Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-10-01
2001-08-07
Stucker, Jeffrey (Department: 1647)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S069100, C435S070100, C435S320100, C435S325000, C530S350000
Reexamination Certificate
active
06271366
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a novel secretory membrane protein involved in differentiation of osteocytes, a DNA encoding said protein, a vector comprising said DNA, a host cell carrying said vector, an antibody against said protein, a method for screening a compound using said protein, and a compound obtainable by said screening method.
BACKGROUND ART
Regeneration of bones by the action of osteoblasts, bone formation, is an important phenomenon in vertebrates for maintaining a living body. Factors involved in bone formation include hormones such as estrogen, calcitonin, and parathyroid hormone (PTH); growth factors such as bone morphologenic protein (BMP); and chemicals such as active vitamin D, calcium preparations, and vitamin K2. Estrogen, calcitonin, active vitamin D, and calcium preparations are used as medicine for controlling bone mass in osteoporosis or similar cases. Any of these drugs can be used to prevent decreases of bone mass by inhibiting bone resorption, rather than for increasing bone mass. An effective remedy for enhancing bone formation has thus not been developed yet.
BMP, which is expected to be a new medicine for treating bone disorders, is a unique cytokine that functions as an ectopic formation signal. BMP is thought to effectively form bone (cartilaginous ossification) by replacing cartilaginous callus with new bone cells in repairing fractures or bone deficits (Duprez D. M., Coltey M., Amthor H., Brickell P. M., Tickle, C. (1996) Dev. Biol. 174, 448-452, Bone morphogenetic protein-2 (BMP-2) inhibits muscle development and promotes cartilage formation in chick limb bud cultures; Nakase, T., Nomura, S., Yoshikawa, H., Hashimoto, J., Hirota, S., Kitamura, Y., Oikawa, S., Ono, K., Takaoka, K. (1994) J. Bone Miner. Res. 9, 651-659, Transient and localized expression of bone morphogenetic protein 4 messenger RNA during fracture healing).
There is no conclusive evidence to show that BMP is involved in bone formation caused in conjunction with bone resorption during constant bone formation. Therefore, it is doubtful that BMP can be used as a medicine to promote bone formation by activating and promoting differentiation of osteoblasts that are essential for constant bone formation. Factors involved in constant bone formation have not been reported yet.
SUMMARY OF THE INVENTION
An objective of the present invention is to provide a novel protein involved in constant bone formation and a gene encoding it. Another objective of the invention is to provide a vector in which the gene is inserted, a host cell carrying the vector, and an antibody that binds to the protein. Still another objective of this invention is to provide a method for screening a compound that binds to the protein, such as a ligand, using the protein.
The present inventors investigated how best to achieve the above objectives and succeeded in isolating three types of genes each encoding secretory membrane proteins from an osteoblast-like cell line by specifically cloning genes encoding secretory membrane proteins. The analysis of one of the isolated genes revealed that a protein encoded by the gene is a novel receptor protein with only the extracellular region, which binds to the plasma membrane through a GPI anchor, and contains cysteine-rich, repetitive regions conserved in TNF receptor super family molecules. Furthermore, the inventors found that high-level expression of the protein in the osteoblast-like cell line inhibited cell proliferation, altered the cells morphologically, and enhanced alkaline phosphatase activity that is one of indicators of the differentiation of osteoblasts. Based on this finding that the isolated protein is involved in the differentiation of osteocytes, the inventors also found that a drug candidate compound for treating bone disorders can be screened using the protein.
The present invention relates to novel secretory membrane proteins, genes encoding them, and a method for screening a drug candidate compound using the proteins. More specifically, the invention relates to:
(1) a protein comprising the amino acid sequence of SEQ ID NO:1 or 2, or the same sequence in which one or more amino acids are replaced, deleted, or added, and having activity to induce differentiation of osteocytes;
(2) a protein encoded by a DNA hybridizing the DNA comprising the nucleic acid sequence of SEQ ID NO:3, and having activity to induce differentiation of osteocytes;
(3) a DNA encoding the protein of SEQ ID NO:1 or 2;
(4) a vector in which the DNA of (3) is inserted;
(5) a host cell carrying the vector of (4);
(6) an antibody binding to the protein of (1) or (2);
(7) a method for screening a compound that binds to the protein of (1) or (2), wherein said method comprises the steps of
(a) bringing a test compound into contact with the protein of (1) or (2), and
(b) screening a compound that binds to the protein of (1) or (2);
(8) a method for screening a compound that promotes or inhibits the osteocyte differentiation-inducing activity of the protein of (1) or (2), wherein said method comprises the steps of
(a) bringing a test compound into contact with the protein of (1) or (2) expressed on the cell surface,
(b) detecting the osteocyte differentiation inducing activity of the protein of (1) or (2), and
(c) screening a compound that promotes or inhibits the osteocyte differentiation inducing activity of the protein of (1) or (2), in comparison with the assay in the absence of the test compound;
(9) a compound that binds to the protein of (1) or (2), wherein said protein is obtainable by the method of (7);
(10) a compound that promotes or inhibits the osteocyte differentiation inducing activity of the protein of (1) or (2), wherein said protein is obtainable by the method of (8);
(11) the compound of (9) or (10), wherein said compound is naturally occurring;
(12) the compound of (9) or (10), wherein said compound is a ligand;
(13) the compound of (9) or (10), wherein said compound is an agonist; and
(14) the compound of (9) or (10), wherein said compound is an antagonist.
The invention also includes a substantially pure polypeptide (1) having an amino acid sequence at least 60% (e.g., at least 70, 80, 90, 95, or 99%) identical to SEQ ID NO:2, (2) having an amino acid sequence that is SEQ ID NO:2 containing at least one conservative amino acid substitution (preferably between 1-30, and more preferably 15 or fewer (e.g., 5 or fewer or even 3 or fewer) substitutions, or (3) encoded by a first nucleic acid that hybridizes under stringent conditions to a second nucleic acid consisting of SEQ ID NO:3. The polypeptide can induce differentiation of an osteocyte (e.g., a human osteocyte).
Also featured in the invention is an isolated nucleic acid encoding a polypeptide of the invention. In addition, nucleic acid fragments that hybridize under stringent conditions to SEQ ID NO:3, such as hybridization probes and PCR primers, are also included in the invention.
The invention further features nucleic acid and viral vectors, as well as transformed host cells, containing a nucleic acid of the invention. Also included in the invention is an antibody (e.g., a monoclonal antibody), or a composition of polyclonal antibodies (e.g., an antiserum) that specifically binds to a polypeptide of the invention.
The invention also features a method of screening for a compound that binds to a polypeptide by (1) contacting a compound with a polypeptide of the invention, and (2) determining whether the compound has bound to the polypeptide. Other methods included in the invention include: (1) a method of screening for a compound that induces osteocyte differentiation by contacting a compound with a polypeptide of the invention, and determining whether the ability of the polypeptide to induce osteocyte differentiation in the presence of the compound is greater than in the absence of the compound, such being an indication that the compound induces osteocyte differentiation; and (2) a method of screening for a compound that inhibits osteocyte differentiation by contacting a compound with a polypepti
Kimura Naoki
Toyoshima Tomoko
Chugai Research Institute for Molecular Medicine, Inc.
Fish & Richardson P.C.
Seharaseyon Jegatheesan
Stucker Jeffrey
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