Protein C16 and C16N or genes encoding the same

Details Protein C16 and C16N or genes encoding the same Protein C16 and C16N or genes encoding the same

1998-10-23

2001-11-27

Priebe, Scott D. (Department: 1632)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C435S320100, C435S325000, C435S070100, C536S023500

Reexamination Certificate

active

06323330

ABSTRACT:

TECHNICAL FIELD
The present invention relates to novel proteins C16 and C16N or variant proteins thereof as well as genes encoding the same. More particularly, the present invention relates to novel proteins C16 and C16N which have activities of inducing cells to become capable of resorbing hydroxyapatite (this activity is hereinafter sometimes briefly referred to as “hydroxyapatite-resorbing activity”), supporting survival of neuron, inhibiting proliferation of osteoblast, and/or promoting expression of type I collagen in osteoblast and which can therefore be used as pharmaceutical agents or in screening for developing new drugs, or to variant proteins thereof, and to genes encoding the same.
BACKGROUND ART
Metabolic regulation of calcium in blood is absolutely essential for survival, and the concentration is maintained constant quite strictly. When the blood calcium concentration is increased by some reason, it causes various diseases such as hypertension, arteriosclerosis, diabetes, myocardial infarction, and hypercalcemia. On the other hand, decrease in blood calcium concentration also results in diseases such as hypocalcemia. In addition, substantial decrease in blood calcium concentration due to, for example, massive hemorrhage or radiation exposure may sometimes lead to death.
As cells responsible for quite important regulation of blood calcium concentration, which is quite important in vivo, osteoclast is presently known. Osteoclast is considered to directly regulate the calcium concentration in blood by resorbing bone (bone matrix) and releasing calcium into blood. In mammals, however, there are only about 50,000 osteoclasts in vivo. On the contrary, there are about 25,000 osteocytes, even in 1 mm
3
of bone, embedded in calcified hard tissues reserving calcium. Population of osteoclasts is therefore considered too small to regulate the calcium concentration in blood (Kumegawa et al.,
Molecular Medicine
, 30, p.1254 (1993) and Ozawa et al.,
Nihon
-
Rinsho
, vol. 52, No. 9, p. 2246 (1994)).
Furthermore, because there are no osteoclasts in op/op mouse which is a model animal for osteopetrosis, the primary function of osteoclast, bone remodeling, scarcely occurs in this animal. Despite the absence of osteoclast, however, the blood calcium concentration is maintained normally in this animal (
Molecular Medicine
, Vol. 30, No. 10, p. 1240 (1993)).
This fact suggests that the regulation of the blood calcium concentration by osteoclast is just supplemental and there may exist some other cells which dominantly regulate the calcium concentration in blood. Although such cells have not yet been identified, if any factors which grow such cells or which induce cells to produce such cells are found, the factors themselves or inhibitors thereof are expected to be useful as therapeutic agents for various diseases caused by abnormality in blood calcium concentration as described above. Although identification of such factor has been desired, its successful cloning has not yet been reported.
DISCLOSURE OF THE INVENTION
The present inventors have long investigated for factors inducing cells into osteoclast, by expression cloning from BW5147 which is a cancer cell having bone metastasis ability (hereinafter simply referred to as “bone metastatic cell”). Specifically, the investigation is carried out as follows: mRNA isolated from BW5147 cell is injected into Xenopus oocyte to be translated, and the translated product (protein) is applied to mouse bone marrow cells in order to determine whether or not the differentiation of the bone marrow cell into osteoclast is induced. In particular, for the purpose of our study, cells which have the four known properties of osteoclast, i.e. (1) TRAP stainability, (2) the presence of calcitonin receptor, (3) resorbing activity on dentine slice, and (4) hydroxyapatite-resorbing activity are identified as “osteoclast”, and factors which induce cells to become said osteoclast are identified as “differentiation-inducing factors for osteoclast” in our study.
In the process of this expression cloning, we found a novel proteinaceous factor of great interest. Specifically, the factor exhibits only the activity (4) among the above-noted activities (1)-(4), and does not have the other activities (1)-(3). This is a factor different from differentiation-inducing factors for osteoclast because it exhibits only the activity (4). In addition, since it has the activity (4), i.e. the activity of inducing cells to become capable of resorbing hydroxyapatite (crystalline calcium in bone), the factor is expected to be a novel factor capable of inducing cells regulating the blood calcium concentration, which has not yet been identified (such factor is hereinafter sometimes simply referred to as “regulating factor for blood calcium concentration”).
We designated this novel proteinaceous factor as “C16”. Surprisingly, further investigations on this factor C16 revealed that the gene corresponding to the factor C16 is exclusively expressed in bone and brain, and that the factor C16 also has activities of supporting survival of neuron, inhibiting proliferation of osteoblast, and promoting expression of type I collagen in osteoblast.
An example of such multifunctional proteinaceous factors having two or more activities is FGF (Fibroblast Growth Factor). Since relatively large amount of FGF is present in embryo, postnatal brain (Risau et al.
EMBO J
., 7, p. 959 (1988); Gospadarowicz et al.,
Methods Enzymol
., 147, p. 106 (1987)) and hypophysis, it has been considered that FGF plays some role also in central nervous system in addition to its function as a fibroblast growth factor. In recent years, many studies have been reported, showing that FGF has a neurotrophic activity. In studies using primary neuron culture, it has been shown that FGF has an activity of supporting survival of neuron in hippocampus, cerebral cortex, corpus striatum, septulum, thalamus, midbrain, and spinal cord (Naruo et al.,
J. Biol. Chem
., 268, p. 2857 (1993)). In the light of such versatility of FGF, it is expected that the factor C16 which we found may also be a versatile factor having multiple functions, such as function inducing cells to become capable of resorbing hydroxyapatite, function supporting survival of neuron, function inhibiting proliferation of osteoblast, and function promoting expression of type I collagen in osteoblast.
Furthermore, we also screened cDNA libraries for factors analogous to C16 using C16 DNA as a probe or primer, and found a new proteinaceous factor which comprises the region from position 1 to 245 of the amino acid sequence of C16 (SEQ ID NO: 2) and additional 334 amino acids linked to its C-terminus. We designated this factor as “C16N”. Like the factor C16, C16N also has the hydroxyapatite-resorbing activity, and activities of supporting survival of neuron, inhibiting proliferation of osteoblast, and promoting expression of type I collagen in osteoblast. Thus, it was shown that C16N also falls within the scope of the present invention as a factor of the same kind as C16.
The present invention has been accomplished on the basis of the findings as described above.
Thus, the gist of the present invention is:
(1) C16 DNA comprising the base sequence shown in SEQ ID NO: 1;
(2) C16 protein comprising the amino acid sequence shown in SEQ ID NO: 2;
(3) DNA encoding a protein which contains insertion, deletion, or substitution of one or more amino acids in the protein of the above item (2), and which protein has the following properties (i), (ii), (iii), and/or (iv):
(i) having activity of inducing cells to become capable of resorbing hydroxyapatite;
(ii) having activity of supporting survival of neuron;
(iii) having activity of inhibiting proliferation of osteoblast;
(iv) having activity of promoting expression of type I collagen in osteoblast.
(4) DNA which hybridizes under stringent conditions to DNA of the above item (1) and which encodes a protein having the following properties (i), (ii), (iii), and/or (iv):
(i) having activity of inducing cells to become capable of r

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