WO2003020307A1 - Genes induits par la proteine 2 morphogenique osseuse et polypeptides, utilisations de ceux-ci dans des techniques diagnostiques et therapeutiques - Google Patents

Genes induits par la proteine 2 morphogenique osseuse et polypeptides, utilisations de ceux-ci dans des techniques diagnostiques et therapeutiques Download PDF

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WO2003020307A1
WO2003020307A1 PCT/US2002/027671 US0227671W WO03020307A1 WO 2003020307 A1 WO2003020307 A1 WO 2003020307A1 US 0227671 W US0227671 W US 0227671W WO 03020307 A1 WO03020307 A1 WO 03020307A1
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big
polypeptide
nucleic acid
cells
seq
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Marie B. Demay
Francesca Gori
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The General Hospital Corporation
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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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • This invention relates to genes induced by bone morphogenic proteins, polypeptides encoded by these genes, and diagnostic and therapeutic methods employing these genes and polypeptides.
  • Bone is a very dense, specialized form of connective tissue, consisting of tough fibers (type I collagen fibrils), which resist pulling forces, and solid particles (calcium phosphate particles), which resist compression.
  • tough fibers type I collagen fibrils
  • solid particles calcium phosphate particles
  • bone is by no means a permanent and immutable tissue.
  • channels and cavities occupied by living cells that are engaged in an unceasing process of remodeling, which involves a coupled process of bone resorption and bone formation.
  • Osteoclasts degrade bone during the resorption phase by attaching to the mineralized bone matrix and excavating small pits on the bone surface, thereby releasing bone collagen and minerals into circulation.
  • osteoblasts replace the bone collagen removed by osteoclasts by depositing new collagen at the resorbed areas.
  • levels of bone resorption and bone formation generally are balanced. Net bone growth occurs when formation outpaces resorption, for example, in a child during growth or in the healing of a bone fracture, while net bone loss occurs when resorption outpaces formation, for example, in osteoporosis.
  • Bone formation in vivo occurs via two major processes, intramembranous and endochondral ossification. Although endochondral bone formation in vivo and in vitro has been the focus of several studies, the molecular signals that control this process remain to be defined. A number of factors have been shown to play critical roles in endochondral bone formation, including transforming growth factor (TGF)- ⁇ superfamily members, most notably, the subfamily of the bone morpho genetic proteins (BMPs) (Centrella et al, Endocrine Rev. 15:27-39, 1994; Celeste et al., Proc. Natl. Acad. Sci. U.S.A. 87:9843-9847, 1990).
  • TGF transforming growth factor
  • BMPs bone morpho genetic proteins
  • BMPs have high homology to decapentaplegic (dpp) and to Ng-1, which are responsible for early embryo pattern formation in Drosophila and Xenopus, respectively (Centrella et al., Endocrine Rev. 15:27-39, 1994). BMPs are expressed in a spatial and temporal pattern during embryogenesis in vertebrates, and are required for the development of many organs and tissues, including the lung, kidney, eye, testis, teeth, skin, and heart (Hogan, Genes Dev. 10:1580-1594, 1996). They are also expressed at sites of new bone formation early in development, suggesting that they are key signaling molecules for limb formation and patterning, as well as for osteogenesis (Hogan, Genes Dev.
  • BMPs were originally identified as bone derived factors capable of inducing ectopic bone formation when injected subcutaneously in animal models (Urist, Science 150:893-899, 1965; Sasano et al., Anat. Rec. 236:373-380, 1993).
  • BMPs and, in particular BMP-2. have been reported to induce the expression of many osteoblast genes (Ahrens et al., D ⁇ A Cell Biol. 12:871-880, 1993; Thies et al., Endocrinology 130:1318-1324, 1992; Yamaguchi et al., Biochem. Biophys. Res. Commun. 220:336- 371, 1996; Harris et al., Mol. Cell. Diff.
  • WD-40 proteins comprise a family of proteins that have been shown to play a role in numerous cellular functions including signal transduction, mR ⁇ A processing, gene regulation, vesicular trafficking, and regulation of the cell cycle ( ⁇ eer et al., Nature 371:297-300, 1994; Smith et al., Trends Biochem. Sci. 24:181-185, 1999; Neer et al, Proc. Natl. Acad. Sci. U.S.A. 97:1096-1100, 2000).
  • these proteins contain a conserved Trp-Asp motif (the so-called WD-40 repeat), which contains a GH dipeptide at the N-terminus and a WD dipeptide at the C-terminus, separated by a region of variable length (Neer et al., Nature 371:297-300, 1994; Smith et al, Trends Biochem. Sci. 24:181-185, 1999).
  • the most extensively characterized WD repeat protein is the G protein ⁇ subunit, which contains seven WD repeats (Neer et al., Cell 84:175-178, 1996; Fong et al., Proc. Natl. Acad. Sci. U.S.A. 83:2162-2166, 1986).
  • the tertiary structure of the G protein ⁇ subunit demonstrates that each of the seven WD repeats folds into four antiparallel ⁇ strands radiating outward from a central axis (Neer et al, Cell 84:175-178, 1996), leading to the description of " ⁇ -propeller.”
  • the present invention provides substantially pure bone morphogenic protein-2
  • polypeptides can include amino acid sequences that are substantially identical to the amino acid sequence of SEQ ID NO:2 (mouse) or SEQ ID NO:4 (human), or an amino acid sequence that is substantially identical to that encoded by the nucleic acid sequence of SEQ ID NO: 1 (mouse) or SEQ ID NO: 3 (human), or a complement thereof.
  • the polypeptides can also include amino acid sequences that are identical to the amino acid sequences of SEQ ID NO: 2 or SEQ ID NO: 4, or the amino acid sequence encoded by the nucleic acid sequence of SEQ JD NO:l or SEQ ID NO:3.
  • the invention also provides substantially purified polypeptides that include the sequence of SEQ ID NO:2 or SEQ ID NO:4 and variants thereof that include sequences that are at least 70%, 75%, 80%, 90%, or 95% identical to these sequences, and which accelerate osteoblastic differentiation of prechondroblastic cells.
  • These polypeptides can, for example, increase alkaline phosphatase activity, cyclic AMP production in response to parathyroid hormone, Cbfal mRNA levels, type I collagen mRNA levels, osteocalcin mRNA levels, or mineralized nodule formation in these cells.
  • These polypeptides can also have other activities of BIG-3, as described herein.
  • the polypeptides described herein can be derived from any mammal such as, for example, a human or a mouse.
  • fragments of the polypeptides described herein include a WD-40 repeat as described herein, which preferably includes 10 or fewer mismatches (e.g., 1, 2, 3, 4, or 5) to the WD-40 repeat sequences described herein or, preferably, additional (e.g., 2-7 or more) WD-40 repeats, at least one of which having three or fewer mismatches. Additional details concerning WD-40 repeats that can be included in the polypeptide fragments of the invention, are provided below.
  • the invention also provides substantially pure or isolated nucleic acid (e.g., DNA, RNA, or modified forms thereof) molecules having sequences encoding BIG-3 polypeptides.
  • nucleic acid molecules can encode an amino acid sequence selected from the group consisting of SEQ ID NO:2 or SEQ ID NO:4, an amino acid sequence that is substantially identical to that which is encoded by the nucleic acid sequence of SEQ ID NO:l or SEQ ID NO:3, or a complement or fragment (see above) thereof.
  • the nucleic acid molecule can have, for example, at least 55% (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) nucleic acid sequence identity to a sequence encoding a BIG-3 polypeptide or a fragment thereof having at least six (e.g., ten, fifteen, twenty, twenty-five, thirty, forty, or fifty) amino acids.
  • the nucleic acid molecule preferably hybridizes under high stringency conditions to at least a portion of a nucleic acid molecule encoding a BIG-3 polypeptide.
  • the invention further includes an isolated nucleic acid molecule that specifically hybridizes under high stringency conditions to the complement of the sequence set forth in SEQ ID NO: 1 or SEQ ID NO:3, and encodes a protein that accelerates osteoblastic differentiation of prechondroblastic cells.
  • the protein can, for example, increase alkaline phosphatase activity, cyclic AMP production in response to parathyroid hormone, Cbfal mRNA levels, type I collagen mRNA levels, osteocalcin mRNA levels, mineralized nodule formation in these cells, or other BIG-3 activities, such as those described herein.
  • the invention also provides vectors including nucleic acid molecules encoding BIG-3 polypeptides, cells containing such vectors, non-human transgenic animals including nucleic acid molecules encoding BIG-3 polypeptides, and antibodies that specifically bind to BIG-3 polypeptides.
  • the invention features a method of detecting a BIG-3 polypeptide in a sample.
  • a sample is contacted with an antibody that specifically binds to a BIG-3 polypeptide, and binding of the antibody to the polypeptide is detected.
  • tissue or organ formation for example, connective tissue, such as bone
  • an effective amount of a BIG-3 polypeptide is administered to the patient.
  • These methods can also be used to potentiate growth of other tissues in patients, such as, for example, cartilage, tendon, ligament, nerve, muscle, and epidermal tissues, as well as to potentiate organ (e.g., pancreas, heart, liver, lung, or kidney) development.
  • These methods can also include administration of a BIG-3 nucleic acid molecule or a compound that modulates (e.g., increases or decreases) BIG-3 activity, such as a BIG-3 activity described herein.
  • the invention also provides methods of identifying compounds that modulate BIG-3 activity or expression. These methods involve contacting a cell that expresses BIG-3 with a candidate compound and determining the effect of the compound on expression or an activity of BIG-3.
  • This activity can be selected from, for example, increases in osteoblastic differentiation of a prechondroblastic cell line, alkaline phosphatase activity, cyclic AMP production in response to parathyroid hormone, Cbfal mRNA levels, type I collagen mRNA levels, osteocalcin mRNA levels, mineralized nodule formation in the cell, or other BIG-3 activities such as those described herein.
  • the cell can be present in an animal or a cultured cell.
  • the invention also includes methods of using compounds identified using these methods in the prevention and treatment of diseases characterized by abnormalities in bone growth, such as those described elsewhere herein. Also included in the invention is the use of the proteins, polypeptides, antibodies, nucleic acid molecules, and identified compounds described herein in the prevention or treatment of diseases characterized by abnormalities in bone growth, such as those described herein, and the use of these agents in the manufacture of medicaments for these purposes.
  • the BIG-3 polypeptide includes a portion having at least 45%, 55%, 70%, 75%, 80%, 85%, 90%, or 95% (e.g., 100%) amino acid identity to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4, an amino acid sequence that is encoded by the nucleic acid sequence of SEQ ID NO:l or SEQ ID NO:3, or a complement or fragment thereof.
  • polypeptide products from splice variants of BIG-3 gene sequences are also included in this definition. Fragments of these proteins or polypeptides are also included in the invention and, preferably, include at least one WD-40 repeat.
  • the proteins or polypeptides can include an Aspartic Acid residue 6 amino acids amino terminal to conserved WD dipeptides in, e.g., 1, 2, 3, 4, 5, 6, or 7 of the WD-40 motifs present in the protein.
  • a "BIG-3 gene” or a "nucleic acid molecule encoding a BIG-3 polypeptide” is a nucleic acid molecule, such as genomic DNA, cDNA, or mRNA, that encodes a BIG-3 polypeptide or a portion thereof, as defined above.
  • sequence identity is used to indicate that a first polypeptide or nucleic acid molecule possesses the same amino acid or nucleotide residue at a given position, compared to a reference polypeptide or nucleic acid molecule to wliich the sequence of the first molecule is aligned.
  • Sequence identity can be measured using sequence analysis software with the default parameters specified therein, such as the introduction of gaps to achieve an optimal alignment. For example, the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705, can be used.
  • the term "substantially identical" is used in reference to a polypeptide or nucleic acid molecule to indicate that the molecule exhibits, over its entire length, at least 50%, preferably at least 60% or 65%, and most preferably 75%, 85%, 90%, or 95% identity to a reference amino acid or nucleic acid sequence.
  • the length of comparison sequences can be, for example, at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably at least 35 amino acids.
  • the length of comparison sequences can be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably at least 110 nucleotides.
  • probe or “primer” is meant a single-stranded DNA or RNA molecule of defined sequence that can base pair to a second DNA or RNA molecule that contains a complementary sequence (the “target”).
  • target a complementary sequence
  • the stability of the resulting hybrid depends upon the extent of the base pairing that occurs, which is affected by parameters such as the degree of complementarity between the probe and the target molecules and the degree of stringency of the hybridization conditions.
  • the degree of hybridization stringency is affected by parameters such as temperature, salt concentration, and the concentration of organic molecules, such as formamide, and appropriate conditions can readily be determined by those skilled in the art.
  • Probes or primers specific for nucleic acid molecules encoding BIG-3 polypeptides can have, preferably, greater than 50% sequence identity, more preferably at least 55-75% sequence identity, still more preferably at least 75-85% sequence identity, yet more preferably at least 85-99% sequence identity, and most preferably 100% sequence identity. Probes can be detectably-labeled, either radioactively, or non-radioactively, by methods well-known to those skilled in the art.
  • Probes are used for methods involving nucleic acid hybridization, such as nucleic acid amplification by polymerase chain reaction (PCR), single stranded conformational polymorphism (SSCP) analysis, restriction fragment polymorphism (RFLP) analysis, Southern hybridization, Northern hybridization, in situ hybridization, and electrophoretic mobility shift assay (EMSA).
  • PCR polymerase chain reaction
  • SSCP single stranded conformational polymorphism
  • RFLP restriction fragment polymorphism
  • Southern hybridization Southern hybridization
  • Northern hybridization in situ hybridization
  • ESA electrophoretic mobility shift assay
  • detectably-labeled is used to denote any means for marking and identifying the presence of a molecule, e.g., an oligonucleotide probe or primer, a gene or fragment thereof, a cDNA molecule, or an antibody.
  • Methods for detectably- labeling molecule are well known in the art and include, without limitation, radioactive labeling (e.g., with an isotope such as 32 P or 35 S) and nonradioactive labeling (e.g., with a fluorescent label, such as fluorescein).
  • substantially pure polypeptide is meant a polypeptide (or a fragment thereof) that has been separated from the components that accompany it in its natural state.
  • the polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated.
  • the polypeptide is a BIG-3 polypeptide that is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, pure.
  • a substantially pure BIG-3 polypeptide can be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding a BIG-3 polypeptide, or by chemically synthesizing the polypeptide.
  • Purity can be measured by any appropriate method, e.g., by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
  • a protein is substantially free of naturally associated components when it is separated from those contaminants that accompany it in its natural state.
  • a protein that is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates can be considered substantially free from its naturally associated components.
  • substantially pure polypeptides not only include those derived from eukaryotic organisms but also those synthesized in E. coli and other prokaryotes.
  • isolated nucleic acid molecule is meant a nucleic acid molecule that is removed from the environment in which it naturally occurs.
  • a naturally- occurring nucleic acid molecule present in the genome of cell or as part of a gene bank is not isolated, but the same molecule, separated from the remaining part of the genome, as a result of, e.g., a cloning event (amplification), is “isolated.”
  • an isolated nucleic acid molecule is free from nucleic acid regions (e.g., coding regions) with which it is immediately contiguous, at the 5' or 3' ends, in the naturally occurring genome.
  • nucleic acid molecules can be part of a vector or a composition and still be isolated, as such a vector or composition is not part of its natural environment.
  • a nucleic acid molecule of the invention can consist of RNA or DNA (e.g., cDNA, genomic DNA, or synthetic DNA), or modifications or combinations of RNA or DNA.
  • the nucleic acid molecules can be double-stranded or single-stranded and, if single-stranded, can be the coding (sense) strand or the non-coding (anti-sense) strand.
  • An antibody is said to "specifically bind" to an antigen, such as a BIG-3 peptide or polypeptide, if it recognizes and binds to the BIG-3 peptide or polypeptide, but does not substantially recognize and bind other molecules (e.g., other peptides or polypeptides) in a sample, e.g., a biological sample, which naturally includes the polypeptide.
  • an antigen such as a BIG-3 peptide or polypeptide
  • high stringency conditions is meant a set of conditions that allow hybridization comparable to hybridization that occurs using a DNA probe of at least 500 nucleotides in length, in a buffer containing 0.5 M NaHPO 4 , pH 7.2, 7% SDS, 1 mM EDTA, and 1% BSA, at a temperature of 65°C, or a buffer containing 48% formamide, 4.8 x SSC, 0.2 M Tris-Cl, pH 7.6, 1 x Denhardt's solution, 10% dextran sulfate, and 0.1% SDS, at a temperature of 42°C (these are typical conditions for high stringency Northern or Southern hybridizations).
  • High stringency hybridization is also relied upon for the success of numerous techniques routinely performed by molecular biologists, such as high stringency PCR, single strand conformational polymorphism analysis, and in situ hybridization, hi contrast to Northern and Southern hybridizations, these techniques are usually performed with relatively short probes (e.g., usually 16 nucleotides or longer for PCR or sequencing, and 40 nucleotides or longer for in situ hybridization).
  • the high stringency conditions used in these techniques are well known to those skilled in the art of molecular biology, and can be found, for example, in Ausubel et al. Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1998, which is hereby incorporated by reference.
  • transformation is used herein to denote any method for introducing a foreign molecule, such as a nucleic acid molecule, into a cell.
  • Lipofection, DEAE- dextran-mediated transfection, microinjection, protoplast fusion, calcium phosphate precipitation, retroviral delivery, electroporation, and biolistic transformation are just a few of the standard methods known to those skilled in the art that can be used.
  • biolistic transformation is a method for introducing foreign molecules into a cell using velocity driven microprojectiles such as tungsten or gold particles. Such velocity-driven methods originate from pressure bursts that include, but are not limited to, helium-driven, air-driven, and gunpowder-driven techniques.
  • Biolistic transformation can be applied to the transformation or transfection of a wide variety of cell types and intact tissues including, without limitation, intracellular organelles (e.g., mitochondria and chloroplasts), bacteria, yeast, fungi, algae, animal tissue, and cultured cells.
  • intracellular organelles e.g., mitochondria and chloroplasts
  • bacteria e.g., yeast, fungi, algae, animal tissue, and cultured cells.
  • transformed cell By “transfected cell,” or “transduced cell,” is meant a cell (or a descendent of a cell) into which a nucleic acid molecule encoding a polypeptide of the invention has been introduced, by means of recombinant techniques.
  • promoter is meant a sequence sufficient to direct transcription.
  • constructs of the invention can include promoter elements that are sufficient to render promoter-dependent gene expression controllable in a cell type-specific, tissue- specific, or temporal-specific manner, or inducible by external signals or agents; such elements can be located in the 5' or 3' or intron sequence regions of the native gene.
  • operably linked is used herein to indicate that a gene and one or more regulatory sequences are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequences.
  • sample is meant a specimen containing a tissue biopsy, cell, blood, serum, urine, stool, or other specimen obtained from a patient or test subject.
  • a sample can be analyzed for the presence of a BIG-3 gene, a BIG-3 polypeptide, or an antibody that binds to a BIG-3 polypeptide to detect, for example, expression levels of a BIG-3 gene or polypeptide using methods that are well known in the art.
  • nucleic acid hybridization such as polymerase chain reaction (PCR), reverse transcriptase/polymerase chain reaction (RT/PCR), and Northern hybridization can be used to detect BIG-3 nucleic acid molecules (e.g., mRNA), and standard immunoassays, such as ELISAs, can be used to measure levels of BIG-3 polypeptides.
  • PCR polymerase chain reaction
  • RT/PCR reverse transcriptase/polymerase chain reaction
  • ELISAs standard immunoassays
  • the invention provides several advantages.
  • the invention provides methods for the treatment and prevention of diseases and conditions of tissues such as the bone, as well as facilitates the identification of compounds that can be used in such methods.
  • Other features and advantages of invention will be apparent from the following detailed description, the drawings, and the claims.
  • MLB13MYC clone 17 cells were treated for 8, 16, and 24 hours with 0, 200, or 500 ng/ml of BMP-2. RT-PCR reactions were performed using 0.1 ⁇ g/ml total RNA. The arrow indicates the candidate cDNA that appears to be differentially expressed, a: 0 ng/ml BMP-2, b: 200 ng/ml BMP-2, c: 500 ng/ml BMP-2.
  • RNA isolated from cells of the osteoblast and chondroblast lineage and from tissues isolated from 3 month-old C57BL/6 mouse, was hybridized with an ⁇ 32 P-labeled cDNA probe for BIG-3. Conditionally immortalized cells were cultured under differentiating conditions for 5 days prior harvesting RNA.
  • lane 1 Conditionally immortalized primary osteocytes from 18.5 dpc embryos
  • lane 2 conditionally immortalized primary osteoblasts from 18.5 dpc embryos
  • lane 3 calvaria from 18.5 dpc embryos
  • lane 4 conditionally immortalized murine marrow stromal cells
  • lane 5 primary calvarial osteoblasts from 18.5 dpc embryos.
  • C Mouse tissues. The experiments were carried out twice and a representative blot is shown. Control hybridization with an 18S rRNA probe verified the amount of RNA loaded.
  • Fig. 3 cDNA sequence, WD-40 repeats, and modeled structure of BIG-3.
  • A) cDNA and deduced amino acid sequence of BIG-3. Blue letters represent the seven WD-40 repeat domains. Red letters indicate the mismatches with the WD-40 repeat consensus sequence.
  • Fig. 4. hnmunohistochemistry of 15.5 dpc mouse calvaria. A Anti BIG-3, B H&E, and C non immune rabbit serum.
  • Fig. 5 Alkaline Phosphatase Activity. Alkaline phosphatase activity of MC3T3E1-EN and MC3T3E1-BIG-3 pooled clones was assessed from 3 to 25 days in culture. Filled square: MC3T3E1-EN pool 1; filled circle: MC3T3E1-EN pool 2; filled triangle: MC3T3E1-EN pool 3; open circle: MC3T3E1-BIG-3 pool 1; open triangle: MC3T3E1-BIG-3 pool 2; open square: MC3T3E1-BIG-3 pool 3. The results are the means ⁇ SEM of two experiments carried out in triplicate, p ⁇ 0.00001 by multiple measures A ⁇ ONA between MC3T3E1-EN pools and MC3T3E1-BIG-3 pools over time.
  • Fig. 6 Cyclic AMP accumulation and PTH 1 receptor binding.
  • RNA isolated from MC3T3E1-EV and MC3T3E1- BIG-3 pooled clones was transferred to nylon membranes and hybridized with ⁇ 32 P- labeled cDNA probes for Cbfal and type I collagen. Control hybridization with an 18S rRNA probe verified the amount of RNA loaded. The experiments were carried out twice, and a representative blot is shown.
  • Fig. 9 Mineral deposition.
  • MC3T3E1-EN and MC3T3E1-BIG-3 pooled clones were cultured for periods ranging from 14 to 35 days. Following staining, the alizarin red-S dye was eluted and measured spectrophotometrically. Results are expressed as nanomoles of Alizarin Red S per well. The results shown are representative of two separate experiments ⁇ SEM carried out in quadruplicate. Filled bar: MC3T3E1-EN clones; Open bar MC3T3E1 -BIG-3 clones, p ⁇ 0.05 by multiple measures A ⁇ ONA.
  • Fig. 10 Comparison of the deduced amino acid sequence of BIG-3 and those of the mouse G protein ⁇ 2 subunit (mG ⁇ 2) and TUP -I. Deduced amino acid sequenced of BIG-3 in black, mG ⁇ ( ⁇ P_034442) in blue and amino acid sequence of TUP I (M31733) in green. Bars indicate conservations of amino acid residues.
  • Fig. 11. Graph showing alkaline phosphatase activity in BIG-3 rransfected cells, in the presence of increasing amounts of noggin.
  • Fig. 12. Graph showing formation of mineralized matrix in BIG-3 transfected cells in the presence and absence of noggin.
  • the invention provides bone morphogenic protein 2 (BMP-2)-induced gene 3 kilobase (BIG-3) protein, which is induced as a prechondroblastic cell line,
  • BMP-2 treatment When treated with BMP-2 these cells rapidly lose chondrocytic markers and express osteoblastic markers, including type I collagen and osteocalcin mRNA. Because the BMP-2 -induced expression of osteoblastic markers by MLB13MYC clone 17 cells parallels molecular events seen during endochondral bone formation in vivo, these cells are suitable models for identifying genes regulated by
  • BIG-3 genes and polypeptides, as well as compounds that modulate BIG-3 activity can be used in methods to diagnose, prevent, and treat diseases and conditions associated with tissue (e.g., bone; also see below) growth and development.
  • the BIG-3 polypeptides of the invention can be purified from cells in which they are naturally expressed or produced using recombinant methods, which are described as follows.
  • cell lines can be produced that overexpress BIG-3 polypeptides, allowing their purification for biochemical characterization, large-scale production, antibody production, or patient therapy.
  • eukaryotic and prokaryotic expression systems can be generated in which nucleic acid molecules containing BIG-3 genes are introduced into a plasmid or other vector, which is then used to transform living cells.
  • Constructs in which BIG-3 cDNAs containing entire open reading frames inserted in the correct orientation into an expression plasmid can be used for protein expression.
  • portions of the BIG-3 gene sequences can be inserted.
  • Prokaryotic and eukaryotic expression systems allow various important functional domains of BIG-3 polypeptides to be recovered, if desired, as fusion proteins, and then used for binding, structural, and functional studies, and also for the generation of appropriate antibodies.
  • Typical expression vectors contain promoters that direct the synthesis of large amounts of mRNA corresponding to the inserted BIG-3 nucleic acid molecule in the plasmid-bearing cells.
  • Stable long-term vectors can be maintained as freely replicating entities by using regulatory elements of, for example, viruses (e.g., the OriP sequences from the Epstein Barr Virus genome).
  • Cell lines can also be produced that have integrated the vector into the genomic DNA, and in this manner the gene product is produced in the cell lines on a continuous basis.
  • plasmid vectors contain several elements required for the propagation of the plasmid in bacteria, and for expression of the DNA inserted into the plasmid. Propagation of only plasmid-bearing bacteria can be achieved by introducing into the plasmid selectable marker-encoding sequences that allow plasmid-bearing bacteria to grow in the presence of otherwise toxic drugs.
  • the plasmid also contains a transcriptional promoter capable of producing large amounts of mRNA from the cloned gene. Such promoters can be, but are not necessarily, inducible promoters that initiate transcription upon induction with a particular compound.
  • the plasmid also preferably contains a polylinker to simplify insertion of the gene in the correct orientation within the vector.
  • the appropriate expression vectors containing a BIG-3 gene, fragment, fusion, or mutant thereof are constructed, they are introduced into appropriate host cells by a transformation technique, such as, for example, calcium phosphate transfection, DEAE-dextran transfection, electroporation, microinjection, protoplast fusion, or liposome-mediated transfection.
  • the host cells that are transfected with the vectors of the invention can include, but are not limited to, E. coli or other bacteria, yeast, fungi, insect cells (using, for example, baculoviral vectors for expression), or cells derived from mice, humans, or other animals.
  • Mammalian cells can also be used to express the BIG-3 protein using a vaccinia virus expression system described, for example, by Ausubel et al, supra.
  • T7 late promoter expression system In vitro expression of BIG-3 polypeptides, fusions, or fragments encoded by cloned DNA is also possible using the T7 late promoter expression system.
  • This system depends on the regulated expression of T7 RNA polymerase, an enzyme encoded in the D A of bacteriophage T7.
  • the T7 RNA polymerase initiates transcription at a specific 23 basepair promoter sequence called the T7 late promoter. Copies of the T7 late promoter are located at several sites on the T7 genome, but none is present in E. coli chromosomal DNA.
  • T7 RNA polymerase catalyzes transcription of viral genes, but not of E. coli genes.
  • E. coli cells are first engineered to carry the gene encoding T7 RNA polymerase next to the lac promoter. In the presence of IPTG, these cells transcribe the T7 polymerase gene at a high rate and synthesize abundant amounts of T7 RNA polymerase. These cells are then transformed with plasmid vectors that carry a copy of the T7 late promoter protein. When IPTG is added to the culture medium containing these transformed E. coli cells, large amounts of T7 RNA polymerase are produced. The polymerase then binds to the T7 late promoter on the plasmid expression vectors, catalyzing transcription of the inserted cDNA at a high rate. Since each E.
  • coli cell contains many copies of the expression vector, large amounts of mRNA corresponding to the cloned cDNA can be produced in this system and the resulting protein can be radioactively labeled.
  • Plasmid vectors containing late promoters and the corresponding RNA polymerases from related bacteriophages such as T3, T5, and SP6 can also be used for in vitro production of proteins from cloned DNA.
  • E. coli can also be used for expression using an M13 phage, such as mGPI-2.
  • vectors that contain phage lambda regulatory sequences or vectors that direct the expression of fusion proteins, for example, a maltose-binding protein fusion protein or a glutathione-S-transferase fusion protein, also can be used for expression in E. coli.
  • Eukaryotic expression systems are also useful for expressing BIG-3 polypeptides, particularly for obtaining appropriate post-translational modification of expressed proteins. Transient transfection of a eukaryotic expression plasmid allows the transient production of BIG-3 polypeptides by a transfected host cell.
  • BIG-3 polypeptides can also be produced by a stably-transfected mammalian cell line.
  • cDNA encoding a BIG-3 polypeptide, fusion, or fragment is cloned into an expression vector that includes the dihydrofolate reductase (DHFR) gene.
  • DHFR dihydrofolate reductase
  • Integration of the plasmid and, therefore, integration of the BIG-3 polypeptide- encoding gene into the host cell chromosome is selected for by inclusion of 0.01-300 ⁇ M methotrexate in the cell culture medium (as is described, for example, by Ausubel et al, supra). This dominant selection can be accomplished in most cell types. Recombinant protein expression can be increased by DHFR-mediated amplification of the transfected gene. Methods for selecting cell lines bearing gene amplifications are described by Ausubel et al, supra. These methods generally involve extended culture in medium containing gradually increasing levels of methotrexate.
  • DHFR-containing expression vectors are pCNSEII-DHFR and pAdD26SN(A) (described, for example, in Ausubel et al, supra).
  • the host cells described above or, preferably, a DHFR-deficient CHO cell line are among those most preferred for DHFR selection of a stably-transfected cell line or DHFR-mediated gene amplification.
  • Eukaryotic cell expression of BIG-3 polypeptides facilitates studies of BIG-3 genes and gene products, including determination of proper expression and post- translational modifications for biological activity, identifying regulatory elements located in the 5', 3', and intron regions of BIG-3 genes, and determining their roles in tissue regulation of BIG-3 polypeptide expression. It also permits the production of large amounts of these polypeptides for isolation and purification, and the use of cells expressing BIG-3 polypeptides as a functional assay system for antibodies generated against the proteins. Eukaryotic cells expressing BIG-3 polypeptides can be used to test the effectiveness of pharmacological agents on BIG-3 polypeptide associated diseases or as means by which to study BIG-3 polypeptides as components of a transcriptional activation system.
  • BIG-3 polypeptides, fusions, and polypeptide fragments in eukaryotic cells also enables the study of the function of the normal complete protein, specific portions of the protein, or of naturally occurring polymorphisms and artificially-produced mutated proteins.
  • the BIG-3 DNA sequences can be altered using procedures known in the art, such as restriction endonuclease digestion, DNA polymerase fill-in, exonuclease deletion, terminal deoxynucleotide transferase extension, ligation of synthetic or cloned DNA sequences, and site-directed sequence alteration using specific oligonucleotides, together with PCR.
  • Another preferred eukaryotic expression system is the baculovirus system using, for example, the vector pBacPAK9, which is available from Clontech (Palo Alto, CA). If desired, this system can be used in conjunction with other protein expression techniques, for example, the myc tag approach described by Evan et al. (Mol. Cell Biol. 5:3610-3616, 1985).
  • pBacPAK9 the vector pBacPAK9
  • this system can be used in conjunction with other protein expression techniques, for example, the myc tag approach described by Evan et al. (Mol. Cell Biol. 5:3610-3616, 1985).
  • the recombinant protein Once the recombinant protein is expressed, it can be isolated from the expressing cells by cell lysis followed by protein purification techniques, such as affinity chromatography.
  • an anti-BIG-3 polypeptide antibody which can be produced by the methods described herein, can be attached to a column and used to isolate the re
  • Lysis and fractionation of BIG-3 polypeptide-harboring cells prior to affinity chromatography can be performed by standard methods (see, e.g:, Ausubel et al, supra).
  • the recombinant protein can, if desired, be purified further by, e.g, high performance liquid chromatography (HPLC; e.g, see Fisher, Laboratory Techniques In Biochemistry and Molecular Biology, Work and Burdon, Eds, Elsevier, 1980).
  • Polypeptides of the invention can also be produced by chemical synthesis (e.g, by the methods described in Solid Phase Peptide Synthesis, 2 nd ed, 1984, The Pierce Chemical Co, Rockford, EL). These general techniques of polypeptide expression and purification can also be used to produce and isolate useful BIG-3 polypeptide fragments or analogs, as described herein. Those skilled in the art of molecular biology will understand that a wide variety of expression systems can be used to produce the recombinant BIG-3 polypeptides. The precise host cell used is not critical to the invention.
  • the BIG-3 polypeptides can be produced in a prokaryotic host (e.g, E. coli) or in a eukaryotic host (e.g, S. cerevisiae, insect cells, such as Sf9 cells, or mammalian cells, such as COS-1, NTH 3T3, or HeLa cells). These cells are commercially available from, for example, the American Type Culture Collection, Rockville, Maryland (see also Ausubel et al, supra).
  • the method of transformation and the choice of expression vehicle (e.g, expression vector) will depend on the host system selected. Transformation and transfection methods are described, e.g, in Ausubel et al, supra, and expression vehicles can be chosen from those provided, e.g, in Pouwels et al. Cloning Nectors: A Laboratory Manual, 1985, Supp. 1987. Polypeptides of the invention can be tested for bone inducing activity using any of the assays described, for example, in U.S. Patent No. 5,728,679.
  • Polypeptide fragments that incorporate various portions of BIG-3 polypeptides are useful in identifying the domains important for the biological activities of BIG-3 polypeptides, such as protein-protein interactions. These fragments can be used alone or as chimeric fusion proteins.
  • BIG-3 polypeptide fragments can also be used to raise antibodies specific for various regions of BIG-3 polypeptides. Methods for generating such fragments are well known in the art (see, for example, Ausubel et al, supra) using the nucleotide sequences provided herein.
  • a BIG-3 polypeptide fragment can be generated by PCR amplifying the desired fragment using oligonucleotide primers designed based upon the BIG-3 nucleic acid sequences provided herein.
  • the oligonucleotide primers include unique restriction enzyme sites that facilitate insertion of the fragment into the cloning site of a mammalian expression vector.
  • This vector can then be introduced into a mammalian cell by artifice by various techniques known in the art, for example, those described herein, resulting in the production of a BIG-3 gene fragment.
  • BIG-3 polypeptides and fragments can also be produced in animal systems, such as transgenic mice, pigs, goats, cows, and the like. Using method known in the art, these polypeptides and fragments can be produced in the milk of such animals. (See, e.g, Simons et al, Bio/Technology 6:179-183, 1988; Wright et al, Bio/Technology 9:830-834, 1991; U.S. Patent No. 4,873,191; U.S. Patent No. 5,322,775; and Hogan et al, "Manipulating the Mouse Embryo; A Laboratory Manual," Cold Spring Harbor Laboratory, 1986, for descriptions of these methods.)
  • BIG-3 polypeptides, fragments of BIG-3 polypeptides, or fusion proteins containing defined portions of BIG-3 polypeptide can be synthesized in bacteria by expression of corresponding DNA sequences in a suitable cloning vehicle. Fusion proteins are commonly used as a source of antigen for producing antibodies. Two widely used expression systems for E. coli are lacZ fusions using the pUR series of vectors and trpE fusions using the pATH vectors. The proteins can be purified, coupled to a carrier protein and mixed with Freund's adjuvant, to enhance stimulation of the antigenic response in an innoculated animal, and injected into rabbits or other laboratory animals. Alternatively, protein can be isolated from BIG-3 polypeptide-expressing cultured cells.
  • the rabbits or other laboratory animals are bled and sera isolated.
  • the sera can be used directly or can be purified prior to use by various methods, including affinity chromatography employing reagents such as Protein A- Sepharose, antigen-Sepharose, or anti-mouse-Ig-Sepharose.
  • affinity chromatography employing reagents such as Protein A- Sepharose, antigen-Sepharose, or anti-mouse-Ig-Sepharose.
  • the sera can then be used to probe protein extracts from BIG-3 polypeptide-expressing tissue electrophoretically fractionated on a polyacrylamide gel to identify BIG-3 polypeptides.
  • synthetic peptides can be made that correspond to the antigenic portions of the protein and used to innoculate the animals.
  • a BIG-3 polypeptide coding sequence can be expressed as a C-tenninal fusion with glutathione S-transferase (GST; Smith et al. Gene 67:31-40, 1988).
  • GST glutathione S-transferase
  • the fusion protein can be purified on glutathione-Sepharose beads, eluted with glutathione, cleaved with thrombin (at the engineered cleavage site), and purified to the degree required to successfully immunize rabbits.
  • Primary immunizations can be carried out with Freund's complete adjuvant and subsequent immunizations performed with Freund's incomplete adjuvant.
  • Antibody titers are monitored by Western blot and immunoprecipitation analyses using the thrombin- cleaved BIG-3 polypeptide fragment of the GST-BIG-3 polypeptide fusion protein. Immune sera are affinity purified using CNBr-Sepharose-coupled BIG-3 polypeptide. Antiserum specificity can be determined using a panel of unrelated GST fusion proteins.
  • monoclonal BIG-3 polypeptide antibodies can be produced by using, as an antigen, a BIG-3 polypeptide isolated from BIG-3 polypeptide-expressing cultured cells or BIG-3 polypeptide isolated from tissues.
  • the cell extracts, or recombinant protein extracts containing BIG-3 polypeptide can, for example, be injected with Freund's adjuvant into mice.
  • the mouse spleens are removed, the tissues are disaggregated, and the spleen cells are suspended in phosphate-buffered saline (PBS).
  • PBS phosphate-buffered saline
  • the spleen cells serve as a source of lymphocytes, some of which are producing antibody of the appropriate specificity.
  • tissue culture wells in the presence of a selective agent such as hypoxanthine, aminopterine, and thymidine (HAT).
  • a selective agent such as hypoxanthine, aminopterine, and thymidine (HAT).
  • HAT thymidine
  • the wells are then screened by ELIS A to identify those containing cells that make antibody, which is capable of binding to a BIG-3 polypeptide or polypeptide fragment or mutant thereof.
  • these wells are again screened to identify antibody-producing cells.
  • Several cloning procedures are carried out until over 90% of the wells contain single clones that are positive for antibody production. From this procedure a stable line of clones that produce the antibody is established.
  • the monoclonal antibody can then be purified by affinity chromatography using Protein A Sepharose, ion-exchange chromatography, as well as variations and combinations of these techniques.
  • Truncated versions of monoclonal antibodies can also be produced by recombinant methods in which plasmids are generated that express the desired monoclonal antibody fragment(s) in a suitable host.
  • peptides corresponding to relatively unique hydrophilic regions of BIG-3 polypeptide can be generated and coupled to keyhole limpet hemocyanin (KLH) through an introduced C- terminal lysine.
  • KLH keyhole limpet hemocyanin
  • Antiserum to each of these peptides is similarly affinity-purified on peptides conjugated to BSA, and specificity is tested by ELISA and Western blotting using peptide conjugates, and by Western blotting and immunoprecipitation using a BIG-3 polypeptide, for example, expressed as a GST fusion protein.
  • monoclonal antibodies can be prepared using the BIG-3 polypeptides described above and standard hybridoma technology (see, e.g, Kohler et al. Nature 256:495, 1975; Kohler et al, Eur. J. Immunol. 6:511, 1976; Kohler et al, Eur. J. Immunol. 6:292, 1976; Hammerling et al. In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, New York, NY, 1981; Ausubel et al, supra). Once produced, monoclonal antibodies are also tested for specific BIG-3 polypeptide recognition by Western blot or immunoprecipitation analysis (for example, by the methods described in Ausubel et al, supra). Monoclonal and polyclonal antibodies that specifically recognize a BIG-3 polypeptide (or a fragment thereof) are considered useful in the invention.
  • Antibodies of the invention can be produced using BIG-3 polypeptide amino acid sequences that do not reside within highly conserved regions, and that appear likely to be antigenic, as analyzed by criteria such as those provided by the Peptide Structure Program (Genetics Computer Group Sequence Analysis Package, Program Manual for the GCG Package, Version 7, 1991) using the algorithm of Jameson and Wolf (CABIOS 4:181, 1988). These fragments can be generated by standard techniques, e.g, by PCR, and cloned into the pGEX expression vector (Ausubel et al, supra). GST fusion proteins are expressed in E. coli and purified using a glutathione- agarose affinity matrix as described in Ausubel et al, supra.
  • two or three fusions are generated for each protein, and each fusion is injected into at least two rabbits.
  • Antisera are raised by injections in series, preferably including at least three booster injections.
  • the invention features various genetically engineered antibodies, humanized antibodies, and antibody fragments, including F(ab')2, Fab', Fab, Fv, and sFv fragments.
  • Antibodies can be humanized by methods known in the art, e.g, monoclonal antibodies with a desired binding specificity can be commercially humanized (Scotgene, Scotland; Oxford Molecular, Palo Alto, CA). Fully human antibodies, such as those expressed in transgenic animals, are also features of the invention (Green et al. Nature Genetics 7:13-21, 1994).
  • Ladner U.S. Patent Nos. 4,946,778 and 4,704,692 describes methods for preparing single polypeptide chain antibodies.
  • Ward et al. (Nature 341:544-546, 1989) describe the preparation of heavy chain variable domains, which they term "single domain antibodies," and which have high antigen-binding affinities.
  • McCafferty et al. (Nature 348:552-554, 1990) show that complete antibody V domains can be displayed on the surface of fd bacteriophage, that the phage bind specifically to antigen, and that rare phage (one in a million) can be isolated after affinity chromatography.
  • Boss et al. (U.S. Patent No.
  • Antibodies to BIG-3 polypeptide can be used, as noted above, to detect BIG-3 polypeptides or to inhibit the biological activities of BIG-3 polypeptides.
  • a nucleic acid molecule encoding an antibody or a portion of an antibody can be expressed within a cell to inhibit BIG-3 polypeptide function.
  • the antibodies can be coupled to compounds for diagnostic and/or therapeutic uses, such as radionuclides and liposomes carrying therapeutic compounds.
  • BIG-3 genes and polypeptides also find use in therapeutic and diagnostic applications involving growth potentiation in diverse tissues, including, for example, cartilage, tendon, ligament, nerve, muscle, and epidermal tissues, as well as organs such as pancreas, heart, liver, lung, and kidney.
  • therapies can be designed to circumvent or overcome inadequate or excessive expression of BIG-3 genes.
  • Inadequate expression of such genes maybe a characteristic of conditions associated with bone loss, such as osteoporosis and osteogenesis imperfecta, which can thereby be treated by administration of BIG-3 polypeptides or genes that encode them.
  • These molecules can also be used to treat diseases and conditions associated with abnormal growth, such as diseases and conditions characterized by excessive or insufficient bone growth, or bone weakening (e.g, osteoporosis, osteogenesis imperfecta, short stature (caused by, e.g, chondrodysplasias) Paget's disease, gigantism, acromegaly, hypertrophic arthritis, Marfan syndrome, sclerosteosis, osteopetrosis, excessive bone growth caused by excessive growth hormone produced by pituitary tumors, and bone cancer). Further, the methods can be used to promote tissue (e.g, bone) healing and in approaches to promote growth of and strengthen teeth. These diseases and conditions (and others) can also be prevented or treated by the use of compounds that modulate BIG-3 expression or activity, as discussed below.
  • bone weakening e.g, osteoporosis, osteogenesis imperfecta, short stature (caused by, e.g, chondrodysplasias) Paget's disease, giganti
  • Reagents that modulate BIG-3 polypeptide biological activity include, without limitation, full length BIG-3 polypeptides, or fragments thereof, BIG-3 polypeptide mRNA or antisense RNA, or any compound that modulates BIG-3 polypeptide biological activity, expression, or stability (see below).
  • Multiple active components can be administered together, for example, molecules of the invention can be administered with each other or with, e.g, BMPs or related molecules.
  • Treatment or prevention of diseases that would benefit from BIG-3 polypeptide expression can be accomplished by replacing a mutant BIG-3 polypeptide gene with a normal BIG-3 polypeptide gene, by modulating the function a mutant protein, by delivering normal BIG-3 polypeptide to the appropriate cells, or by altering the levels of normal or mutant protein. It is also possible to modify the pathophysiologic pathway in which the protein participates to correct the physiological defect. To replace a mutant protein with normal protein, or to add protein to cells that no longer express sufficient BIG-3 polypeptides, it may be necessary to obtain large amounts of pure BIG-3 polypeptide from cultured cell systems that can express the protein. Delivery of the protein to the effected tissue can then be accomplished using appropriate packaging or administration systems. Alternatively, small molecule analogs that act as BIG-3 polypeptide agonists or antagonists can be administered to produce a desired physiological effect.
  • Gene therapy is another therapeutic approach for preventing or ameliorating diseases related to BIG-3 polypeptide expression.
  • Nucleic acid molecules encoding BIG-3 polypeptides can be delivered to cells, where it must be in a form in which it can be taken up and direct expression of sufficient protein to provide effective function.
  • Transducing retroviral, adenoviral, and human immunodeficiency viral (HIV) vectors can be used for somatic cell gene therapy especially because of their high efficiency of infection and stable integration and expression; see, e.g, Cayouette et al. Hum. Gene Therapy 8:423-430, 1997; Kido et al, Curr. Eye Res. 15:833-844, 1996; Bloomer et al, J. Virol.
  • a full length BIG-3 polypeptide gene can be cloned into a retroviral vector and driven from its endogenous promoter or from the retroviral long terminal repeat or from a promoter specific for the target cell type of interest (such as neurons).
  • viral vectors which can be used include adenovirus, adeno- associated virus, vaccinia virus, bovine papilloma virus, or a herpes virus such as Epstein-Barr virus. Gene transfer can also be achieved using non- viral means requiring infection in vitro. This would include calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes can also be beneficial for delivery of DNA into a cell. Transplantation of normal genes into the affected tissues of a patient can also be accomplished by transferring a normal BIG-3 polypeptide gene into a cultivatable cell type ex vivo, after which the cells are injected into the targeted tissue(s).
  • Retroviral vectors, adenoviral vectors, adenovirus-associated viral vectors, or other viral vectors with the appropriate tropism for cells likely to be involved in BIG-3 polypeptide-related diseases can be used as gene transfer delivery systems for therapeutic BIG-3 polypeptide gene constructs.
  • Numerous vectors useful for this purpose are generally known (see, for example, Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244:1275-1281, 1989; Eglitis et al, BioTechniques 6:608- 614, 1988; Tolstoshev and Anderson, Curr. Opin. Biotech. 1:55-61, 1990; Sharp, The Lancet 337:1277-1278, 1991; Cornetta et al, Nucl. Acid Res. and Mol.
  • Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al, N. Engl. J. Med. 323:370, 1990; Anderson et al, U.S. Patent No. 5,399,346).
  • Non- viral approaches can also be employed for the introduction of therapeutic
  • a BIG-3 polypeptide nucleic acid molecule or antisense nucleic acid molecule can be introduced into a cell by lipofection (Feigner et al, Proc. Natl. Acad. Sci. U.S.A. 84:7413, 1987; Ono et al, Neurosci. Lett. 117:259, 1990; Brigham et al, Am. J. Med. Sci. 298:278, 1989; Staubinger et al, Meth. Enz. 101:512, 1983), asialoorosomucoid-polylysine conjugation (Wu et al, J. Biol. Chem. 263:14621, 1988; Wu et al, J. Biol. Chem. 264:16985, 1989), or, less preferably, micro-injection under surgical conditions (Wolff et al. Science 247:1465, 1990).
  • BIG-3 polypeptide cDNA expression can be directed from any suitable promoter (e.g, the human cytomegalovirus (CMV), simian virus 40
  • CMV human cytomegalovirus
  • simian virus 40 e.g., the human cytomegalovirus (CMV), simian virus 40
  • enhancers known to preferentially direct gene expression in specific cell types can be used to direct BIG-3 polypeptide expression.
  • the enhancers used include, without limitation, those that are characterized as tissue- or cell-specific enhancers.
  • a BIG-3 polypeptide genomic clone is used as a therapeutic construct (such clones can be identified by hybridization with the BIG-3 polypeptide cDNA described above), regulation can be mediated by the cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source, including any of the promoters or regulatory elements described above.
  • Antisense-based strategies can be employed to explore BIG-3 polypeptide gene function and as a basis for therapeutic drug design.
  • the principle is based on the hypothesis that sequence-specific suppression of gene expression (via transcription or translation) can be achieved by intracellular hybridization between genomic DNA or mRNA and a complementary antisense species.
  • sequence-specific suppression of gene expression via transcription or translation
  • a complementary antisense species can be achieved by intracellular hybridization between genomic DNA or mRNA and a complementary antisense species.
  • the formation of a hybrid RNA duplex interferes with transcription of the target BIG-3 polypeptide-encoding genomic DNA, or processing, transport, translation, and/or stability of the target BIG-3 polypeptide mRNA.
  • Antisense molecules can be delivered by a variety of approaches.
  • antisense oligonucleotides or antisense RNA can be directly administered (e.g, by intravenous injection) to a subject in a form that allows uptake into cells.
  • viral or plasmid vectors that encode antisense RNA (or RNA fragments) can be introduced into a cell in vivo or ex vivo.
  • Antisense effects can be induced by control (sense) sequences; however, the extent of phenotypic changes are highly variable. Phenotypic effects induced by antisense effects are based on changes in criteria such as protein levels, protein activity measurement, and target mRNA levels.
  • BIG-3 polypeptide gene therapy can also be accomplished by direct administration of antisense BIG-3 polypeptide mRNA to a cell that is expected to be adversely affected by the expression of wild type or mutant BIG-3 polypeptide.
  • the antisense BIG-3 polypeptide mRNA can be produced and isolated by any standard technique, but is most readily produced by in vitro transcription using an antisense BIG-3 polypeptide cDNA under the control of a high efficiency promoter (e.g, the T7 promoter).
  • Administration of antisense BIG-3 polypeptide mRNA to cells can be carried out by any of the methods for direct nucleic acid administration described above.
  • An alternative strategy for inhibiting BIG-3 polypeptide function using gene therapy involves intracellular expression of an anti-BIG-3 polypeptide antibody or a portion of an anti-BIG-3 polypeptide antibody.
  • the gene (or gene fragment) encoding a monoclonal antibody that specifically binds to BIG-3 polypeptide and inhibits its biological activity can be placed under the transcriptional control of a tissue-specific gene regulatory sequence.
  • Another therapeutic approach within the invention involves administration of recombinant BIG-3 polypeptide, either directly to the site of a potential or actual disease-affected tissue (for example, by injection) or systemically (for example, by any conventional recombinant protein administration technique).
  • the dosage of BIG-3 polypeptide depends on a number of factors, including the size and health of the individual patient, but, generally, between 0.1 mg and 100 mg inclusive are administered per day to an adult in any pharmaceutically acceptable formulation.
  • the methods of the instant invention can be used to diagnose or treat the disorders described herein in any mammal, for example, humans, domestic pets, or livestock. Where a non-human mammal is treated or diagnosed, the BIG-3 polypeptide, nucleic acid, or antibody employed is preferably specific for that species.
  • a BIG-3 polypeptide, gene, or modulator of a BIG-3 polypeptide can be administered within a pharmaceutically-acceptable diluent, carrier, or excipient, in unit dosage form.
  • Formulations for parenteral administration can, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes.
  • Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers can be used to control the release of the compounds.
  • Formulations for inhalation can contain excipients, for example, lactose, or can be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or can be oily solutions for administration in the form of nasal drops, or as a gel.
  • BIG-3 genes and polypeptides have a number of diagnostic uses. For example, as discussed above, maintenance of bone structure requires a balance between the bone resorbing activity of osteoclasts and the bone formation activity of osteoblasts. Throughout life, a dynamic process of bone remodeling takes place through the opposing activities of these cells, with the net effect, under normal circumstances, of maintaining bone structure. Disruption of this balance is a hallmark of bone injury, as well as several diseases of bones, such as osteoporosis, Paget's disease, and osteogenesis imperfecta.
  • the genes of the invention are induced in the differentiation of the bone formation cells, osteoblasts.
  • the genes of the invention and, in particular, the gene products they encode can be used as markers of bone formation and resorption, facilitating diagnosis of, for example, pathological alterations in bone turnover and to measure responses to therapy. This is of significant medical relevance, as numerous and prevalent debilitating diseases are associated with loss of the balance between bone loss and formation. In particular, osteoporosis and Paget's disease are associated with net bone loss.
  • Standard diagnostic methods such as immunoassays, including enzyme-linked immunosorbent assays (ELISAs), quantitative PCR, and reverse transcriptase/polymerase chain reaction (RT/PCR)-based assays can be readily adapted for use in the diagnostic methods of the invention (see, e.g, Ausubel et al, supra; Ehrlich (Ed.) PCR Technology: Principles and Applications for DNA Amplification, Stockton Press, NY; Yap et al, Nucl. Acids. Res. 19:4294, 1991).
  • Materials from which samples may be obtained for use in diagnosis include, for example, urine, blood samples (e.g, serum) and bone marrow, which can be obtained using standard methods.
  • Standard immunoassays can be used to detect or to monitor BIG-3 polypeptide expression in a biological sample.
  • BIG-3 polypeptide-specific polyclonal or monoclonal antibodies produced as described above can be used in any standard immunoassay format (e.g, ELISA, Western blot, or RIA) to measure BIG-3 polypeptide levels. These levels can be compared to wild-type BIG-3 polypeptide levels in control samples. For example, a decrease in BIG-3 polypeptide production can indicate a condition or a predisposition to a condition involving insufficient BIG-3 polypeptide biological activity.
  • Immunohistochemical techniques can also be utilized for BIG-3 polypeptide detection.
  • a tissue sample can be obtained from a patient, sectioned, and stained for the presence of BIG-3 polypeptides using an anti-BIG-3 polypeptide antibody and any standard detection system (e.g, one which includes a secondary antibody conjugated to horseradish peroxidase).
  • any standard detection system e.g, one which includes a secondary antibody conjugated to horseradish peroxidase.
  • diagnosis of BMP-related disorders may be evaluated by an examination of BIG-3 genes and a determination of whether such genes include mutations characteristic of disease.
  • such BIG-3 genes and polypeptides may be used for diagnostic purposes for detecting alterations in any of a variety of issues including, for example, cartilage, tendon, ligament, nerve, muscle, and epidermal tissues, as well as organs such as pancreas, heart, liver, lung, and kidney.
  • the invention also includes methods for identifying compounds that modulate BIG-3 activity. These compounds can be used, for example, in the prophylactic and treatment methods described above.
  • Candidate compounds can be screened by contacting cells (e.g, transfected MLB13MYC clone 17 cells) that express BIG-3 with the compounds, and then by determining the effect of the compounds on BIG-3 activity, as described herein.
  • the screening methods can also be carried out in model animal systems, such as transgenic mice.
  • novel drugs for the prevention or treatment of diseases related to BIG-3 can be identified from large libraries of natural products, synthetic (or semi-synthetic) extracts, and chemical libraries using methods that are well known in the art.
  • test extracts or compounds are not critical to the screening methods of the invention.
  • Compounds that are found to increase expression and/or activity of BIG-3 can be used in the prevention and treatment of diseases and conditions in which it would be desirable to increase rates of bone formation, while compounds that are found to decrease such expression and/or activity can be used in the prevention and treatment of diseases and conditions in which it would be desirable to decrease rates of bone formation. Examples of such diseases and conditions are provided above.
  • BIG-3 encodes a novel 328 amino acid, 34 kDa protein belonging to the family of WD-40 repeat proteins.
  • this family of proteins has been shown to play a role in numerous cellular functions including signal transduction, mRNA processing, gene regulation, vesicular trafficking, and regulation of the cell cycle, and structurally they contain a conserved Trp-Asp motif (the so-called WD-40 repeat), that contains the GH dipeptide at the N-terminus and the WD dipeptide at the C-terminus separated by a region of variable length.
  • Trp-Asp motif the so-called WD-40 repeat
  • RNA isolated from MLB13MYC clone 17 cells treated with 0, 200, or 500 ng/ml of BMP-2 for 8, 16, and 24 hours, was used for ddPCR experiments. After overnight autoradiography, bands representing differentially expressed mRNAs were identified. One of the differentially expressed PCR products (Fig. 1 A, arrow), was excised, reamplified, and used as a probe in Northern analyses to confirm differential expression. The ddPCR product was then subcloned into pGEM T-easy. Probing of Northern blots with this subcloned cDNA revealed a single mRNA transcript of 3 kb (Fig.
  • the full-length cDNA sequence for BIG-3 was obtained by screening a MLB13MYC clone 17 cDNA library using the 210 bp ddPCR product as a radiolabeled probe. The screening of 1 x 10 6 pfu resulted in the isolation of three independent overlapping phage clones.
  • the acquisition of the mature osteoblast phenotype is characterized by the ability of these cells to synthesize alkaline phosphatase, and an increase in the specific activity of this enzyme is directly correlated with a shift to a more differentiated state (Stein et al. Endocrine Rev. 14:424-442, 1993). Therefore, three independent pools of 8-12 clones of both MC3T3E1-BIG-3 and MC3T3E1-EN were examined for the time course of onset of, and magnitude of increase in, alkaline phosphatase (AP) activity. In the MC3T3E1-EN pools, AP activity did not increase significantly until 11 days (p ⁇ 0.0005 by Student's t-test).
  • PTH 1 receptor The receptor for parathyroid hormone (PTH 1 receptor) has been shown to be induced during osteoblastic differentiation (Bos et al. Tissue Int. 58:95-100, 1996; Lee et al. Bone 14:341-345, 1993). Cyclic AMP production in response to PTH, an indicator of PTH 1 receptor activity, was increased 10- and 14-fold at 7 days and 14 days, respectively, in the three independent pools of 8-12 clones of MC3T3E1-BIG-3, relative to the three independent MC3T3E1-EN pooled clones (Fig. 6A).
  • MC3T3E1-BIG-3 clones compared to the MC3T3E1-EN clones.
  • Stable expression of BIG-3 did not alter the proliferation of MC3T3-E1 cells, as assessed by counting pooled clones at regular intervals from the time of plating until they achieved confluence. Dramatic overexpression of proteins can result in a phenotype that reflects interference with normal cellular actions, rather than enhancing the normal effects of the protein of interest.
  • the final step in the program of osteoblast differentiation is thought to be the formation of mineralized bone nodules.
  • Mineralized nodules were first seen at 21 days in the MC3T3E1-BIG-3 clones, 14 days before they were detectable in the MC3T3E1-EN clones.
  • Quantitation of mineral deposition by elution of Alizarin Red S from stained mineral deposits at 25 days demonstrated a 3- fold increase in the dye content of the MC3T3E1-BIG-3 clones compared to MC3T3E1-EN clones (Fig. 9).
  • MC3T3-E1 cells stably transfected with empty vector (EN) or BIG-3, were treated, or not, with noggin 3 times weekly, starting the day after plating.
  • EN empty vector
  • the acquisition of alkaline phosphatase activity in the EN transfected cells was inhibited by a dose as low as 100 ng/ml of noggin, whereas doses as high as 1000 ng/ml of noggin did not effect the time of onset or magnitude of increase in alkaline phosphatase activity in the BIG-3 transfected cells (Fig. 11).
  • the prechondroblastic cell line, MLB13MYC clone 17, was maintained as previously reported (Rosen et al, J. Bone Min. Res. 9:1759-1768, 1994). At confluence, the cells were treated with 0, 200, and 500 ng/ml BMP-2 (Genetics Institute, Cambridge MA) in DMEM, 1% HI-FCS, and 1% penicillin/streptomycin (Gibco/BRL, Grand Island, ⁇ Y) for 8 to 72 hours. All cells were harvested 72 hours post confluence. Conditionally immortalized murine bone marrow stromal cells, osteoblasts, osteocytes, and growth plate chondrocytes (Dr. F.R.
  • MC3T3E1 cells obtained from the American Type Culture Collection (ATCC, Manassas, Virginia), were cultured in ⁇ MEM, 10% FBS, and 1% penicillin/streptomycin for periods ranging from 3 to 35 days.
  • ATCC American Type Culture Collection
  • FBS FBS
  • penicillin/streptomycin for periods ranging from 3 to 35 days.
  • medium was supplemented with ⁇ -glycerol phosphate and ascorbic acid (Quarles et al. Endocrinology 128:3144-3151, 1991) (Sigma Chemical Co, St. Louis, MO). Fresh medium was added twice a week.
  • RNA isolated from MLB13MYC clone 17 cells (Tri reagent, Sigma Chemical Co, St. Louis, MO) treated with BMP-2 (200 and 500 ng/ml) for 8, 16, or 24 hours, was first treated with RNase-free DNase I to eliminate contaminating chromosomal DNA (MessageClean® kit, GenHunter Corp, Arlington, TN).
  • RNA isolated from parallel, untreated cultures was used as control for each time point.
  • Two independent ddPCR reactions were performed according to the manufacturer's instructions (RNAimage® kit, GenHunter Corp, Nashville, TN).
  • the radioactive PCR products were resolved on denaturating 6% polyacrylamide/urea sequencing gels. After overnight autoradiography, differentially expressed bands were identified by comparison of ddPCR products representing cDNA species from BMP-2 treated and untreated cells. ddPCR products that were reproducibly differentially expressed were excised from the gel, reamplified, and subcloned into PGEM-T easy (Promega, Norwalk, CT).
  • RNA Ten ⁇ g of total RNA (or two ⁇ g of Poly A + mRNA for osteocalcin detection) was isolated from the cell types indicated and from tissues of a 3 month-old male C57BL6 mouse, were resolved on a 1% agarose/formaldehyde gel and transferred to a nylon membrane (Biotrans ICN, Aurora, OH) by capillary blotting. Probes were radiolabeled with [ ⁇ - 32 P] dATP (Dupont New England Nuclear, Boston, MA) to a specific activity of > 10 8 cpm/ng DNA (MegaprimeTM DNA labeling systems, Amersham Piscataway, NJ). Membranes were exposed for equivalent lengths of time to permit assessment of the relative level of expression of BIG-3 in the cell lines and tissues examined. Control hybridization with an 18S rRNA antisense oligonucleotide or GAPDH verified that equal amounts of RNA were loaded. Cloning and sequencing
  • MC3T3-E1 cells were plated at a density of 3 x 10 3 cells/cm 2 . After 24 hours, they were stably transfected with the full-length coding region of BIG-3 (MC3T3E1- BIG-3) cloned into pcDNA3.1 (Invitrogen Carlsbad, CA) or with the empty vector (MC3T3E1-EV) using calcium-phosphate precipitation. After 24 hours, and every 48 hours thereafter, the medium was replaced with fresh media containing 300 ⁇ g/ml of G418 for 14 days. This G418 dose was demonstrated to kill 100% of untransfected cells in 10 days. Three independent pools of 8-12 clones of MC3T3E1-BIG3 and MC3T3E1-EV were isolated. Pools of clones between passages 2 and 5 were used for the experiments reported.
  • MC3T3E1-EV and MC3T3E1-BIG-3 pooled clones were plated at a density of 5 x 10 3 cells/cm 2 and cultured for periods ranging from 2 to 7 days. At each time point cells were harvested and counted using a hemocytometer. Duplicate measurements were perfonned on four independent wells for each time point.
  • MC3T3E1-EV and MC3T3E1 -BIG-3 pooled clones were plated at a density of 5 x 10 3 cells/cm 2 and cultured for periods ranging from 3 to 25 days.
  • the alkaline phosphatase activity in cell lysates was assessed in assay buffer (50 mM Tris-HCl pH 7.6 and 0.1% Triton X-100) containing 1.5 M 2-amino-2-methyl-l- propanol for 1 hour at 37°C using p-nitrophenylphosphate as a substrate.
  • the release of p-nitrophenol was monitored by measuring absorbance at 405 nm.
  • MC3T3E1-EV and MC3T3E1-BIG-3 pooled clones were plated at a density of 5 x 10 3 cells/cm 2 and cultured for periods ranging from 5 to 25 days.
  • assay buffer Dulbecco's modified Eagle's medium containing 2 lnM isobutylmethylxanthine, lmg/ml BSA, 35 mM HEPES-NaOH pH 7.4
  • MC3T3E1-EV and MC3T3E1-BIG-3 pooled clones were plated at a density of 50 x 10 3 cells/cm 2 and cultured for periods ranging from 7 to 21 days. Measurement of PTH 1 receptor specific binding was performed as previously described (Bringhurst et al. Endocrinology 132:2090-2098, 1993). In brief, cell monolayers in 24-well dishes
  • 125 were washed twice with 0.5 ml ice-cold binding buffer before incubation with I- labeled [Nle ' , Tyr ] rat PTH-(l-34)amide (100,000 - 200,000 cpm/well) for 4 hours at 15°C. Cells were then washed with ice-cold binding buffer, before assessment of cell-associated radioactivity. Receptor number was determined by Scatchard analysis,
  • MLB13MYC clone 17 cells were plated at a density of 2.5 x 10 4 cells/cm 2 and treated with 0 or 200 ng/ml of BMP-2 for 24 hours.
  • Pooled MC3T3E1-EV and MC3T3E1-BIG-3 clones were plated at a density of 5 x 10 cells/cm and cultured for periods ranging from 3 to 30 days.
  • Cells were lysed in 25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton and 10 ⁇ g of protein was subjected to SDS -PAGE under reducing conditions.
  • MC3T3E1-EV and MC3T3E1-BIG-3 pooled clones were plated at a density of 5 x 10 3 cells/cm 2 and cultured for periods ranging from 14 to 35 days.
  • the formation of mineralized matrix nodules was assessed by alizarin red-S staining (Gori et al, J. Bone Min. Res. 14:1522-1535, 1999).
  • the alizarin red-S dye was eluted and measured spectrophotometrically (Gori et al, J. Bone Min. Res. 14:1522-1535, 1999).
  • Proteins in the WD-40 family contain four to sixteen conserved WD-40 repeats characterized by a variable length region followed by a core sequence delimited by two characteristic dipeptides, GH and WD (Neer et al. Nature 371 :297-300, 1994; Smith et al. Trends Biochem. Sci. 24:181-185, 1999; Neer et al, Proc. Natl. Acad. Sci. U.S .A. 97: 1096-1100, 2000).
  • the most highly conserved feature of the WD repeat proteins is an aspartic acid residue 6 positions N-terminal to the WD, present in 86% of WD repeats (Garcia-Higuera et al.
  • the tertiary structure of the G protein ⁇ subunit demonstrates that each of the seven WD repeats folds into four antiparallel ⁇ strands radiating outward from a central axis (Neer et al. Cell 84:175- 178, 1996), leading to the description of " ⁇ -propeller.”
  • Computerized structure homology searches confirm that BIG-3 is closely related to the G-protein ⁇ subunit as well as to Tupl (Sprague et al, EMBO J. 19:3016-3027, 2000), which forms a corepressor complex with Ssn6, regulating at least 3% of the genes expressed in S.
  • the WD-40 family of proteins participates in a wide variety of cellular functions including signal transduction, mRNA processing, gene regulation, and the cell cycle (Neer et al. Nature 371:297-300, 1994; Neer et al, Proc. Natl. Acad. Sci. U.S.A. 97:1096-1100, 2000).
  • BIG-3 did not alter the proliferation rate of MC3T3-E1 cells, but dramatically accelerated the rate of osteoblast differentiation.
  • the 2-fold increase in BIG-3 protein observed at 3 days in MC3T3E1 -BIG-3 clones appears to be sufficient to initiate acceleration in the program of osteoblastic differentiation.
  • the level of BIG-3 protein decreases once the MC3T3-E1 cells are terminally differentiated and mineralize. Since the WD-40 repeat proteins are involved in recruitment of other proteins, the ⁇ - propeller structure of BIG-3 appears to be involved in the assembly of a multimeric protein complex that interacts with osteogenic factors and thereby modulates the process of osteoblast differentiation. Some of the amino and carboxy terminal regions of non- WD-40 repeat sequences have been shown to be important for the function of protein in which they occur. For example the glutamine-rich amino terminal extension of Tup 1 is important for its role as transcription factor (Chen et al, Mol. Cell Biol.

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Abstract

La présente invention concerne un polypeptide 3 kilobases (BIG-3) à gène induit par la protéine 2 morphogénique osseuse, des gènes codant pour ces polypeptides et des techniques diagnostiques et thérapeutiques utilisant ces molécules.
PCT/US2002/027671 2001-08-31 2002-08-29 Genes induits par la proteine 2 morphogenique osseuse et polypeptides, utilisations de ceux-ci dans des techniques diagnostiques et therapeutiques WO2003020307A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000061629A1 (fr) * 1999-04-09 2000-10-19 Human Genome Sciences, Inc. 49 proteines humaines secretees

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000061629A1 (fr) * 1999-04-09 2000-10-19 Human Genome Sciences, Inc. 49 proteines humaines secretees

Non-Patent Citations (1)

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Title
DATABASE GENBANK [online] 25 January 2001 (2001-01-25), YUE H. ET AL.: "Gtp-binding protein associated factors", XP002960852, accession no. EMBL Database accession no. (AX077640) *

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