GPCRs and is invariant). Moreover, an almost invariant proline is present in the seventh transmembrane domain of STMST-2 (pro294 of SEQ ID NO:5).
As such, the STMST family of proteins, like the A-2 family of proteins, are refered to herein as G protein-coupled receptor-like proteins.
STMST-1 is also predicted to contain the following sites: cAMP and cGMP- dependent protein kinase phosphorylation site at aa 225-228 (KRRS); Protein kinase C phosphorylation sites at aa 153-155 (SER) and at aa 290-292 (SSR); Casein kinase II phosphorylation sites at aa 228-231 (SSID) and at aa 291-294 (SRQD); N- myristoylation sites at aa 9-14 (GSAVGW), aa 169-174 (GLGFGV), aa 181-186 (GGSVAM), aa 187-192 (GVICTA), aa 232-237 (GSEPAK), and at aa 244-249 (GLVTTI); Amidation site at aa 223-226 (QGKR). Likewise, STMST is predicted to contain the following sites: cAMP- and cGMP- dependent protein kinase phosphorylation sites at aa 225-228 (KRRS), aa 393-396 (RRFS), aa 436-439 (RRAS), and at aa 453-456 (RRRS); Protein kinase C phosphorylation sites at aa 253-255 (SER), aa 268-270 (SLR), aa 392-394 (TRR), aa 462-464 (SLR), aa 482-484 (SPR), and at aa 560-562 (SLR); Casein kinase II phosphorylation sites at aa 228-231 (SSID), aa 324-327 (SDDE), aa 328-331 (TSLE), aa 364-367 (SALE), aa 396-399 (SHDD), aa 417-420 (SGED), aa 466-469 (SALD), aa 506-509 (TAFE), aa 568-571 (SWGE), and at aa 590-593 (SPSE); Tyrosine kinase phosphorylation site at aa 342-348 (RSLDYGY); N-myristoylation sites at aa 9-14
(GSAVGW), aa 169-174 (GLGFGV), aa 181-186 (GGSVAM), aa 187-192 (GVICTA), aa 232-237 (GSEPAK), aa 244-249 (GLVTTI), aa 531-536 (GADPGE), aa 564-569 (GLSASW), aa 573-578 (GGLRAA), and at aa 579-584 (GGGGST); Amidation site at aa 223-226 (QGKR).
Tissue Distribution of STMST-1 mRNA
This Example describes the tissue distribution of STMST mRNA, as determined by Northern blot hybridization.
Northern blot hybridizations with the various RNA samples were performed (Clontech Multi-tissue Northern I and human fetal tissue northern) under standard conditions and washed under stringent conditions. A 4.5 Kb mRNA transcript was detected in heart, brain, placenta, lung, liver, skeletal muscle, fetal brain, fetal lung, and fetal kidney. Expression was highest in fetal brain.
Northern blot hybridization of poly A+ mRNA samples were also performed (Human Clonetech poly A+ northern). A -4.5 mRNA transcript was expressed in the following tissues at relative levels of heart > brain > placenta > liver > kidney.
Example 2 : Characterization of STMST Expression by RT-PCR
In this example, STMST expression levels were measured in a variety of tissue and cell samples using the Taqman™ procedure. The Taqman™ procedure is a quantitative, real-time PCR-based approach to detecting mRNA. The RT-PCR reaction exploits the 5' nuclease activity of AmpliTaq Gold™ DNA Polymerase to cleave a TaqMan™ probe during PCR. Briefly, cDNA is generated from the samples of interest and serves as the starting materials for PCR amplification. In addition to the 5' and 3' gene-specific primers, a gene-specific oligonucleotide probe (complementary to the region being amplified) is included in the reaction (i.e., the Taqman™ probe). The TaqMan™ probe incldes the oligonucleotide with a fluorescent reporter dye covalently linked to the 5' end of the probe (such as FAM (6-carboxyfluorescein), TET (6-carboxy- 4,7,2',7'-tetrachlorofluorescein), JOE (6-carboxy-4,5-dichloro-2,7- dimethoxyfluorescein), or VIC) and a quencher dye (TAMRA (6-carboxy-N,N,N',N'- tetramethylrhodamine) at the 3' end of the probe.
During the PCR reaction, cleavage of the probe separates the reporter dye and the quencher dye, resulting in increased fluorescence of the reporter. Accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the reporter dye. When the probe is intact, the proximity of the reporter dye to the quencher dye results in suppression of the reporter fluorescence. During PCR, if the target of interest is present, the probe specifically anneals between the forward and reverse primer sites. The 5 '-3' nucleolytic activity of the AmpliTaq™ Gold DNA Polymerase cleaves the probe between the reporter and the quencher only if the probe hybridizes to the target. The probe fragments are then displaced from the target, and polymerization of the strand continues. The 3' end of the probe is blocked to prevent extension of the probe during PCR. This process occurs in every cycle and does not interfere with the exponential accumulation of product. Table II sets forth the relative expression of STMST mRNA in a variety of tissues.
TABLE II: STMST Expression in Human Tissues
Figure imgf000080_0001
Figure imgf000080_0002
Figure imgf000081_0001
Figure imgf000081_0002
The highest expression was observed in epithelial cells, brain (cortex/hypothalamus), osteoblasts and dermal fibroblasts in the human tissue panel tested. To further investigate the high expression in primary osteoblasts, STMST expression levels were measured by quantitative PCR using the Taqman™ procedure as described above. The relative levels of STMST expression in various cell lines is depicted in Figure 4A. The data demonstrate that at least three-fold STMST expression is seen in the ZB Osteo D18 cell line as well as in HUBCOB6 cells. Expression was also significantly increased in Clonetics D7 cells (i.e., differentiated osteoblasts). The data presented in Figure 4B depict relative STMST expression levels in primary osteoblasts treated for 0, 1, 6 or 24 hours with either parathyroid hormone (PTH), interleukin- 1 (IL- 1 ) or dexamethasone (DEX). As clearly demonstrated by the data in Figure 4B, expression of STMST is upregulated in primary human osteoblasts stumulated for 24 hours with PTH. Transcriptional profiling analysis of a cDNA array (Figure 4C) confirms that expression in primary human osteobasts is inducible by treatment of cells with parathyroid hormone (PTH). These data suggest that STMST and/or STMST agonism may mimic PTH anabolic effects on bone.
Example 3: Characterization of STMST Expression in Osteogenic Cells by Northern Blot Analysis and in situ Analysis
Northern blot hybridization of poly A+ from the following samples was performed under standard hybridization and wash conditions: human bone (total mRNA), human bone (poly A+ RNA), HuBCOBό (primary osteoblasts), HuBCOBl 1 (primary osteoblasts), huBCOB12 (primary osteoblasts), U2OS (osteoblast cell line), human spleen control (total mRNA) and human skeletal muscle (total mRNA). STMST transcript was also detected in human spleen mRNA.
In situ analysis was performed according to standard methodologies on tissue sections of human fetal bone. To generate a sense probe the following primers were used; forward primer: AGATGCCACCTTCCAGGCT (SEQ ID NO: 17) and reverse primer: GGAGAAGTGCATGGCCCTC (SEQ ID NO: 18) resulting in a sense probe having the following sequence: TCTCATCGTCTGACTCCTCGTCGTTGG (SEQ ID NO:19). Sense STMST probe hybridized particularly to osteoblasts within human fetal bone sections, consistent with coexpression of STMST with PTH-R positive osteoblasts.
Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

What is claimed:
1. An isolated nucleic acid molecule selected from the group consisting of: a) a nucleic acid molecule comprising a nucleotide sequence which is at least 75% homologous to the nucleotide sequence of SEQ ID NO: 1 , the nucleotide sequence of SEQ ID NO: 3 or a complement thereof; b) a nucleic acid molecule comprising a nucleotide sequence which is at least 80% homologous to the nucleotide sequence of SEQ ID NO:4, the nucleotide of SEQ ID NO: 6, or a complement thereof; c) a nucleic acid molecule comprising a fragment of at least 1000 nucleotides of a nucleic acid comprising the nucleotide sequence of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6 or a complement thereof; d) a nucleic acid molecule which encodes a polypeptide comprising an amino acid sequence at least about 75% homologous to the amino acid sequence of SEQ ID NO:2; e) a nucleic acid molecule which encodes a polypeptide comprising an amino acid sequence at least about 65% homologous to the amino acid sequence of SEQ ID NO:5; f) a nucleic acid molecule which encodes a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO:2 or the amino acid sequence of SEQ ID NO:5, wherein the fragment comprises at least 15 contiguous amino acid residues of the amino acid sequence of SEQ ID NO':2 or the amino acid sequence of SEQ ID NO:5; and g) a nucleic acid molecule which encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID
NO: 2 or the amino acid sequence of SEQ ID NO: 5, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule comprising SEQ ID NO:l or SEQ ID NO:4 under stringent conditions.
2. The isolated nucleic acid molecule of claim 1 which is selected from the group consisting of: a) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l or SEQ ID NO:4 or a complement thereof; and b) a nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or the amino acid sequence of SEQ ID
NO:5.
3. The nucleic acid molecule of claim 1 further comprising vector nucleic acid sequences.
4. The nucleic acid molecule of claim 1 further comprising nucleic acid sequences encoding a heterologous polypeptide.
5. A host cell which contains the nucleic acid molecule of claim 1.
6. The host cell of claim 5 which is a mammalian host cell.
7. A non-human mammalian host cell containing the nucleic acid molecule of claim 1.
8. An isolated polypeptide selected from the group consisting of:
a) a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO:2 or the amino acid sequence of SEQ ID NO:5, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NO:2 or SEQ ID NO:5; b) a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:2, the amino acid sequence of SEQ ID NO:5, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule comprising SEQ ID NO:l or SEQ ID NO:4 under stringent conditions; and c) a polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 75% homologous to a nucleic acid comprising the nucleotide sequence of SEQ ID NO:l or the nucleotide sequence of SEQ ID NO:3; d) a polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 80% homologous to a nucleic acid comprising the nucleotide sequence of SEQ ID NO:4 or the nucleotide sequence of SEQ ID NO: 6; e) a polypeptide comprising an amino acid sequence which is at least 75% homologous to the amino acid sequence of SEQ ID NO:2; and f) a polypeptide comprising an amino acid sequence which is at least
65% homologous to the amino acid sequence of SEQ ID NO:5.
9. The isolated polypeptide of claim 8 comprising the amino acid sequence of SEQ ID NO:2 or the amino acid sequence of SEQ ID NO:5.
10. The polypeptide of claim 8 further comprising heterologous amino acid sequences.
11. An antibody which selectively binds to a polypeptide of claim 8.
12. A method for producing a polypeptide selected from the group consisting of: a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or the amino acid sequence of SEQ ID NO:5; b) a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or the amino acid sequence of SEQ ID NO: 5, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NO:2 or SEQ
ID NO:5; and c) a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:2 or the amino acid sequence of SEQ ID NO:5, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule comprising SEQ ID NO: 1 or SEQ ID NO:4 under stringent conditions; comprising culturing the host cell of claim 5 under conditions in which the nucleic acid molecule is expressed.
13. A method for detecting the presence of a polypeptide of claim 8 in a sample comprising: a) contacting the sample with a compound which selectively binds to the polypeptide; and b) determining whether the compound binds to the polypeptide in the sample to thereby detect the presence of a polypeptide of claim 8 in the sample.
14. The method of claim 13, wherein the compound which binds to the polypeptide is an antibody.
15. A kit comprising a compound which selectively binds to a polypeptide of claim 8 and instructions for use.
16. A method for detecting the presence of a nucleic acid molecule in claim 1 in a sample comprising: a) contacting the sample with a nucleic acid probe or primer which selectively hybridizes to the nucleic acid molecule; and b) determining whether the nucleic acid probe or primer binds to a nucleic acid molecule in the sample to thereby detect the presence of a nucleic acid molecule of claim 1 in the sample.
17. The method of claim 16, wherein the sample comprises mRNA molecules and is contacted with a nucleic acid probe.
18. A kit comprising a compound which selectively hybridizes to a nucleic acid molecule of claim 1 and instructions for use.
19. A method for identifying a compound which binds to a polypeptide of claim 8 comprising: a) contacting the polypeptide, or a cell expressing the polypeptide with a test compound; and b) determining whether the polypeptide binds to the test compound.
20. The method of claim 19, wherein the binding of the test compound to the polypeptide is detected by a method selected from the group consisting of: a) detection of binding by direct detection of test compound/polypeptide binding; b) detection of binding using a competition binding assay; and c) detection of binding using an assay for STMST activity.
21. A method of modulating the activity of a polypeptide of claim 8 comprising contacting the polypeptide or a cell expressing the polypeptide with a compound which binds to the polypeptide in a sufficient concentration to modulate the activity of the polypeptide.
22. The method of claim 21 , wherein the activity is a hypothalamic activity.
23. The method of claim 21 , wherein the activity is an osteogenic-related activity.
24. A method for identifying a compound which modulates the activity of a polypeptide of claim 8 comprising: a) contacting the polypeptide with a test compound; and b) determining the effect of the test compound on the activity of the polypeptide to thereby identify a compound which modulates the activity of the polypeptide.
25. A method for identifying a compound which modulates the activity of a polypeptide of claim 8 comprising: a) administering a test compound to an animal which expresses the polypeptide; and b) determining the effect of the test compound on the activity of the polypeptide to thereby identify a compound which modulates the activity of the polypeptide.
26. The method of claim 23, wherein determining the effect of the test compound on the activity of the polypeptide inlcudes determining the effect of the test compound on a hypothalamic activity.
27. The method of claim 23 , wherein determining the effect of the test compound on the activity of the polypeptide inlcudes determining the effect of the test compound on an osteogenic-related activity.
28. The method of claim 26, wherein the hypothalamic activity is selected from the group consisting of regulation of food intake, regulation of feeding behavior, regulation of body temperature, regulation of the sleep- wake cycle, regulation of memory, regulation of behavior, control of thirst, and regulation of autonomic nervous system function.
29. A method for affecting a bone-related disorder in a patient, comprising administering to said patient a therapeutically effecting amount of a compound which modulates STMST expression or activity such that the bone-related disorder is affected.
30. The method of claim 29, wherein the bone-related disorder is selected form the group consisting of osteoporosis, Paget's disease, osteoarthritis, degenerative arthritis, osteogenesis imperfecta, fibrous displasia, hypophosphatasia, bone sarcoma, myeloma bone disorder, osteolytic bone lesions and hypercalcemia.
PCT/US2001/013795 2000-04-28 2001-04-27 Stmst protein and nucleic acid molecules and uses therefor WO2001083523A2 (en)

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GB2367823A (en) * 2000-07-24 2002-04-17 Smithkline Beecham Corp AXOR28 polypeptides and polynucleotides
WO2003104279A1 (en) * 2002-06-10 2003-12-18 Bayer Healthcare Ag Regulation of a human g protein-coupled receptor
EP1454982A1 (en) * 2001-12-14 2004-09-08 Takeda Chemical Industries, Ltd. Mtehod of analyzing gene expression
JPWO2006006722A1 (en) * 2004-07-13 2008-05-01 武田薬品工業株式会社 How to regulate cell function

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WO2001048015A2 (en) * 1999-12-28 2001-07-05 Pharmacia & Upjohn Company G protein-coupled receptors
WO2001064882A2 (en) * 2000-02-29 2001-09-07 Millennium Pharmaceuticals, Inc. 1983, 52881, 2398, 45449, 50289, and 52872, g protein-coupled receptors and uses therefor

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
WO1999061463A1 (en) * 1998-05-29 1999-12-02 Millennium Pharmaceuticals, Inc. Novel secreted and membrane-associated proteins and uses therefor
WO2001048015A2 (en) * 1999-12-28 2001-07-05 Pharmacia & Upjohn Company G protein-coupled receptors
WO2001064882A2 (en) * 2000-02-29 2001-09-07 Millennium Pharmaceuticals, Inc. 1983, 52881, 2398, 45449, 50289, and 52872, g protein-coupled receptors and uses therefor

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2367823A (en) * 2000-07-24 2002-04-17 Smithkline Beecham Corp AXOR28 polypeptides and polynucleotides
EP1454982A1 (en) * 2001-12-14 2004-09-08 Takeda Chemical Industries, Ltd. Mtehod of analyzing gene expression
EP1454982A4 (en) * 2001-12-14 2005-12-28 Takeda Pharmaceutical Mtehod of analyzing gene expression
WO2003104279A1 (en) * 2002-06-10 2003-12-18 Bayer Healthcare Ag Regulation of a human g protein-coupled receptor
JPWO2006006722A1 (en) * 2004-07-13 2008-05-01 武田薬品工業株式会社 How to regulate cell function

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WO2001083523A3 (en) 2002-06-13

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