U.S. patent application number 10/344339 was filed with the patent office on 2004-04-29 for polypeptides controlling phosphoric acid metabolism, calcium metabolism, calcification and vitamin d metabolism and dnas encoding the same.
Invention is credited to Fukumoto, Seiji, Mizutani, Satoru, Shimada, Takashi, Yamashita, Takeyoshi.
Application Number | 20040082506 10/344339 |
Document ID | / |
Family ID | 27481531 |
Filed Date | 2004-04-29 |
United States Patent
Application |
20040082506 |
Kind Code |
A1 |
Yamashita, Takeyoshi ; et
al. |
April 29, 2004 |
Polypeptides controlling phosphoric acid metabolism, calcium
metabolism, calcification and vitamin d metabolism and dnas
encoding the same
Abstract
A DNA, which encodes the following polypeptide (a), (b), (c) or
(d): (a) a polypeptide consisting of an amino acid sequence
represented by SEQ ID NO: 2 or 4, (b) a polypeptide consisting of
an amino acid sequence derived from the amino acid sequence
represented by SEQ ID NO: 2 or 4 by deletion, substitution or
addition of one or several amino acids, and having
hypophosphatemia-inducing activity, phosphate transport-suppressing
activity, calcification-suppressing activity or in vivo vitamin D
metabolism-regulating activity, (c) a polypeptide consisting of a
partial sequence of the amino acid sequence represented by SEQ ID
NO: 2, wherein the above partial sequence contains an amino acid
sequence at least ranging from the 34.sup.th to 201.sup.st amino
acids in the above amino acid sequence, or (d) a polypeptide
consisting of a partial sequence of the amino acid sequence
represented by SEQ ID NO: 2, wherein the above partial sequence:
(i) contains an amino acid sequence ranging from at least the
34.sup.th to 201.sup.st amino acids in said amino acid sequence,
(ii) consists of an amino acid sequence derived from the partial
sequence by deletion, substitution, or addition of one or several
amino acids, and (iii) has hypophosphatemia-inducing activity,
phosphate transport-suppressing activity, calcification-suppressing
activity or in vivo vitamin D metabolism-regulating activity.
Inventors: |
Yamashita, Takeyoshi;
(Gunma, JP) ; Shimada, Takashi; (Gunma, JP)
; Mizutani, Satoru; (Kanagawa, JP) ; Fukumoto,
Seiji; (Tokyo, JP) |
Correspondence
Address: |
Foley & Lardner
Washington Harbour
Suite 500
3000 K Street NW
Washington
DC
20007-5109
US
|
Family ID: |
27481531 |
Appl. No.: |
10/344339 |
Filed: |
July 21, 2003 |
PCT Filed: |
August 10, 2001 |
PCT NO: |
PCT/JP01/06944 |
Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 514/15.4; 514/16.7; 530/358; 536/23.5 |
Current CPC
Class: |
A61P 19/08 20180101;
A61K 39/00 20130101; C07K 14/705 20130101; A61P 35/00 20180101;
A61P 3/14 20180101; A61P 19/10 20180101; A61K 38/00 20130101; A61P
19/00 20180101; C07K 14/47 20130101; C07K 14/50 20130101; A61P 3/02
20180101; A61P 13/12 20180101; A61K 48/00 20130101 |
Class at
Publication: |
514/012 ;
530/358; 536/023.5; 435/069.1; 435/320.1; 435/325 |
International
Class: |
A61K 038/17; C07H
021/04; C07K 014/72; C12P 021/02; C12N 005/06 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 11, 2000 |
JP |
2000-245144 |
Sep 21, 2000 |
JP |
2000-287684 |
Dec 22, 2000 |
JP |
2000-391077 |
Apr 19, 2001 |
JP |
2001-121527 |
Claims
1. A DNA, which encodes the following polypeptide (a), (b), (c) or
(d): (a) a polypeptide consisting of an amino acid sequence
represented by SEQ ID NO: 2 or 4, (b) a polypeptide consisting of
an amino acid sequence derived from the amino acid sequence
represented by SEQ ID NO: 2 or 4 by deletion, substitution or
addition of one or several amino acids, and having
hypophosphatemia-inducing activity, phosphate transport-suppressing
activity, calcification-suppressing activity or in vivo vitamin D
metabolism-regulating activity, (c) a polypeptide consisting of a
partial sequence of the amino acid sequence represented by SEQ ID
NO: 2, wherein said partial sequence contains an amino acid
sequence at least ranging from the 34.sup.th to 201.sup.st amino
acids in said amino acid sequence, or (d) a polypeptide consisting
of a partial sequence of the amino acid sequence represented by SEQ
ID NO: 2, wherein said partial sequence: (i) contains an amino acid
sequence at least ranging from the 34.sup.th to 201.sup.st amino
acids in said amino acid sequence, (ii) consists of an amino acid
sequence derived from the partial sequence by deletion,
substitution or addition of one or several amino acids, and (iii)
has hypophosphatemia-inducing activity, phosphate
transport-suppressing activity, calcification-suppressing activity
or in vivo vitamin D metabolism-regulating activity.
2. A DNA, which contains the following DNA (e) or (f): (e) a DNA
consisting of a nucleotide sequence ranging from the 133.sup.rd to
885.sup.th nucleotides in the nucleotide sequence represented by
SEQ ID NO: 1 or a nucleotide sequence ranging from the 1.sup.st to
681.sup.st nucleotides in the nucleotide sequence represented by
SEQ ID NO: 3, or (f) a DNA hybridizing under stringent conditions
to a probe prepared from a DNA consisting of the whole or a part of
the nucleotide sequence represented by SEQ ID NO: 1 or 3, and
encoding a polypeptide having hypophosphatemia-inducing activity,
phosphate transport-suppressing activity, calcification-suppressing
activity or in vivo vitamin D metabolism-regulating activity.
3. A recombinant vector, which contains the DNA of claim 1 or
2.
4. A transformant, which contains the recombinant vector of claim
3.
5. A polypeptide, which is the following polypeptide (a), (b), (c)
or (d): (a) a polypeptide consisting of an amino acid sequence
represented by SEQ ID NO: 2 or 4, (b) a polypeptide consisting of
an amino acid sequence derived from the amino acid sequence
represented by SEQ ID NO: 2 or 4 by deletion, substitution or
addition of one or several amino acids, and having
hypophosphatemia-inducing activity, phosphate transport-suppressing
activity, calcification-suppressing activity or in vivo vitamin D
metabolism-regulating activity, (c) a polypeptide consisting of a
partial sequence of the amino acid sequence represented by SEQ ID
NO: 2, wherein said partial sequence contains at least an amino
acid sequence ranging from the 34.sup.th to 201.sup.st amino acids
in said amino acid sequence, or (d) a polypeptide consisting of a
partial sequence of the amino acid sequence represented by SEQ ID
NO: 2 wherein said partial sequence: (i) contains an amino acid
sequence ranging from at least the 34.sup.th to 201.sup.th amino
acids in said amino acid sequence, (ii) consists of an amino acid
sequence derived from the partial sequence by deletion,
substitution, or addition of one or several amino acids, and (iii)
has hypophosphatemia-inducing activity, phosphate
transport-suppressing activity, calcification-suppressing activity
or in vivo vitamin D metabolism-regulating activity.
6. The polypeptide of claim 5, which is modified by at least one
substance selected from the group consisting of polyethylene
glycol, dextran, poly(N-vinyl-pyrrolidone), polypropylene glycol
homopolymer, copolymer of polypropylene oxide and polyethylene
oxide, polyoxyethylated polyol and polyvinyl alcohol.
7. A pharmaceutical composition, which contains the polypeptide of
claim 5 or 6 as an active ingredient.
8. A pharmaceutical composition, which contains the polypeptide of
claim 5 or 6 as an active ingredient and is capable of regulating
in vivo calcium metabolism, phosphate metabolism, calcification or
vitamin D metabolism.
9. A pharmaceutical composition, which contains the polypeptide of
claim 5 or 6 as an active ingredient and is effective against at
least one disease selected from the group consisting of
hyperphosphatemia, hyperparathyroidism, renal osteodystrophy,
ectopic calcification, osteoporosis and hypervitaminosis D.
10. An antibody, which reacts with the polypeptide of claim 5 or 6
or partial fragments thereof.
11. A method for producing the antibody of claim 10, which
comprises the step of immunizing an animal with the polypeptide of
claim 5 or 6, or partial fragments thereof as an antigen.
12. A pharmaceutical composition, which contains the antibody of
claim 10 as an active ingredient.
13. A pharmaceutical composition, which contains the antibody of
claim 10 as an active ingredient, and is capable of regulating in
vivo calcium metabolism, phosphate metabolism, calcification or
vitamin D metabolism.
14. A pharmaceutical composition, which contains the antibody of
claim 10 as an active ingredient, and is effective against bone
diseases.
15. The pharmaceutical composition of claim 14, wherein the bone
disease is at least one disease selected from the group consisting
of osteoporosis, vitamin D-resistant rickets, renal osteodystrophy,
dialysis-associated bone diseases, osteopathy with
hypocalcification, Paget's disease and tumor-induced
osteomalacia.
16. A diagnostic agent, which contains the antibody of claim 10,
and is for a disease which develops at least one abnormality of
abnormal calcium metabolism, abnormal phosphate metabolism,
abnormal calcification and abnormal vitamin D metabolism.
17. The diagnostic agent of claim 16, wherein the disease is at
least one disease selected from the group consisting of renal
failure, renal phosphate leak, renal tubular acidosis and Fanconi's
syndrome.
18. A diagnostic agent for a bone disease, which contains the
antibody of claim 10.
19. The diagnostic agent of claim 18, wherein the bone disease is
at least one disease selected from the group consisting of
osteoporosis, vitamin D-resistant rickets, renal osteodystrophy,
dialysis-associated bone diseases, osteopathy with
hypocalcification, Paget's disease and tumor-induced
osteomalacia.
20. A diagnostic agent, which contains a DNA having a nucleotide
sequence represented by SEQ ID NO: 11 or partial fragments thereof,
and is for a disease which develops at least one abnormality of
abnormal calcium metabolism, abnormal phosphate metabolism and
abnormal calcification.
21. The diagnostic agent of claim 20, wherein the partial fragment
has a sequence ranging from the 498.sup.th to 12966.sup.th
nucleotides of the nucleotide sequence represented by SEQ ID NO:
11.
22. The diagnostic agent of claim 20 or 21, wherein the disease is
autosomal dominant hypophosphatemic rickets/osteomalacia.
Description
TECHNICAL FIELD
[0001] The present invention relates to a polypeptide that
regulates phosphate metabolism, calcium metabolism, calcification
and/or vitamin D metabolism, a DNA encoding the polypeptide, and a
pharmaceutical composition containing the polypeptide as an active
ingredient, and an antibody recognizing the polypeptide, a
pharmaceutical composition containing the antibody as an active
ingredient, a diagnostic method using the antibody, and a
diagnostic composition.
BACKGROUND ART
[0002] Inorganic phosphates (hereinafter, may be referred to as
"phosphate") are essential in energy metabolism in vivo and
maintenance of cellular functions, and play an important role in
tissue calcification in cooperation with calcium. Supply of
phosphate to an organism depends mainly on absorption in the
intestinal tract, and phosphate excretion depends on urinary
excretion in the kidney and fecal excretion in the intestinal
tract. In living organisms, phosphate is distributed in body fluid,
intracellular fractions and calcified tissues. The level of
excretion of inorganic phosphate in an adult is maintained at
almost the same level of absorption of inorganic phosphate,
suggesting the presence of a regulatory mechanism which maintains
homeostasis of the phosphate metabolism. It is known that the
metabolism of calcium, which shares similarlity with the phosphate
metabolism in terms of a distribution and homeostatic control of
blood level, is controlled in a co-operative manner in mammals by
regulatory factors, such as, at least parathyroid hormone,
calcitonin and 1.alpha.,25-dihydroxyvitamin D3.
[0003] In the regulation of phosphate metabolism it is known that
parathyroid hormone promotes phosphate excretion, and that
1.alpha.,25-dihydroxyvitamin D3 promotes phosphate absorption in
the intestinal tract. This clearly suggests close association
between phosphate metabolism and calcium metabolism. However, a
substance primarily controlling phosphate has not yet been
elucidated.
[0004] Now, examples of a disease which is associated with the loss
of the homeostasis of phosphate metabolism and lower inorganic
phosphate levels in the blood include primary hyperparathyroidism,
hereditary hypophosphatemic rickets, and tumor-induced
osteomalacia.
[0005] Primary hyperparathyroidism is a disease characterized by an
overproduction of parathyroid hormone in the parathyroid glands,
and is known to develop hypophosphatemia with increased phosphate
excretion because overproduced parathyroid hormone suppresses
reabsorption of inorganic phosphate in the kidney.
[0006] Further, known examples of hypophosphatemia resulting from
hereditary diseases include type I vitamin D-dependent rickets,
type II vitamin D-dependent rickets and vitamin D-resistant
rickets. Type I vitamin D-dependent rickets is a disease caused by
hereditary dysfunction of the synthase to produce active vitamin D
metabolites, and type II vitamin D-dependent rickets is a disease
caused by hereditary dysfunction of vitamin D receptor. Both
diseases develop hypophosphatemia together with hypocalcemia due to
attenuated action of vitamin D3 metabolites. In contrast, for
vitamin D-resistant rickets, at least 2 types of clinical
conditions, X-linked chromosomal and autosomal hypophosphatemic
rickets resulting from different causes are known to exist.
[0007] Both of the above-mentioned clinical conditions of vitamin
D-resistant rickets lead to hypophosphatemia characterized by renal
phosphate. Recently, it has been shown in patients with X-linked
hypophosphatemic rickets (hereinafter, also referred to as "XLH")
that the disease is induced by mutations in the gene encoding an
endopeptidase-like protein, named PHEX, on X chromosome. However, a
mechanism how dysfunction of PHEX protein induces hypophosphatemia
has not been elucidated. Interestingly, gene analysis of a
naturally occurring mutant mouse (Hyp) which developed
hypophosphatemia has revealed the partial deletion of the gene
encoding PHEX in this mouse. Experiments using these mice have
revealed that PHEX deficient mice have normal renal function, and a
humoral factor, which is different from parathyroid hormone, but
induces hypophosphatemia, is present in the body fluid of Hyp mice.
Concerning autosomal dominant hypophosphatemic rickets/osteomalacia
(hereinafter also referred to as ADHR), a gene responsible for this
disease has been pursued, and the presence of such a gene in 12p13
region has been indicated by linkage analysis. However, the region
that has been narrowed down so far is still wide and contains many
genes, so that no candidate gene has been specified yet.
[0008] Tumor-induced osteomalacia is a disease which develops
hypophosphatemia with increased renal phosphate in association with
tumorigenesis, and is characterized in that the hypophosphatemia is
eliminated by irradiation to tumor or removal of tumor. In this
disease, it is thought that tumor produces a factor which induces
hypophosphatemia due to suppressed reabsorption of phosphate in the
kidneys.
[0009] It has not been confirmed whether a putative causative
molecule for vitamin D-resistant rickets is identical to that for
tumor-induced osteomalacia. However, the two factors are identical
in that they clearly are unknown phosphate metabolism factors which
promote urinary phosphate excretion. The putative phosphate
metabolism regulatory factor is often referred to as, the name
Phosphatonin. The relationship of this unknown phosphate metabolism
regulatory factor and vitamin D-resistant rickets or tumor-induced
osteomalacia has been summarized as general remarks (Neison, A. E.,
Clinical Endocrinology, 47:635-642, 1997; Drezner, M. K., Kidney
Int., 57:9-18, 2000).
[0010] Another characteristic of vitamin D-resistant rickets or
tumor-induced osteomalacia is impairment of bone calcification.
This impaired bone calcification could be thought to be secondarily
developed by hypophosphatemia. However, since abnormal bone
calcification in experiments using Hyp mice, the model mice for
vitamin D-resistant rickets is shown to develop independently from
phosphate levels (Ecarot, B., J. Bone Miner. Res., 7:215-220, 1992;
Xiao, Z. S., Am. J. Physiol., E700-E708, 1998), it is conceivable
that the above unknown regulatory factor for phosphate metabolism
can directly regulate calcification in bone tissue.
[0011] As described above, research data have strongly been
suggesting the presence of an unknown factor which regulates
phosphate metabolism, but there has been no case that can elucidate
at a molecular level, an entity which exhibits the putative
activity. While WO99/60017 discloses a novel polypeptide sequence
as a novel polypeptide hormone, Phosphatonin, however, it does not
disclose the characteristic activity of phosphatonin which concerns
induction of hypophosphatemia. Thus, it is conceivable that an
unidentified intrinsic factor regulating phosphate metabolism may
exist in organisms.
[0012] Vitamin D2 and vitamin D3 ingested from foods, or vitamin D3
synthesized in the skin is hydrolyzed by vitamin D-25-hydroxylase
existing mainly in the liver to produce 25-hydroxyvitamin D. Then,
25-hydroxyvitamin D is hydrolyzed by 25-hydroxyvitamin
D-1.alpha.-hydroxylase existing in renal epithelial cells of
proximal tubules in the kidney to produce
1.alpha.,25-dihydroxyvitamin D. This 1.alpha.,25-dihydroxyvitamin D
is a mineral regulatory hormone having physiological activities
that increase serum calcium and phosphate levels, and is known to
be responsible for inhibiting the secretion of parathyroid hormone
and to be involved in the promotion of bone resorption.
1.alpha.,25-dihydroxyvitamin D is then converted into metabolites
in vivo which has not the above physiological activities by
24-hydroxylase existing mainly in the kidney or small intestine. In
this regard, 24-hydroxylase is thought to be an enzyme which is
responsible for the inactivation of 1.alpha.,25-dihydroxyvitamin D.
On the other hand, 24-hydroxylase is known to also act on
25-hydroxy vitamin D and convert it into 24,25-dihydroxyvitamin D.
The 24,25-dihydroxyvitamin D has been reported to have
physiological effects that increase bone mass or promote
differentiation of cartilage, suggesting that this enzyme has an
aspect for generating biological active vitamin D metabolites.
[0013] Known factors that regulate the expression level of
1.alpha.-hydroxylase, which has an important role in the activation
of vitamin D, include parathyroid hormone (PTH), calcitonin,
1.alpha.,25-dihydroxyvitamin D and the like. PTH whose secretion is
promoted by decreases in blood calcium levels acts on PTH receptors
existing in epithelial cells of the renal proximal tubules to
promote transcription of 1-hydroxylase gene through an elevated
intracellular cAMP level, so as to increase blood
1.alpha.,25-dihydroxyvitamin D concentration.
1.alpha.,25-dihydroxyvitamin D promotes absorption of calcium from
the intestinal tract and calcium reabsorption in the kidney,
thereby increasing the blood calcium level. Further, it has been
reported that the binding of 1.alpha.,25-dihydroxyvitamin D to
vitamin D receptor (VDR) acts on a promoter region of
1.alpha.-hydroxylase gene or PTH gene to suppress the transcription
of such genes. Specifically, 1.alpha.,25-dihydroxyvitamin D has a
feedback control mechanism for its activation factor, PTH and
1.alpha.-hydroxylase. This mechanism plays an important role in
maintaining homeostasis of calcium metabolism.
[0014] Recently, it has been reported that a decrease in serum
phosphate level enhances the expression of 1.alpha.-hydroxylase
gene. In phosphate metabolism, the presence of a mechanism is also
assumed that enhancement in the expression of 1.alpha.-hydroxylase
gene association with decreased serum phosphate level elevates
serum 1.alpha.,25-dihydroxyvitamin D level and, consequently,
corrects the serum phosphate level by promoting absorption of
phosphate from the small intestine.
[0015] Examples of a factor responsible for regulating the
expression of 24-hydroxylase gene include
1.alpha.,25-dihydroxyvitamin D and PTH. It has been shown that
1.alpha.,25-dihydroxyvitamin D interact with the vitamin D receptor
(VDR) and the complex binds to a vitamin D receptor response
sequence existing in the promoter region of 24-hydroxylase gene so
as to promote transcription. 1.alpha.,25-dihydroxyvitamin D is
thought to activate 24-hydroxylase, and then to induce a decrease
in the 1.alpha.,25-dihydroxyvitamin D level due to the activated
catabolic pathway. It is known that the expression of
24-hydroxylase gene is suppressed by PTH, but its detailed
molecular mechanism is unknown.
DISCLOSURE OF THE INVENTION
[0016] The purpose of the present invention is to provide a novel
tumor-derived factor which is capable of inducing decreases in
blood phosphate levels.
[0017] It is conceivable that a tumor identified in tumor-induced
osteomalacia secretes a soluble factor having physiological
activity, so that the blood phosphate levels decrease. The
tumor-derived factor causes the homeostasis of phosphate metabolism
to fail. Therefore, the factor may be characterized by any one of
(1) the factor which is not originally produced in vivo is produced
tumor-specifically, (2) the factor is overproduced in tumor, though
it is also produced in normal tissue, and (3) the factor is
produced in tumor without being physiologically controlled.
[0018] Based on an assumption that a tumor-derived
hypophosphatemia-induci- ng factor is characteristically produced
in a tumor-induced osteomalacia-derived tumor as described above,
we have anticipated enhanced transcription of a gene encoding the
hypophosphatemia-inducing factor or enhanced stability of mRNA of
the factor in a tumor. Hence, after extraction of RNA from a part
of the tumor tissues that were excised from a patient with
tumor-induced osteomalacia for therapeutic purposes, we prepared
cDNA library using phage vectors and plasmid vectors, and then
screened for gene fragments that were specifically expressed in the
tumor. Methods performed for screening were a method which selects
cDNA fragments determined to be specifically expressed in the
tumor, and a method which selects cDNA fragments in a tumor-derived
cDNA library which do not cross-react with cDNA probes derived from
a cell line of epithelial cells of the renal proximal tubules. We
further narrowed down the selected cDNA fragments by confirming for
novelty in sequences and characteristic expression in the tumor,
thereby obtaining a plurality of cDNA fragments expected to encode
the hypophosphatemia-inducing factor. From the sequence
information, we attempted to clone cDNAs containing ORF to which
each fragment belongs and successfully obtained DNAs encoding novel
polypeptides. We further thoroughly studied to find biological
activities of the novel polypeptides, so that we have completed the
present invention by elucidating that the novel polypeptide has
activities to suppress phosphate transport, to induce
hypophosphatemia and to suppress calcification of bone tissue in
animals.
[0019] Specifically, the present invention is as follows.
[0020] (1) A DNA, which encodes the following polypeptide (a), (b),
(c) or (d):
[0021] (a) a polypeptide consisting of an amino acid sequence
represented by SEQ ID NO: 2 or 4,
[0022] (b) a polypeptide consisting of an amino acid sequence
derived from the amino acid sequence represented by SEQ ID NO: 2 or
4 by deletion, substitution or addition of one or several amino
acids, and having hypophosphatemia-inducing activity, phosphate
transport-suppressing activity, calcification-suppressing activity
or in vivo vitamin D metabolism-regulating activity,
[0023] (c) a polypeptide consisting of a partial sequence of the
amino acid sequence represented by SEQ ID NO: 2, wherein the above
partial sequence contains an amino acid sequence at least ranging
from the 34.sup.th to 201.sup.st amino acids in the above amino
acid sequence, or
[0024] (d) a polypeptide consisting of a partial sequence of the
amino acid sequence represented by SEQ ID NO: 2, wherein the
partial sequence:
[0025] (i) contains an amino acid sequence at least ranging from
the 34.sup.th to 201.sup.th amino acids in the above amino acid
sequence,
[0026] (ii) consists of an amino acid sequence derived from the
partial sequence by deletion, substitution or addition of one or
several amino acids, and
[0027] (iii) has hypophosphatemia-inducing activity, phosphate
transport-suppressing activity, calcification-suppressing activity
or in vivo vitamin D metabolism-regulating activity.
[0028] (2) A DNA, which contains the following DNA (e) or (f):
[0029] (e) a DNA consisting of a nucleotide sequence ranging from
the 133.sup.rd to 885.sup.th nucleotides in the nucleotide sequence
represented by SEQ ID NO: 1 or a nucleotide sequence ranging from
the 1.sup.st to 681.sup.st nucleotides in the nucleotide sequence
represented by SEQ ID NO: 3, or
[0030] (f) a DNA hybridizing under stringent conditions to a probe
prepared from a DNA consisting of the whole or a part of the
nucleotide sequence represented by SEQ ID NO: 1 or 3, and encoding
a polypeptide having hypophosphatemia-inducing activity, phosphate
transport-suppressing activity, calcification-suppressing activity
or in vivo vitamin D metabolism-regulating activity.
[0031] Here, the term "stringent conditions" satisfies conditions
of a sodium concentration of 750 mM or more, preferably 900 mM or
more, a temperature of 40.degree. C. or more, preferably,
42.degree. C. Specifically, stringent conditions consist of
6.times.SSC, 5.times. Denhardt, 0.5% SDS, 50% Formamide and
42.degree. C.
[0032] (3) A recombinant vector, which contains the above DNA.
[0033] (4) A transformant, which contains the above recombinant
vector.
[0034] (5) A polypeptide, which is the following polypeptide (a),
(b), (c) or (d):
[0035] (a) a polypeptide consisting of an amino acid sequence
represented by SEQ ID NO: 2 or 4,
[0036] (b) a polypeptide consisting of an amino acid sequence
derived from the amino acid sequence represented by SEQ ID NO: 2 or
4 by deletion, substitution or addition of one or several amino
acids, and having hypophosphatemia-inducing activity, phosphate
transport-suppressing activity, calcification-suppressing activity
or in vivo vitamin D metabolism-regulating activity,
[0037] (c) a polypeptide consisting of a partial sequence of the
amino acid sequence represented by SEQ ID NO: 2, wherein the above
partial sequence contains at least an amino acid sequence ranging
from the 34.sup.th to 201.sup.st amino acids in the above amino
acid sequence, or
[0038] (d) a polypeptide consisting of a partial sequence of the
amino acid sequence represented by SEQ ID NO: 2 wherein the partial
sequence:
[0039] (i) contains an amino acid sequence ranging from at least
the 34.sup.th to 201.sup.st amino acids in the above amino acid
sequence,
[0040] (ii) consists of an amino acid sequence derived from the
partial sequence by deletion, substitution, or addition of one or
several amino acids, and
[0041] (iii) has hypophosphatemia-inducing activity, phosphate
transport-suppressing activity, calcification-suppressing activity
or in vivo vitamin D metabolism-regulating activity.
[0042] The above polypeptide also includes a polypeptide modified
by at least one substance selected from the group consisting of
polyethylene glycol, dextran, poly (N-vinyl-pyrrolidone),
polypropylene glycol homopolymer, copolymer of polypropylene oxide
and polyethylene oxide, polyoxyethylated polyol and polyvinyl
alcohol.
[0043] (6) A pharmaceutical composition which contains the above
polypeptide as an active ingredient.
[0044] The above pharmaceutical composition can be used to enable
in vivo regulation of calcium metabolism, phosphate metabolism,
calcification or vitamin D metabolism. Further, the above
pharmaceutical composition is effective against at least one
condition selected from the group consisting of hyperphosphatemia,
hyperparathyroidism, renal osteodystrophy, ectopic calcification,
osteoporosis and hypervitaminosis D.
[0045] (7) An antibody, which reacts with the above polypeptide or
partial fragments thereof.
[0046] The above antibody can be obtained by a method comprising
the steps of immunizing an animal with the polypeptide of the
present invention or partial fragments thereof, as an antigen.
[0047] (8) A pharmaceutical composition, which contains the above
antibody as an active ingredient.
[0048] The above pharmaceutical composition can regulate in vivo
calcium metabolism, phosphate metabolism, calcification or vitamin
D metabolism, or be effective against bone diseases. Here, the bone
disease is at least one disease selected from the group consisting
of osteoporosis, vitamin D-resistant rickets, renal osteodystrophy,
dialysis-associated bone diseases, osteopathy with
hypocalcification, Paget's disease and tumor-induced
osteomalacia.
[0049] (9) A diagnostic agent, which contains the above antibody
and is for a disease which develops at least one abnormality of
abnormal calcium metabolism, abnormal phosphate metabolism,
abnormal calcification and abnormal vitamin D metabolism (for
example, a disease selected from the group consisting of renal
failure, renal phosphate leak, renal tubular acidosis and Fanconi's
syndrome).
[0050] (10) A diagnostic agent for a bone disease, which contains
the above antibody, wherein the bone disease is at least a disease
selected from the group consisting of osteoporosis, vitamin
D-resistant rickets, renal osteodystrophy, dialysis-associated bone
diseases, osteopathy with hypocalcification, Paget's disease and
tumor-induced osteomalacia.
[0051] (11) A diagnostic agent, which contains a DNA having a
nucleotide sequence represented by SEQ ID NO: 11 or partial
fragments thereof, and is for a disease which develops at least one
abnormality of abnormal calcium metabolism, abnormal phosphate
metabolism, abnormal calcification and abnormal vitamin D
metabolism.
[0052] An example of the partial sequence has a sequence ranging
from the 498.sup.th to 12966.sup.th nucleotides of the nucleotide
sequence represented by SEQ ID NO: 11. An example of the disease is
autosomal dominant hypophosphatemic rickets/osteomalacia.
[0053] The present invention is explained in detail as follows.
This specification includes part or all of the contents disclosed
in the specification and/or drawings of Japanese Patent Application
Nos. 2000-245144, 2000-287684, 2000-391077 and 2001-121527, which
are priority documents of the present application.
[0054] The terms used in the present specification are defined as
follows.
[0055] The term "activity to decrease blood 1,25-dihydroxyvitamin
D3 levels" indicates an activity which acts to decrease blood
levels of 1,25-dihydroxyvitamin D3.
[0056] The term "hypophosphatemia-inducing activity" indicates an
activity which acts to decrease blood phosphate levels. Blood
phosphate level is defined by the balance between (i) absorption
from the intestinal tract and excretion into urine and feces, and
(ii) in vivo distribution of phosphate to cells or calcified
tissues as represented by bone tissues. Therefore, the term
"hypophosphatemia-inducing activity" used in the present
specification means an activity to lower blood phosphate levels in
a healthy living organism, and does not necessarily mean an
activity to cause pathologic hypophosphatemia. The
hypophosphatemia-inducing activity may be equivalent, on a tissue
level, to phosphate absorption-suppressing activity in the
intestinal tract, phosphate excretion-promoting activity in the
kidney or the intestinal tract, or activity which promotes transfer
of phosphate into cells.
[0057] Further, the term "phosphate transport-suppressing activity"
in the present invention means an activity which acts on a target
cell so as to suppress activity of a phosphate transport carrier
existing on the cell membrane. Possible target cells are mainly
epithelial cells of the renal tubules, epithelial cells of the
intestines or osteoblasts.
[0058] Furthermore, the term "calcification-suppressing activity"
in the present invention means an activity which suppresses the
process to generate or accumulate crystal substances containing
calcium and phosphate as compositions in bone tissues and soft
tissues.
[0059] Furthermore, the term "in vivo vitamin D
metabolism-regulating activity" indicates a potency to regulate
changes in the absolute amounts or in the abundance ratio of
vitamin D existing in vivo or of the metabolites synthesized in
vivo therefrom. In vivo regulation of the vitamin D and of the
metabolite thereof is ruled by mainly (i) absorption or excretion
in the intestinal tract and (ii) reabsorption or excretion in the
kidney, followed by (iii) in vivo synthesis of vitamin D, and (iv)
metabolic conversion mainly led by hydroxylation reaction. Known,
main metabolites resulting from the metabolic conversion (iv) are
as follows: 25-hydroxyvitamin D which is produced by hydroxylation
at position 25 of vitamin D by vitamin D-25-hydroxylase;
1.alpha.,25-dihydroxyvitamin D which is produced by hydroxylation
at position 1.alpha. of hydroxyvitamin D by 25-hydroxyvitamin
D-10-hydroxylase; or 24,25-dihydroxyvitamin D or 1.alpha.
24,25-trihydroxyvitamin D which is produced by introduction of a
hydroxyl group at position 24 of the metabolite by 24-hydroxylase.
Vitamin D metabolism-regulating activity can be represented as an
activity to regulate an enzymatic activity, gene expression or
changes in expressed protein levels of enzymes involved in the
generation of such vitamin D metabolites.
[0060] 1. DNA Encoding Polypeptide which Regulates Phosphate
Metabolism, Calcium Metabolism, Calcification and Vitamin D
Metabolism
[0061] (1) DNA Cloning
[0062] A DNA represented by SEQ ID NO: 1, which is one of DNAs of
the present invention, is obtained by screening a cDNA library
prepared using a part of a tumor excised from a patient suspected
of having tumor-induced osteomalacia.
[0063] Tumor-induced osteomalacia is a disease which develops
hypophosphatemia and osteomalacia due to insufficient calcification
of bone tissues in association with the presence of tumors, and is
characterized in that the removal of the tumor causes these
symptoms to disappear. There have been reports that tumor extracts
promote urinary phosphate excretion in rats (Popvtzer, M. M. et
al., Clinical Research 29: 418A, 1981), and that hypophosphatemia
was induced in an experiment of transplanting excised tumors into
mice (Miyauchi, A. et al., J. Clin. Endocrinol. Metab. 67:46-53,
1988). Thus, it has been considered that tumors produce and secrete
a systemic unknown factor.
[0064] We used a case relating to the tumor described in Fukumoto,
S. et al., Bone 25: 375-377, 1999. In this case, a significant
recovery from hypophosphatemia was achieved by operative excision
of the tumor. Further, the tumor size in this case was as small as
about 1 cm in diameter. Based on an inference that such a small
tissue produces and secretes an active substance which induces
hypophosphatemia and systemic osteomalacia, we have anticipated
that the tumor-derived cDNA library that we have prepared contains,
at a higher frequency compared to another tissue-derived cDNA
library, at least a partial fragment of the gene encoding the
active substance involved in the induction of such clinical
conditions. Accordingly, to identify a sequence of the fragment of
the gene encoding the tumor-derived active substance, cDNA
fragments that are specifically abundant in cDNA library of the
tumor were extracted by a differential screening method.
[0065] Next, the nucleotide sequences of the obtained cDNA
fragments were identified, and compared to each other. Then based
on overlap of the nucleotide sequences, contigs were prepared to
classify each sequence thought to be derived from the same gene.
Homology searches were performed for the thus obtained nucleotide
sequences with the nucleotide sequences registered at Genbank which
is the database provided by National Center for Biotechnology
Information (USA) (hereinafter, may also referred to as "NCBI"). In
this way, the nucleotide sequence that is specifically abundant in
the tumor cDNA library, that is, a sequence ranging from nucleotide
Nos. 1522 to 2770 of the nucleotide sequence represented by SEQ ID
NO: 1 was obtained. This sequence was identical to a part of the
human sequence, 12p13 BAC RPCI11-388F6 registered at Genbank under
Accession No. AC008012. This registered sequence is thought to
represent a partial sequence of 12p13 region of a human chromosome
sequence. While the locations of estimated genes within the
registered sequence were shown with the nucleotide sequence
information, the sequence ranging from nucleotide Nos. 1522 to 2770
of the nucleotide sequence represented by SEQ ID NO: 1, and the
nucleotide sequence represented by SEQ ID NO: 1 of the present
invention were not included in the any of specified regions of
estimated genes.
[0066] Probes and PCR primers were then designed based on the
nucleotide sequence ranging from nucleotide Nos. 1522 to 2770 of
the nucleotide sequence represented by SEQ ID NO: 1, and then a
continuous nucleotide sequence contained in the tumor cDNA library
was isolated and identified, thereby obtaining the nucleotide
sequence represented by SEQ ID NO: 1 of the present invention. The
nucleotide sequence of SEQ ID NO: 1 had an open reading frame
(hereinafter, may also be referred to as "ORF") encoding a
polypeptide consisting of an amino acid sequence represented by SEQ
ID NO: 2 of the present invention that is inferred to have a
secretion signal. We have considered that this is a polypeptide
having a novel sequence, because the amino acid sequence of the
polypeptide has not been registered at Genbank amino acid sequence
database. After a search using the nucleotide sequence represented
by SEQ ID NO: 1 and the amino acid sequence represented by SEQ ID
NO: 2, we have identified a nucleotide sequence represented by SEQ
ID NO: 9 and an amino acid sequence represented by SEQ ID NO: 10,
which are thought to be murine orthologs of the molecule. As
described later, a recombinant protein prepared according to a
human amino acid sequence shows activity in mice. Thus, the amino
acid sequence represented by SEQ ID NO: 2 was compared with the
amino acid sequence represented by SEQ ID NO: 10 or murine
full-length sequence (Biochem. Biophys. Res. Commun. 2000, 277(2),
494-498), so that the present invention makes it possible to easily
assess whether proteins, wherein an amino acid, other than amino
acids conserved between the two has been substituted, has
biological activity equivalent to, or similar to that of the
polypeptide of the present invention.
[0067] (2) Determination of Nucleotide Sequence
[0068] The nucleotide sequence of DNA obtained as described in (1)
above is determined. The nucleotide sequence can be determined by
known techniques, such as the Maxam-Gilbert's chemical modification
method or a dideoxynucleotide chain termination method using M13
phage. Normally, sequencing is performed using an automatic
sequencer (for example, 373A DNA sequencer manufactured by
PERKIN-ELMER).
[0069] The nucleotide sequence of the DNA of the present invention
is exemplified in SEQ ID NO: 1, and the amino acid sequence of the
polypeptide of the present invention is exemplified in SEQ ID NO:
2. As long as the polypeptide consisting of the amino acid sequence
has hypophosphatemia-inducing activity, phosphate
transport-suppressing activity, calcification-suppressing activity
or vitamin D metabolite-regulating activity, the amino acid
sequence may contain a mutation, such as deletion, substitution or
addition of one or several amino acids.
[0070] For example, 1 or several, preferably 1 to 10, more
preferably 1 to 5 amino acids may be deleted from the amino acid
sequence represented by SEQ ID NO: 2; 1 or several, preferably 1 to
10, more preferably 1 to 5 amino acids may be added to the amino
acid sequence represented by SEQ ID NO: 2; or 1 or several,
preferably 1 to 10, more preferably 1 to 5 amino acids may be
substituted with (an)other amino acids in the amino acid sequence
represented by SEQ ID NO: 2.
[0071] Further, as a method of substitution, conservative
substitution may be performed within a family which retains the
characteristics of the amino acids to some extent. Examples of
families generally classified according to the characteristics of
amino acid side chains are as follows.
[0072] (i) Acidic amino acid family: aspartic acid, glutamic
acid
[0073] (ii) Basic amino acid family: lysine, arginine,
histidine
[0074] (iii) Nonpolar amino acid family: alanine, valine, leucine,
isoleucine, proline, phenylalanine, methionine, tryptophan
[0075] (iv) Non-charged polar amino acid family: glycin,
asparagine, glutamine, cysteine, serine, threonine, tyrosine
[0076] (v) Aliphatic hydroxyamino acid family: serine,
threonine
[0077] (vi) Amide-containing amino acid family: asparagine,
glutamine
[0078] (vii) Aliphatic amino acid: alanine, valine, leucine,
isoleucine
[0079] (Viii) Aromatic amino acid family: phenylalanine,
tryptophan, tyrosine
[0080] (ix) Hydrophobic amino acid family: leucine, isoleucine,
valine
[0081] (x) Small amino acid family: alanine, serine, threonine,
methionine, glycin
[0082] Examples of substitution are sequences which are derived
from the amino acid sequence represented by SEQ ID NO: 2 by
substitution of the 176.sup.th Arg and/or 179.sup.th Arg with
(an)other amino acids, preferably, Ala, Gln or Trp, so that
cleavage is inhibited or suppressed therein. Further, the
polypeptide of the present invention also encompasses a polypeptide
consisting of an amino acid sequence derived from the amino acid
sequence represented by SEQ ID NO: 2 by deletion of 10 or more
amino acids on the N-terminal side, C-terminal side or both sides
(terminal-deleted type). Examples of such terminus-deleted forms
include a sequence derived from the amino acid sequence represented
by SEQ ID NO: 2 by deletion of 20, 40, 45 or 50 amino acids on the
C-terminal side, and/or 24 or 33 amino acids on the N-terminal
side. Embodiments of the terminal-deleted types are as shown
below.
1 Number of amino Number of amino Position in the amino acid
sequence acids deleted on the acids deleted on the represented by
SEQ ID NO: 2 N-terminal side C-terminal side (Nucleotide No. in SEQ
ID NO: 1) 33 on the N- No deletion 34-251(232-885) terminal side 33
on the N- 20 on the C-terminal 34-231(232-825) terminal side side
33 on the N- 40 on the C-terminal 34-211(232-765) terminal side
side 33 on the N- 45 on the C-terminal 34-206(232-750) terminal
side side 33 on the N- 50 on the C-terminal 34-201(232-735)
terminal side side 24 on the N- No deletion 25-251(205-885)
terminal side (corresponding to the 1.sup.st to the 681.sup.st in
SEQ ID NO: 3, and SEQ ID NO: 4) 24 on the N- 20 on the C-terminal
25-231(205-825) terminal side side 24 on the N- 40 on the
C-terminal 25-211(205-765) terminal side side 24 on the N- 45 on
the C-terminal 25-206(205-750) terminal side side 24 on the N- 50
on the C-terminal 25-201(205-735) terminal side side No deletion 20
on the C-terminal 1-231(133-825) side No deletion 40 on the
C-terminal 1-211(133-765) side No deletion 45 on the C-terminal
1-206(133-750) side No deletion 50 on the C-terminal 1-201(133-735)
side
[0083] In addition to partial fragments (terminal-deleted type
partial fragments) of the amino acid sequence represented by SEQ ID
NO: 2 above, the polypeptide of the present invention encompasses
mutated fragments that are derived from these terminal-deleted type
polypeptides by deletion, substitution or addition of one or
several amino acids. Figures in parentheses following the position
numbers of amino acids shown in SEQ ID NO: 2 in the above list
indicate the position numbers of nucleotides in the nucleotide
sequence represented by SEQ ID NO: 1. Hence, the present invention
also encompasses DNAs consisting of the nucleotide sequences shown
by these positions, or DNAs hybridizing under stringent conditions
to these DNAs.
[0084] In the present invention, to introduce a mutation into at
least a part of the amino acid sequence of the polypeptide of the
present invention, a technique which introduces a mutation into the
nucleotide sequence of a DNA encoding the amino acid is
employed.
[0085] Mutations can be introduced into DNA by a known technique,
such as the Kunkel method or the Gapped duplex method, or a method
according thereto. For example, a mutation is introduced based on
the site-directed mutagenesis method using a mutant oligonucleotide
as a primer. Further, a mutation can also be introduced using a kit
for introducing mutations, such as Mutan-K (TAKARA), Mutan-G
(TAKARA), LA PCR in vitro Mutagenesis series kit (TAKARA) or the
like.
[0086] Furthermore, the DNA of the present invention also
encompasses a DNA hybridizing under stringent conditions to a probe
prepared from the above DNA of the present invention (SEQ ID NO: 1,
3, 5, 7 and 9) and encoding a polypeptide having
hypophosphatemia-inducing activity, phosphate transport-suppressing
activity, calcification-suppressing activity or vitamin D
metabolism-regulating activity. The probe used herein has a
sequence which is complementary to the entire sequence of or a
sequence (partial sequence) of continuous 17 nucleotides or more of
the sequence represented by SEQ ID NO: 1, 3, 5, 7 or 9.
[0087] Here, the term "stringent conditions" satisfies conditions
of sodium concentration of 750 mM or more, preferably, 900 mM or
more, and temperature of 40.degree. C. or more, preferably
42.degree. C. Specifically, the stringent conditions used herein
indicate the conditions consisting of 6.times.SSC, 5.times.
Denhardt, 0.5% SDS, 50% Formamide and 42.degree. C. In addition,
6.times.SSC means 900 mM NaCl and 90 mM sodium citrate. Denhardt's
solution (Denhardt) contains BSA (bovine serum albumin),
polyvinylpyrrolidone and Ficoll 400. 50.times. Denhardt consists of
a composition of 1% BSA, 1% polyvinylpyrrolidone and 1% Ficoll 400
(5.times. Denhardt means a one-tenth concentration of 50.times.
Denhardt).
[0088] Once the nucleotide sequence of the DNA of the present
invention is determined, the DNA of the present invention can be
obtained by chemical synthesis or PCR using primers synthesized
from the determined nucleotide sequence.
[0089] 2. Recombinant Vector Containing the DNA of the Present
Invention and Preparation of Transformant
[0090] (1) Preparation of Recombinant Vector
[0091] The recombinant vector of the present invention can be
obtained by ligating (inserting) the DNA of the present invention
into an appropriate vector. A vector for inserting the DNA of the
present invention is not specifically limited, as long as it can be
replicated in a host. Examples of such a vector include plasmid DNA
and phage DNA.
[0092] Examples of plasmid DNAs include plasmids derived from
Escherichia coli (for example, pBR322, pBR325, pUC118 and pUC119),
plasmids derived from Bacillus subtilis (for example, pUB110 and
pTP5), plasmids derived from yeast (for example, YEp13, YEp24 and
YCp50). An example of a phage DNA is .lambda. phage. Further,
animal virus vectors such as a retrovirus, adenovirus or vaccinia
virus, or insect virus vectors such as baculovirus can be used.
Furthermore, a fusion plasmid to which GST, His-tag and the like
are ligated can be used.
[0093] In order to insert the DNA of the present invention into a
vector, a method can be employed, which comprises cleaving the
purified DNA using an appropriate restriction enzyme at first, and
ligating to the obtained cleaved DNA to the vector by inserting the
cleaved DNA into a restriction enzyme site or multi-cloning site of
an appropriate vector DNA.
[0094] The DNA of the present invention is required to be
incorporated into a vector so that the DNA can exert its function.
To the vector of the present invention, in addition to a promoter
and the DNA of the present invention, cis element such as an
enhancer, splicing signal, poly A addition signal, a selection
marker and ribosome binding sequence (SD sequence) may be ligated,
if necessary. In addition, examples of a selection marker include a
dihydrofolate reductase gene, ampicillin resistance gene and
neomycin-resistance gene.
[0095] (2) Preparation of Transformant
[0096] The transformant of the present invention can be obtained by
introducing the recombinant vector of the present invention into a
host, such that the target gene can be expressed. A host to be used
herein is not specifically limited, as long as it can express the
DNA of the present invention. Examples of such a host include
bacteria of: the genus Escherichia, such as Escherichia coli; the
genus Bacillus, such as Bacillus subtilis; the genus Pseudomonas,
such as Pseudomonas putida; or yeast such as Saccharomyces
cerevisiae or Schizosaccharomyces pombe. Further, animal cells,
such as COS cells, CHO cells or HEK293 cells, and insect cells,
such as Sf9 or Sf21 can also be used.
[0097] When bacteria, such as Escherichia coli is used as a host,
it is preferable that the recombinant vector of the present
invention can autonomously replicate in the bacteria, and comprises
a promoter, ribosome binding sequence, the DNA of the present
invention and transcription termination sequence. In addition, a
gene regulating a promoter may also be contained. Examples of
Escherichia coli include JM109 and HB 101, and an example of
Bacillus subtilis is Bacillus subtilis. Any promoter may be used,
as long as it can be expressed in a host, such as Escherichia coli.
For example, promoters derived from Escherichia coli, such as a trp
promoter, lac promoter, PL promoter or PR promoter or a
phage-derived T7 promoter or the like may be used. An artificially
designed and modified promoter, such as a tac promoter may be used.
A method to be employed herein for introducing a recombinant vector
into bacteria is not specifically limited, as long as it is a
method for introducing DNA into bacteria. Examples of such a method
include a method which uses calcium ion, and electroporation
method.
[0098] When yeast is used as a host, for example, Saccharomyces
cerevisiae, Schizosaccharomyces pombe and Pichia pastoris are used.
A promoter to be used in this case is not specifically limited, as
long as it can be expressed in yeast. Examples of such a promoter
include a gall promoter, gal10 promoter, heat-shock protein
promoter, MF.alpha. 1 promoter, PHO5 promoter, PGK promoter, GAP
promoter, ADH promoter and AOX1 promoter. A method for introducing
a recombinant vector into yeast is not specifically limited, as
long as it is a method for introducing DNA into yeast. Examples of
such a method include an electroporation method, spheroplast method
and lithium acetate method.
[0099] When an animal cell is used as a host, monkey cells COS-7,
Vero, Chinese hamster ovary cells (CHO cells), mouse L cells, rat
GH3 cells, human FL, HEK293, HeLa, Jurkat cells or the like are
used. As a promoter, SR.alpha. promoter, SV 40 promoter, LTR
promoter, .beta.-actin promoter or the like is used. In addition,
an early gene promoter of human cytomegalovirus or the like may
also be used. Examples of a method for introducing a recombinant
vector into animal cells include an electroporation method, calcium
phosphate method and lipofection method.
[0100] When an insect cell is used as a host, Sf9 cells, Sf21 cells
or the like are used. As a method for introducing a recombinant
vector to an insect cell, a calcium phosphate method, lipofection
method, electroporation method or the like is used.
[0101] 3. Polypeptide Having Hypophosphatemia-Inducing Activity
[0102] Several attempts have been made to isolate and identify a
tumor-derived factor having hypophosphatemia-inducing activity in
tumor-induced osteomalacia. Thus, the polypeptide of the present
invention has been shown to have characteristics of being a novel
secretion factor which is produced by tumor-induced osteomalacia
tumors. The predicted biological activities of the
hypophosphatemia-inducing factor have been reported as follows.
[0103] Effect on the Promotion of Phosphate Excretion into
Urine:
[0104] Aschinberg, L. C. et al., Journal of Pediatrics 91:56-60,
1977, Lau, K. et al., Clinical Research 27:421A, 1979, Miyauchi, A.
et al., J. Clin. Endocrinol. Metab. 67:46-53, 1988
[0105] Suppression of Phosphate Transport Activity of Epithelial
Cells of the Renal Tubules:
[0106] Cai, Q. et al., N. Engl. J. Med. 330: 1645-1649, 1994,
Wilkins, G. E. et al., J. Clin. Endocrinol. Metab. 80:1628-1634,
1995, Rowe, P. S. N. et al., Bone 18:159-169, 1996
[0107] Suppression of 25-Hydroxyvitamin D-1.alpha.-Hydroxylase
Activity:
[0108] Miyauchi, A. et al., J. Clin. Endocrinol. Metab. 67:46-53,
1988
[0109] In particular, it has been proposed that an unknown molecule
directly having activity to suppress reabsorption of phosphate in
the kidney has been referred to as "Phosphatonin" (Econs, M. J.
& Drezner, M. K., N. Engl. J. Med 330: 1679-1681, 1994). It has
also been suggested that an unknown molecule having such a
biological activity is also present in XLH. Clinical findings for
XLH patients are characterized by hypophosphatemia with enhanced
urinary phosphate excretion, which is the same as tumor-induced
osteomalacia patients, and XLH develops osteomalacia or rickets due
to calcification insufficiency in bone tissues. A gene responsible
for XLH has been shown to be a gene encoding an endopeptidase-like
protein, called PHEX. Recently, Hyp mice, the natural mutant mice,
known to express a phenotypic trait similar to that of XLH, have
been shown to have a partial deletion in the gene encoding PHEX,
thereby suggesting that determining Hyp mice to be XLH model mice
is valid (Strom, T. M. et al. Human Molecular Genetics 6:165-171,
1997). That the hypophosphatemia-inducing factor in Hyp mice is a
humoral factor has been shown by a parabiosis experiment using Hyp
mice and normal mice (Meyer, R. A. et al., J. Bone Miner. Res. 4:
493-500, 1989). In this experiment, blood phosphate levels of
normal mice decreased, and urinary phosphate excretion increased.
Hence, it has been considered that the humoral
hypophosphatemia-inducing factor existing in the Hyp mice acted on
the normal mice. So far the relationship between PHEX expected to
have a peptide cleavage activity and this unknown
hypophosphatemia-inducing factor has not been clear. However, some
hypotheses concerning a relationship that PHEX may regulate an
activity of an unknown hypophosphatemia-inducing factor and a
possibility that a hypophosphatemia-inducing factor found in
tumor-induced osteomalacia may be identical to that found in XLH
each other have been proposed (Drezner, M. K. Kidney Int 57:9-18,
2000). According to this hypothesis, PHEX and a
hypophosphatemia-inducing factor are both normally expressed in the
same cell, and PHEX acts suppressively on the
hypophosphatemia-inducing factor. The functions of PHEX decrease or
disappear in XLH patient, so that activity of
hypophosphatemia-inducing factor is strongly expressed. It is
presumed that in tumor-induced osteomalacia, both PHEX and
hypophosphatemia-inducing factor are elevated, and finally active
hypophosphatemia-inducing factor quantitatively exceeds the normal
level. It is also presumed that this hypophosphatemia-inducing
factor acts suppressively on the phosphate transport activity of
NPT2 which is one of phosphate transporters in the kidney. Many
attempts to search for such an unknown hypophosphatemia-inducing
factor have been made, but none were able to identify the molecule.
According to a study by Cai et al, it has been presumed that the
molecular weight of a hypophosphatemia-inducing factor is between 8
kDa to 25 kDa (Cai, Q. et al., N. Engl. J. Med. 330: 1645-1649,
1994), while Rowe et al have proposed 56 kDa and 58 kDa proteins as
candidate molecules. Recently, Rowe et al have filed a patent
application (WO99/60017) for a polypeptide consisting of 430 amino
acid residues as a tumor-derived phosphate metabolism-regulating
factor for tumor-induced osteomalacia. However, the polypeptide
disclosed in this application was a partial sequence of a protein
which was originally present, and no biological activity relating
to hypophosphatemia-inducing activity was disclosed. Recently, a
polypeptide corresponding to the full-length molecule as disclosed
by the name of MEPE in this patent has been reported, but no
activity to induce hypophosphatemia was also disclosed (Rowe, P. S.
N. et al, Genomics 67:54-68, 2000). In addition, no sequence or
structural similarity between this molecule and the polypeptide of
the present invention has been recognized.
[0110] As described above, the presence of a physiologically active
factor having an activity to induce hypophosphatemia is inferred,
but the entity thereof has not been shown so far. In the present
invention, we have clarified the entity of the polypeptide, and a
gene sequence encoding the polypeptide. Further, as described
later, we have produced the polypeptide of the present invention,
showed that the product acts as a regulatory factor for phosphate
metabolism, calcium metabolism and vitamin D metabolism or
calcification and osteogenesis, and the product is useful as a
pharmaceutical composition. Furthermore, we have shown that the
antibody of the present invention is useful not only for therapy,
but also for clinical examination and diagnosis. Moreover, we have
shown that the DNA encoding the polypeptide of the present
invention is useful for the diagnosis of hereditary diseases, and
for polymorphic diagnosis of phosphate metabolism, calcium
metabolism and bone metabolism.
[0111] The polypeptide having a hypophosphatemia-inducing activity
of the present invention can be produced by introducing, for
example, a sequence containing nucleotide Nos. 133.sup.rd to
885.sup.th of the nucleotide sequence represented by SEQ ID NO: 1
into an appropriate host cell in a form capable of being expressed
to prepare a transformant cell, and then allowing the DNA
introduced into the transformant cell to be expressed. In addition,
the polypeptide chain that is produced in this manner may be
modified by a protein modification mechanism of the host, such as
cleavage or addition of sugar chains.
[0112] The polypeptide of the present invention can be obtained by
culturing the above transformants, and then collecting from the
culture product. The term "culture product" means, in addition to
culture supernatant, any cultured cells or cultured microorganisms,
or disrupted cells or disrupted microorganisms.
[0113] The transformant of the present invention may be cultured by
any ordinary method for culturing a host.
[0114] Either natural or synthetic medium can be used for culturing
the transformant that is obtained using a microorganism, such as
Escherichia coli or yeast as a host, as long as it contains a
carbon source, a nitrogen source, inorganic salts and the like that
are assimilable by microorganisms and allows efficient culturing of
transformants. Examples of a carbon source include carbohydrates
such as glucose, fructose, sucrose or starch, organic acid such as
acetic acid or propionic acid, and alcohols such as ethanol or
propanol. Examples of a nitrogen source include ammonia, ammonium
salts of organic or inorganic acid such as ammonium chloride,
ammonium sulfate, ammonium acetate or ammonium phosphate, or other
nitrogen-containing compounds, and peptone, meat extract, and corn
steep liquor. Examples of minerals include potassium primary
phosphate, potassium secondary phosphate, magnesium phosphate,
magnesium sulfate, sodium chloride, ferrous sulfate, manganese
sulfate, copper sulfate and calcium carbonate.
[0115] Culturing is normally performed under aerobic conditions,
such as shake culture or aeration-agitation culture, at 37.degree.
C. for 4 to 48 hours. During a culturing period, pH is maintained
within 6.0 to 8.0. pH is adjusted using inorganic or organic acid,
alkali solution or the like. While culturing, antibiotics, such as
ampicillin or tetracycline, may be added to media, if
necessary.
[0116] When a microorganism transformed with an expression vector
using an inducible promoter is cultured, an inducer may be added to
a medium, if necessary. For example, when a microorganism
transformed with an expression vector having T7 promoter which can
be induced with isopropyl-.beta.-D-thiogalactopyranoside (IPTG) is
cultured, IPTG or the like may be added to the medium. In addition,
when a microorganism transformed with an expression vector using
trp promoter which can be induced with indoleacetic acid (IAA) is
cultured, IAA or the like can be added to a medium.
[0117] Examples of a medium for culturing a transformant that is
obtained using an animal cell as a host include a generally
employed RPMI1640 medium, DMEM medium or a medium supplemented with
fetal calf serum or the like. Culturing is performed normally under
5% CO.sub.2 at 37.degree. C. for 1 to 10 days. While culturing,
antibiotics, such as kanamycin or penicillin, may be added to
media, if necessary. After culturing, when the polypeptide of the
present invention is produced within microorganisms or cells, the
target polypeptide is collected by ultrasonication, repetitive of
freeze-thawing, homogenizing treatment or the like to disrupt the
microorganisms or cells. Further, when the polypeptide of the
present invention is produced outside bacteria or cells, the
culture solution is used intact, or centrifugation or the like is
performed to remove the bacteria or cells. Then, the polypeptide of
the present invention can be isolated and purified from the above
culture product by a singular or a combined use of general
biochemical methods for isolation and purification of protein, such
as ammonium sulfate precipitation, gel chromatography, ion exchange
chromatography and affinity chromatography.
[0118] As described above about XLH, PHEX which is thought to be an
endopeptidase, has an important meaning in regulation of the
hypophosphatemia-inducing factor. Hence, it is possible that the
polypeptide having the amino acid sequence of SEQ ID NO: 2 of the
present invention may have varied activities as a result of further
modification and cleavage. In the present invention, using CHO
ras-clone 1 cells as a host, a cloned cell line producing a
recombinant polypeptide conferred with six continuous His at the
C-terminus of the polypeptide chain of the present invention was
prepared. This cell line was deposited at the National Institute of
Advanced Industrial Science and Technology, International Patent
Organism Depositary (Chuo 6, 1-1-1, Higashi, Tsukuba-shi, Ibaraki,
Japan) (Accession number FERM BP-7273, original deposit date: Aug.
11, 2000).
[0119] When the polypeptide of the present invention produced by
the cell line and secreted into the culture solution was examined,
gene products having different sizes were detected by Western
blotting using an antibody recognizing His-tag sequence, as shown
in FIG. 2. Proteins corresponding to respective bands were isolated
and the determination of N-terminal amino acid sequences was
performed. Thus, the N-terminal amino acid sequences were identical
to the N-terminal amino acid sequence represented by SEQ ID NO: 4
and that represented by SEQ ID NO: 8, respectively. It is
considered that the protein having the former sequence corresponds
to a protein of which signal sequence is removed, and the protein
having the latter sequence corresponds to a protein cleaved with an
enzyme, such as an endopeptidase.
[0120] Now, furin is known as one of proteolytic enzymes which
recognize RXXR. Actually, when the polypeptide of the present
invention was expressed in a furin-deficient cell line, no fragment
was detected. Further, when a recombinant protein .alpha.1-PDX
having furin inhibition activity was co-expressed with the
polypeptide of the present invention, cleaved products in the
supernatant decreased significantly.
[0121] Hence, the present invention also encompasses a method for
producing the polypeptide of the present invention which comprises
the step of using furin-deficient cells upon culturing, or allowing
co-existence with a substance which suppresses furin activity.
[0122] Cai et al have suggested that phosphate
transport-suppressing activity in the culture supernatant obtained
by culturing tumor-derived cells of tumor-induced osteomalacia
exhibited within the molecular weight range of 8 kDa to 25 kDa when
measured by a fractionation method with dialysis membranes. It is
also conceivable that the activity may be varied by converting the
polypeptide having the amino acid sequence of SEQ ID NO: 4 of the
present invention into polypeptides resulting from cleavage between
residue No. 179, Arg, and residue No. 180, Ser, of SEQ ID NO:
2.
[0123] The polypeptide of the present invention has an activity to
suppress the phosphate transport activity of epithelial cells of
the renal proximal tubules, which is a form of the effect of
hypophosphatemia-inducing activity, as shown in Table 2 (Example
7). Most free inorganic phosphate existing in the blood is filtered
in the glomeruli of the kidneys, wherein approximately 80 to 90% of
the inorganic phosphate is reabsorbed in the renal proximal
tubule.
[0124] This reabsorption is performed by phosphate transport by
type II Na-dependent phosphate transporter existing on the lumen
side of the proximal tubule. The polypeptide of the present
invention has an activity to suppress phosphate transport activity.
This means that the polypeptide of the present invention promotes
urinary excretion of phosphate in vivo. Thus, it can be considered
that the polypeptide of the present invention induces
hypophosphatemia by exerting its activity to suppress phosphate
reabsorption in the kidney, in particular, phosphate transport in
renal cells of proximal tubules, so that the polypeptide of the
present invention is expected to be the same substance as the above
Phosphatonin.
[0125] Recently, Na-dependent phosphate transporter in the
intestinal tract has been identified. The transporter is named type
IIb, because it has a high homology with that of type II
Na-dependent phosphate transporter existing in the kidney. It is
conceivable that the polypeptide of the present invention may also
be responsible for suppressing type IIb Na-dependent phosphate
transporter existing on the lumen side of the intestinal tract,
similarly for type II Na-dependent phosphate transporter in the
kidney. This can be regarded as a form of the effect of the
hypophosphatemia-inducing activity.
[0126] The in vivo activity of the polypeptide of the present
invention was evaluated by an experiment wherein the above
recombinant cells expressing the polypeptide of the present
invention had been transplanted subcutaneously to nude mice.
[0127] The transplanted cells in this experiment were grown in
subcutaneous space of nude mice, and then allowed to form tumors.
The polypeptide of the present invention produced and secreted by
the cell with the tumor formation is characterized by being
released into the body fluid of the mice, so that releasing of
tumor-derived humoral factor in tumor-induced osteomalacia can be
reproduced in this animal model. In this model experiment, as shown
in Table 4 (Example 11), mice transplanted with cells expressing
the polypeptide of the present invention developed evident
hypophosphatemia, compared to control individual mice allowed to
generate tumors by transplantation with CHO cells into which no DNA
of the present invention had been introduced, or individual mice
that have generated no tumor. Accordingly, the polypeptide of the
present invention was shown to have the hypophosphatemia-inducing
activity. In addition, it was also shown that the phosphate
reabsorption rate also decreased, and that phosphate reabsorption
in the kidneys was suppressed. Therefore, it was concluded that the
polypeptide of the present invention is the
hypophosphatemia-inducing factor in tumor-induced osteomalacia.
[0128] On the other hand, in the above model experiment,
hypocalcemia was found in the mice transplanted with the
recombinant cells producing the polypeptide of the present
invention. Thus, it was also shown that the polypeptide of the
present invention is also an hypocalcemia-inducing factor. In the
experiment described in Example 16, wherein CHO cells expressing
the polypeptide of the present invention were transplanted into
nude mice, it was shown that serum 1.alpha.,25-dihydroxyvitamin D
levels continuously decreased. As described in Examples 19 and 20,
it was shown that when a mutation-introduced polypeptide of the
present invention or a wild type full-length polypeptide of the
present invention was administered three times to normal mice,
serum 1.alpha.,25-dihydroxyvitamin D levels decreased in both
cases. Further, as described in Example 24, after a single
administration of the polypeptide of the present invention,
decreased serum 1.alpha.,25-dihydroxyvitamin D levels were observed
within several hours. Hence, it is conceivable that this activity
which causes such a decrease of 1.alpha.,25-dihydroxyvitamin D
level is a major biological or physiological effect of the
polypeptide of the present invention.
[0129] As described above, serum 1.alpha.,25-dihydroxyvitamin D
levels are ruled by 1.alpha.-hydroxylase and 24-hydroxylase. As
described in Example 16, it was shown that the effect of the
polypeptide of the present invention to decrease serum
1.alpha.,25-dihydroxyvitamin D levels is accompanied by
fluctuations in expression of these metabolic enzymes. Further, as
described in Example 24, at 1 hour after administration of the
polypeptide of the present invention, decreased gene transcription
products of 1.alpha.-hydroxylase which is responsible for
production of active vitamin D metabolites, and increased gene
transcription products of 24-hydroxylase which catabolizes active
vitamin D metabolites were observed. The serum
1.alpha.,25-dihydroxyvitamin D levels gradually decreased after
these fluctuations in expression, suggesting that the effect of the
polypeptide of the present invention to decrease serum
1.alpha.,25-dihydroxyvitamin D levels is at least due to suppressed
expression of 1.alpha.-hydroxylase gene and enhanced expression of
24-hydroxylase gene.
[0130] In contrast, in a long-term transplantation experiment (from
44 to 46 days after transplantation) wherein CHO cells expressing
the polypeptide of the present invention was transplanted into nude
mice as described in Example 11, expression of the
1.alpha.-hydroxylase gene was elevated. The mouse serum PTH levels
at this time point was proved to be significantly elevated compared
to a control group. Thus, it can be presumed that enhanced
expression of 1.alpha.-hydroxylase gene was caused by a PTH effect
at a high level. However, interestingly, even in the presence of
high PTH level, expression of 24-hydroxylase gene was kept
elevated, it can be understood that the high PTH level failed to
interfere with the regulation by the polypeptide of the present
invention of expression of 24-hdyroxylase gene. As described in
Example 11, serum 1.alpha.,25-dihydroxyvitamin D3 levels was not
increased although the mice expressed severe hypophosphatemia- or
rickets-like clinical findings. This suggests the affect of
continuous enhancement of expression of 24-hydroxylase gene by the
polypeptide of the present invention.
[0131] A. Nykjaer et al have revealed that 25-hydroxyvitamin D is
reabsorbed in the renal proximal tubule (Cell, Vol. 96, p507-515,
1999). While the present specification does not describe it, but in
an experiment wherein CHO cells expressing the polypeptide of the
present invention had been transplanted into nude mice, no
significant change was found in the serum 25-hydroxyvitamin D
level. In addition, it was not recognized that the action of the
polypeptide of the present invention affected the fractional
excretion (calculated with urinary level/serum level/GFR) of main
electrolytes, such as sodium, potassium or chloride, main amino
acids or glucose, supporting the fact that the reabsorption
function of the renal tubule was undamaged (T. Shimada et al.,
Proc. Natl. Acad. Sci, in press). Therefore, it was suggested that
the polypeptide of the present invention does not decrease serum
1.alpha.,25-dihydroxyvitamin D levels by inhibiting reabsorption of
25-hydroxyvitamin D in the renal tubule, but by specifically acting
on the synthetic pathway of 1.alpha.,25-dihydroxyvitamin D.
[0132] It is known that the serum 1.alpha.,25-dihydroxyvitamin D
level is significantly decreased in tumor-induced osteomalacia.
Further, in hypophosphatemic vitamin D-resistant rickets (XLH) or
Hyp, the model mice expressing the clinical conditions of XLH,
serum 1.alpha.,25-dihydroxyvit- amin D levels are within the normal
range or within a range somewhat below the lower limit of the
normal range, regardless of severely decreased serum phosphate
levels. It is also known that the expression of 24-hydroxylase gene
is elevated in Hyp mice. In these clinical conditions developing
hypophosphatemia, normally, as serum phosphate level decreases,
1.alpha.-hydroxylase gene expression rises, thereby increasing
serum 1.alpha.,25-dihydroxyvitamin D levels. Thus, it is thought
that a failure in any of regulatory systems disabling such normal
physiological response is at least one of the causes of the
clinical conditions. These phenomena are analogous to the
physiological responses observed in the mice described in Example
11, 19 or 20, strongly suggesting that the polypeptide of the
present invention functions to decrease serum
1.alpha.,25-dihydroxyvitamin D3 levels in the above clinical
conditions.
[0133] It is clear from the X-ray images shown in FIG. 5 that the
degree of calcification in bone tissues of the mice transplanted
with the recombinant cells expressing the polypeptide of the
present invention was significantly decreased when compared to that
in the control group. Thorax deformation or the like was also
observed, suggesting that the polypeptide of the present invention
had an effect on skeletal formation.
[0134] In other words, it is conceivable that the polypeptide of
the present invention has an effect on suppressing calcification of
bone tissues, or effect on promoting recruitment of calcium and
phosphate from bone tissues. It is also conceivable that
significant decreases in both blood phosphate and calcium levels
caused secondary suppression of bone tissue calcification.
[0135] In Hyp mice, it is thought that bone-derived cells produce a
factor which suppresses phosphate transport activity in the renal
proximal tubule (Lajeunesse, D. et al., Kidney Int. 50: 1531-1538,
1996). Further, it has been reported that osteoblasts of Hyp mice
release calcification-suppressing factors (Xiao, Z. S., Am. J.
Physiol., E700-E708, 1998). As described above, in XLH and
tumor-induced osteomalacia, clinical findings such as
hypophosphatemia and insufficient calcification in bone tissues
closely resembles from each other, and these clinical findings
would likely be induced by a single humoral factor. Taken together
these facts suggest the possibility that the renal phosphate
transport-suppressing activity and the bone
calcification-suppressing activity reported in these studies of Hyp
mice can be caused by the same factor. In addition, it has also
been reported that the osteoblasts of Hyp mice exhibit abnormal
osteogenesis even in the state wherein calcium and phosphate levels
are in the normal range (Ecarot, B. et al., J. Bone Miner. Res. 7:
215-220, 1992). The polypeptide of the present invention has
activities that is similar to those of a putative factor of the
above Hyp mice. Thus, it is conceivable that the polypeptide of the
present invention has, in addition to the hypophosphatemia-inducing
activity, an effect of directly regulating calcification of bone
tissues not mediated by the abnormalities in calcium or phosphate
metabolism.
[0136] In the course of completing the present invention, in
addition to a gene encoding the polypeptide of the present
invention, dentin matrix protein-1 (DMP-1) was also obtained as
shown in Table 1 of Example 3. This gene is abundantly expressed in
the dentin of teeth, and a protein encoded by this gene is thought
to have an important role, as an extracellular matrix protein of
dentin, in the formation of calcified matrix of dentin. Similarly,
a gene encoding a matrix extracellular phosphorylation protein
(MEPE) was obtained as OST190. Detailed functions of the molecule
are unknown. Further similarly, a gene encoding osteopontin was
also obtained. MEPE, DMP-1 and osteopontin have common
characteristics in that they are phosphorylation proteins having
RGD motif sequence, are rich in serine and threonine that can be
phosphorylated, have high contents of glutamic acid and aspartic
acid that are acidic amino acids, and show intense acidic protein
characteristics. A characteristic acidic region, named ASARM
sequence, is conserved between MEPE and DMP-1 (Rowe, P. S. N. et
al, Genomics 67: 54-68, 2000), suggesting similarity in their
physiological or functional significance. Interaction with
inorganic calcium and/or phosphate upon the start of calcification
is thought to be one of the functions of such a characteristic
protein. Expression of osteopontin gene in a variety of cells has
been reported, such as in macrophages, in addition to osteoblasts
and osteoclasts. On the other hand, expression of DMP-1 in bone
tissues, particularly in osteocytes has been reported recently. The
gene expression of MEPE in the myeloid tissue or in osteosarcoma
cells such as SaOS-2 are known. The fact that such an acidic matrix
protein found in the calcified tissue has been found together with
the polypeptide of the present invention in the course of the
present invention represents an aspect of a type of effect of the
polypeptide of the present invention. Specifically, there are
possibilities that the polypeptide of the present invention induces
expression of calcified tissue-specific molecules which are
represented by the above molecules, so that the polypeptide
regulates calcification, calcium metabolism and phosphate
metabolism in a cooeprative manner, or the induced molecule
secondarily regulates calcification, calcium metabolism, and
phosphate metabolism. It is also conceivable that the polypeptide
of the present invention can regulate bone metabolism by directly
acting on osteoblasts, osteocytes and osteoclasts. Hence, the
polypeptide of the present invention may be effective in the
therapy for metabolic bone diseases as represented by
osteoporosis.
[0137] Recently, cells having osteoblast-like phenotype have been
shown to appear at ectopic calcification sites, suggesting that
calcification may occur by the mechanism similar to that of the
process of calcification in bone tissue. Therefore, it is also
conceivable that the polypeptide of the present invention is
effective in the therapy of ectopic calcification by suppressing
the appearance or function of such cells in charge of
calcification.
[0138] In the present invention, the above polypeptide can be
modified. For example, polyethylene glycol, dextran, poly
(N-vinyl-pyrrolidone), polypropylene glycol homopolymer, copolymer
of polypropylene oxide/ethylene oxide, polyoxyethylated polyol,
polyvinyl alcohol and the like are appropriately selected for use.
As a modification method, any known technique can be employed. For
example, one such technique is disclosed in detail in JP Patent
Publication (PCT translation) No. 10-510980.
[0139] 4. Antibody Against Polypeptide of the Present Invention
[0140] An antibody of the present invention specifically reacts
with the above polypeptide of the present invention. In the present
invention, the term "antibody" means the entire antibody molecule
or the fragment thereof (for example, Fab or F(ab').sub.2 fragment)
which are capable of binding to the antigenic polypeptide or
fragments thereof, and may be a polyclonal or a monoclonal
antibody.
[0141] The antibody of the present invention can be prepared
according to a standard method. For example, the antibody can be
prepared by either an in vivo method, which involves immunization
of animals once or several times (booster immunization) at an
interval of several weeks using an antigen together with an
adjuvant, or an in vitro method, which involves isolating
immunocytes and allowing the immunocytes to be sensitized using an
appropriate culture system. Examples of immunocytes capable of
producing the antibody of the present invention include spleen
cells, tonsil cells and lymphoid cells.
[0142] A polypeptide to be used as an antigen does not have to be
the above entire polypeptide of the present invention. A part of
the polypeptide may be used as an antigen. To use a short peptide
as an antigen, particularly, a peptide having as short as
approximately 20 amino acid residues, such a peptide is bound by
chemical modification or the like to a carrier protein with high
antigenicity, such as keyhole limpet hemocyanin or bovine serum
albumin, or covalently bound to a peptide having the branched
skeleton, such as a lysine core MAP peptide, instead of a carrier
protein (Posnett et al., J. Biol. Chem. 263, 1719-1725, 1988; Lu et
al., Mol. Immunol. 28, 623-630, 1991; Briand et al., J. Immunol.
Methods 156, 255-265, 1992).
[0143] As an adjuvant, for example, Freund's complete or incomplete
adjuvant, aluminum hydroxide gel or the like is used. As animals to
be administered with an antigen, for example, a mouse, rat, rabbit,
sheep, goat, chicken, cattle, horse, guinea pig and hamster are
used.
[0144] Polyclonal antibodies can be obtained by collecting blood
from these immunized animals, separating the serum, and purifying
immunoglobulins using one of or an appropriate combination of
ammonium sulfate precipitation, anion exchange chromatography, and
protein A or G chromatography. When the above animal is a chicken,
antibodies can be purified from the eggs.
[0145] Monoclonal antibodies can be prepared by purification from
the culture supernatant of hybridomas prepared by allowing
immunocytes, which have been sensitized in vitro or of the above
animals, to fuse with parent cells that can be cultured, or from
ascites obtained by intraperitoneal inoculation of the hybridomas
into the animals. As parent cells, a generally available
established cell line of an animal, such as a mouse, can be used. A
preferred cell line to be used herein has drug selectivity, and has
a characteristic such that it cannot survive in HAT selection
medium (hypoxantine, aminopterin and thymidine are contained) when
it is in unfused state, but can survive only in its fused state
with antibody-producing cells. Examples of such a cell line include
X63, NS-1, P3U1, X63.653, SP2/0, Y3, SKO-007, GM1500, UC729-6,
HM2.0 and NP4-1 cells.
[0146] Specific techniques to prepare monoclonal antibodies are as
follows.
[0147] The polypeptide or the fragment thereof prepared as
described above is administered as an antigen to the above animal.
Antigen dosage per animal is 1 to 100 .mu.g when an adjuvant is
used. Immunization is performed mainly by intravenous, subcutaneous
or intraperitoneal injection. In addition, the immunization
interval is not specifically limited. At an interval of several
days to several weeks, preferably 1 to 3 weeks, immunization is
performed 1 to 10 times, preferably 2 to 5 times. 1 to 10 days
later, preferably 1 to 4 days later the final immunization date,
antibody-producing cells are collected.
[0148] To obtain hybridomas, cell fusion of antibody-producing
cells and parent cells (myeloma cells) is performed. Cell fusion is
performed in a serum-free medium for culturing animal cells, such
as DMEM or RPMI-1640 medium, by mixing 5.times.10.sup.6 to
1.times.10.sup.8 cells/ml antibody-producing cells with
1.times.10.sup.6 to 2.times.10.sup.7 cells/ml myeloma cells (a
preferred ratio of antibody-producing cells to myeloma cells is
5:1), and performing fusion reaction under the presence of a cell
fusion-promoting agent. As a fusion-promoting agent, polyethylene
glycol or the like having a mean molecular weight of 1000 to 6000
daltons can be used. In addition, antibody-producing cells and
myeloma cells can also be fused with a commercial cell fusion
device using electric stimulation (for example,
electroporation).
[0149] Following the treatment to accomplish cell fusion,
hybridomas of interest are selected from the cells. The cell
suspension is appropriately diluted in, for example, RPMI-1640
medium containing fetal calf serum, inoculated on a microtiter
plate at a concentration of approximately 5.times.10.sup.5
cells/well. The selection medium is added to each well, and then
culturing is performed while properly exchanging the selection
medium. As a result, cells that have proliferated at around 14 days
after the start of culturing in the selection medium can be
obtained as hybridomas. The culture supernatant of the hybridomas
that have proliferated is screened for the presence of antibodies
which react with the polypeptide of the present invention.
Screening for hybridomas may be performed according to an ordinary
method, and the screening method is not specifically limited. For
example, a part of the culture supernatant contained in wells in
which hybridomas are grown is collected, and then screened by
enzyme immunoassay, radioimmunoassay or the like.
[0150] Alternatively, monoclonal antibodies can be prepared by
culturing immortalized antibody-producing cells which are obtained
by allowing an appropriate virus, such as EB virus, to infect
immunocytes sensitized in vitro or of the above immunized
animal.
[0151] Aside from these cell engineering techniques, monoclonal
antibodies can also be obtained by gene engineering techniques. For
example, such antibody genes can also be amplified and obtained by
PCR (polymerase chain reaction) from immunocytes sensitized in
vitro or of the above animal. The gene is introduced into a
microorganism, such as Escherichia coli, so as to allow it to
produce antibodies, or used to allow a phage to express the
antibody as a fusion protein on the surface.
[0152] Quantitative determination of the amount of the polypeptide
of the present invention in vivo using the antibody of the present
invention makes it possible to elucidate the relationship between
the polypeptide of the present invention and clinical conditions of
various diseases. Moreover, the antibody can be applied to
diagnosis or therapy and subjected to perform efficient affinity
purification of the polypeptide of the present invention.
[0153] It is assumed that there are some diseases of which cause
resides in a decreased of serum 1.alpha.,25-dihydroxyvitamin D
levels induced by the excessive action of the polypeptide of the
present invention. For example, although hypophosphatemic vitamin
D-resistant rickets (XLH) develops severe hypophosphatemia, no
increase is found in serum 1.alpha.,25-dihydroxyvitamin D3 levels.
The reason is thought to be abnormality in groups of vitamin D
metabolizing enzyme genes. In this disease, excessive action of the
polypeptide of the present invention may be involved. In Hyp, which
is a mouse model of XLH, enhanced expression of 24-hydroxylase gene
has been reported. This agrees with the effect of the polypeptide
of the present invention to induce enhanced 24-hydroxylase gene
expression. Therefore, it is expected that this disease can be
treated by administering the antibody against the polypeptide of
the present invention to normalize vitamin D metabolism and then to
correct the serum 1.alpha.,25-dihydroxyvitamin D3 levels. An
example of a disease that presents clinical findings similar to
that of XLH is autosomal dominant hypophosphatemic rickets (ADHR).
Upon cloning of the polypeptide of the present invention, we have
inferred that a gene encoding the polypeptide is a gene responsible
for ADHR, based on its location on the chromosome. Recently, a gene
responsible for ADHR has been analyzed and reported that the
disease is caused by a missense mutation in the gene encoding the
polypeptide. We have further clarified that this mutation confers
resistance against enzymatic cleavage, and showed that excessive
effect of this molecule is a cause of the disease. It is
conceivable that the antibody against the polypeptide of the
present invention is effective in treating this disease through its
suppressing effect. ADHR develops osteomalacia, disordered mineral
metabolism and disordered vitamin D metabolism. Thus, among
metabolic bone disases which closely relate to such metabolic
pathway, there may be diseases on which the polypeptide of the
present invention acts as a cause of the disease. It can be
expected that the antibody against the polypeptide of the present
invention is effective for such a disease. As one effect of
1.alpha.,25-dihydroxyvitamin D, suppression of differentiation into
adipocytes is known. It is known that adipocytes in the bone marrow
increases with age. In this case, there may be enhanced
differentiation into adipocytes from common precursor cells of
osteoblasts, adipocytes and stromal cells supporting hematopoiesis
existing in the bone marrow. It is conceivable that in this
process, the polypeptide of the present invention may excessively
act to decrease local 1.alpha.,25-dihydroxyvitamin D levels.
Therefore, it can be expected that the use of the antibody against
the polypeptide of the present invention enables to increase blood
or local 1.alpha.,25-dihydroxyvitamin D levels, suppress
differentiation into adipocytes, and improve decreased ability of
bone formation or hematopoiesis. In addition, the antibody is also
expected to be effective against obesity. It is conceivable that
there are other such diseases in which the polypeptide of the
present invention is involved. Such a disease can be screened by an
immunological quantitative determination method as represented by
ELISA combined with the use of the antibody against the polypeptide
of the present invention. Accordingly, the physiological normal
range of the polypeptide of the present invention can be
established, and disease groups deviated from the range can be
clarified. It can be expected that the antibody against the
polypeptide of the present invention is used for therapeutic
purposes against diseases showing abnormally high blood levels of
the polypeptide of the present invention as measured by the above
method.
[0154] 5. Pharmaceutical Composition
[0155] (1) Pharmaceutical Composition Comprising the Polypeptide of
the Present Invention
[0156] The polypeptide of the present invention can be used as a
pharmaceutical composition for diseases with unfavorably elevated
blood phosphate levels. In chronic renal failure, decreased levels
of phosphate excretion from the kidney result in elevated blood
phosphate levels. Hyperphosphatemia further aggravates renal
functions, and promotes secretion of parathyroid hormone from the
parathyroid gland, thereby inducing secondary hyperparathyroidism.
This disease causes itching of the skin, as well as decreased Ca
absorption from the intestinal tract due to disordered synthesis of
1.alpha.,25-dihydroxyvitamin D3 in the kidney. In addition, the
state of oversecretion of parathyroid hormone due to the retention
of blood phosphate promotes Ca mobilization from bone tissues. When
this state continues, osteitis fibrosa or hyperplasia of the
parathyroid glands that is one of the clinical conditions of renal
osteodystrophy. One preferred method to evade this state is to
improve the above-mentioned hyperphosphatemia, but current medical
treatment cannot sufficiently control hyperphosphatemia. In chronic
renal failure at a stage wherein the urination function is
retained, the polypeptide of the present invention has an activity
to correct blood phosphate levels by suppressing type II
Na-dependent phosphate transporter existing in epithelial cells of
the renal proximal tubules to promote phosphate excretion into
urine (phosphate transport-suppressing activity). In addition, the
polypeptide of the present invention can correct blood phosphate
levels by acting on the intestinal tract in a way similar to that
in the kidneys to suppress type IIb Na-dependent phosphate
transporter and thus reduce phosphate absorption into the body.
[0157] The polypeptide of the present invention can also be used as
a pharmaceutical composition for diseases resulting from abnormal
calcium metabolism and phosphate metabolism. The term "abnormal
calcium metabolism" indicates a state at which serum calcium levels
deviate from clinically defined normal range, or a state at which
serum calcium levels are within the normal range, but the functions
of the kidney, intestinal tract, bone tissue and parathyroid glands
are abnormally increased or decreased to maintain serum calcium
levels, or a state at which hormones regulating the serum calcium,
such as parathyroid hormone, 1.alpha.,25-dihydroxyvitamin D3 or
calcitonin exhibit abnormal values. In addition, the term "abnormal
phosphate metabolism" indicates a state at which serum phosphate
levels deviate from the clinically-defined normal range, or a state
in which serum phosphate levels are within the normal range, but
phosphate-balancing functions are abnormally increased or decreased
in the kidney, intestinal tract and bone tissue.
[0158] Renal osteodystrophy along with the above secondary
hyperparathyroidism takes various clinical forms, such as adynamic
bone disease, osteitis fibrosa or the mixed type thereof. Against
secondary hyperparathyroidism, 1.alpha.,25-dihydroxyvitamin D3,
1.alpha.-hydroxyvitamin D3 or the like is generally used to
suppress parathyroid hormone. When the value of parathyroid hormone
is not sufficiently suppressed, a pulse therapy (hereinafter, may
also be referred to as "vitamin D pulse therapy") which involves
administering over dose of 1.alpha.,25-dihydroxyvitamin D3 or
1.alpha.-hydroxyvitamin D3 may be performed. The normal serum
parathyroid hormone level is 65 pg/ml or less. When parathyroid
hormone levels are at the normal levels in this disease condition,
adynamic bone disease, which is a form of renal osteodystrophy, is
caused. Moreover, when parathyroid hormone levels increase,
osteitis fibrosa, which is contrary to the above clinical
condition, occurs. As a recent medical guideline for such diseases,
it has been proposed that parathyroid hormone levels be maintained
at approximately 130 to 260 pg/ml. However, fundamental causes of
abnormal metabolism remain unknown. It is known that parathyroid
hormone is induced by elevated blood phosphate levels, and is
suppressed by elevated serum calcium levels. Since the polypeptide
of the present invention lowers blood phosphate levels and blood
calcium levels, it can be inferred that the polypeptide can modify
the functions of parathyroid hormone. Further, there has been
reported that in tumor-induced osteomalacia patients,
1.alpha.,25-dihydroxyvitamin D3 falls to the detection limit or
below. Thus, it can also be inferred that the polypeptide of the
present invention may be involved in regulating the activity of
1.alpha.,25-dihydroxyvitamin D3. It is conceivable that in renal
osteodystrophy with the above abnormal regulation or impaired
activity of parathyroid hormone, the polypeptide of the present
invention can be used as a clinically useful pharmaceutical
composition for either adynamic bone disease or osteitis fibrosa.
Therefore, it is conceivable that administration of the polypeptide
of the present invention provides a useful therapy for either
osteitis fibrosa or adynamic bone disease, which are contrary to
each other, in renal osteodystrophy.
[0159] Furthermore, the polypeptide of the present invention can be
used as a pharmaceutical composition for ectopic calcification.
Calcification of tissues other than bone tissues damages
biofunctions. In particular, dysfunction due to calcification of
the heart or blood vessel threatens life. A risk factor of this
ectopic calcification is an increase in the product of blood
calcium ion and phosphate ion levels (hereinafter, may also be
referred to as "the calcium-phosphate product"). In the above
therapy for secondary hyperparathyroidism, when the vitamin D pulse
therapy is performed against clinical conditions of
hyperphosphatemia, the calcium-phosphate product can increase to
cause ectopic calcification. Calcification of the cardiovascular
system in hemodialysis patients is a serious problem. The
polypeptide of the present invention has an activity to
significantly decrease serum calcium levels and phosphate levels as
shown in Table 4, so that it can be expected to be effective
against ectopic calcification along with various diseases.
[0160] Furthermore, the polypeptide of the present invention can be
used as a pharmaceutical composition against metabolic bone
diseases. The polypeptide of the present invention has a strong
regulatory activity for calcium metabolism, phosphate metabolism,
calcification or vitamin D metabolism. Examples of hormones
involved in calcium metabolism and phosphate metabolism include
calcitonin, parathyroid hormone and 1.alpha.,25-dihydroxyvitamin
D3. Calcitonin has an activity to decrease serum calcium, and
parathyroid hormone and 1.alpha.,25-dihydroxyvitamin D3 have an
activity to increase serum calcium. Parathyroid hormone has been
reported to have an effect on phosphate excretion to urine, and
calcitonin has also been reported to have similar activity.
1.alpha.,25-dihydroxyvitamin D3 has an activity to promote
phosphate absorption from the intestinal tract. As described above,
each hormone has a different activity for maintaining the balance
between blood calcium and phosphate levels, but calcitonin and
1.alpha.,25-dihydroxyvit- amin D3 are used as therapeutic agents
for osteoporosis. In addition, parathyroid hormone is being
developed as a therapeutic drug for osteoporosis.
[0161] Bone metabolism is characterized in that catabolism and
anabolism of bone tissues, that is, bone resorption and bone
formation, are coupling. Continuous administration with parathyroid
hormone causes bone mass to decrease. However, when allowed to act
intermittently, parathyroid hormone is known to promote bone
formation. Since the polypeptide of the present invention has an
effect of regulating calcium metabolism and phosphate metabolism,
it can be expected that the polypeptide can be effective against
metabolic bone diseases including osteoporosis, when an appropriate
method for using the polypeptide is selected.
[0162] Furthermore, the polypeptide of the present invention can be
used as a pharmaceutical composition for diseases or clinical
conditions with unfavorably elevated serum
1.alpha.,25-dihydroxyvitamin D levels, or for clinical conditions
with unfavorable physiological responses induced by serum
1.alpha.,25-dihydroxyvitamin D.
[0163] 1.alpha.,25-dihydroxyvitamin D is known to act on the
parathyroid glands to suppress secretion of parathyroid hormone
(PTH). Therefore, a therapy, which has been clinically established
for secondary hyperparathyroidism in chronic renal failure,
particularly for cases with high serum PTH levels, involves
intermittent administration of a high concentration of
1.alpha.,25-dihydroxyvitamin D. A disadvantage of this therapy is
that it easily induces ectopic calcification. In chronic renal
failure patients with high serum phosphate levels, unfavorable
calcification due to administration of 1.alpha.,25-dihydroxyvitamin
D is often observed in tissues other than bone tissues. Since the
polypeptide of the present invention has an effect to promote a
quick decrease in serum 1.alpha.,25-dihydroxyvitamin D within
several hours after administration of the polypeptide, the
polypeptide is useful in therapy and in preventing ectopic
calcification that is caused by excessive
1.alpha.,25-dihydroxyvitamin D levels.
[0164] Moreover, intermittent administration of high concentrations
of 1.alpha.,25-dihydroxyvitamin D causes excessively suppressed PTH
secretion, so that it can induce adynamic bone disease developing
clinical conditions such as arrested bone metabolism. For such a
case, it can be expected that the administration of the polypeptide
of the present invention would cause 1.alpha.,25-dihydroxyvitamin D
in serum to decrease, promote proper PTH secretion in the
parathyroid glands, and provide recovery from adynamic bone
disease.
[0165] For calcification of blood vessels, involvement of
1.alpha.,25-dihydroxyvitamin D as a calcification-promoting factor
has been reported. The polypeptide of the present invention can be
used therapeutically or prophylactically against clinical
conditions associated with calcification of blood vessels, such as
arteriosclerosis due to aging, diabetic angiopathy, or
calcification of the cardiovascular system in dialysis
patients.
[0166] Calcium absorption from the intestinal tract is known to be
promoted by 1.alpha.,25-dihydroxyvitamin D in serum. The
polypeptide of the present invention can be used to correct
hypercalcemia by decreasing serum 1.alpha.,25-dihydroxyvitamin D
levels. Examples of a cause of hypercalcemia include overproduction
of PTH due to primary hyperparathyroidism, a high concentration of
1.alpha.,25-dihydroxyvitamin D along with chronic granuloma, such
as sarcoidosis or tuberculosis, or accelerated bone resorption due
to PTHrP produced by malignant tumors. In hypercalcemia which is
mainly caused not only by excessive 1.alpha.,25-dihydroxyvitamin D,
but also by excessive PTH or PTHrP, it can be expected that
administration of the polypeptide of the present invention causes
the serum 1.alpha.,25-dihydroxyvitamin D level to decrease, so as
to improve hypercalcemia. In particular, activated macrophages in
chronic granuloma, such as sarcoidosis or tuberculosis, has
1.alpha.-hydroxylase activity and excessively produces
1.alpha.,25-dihydroxyvitamin D3. It is expected that this
1.alpha.-hydroxylase activity is directly lowered by the
polypeptide of the present invention.
[0167] 1.alpha.,25-dihydroxyvitamin D is known to promote
differentiation of osteoclasts, and administration of the
polypeptide of the present invention is expected to suppress bone
resorption. In vitro, 1.alpha.,25-dihydroxyvitamin D is known to be
a strong osteoclast differentiation-inducing factor. Excessive bone
resorption by osteoclasts causes osteopenia as represented by
osteoporosis. In such diseases for which promoted bone resorption
is observed, the polypeptide of the present invention is expected
to restore bone turnover to the normal condition by transiently
lowering serum 1.alpha.,25-dihydroxyvitamin D levels. In addition
to suppressing the differentiation of osteoblasts in vitro,
1.alpha.,25-dihydroxyvitamin D has also been suggested to be a
factor of suppressing bone formation in vivo in a bone
transplantation experiment using vitamin D receptor-deficient mice.
From such view points, it can be expected that the polypeptide of
the present invention, which is capable of lowering
1.alpha.,25-dihydroxyvitamin D, is effective against metabolic bone
diseases with decreased bone mass. Further, there has been a report
confirming that administration of 24,25-dihydroxyvitamin D, which
is one of vitamin D3 metabolites, causes increased bone mass.
24,25-dihydroxyvitamin D is a product of hydroxylation of
25-hidroxyvitamin D by 24-hydroxylase. Since the polypeptide of the
present invention has an effect to enhance the expression of
24-hydroxylase gene significantly, it can be expected that
administration of this peptide causes blood 24,25-dihydroxyvitamin
D levels to increase, and to increase bone mass under clinical
conditions in bone diseases, such as osteoporosis or skeletal
dysplasia.
[0168] PTH is known to have a strong bone resorption-promoting
effect. However, bone turnover can be stimulated by intermittent
administration of PTH, and finally, an effect of increasing bone
mass can be expressed. Physiological or biological activities of
the polypeptide of the present invention that have been shown so
far include: an effect of regulating 1.alpha.,25-dihydroxyvitamin
D; effects of regulating serum calcium and serum phosphate levels;
and effect of regulating calcification. Thus, it is conceivable
that bone turnover can be regulated by allowing the polypeptide to
effectively act on bone tissues. Therefore, the polypeptide can be
expected to be effective against postmenopausal osteoporosis with
enhanced bone turnover and senile osteoporosis with lowered bone
turnover. There may be other diseases that the polypeptide of the
present invention is involved in. Such a disease can be screened by
an immunochemical assay as represented by ELISA combined with the
use of one or more antibodies against the polypeptide of the
present invention.
[0169] Accordingly, the physiological normal range of the
polypeptide of the present invention can be set, and disease groups
with the polypeptide levels which deviate from the range can be
clarified. It can be expected that the polypeptide of the present
invention is therapeutically used for diseases showing abnormally
low blood levels of the polypeptide of the present invention as
measured by the above method.
[0170] (2) Pharmaceutical Composition Containing the Antibody of
the Present Invention
[0171] The antibody of the present invention can be used as a
pharmaceutical composition for vitamin D-resistant rickets and
tumor-induced osteomalacia. The neutralizing antibody of this
polypeptide can be obtained as a polyclonal antibody or a
monoclonal antibody by the above-mentioned method for preparing
antibodies. As a method for more properly using the antibody as a
medicine, a human-type antibody or a humanized antibody can be
prepared. Hypophosphatemia and osteomalacia in tumor-induced
osteomalacia can be treated or improved by suppressing excessive
activity of the polypeptide of the present invention. It can be
expected that administration of the neutralizing antibody against
the polypeptide of the present invention improves tumor-induced
osteomalacia symptoms. Further, since hypophosphatemia-inducing
factor and calcification-suppressing factor in XLH are thought to
be equivalent to the polypeptide of the present invention, the
neutralizing antibody can also be a therapeutic agent against
vitamin D-resistant rickets including XLH.
[0172] The antibody of the present invention can be used as a
pharmaceutical composition for diseases with abnormal calcium or
phosphate metabolism, or metabolic bone diseases associated with
the presence of the polypeptide of the present invention in an
excessive amount. As described above, in chronic renal failure or
hemodialysis patients, abnormalities occur in the mechanism for
maintaining the homeostasis of calcium metabolism or phosphate
metabolism, and these abnormalities may be due to overproduction or
accumulation of the polypeptides of the present invention. It has
been shown that the polypeptide of the present invention regulates
bone metabolism. Hence, it is conceivable that metabolic bone
diseases which are caused by the presence of excessive amounts of
the polypeptide of the present invention, may also exist. In this
case, it can be expected that the clinical conditions can be
treated or improved by the antibody against the polypeptide of the
present invention.
[0173] Examples of diseases to which the antibody of the present
invention can be applied include at least one kind of bone disease,
such as osteoporosis, vitamin D-resistant rickets, renal
osteodystrophy, dialysis-associated bone diseases, osteopathy with
hypocalcification, Paget's disease and tumor-induced osteomalacia.
Here, the bone disease may be a single disease, a complication
thereof, or the bone disease complicated with any disease other
than the above diseases.
[0174] (3) Administration Protocol
[0175] A pharmaceutical composition which contains the polypeptide
of the present invention or the antibody thereof, as an active
ingredient, may contain a pharmaceutically acceptable carriers and
additives. Examples of such carriers and additives include water,
pharmaceutically acceptable organic solvent, collagen, polyvinyl
alcohol, polyvinylpyrrolidone, carboxy vinyl polymer, sodium
alginate, water-soluble dextran, sodium carboxymethyl starch,
pectin, xanthan gum, gum arabic, casein, gelatine, agar, glycerin,
propylene glycol, polyethylene glycol, vaseline, paraffin, stearyl
alcohol, stearic acid, human serum albumin, mannitol, sorbitol,
lactose, and surfactant which is a pharmaceutically acceptable
additives. The additives to be used herein are properly selected
from the above items either singly or in combination depending on
the employed dosage form of the present invention.
[0176] When the polypeptide or the antibody of the present
invention is used as a prophylactic or therapeutic agent for bone
diseases, the subject for which the polypeptide or the antibody is
used is not specifically limited.
[0177] The polypeptide of the present invention can be used as a
pharmaceutical composition which is capable of regulating calcium
metabolism, phosphate metabolism, calcification or vitamin D
metabolism in organisms.
[0178] The antibody of the present invention can be used
specifically to treat or prevent at least one kind of bone disease,
such as osteoporosis, vitamin D-resistant rickets, renal
osteodystrophy, dialysis-associated bone diseases, osteopathy with
hypocalcification, Paget's disease, tumor-induced osteomalacia, as
described above. The bone disease, for which the polypeptide or the
antibody of the present invention can be used, may be a single
disease, a complication thereof, or the bone disease complicated
with any disease other than the above diseases.
[0179] A prophylactic agent or therapeutic agent which contains the
polypeptide or the antibody of the present invention may be
administered orally or parenterally in the case of the polypeptide,
and parenterally in the case of the antibody.
[0180] When the polypeptide of the present invention is
administered orally, the dosage form to be applied thereto may be a
solid preparation, such as a tablet, granule, powder or pill, or a
liquid preparation, such as a liquid drug or syrup, or the like. In
particular, a granule and powder can be formulated into a unit dose
form, that is, a capsule. In the case of a liquid preparation, it
can be formulated into a dry product which is re-dissolved for
use.
[0181] Of these dosage forms, a solid preparation for oral
administration normally contains in its composition additives,
which are generally pharmaceutically employed, such as a binder,
excipient, lubricant, disintegrating agent or wetting agent. In
addition, a liquid preparation for oral administration normally
contains in its composition an additives, which is pharmaceutically
generally employed, such as a stabilizer, buffer, corrigent,
preservative, flavoring agent or colorant.
[0182] When the polypeptide or the antibody of the present
invention is administered parenterally, it can be formulated into
an injection, suppository or the like.
[0183] In the case of an injection, it is normally provided in a
unit dose ampule or a vessel for multiple dose, or may be in a
powdery form which is re-dissolved, when used, in an appropriate
carrier, such as sterilized water containing no pyrogenic
substance. These dosage forms normally contain in their
compositions, additives which are pharmaceutically generally
employed, such as an emulsifier or suspension. Examples of
injection procedures include drip intravenous infusion, intravenous
injection, intramuscular injection, intraperitoneal injection,
subcutaneous injection or intradermal injection. In addition, doses
differ depending on the age of the subject, route for
administration and dosage frequency, and can be varied widely.
[0184] In this case, the effective dose to be administered is a
combination of the effective dose of the peptide or the antibody of
the present invention with an appropriate diluent, and a
pharmacologically usable carrier, in the case of the polypeptide,
is 0.01 to 100 .mu.g, preferably, 0.5 to 20 .mu.g/administration/kg
of body weight. Further, in the case of the antibody, the effective
dose is 0.1 .mu.g to 2 mg, preferably, 1 to 500
.mu.g/administration/kg of body weight.
[0185] 6. Diagnostic Agent of Disease
[0186] (1) The Antibody or the Polypeptide of the Present
Invention
[0187] The antibody of the present invention is used for detection
or quantitative determination of the polypeptide of the present
invention or of the metabolites existing in blood or urine, so that
the relationship between the polypeptide of the present invention
and clinical conditions can be elucidated, and the antibody can be
applied as a diagnostic agent for associated diseases.
[0188] The term "associated disease" means a bone disease or a
disease developing at least an abnormality from among: abnormal
calcium metabolism, abnormal phosphate metabolism, abnormal
calcification and abnormal vitamin D metabolism. Examples of such a
disease include osteoporosis, vitamin D-resistant rickets, renal
osteodystrophy, dialysis-associated bone diseases, osteopathy with
hypocalcification, Paget's disease, renal failure, renal phosphate
leak, renal tubular acidosis and Fanconi's syndrome.
[0189] Methods for quantitatively determining bound molecules using
antibodies have been generalized, such as radioimmunoassay or
enzyme immunoassay. Levels of the polypeptide of the present
invention in blood or urine measured by these methods can be
indicators for new clinical judgement. For example, when a rickets
patient shows high blood levels of the polypeptide of the present
invention compared to a normal subject, XLH or ADHR can be strongly
suspected. Further, based on changes in blood levels of the
polypeptide of the present invention, prognosis of a progress into
secondary hyperparathyroidism in a chronic renal failure patient
can be made.
[0190] For tumor-induced osteomalacia, generally, it is often
difficult to find a tumor. However, the use of the antibody of the
present invention enables to establish useful diagnostic measures.
For example, when a patient with no family history of rickets or
osteomalacia shows significantly higher blood levels of the
polypeptide of the present invention compared to a normal
individual, tumor-induced osteomalacia can be suspected.
[0191] (2) DNA of the Present Invention
[0192] In the present invention, detection of abnormal DNA of the
present invention from a patient with abnormal phosphate metabolism
or abnormal calcium metabolism, or a patient with a metabolic bone
disease makes it possible to diagnose and prevent the disease.
[0193] A search for the nucleotide sequence of the DNA of the
present invention over the Genbank nucleotide sequence database
reveals that the nucleotide sequence (in the form of three
fragments) matches with a sequence of human 12p13 BAC RPCI11-388F6
(Accession No. AC008012). This fragmentation indicates that the
nucleotide sequence of the DNA of the present invention is provided
as a splicing product from a chromosome sequence. Thus it is clear
that the DNA encoding the polypeptide of the present invention
contains at least a sequence ranging from the 498.sup.th to
12966.sup.th nucleotides of a sequence represented by SEQ ID NO: 11
or partial fragments thereof. Substitution, insertion or deletion
of nucleotides within the range causes increases, decreases or
disappearance of the biological and physiological activities of the
polypeptide of the present invention. The polypeptide of the
present invention has a strong effect on phosphate metabolism,
calcium metabolism, bone metabolism, and vitamin D metabolism.
Therefore, when gene polymorphism or mutation due to substitution,
insertion, deletion and the like of partial nucleotides of the
nucleotide sequence represented by SEQ ID NO: 11 or partial regions
thereof (for example, a sequence from the 498.sup.th to the
12966.sup.th) is shown, diagnosis and prevention of a disease
containing abnormal phosphate metabolism and calcium metabolism, or
a disease developing abnormal bone metabolism, or a disease
developing abnormal vitamin D metabolism become possible.
[0194] Now, it has been reported that the gene responsible for ADHR
is present at 12p13 as a result of linkage analysis of families
having ADHR (Econs, M. J. et al., J. Clin. Invest. 100: 2653-2657,
1997). In this report, it has also been shown that the responsible
gene is present within a 18 cM range between micro satellite
markers D12S100 and D12S397. We confirmed that the location at
which the DNA of the present invention is present on the chromosome
by comparing with the reported location. The region at which the
DNA encoding the polypeptide of the present invention is present
was identical to the region at which the gene responsible for ADHR
is present. Based on the biological activities of the polypeptide
of the present invention and the location of the gene on the
chromosome, it is conceivable that the polypeptide of the present
invention is encoded by the responsible gene of ADHR. This can be
confirmed by separating cellular components from the blood of ADHR
patients, isolating chromosomal DNAs from the cellular components,
and then finding mutations in the nucleotide sequence within the
region represented by SEQ ID NO: 11. Hence, the gene having the
above nucleotide sequence region is used as a diagnostic agent for
autosomal dominant hypophosphatemic rickets, X-linked
hypophosphatemic rickets, hypophosphatemic bone disease,
osteoporosis and the like.
[0195] Between exon 1 and exon 2 of the DNA region encoding the
polypeptide of the present invention that we have specified, STS
sequence, which has been registered at NCBI Genbank under Accession
No. G19259, is present. This marker is thought to be very important
in studying the relationship between the DNA and hereditary
characters.
[0196] The present invention will bring a big change in
understanding conventional calcium metabolism, phosphate
metabolism, bone metabolism and vitamin D metabolism. According to
the present invention, there is provided a delayed progress to the
stage of hemodialysis in chronic renal failure, or a new
therapeutic method and diagnostic method for phosphate
metabolism-associated and calcium metabolism-associated diseases,
and metabolic bone diseases. Further, the present invention is also
useful to improve or support existing therapeutic methods.
BRIEF DESCRIPTION OF DRAWINGS
[0197] FIG. 1 includes photographs showing the amplification
products analyzed using agarose electrophoresis. To study the tumor
specificity of OST311, PCR was performed using, as templates,
first-strand cDNA extracted from tumor tissues and first-strand
cDNA extracted from control bone tissues, and using OST311 specific
primers represented by SEQ ID NOS: 22 and 23, and G3PDH specific
primers represented by SEQ ID NOS: 26 and 27.
[0198] FIG. 2 is a photograph showing that recombinant OST311 was
detected by performing Western blotting for elution fractions which
had been prepared by subjecting recombinant OST311 to affinity
purification using nickel resin, and then isolated and purified
using strong cation-exchange resin SP-5PW.
[0199] FIG. 3A shows the results of predicting sites of the
polypeptide having the amino acid sequence represented by SEQ ID
NO: 2, which are appropriate for preparation of a peptide antibody,
using a computing function of MacVector version 6.5.1.
[0200] FIG. 3B shows the results of predicting the degree of
hydrophobicity of the polypeptide having the amino acid sequence
represented by SEQ ID NO: 2 using the computing function of
MacVector version 6.5.1.
[0201] FIG. 4 shows time-course changes in average body weight for
31 days after transplantation of CHO-OST311H cells between a non
tumor-bearing group (a line in the graph indicated with avr.-) and
a CHO-OST311H cells-transplanted tumor-bearing group (a line in the
graph indicated with avr.+).
[0202] FIG. 5 includes X-ray pictures showing the whole skeletal
soft roentgenogram of a control CHO ras clone-1 cells-transplanted
tumor-bearing individual, a CHO-OST190H cells-transplanted
tumor-bearing individual, and a CHO-OST311H cells-transplanted
tumor-bearing individual.
[0203] FIG. 6 shows the results of alignment of amino acid sequence
between human OST311 polypeptide and mouse OST311 polypeptide.
[0204] FIG. 7 shows the results of measuring serum phosphate
levels, serum calcium levels and serum alkaline phosphatase
activities of a non tumor-bearing group (n=6), a CHO ras clone-1
tumor-bearing group (n=10), a CHO-OST190H tumor-bearing group
(n=10), and a CHO-OST311H tumor-bearing group-1 (n=6), and a
CHO-OST311H tumor-bearing group-2 (n=6). Blood was collected from
the heart of each individual on days 44 to 46.
[0205] FIG. 8 includes photographs showing comparison of expression
levels of sodium-phosphate cotransporter (NaPi-7) as measured by
the Western blotting method. Specifically, brush border membranes
of proximal tubular epithelial cells were prepared from the kidneys
excised from the CHO-OST311H tumor-bearing individuals, and non
tumor-bearing individuals, and then the expression levels of NaPi-7
were compared by the Western blotting method.
[0206] FIG. 9A includes photographs showing changes in mRNA levels,
as detected by Northern blotting, of renal sodium-phosphate
cotransporters (NaPi-7, NPT-1), . The kidneys were collected from
the mice sacrificed on days 44 to 46 after tumor
transplantation.
[0207] FIG. 9B includes photographs showing changes in mRNA levels,
as detected by Northern blotting, of a sodium-phosphate
cotransporter (NaPi-IIb) in the small intestines of the mice. The
small intestines were collected from the mice sacrificed on days 44
to 46 after tumor transplantation.
[0208] FIG. 9C includes photographs showing changes in mRNA levels,
as detected by Northern blotting, of vitamin D-metabolizing enzymes
(1.alpha.OHase, 24OHase) in the kidneys of the mice. The kidneys
were collected from the mice sacrificed on days 44 to 46 after
tumor transplantation.
[0209] FIG. 10 shows X-ray pictures showing the femora collected
from the mice that were sacrificed on days 44 to 46 after tumor
transplantation.
[0210] FIG. 11A includes graphs showing comparisons of serum
phosphate levels and serum calcium levels, on day 2 after
transplantation, among PBS, CHO ras clone-1 cells, and CHO-OST311H
cells-transplanted nude mice (6-week-old, BALB/c, male).
[0211] Each value is shown in terms of mean.+-.standard
deviation.
[0212] FIG. 11B includes graphs showing comparisons of serum
phosphate levels and serum calcium levels, on day 6 after
transplantation, among PBS, CHO ras clone-1 cells, and CHO-OST311H
cells-transplanted nude mice (6-week-old, BALB/c, male).
[0213] Each value is shown in terms of mean.+-.standard
deviation.
[0214] FIG. 12 includes photographs showing the results obtained by
purifying the culture supernatant of CHO-OST311H cells and
subjecting the elution fractions to Western blotting using
anti-His6 antibody and antiOST311 peptide antibody (311-114). The
left panel shows the detection of 311:26-251, the center panel 311:
25-179, and the right panel 311: 180-251. Fractions shown with "*"
(placed on the upper portion of the gel photographs) were used for
a single dose examination conducted on normal mice.
[0215] FIG. 13 includes photographs showing undemineralized slices
stained with Villanueva-Goldner. The undemineralized sections were
of the proximal metaphysis of tibia extracted from CHO-OST311H
cells-transplanted mice and non tumor-bearing mice.
[0216] FIG. 14 includes photographs showing the results of
performing Northern blotting for vitamin D-metabolizing enzyme gene
products in the kidneys excised from CHO-OST311H cells-transplanted
mice and control mice.
[0217] FIG. 15A shows the time schedule of experiment 1 wherein
CHO-producing recombinant OST311H full-length protein was
administered to normal mice. FIG. 15B includes graphs showing serum
phosphate levels at each time point of blood collection, and FIG.
15C includes graphs showing serum calcium levels at the same points
in time.
[0218] FIG. 16A shows the time schedule of experiment 2 wherein
CHO-producing recombinant OST311H full-length protein was
administered to normal mice. FIG. 16B includes graphs showing serum
phosphate levels at each time point of blood collection, and FIG.
16C includes graphs showing serum calcium levels at the same points
in time.
[0219] FIG. 17 is a photograph showing the recombinant protein
detected in a culture supernatant when the culture supernatant of
mutant recombinant OST311RQH-producing CHO-OST311RQH cells and
OST311RQH/pEAK rapid cells were subjected to Western blotting.
[0220] FIG. 18A shows the time schedule of an experiment wherein
mutant recombinant OST311RQH was administered to normal mice. FIG.
18B includes graphs showing serum phosphate levels at each time
point of blood collection, and FIG. 18C includes graphs showing
serum calcium levels at the same points in time.
[0221] FIG. 19A shows serum phosphate levels on day 2 after
transplantation in a CHO-OST311RQH cells-transplanted experiment.
FIG. 19B shows serum calcium levels in the same experiment.
[0222] FIG. 20 includes photographs showing recombinant OST311H in
serum-free culture supernatant of CHO-OST311H cells detected by
Western blotting using anti-OST311 partial peptide rabbit
anti-serum.
[0223] FIG. 21A is a table showing recombinant OST311-detected
levels in each combination of 6 types of anti-OST311 peptide
polyclonal antibodies, when sandwich ELISA was constructed using
these polyclonal antibodies.
[0224] FIG. 21B is a graph of plotting the relations between
concentrations of purified recombinant OST311H and measured values
corresponding thereto that were obtained using ELISA system
combined with the use of 311-48 antibody or 311-180 antibody as an
immobilized antibody, and 311-148 antibody as an antibody for
detection.
[0225] FIG. 22A shows the expression of renal NaPi-7 analyzed by
Western blotting at 1, 3 and 8 hours after administration of
recombinant OST311 protein or vehicle to mice. FIG. 22B shows the
expression of NaPi-7 analyzed by Northern blotting using total RNA
of kidney following the similar treatment.
[0226] FIG. 23 shows the changes in serum 1,25-dihydroxyvitamin D3
levels at 1, 3, and 8 hours after administration of recombinant
OST311 protein or a vehicle to mice.
[0227] FIG. 24 shows the expression of 25-hydroxyvitamin
D-1-.alpha.-hydroxylase (1.alpha.OHase) or 25-hydroxyvitamin
D-24-hydroxylase (24OHase) gene analyzed by Northern blotting using
total RNA of kidney at 1, 3 and 8 hours after administration of
recombinant OST311 protein or a vehicle to mice.
[0228] FIG. 25 shows mean serum phosphate levels in each group,
when the mean blood serum phosphate level on day 3 after cell
transplantation in a CHO-ras clone-1 cells-transplanted group is
considered as 100%.
[0229] FIG. 26 shows the nucleotide sequence and amino acid
sequence of recombinant His-OST311 encoded by plasmid OST311/pET28,
and the DNA sequence and amino acid sequence of recombinant
MK-OST311 encoded by plasmid pET22-MK-OST311.
[0230] FIG. 27 shows an elution pattern when recombinant refolded
His-OST311 was subjected to HPLC purification using cation-exchange
column SP-5PW (TOSOH, Japan).
[0231] FIG. 28 shows an elution pattern when recombinant refolded
MK-OST311 was subjected to HPLC purification using cation-exchange
column SP-5PW (TOSOH, Japan).
[0232] FIG. 29 shows an elution pattern when PEGylated recombinant
MK-OST311 was subjected to HPLC purification using cation-exchange
column SP-5PW (TOSOH, Japan).
[0233] FIG. 30 includes graphs showing serum phosphate levels at 8
or 9 hours after single administration of (A) Escherichia
coli-producing His-OST311 recombinant or (B) PEGylated MK-OST311
recombinant.
[0234] FIG. 31 shows the results of analysis by the Northern
blotting method on changes in the expression of vitamin
D-metabolizing enzyme gene in the kidney at 1 and 4 hours after
single administration of Escherichia coli-producing His-OST311
recombinant.
[0235] FIG. 32 shows changes in serum 1,25-dihydroxyvitamin D3
levels at 1, 4 and 9 hours after single administration of
Escherichia coli-producing His-OST311 recombinant.
[0236] FIG. 33 shows a mutant OST311 recombinant detected by
Western blotting, when mutation was introduced into amino acid Nos.
174 to 180 of OST311, and then the gene was expressed in pEAK cells
so as to secrete the mutant OST311 recombinant in the cell
supernatant.
BEST MODE FOR CARRYING OUT THE INVENTION
[0237] The present invention will be described more specifically by
the following examples. These examples are not intended to limit
the scope of the present invention.
EXAMPLE 1
Construction of Human Tumor-Induced Osteomalacia-Derived Tumor cDNA
Library
[0238] Tumor tissues frozen by liquid nitrogen were homogenized in
5 ml of ISOGEN (NIPPON GENE, Japan) solvent, and then approximately
0.13 mg of total RNA was prepared according to the attached
manufacturer's manual. cDNA was synthesized from 1.5 .mu.l of the
total RNA using a SMART cDNA library preparation kit (CLONTECH,
USA) according to the attached manufacturer's manual. Hereinafter,
this cDNA is denoted as cDNA#2. EcoR I adapter was ligated to this
cDNA#2, and then inserted into ?ZAPII phage vector (STRATAGENE,
USA) that had been previously digested with a restriction enzyme
EcoR I. Using a Gigapack III Gold phage packaging kit (STRATAGENE,
USA), a tumor-induced osteomalacia tumor phage library was
constructed according to the attached manufacturer's manual. The
obtained library contained a total of approximately 600,000
independent clones. Further, the above phage library was allowed to
infect Escherichia coli strain XLI-Blue MRF', and then poured onto
20 petri dishes (15 cm). The petri dishes were incubated at
37.degree. C. for 10 hours for plaque formation. All the plaques
were extracted into an SM buffer, so that tumor-induced
osteomalacia tumor cDNA phage library was constructed.
EXAMPLE 2
Implementation for Positive Screening of Tumor cDNA Outline
[0239] The fact that tumor-induced osteomalacia is curable by
surgically excising a tumor suggests a possibility of high and
specific expression of the causative gene in tumors. In addition,
it has been reported so far that tumor-induced osteomalacia tumors
are often constituted of mesoblastic, in particular mesenchymal
cells. Therefore, it is necessary to identify a gene group that
expressed lowly in normal mesoblast-derived tissues, but expressed
specifically and highly only in tumor tissues. Hence, as described
below, positive screening to which a cDNA subtraction technique was
applied was performed. Subtraction of tumor tissue-derived cDNA and
cDNA isolated from a bone tissue as a control was performed, so as
to enrich a gene group that specifically and highly expressed only
in tumor tissues, but not expressed in bone tissues. Hybridization
was performed for tumor cDNA phage library using the subtracted
cDNA group as a probe, thereby obtaining gene fragments
specifically expressed in tumors.
[0240] (1) Construction of Control Human Bone Tissue cDNA
[0241] Human bone tissues frozen by liquid nitrogen were
homogenized in 5 ml of ISOGEN (NIPPON GENE, Japan) solvent, and
then approximately 0.011 mg of total RNA was prepared according to
the attached manufacturer's manual. cDNA was synthesized from 3
.mu.l of the total RNA using SMART cDNA library preparation kit
(CLONTECH, USA) according to the attached manufacturer's manual.
Hereinafter, the thus obtained cDNA is referred to as cDNA # 4.
[0242] (2) Subtraction of Tumor-Induced Osteomalacia Tumor cDNA and
Control Bone Tissue cDNA
[0243] To enrich a gene highly expressed in cDNA#2 described in
Example 1, hybridization of cDNA#2 and cDNA#4 described in Example
2(1) was performed using a PCR-Select cDNA subtraction kit
(CLONTECH, USA) according to the attached manufacturer's manual,
thereby subtracting gene fragments contained in cDNA#4 from cDNA#2.
Then, the subtracted cDNA#2 was amplified by PCR according to the
attached manufacturer's manual, so that a subtracted cDNA group (A)
was obtained.
[0244] On the other hand, because of characteristics of the
subtraction kit, such that its hybridization process is performed
only twice, which is less than that of common techniques, it is
difficult by this kit to completely subtract genes existing in many
numbers in both subjects. Accordingly, when hybridization of
tumor-induced osteomalacia tumor cDNA library with only subtracted
cDNA group (A) as a probe, is performed, genes that cannot be
subtracted completely are also obtained as positive clones. Thus,
cDNA#2 was subtracted from cDNA#4 described in Example 2 (1) by the
same method, and then the subtracted cDNA#4 was amplified by PCR,
thereby preparing a subtracted cDNA group (B) as a control probe.
Hybridization of tumor cDNA library respectively with the
subtracted cDNA group (B) and the previously described subtracted
cDNA group (A), and comparison of both signals make it possible to
isolate gene fragments specifically contained in tumor-induced
osteomalacia tumors.
[0245] (3) Differential Hybridization of Tumor-Induced Osteomalacia
Tumor cDNA Library
[0246] After infecting Escherichia coli strain XLI-Blue with the
tumor-induced osteomalacia tumor cDNA phage library described in
Example 1, the infected E. coli was inoculated again so as to form
3,000 plaques per petri dish (15 cm), and then incubated at
37.degree. C. for 8 hours. Then, plaques on each petri dish were
transferred to two Hybond N+ (Amersham Pharmacia Biotech, USA)
nylon filters. The nylon filters to which plaques are transferred
were subjected to DNA immobilization treatment according to the
attached manufacturer's manual, and then screening was performed
using the subtracted cDNA(A) described in Example 2(2) and the
subtracted cDNA(B) as probes, respectively.
[0247] Probe labeling, hybridization and signal detection were
performed using Alphos Direct system (Amersham Pharmacia Biotech,
USA) according to the attached manufacturer's manual. The
subtracted cDNA(A) and the subtracted cDNA(B) described in Example
2(2) were used as probes at 100 ng each, and then the probes were
labeled with fluorescence according to protocols. The probes were
respectively added to 50 ml of hybridization buffer supplied with
Alphos Direct system, and at the same time, 2 sets of each of the 8
nylon filters which plaques had been transferred to were
respectively hybridized and washed according to protocols. After
washing, the nylon filters were subjected to luminescence reaction,
exposed to ECL film (Amersham Pharmacia Biotech, USA) for 2 hours,
developed with an automatic processor (FUJI FILM, Japan), and then
the results were analyzed.
[0248] As a result, independent plaques placed in a portion which
were strongly burnt after exposure when the subtracted cDNA (A) was
used as a probe, but not burnt when the subtracted cDNA (B) was
used as a probe were visually selected, scraped off from the petri
dish, and then suspended in 0.5 ml SM buffer. The suspensions were
allowed to stand at 4.degree. C. for 2 hours or more, thereby
extracting phages.
[0249] (4) Nucleotide Sequence Analysis of Positive Clone
[0250] Using 0.5 .mu.l of the phage solution obtained in Example
2(3) containing positive clones as a template, T7 primer
(TAATACGACTCACTATAGGG) (SEQ ID NO: 24) and T3 primer
(ATTAACCCTCACTAAAGGGA) (SEQ ID NO: 25) that were internal sequences
of the phage vector, and LA-taq polymerase (TAKARA SHUZO, Japan),
PCR was performed for 35 cycles, each cycle (process) consisting of
96.degree. C. for 30 seconds, 55.degree. C. for 30 seconds, and
72.degree. C. for 30 seconds. The PCR products were subjected to
0.8% agarose gel electrophoresis. Clones for which a clear single
band was observed were sequenced by ABI377 DNA sequencer (PE
Applied systems, USA) using PCR amplification fragments as
templates.
[0251] When two clear bands were observed, PCR products were
respectively extracted from each gel portion of the corresponding
bands using QIAquick Gel Extraction kit (QIAGEN, Germany), and then
sequenced by ABI377 DNA sequencer.
[0252] As a result of differential hybridization for 341,000
plaques of tumor-inducced osteomalacia phage library, 456 positive
plaques were identified, and the nucleotide sequences of all of
these plaques were determined.
EXAMPLE 3
Narrowing Down the Candidate Genes of Human
Hypophosphatemia-Inducing Factor
[0253] Homology search for the sequence information of 456 positive
clones obtained in Example 2 against nucleotide sequences
registered at Genbank, the nucleotide sequence database provided by
NCBI was performed. As a result, a group of genes listed on Table 1
was obtained as existing genes that had appeared at high
frequencies. Further, as a result of database search, there are 100
clones of unknown gene fragments of which biological activities
were not known. As to nucleotide sequence information of these
unknown gene fragments, overlaps among clones was further extracted
as frequency information. Among them, the most frequently
overlapping gene, OST311 sequence consisting of 7 clones
sequentially forming one contig, was obtained. The nucleotide
sequence obtained at this time ranged from nucleotide Nos. 1522 to
2770 of SEQ ID NO: 1. When a search for the gene fragment of OST311
was performed over the existing database, it was not registered as
cDNA or EST, and corresponded only with a genomic sequence. The
relevant genomic sequence is AC008012, and it has already been
reported to be located at 12p13 on the chromosome. However, a
region encoding a protein (ORF) was not found within the obtained
sequence, so that the sequence was predicted to correspond to a
3'-untranslated region. Hence, cloning of full-length cDNA from
tumor-induced osteomalacia tumor cDNA library was performed. In
addition, the ORF-predicting function of DNASIS-Mac version 3.7 was
used to predict ORF.
2TABLE 1 Clone ID Frequency Description OST 131 236 Dentin matrix
protein-1(DMP1) OST 1 35 Heat shock protein-90 (HSP90) OST 2 13
Osteopontin OST 311 7 Unknown/genomic DNA 12p13 OST 1001 4 CD44
antigen OST 584 3 Fibronectin OST 666 3 Translational regulatory
tumor protein OST 133 2 Beta 2 microglobulin OST 837 2 Fibroblast
growth factor (FGF) OST 562 2 Annexin II/lipocortin II OST 1002 2
Cytochrome c oxygenase subunit 2 OST 1003 2 Stathmin OST 1004 2
Unknown OST 903 2 Unknown
EXAMPLE 4
Cloning of Full-Length OST311
[0254] Based on the OST311 sequence obtained in Example 3, the
following primers were synthesized. Then, PCR was performed, using
a phage solution of tumor-induced osteomalacia tumor cDNA library
as a template, for 35 cycles, each cycle (process) consisting of
96.degree. C. for 30 seconds, 55.degree. C. for 30 seconds, and
72.degree. C. for 30 seconds.
3 311-U65: TTCTGTCTCGCTGTCTCCC (SEQ ID NO: 12) 311-L344:
CCCCTTCCCAGTCACATTT (SEQ ID NO: 13)
[0255] PCR products were subjected to 2% agarose gel
electrophoresis, amplification of PCR products of predicted sizes
was confirmed, and then the PCR products were purified using
MicroSpin column S-300 HR (Amersham Pharmacia Biotech, USA). The
resulting PCR products were fluorescence-labeled using Alphos
Direct system (Amersham Pharmacia Biotech, USA) according to the
attached manufacturer's manual. Then, plaque hybridization for
20,000 clones of tumor-induced osteomalacia tumor cDNA library was
performed using these labeled products as probes.
[0256] The thus obtained 40 positive clones were amplified by PCR
in the manner as described in Example 2(4) using T7 and T3. Based
on the nucleotide sequence of the resulting PCR product, a primer
311-L296 (SEQ ID NO: 14, GGGGCATCTAACATAAATGC) was synthesized.
Again, plaque hybridization for 20,000 clones of tumor-induced
osteomalacia tumor cDNA library was performed using as probes the
PCR products amplified using 311-U65 (SEQ ID NO: 12) and 311-L344
(SEQ ID NO: 13) primers. For 62 positive clones, the nucleotide
sequence of a PCR product amplified using T7 and 311-L296 (SEQ ID
NO: 14) primers was determined. The determined sequence was linked
to the nucleotide sequences that had been determined so far. Thus,
the nucleotide sequence represented by SEQ ID NO: 1 was obtained.
It became clear that ORF of OST311 starts from an initiation codon
located at nucleotide No. 133 of SEQ ID NO: 1. Furthermore, the
following primers were synthesized to finally determine the
sequence of ORF.
4 311-F1: AGCCACTCAGAGCAGGGCAC (SEQ ID NO: 15, Nucleotide Nos. 112
to 131) 311-F2: GGTGGCGGCCGTCTAGAACTA (SEQ ID NO: 16, Vector
sequence) 311-F3: TCAGTCTGGGCCGGGCGAAGA (SEQ ID NO: 17, Nucleotide
Nos. 539 to 559) 311-L1: CACGTTCAAGGGGTCCCGCT (SEQ ID NO: 18,
Nucleotide Nos. 689 to 708) 311-L3: TCTGAAATCCATGCAGAGGT (SEQ ID
NO: 19, Nucleotide Nos. 410 to 429) 311-L5: GGGAGGCATTGGGATAGGCTC
(SEQ ID NO: 20, Nucleotide Nos. 200 to 220) 311-L6:
CTAGATGAACTTGGCGAAGGG (SEQ ID NO: 21, Nucleotide Nos. 868 to
888)
[0257] Using 311-F2 (SEQ ID NO: 16) and 311-L6 (SEQ ID NO: 21)
primers, tumor-induced osteomalacia tumor cDNA library as a
template, and Pyrobest DNA polymerase (TAKARA SHUZO, Japan), PCR
was performed for 35 cycles, each cycle (process) consisting of
96.degree. C. for 30 seconds, 55.degree. C. for 30 seconds and
72.degree. C. for 30 seconds.
[0258] When the PCR products were subjected to 2% agarose gel
electrophoresis, a single fragment of approximately 980 nucleotide
pairs was confirmed. Then, the nucleotide sequence of the amplified
fragment was determined using the above primers (SEQ ID NOS. 15 to
21). The thus determined ORF region (SEQ ID NO: 1) encoding the
polypeptide represented by SEQ ID NO: 2 was placed between the
initiation codon ATG located at nucleotide No. 133 and the
termination codon TAG located at nucleotide No. 886 of SEQ ID NO:
1.
EXAMPLE 5
Specificity of OST311 Against Tumor-Induced Osteomalacia Tumor
[0259] To study the tumor specificity of OST311, PCR was performed
for 35 cycles using as templates first-strand cDNAs extracted from
tumor tissues and from control bone tissues, and using OST311
specific primers shown below (SEQ ID NOS: 22 and 23). Each PCR
cycle is a process consisting of 96.degree. C. for 30 seconds,
55.degree. C. for 30 seconds and 72.degree. C. for 30 seconds. In
addition, DMSO was added into both reaction solutions to have a
final concentration of 2%, and LA-taq DNA polymerase (TAKARA SHUZO,
Japan) was used as an enzyme. Further, as an internal standard, PCR
was performed under similar conditions using primers specific to
G3PDH (FW: ACCACAGTCCATGCCATCAC (SEQ ID NO: 26), RV:
TCCACCACCCTGTTGCTGTA (SEQ ID NO: 27)).
5 311F1EcoRI: CCGGAATTCAGCCACTCAGAGCAGGGCACG (SEQ ID NO: 22)
311LHisNot: ATAAGAATGCGGCCGCTCAATGGTGATGGTGATGATGGATGAACTTGGCGA- A
(SEQ ID NO: 23)
[0260] As shown in FIG. 1, these PCR products were subjected to 2%
agarose gel electrophoresis. When OST311 primers were used, PCR
products with the predicted size were observed only when the tumor
tissue was used as a template. In contrast, when G3PDH primers were
used, PCR products with the predicted size were observed at the
same level in both cases of the tumor tissues and control bone
tissues. From these results, tumor tissue-specific expression of
OST311 was confirmed.
EXAMPLE 6
Isolation of the CHO Cells Stably Expressing OST311
(1) Construction of OST311 Expression Vector
[0261] Using 311F1EcoRI (SEQ ID NO: 22) and 311LHisNot (SEQ ID NO:
23) primers shown in Example 5, and tumor-induced osteomalacia
tumor cDNA library as a template, PCR was performed for 35 cycles,
each cycle (process) consisting of 96.degree. C. for 30 seconds,
55.degree. C. for 30 seconds and 72.degree. C. for 30 seconds. In
addition, DMSO was added to the reaction solution to have a final
concentration of 2%, and LA-taq DNA polymerase (TAKARA SHUZO,
Japan) was used as an enzyme. 311F1EcoRI primer was annealed to
nucleotide No. 111 of SEQ ID NO: 1 located upstream from Kozak
sequence, and 311LHisNot primer was annealed to nucleotide No. 871
of SEQ ID NO: 1. A region encoding the full-length polypeptide
represented by SEQ ID NO: 2 can be amplified by performing PCR
using both primers. In addition, 311LHisNot primer contains a
nucleotide sequence that adds six histidine residues after the
amino acid No. 251 of SEQ ID NO: 2 and also adds a termination
codon after the last histidine codon. Thus, the translated
recombinant protein has a His6 tag sequence at the C-terminus, so
that it is useful for recognition of a recombinant by an antibody,
and purification of the recombinant using nickel resin.
[0262] After being digested with restriction enzymes EcoR I and Not
I, the PCR product was ligated to plasmid vector pcDNA3.1Zeo
(INVITROGEN, USA) for expression in animal cells that had been
digested with EcoR I and Not I similarly. The thus obtained
recombinant vector was introduced into Escherichia coli strain
DH5.alpha.. E. coli was cultured in 3 ml of LB medium containing
100 mg/ml ampicillin, and then the plasmid was purified using GFX
plasmid purification kit (Amersham Pharmacia Biotech, USA). The
sequence of the inserted gene was determined by a standard method.
Thus, it was confirmed that the sequence was identical to an
equivalent portion of SEQ ID NO: 1, and a nucleotide sequence
encoding the His6 tag sequence had been added immediately before
the termination codon.
[0263] (2) Isolation of the CHO Cells which Stably Express
OST311
[0264] Approximately 20 .mu.g of the plasmid, to which OST311 ORF
portion prepared in Example 6 (1) had been inserted, was digested
with a restriction enzyme Fsp I so as to cleave a site of the
ampicillin resistance gene within the vector. Then the cleaved
vector was subjected to ethanol precipitation, and then dissolved
in 10 .mu.l of ultrapure water. Subsequently, the total volume of
the solution was introduced into host cells by an electroporation
using Gene Pulser II (Bio Rad, USA). CHO Ras clone-1 cells
(Shirahata, S., Biosci. Biotech. Biochem, 59(2): 345-347, 1995)
were used as host cells. CHO Ras clone-1 was cultured in a 75
cm.sup.2 culture flask (CORNING USA) containing MEM.alpha. medium
supplemented with 10% FCS at 37.degree. C. under 5% CO.sub.2 and
100% humidity until the cells grew to cover approximately 90% of
culturing area. Then, adherent cells were removed by trypsin
treatment, so that approximately 1.times.10.sup.7 cells were
obtained. The obtained cells were resuspended in 0.8 ml of PBS,
mixed with the plasmid digested with Fsp I, and then cooled on ice
for 10 minutes. The cells containing the plasmid were transferred
into the cuvette four millimeter in width. After electric pulse was
applied at set values (0.25 kV and 975 .mu.F), the cuvette was
cooled again for 10 minutes. The gene-transferred cells were
cultured in 10% FCS-containing MEM.alpha. medium for 24 hours.
Then, Zeosin (INVITROGEN, USA) was added to the culture medium to
have a final concentration of 0.5 mg/ml, and then further cultured
for 1 week. Subsequently, for cloning cells showing drug resistance
ability, the cells were re-inoculated in a 96-well plate (CORNING,
USA) to 0.2 cells/well by a limited dilution method, and then
cultured in the presence of zeosin with a final concentration of
0.3 mg/ml for about 3 weeks, thereby obtaining 35 clones of the
drug-resistant strain.
[0265] (3) Confirmation of Production of Recombinant by CHO Cells
which Stably Express OST311
[0266] For 35 clones showing drug resistance, the presence of
recombinant OST311 in the conditioned medium was confirmed by the
Western blotting method.
[0267] 0.2 ml of collected the conditioned medium was concentrated
to about 40 to 50 .mu.l using an ultra-free MC M.W. 5,000 cut
membrane system (MILLIPORE, USA). 10 .mu.l of a sample buffer
containing 1 M Tris-Cl pH6.8, 5% SDS, 50% glycerol, and 100 mM DTT
was added to the concentrate, and then it was heated at 95.degree.
C. for 5 minutes. Then the protein in the conditioned medium was
separated by polyacrylamide electrophoresis with a 10 to 20%
gradient. Thereafter, the protein in the gel was transferred to
Immobilon PVDF membrane (MILLIPORE, USA) using a semi-dry blotting
system (Owl Separation Systems, USA). This PVDF membrane was
incubated with anti-His (C-terminus) antibody (INVITROGEN, USA)
that had been diluted {fraction (1/5000)} in TTBS buffer (Sigma,
USA) at room temperature for 1 hour. Then, the membrane was exposed
to film for 5 minutes using ECL system (Amersham Pharmacia Biotech,
USA), and developed using an automatic processor (FUJI FILM,
Japan). As a result, clone # 20, for which the most intense signals
had been observed at approximately 32 kDa and 10 kDa, was isolated.
Hereinafter, # 20 cell was named CHO-OST311H, and deposited with
National Institute of Advanced Industrial Science and Technology,
International Patent Organism Depositary (Higashi, Tsukuba-shi,
Ibaraki 1-1-1) (Accession No. FERM BP-7273).
EXAMPLE 7
Measurement of Inhibitory Activity of Phosphate Uptake by
CHO-OST311H Conditioned Medium
[0268] CHO-OST311H was cultured in a 225 cm.sup.2 culture flask
(CORNING, USA) containing MEM .alpha. medium supplemented with 10%
FCS at 37.degree. C. and under 5% CO.sub.2, and 100% humidity until
the cells grew to cover about 80% of the flask area. Then, the
medium was replaced with 30 ml of serum-free medium CHO-S-SFM II
(LIFE TECHNOLOGY, USA). 48 hours later, the conditioned medium was
collected. The conditioned medium was centrifuged at 1,200 g for 5
minutes to remove suspended cells and the like, and then filtered
using a Minisart-plus 0.22 .mu.m filter (Sartorius, Germany).
[0269] Using this conditioned medium, the effect on phosphate
uptake activity of a human renal proximal tubular cell line (CL-8
cells) was examined. The human renal proximal tubular cell line was
cultured under 5% CO.sub.2 and 100% humidity at 37.degree. C. in
DMEM medium containing 10% FCS (LIFE TECHNOLOGY). To measure
phosphate uptake activity, the human renal proximal tubular cell
line was first cultured in DMEM medium containing 10% FCS in a
48-well plate (CORNING, USA). When the cells grew to cover the
entire bottom surface of the plate at 3 days after the start of
culturing, the culture medium was replaced with 200 .mu.l of
serum-free medium CHO-S-SFM II (LIFE TECHNOLOGY, USA), followed by
further culturing for 20 to 24 hours. Using the cells in this
state, the following experiments for measuring phosphate uptake
activity (Experiments 1 and 2) were conducted.
[0270] (1) Experiment 1:
[0271] CHO-S-SFM II medium was removed, and then 200 .mu.l of the
above conditioned medium of CHO-OST311H cells prepared in CHO-S-SFM
II medium was added per well. At this time as control wells, 3
wells containing media which had not been replaced with CHO-S-SFM
II, and 3 wells containing media which had been supplemented
respectively with 200 .mu.l of the conditioned medium of
CHO-OST190H cells prepared in a manner similar to that for
CHO-OST311H were prepared. The above-described CHO-OST190H cells
are recombinant cells that have been prepared by introducing
OST190H, which we have cloned in a manner similar to that for
CHO-OST311H cells, into CHO ras clone-1 such that OST190H can be
expressed. Similar to OST311H, expressed CHO-OST190H contains a
His6 tag sequence added to the C-terminus of a polypeptide, which
is same as the polypeptide named MEPE as reported by Rowe, P. S. N.
et al, Genomics 67:54-68, 2000. After each sample was added,
incubation was further performed in a CO.sub.2-incubator for 26
hours, phosphate uptake activity of the cells in each well was
measured by the following method for measuring phosphate uptake
activity.
[0272] (2) Experiment 2:
[0273] 100 .mu.l of cultured midium was removed from 200 .mu.l of
the culturing CHO-S-SFM II medium. Into these culture, 100 .mu.l of
the conditioned medium of CHO ras clone-1 cells was added
respectively to 3 wells, and 100 .mu.l of the conditioned medium of
CHO-OST311H cells was added respectively to 3 wells. Then,
incubation was performed in a CO.sub.2 incubator for 24 hours.
Subsequently, phosphate uptake activity of the cells in each well
was measured by the following method for measuring phosphate
transport.
[0274] Measurement Method of Phosphate Uptake Activity:
[0275] After the addition of the conditioned medium and incubation,
the cells were washed with a buffer containing no phosphoric acid
(150 mM NaCl, 1 mM CaCl.sub.2, 1.8 mM MgSO.sub.4, 10 mM HEPES,
pH7.4), and then incubated in the same solution at room temperature
for 10 minutes. The solution was removed, and then an assay
solution, which had been prepared by adding radioactive
KH.sub.2PO.sub.4 (NEN) to have 0.105 mM to the buffer, was added.
The solution was subjected to incubation at room temperature for 10
minutes. After incubation, the cells were washed three times with a
stop solution which had been ice-cooled immediately after the
removal of the assay solution (150 mM choline chloride, 1 mM
CaCl.sub.2, 1.8 mM MgSO.sub.4, 10 mM HEPES, pH7.4). This washing
solution was removed, 80 .mu.l of 0.2 N NaOH was added to the
cells, and then the solution was incubated at room temperature for
10 minutes, so that the cells were lysed. To measure radioactivity
in the cell lysis solution, the solution was transferred to
ReadyCap (Beckman), dried at 50.degree. C., and then placed in
glass vial. Then, radioactivity was measured using a scintillation
counter (Wallac1410, Pharmacia). Phosphate uptake activity in each
experiment is shown in Table 2 wherein mean uptake in the control
not supplemented with conditioned medium is considered as 100%. The
conditioned medium of CHO-OST311H cells significantly suppressed
phosphate uptake activity of the human epithelial cells of renal
proximal convoluted tubules.
6TABLE 2 OST311 activity against phosphate uptake by renal tubular
epithelial cells Experiment 1 Phosphate uptake Sample activity .+-.
SEM t-test Not supplemented 100 .+-. 1.9 -- CHO-OST190H 103.8 .+-.
0.9 Not significant CHO-OST311H 87.4 .+-. 0.2 p < 0.01
Experiment 2 Phosphate uptake activity .+-. SEM t-test Conditioned
media of CHO 100 .+-. 1.5 -- CHO-OST311H 87.2 .+-. 1.2 p <
0.01
EXAMPLE 8
Partial Purification of Recombinant OST311 from CHO-OST311H
Conditioned Medium
[0276] Recombinant OST was partially purified from the conditioned
medium prepared in the manner described in Example 7 by the
following method. Processes 1) to 4) were performed in a
chromatochamber at 4.degree. C.
[0277] 1) A disposable polypropylene column was filled with ProBond
nickel resin (INVITROGEN, USA) to have a bed volume of 3 ml, and
then the column was washed and equilibrated with 30 ml of buffer 1
(Table 3).
[0278] 2) 120 ml of the conditioned medium prepared in the manner
described in Example 7 was applied to the above nickel column by
free-fall, allowing recombinant OST311 to bind.
[0279] 3) 30 ml of buffer 2 shown in Table 3 was used to remove
non-specifically adsorbed proteins.
[0280] 4) 3 ml of buffer 3 shown in Table 3 was respectively added
at four separate times, so that recombinant OST311 was eluted. 20
.mu.l each of these four fractions was subjected directly (as
original concentration) to Western blotting in the manner described
in Example 6(3). Thus, detection of OST311 was attempted with
anti-His antibody.
[0281] As a result, approximately 32 kDa and 10 kDa signals were
intensively observed in the second fraction.
[0282] 5) The above second fraction was applied to NAP25 and NAP10
columns (Amersham Pharmacia Biotech, USA), and the solvent was
replaced with buffer 4 shown in Table 3.
[0283] 6) Recombinant OST311 for which the solvent had been
replaced with buffer 4 was applied to SP-5PW (strong
cation-exchange resin, TOSOH, Japan) at a flow rate of 1 ml/minute
using high performance liquid chromatography (Hitachi, Japan).
Buffer 5 shown in Table 3 was added with a 1%/minute gradient for
elution, thereby sampling 2 ml each of fractions. As shown in FIG.
2, Western blotting was performed for each elution fraction in the
manner described in Example 8(5), so that detection of OST311 was
attempted. Approximately 10 kDa signal was eluted with about 280 mM
NaCl, and approximately 32 kDa signal was eluted with about 400 mM
NaCl. The corresponding fractions were subjected to
SDS-polyacrylamide gel electrophoresis, and then stained using a
silver staining kit (Daiichi Chemicals, Japan). The purity of the
fraction containing approximately 10 kDa and 32 kDa was 70% or
more.
7TABLE 3 Buffer 1 Buffer 2 Buffer 3 Buffer 4 Buffer 5 10 mM Na/Pi
10 mM Na/Pi 10 mM Na/Pi 10 mM Na/Pi 10 mM Na/Pi pH6.5 pH6.5 pH6.5
pH6.5 pH6.5 0.5 M NaCl 10 mM 0.5 M 5 mM CHAPS 1 M NaCl imidazole
imidazole 5 mM CHAPS 0.5 M NaCl 0.5 M NaCl 5 mM CHAPS Na/Pi: sodium
phosphate buffer
EXAMPLE 9
N-Terminal Amino Acid Sequence Analysis of Partially Purified
Recombinant OST311
[0284] Approximately 10 kDa and 32 kDa of partially purified
fractions recognized by anti-His antibody that had been obtained by
the method described in Example 8 were subjected to
SDS-polyacrylamide gel electrophoresis. Next, using a semi-dry
blotting system (Owl Separation Systems, USA), protein in the gel
was transferred to an Immobilon PVDF membrane (MILLIPORE, USA). The
PVDF membrane was stained with CBB, approximately 10 kDa and 32 kDa
bands were excised, and then the N-terminal amino acid sequences
were determined using a protein sequencer Model 492 (PE Applied
Systems, USA).
[0285] As a result, it became clear that the N-terminal amino acid
sequence of approximately 35 kDa band was an OST311 sequence
starting from residue No. 25, Tyr, of SEQ ID NO: 2. From this
result, it was confirmed that a sequence ranging from residue No.
1, Met, to residue No. 24, Ala, of SEQ ID NO: 2 had been cleaved as
a secretion signal sequence. On the other hand, it became clear
that the N-terminal amino acid sequence of approximately 10 kDa
band was an OST311 sequence starting from residue No. 180, Ser, of
SEQ ID NO: 2. The presence of a motif consisting of RRXXR
immediately before Ser at residue No. 180 revealed that recombinant
OST311 had been cleaved by some protease derived from CHO
cells.
[0286] As described above, recombinant OST311 produced by
CHO-OST311 cells was shown to be present as at least 3 types of
polypeptides after secretion: a polypeptide (SEQ ID NO: 4) from
residue No. 25, Tyr, to No. 251, Ile, a polypeptide (SEQ ID NO: 6)
from residue No. 25, Tyr, to No. 179, Arg, and a polypeptide (SEQ
ID NO: 8) from residue No. 180, Ser, to No. 251, Ile of SEQ ID NO:
2.
EXAMPLE 10
Preparation of Anti-OST311 Partial Peptide Polyclonal Antibody
[0287] The degree of hydrophobicity of the polypeptide of SEQ ID
NO: 2 was predicted using a computing function of MacVector version
6.5.1., so that sites suitable for antigen preparing peptide
antibodies were predicted (FIGS. 3A and B). Here, the suitable site
was predicted from the perspective that the sites have high degree
of hydrophilicity, and are less subject to sugar chain modification
and phosphorylation. Hence, selected and synthesized as antigens
were 311-48 (SEQ ID NO: 28) prepared by artificially adding a
cysteine residue at the synthetic stage to the C-terminus of a
peptide consisting of 20 amino acids starting from residue No. 48,
Arg, of SEQ ID NO: 2, and 311-114 (SEQ ID NO: 29) prepared by
similarly adding a cysteine residue to a peptide consisting of 20
amino acids starting from residue No. 114, Arg. Specifically,
cysteine residues were artificially added to the C-termini of both
peptides at the synthetic stage, so that the products could be
coupled with carrier proteins (bovine thyroglobulin). Coupling with
carrier proteins and immunization of rabbits were consigned to IBL,
Co., Ltd. (1091-1, Fujioka-shi, Gunma, Japan) (Assignment number:
1515).
8 311-48: RNSYHLQIHKNGHVDGAPHQC (SEQ ID NO: 28) 311-114:
RFQHQTLENGYDVYHSPQYHC (SEQ ID NO: 29)
EXAMPLE 11
Experiment of Transplanting CHO-OST311H Cells into Nude Mice
[0288] To test whether OST311 is a causative factor for
tumor-induced osteomalacia CHO-OST311H cells were transplanted into
6-week-old BALB/c nude mice (male) for tumor generation, thereby
developing a murine tumor-induced osteomalacia model which
constantly secrets recombinant OST311 from the tumors. As a control
for the experiment, CHO ras clone-1 cells and CHO-OST190H described
in Example 7 were used similarly for the transplantation
experiment.
[0289] (1) Transplantation of CHO Cells
[0290] CHO-OST311H cells and CHO-OST190 cells were scattered by
trypsin treatment from culture flasks, suspended to
1.times.10.sup.8 cells/ml in PBS. The suspension was subcutaneously
injected, 0.1 ml each, to both latera of the nude mice
(2.times.10.sup.7 cells/mouse). In addition, as a control group,
subcutaneous injection of the same number of CHO ras clone-1 cells
were performed in the same manner. For about 1 month after
injection, five nude mice were housed in a plastic cage and allowed
access to solid food CE-2 (CLEA JAPAN, Japan) and tap water, ad
libitum. 2 weeks after transplantation, tumor generation was
observed for 75% of the control group and 66.7% of OST311
group.
[0291] (2) Comparison of Changes in Body Weight
[0292] Time-course changes in average body weight for 31 days after
transplantation of CHO-OST311H cells were compared between a non
tumor-bearing group (a line indicated with avr.- in the graph) and
a CHO-OST311H cell tumor-bearing group (a indicated with avr.+ line
in the graph). As shown in FIG. 4, the CHO-OST311H cell
tumor-bearing group showed suppressed increases in body weight
compared to the non tumor-bearing group, and there were clear
significant differences between the two groups (24.1.+-.1.5 g vs.
26.7.+-.1.0 g, p<0.001, day 31). In contrast, similar
differences were not observed in average body weight between the
CHO ras clone-1 cell tumor-bearing control group and the non
tumor-bearing group (27.0.+-.1.8 g vs. 26.7.+-.1.0 g, no
significant difference, day 31).
[0293] (3) Measurement of Serum Phosphate and Calcium, and Urine
Phosphate and Calcium
[0294] On days 30 to 40 after cell transplantation, nude mice were
housed in metabolic cages for 24 hours. After urine was collected,
blood was collected from the heart or orbital cavity of the mice
under the anesthetized condition using diethylethel. The peripheral
blood was subjected to preparation of serum using Microtainer
(Beckton Dickinson, USA). After the volume was measured, the urine
was centrifuged to collect supernatant. Serum and urine phosphate
levels were measured using P-test Wako (Wako Pure Chemical
Industries, Japan), and serum and urine calcium levels were
measured using calcium-test Wako (Wako Pure Chemical Industries,
Japan), and serum and urine creatinine levels were measured using
CRE-EN KAINOS (KAINOS, Japan).
[0295] Experiment 1:
[0296] On day 34 after cell transplantation, serum phosphate levels
of the non tumor-bearing group, the CHO ras clone-1 cell
tumor-bearing group, the CHO-OST190H cell tumor-bearing group, and
the CHO-OST311H cell tumor-bearing group were measured.
[0297] Experiment 2:
[0298] On days 44 to 46 after cell transplantation, serum and urine
phosphate levels, calcium levels and creatinine levels of the non
tumor-bearing group and the CHO-OST311H cell tumor-bearing group
were measured. Renal fractional excretion of phosphate and calcium
were determined by dividing phosphate or calcium clearance by
creatinine clearance.
[0299] Measurement results are shown in Table 4 below.
9TABLE 4 Serum and urine phosphate and calcium in
cells-transplanted mice Experiment 1 Serum phosphate Group Number
of mouse level .+-. SEM (mg/dl) t-test Non tumor-bearing 7 8.17
.+-. 0.60 -- mice CHO ras clone-1 4 8.50 .+-. 0.38 No significant
difference CHO-OST190H 4 9.49 .+-. 0.52 No significant difference
CHO-OST311H 9 4.39 .+-. 0.23 p < 0.001 Experiment 2 Group Non
tumor CHO-OST311H t-test Number of mouse 4 6 -- Serum phosphate
level .+-. SEM 8.29 .+-. 0.59 4.25 .+-. 0.15 p < 0.001 (mg/dl)
Renal fractional excretion of 0.23 .+-. 0.02 0.44 .+-. 0.06 p <
0.05 phosphate Serum Ca level .+-. SEM (mg/dl) 6.72 .+-. 0.27 4.61
.+-. 0.19 p < 0.001 Renal fractional excretion of 0.0040 .+-.
0.0006 0.0059 .+-. 0.0010 No significant difference calcium
[0300] (4) Soft Roentgenogram of Whole Skeleton
[0301] After transplantation of the CHO-OST311H cells, individuals
in which formation of tumors is recognized exhibited significant
abnormalities in their physical constitutions and walking
functions, compared to non tumor-bearing individuals or the control
CHO ras clone-1-transplanted individuals. Hence, the tumor-bearing
individuals were predicted to have skeletal abnormalities. Then,
individuals in which formation of tumors is recognized were
selected at random from the control CHO ras clone-1
cells-transplanted group, the CHO-OST190H cells-transplanted group
and CHO-OST311H cells-transplanted group, and then X-ray pictures
were taken therefor using a radiography system .mu.FX-100 (FUJI
FILM, Japan) according to the attached manufacturer's manual. X-ray
pictures were taken under conditions of X-ray tube voltage of 25
kV, X-ray tube current of 0.1 mA and exposure time of 10 seconds.
The individuals were exposed to a imaging plate, and then image
analysis was performed using BAS2000 (FUJI FILM, Japan).
[0302] As a result, as shown in FIG. 5, reduced brightness of soft
roentgenogram of the bone was recognized in the whole skeleton of
CHO-OST311H cells-transplanted individual, so that defect of
mineralization was recognized. In addition, skeletal deformity,
such as distortion of rib cage was also recognized.
[0303] (5) Measurement of Serum Phosphate and Calcium Levels and
Alkaline Phosphatase Activities
[0304] The serum obtained by collecting blood from the heart on
days 44 and 46 after cell transplantation was stored at -20.degree.
C. once. The serum samples were thawed together, phosphate and
calcium levels contained in each serum were measured again, and
alkaline phosphatase activities were also measured. Phosphate
levels were measured using P-test Wako (Wako Pure Chemical
Industries, Japan), calcium levels were measured using calcium-test
Wako (Wako Pure Chemical Industries, Japan), and alkaline
phosphatase activities were measured using calcium alkaline
phosphor B-test Wako (Wako Pure Chemical Industries, Japan). The
results were classified into a non tumor-bearing group (n=6), a CHO
tumor-bearing group (n=10), a CHO-OST190H cell tumor-bearing group
(n=10) and a CHO-OST311H tumor group (n=6.times.2). The CHO-OST311H
group was classified into two groups: a group sacrificed on day 44
(CHO-OST311H-1: n=6) and a group sacrificed on day 46
(CHO-OST311-2: n=6). As shown in FIG. 7, significant changes
including decreased serum phosphate level (FIG. 7A), decreased
serum calcium level (FIG. 7B) and increased serum alkaline
phosphatase activity (FIG. 7C) were recognized in the CHO-OST311H
tumor group.
[0305] (6) Expression of Sodium-Phosphate Cotransporter (NaPi-7) on
Renal Proximal Tubule
[0306] i) Preparation of Brush Border Membranes (Hereinafter,
Referred to as BBM) of Proximal Tubular Epithelial Cell
[0307] Kidneys were excised from the CHO-OST311H tumor-bearing
individuals and non tumor-bearing individuals under the
anesthetized condition using diethylether. Each kidney was cut in
half to obtain coronal sections (Experiment 1: 6 numbers of
CHO-OST311H tumor-bearing individuals and 4 numbers of non
tumor-bearing individuals. Experiment 2: 6 numbers of CHO-OST311H
tumor-bearing individuals, and 2 numbers of non tumor-bearing
individuals). Using half of each of the respectively excised
kidneys from the individuals, BBM was prepared according to the
protocols as reported by Kessler et al (Biochem. Biophys. Acta.
506, pp.136-154).
[0308] The kidney was homogenized in 3 ml of a homogenizing buffer
(50 mM mannitol, 2 mM Tris/HEPES pH 7.5) using a glass-made
homogenizer at 1,300 rpm for 2 minutes so that homogenous kidney
extracts were obtained. After CaCl.sub.2 was added to the extract
to have a final concentration of 10 mM, the solution was agitated
at 4.degree. C. for 15 minutes, and then centrifuged at 4,900 g for
15 minutes at 4.degree. C. The thus obtained supernatant was
filtered using a Kimwipe, and then centrifuged at 16,200 g for 60
minutes at 4.degree. C., thereby allowing fractions containing many
BBM to precipitate. The precipitate was resuspended in 5 ml of a
suspension buffer (50 mM mannitol, 2 mM Tris/HEPES, pH 7.5), and
then the solution was centrifuged again at 16,200 g for 60 minutes
at 4.degree. C. This procedure was repeated twice, and then the
product was resuspended in 0.1 ml of a suspension buffer. The
protein concentration of the thus obtained solution was 3 to 4
mg/ml as determined by standard methods.
[0309] ii) Western Blotting of BBM Protein
[0310] As described above, BBM proteins prepared from each mouse
were diluted separately with a suspension buffer to 10 .mu.g/.mu.l.
Then, 2.5 .mu.l of a sample buffer containing 1 M Tris-Cl pH 6.8,
5% SDS, 50% glycerol and 100 mM DTT was added to the diluted
solution. After the solution was heated at 95.degree. C. for 5
minutes, protein in BBM solution was separated by polyacrylamide
electrophoresis with a 10 to 20% gradient. Subsequently, the
protein in the gel was transferred to Immobilon PVDF membrane
(MILLIPORE, USA) using a semi-dry blotting system (Owl Separation
Systems, USA). The PVDF membrane was incubated with anti-NaPi-7
polyclonal antibody diluted {fraction (1/2000)} in TTBS buffer
(Sigma, USA) at room temperature for 3 hours. The antibody is a
polyclonal antibody which has been obtained by immunizing a rabbit
with a synthetic peptide (LALPAHHNATRL) corresponding to the
C-terminal site of mouse NaPi-7 by standard methods in KIRIN
BREWERY CO., LTD., Pharmaceutical Research Laboratories,
Pharmaceutical Division. After reaction with this antibody, the
reaction product was further incubated with anti-rabbit IgG
secondary antibody (DAKO, Denmark) bound to horseradish peroxidase
(HRP), and then bands were detected using ECL system (Amersham
Pharmacia Biotech, USA).
[0311] Under reduction conditions, approximately 80 kDa and 35 kDa
of bands and 170 to 200 kDa of high molecular smears were detected
with the antibody (FIG. 8). These band patterns are the same as the
cases reported by Tatsumi et al in J. Biol. Chem. Vol.273, pp
28568-28575, 1998, and these bands had been confirmed to uniformly
change depending on the amount of phosphate intake from food by
mice or rats. From these facts, the bands proved to the
polypeptides derived from NaPi-7. As shown in FIG. 8, for all the
above fragments (bands indicated with arrows), the NaPi-7 signals
contained in BBM proteins that had been prepared from CHO-OST311H
tumor-bearing individuals were significantly reduced compared to
those prepared from non tumor-bearing individuals. These results
were reproduced in individually conducted Experiment 1 and 2. On
the other hand, these BBM proteins were separated by polyacrylamide
electrophoresis with a 10 to 20% gradient, and then stained with
CBB. BBM proteins of individuals were equally stained, suggesting
that reduced signals in Western blotting are specifically observed
for NaPi-7 (FIG. 8). Based on this fact, it is inferred that OST311
protein acts on renal proximal tubular cells, and downregulates the
expression level of NaPi-7 at the protein level, so as to induce
hypophosphatemia.
[0312] (7) Analysis of Changes in mRNAs of Phosphate Transporters
and Vitamin D-Metabolizing Enzymes in the Kidney and Small
Intestine
[0313] i) Preparation of Total RNA
[0314] Small intestines and kidneys were excised from the mice
sacrificed on days 44 to 46 after cell transplantation. The kidneys
were rapidly frozen in dry ice. The frozen kidneys were
cryopreserved in a deep freezer at -80.degree. C. until use. One
frozen kidney was homogenized in 5 ml of ISOGEN (Nippon Gene,
Japan), and then total RNA was prepared according to the attached
manufacturer's manual. 15 .mu.g of the prepared total RNA was
electrophoresed by formaldehyde-containing denatured gel with 1%
agarose concentration according to standard methods, and then
transferred to Hybond-N+ (Amersham Pharmacia, USA) overnight by a
capillary transfer method. The filter transferred with RNA was
irradiated with UV using a Stratalinker (STRATAGENE, USA) for
immobilization of the transferred RNA, washed with 2.times.SSC,
air-dried, and then stored at room temperature until use. The small
intestines were washed with physiological saline to remove the
content, and then reversed. Next, the epithelia of the small
intestine were collected by scratching with a preparation, and were
then rapidly frozen with liquid nitrogen. The frozen small
intestinal epithelia were cryopreserved in a deep freezer at
-80.degree. C. until use. Frozen small intestinal epithelia were
homogenized in 5 ml of ISOGEN (Nippon Gene, Japan), and then total
RNA was prepared according to the attached manufacturer's manual.
20 .mu.g of the prepared total RNA was electrophoresed by
formaldehyde-containing denatured gel with 1% agarose concentration
according to standard methods, and then transferred to Hybond-N+
(Amersham Pharmacia, USA) overnight by a capillary transfer method.
The filter transferred with RNA was irradiated with UV using a
Stratalinker (STRATAGENE, USA) for immobilization of the
transferred RNA, washed with 2.times.SSC, air-dried, and then
stored at room temperature until use.
[0315] ii) Preparation of Template DNA for Probe
[0316] 5 .mu.g of the total RNA prepared from a mouse (individual
mouse No. 1) was used to synthesize cDNA in 20 .mu.L of a reaction
solution (50 mM Tris (pH8.3), 75 mM KCl, 3 mM MgCl.sub.2, 10 mM
DTT, 25 g/mL (dT) 18, 2.5 mM dNTP, 200 units of MMLV reverse
transcriptase (TOYOBO, Japan)) at 37.degree. C. for 1 hour, and
then the reaction solution was treated at 70.degree. C. for 15
minutes to inactivate the enzyme. The synthesized cDNA was diluted
5 fold, and then used in the following reaction.
[0317] The following primers were synthesized from the sequences
registered at GenBank (NCBI, USA), and then used for PCR
reaction:
[0318] Synthetic Primers for Obtaining Mouse GAPDH cDNA
[0319] mGAPDHFW TGAAGGTCGGTGTGAACGGATTTGGC (SEQ ID NO: 30)
[0320] mGAPDHRV CATGTAGGCCATGAGGTCCACCAC (SEQ ID NO: 31)
[0321] Synthetic Primers for Obtaining Mouse Npt-1 cDNA
[0322] mNPt1FW GTAAAGAACCCTGTGTATTCC (SEQ ID NO: 32)
[0323] mNpt1RV CTGCCTTAAGAAATCCATAAT (SEQ ID NO: 33)
[0324] Synthetic Primers for Obtaining Mouse NaPi-7 cDNA
[0325] mNaPi7FW GAGGAATCACAGTCTCATTC (SEQ ID NO: 34)
[0326] nNaPi7RV CTTGGGGAGGTGCCCGGGAC (SEQ ID NO: 35)
[0327] Synthetic Primers for Obtaining Mouse NaPi-2b cDNA
[0328] mNaPi2bFW TCCCTCTTAGAAGACAATACA (SEQ ID NO: 36)
[0329] mNaPi2bRV GTGTTTAAAGGCAGTATTACA (SEQ ID NO: 37)
[0330] Synthetic Primers for Obtaining Mouse Vitamin D1.alpha.
Hyroxylase cDNA
[0331] m1aOHaseFW CAGACAGAGACATCCGTGTAG (SEQ ID NO: 38)
[0332] m1aOHaseRV CCACATGGTCCAGGTTCAGTC (SEQ ID NO: 39)
[0333] Synthetic Primers for Obtaining Mouse Vitamin D 24
Hydroxylase cDNA
[0334] m24OhaseFW GACGGTGAGACTCGGAACGT (SEQ ID NO: 40)
[0335] m24OhaseRV TCCGGAAAATCTGGCCATAC (SEQ ID NO: 41)
[0336] A reaction solution was prepared according to the attached
manufacturer's manual of TakaLa LA-Taq (TAKARA SHUZO, Japan). 1
.mu.L of cDNA and 10 pmol of each of the primer as described above
were added to 50 mL of the reaction solution. The solution was
maintained at 94.degree. C. for 1 minute, and then amplification
was performed for 40 cycles, each incubation cycle consisting of
94.degree. C. for 30 seconds, 55.degree. C. for 30 seconds and
72.degree. C. for 1 minute. Then the amplified bands were separated
by 0.8% agarose gel electrophoresis, and then target fragments were
collected using Gene Clean II (Bio101, USA). Concerning GAPDH,
.sup.32P-labeled probe was prepared using as a template the
fragment obtained by the procedure and a Megaprimer Labeling kit
(Amersham Pharmacia Biotech, USA), and then used for the following
hybridization. Concerning other genes, the obtained PCR fragments
were incorporated into pGEM-T vector (Promega, USA), and then
introduced into Escherichia coli DH5.alpha.. T7 (SEQ ID NO: 42) and
SP6 primers (SEQ ID NO: 43) were added, 10 pmol each, to a PCR
reaction solution, and then transformed Escherichia coli was added
to the same. After the solution was maintained at 94.degree. C. for
10 minutes, amplification was performed for 40 cycles, each cycle
consisting of 94.degree. C. for 30 seconds, 55.degree. C. for 30
seconds and 72.degree. C. for 1 minute. The reaction solution was
subjected to 0.8% agarose gel electrophoresis to separate amplified
bands, and then a target fragment was extracted using Gene Clean II
(Bio 101, USA).
10 T7 TAATACGACTCACTATAGGG (SEQ ID NO: 42) SP6 GATTTAGGTGACACTATAG
(SEQ ID NO: 43)
[0337] The nucleotide sequences of the amplified fragments obtained
by the above procedures were determined using ABI377 DNA sequencer
(PE Applied System, USA), thereby confirming that each target
fragment was obtained. The thus obtained DNA fragments were
.sup.32P-labelled using a Megaprimer Labeling kit (Amersham
Pharmacia, USA), and then used as probes in the following
hybridization.
[0338] iii) Hybridization
[0339] Hybridization was performed according to the attached
manufacturer's manual using ExpressHyb hybridization solution
(CLONTECH, USA) or Perfecthyb hybridization solution (TOYOBO,
Japan). After hybridization and washing, an imaging plate (FUJI
FILM, Japan) was exposed for 30 minutes to overnight, and then
analysis was made using BAS2000 image analyzer (FUJI FILM, Japan)
(FIG. 9A to C). In addition, signal intensities of target bands
were measured. After the signal intensities of each gene were
corrected by the signal intensity of GAPDH, the ratio of the mean
values of the non tumor-bearing group (Individual mice No. 1 to 4)
to the tumor-bearing group (individual No. 5 to 10) was obtained.
The following Table 5 shows the ratios. As tumors were developed in
the group transplanted with CHO-OST311H, mRNA levels of NaPi-7, the
type II phosphate transporter in the kidney, significantly
decreased, while NPT-1, the type I phosphate transporter in the
kidney, did not largely change. Further, a significant decrease was
observed in mRNA of NaPi-IIb, the phosphate transporter in the
small intestine. In contrast, for renal vitamin D metabolic enzyme,
both mRNAs of 25-hydroxyvitamin D-1-.alpha.-hydroxylase
(1.alpha.OHase) and 25-hydroxyvitamin D-24-hydroxylase (24OHase)
increased.
11TABLE 5 Each mRNA ratio of the tumor-bearing group to the non
tumor-bearing group (Tumor-bearing group/non tumor-bearing group)
NPT-1 0.88 NaPi-7 0.50 NaPi-2b 0.23 Vitamin D1.alpha.hydroxylase
3.90 Vitamin D24 hydroxylase 1.94
[0340] (8) Measurement of Serum 1,25-dihydroxyvitamin D Levels
[0341] The sera from mice of the control group and the sera of the
mice from the OST311H group were collected in equivalent amounts
from each individual on days 44 and 46 after tumor transplantation.
The sera collected from each group (0.5 ml of each total amount)
were submitted to Mitsubishi Kagaku Bio-Clinical Laboratories, Inc,
and then the 1,25-dihydroxyvitamin D levels contained in the sera
were measured in a manner similar to clinical examination. As a
result, serum 1,25-dihydroxyvitamin D levels of the control group
and OST311 group were 28.0 pg/ml and 23.9 pg/ml, respectively. As
described above, 1,25-dihydroxyvitamin D levels did not increase
even when hypophosphatemia and hypocalcemia were observed. This
result clearly suggests that vitamin D metabolism was affected as a
result of the effect of OST311.
[0342] (9) Soft Roentgenogram of Femora
[0343] Non tumor-bearing mice and CHO ras clone-1, CHO-OST190H or
CHO-OST311H-transplanted mice were sacrificed on days 44 to 46
after transplantation, the femora were collected, and then the
femora were fixed in 4% neutral formalin for 3 days. Next, the soft
tissue surrounding the bone was removed. Two individuals were
selected at random from each group, and then irradiated with X-ray
using a radiography system .mu.FX-100 (FUJI FILM, Japan) under the
following conditions: X-ray tube voltage of 25 kV, X-ray tube
current of 0.1 mA and exposure time of 5 seconds, and then exposed
to an imaging plate. The results are shown in FIG. 10. Decreased
bone trabecula of the cortical bone was observed in CHO-OST311H
group.
EXAMPLE 12
Analysis of Nucleotide Sequence Homology and Genomic Region of
OST311
[0344] Using at least a part of the amino acid sequence represented
by SEQ ID No: 2 and of the nucleotide sequence represented by SEQ
ID NO: 1, molecules corresponding to OST311 can be searched from
various species. The mouse genome sequence database was searched
using a partial nucleotide sequence of SEQ ID NO: 1, thus, a
sequence having high homology with OST311 was found in the sequence
of mouse chromosome 6 registered at Genbank under Accession No. AC0
15538. An amino acid sequence of a partial polypeptide of mouse
OST311 obtained from the sequence is shown in SEQ ID NO: 10, and a
nucleotide sequence corresponding to a partial sequence of cDNA is
shown in SEQ ID NO: 9. FIG. 6 shows the results of comparing amino
acid homology between human OST311 polypeptide and mouse OST311
polypeptide. As shown in Example 11, it became clear that OST311
polypeptide having the human amino acid sequence had clear
biological activities in mice. These results suggest that the
activity can be easily retained, even when these amino acids are
substituted, deleted, or inserted within a region having low
homology in the amino acid sequence as shown in FIG. 6.
[0345] The nucleotide sequence shown in SEQ ID NO: 1 and human
12p13 BAC RPCI11-388F6 (Accession No. AC008012) which had been
recognized using the database to have a region that matches with a
part of SEQ ID NO: 1 were compared, so that the sequence of the
region encoding OST311 was determined. The nucleotide sequence
neighboring OST311 gene is shown in SEQ ID NO: 11. TATAA box is
present between nucleotide No. 498 to 502 of SEQ ID NO: 11. The
first sequence matching with cDNA sequence (SEQ ID NO: 1) which we
have determined, starts from nucleotide No. 1713 of SEQ ID NO: 11
and continues to nucleotide No. 2057. The next section matching
with the nucleotide sequence represented by SEQ ID NO: 1 ranges
from nucleotide No. 8732 to 8833 of SEQ ID NO: 11. This section is
considered to be exon 2. The last section matching with the
nucleotide sequence represented by SEQ ID NO: 1 starts from
nucleotide No. 10644 and ends at nucleotide No. 12966 of SEQ ID NO:
11. The sequence ranging from nucleotide No. 498 to 12966 of SEQ ID
NO: 11 can be considered to be at least a part of the gene encoding
OST311. Further, it became clear that the STS sequence registered
at Genbank under Accession No. G19259 is present between exon 1 and
exon 2. OST311 is present in 12p13 region. According to Econs, M.
J. et al., J. Clin. Invest. 100:2653-2657, 1997, it is inferred
that the gene responsible for autosomal vitamin D-resistant rickets
(ADHR) is present within a 18 cM region between D12S100 and
D12S397, the microsatellite markers of 12p13 (particularly, within
approximately a 10 cM region between D12S314 and D12S397), from a
result of chain analysis. We evaluated the physical locations of
the OST311 gene and the above microsatellite marker on the
chromosome 12. As a result, D12S100 and D12S314 were in a 4602 to
6129 kb region, OST311 was in a 8958 to 9129 kb region, and D12S397
was in a 16280 to 16537 kb region. Based on these results and the
strong phosphate metabolism-regulating activity of OST311, it was
found that OST311 is the gene responsible for ADHR.
EXAMPLE 13
Short-Term Experiment of Transplantation of CHO-OST311H Cells into
Nude Mice
[0346] CHO-OST311H cells were transplanted subcutaneously into the
dorsa of nude mice (6-week-old, BALB/c, male). On days 2 and 6
after transplantation, serum phosphate and calcium levels were
measured, and the effect of recombinant OST311 within the short
term was examined. CHO ras clone-1 cells were used similarly, as a
control for this transplantation experiment.
[0347] (1) Transplantation of CHO Cells
[0348] In a manner similar to the method described in Example 11
(1), 2.times.10.sup.7 CHO-OST311H cells and 2.times.10.sup.7 CHO
ras clone-1 cells were subcutaneously transplanted, each, into nude
mice (n=6 each). Further, equivalent amounts of PBS were similarly
subcutaneously transplanted (n=6). Each group of 6 nude mice was
housed in a plastic cage and allowed to access to solid food CE-2
(CLEA JAPAN, Japan) and tap water ad libitum. At 6 days after
transplantation, no significant tumor development was observed.
[0349] (2) Measurement of Serum Phosphate and Calcium Levels on Day
2 after Cell Transplantation
[0350] On the next day after cell transplantation, blood was
collected from the orbital cavity of the mice under the
anesthetized condition using diethylethel. The peripheral blood was
subjected to serum separation using Microtainer (Beckton Dickinson,
USA). Serum phosphate levels were measured using P-test Wako (Wako
Pure Chemical Industries, Japan), and serum calcium levels were
measured using calcium-test Wako (Wako Pure Chemical Industries,
Japan). As shown in FIG. 11A, compared to the PBS-administered
group and the CHO ras clone-1 cells-transplanted group, significant
decreases in serum phosphate levels were observed in all cases of
the CHO-OST311H cells-transplanted group. In contrast, no change
was observed in serum calcium levels. These results clearly showed
that OST311 caused decreases only in serum phosphate levels on day
2 after administration.
[0351] (3) Measurement of Serum Phosphate and Calcium Levels on Day
6 after Cell Transplantation
[0352] On day 6 after cell transplantation, blood was collected
from the hearts of the mice under the anesthetized condition using
diethylethel. As described above, serum phosphate and calcium
levels of each group were measured. As shown in FIG. 11B, similar
to the results on day 2 after transplantation, compared to the
PBS-administered group and the CHO ras clone-1 cells-transplanted
group, significant decreases in serum phosphate levels were
observed in all cases of the CHO-OST311H cells-transplanted group.
In contrast, slight decreases were observed in serum calcium levels
of among the CHO-OST311H cells-transplanted group.
EXAMPLE 14
Purification of Recombinant OST311
[0353] CHO-OST311H cells were allowed to grow in 10% FCS-containing
MEM.alpha. medium within a 225 cm.sup.2 culture flask (CORNING,
USA) at 37.degree. C. under 5% CO.sub.2 and 100% humidity. When the
cells grew to cover approximately 80% of the area of the flask, the
medium was replaced with 50 ml of a serum-free medium, CHO-S-SFM II
(LIFE TECHNOLOGY, USA), and 48 hours later, conditioned medium was
collected. Recombinant OST311 was purified by the following method
using 1,000 ml in total of the conditioned medium obtained in this
manner.
[0354] 1000 ml of the conditioned medium was centrifuged at 16,200
g for 15 minutes at 4.degree. C. to remove the suspended cells, and
then the supernatant was subjected to SP-sepharose FF (Amersham
Pharmacia, USA) packed in a glass column (30 mm in internal
diameter.times.200 mm in length). The fraction that had passed
through the column was adsorbed to Talon Superflow (metal chelate
resin, CLONTECH, USA). Non-specific adsorbate was removed using a
washing buffer consisting of 50 mM sodium phosphate buffer (pH 6.6)
and 0.3 M NaCl, and then elution was performed using 50 mM sodium
phosphate buffer (pH 6.7) and 0.2 M Imidazole. The right panel in
FIG. 12 shows the elution fractions as detected by Western blotting
using an anti-His6 antibody (INVITROGEN, USA). These fractions
contained a partial polypeptide (SEQ ID NO: 8) consisting of the
amino acid residue No. 180, Ser, to No. 251, Ile, described in
Example 9. On the other hand, the above protein contained in the
conditioned medium adsorbed to SP-sepharose FF was eluted in a 50
mM sodium phosphate buffer (pH 6.7) using a 0 to 0.7 M NaCl
concentration gradient. The left panel in FIG. 12 shows the elution
fractions as detected by Western blotting using an anti-His6
antibody (INVITROGEN, USA). These fractions eluted at approximately
0.3 M NaCl contained a partial polypeptide (SEQ ID NO: 4)
consisting of the amino acid residue No. 25, Tyr, to No. 251, Ile
described in Example 9. Further the center panel in FIG. 12 shows
the elution fractions as detected by Western blotting using a
polyclonal antibody (311-114) prepared using the OST311 partial
peptide (SEQ ID NO: 29) described in Example 10. These fractions
eluted at a concentration of approximately 0.4 M NaCl contained a
partial peptide (SEQ ID NO: 6) which comprises amino acid residue
No. 25, Tyr to No. 179, Arg, described in Example 9. Thus, the
fractions containing three types of OST311 partial peptide,
specifically, SEQ ID NO: 4 (hereinafter, referred to as 311:
25-251), SEQ ID NO: 6 (hereinafter, referred to as 311: 25-179) and
SEQ ID NO: 8 (hereinafter, referred to as 311:180-251) were
purified and separated, and then concentrated using a VIVASPIN
column with a molecular weight of 10,000 (Sartorius, USA) for
ultrafiltration, followed by replacement with a solvent consisting
of 1 ml of 5 mM HEPES (pH 6.9) and 0.1 M NaCl.
EXAMPLE 15
Histological Analysis of Undemineralized Bone Section
[0355] When CHO-OST311H cells-transplanted mice and non
tumor-bearing mice prepared in Example 11 were sacrificed, the
right femora and tibiae were excised, leaving the connection at the
knee joint intact. Immediately after cutting the shaft of the
tibiae and the shaft of femora, the femora and tibiae were stored
in previously prepared ice-cooled neutral formalin. Thus,
undemineralized specimens were prepared. The method for preparing
the unmineralized specimens is described below.
[0356] The bone tissues were pre-stained with Villanueva bone stain
for 3 to 7 days. The tissues were dehydrated through a graded
alcohol series, and then the solvent was replaced with acetone.
After acetone monomer and then monomer were applied, the tissue
samples were embedded in resin. Methyl methacrylate (MMA) resin was
used for embedding the samples. The tissue samples were placed in
an incubator at about 35.degree. C. for complete polymerization. At
this time, the tissues were kept embedded sufficiently within the
resin by appropriate addition of MMA. MMA used herein for embedding
samples was prepared by adding and completely dissolving 40 g of
MMA polymer (Wako Pure Chemical Industries, Japan) in 100 ml of MMA
monomer (Wako Pure Chemical Industries, Japan), and then adding and
completely dissolving Benzoyl peroxide (Nacalai Tesque, Japan) at a
rate of 1 g per solution. The specimens were prepared for tibia. To
be able to observe the cancellous bone of the tibia, the frontal
section was trimmed, and then 4 .mu.m-thick frontal section samples
were prepared using a microtome for hard tissues (type RM2065 super
cut, Leica). Post-staining with Villanueva-Goldner was carried out.
The thus obtained sections were cleared in xylene, and then sealed
in using CLEAR SEAL (MARUTO, Japan) and ONE LIGHT (MARUTO,
Japan).
[0357] Microscopic images of the sections are shown in FIG. 13.
Increased width of the growth plate was observed in CHO-OST311H
cells-transplanted tumor-bearing mice, compared to the control
group. Further, significantly increased osteoid and decreased
mineralized area were observed in the metaphysis. There was no
evidence of ostitis fibrosa, and the bone collected from CHO-OST311
cells-transplanted tumor-bearing mouse exhibited typical features
of osteomalacia.
EXAMPLE 16
Examination of Vitamin D Metabolism at an Early Stage after
Transplantation with CHO-OST311H Cells
[0358] To examine the effect of OST311 on vitamin D metabolism, an
experiment of transplanting CHO-OST311H cells into nude mice
(6-week-old, BALB/c, male) was conducted in a manner similar to the
method described in Example 13. Two experimental control groups
consisting of a group which was transplanted similarly with CHO ras
clone-1 cells, and a group administered with PBS in an equivalent
dose with the cell suspension solution were established and used
for comparison. Each group consisting of 6 mice was housed in a
plastic cage with free access to tap water and solid food CE2
containing 1.03% inorganic phosphate and 1.18% calcium (CLEA JAPAN,
Japan). Fluctuations in serum 1,25-hydroxyvitamin D levels and
changes in the expression of vitamin D-metabolizing enzyme groups
on days 1, 2, 3 and 6 after transplantation were examined.
[0359] (1) Measurement of Serum 1,25-dihydroxyvitamin D Levels
[0360] On days 1, 2, 3 and 6 after cell transplantation, blood was
collected from the heart of the mice of the PBS-administered group,
the CHO-ras clone-1 cells-transplanted group and the CHO-OST311H
cells-transplanted group, respectively under the anesthetized
condition with diethylethel, and then sera were separated using a
Microtainer (Beckton Dickinson, USA). Equivalent volumes of the
sera collected from each mouse were mixed together to have a total
volume of 0.25 ml per group. 1,25-dihydroxyvitamin D levels
contained therein were measured using 1,25(OH)2D RIA-Kit, "TFB"
(TFB, Japan). As a result, as shown in Table 6, significant
decreases in 1,25-dihydroxyvitamin D levels were already observed
on day 1 after transplantation in the CHO-OST311H
cells-transplanted group, compared to the PBS-administered group
and the CHO-ras clone-1 cells-transplanted group. This decreasing
effect was also observed on days 2, 3 and 6 after transplantation.
These results were consistent with decreases in serum
1,25-dihydroxyvitamin D levels, which is a typical clinical finding
for tumor-induced osteomalacia.
12TABLE 6 Serum 1,25-dihydroxyvitamin D levels in
cells-transplanted mice Days after transplantation 1 2 3 6
PBS-administered group (pmol/L) 338 164.3 164.5 273.7 n = 5 CHO-ras
clone-1 cells-transplanted 271.9 178.3 182.9 184.6 group (pmol/L) n
= 6 CHO-OST311cells-transplan- ted 46.7 36.3 34.5 49.1 group
(pmol/L) n = 6
[0361] (2) Expression Analysis of Vitamin D-Metabolizing Enzyme
Genes in the Kidney
[0362] To study whether the above effect of decreasing
1,25-dihydroxyvitamin D3 was due to fluctuations in
25-hydroxyvitamin D-1-.alpha.-hydroxylase (1.alpha.OHase) gene or
in 25-hydroxyvitamin D-24-hydroxylase (24OHase) gene, 3 or 4 mice
were selected at random from each of the PBS-administered group,
the CHO-ras clone-1 cells-transplanted group and the CHO-OST311H
cells-transplanted group on day 3 after transplantation. The
kidneys were excised, total RNAs were prepared according to the
procedures described in Example 11 (7), and then the Northern
blotting was performed using the probes described in the same. FIG.
14 shows the results. mRNA expression levels of 1.alpha.OHase gene
were observed to be significantly attenuated in the CHO-OST311H
cells-transplanted group, compared to the PBS-administered group
and the CHO-ras clone-1 cells-transplanted group. This result
suggests a possibility that OST311 directly or indirectly
suppresses the expression of this gene, so as to suppress
biosynthesis of serum 1,25-dihydroxyvitamin D. On the other hand,
mRNA expression levels of 24OHase gene were significantly enhanced
in the CHO-OST311H cells-transplanted group, compared to the
PBS-administered group and the CHO-ras clone-1 cells-transplanted
group. This result suggests a possibility that OST311 directly or
indirectly enhances the expression of this gene, so as to promote
inactivation of serum 1,25-dihydroxyvitamin D.
[0363] In example 11(8), no significant difference in serum
1,25-dihydroxyvitamin D levels on days 44 and 46 after
transplantation was observed, compared to the control group.
Another result was different from the result in this example in
that mRNA expression levels of 1.alpha.OHase tended to increase. At
least one possible explanation for the difference is due to the
effect of serum parathyroid hormones described in Example 17.
EXAMPLE 17
Examination of Serum Parathyroid Hormone Levels at an Early Stage
after Transplantation with CHO-OST311H Cells
[0364] Each mouse serum collected in equivalent volumes on days 1,
2, 3, 6 and 45 after transplantation with CHO cells described in
Example 11, 13 and 16 was well mixed together to have a total
volume of 0.15 ml. Then, serum parathyroid hormone levels were
measured using a Rat PTH IRMA kit (Nihon Medi-Physics, Japan)
according to the attached manufacturer's manual. As shown in Table
7, significantly increased levels of serum parathyroid hormone were
observed in CHO-OST311-transplanted group, and the difference was
significant on day 45 after transplantation.
13TABLE 7 Parathyroid hormone levels in cells-transplanted mice
Days after transplantation 1 2 3 6 45 PBS-administered group
(pg/ml) 45.2 23.8 28.2 19.7 .sup.141.9 n = 5 CHO-ras clone-1 15.8
26.6 15.7 13.8 .sup.240.4 cells-transplanted group (pg/ml) n = 6
CHO-OST311 cells-transplanted 13.8 20.6 44 57.8 .sup.3211.7 group
(pg/ml) n = 6 .sup.1Measured value when non tumor-bearing mice (n =
6) were used. .sup.2n = 10. .sup.3n = 12.
EXAMPLE 18
Experiment of Administering CHO-Producing Recombinant OST311H
Full-Length Protein to Normal Mice
[0365] To study the effect of CHO-producing recombinant OST311H
full-length protein on normal mice (BALB/c, male, 6-week-old), a
polypeptide having a histidine tag added to the C-terminus of the
251.sup.th amino acid residue, Tyr, to the 251.sup.st Ile (SEQ ID
NO: 4) was partially purified by the purification method described
in Example 14 (1). This purified fraction was intraperitoneally
administered to normal mice at 0.1 ml per administration. It was
assumed that this purified fraction contained approximately 0.15 to
0.75 .mu.g of recombinant OST311, based on the fluorescent
intensity obtained by Western blotting. Similar to Example 14, 0.1
ml each of a solvent (5 mM HEPES buffer/0.1 M NaCl, pH=7.0) was
intraperitoneally administered to the control group. The
OST311-administrated group and the control group consisted
respectively of five mice. Each group of 5 mice was housed in a
plastic cage and allowed to access to tap water and solid food CE2
(CLEA JAPAN, Japan) containing 1.03% inorganic phosphate and 1.18%
calcium ad libitum.
[0366] [Experiment 1]
[0367] The experimental outline is shown in FIG. 15A. Additional
administration was performed at 5, 10, 23, 28 and 33 hours after
the 1st intraperitoneal administration. Thus, intraperitoneal
administration was performed 6 times in total. Subsequently, blood
was collected from the orbital cavity using glass-made capillaries
at 36, 47 and 71 hours after the 1st intraperitoneal
administration, and then sera were separated using a Microtainer
(Beckton Dickinson, USA).
[0368] Phosphate and calcium levels in the thus obtained sera were
determined using P-test Wako or calcium-test Wako (Wako Pure
Chemical Industries, Japan) according to the attached
manufacturer's manual. Thus, as shown in FIG. 15B, effects of
significantly decreasing serum phosphate levels were observed
(t-test **p<0.001, *p<0.01) in the OST311-administered group
at 36 hours after the 1st administration. In addition, this effect
was further maintained at 11 hours after this time point (47 hours
after the 1st administration). On the other hand, this activity
disappeared at 71 hours after the 1st administration (38 hours
after the final administration). Moreover, no significant change
was found in serum calcium levels at any time (FIG. 15C).
[0369] [Experiment 2]
[0370] The experimental outline is shown in FIG. 16A. Additional
administration was performed at 5 and 11 hours after the 1st
intraperitoneal administration. Thus, intraperitoneal
administration was performed 3 times in total. Subsequently, blood
was collected from the orbital cavity using glass-made capillaries
at 13 and 24 hours after the 1st intraperitoneal administration,
and then sera were separated using a Microtainer (Beckton
Dickinson, USA). Phosphate and calcium levels in the thus obtained
sera were measured respectively using P-test Wako and calcium-test
Wako according to the attached manufacturer's manual. Thus, as
shown in FIG. 16B, effects of significantly decreasing serum
phosphate levels were observed (t-test **p<0.05, *p<0.01) in
the OST311-administered group at 13 hours after the 1 st
administration. Further, this effect was maintained at 11 hours
following this time point. Furthermore, no significant change in
serum calcium levels was observed at any time (FIG. 16C).
[0371] The results of Experiments 1 and 2 revealed that
intraperitoneal administration into normal mice with the
full-length fraction of CHO-producing recombinant OST311 protein
induces hypophosphatemia, the effect of decreasing serum phosphate
levels was already observed at 13 hours after the first
administration, and the activity was maintained at least for 11
hours after the administration was stopped.
EXAMPLE 19
Introduction of Amino Acid Mutation into OST311
[0372] As described in Example 9, it was shown that a part of
recombinant OST311 produced by CHO-OST311H cells is cleaved during
its secreting process by a polypeptide (SEQ ID NO: 6) having a
sequence from the 25.sup.th Tyr to the 179.sup.th Arg amino acid
residues and a polypeptide (SEQ ID NO: 8) having a sequence from
the 180.sup.th Ser to the 251.sup.st Ile amino acid residues.
[0373] This cleavage may be due to a protease which recognizes a
motif consisting of RXXR sequence or RRXXR sequence located
immediately before the 180.sup.th Ser amino acid residue. When the
full-length recombinant is administered into a living organism, it
is considered that the recombinant has a possibility to undergo
this cleavage or degradation similar to this cleavage. Hence, gene
OST311RQ encoding a sequence which can substitute both the
176.sup.th Arg and the 179.sup.th Arg amino acid residues with Gln
was prepared for introducing the mutation.
[0374] (1) Preparation of OST31 I/pCAGGS Plasmid
[0375] PCR was performed by LA Taq polymerase (TAKARA SHUZO, Japan)
using OST311H/pcDNA3.1 plasmid as a template, and 311F1EcoRI (SEQ
ID NO: 22) and 311LNot (SEQ ID NO: 44) as primers. After
maintaining the temperature at 94.degree. C. for 1 minute, reaction
was performed for 25 cycles, each cycle consisting of 94.degree. C.
for 30 seconds, 55.degree. C. for 30 seconds and 72.degree. C. for
1 minute. After reaction, the PCR products were blunt-ended using
T4 DNA polymerase (Roche, Swiss), and then phenol-chloroform
treatment was performed to inactivate the enzyme. DNA was
precipitated using ethanol, and then the DNA ends were
phosphorylated using polynucleotide kinase (Roche, Swiss). Target
DNA fragments were separated by 0.8% agarose gel electrophoresis,
and then collected using Gene clean II (BIO101, USA). Plasmid
vector pCAGGS (Niwa H, et al., Gene. 1991 Dec. 15; 108(2): 193-9.)
was digested with EcoR I, and then blunt-ended using Klenow
fragments (Roche, Swiss). Subsequently, dephosphorylation of DNA
ends was performed using bovine small intestine alkaline
phosphatase (TAKARA SHUZO, Japan). The target DNA fragments were
separated by 0.8% agarose gel electrophoresis, and then collected
using Gene Clean II (BIO101, USA). The thus obtained OST311cDNA was
ligated to pre-digested pCAGGS plasmid using a DNA ligation kit
(version 2) (TAKARA SHUZO, Japan) according to the attached
manufacturer's manual. The product was introduced into Escherichia
coli DH5.alpha. for cloning, so that the relevant plasmid was
obtained. This plasmid DNA was used for preparing OST311RQH
gene.
[0376] 311LNot: ATAAGAATGCGGCCGCTCAGATGAACTTGGCGAA (SEQ ID NO:
44)
[0377] (2) Preparation of OST311RQH Gene
[0378] The following primers were synthesized.
14 OST311ME1: (SEQ ID NO: 45) ATGAATTCCACCATGTTGGGGGCCCGCCTCAGG
OST311HNt: (SEQ ID NO: 46)
ATGCGGCCGCCTAATGATGATGATGATGATGGATGAACTTGGCGAAGGG OST311RQF: (SEQ
ID NO: 47) ATACCACGGCAGCACACCCAGAGCGCCGAG OST311RQR: (SEQ ID NO:
48) CTCGGCGCTCTGGGTGTGCTGCCGTGGTAT
[0379] OST311ME1 is a forward primer which is a section containing
the initiation methionine of OST311, OST311HNt is a reverse primer
which adds 6 histidines to the 3' terminus of OST311, OST311RQF and
OST311RQR are a forward primer and a reverse primer for introducing
mutations, respectively by substituting the 527.sup.th and
536.sup.th guanines (corresponding to the 659.sup.th and 668.sup.th
guanines in SEQ ID NO: 1) in the coding region of OST311 cDNA with
adenines, so as to substitute the arginines at amino acid No. 176
and 179 with glutamines. 2 types of reaction solutions were
prepared 20 .mu.L each according to the attached manufacturer's
manual using pfu DNA polymerase (Promega, USA). On the one hand,
OST311ME1 and OST311RQR were used as primers at a final
concentration of 0.2 .mu.M, and 10 ng of OST311 expression vector
described in Example 6 (1) was used as a template. After the
temperature was maintained at 94.degree. C. for 1 minute, PCR
reaction was performed for 25 cycles, each cycle consisting of
94.degree. C. for 30 seconds, 55.degree. C. for 30 seconds and
72.degree. C. for 1 minute. On the other hand, OST311RQF and
OST311HNt were used as primers at a final concentration of 0.2
.mu.M, and 10 ng of OST311/pCAGGS plasmid was used as a template.
After the temperature was maintained at 94.degree. C. for 1 minute,
PCR reaction was performed for 35 cycles, each cycle consisting of
94.degree. C. for 30 seconds, 55.degree. C. for 30 seconds and
72.degree. C. for 1 minute. The above two types of reaction
products were diluted 10-fold respectively, and then 1 .mu.L of
each solution was added to 50 .mu.L of a reaction solution prepared
using LA Taq polymerase (TAKARA SHUZO, Japan) according to the
attached manufacturer's manual. After the temperature was
maintained at 94.degree. C. for 1 minute, PCR reaction was
performed using LA Taq polymerase (TAKARA SHUZO, Japan) and OST311
ME1 and OST311HNt as primers at a final concentration of 0.2 .mu.M
for 25 cycles, each cycle consisting of 94.degree. C. for 30
seconds, 55.degree. C. for 30 seconds, and 72.degree. C. for 1
minute. After PCR reaction, the solution was maintained at
72.degree. C. for 7 minutes. The thus obtained reaction product was
subjected to phenol/chloroform treatment, deproteinization, ethanol
precipitation, and then digestion with EcoR I and Not I. An
approximately 800 bp DNA fragment was separated by 2% agarose gel
electrophoresis, and then collected using Gene Clean II (BIO101,
USA). The thus obtained DNA fragment was inserted to the EcoR I,
Not I site of a vector, IRES-EGFP-pEAK8 that had been prepared by
ligating an internal ribosomal entry site (IRES) and enhanced green
fluorescent protein (EGFP) to a plasmid pEAK8 (EdgeBioSystems,
USA), thereby obtaining OST311RQH/IRES-EGFP/pEAK8 plasmid. Plasmid
DNA was prepared according to the standard methods, and then the
nucleotide sequence was determined using ABI3700 fluorescence DNA
sequencer (PE Applied Systems, USA), thereby confirming that the
sequence encodes a polypeptide wherein relevant mutations R176Q and
R179Q are introduced and the histidine tag is added to the
C-terminus. The polypeptide encoded by this gene is hereinafter
referred to as OST311RQH.
[0380] (3) Isolation of the CHO Cells which Stably Express
OST311RQH
[0381] OST311RQH/IRES-EGFP/pEAK8 plasmid was introduced into
CHO-ras clone-1 cell using Transfectam (Promega, USA) according to
the attached manufacturer's manual. Drug resistant cells were
selected in MEM.alpha. medium containing 5 .mu.g/mL puromycin and
10% FCS. Then, cells with strong fluorescence intensity of GFP
(Green Fluorescent Protein) were sorted using FACS vantage (Beckton
Dickinson, USA), and then cloned. When the cloned cells reached
confluent, the medium was replaced with serum-free DF (DMEM/F-12)
medium, and then conditioned medium was collected 2 days after
replacement. 50 .mu.L of the collected conditioned medium was
adsorbed to immobilon P filter (Millipore, USA) using a 96 well
convertible filter system (Lifetechoriental, USA). The prepared
filter was washed with TBS and TTBS, and then subjected to blocking
using Blockace (Daiichi Pharmaceutical, Japan) for 1 hour at room
temperature. After blocking, the filter was allowed to react for 1
hour with HRP-labeled anti-His6 monoclonal antibody (Invitrogen,
USA) diluted 5000 fold with Blockace. After reaction, the filter
was washed with TTBS and TBS, and then signal detection was
performed using ECL (Amersham Pharmacia, USA) according to the
attached manufacturer's manual. Based on signal intensity, high
expression clone CHO-OST311RQH was selected.
[0382] (4) Preparation of Conditioned Medium of the OST311RQH pEAK
Rapid Cells
[0383] pEAK rapid cells (EdgeBioSystems, USA) were inoculated in 20
flasks for tissue culture (225 cm.sup.2, CORNING, USA). 0.48 mg of
OST311RQH/IRES-GFP/pEAK8 plasmid was transfected to the cells by a
phosphate calcium method according to the attached manufacturer's
manual of pEAK system (EdgeBioSystems, USA). The cells were allowed
to stand for 4 hours. Next, the medium of each flask was replaced
with 50 mL of serum-free MEM.alpha. medium, the cells were cultured
for 2 days at 37.degree. C., and then the conditioned medium was
collected.
[0384] (5) Confirmation of Expression of Recombinant OST311RQH
[0385] The conditioned medium resulting from the transient
expression of the 2 types of the above CHO-OST311RQH cell clones
and pEAK rapid cells, 10 .mu.L each, were subjected to the Western
blotting in the manner as described in Example 6(3), so that the
presence of recombinant OST311RQH in the culture supernatant was
examined. Anti-His (C-terminus) antibody (Invitrogen, USA) was used
as a detection antibody. Thus as shown in FIG. 17, a strong signal
located at the same position with approximately 32 kDa band
described in Example 6(3) was observed for all the culture
supernatants. Moreover, for all the conditioned medium, an
approximately 10 kDa signal which was present in the CHO-OST311H
conditioned medium was not observed by Western blotting. It can be
inferred from these results that introduction of mutations R176Q
and R179Q caused inhibited or attenuated cleavage of the
polypeptide that was predicted to occur at these positions, so that
the ratio of the presence of the polypeptide (SEQ ID NO: 8) having
a sequence from the 180.sup.th Ser to the 251 Ile amino acids
decreased significantly.
EXAMPLE 20
Administration Experiment of Recombinant OST311RQH to Normal
Mice
[0386] A purified fraction containing approximately 2.8 pg/ml
recombinant OST311RQH protein was obtained from 500 ml of the
culture supernatant prepared in Example 19 (5) according to the
method described in Example 14 (1). The purified fraction was
successively administered, 0.1 ml/administration, intraperitoneally
to normal mice (BALB/c, male, 6-week-old), and then serum
phosphate, calcium and 1,25-dihydroxyvitamin D levels were
measured. To a control group, a vehicle (5 mM HEPES buffer/0.1 M
NaCl pH=7.0) was similarly administered, 0.1 ml each,
intraperitoneally. The OST311RQH-administrated group and the
control group consisted respectively of 6 mice. Each group of 6
mice was housed in a plastic cage and allowed to access to tap
water and solid food CE2 (CLEA JAPAN, Japan) containing 1.03%
inorganic phosphate and 1.18% calcium ad libitum.
[0387] Experimental protocols are shown in FIG. 18A. Additional
administration was performed at 5, 10, 24, 29 and 34 hours after
the 1st administration. Thus, administration was successively
performed 6 times in total. In the process of the above procedures,
under the anesthetized condition with diethylethel, blood was
collected from the orbital cavity using glass-made capillaries at
24 hours after the first administration (immediately before
4.sup.th administration), and blood was collected from the heart at
48 hours after the first administration.
[0388] (1) Measurement of Serum Phosphate and Calcium Levels
[0389] Serum phosphate levels of the serum collected at 24 and 48
hours after the first administration were measured by the method
described in Example 14 (3). As a result, at any time of blood
collection, the OST311RQH-administered group showed significant
hypophosphatemia, as shown in FIG. 18B (t-test **p<0.01,
*p<0.05). In contrast, no significant fluctuation was observed
in serum calcium levels (FIG. 18C).
[0390] (2) Measurement of Serum 1,25-dihydroxyvitamin D Levels
[0391] Equivalent volumes of sera collected from each mouse at 48
hours after the first administration were mixed together by each
group. Then, serum 1,25-dihydroxyvitamin D levels were measured by
the method described in Example 16(1). As a result, while the
control group showed 244.7 pmol/L, the OST311RQH-administered group
showed a significant decrease, 24.6 pmol/L.
EXAMPLE 21
CHO-OST311RQH Cell Transplantation Experiment
[0392] An experiment, wherein CHO-OST311RQH that stably expresses
OST311RQH cells as established in Example 19 (3) were transplanted
into nude mice (7-week-old, BALB/c-nude, male, n=8) was carried out
similarly to the method described in Example 13. CHO ras clone-1
cells were similarly transplanted as a control group (n=6). Each
group of the nude mice was housed in a plastic cage and allowed to
access to tap water and solid food CE2 (CLEA JAPAN, Japan) 1.03%
inorganic phosphate and 1.18% calcium ad libitum.
[0393] On day 2 after cell transplantation, blood was collected
from the orbital cavity using glass-made capillaries, and then
serum phosphate and calcium levels were measured by a method
similar to the method described in Example 14(3). As shown in FIG.
19A, a significant decrease in serum phosphate levels was observed
in the CHO-OST311RQH cells-transplanted group (t-test *p<0.001),
while no significant change was observed in serum calcium levels
(FIG. 19B)
EXAMPLE 22
Preparation of Anti-OST311 Partial Peptide Polyclonal Antibody
(2)
[0394] 4 types of partial OST311 peptides were prepared (SEQ ID NO:
49 to 52) in the manner described in Example 10. Rabbits were
immunized with these peptides as antigens, and then Western
blotting was performed according to the method described in Example
6(3) using the resulting anti-sera, so that recombinant OST311H was
detected in the serum-free conditioned medium of CHO-OST311H cells.
Antibody reaction was performed in a solution, which had been
prepared by diluting the anti-sera for each peptide 250-fold with
TTBS, at 4.degree. C. with agitation overnight. After washing,
alkaline phosphatase-labeled goat anti-rabbit antibody (DAKO,
Denmark) was added to the solution for binding, and then
recombinant OST311 was detected using an alkaline phosphatase
coloring kit (BIO-RAD, USA) (FIG. 20).
[0395] Partial Peptides
15 311-148: GMNPPPYSQFLSRRNEC (SEQ ID NO: 49) 311-170: CNTPIPRRHTR
(SEQ ID NO: 50) 311-180: SAEDDSERDPLNVLKC (SEQ ID NO: 51) 311-210:
LPSAEDNSPMASDC (SEQ ID NO: 52)
EXAMPLE 23
Construction of ELISA System for Detecting OST311 Protein
[0396] (1) Purification of Antibody from Anti-OST311 Partial
Peptide Rabbit Anti-Serum
[0397] Econo-Pac disposable chromatography column (BIO-RAD, USA)
was filled with 3 ml slurry of protein A sepharose 4FF (Amersham
pharmacia, USA), and then washed with 10 ml of 0.1 M glycine
hydrochlorate buffer (pH 3.3) and 20 ml of PBS. 2 types of rabbit
anti-sera described in Example 10 and 4 types of the same described
in Example 22 were added 800 to 900 .mu.l each, so that antibody
fractions were adsorbed to the resin. The column was washed with 9
ml of PBS to remove contaminants, and then 1 ml each of 0.1 M
glycine hydrochlorate buffer (pH 3.3) was added, thereby obtaining
IgG elution fractions. Upon elution, 10 .mu.l of a neutralization
buffer (1 M Tris) was added to each fraction whenever necessary to
neutralize the solutions. The absorbance at 280 nm was measured so
as to determine the concentration of antibody contained in the
elution fraction (absorbance coefficient calculated as: 1.34
(mg/ml).sup.-1.multidot.(cm).sup.-1). Then, some fractions were
together applied to a NAP25 column, and the solvent was replaced
with 50 mM sodium hydrogen carbonate solution. As a result, 5 to 15
mg of antibodies were obtained (these polyclonal antibodies are
hereinafter respectively referred to as 311-48 antibody, 311-114
antibody, 311-148 antibody, 311-170 antibody, 311-180 antibody and
311-210 antibody) from each peptide anti-serum.
[0398] (2) Biotinylation of Anti-OST311 IgG
[0399] All the above 6 types of anti-OST311 peptide polyclonal
antibodies were diluted to 1 mg/ml in 50 mM sodium hydrogen
carbonate solution. Then, 1 mg of each type of antibodies was mixed
well with Biotin-AC5-Osu solution (1.82 .mu.g/ml) (Japan, Dojindo)
dissolved in 10 .mu.l of dimethylformamide by inversion for 2 hours
at 4.degree. C. Subsequently, the mixed solution was subjected to
NAP10 column to remove unreacted Biotin-AC5-Osu and the solvent was
replaced with PBS, thereby obtaining 6 types of biotinylated
anti-OST311 peptide polyclonal antibodies.
[0400] (3) Detection of OST311 in the Conditioned Medium of
OST311-Expressing Cells by the Sandwich ELISA Method Using Anti-OST
Peptide Rabbit Polyclonal Antibody
[0401] A sandwich ELISA system was constructed by combining the 6
types of anti-OST311 peptide polyclonal antibodies for
immobilization and the above 6 types of biotinylated antibodies for
detection. Thus, detection of OST311 protein in the conditioned
medium of OST311-expressing cells was examined.
[0402] The above 6 types of anti-OST311 peptide polyclonal
antibodies for immobilization obtained by Protein A purification
were diluted to 10 .mu.g/ml in 50 mM sodium hydrogen carbonate
solution. 50 .mu.l of each diluted solution was added to each well
of a 96-well ELISA plate Maxisorp (Nunc, USA), and then allowed to
stand for 1 hour at 37.degree. C., thereby immobilizing IgG. Next,
the reaction solution was removed, and then 50 .mu.l of Superblock
blocking buffer in TBS (PIERCE, USA) was added per well to carry
out blocking at room temperature for 10 minutes. After the solution
was removed, OST311RQH peak rapid culture supernatant described in
Example 19(5) or MEM.alpha. medium as a control was added, 50 .mu.l
per well, and then allowed to stand at room temperature for 1 hour
for binding with the immobilized antibodies. After antibody
reaction, the solution was washed three times with TTBS, 50 .mu.l
each of the above 6 types of biotinylated anti-OST311 antibodies
(311-48, 311-114, 311-148, 311-170, 311-180 and 311-210) diluted to
10 .mu.g/ml with TTBS containing 10% Blockace (Dainippon
Pharmaceutical, Japan) was added per well, and then allowed to
stand at room temperature for 30 minutes, thereby performing
secondary antibody reaction. Each well was washed three times with
TTBS, and then 50 .mu.l of HRP-labeled streptavidin (DAKO, Denmark)
diluted 10,000 fold with TTBS containing 10% Blockace was added per
well, and then allowed to stand at room temperature for 30 minutes
for binding with the biotinylated antibodies. Each well was then
washed three times with TTBS, and then 50 .mu.l of
tetramethylbenzidine, the peroxidase chromogenic substrate (DAKO,
Denmark) was added per well, and then allowed to develop color at
room temperature for 5 minutes. Subsequently, 50 .mu.l of 0.5 M
sulfuric acid solution was added per well to stop reaction.
Measurement was performed using an absorbance measurement system
MTP300 (CORONA ELECTRIC, Japan) for a 96-well plate, and the
absorbance at 450 nm was divided by the absorbance at 570 nm. When
only MEM.alpha. was added as a control, each of values obtained by
450 nm/570 nm was 0.02 or less in every cases. In contrast, as
shown in FIG. 21A, with a combination of immobilized 311-48
antibody and detection with 311-180 antibody, or a combination of
immobilized 311-180 antibody and detection with 311-148 antibody,
OST311RQH in the conditioned medium could be detected significantly
more than the control. Moreover, with a combination of immobilized
311-48 antibody and detection with 311-148 antibody, it is inferred
that not only the full-length polypeptide, but also the N-terminal
partial polypeptide fragment can be detected, because the antigenic
sites of both antibodies were contained in the N-terminal partial
peptide (SEQ ID NO: 6) following the cleavage of OST311 protein
described in Example 9. In contrast, with a combination of
immobilized 311-210 antibody and detection with 311-180 antibody,
it is inferred that not only the full-length, but also the
C-terminal partial peptide (SEQ ID NO: 8) following cleavage
described in Example 9 can be detected. Therefore, the multiple use
of these combinations makes it possible to measure the absolute
amount of and the ratio of the presence of OST311 full-length
polypeptide and partial polypeptides in specimens, such as
biological samples.
[0403] (4) Quantitative Determination of Recombinant OST311 Protein
Concentration by Sandwich ELISA Method Using Anti-OST311 Peptide
Rabbit Polyclonal Antibody
[0404] In the above ELISA system, detection of purified recombinant
OST311H consisting of a serial dilution of 1, 0.67, 0.33, 0.1,
0.067, 0.033 and 0.01 .mu.g/ml were examined with a combination of
311-48 antibody or 311-180 antibody as an immobilized antibody and
311-148 antibody as an antibody for detection. As shown in FIG.
21B, fine linearity could thus be obtained within a range of 0.1 to
1 .mu.g/ml (311-48: R=0.990, 311-180: R.sup.2=0.996). This result
revealed that recombinant OST311H at least within this
concentration range can be detected.
EXAMPLE 24
Examination of Effect by Single Administration of Recombinant
OST311 Protein
[0405] To examine the short-term effect of CHO-producing
recombinant OST311H full-length protein on normal mice (BALB/c,
male, 6-week-old), purified recombinant OST311H full-length protein
was administered once, 5.0 .mu.g/0.1 ml per mouse via the caudal
vein. To a control group, 0.1 ml of a vehicle (PBS) was
administered per mouse via the caudal vein. At 1, 3 and 8 hours
after administration, blood collection from the heart and
dissection were performed, serum phosphate, calcium and vitamin D
levels were measured, and then the expression amount of the
sodium-phosphate cotransporter on the renal proximal tubule was
analyzed. The OST311-administrated group and the control group
respectively consisted of 6 mice. 6 mice per group were housed and
allowed to access to tap water and solid food CE2 (CLEA JAPAN,
Japan) containing 1.03% inorganic phosphate and 1.18% calcium ad
libitum.
[0406] (1) Time-Course Changes in Serum Phosphate Levels
[0407] As shown in Table. 8, while no significant change was
observed in serum phosphate levels at 1 and 3 hours after single
administration with OST311 protein, at 8 hours after
administration, a significant decrease was observed. This result
clarified that the effect of OST311 requires 3 to 8 hours to lower
serum phosphate levels. On the other hand, no change was observed
in serum calcium levels at all time.
16TABLE 8 Serum phosphate levels Time 1 3 8 Vehicle-administered
group 9.82 .+-. 0.61 9.99 .+-. 0.20 9.55 .+-. 0.29 (mg/dL)
OST311-administred group 9.61 .+-. 0.51 9.96 .+-. 0.39 7.82 .+-.
0.27 (mg/dL) t-test p > 0.5 p > 0.5 p < 0.005
[0408] (2) Expression of Sodium-Phosphate Cotransporter on Renal
Proximal Tubule
[0409] According to the method described in Example 11(6), the
kidneys collected at 1, 3 and 8 hours after administration were
mixed together per group, and then brush border membranes (BBM) of
proximal tubule were prepared. The ratio of the presence of
sodium-phosphate cotransporter (NaPi7) protein in the obtained BBM
was analyzed by the Western blotting method. As shown in FIG. 22A,
while the expression amounts of NaPi7 at 1 and 3 hours after
administration were equivalent to that of the vehicle-administered
group, NaPi7 in the OST311-administered group at 8 hours after
administration was shown to be significantly decreased compared to
that of the vehicle-administered group. Meanwhile, to examine if
the decreased NaPi7 protein was associated with RNA transcription
regulation, according to the procedure described in Example 11 (7),
total RNA was prepared from the kidneys excised from each mouse and
Northern blotting was performed using the probe described in the
same. Thus, as shown in FIG. 22B, while mRNA levels of NaPi7 at 1
and 3 hours after administration were equivalent to that of the
vehicle-administered group, NaPi7 in the OST311-administered group
at 8 hours after administration was shown to be significantly
decreased compared to that of the vehicle-administered group. The
above results clearly showed that decreases in serum phosphate
levels due to direct or indirect effect of recombinant OST311
protein correlated with downregulation of sodium-phosphate
cotransporter on the renal tubule in their times of fluctuations,
and suppression of NaPi-7 at the mRNA transcription level occurs at
least as a factor contributing to the downregulation at the protein
level.
[0410] (3) Time-course changes in serum 1,25-dihydroxy vitamin D3
levels Serum 1,25-dihydroxyvitamin D3 levels at 1, 3 and 8 hours
after administration were measured by the method described in
Example 16(1). As shown in FIG. 23, in the OST311-administered
group, a significant decrease in serum 1,25-dihydroxyvitamin D3
level was already observed at 3 hours after administration, and a
further decrease in the same was observed at 8 hours after
administration.
[0411] (4) Changes in Expression of Vitamin D-Metabolizing Enzyme
Genes
[0412] To elucidate whether the decreased serum
1,25-dihydroxyvitamin D3 levels were due to fluctuations in
25-hydroxyvitamin D-1-.alpha.-hydroxylase (1.alpha.OHase) or
25-hydroxyvitamin D-24-hydroxylase (24OHase) gene expressed in the
kidney, total RNAs were prepared from the kidneys at 1, 3 and 8
hours after administration according to the procedure described in
Example 11(7), and then Northern blotting was performed using the
probe described in the same. As shown in FIG. 24, already at 1 hour
after administration, decreased mRNA levels of 1.alpha.OHase gene
and increased mRNA levels of 24OHase gene were observed. This
tendency was shown to be more significant at 8 hours after
administration. In FIG. 24, "vehicle" indicates a solvent of
recombinant OST311 protein comprising 20 mM phosphate buffer (pH
6.7) and 0.3 M NaCl.
[0413] These results clearly showed that OST311 lowers serum
1,25-dihydroxyvitamin D3 levels by regulating the expression of
25-hydroxyvitamin D-1.alpha.-hydroxylase (1.alpha.OHase) or
25-hydroxyvitamin D-24-hydroxylase (24OHase) gene expressed in the
kidney.
EXAMPLE 25
Examination of Activity of C-Terminus-Deleted OST 311
[0414] (1) Construction of Expression System for OST311 Lacking
C-Terminal Portion
[0415] The following primers were synthesized.
17 OST311R693 ATGCGGCCGCTATCGACCGCCCCTGACCACCCC (SEQ ID NO: 53)
OST311R633 ATGCGGCCGCTACGGGAGCTCCTGTGAACAG- GA (SEQ ID NO: 54)
OST311R618 ATGCGGCCGCTCAACAGGAGGCCGGGGCCGGGGT (SEQ ID NO: 55)
OST311R603 ATGCGGCCGCTCACGGGGTCATCCGGGCCCGGGG (SEQ ID NO: 56)
[0416] OST311R693, OST311R633, OST311R618 and OST311R603 are
reverse primers for deleting 20, 40, 45 and 50 amino acid residues
from of the 3' terminus of OST311, respectively and introducing a
termination codon and Not I recognition sequence. Each of these
reverse primers and a forward primer OST311ME1 (SEQ ID NO: 45)
containing the initiation methionine of OST311 and EcoR I
recognition sequence described in Example 19 were combined to have
a final concentration of 0.2 .mu.M. Using these primers, Pyrobest
DNA polymerase (TAKARA SHUZO, Japan) and 100 ng of
OST311RQH/IRES-EGFP/pEAK8 plasmid DNA described in Example 19(2) as
a template, PCR reaction was performed for 25 cycles after
maintaining the temperature at 94.degree. C. for 1 minute. Each
reaction cycle consisted of 94.degree. C. for 30 seconds,
55.degree. C. for 30 seconds and 72.degree. C. for 1 minute. The
obtained reaction product was subjected to phenol/chloroform
treatment, deproteinization and then ethanol precipitation. The
reaction product was then digested with EcoR I and Not I, and then
subjected to 2% agarose gel electrophoresis, so that each DNA
fragment was separated and collected using Gene Clean II (BIO110,
USA). The obtained DNA fragment was ligated to pEAK8 vector
(EdgeBioSystems, USA) that had been digested with EcoR I and Not I,
thereby obtaining pPKOST311-.DELTA. C20, -.DELTA. C40, -.DELTA.
C45, -.DELTA. C50 plasmids. Plasmid DNAs were prepared by standard
methods, and then the nucleotide sequences were determined using
ABI3700 fluorescence DNA sequencer (PE Applied Biosystems, USA),
thereby confirming that base pairs had been deleted as desired from
each of the 3' terminus of OST311RQH gene.
[0417] (2) Isolation of the CHO Cells Stably Expressing
Recombinant
[0418] pPKOST311-.DELTA.C20, -.DELTA.C40, -.DELTA.C45, -.DELTA.C50
plasmid DNAs were respectively introduced into CHO ras clone-1
cells using Transfectam (Promega, USA) according to the attached
manufacturer's manual. CHO-OST311RQ-.DELTA.C20, -.DELTA.C40,
-.DELTA.C45 and -.DELTA.C50 cells showing drug resistance in
MEM.alpha. medium containing 5 .mu.g/ml puromycin and 10% FCS were
obtained. These cells were respectively inoculated into 24-well
plates, and then cultured in MEM.alpha. medium containing 5
.mu.g/ml puromycin and 10% FCS to reach confluent. Subsequently,
the medium was replaced with a serum-free DF (DMEM/F-12) medium. 3
days later, conditioned medium was collected. The obtained
conditioned medium was subjected to the Western blotting method
using OST311-specific polyclonal antibody 311-148 or 311-180
described in Example 22, thereby confirming the expression of each
relevant protein at a position corresponding to each predicted
molecular weight.
[0419] (3) Experiment of Transplanting CHO Cells Expressing
C-Terminus-Deleted OST311-
[0420] CHO cells expressing the above 20, 40, 45 and 50
residues-deleted OST311 were separately transplanted subcutaneously
into nude mice (6-week-old, BALB/c-nude, male, 6 mice per group) in
a manner similar to the method described in Example 13. As control
groups, the full-length OST311RQH-expressing CHO cells and CHO ras
clone-1 cells were separately transplanted subcutaneously (n=6).
Each group of mice was housed in a plastic cage and allowed to
access to tap water and solid food CE2 (CLEA JAPAN, Japan) ad
libitum.
[0421] On day 3 after cell transplantation, blood was collected
from the heart, and then serum phosphate and calcium levels, and
1,25-dihydroxyvitamin D3 levels were measured by a method similar
to that described in Example 20. As shown in FIG. 25, in all of
CHO-OST311RQ-.DELTA.C20, -.DELTA.C40, -.DELTA.C45 and -.DELTA.C50
cells-transplanted groups, significant decreases in serum phosphate
levels, equivalent to that of the group transplanted with the cells
expressing the full-length OST311RQH, were observed (t-test,
**p<0.001). Moreover, significant decreases in serum
1,25-dihydroxyvitamin D3 levels were also observed in
CHO-OST311RQ-.DELTA.C40, -.DELTA.C45 and -.DELTA.C50
cells-transplanted groups (when an average serum level of CHO-ras
clone-1-transplanted group was defined as 100%, full-length: 3.1%,
.DELTA.C40: 9.4%, .DELTA.C45: 10.0% and .DELTA.C 50: 68.1%). These
results clearly showed that even when at least 50 amino acids were
deleted from the C-terminus of OST311 protein, serum
phosphate-decreasing activity or serum 1,25-dihydroxyvitamin D3
level-decreasing activity was maintained.
EXAMPLE 26
Examination of Activity of N-Terminus-Deleted OST311
[0422] (1) Construction of Expression System for OST311 Lacking 9
Amino Acid Residues of N-Terminus
[0423] The following oligo DNAs were synthesized.
18 OST311SGFW: (SEQ ID NO: 57)
aattccaccATGTTGGGGGCCCGCCTCAGGCTCTGGGTCTGTGCTTGTGC
AGCGTCTGCAGCATGAGCGTCCTgcatGC OST311SGRV: (SEQ ID NO: 58)
aattGCatgcAGGACGCTCATGCTGCAGACGCTGCACAAGGCACAGACCC
AGAGCCTGAGGCGGGCCCCCAACATggtgg
[0424] OST311SGFW is an oligo DNA which consists of a gene sequence
encoding a signal peptide portion consisting of amino acid residues
from the initiation methionine of OST311 to the 24.sup.th Ala of
SEQ ID NO: 2, and contains an EcoR I recognition sequence on its 5'
terminus. OST311SGRV is a complementary strand of OST311SGFW, and
contains an EcoR I recognition sequence on its 5' terminus. In
addition, the recognition site of a restriction enzyme Sph I has
been introduced into the 3' side of OST311SGFW and the 5' side of
OST311 SGRV. The 23.sup.rd Arg within the signal peptide sequence
is substituted with His by introduction of the Sph I recognition
site. The above oligo DNAs were annealed according to standard
methods, thereby obtaining double-stranded DNA fragments containing
EcoR I recognition sequences on both ends and Sph I recognition
sequence at a position corresponding to the 23.sup.rd amino acid
residue in the signal peptide, and encoding the full-length signal
peptide starting from the initiation methionine of OST311 and
containing one modified residue. The obtained DNA fragments were
inserted into EcoR I-digested pEAK8 vectors (EdgeBioSystems, USA).
Plasmid DNAs wherein both EF1 promoters and the above DNA fragments
existing within the vector in forward direction were then selected,
thereby obtaining plasmid pPKFGSG.
[0425] Next, the following primers were synthesized.
[0426] OST311dN9: ATATGCATGCCTCCAGCTGGGGTGGCCTGATCCAC (SEQ ID NO:
59).
[0427] OST311dN9 is a forward primer designed to contain a Sph I
recognition site on its 5' terminus, and the 24.sup.th Ala residue
of SEQ ID NO: 2 followed by an amino acid sequence starting from
the 34.sup.th Ser residue of the same. Using a combination of this
primer and a reverse primer OST311HNt (Example 19, SEQ ID NO: 46)
which has been designed to have an Not I recognition sequence and 6
histidine residues added to the C-terminal portion followed by a
termination codon, and OST311RQH/IRES-EGFP/pEAK8 plasmid DNA
described in Example 19(2) as a template, PCR amplification was
performed in the manner described in Example 25 (1). The obtained
PCR product was digested with Sph I and Not I, and then inserted
into the above-described, Sph I- and Not I-digested plasmid vector
pPKFGSG according to standard methods.
[0428] The nucleotide sequence of the obtained plasmid
OST311.DELTA.N9-pPKFGSG was determined using ABI3700 fluorescence
DNA sequencer (PE Applied Biosystems, USA), so that the inserted
gene sequence was confirmed to contain a signal peptide from the
initiation methionine to the 24.sup.th Ala of OST311RQH gene
(wherein the 23.sup.rd Arg had been substituted with His), contain
deletion of only a gene sequence corresponding to 9 amino acid
residues from the following 25.sup.th Tyr to the 33.sup.rd Gly, and
encode the whole sequence from the 34.sup.th Ser to the termination
codon containing the histidine tag.
[0429] (2) Isolation of the CHO cells stably expressing recombinant
OST311.DELTA.N9-pPKFGSG plasmid DNA was introduced into CHO ras
clone-1 cells using Transfectam (Promega, USA) according to the
attached manufacturer's manual, and then CHO-OST311RQ-.DELTA.N9
cells showing drug resistance in MEM.alpha. medium containing 5
.mu.g/ml puromycin and 10% FSC were obtained. The conditioned
medium was collected from the obtained cells in the manner
described in Example 25. Western blotting was then performed using
OST311 specific polyclonal antibody 311-148 described in Example 22
or a polyclonal antibody 311-237 newly obtained by immunizing a
rabbit with a partial polypeptide from the 237.sup.th Gly to
251.sup.st Ile of SEQ ID NO: 2. Thus, expression of a relevant
protein at the position corresponding to a predicted molecular
weight was confirmed. These results revealed that OST311 signal
peptide, wherein the 23.sup.rd Arg of SEQ ID NO: 2 had been
substituted with His, functioned properly enough to secrete OST311
recombinant protein, and even when at least a portion from the
25.sup.th Tyr to 33.sup.rd Gly had been deleted, the recombinant
protein could be present stably to some extent in the cultured
medium after secretion.
[0430] (3) Experiment of Transplanting CHO Cells Expressing
N-Terminal 9 Amino Acids-Deleted OST311
[0431] The above CHO-OST311RQ-.DELTA.N9 cells were subcutaneously
transplanted to nude mice (8-week-old, BALB/c-nude, male, 6 mice
per group) in a manner similar to the methods described in Example
13. As control groups, subcutaneous transplantation of full-length
OST311RQH-expressing CHO cells and of CHO ras clone-1 cells were
respectively performed similarly (n=6). Each group of the nude mice
was housed in a plastic cage and allowed to access to tap water and
solid food CE-2 (CLEA JAPAN, Japan) ad libitum.
[0432] On day 4 after cell transplantation, blood was collected
from the orbital cavity using glass-made capillaries, and then
serum phosphate levels were measured in the manner described in
Example 20. Thus, in CHO-OST311RQ-.DELTA.N9 cells-transplanted
group, a significant decrease in serum phosphate levels, which was
equivalent to that of the full-length recombinant-expressing
cells-transplanted group, was observed (CHO-ras clone-1 group:
6.85.+-.0.12 mg/dL, CHO-OST311RQH group: 3.91.+-.0.23 mg/dL
(p<0.001, to CHO-ras clone-1 group), CHO-OST311RQ-.DELTA.N9
group: 4.33.+-.0.15 mg/dL (p<0.001, to CHO-ras clone-1
group).
[0433] These results revealed that even when at least 9 amino acid
residues consisting of the 25.sup.th Tyr to 33.sup.rd Gly of SEQ ID
NO: 2 was deleted, the biological activity of OST311 remained
undamaged.
EXAMPLE 27
Examination of Escherichia coli-Producing OST311 Recombinant
[0434] (1) Construction of OST311 Escherichia coli Expression
Vector OST311/pET3a
[0435] The following primers were synthesized.
19 OST311N: TGTATCCCAATGCCTCCCCACTG (SEQ ID NO: 60) OST311Bm:
ATGGATCCCTAGATGAACTTGGCGAAGGG (SEQ ID NO: 61)
[0436] PCR was performed using as a template OST311/pCAGGS plasmid
prepared in Example 19, OST311N (SEQ ID NO: 60) and OST311Bm (SEQ
ID NO: 61) as primers, and pfu DNA polymerase (Promega, USA). After
the temperature was maintained at 94.degree. C. for 1 minute,
reaction was performed for 35 cycles, each cycle consisting of
94.degree. C. for 30 seconds, 55.degree. C. for 30 seconds and
72.degree. C. for 1 minute. After reaction, phenol/chloroform
treatment was performed to inactivate the enzyme, and then DNA was
collected by ethanol precipitation. The DNA was digested with BamH
I, the target OST311 cDNA fragments were separated by 2% agarose
gel electrophoresis, and then collected using Gene Clean II
(BIO101, USA). Meanwhile, plasmid vector pET3a (Novagen, USA) was
digested with Nde I, and then blunt-ended using Klenow fragments
(Roche, Swiss). The vector was further digested with BamH I, the
obtained plasmid DNA fragment was separated by 0.8% agarose gel
electrophoresis, and then collected using Gene Clean II (BIO101,
USA). The thus obtained OST311 cDNA fragment was ligated to the
digested plasmid pET3a using a DNA ligation kit version 2 (TAKARA
SHUZO, Japan). The product was then introduced into Escherichia
coli DH5.alpha. for cloning, and then a plasmid was extracted. The
nucleotide sequence of the plasmid was confirmed to make sure that
OST311 cDNA had been inserted into pET3a as expected. The plasmid
was named OST311/pET3a.
[0437] (2) Construction of OST311/pET28 Vector for Expression of
OST311 in Escherichia coli
[0438] The following primers were synthesized.
20 OST311Nd: ATCATATGTATCCCAATGCCTCCCCACTG (SEQ ID NO: 62)
OST311Not: ATGCGGCCGCCTAGATGAACTTGGCGAAGGG (SEQ ID NO: 63)
[0439] PCR was performed using OST311/pET3a plasmid as a template,
OST311Nd (SEQ ID NO: 62) and OST311Not (SEQ ID NO: 63) as primers,
and LA Taq (TAKARA SHUZO, Japan). After the temperature was
maintained at 94.degree. C. for 1 minute, reaction was performed
for 35 cycles, each cycle consisting of 94.degree. C. for 30
seconds, 55.degree. C. for 30 seconds and 72.degree. C. for 1
minute. After reaction, phenol/chloroform treatment was performed
to inactivate the enzyme. The amplified DNA fragment was then
collected by ethanol precipitation. The DNA fragment was digested
with Nde I and Not I, the target OST311 cDNA fragment was separated
by 2% agarose gel electrophoresis, and then collected using Gene
Clean II (BIO101, USA). Meanwhile, plasmid vector pET28 (Novagen,
USA) was digested with Nde I and Not I, and then dephosphorylated
using bovine intestinal alkaline phosphatase (TAKARA SHUZO, Japan).
The product was then separated by 0.8% agarose gel electrophoresis,
and then the relevant digested plasmid was collected using Gene
Clean II (BIO101, USA). The thus obtained OST311cDNA was ligated to
the digested pET28 plasmid using a DNA ligation kit version 2
(TAKARA SHUZO, Japan), and then the ligated product was introduced
into Escherichia coli DH5.alpha. for cloning, thereby extracting a
plasmid. The nucleotide sequence of the plasmid was confirmed to
make sure that OST311 cDNA which allowed expression of recombinant
OST311 having a His-Tag sequence added to the N-terminal side had
been inserted into pET28. This plasmid was named OST311/pET28. The
amino acid sequence and the nucleotide sequence of the recombinant
His-OST311 encoded by this vector are shown in FIG. 26.
[0440] (3) Expression of Recombinant His-OST311 in Escherichia coli
and Preparation of the Same
[0441] Plasmid OST311/pET28 was introduced into Escherichia coli
BL21 (DE3) Codon Plus RP (STRATAGENE, USA) for transformation, and
then clones were obtained. The obtained Escherichia coli clones
were inoculated in 100 ml of LB medium containing 10 mg of
kanamycin (SIGMA, USA) and cultured at 37.degree. C. overnight. The
bacterial cell suspension was inoculated in 1 L of LB medium to
A.sub.600=0.1, and then shake-cultured using a 3 L Sakaguchi flask
at 37.degree. C. Absorbance of the culture suspension was measured
with time. When it reached A.sub.600=0.6 to 1.0,
isopropyl-1-thio-.beta.-galactoside (IPTG) (Wako Pure Chemical
Industries, Japan) was added to 1 mM. 4 hours later, the cells were
collected by centrifugation (7700 g.times.15 minutes). The
collected cells were suspended in 20 ml of 0.1 M Tris hydrochloride
buffer (pH 7.5) containing 1 mM DTT, and then disrupted using
French Press. The solution containing the disrupted cells was
centrifuged (7700 g.times.15 minutes), and then the precipitate
fraction was suspended in 15 ml of 0.1 M Tris hydrochloride buffer
(pH 7.5). DNase I (Roche, Swiss) was added to the suspension to 0.1
mg/mL, and then shaken at 4.degree. C. for 1 hour. Next,
centrifugation was performed (23400 g.times.15 minutes), and then
the precipitate fraction was collected as inclusion body. The
obtained inclusion body was washed by suspending in 10 ml of 20 mM
Tris hydrochloride buffer (pH 8) containing 0.75 M urea and 1%
Triton-X, and then centrifuging (23,400 g.times.15 minutes) to
collect precipitate. This washing procedure was repeated twice.
[0442] The washed inclusion body was suspended in 5 ml of a
denaturing solution (50 mM phosphate buffer (pH 8) containing 1 mM
DTT and 6 M guanidine hydrochloride), and then solubilized by
shaking the suspension at 37.degree. C. for 1 hour. Insoluble
matters were removed as precipitate by centrifugation (23,400
g.times.15 minutes), and then the solution was equilibrated with 6
M guanidine hydrochloride-containing 50 mM phosphate buffer (pH 6).
The solubilized sample was applied to a column filled with Ni-NTA
Agarose (QIAGEN, Germany), and then washed with 6 M guanidine
hydrochloride-containing 50 mM phosphate buffer (pH 6). Protein
adsorbed to the column was eluted using 50 mM phosphate buffer (pH
4.5) containing 500 mM imidazole (Nacalai Tesque, Japan) and 6 M
guanidine hydrochloride, thereby purifying denatured His-OST311.
The concentration was obtained based on UV absorbance at 280 nm of
the purified sample, and then 50 mM phosphate buffer (pH 6)
containing 6 M guanidine hydrochloride was added to the sample to
have a final concentration of 2 mg/ml, thereby preparing denatured
His-OST311 solution. Cysteine as a reducing agent was added to the
sample to have a final concentration of 1 mM, diluted 100 fold with
20 mM phosphate buffer (pH 6) containing 0.6 M guanidine
hydrochloride and 0.1% Tween 20 to start refolding. Incubation was
performed at 4.degree. C. for 3 days or more.
[0443] The refolding solution was dialyzed against 0.1 M acetate
buffer (pH 4.8) at 4.degree. C. The dialyzed refolding solution was
concentrated approximately 10 fold using a ultrafiltration
membrane, and then purified by HPLC using cation exchange column
SP-5PW (TOSOH, Japan). Protein was eluted using 10%
glycerol-containing 20 mM phosphate buffer (pH 6) with a linear
NaCl gradient from 0.5 M to 2 M. This elution pattern is shown in
FIG. 27. SDS-PAGE analysis and mass spectrometry measurement
revealed that among eluted two types of protein peaks, His-OST311
was contained in a peak eluted at a lower salt concentration. As
described above, approximately 0.6 mg of the final purified
product, His-OST311, could be prepared from approximately 1 L of
cultured cells.
[0444] (4) Construction of pET22b-MK-OST311 Vector for Expression
of MK-OST311
[0445] The following primers were synthesized.
[0446] OST311MK1: gaattcatatgaaatacccgaacgcttccccgctgctgggctccagctg
(SEQ ID NO: 64)
[0447] OST311MK2: cccaagcttgcggccgcctagatgaacttggc (SEQ ID NO:
65)
[0448] A target sequence was amplified by PCR using the above
His-OST311 expression plasmid OST311/pET28 as a template and
OST311MK1 (SEQ ID NO: 64) and OST311MK2 (SEQ ID NO: 65) as primers.
In the OST311 cDNA obtained by this procedure, 27 nucleotides
following the initiation codon (ATG) had been converted to
Escherichia coli type codons. The PCR product was purified using a
QIAquick PCR purification Kit (QIAGEN, Germany), and then digested
with restriction enzymes Nde I (TAKARA SHUZO, Japan) and Not I
(TAKARA SHUZO, Japan) at 37.degree. C. for 1 hour. The digested PCR
product was separated by agarose electrophoresis, and then purified
using a QIAquick PCR purification Kit (QIAGEN, Germany). The
obtained DNA fragment was digested with restriction enzymes Nde I
and Not I at 37.degree. C. for 1 hour, and then ligated to plasmid
vector pET22b (Novagen, USA), which had been separated and purified
by agarose electrophoresis, using a DNA Ligation kit Ver 2 (TAKARA
SHUZO, Japan) at 16.degree. C. for 15 minutes. The ligated product
was introduced into Escherichia coli JM109 (TAKARA SHUZO, Japan)
for cloning, and then a plasmid was extracted by standard methods.
The nucleotide sequence of the obtained plasmid was determined to
make sure that the obtained OST311 cDNA had been inserted into
pET22b vector as expected. This plasmid was named pET22-MK-OST311.
The nucleotide sequence and amino acid sequence of recombinant
MK-OST311 encoded by the vector are shown in FIG. 26.
[0449] (5) Expression of MK-OST311 in Escherichia coli and
Preparation of the Same
[0450] Plasmid pET22-MK-OST311 was introduced into Escherichia coli
BL21(DE3) Codon Plus RP (STRATAGENE, USA), and then the transformed
clones were obtained. The obtained Escherichia coli clones were
inoculated in 100 ml of LB medium containing 10 mg of ampicillin
and then cultured at 37.degree. C. overnight. The bacterial cell
suspension was inoculated in 1 L of LB medium to A.sub.600=0.1, and
then cultured with shaking using a 3 L Sakaguchi flask at
37.degree. C. IPTG was added to the bacterial cell culture to
induce expression of the recombinant, and then inclusion bodies
were prepared in a manner similar to the above preparation method
of His-OST311.
[0451] The washed inclusion bodies were suspended in 5 ml of
denaturing solution (50 mM phosphate buffer (pH 8) containing 1 mM
DTT and 6 M guanidine hydrochloride), and then solubilized by
shaking the suspension at 37.degree. C. for 1 hour. The solubilized
product was diluted 2 fold using a denaturing solution, and then
diluted 100 fold using 20 mM phosphate buffer (pH 6) containing 0.6
M guanidine hydrochloride and 0.1% Tween 20 to start refolding.
Incubation was performed at 4.degree. C. for 3 days or more. It was
shown that under conditions of addition of oxidant and a pH of 7 or
more, protein was precipitated so that refolding efficiency
decreased significantly. The refolding solution was dialyzed
against 0.1 M acetate buffer (pH 4.8) at 4.degree. C. The dialyzed
refolding solution was concentrated approximately 10 fold using an
ultrafiltration membrane, and then purified by HPLC using cation
exchange column SP-5PW (TOSOH, Japan). Protein was eluted using 10%
glycerol-containing 20 mM phosphate buffer (pH 6) with a linear
NaCl gradient from 0.5 M to 2 M. As shown in FIG. 28, there were 2
peaks of protein elution, and SDS-PAGE analysis and mass
spectrometry measurement revealed that MK-OST311 was contained in a
peak eluted at a lower salt concentration. Thus, approximately 0.6
mg of the final purified product, MK-OST311, could be purified from
approximately 1 L of flask-cultured cells.
[0452] (6) PEGylation of MK-OST311
[0453] 10 ml of MK-OST311 (0.05 mg/ml) purified with an ion
exchange column was adjusted to have pH 4.8 using 10% acetic acid.
25 mg of activated PEG (Sharewater, USA) with a molecular weight of
20,000 dissolved in 10 mM acetate buffer (pH 4.8) was added to this
solution with agitation in ice. 15 minutes later, 1 M sodium cyano
borohydride (Nacalai Tesque, Japan) dissolved in 10 mM acetate
buffer (pH 4.8) was added to the solution to have a final
concentration of 15 mM, and then the solution was agitated at
4.degree. C. for 16 hours. OST311 PEGylated by this reaction was
purified by HPLC using cation exchange column SP-5PW (TOSOH,
Japan). Protein was eluted using 10% glycerol-containing 20 mM
phosphate buffer (pH 6) with a linear NaCl gradient from 0.5 M to 2
M. As shown in FIG. 29, PEGylated MK-OST311 was eluted as a single
peak on the side of lower ion strength compared to that of
MK-OST311.
[0454] (7) Examination of Activity of His-OST311 Recombinant
[0455] To examine the biological activity of the purified
His-OST311 recombinant, single administration of the recombinant
protein, 4.5 .mu.g/0.1 ml each, to normal mice (5-week-old, BALB/c,
male, 6 mice per group) via the caudal vein in the manner described
in Example 24 was performed. 9 hours later, serum phosphate and
1,25-dihydroxyvitamin D3 levels were measured by a method similar
to that described in Example 20. Single administration of the same
dose of CHO-OST311H cell-derived purified recombinant was performed
to a positive control group, and single administration of a vehicle
comprising 20 mM phosphate buffer (pH 6.9) and 0.3 M NaCl was
performed, 0.1 ml each, to a vehicle-administered group, both via
the caudal vein.
[0456] As shown in FIG. 30A, in His-OST311-administered group at 9
hours after administration, a significant effect of decreasing
serum phosphate levels was observed compared to the
vehicle-administered group. The degree of the decrease was
equivalent to that of CHO-producing recombinant
protein-administered group. In addition, the serum
1,25-dihydroxyvitamin D3 levels at 9 hours after administration in
His-OST311-administered group also showed a significant decrease as
shown in FIG. 32.
[0457] As described in Example 24 (3) and (4), significantly
decreased serum 1,25-dihydroxyvitamin D3 levels were already
observed at 4 hours after single administration of CHO
cell-producing OST311 protein. Before this time point, at 1 hour
after administration, decreased expression of 25-hydroxyvitamin
D-1-.alpha.-hydroxylase (1.alpha.OHase) and enhanced expression of
25hydroxyvitamin D-24-hydroxylase (24OHase) were observed in the
kidneys. Hence, single administration of His-OST311, 4.5 .mu.g/0.1
ml per mouse, to BALB/c mice (5-week-old, male) was performed via
the caudal vein, and then the kidneys were excised 1 and 4 hours
later. Changes in the expression of 1.alpha.OHase gene and 24OHase
gene in the kidneys were analyzed by the Northern blotting method.
Single administration of the same dose of CHO-OST311H cell-derived
purified recombinant was performed to a positive control group, and
single administration of a vehicle comprising 20 mM phosphate
buffer (pH 7.0) and 0.3 M NaCl was performed, 0.1 ml each, to a
vehicle-administered group, both via the caudal vein. As shown in
FIG. 31, similar to CHO-producing recombinant, His-OST311 caused
decreased expression of 1.alpha.OHase gene and enhanced expression
of 24OHase gene already at 1 hour after administration. It was
revealed that His-OST311 has activity regulating expression of
vitamin D-metabolizing enzyme genes equivalent to that of the
CHO-producing recombinant. Further, as shown in FIG. 32, changes in
serum 1,25-dihydroxyvitamin D3 levels showed a moderate decrease at
4 hours after administration with recombinant, and showed a more
significant decrease at 8 hours. It was revealed that this manner
of changes was almost consistent with that of changes with time as
observed in the experiment of administering CHO-producing
recombinant described in Example 24 (3).
[0458] From the above results, it was revealed that recombinant
His-OST311 produced by Escherichia coli has biological activities
of at least serum phosphate-decreasing activity and vitamin D
metabolism-regulating activity equivalent to the activities of the
secreted recombinant produced by CHO-OST311H cells.
[0459] (8) Examination of Activity of PEGylated MK-OST311
[0460] The biological activity of PEGylated MK-OST311 was examined
by performing single administration of PEGylated MK-OST311, 5.0
.mu.g/0.1 ml per mouse, to normal mice (5-week-old, BALB/c, male, 8
mice per group) via the caudal vein in the manner described in
Example 24, and then measuring serum phosphate levels at 9 hours
after administration by a method similar to that of Example 20.
Single administration of a vehicle comprising 20 mM PB (pH6.0), 10%
glycerol, 1 M NaCl and 0.1% Tween 20 was performed, 0.1 ml/mouse,
to a vehicle-administered group via the caudal vein. At 8 hours
after administration, blood was collected from the orbital cavity
using glass-made capillaries, and then inorganic phosphate levels
of the obtained sera were measured. As shown in FIG. 30B, a
significant effect of decreasing serum phosphate levels was
observed in PEGylated MK-OST311-administered group, compared to the
vehicle-administered group. The result reveled that when
Escherichia coli-producing recombinant was PEGylated, the
biological activity of it is not inhibited.
EXAMPLE 28
Introduction of Amino Acid Mutation into Cleavage Site
[0461] As described in Example 9, it was shown that OST311 was
cleaved at a position between the 179.sup.th Arg and the 180.sup.th
Ser amino acid residues of SEQ ID NO: 2. Meanwhile, it was
confirmed as described in Example 19 that this cleavage was
inhibited by simultaneous substitution of both 176.sup.th Arg and
179.sup.th Arg amino acid residues of OST311 with Gln. These facts
suggest a possibility that this cleavage is due to some protease
which recognizes a motif consisting of adjacent RXXR or RRXXR
sequence. Moreover, when full-length recombinant consisting of the
polypeptide represented by SEQ ID NO: 4 is administered in vivo,
the same cleavage as described above or similar cleavage thereto
may occur. Hence, the 175.sup.th to the 180.sup.th amino acid
residues of SEQ ID NO: 2 were respectively substituted with Ala,
Gln or Trp, and then how the substitution affected expression and
secretion patterns in each mutant recombinant in CHO cells was
examined.
[0462] (1) Construction of OST311 Gene with Mutations at Cleavage
Site
[0463] The following primers were synthesized.
21 pyh23PA1F AACACCCCCATAGCACGGCGGCACA (SEQ ID NO: 66) pyh23PA1R
TGTGCCGCCGTGCTATGGGGGTGTT (SEQ ID NO: 67) pyh23RA1F
ACCCCCATACCAGCGCGGCACACCCG (SEQ ID NO: 68) pyh23RA1R
CGGGTGTGCCGCGCTGGTATGGGGGT (SEQ ID NO: 69) pyh23RA2F
CCCATACCACGGGCGCACACCCGGAG (SEQ ID NO: 70) pyh23RA2R
CTCCGGGTGTGCGCCCGTGGTATGGG (SEQ ID NO: 71) pyh23HA1F
ATACCACGGCGGGCCACCCGGAGCGC (SEQ ID NO: 72) pyh23HA1R
GCGCTCCGGGTGGCCCGCCGTGGTAT (SEQ ID NO: 73) pyh23TA1F
CCACGGCGGCACGCCCGGAGCGCCG (SEQ ID NO: 74) pyh23TA1R
CGGCGCTCCGGGCGTGCCGCCGTGG (SEQ ID NO: 75) pyh23RA3F
CGGCGGCACACCGCGAGCGCCGAGGA (SEQ ID NO: 76) pyh23RA3R
TCCTCGGCGCTCGCGGTGTGCCGCCG (SEQ ID NO: 77) pyh23SA1F
CGGCACACCCGGGCCGCCGAGGACGA (SEQ ID NO: 78) pyh23SA1R
TCGTCCTCGGCGGCCCGGGTGTGCCG (SEQ ID NO: 79) pyh23RKQ1F
ACCCCCATACCACAGCGGCACACCCG (SEQ ID NO: 80) pyh23RKQ1R
CGGGTGTGCCGCTGTGGTATGGGGGT (SEQ ID NO: 81) pyh23RKQ2F
CCCATACCACGGCAGCACACCCGGAG (SEQ ID NO: 82) pyh23RKQ2R
CTCCGGGTGTGCTGCCGTGGTATGGG (SEQ ID NO: 83) pyh23RKQ3F
CGGCGGCACACCCAGAGCGCCGAGGA (SEQ ID NO: 84) pyh23RKQ3R
TCCTCGGCGCTCTGGGTGTGCCGCCG (SEQ ID NO: 85) pyh23RWF
CGGCGGCACACCTGGAGCGCCGAGG (SEQ ID NO: 86) pyh23RWR
CCTCGGCGCTCCAGGTGTGCCGCCG (SEQ ID NO: 87)
[0464] pyh23PA1F and pyh23PA1R are forward and reverse primers for
introducing a mutation, in which substitution of the 652.sup.nd
cytosine of OST311 cDNA (SEQ ID NO: 1) with guanine causes
substitution of the 174.sup.th Pro amino acid residue of SEQ ID NO:
2 with Ala. Hereinafter, this mutation is referred to as P174A.
[0465] pyh23RA1F and pyh23RA1R are forward and reverse primers for
introducing a mutation, in which substitution of the 655.sup.th
cytosine and the 656.sup.th guanine of OST311 cDNA (SEQ ID NO: 1)
with guanine and cytosine, respectively, causes substitution of the
175.sup.th Arg amino acid residue of SEQ ID NO: 2 with Ala.
Hereinafter, this mutation is referred to as R175A.
[0466] pyh23RA2F and pyh23RA2R are forward and reverse primers for
introducing a mutation, in which substitution of the 658.sup.th
cytosine and the 659.sup.th guanine of OST311 cDNA (SEQ ID NO: 1)
with guanine and cytosine, respectively, causes substitution of the
176.sup.th Arg amino acid residue of SEQ ID NO: 2 with Ala.
Hereinafter, this mutation is referred to as R176A.
[0467] pyh23HA1F and pyh23HA1R are forward and reverse primers for
introducing a mutation, in which substitution of the 661.sup.st
cytosine and the 662.sup.nd adenine of OST311 cDNA (SEQ ID NO: 1)
with guanine and cytosine, respectively, causes substitution of the
177.sup.th His amino acid residue of SEQ ID NO: 2 with Ala.
Hereinafter, this mutation is referred to as H177A.
[0468] pyh23TA1F and pyh23TA1R are forward and reverse primers for
introducing a mutation, in which substitution of the 664.sup.th
adenine of OST311 cDNA (SEQ ID NO: 1) with guanine causes
substitution of the 178.sup.th Thr amino acid residue of SEQ ID NO:
2 with Ala. Hereinafter, this mutation is referred to as T178A.
[0469] pyh23RA3F and pyh23RA3R are forward and reverse primers for
introducing a mutation, in which substitution of the 667.sup.th
cytosine and the 668.sup.th guanine of OST311 cDNA (SEQ ID NO: 1)
with guanine and cytosine, respectively, causes substitution of the
179.sup.th Arg amino acid residue, of SEQ ID NO: 2 with Ala.
Hereinafter, this mutation is referred to as R179A.
[0470] pyh23SA1F and pyh23SA1R are forward and reverse primers for
introducing a mutation, in which substitution of the 670.sup.th
adenine and the 671.sup.st guanine of OST311 cDNA (SEQ ID NO: 1)
with guanine and cytosine, respectively, causes substitution of the
180.sup.th Ser amino acid residue of SEQ ID NO: 2 with Ala.
Hereinafter, this mutation is referred to as S180A.
[0471] pyh23RKQ1F and pyh23RKQ1R are forward and reverse primers
for introducing a mutation, in which substitution of the 656.sup.th
guanine of OST311 cDNA (SEQ ID NO: 1) with adenine causes
substitution of the 175% Arg amino acid residue of SEQ ID NO: 2
with Gln. Hereinafter, this mutation is referred to as R175Q.
[0472] pyh23RKQ2F and pyh23RKQ2R are forward and reverse primers
for introducing a mutation, in which substitution of the 659.sup.th
guanine of OST311 cDNA (SEQ ID NO: 1) with adenine causes
substitution of the 176.sup.th Arg amino acid residue of SEQ ID NO:
2 with Gln. Hereinafter, this mutation is referred to as R176Q.
[0473] pyh23RKQ3F and pyh23RKQ3R are forward and reverse primers
for introducing a mutation, in which substitution of the 668.sup.th
guanine of OST311 cDNA (SEQ ID NO: 1) with adenine causes
substitution of the 179.sup.th Arg amino acid residue of SEQ ID NO:
2 with Gln. Hereinafter, this mutation is referred to as R179Q.
[0474] pyh23RWF and pyh23RWR are forward and reverse primers for
introducing a mutation, in which substitution of the 667.sup.th
cytosine of OST311 cDNA (SEQ ID NO: 1) with thymine causes
substitution of the 179.sup.th Arg amino acid residue of SEQ ID NO:
2 with Trp. Hereinafter, this mutation is referred to as R179W;
[0475] (1)-1 Construction of OST311P174AH Gene
[0476] 2 types of reaction solutions (100 .mu.L each) were prepared
using Pyrobest DNA polymerase (TAKARA SHUZO, Japan) according to
the attached manufacturer's manual. For one reaction solution,
OST311ME1 (SEQ ID NO: 45) and pyh23PA1F (SEQ ID NO: 66) were used
as primers at a final concentration of 0.2 .mu.M, and for the other
reaction solution, pyh23PA1R (SEQ ID NO: 67) and OST311HNt (SEQ ID
NO: 46) were used as primers at a final concentration of 0.2 .mu.M.
To each reaction solution, 10 ng of OST311/pCAGGS plasmid described
in Example 19(1) was added as a template, and then the solution was
maintained at 94.degree. C. for 1 minute. Then, PCR reaction was
performed for 40 cycles, each cycle consisting of 94.degree. C. for
20 seconds, 55.degree. C. for 30 seconds and 72.degree. C. for 1
minute. The two types of reaction solutions were diluted
respectively 10 fold, and then 1 .mu.L of each solution was added
to 100 .mu.L of a reaction solution prepared according to the
document attached to Pyrobest DNA polymerase (TAKARA SHUZO, Japan).
OST311ME1 (SEQ ID NO: 45) and OST311HNt (SEQ ID NO: 46) were added
as primers to the solution to have a final concentration of 0.2
.mu.M, and then the solution was maintained at 94.degree. C. for 1
minute. Then, PCR reaction was performed for 30 cycles, each cycle
consisting of 94.degree. C. for 20 seconds, 55.degree. C. for 30
seconds and 72.degree. C. for 1 minute and 30 seconds. After PCR
reaction, the solution was further maintained at 72.degree. C. for
7 minutes. The thus obtained reaction products were collected using
Gene Clean II (BIO101, USA) according to the attached
manufacturer's manual. The products were then digested with EcoR I
and Not I, and then subjected to 2% agarose gel electrophoresis to
separate approximately 800 bp DNA fragments. The fragment was
collected using Gene Clean II (BIO101, USA). The thus obtained DNA
fragments were inserted into EcoR I and Not I sites of pEAK8 vector
(EdgeBio, USA), thereby obtaining plasmid OST311P174AH-pEAK8.
Plasmid DNA was prepared according to standard methods, and the
nucleotide sequence was determined using ABI3700 fluorescence DNA
sequencer (PE Applied Systems, USA). Thus, it was confirmed that
mutation P174H had been introduced as expected. Moreover, it was
confirmed that a histidine tag had been added to the C-terminus.
The polypeptide encoded by a mutant gene having mutation P174A
introduced therein is referred to as OST311P174AH.
[0477] (1)-2 Preparation of OST311R175AH Gene
[0478] OST311R175AH gene was prepared using pyh23RA1F (SEQ ID NO:
68) and pyh23RA1R (SEQ ID NO: 69) primers by a method similar to
that of (1)-1.
[0479] (1)-3 Preparation of OST311R176AH Gene
[0480] OST311R176AH gene was prepared using pyh23RA2F (SEQ ID NO:
70) and pyh23RA2R (SEQ ID NO: 71) primers by a method similar to
that of (1)-1.
[0481] (1)-4 Preparation of OST311H177AH Gene
[0482] OST311H177AH gene was prepared using pyh23HA1F (SEQ ID NO:
72) and pyh23HA1R (SEQ ID NO: 73) primers by a method similar to
that of (1)-1.
[0483] (1)-5 Preparation of OST311T178AH Gene
[0484] OST311T178AH gene was prepared using pyh23TA1F (SEQ ID NO:
74) and pyh23TA1R (SEQ ID NO: 75) primers by a method similar to
that of (1)-1.
[0485] (1)-6 Preparation of OST311R179AH Gene
[0486] OST311R179AH gene was prepared using pyh23RA3F (SEQ ID NO:
76) and pyh23RA3R (SEQ ID NO: 77) primers by a method similar to
that of (1)-1.
[0487] (1)-7 Preparation of OST311S180AH Gene
[0488] OST311S180AH gene was prepared using pyh23SA1F (SEQ ID NO:
78) and pyh23SA1R (SEQ ID NO: 79) primers by a method similar to
that of (1)-1.
[0489] (1)-8 Preparation of OST311R175QH Gene
[0490] OST311R175QH gene was prepared using pyh23RKQ1F (SEQ ID NO:
80) and pyh23RKQ1R (SEQ ID NO: 81) primers by a method similar to
that of (1)-1.
[0491] (1)-9 Preparation of OST311R176QH Gene
[0492] OST311R176QH gene was prepared using pyh23RKQ2F (SEQ ID NO:
82) and pyh23RKQ2R (SEQ ID NO: 83) primers by a method similar to
that of (1)-1.
[0493] (1)-10 Preparation of OST311R179QH Gene
[0494] OST311R179QH gene was prepared using pyh23RKQ3F (SEQ ID NO:
84) and pyh23RKQ3R (SEQ ID NO: 85) primers by a method similar to
that of (1)-1.
[0495] (1)-11 Preparation of OST311R179WH Gene
[0496] OST311R179WH gene was prepared using pyh23RWF (SEQ ID NO:
86) and pyh23RWR (SEQ ID NO: 87) primers by a method similar to
that of (1)-1.
[0497] (2) Transient Expression of OST311 Genes with Mutation at
Cleavage Site and Preparation of Conditioned Media
[0498] pEAK rapid cells (EdgeBiosystems, USA) were inoculated onto
a 12-well plate. The above 11 types of expression plasmids for
mutant OST311 were transfected into the cells by a phosphate
calcium method according to the document attached to pEAK system
(EdgeBiosystems, USA). The cells were allowed to stand for 4 hours,
the medium was replaced with 1.5 ml of serum-free MEM.alpha.
medium, the cells were cultured at 37.degree. C. for 2 days, and
then the conditioned medium was collected.
[0499] (3) Evaluation of Expression of OST311 Genes with the
Mutation at Cleavage Site
[0500] The thus obtained conditioned medium was subjected to
Western blotting in the manner described in Example 6(3), and then
the presence of OST311 recombinant with the mutation at cleavage
site in the conditioned medium was examined. OST311-specific
polyclonal antibody, 311-148 described in Example 22 was used for
detection. Thus, as shown in FIG. 33, a degradation product
containing the polypeptide represented by SEQ ID NO: 6 was observed
at around 16 kDa, similarly to the wild type, for mutation P174A,
R175A, R175Q, H177A, T178A or S180A-introduced OST311 recombinant
among those substituted at the 174.sup.th to 180.sup.th amino acid
residues. However, no degradation product was observed for any of
the mutations R176A, R179A, R176Q, R179Q or R179W-introduced OST311
recombinant. These results suggest that in particular the
176.sup.th Arg and the 179.sup.th Arg amino acid residues play an
important role in the cleavage that occurs between the 179.sup.th
Arg and the 180.sup.th Ser amino acid residues. That is, cleavage
is inhibited or suppressed by substitution of both residues at
least with any amino acid of Ala, Gln or Trp. Hence, it is expected
that production of the full-length polypeptide is promoted using
this finding.
EXAMPLE 29
[0501] As shown in Example 6, a recombinant product obtained by
expression of OST311 in CHO cells or COS cells is cleaved between
the 179.sup.th arginine and 180.sup.th serine located immediately
after RXXR motif as shown in Example 9. As clearly shown in Example
18, the hypophosphatemia-inducing activity of OST311 protein was
retained in the full-length protein that had not been cleaved at
this site. Further, involvement of RXXR motif in this cleavage was
clear from the mutation introduction experiment shown in Examples
19 and 28. In order to produce and obtain recombinant OST311
efficiently, avoidance of this cleavage is an important problem,
and introduction of the mutation into RXXR motif shown in Example
28 is one of the effective methods. Furin is known as one of
proteinases which recognize RXXR motif. This enzyme is localized in
the Trans-golgi region, and is thought to cleave post-translated
protein by recognizing RXXR motif in the process of secretion.
[0502] (1) Involvement of Furin in Cleavage of OST311
[0503] OST311/pCAGGS plasmid was introduced into furin-deficient
LoVo cells using Transfectum (Promega, USA). Then, the cells were
cultured for 48 hours. When OST311 protein transiently expressed
and secreted in the culture solution was analyzed by Western
blotting using 311-148 antibody, no cleaved product was detected.
This result suggests that furin is involved in the observed
cleavage when OST311 is produced.
[0504] (2) Avoiding Cleavage of OST311
[0505] It was assumed from the above results that inhibition of
furin activity was effective to improve the productivity of OST311,
in which it was not cleaved between the 179.sup.th arginine and the
180.sup.th serine. Accordingly, it is conceivable that OST311 is
expressed in a host, such as Lovo cells having no furin activity,
or under a condition in which furin activity is suppressed by the
addition of a furin inhibitor. In order to suppress furin activity
of CHO-OST311H cells, .alpha.1-antitrypsin Portland (.alpha.1-PDX)
was transiently expressed according to the method reported by
Benjannet S et al (J Biol Chem 272: 26210-8, 1997), and then the
conditioned medium was collected. The ratio of the full-length
polypeptide in the recombinant products was increased, compared to
that in the conditioned medium of the control CHO-OST311H cells
without this gene. Accordingly, it was concluded that the
production efficiency of the full-length OST311 protein can be
elevated by introducing a substance suppressing furin activity
extrinsically or intrinsically.
[0506] All publications, patents and patent applications cited
herein are incorporated herein by reference in their entirety.
INDUSTRIAL APPLICABILITY
[0507] According to the present invention, there are provided a
polypeptide regulating phosphate metabolism, calcium metabolism
and/or calcification, a DNA encoding the polypeptide, and a
pharmaceutical composition containing the polypeptide as an active
ingredient, and an antibody recognizing the polypeptide, a
pharmaceutical composition containing the antibody as an active
ingredient, a diagnostic method using the antibody, and a
diagnostic composition.
Sequence Listing Free Text
[0508] SEQ ID NO: 12: Synthetic DNA
[0509] SEQ ID NO: 13: Synthetic DNA
[0510] SEQ ID NO: 14: Synthetic DNA
[0511] SEQ ID NO: 15: Synthetic DNA
[0512] SEQ ID NO: 16: Synthetic DNA
[0513] SEQ ID NO: 17: Synthetic DNA
[0514] SEQ ID NO: 18: Synthetic DNA
[0515] SEQ ID NO: 19: Synthetic DNA
[0516] SEQ ID NO: 20: Synthetic DNA
[0517] SEQ ID NO: 21: Synthetic DNA
[0518] SEQ ID NO: 22: Synthetic DNA
[0519] SEQ ID NO: 23: Synthetic DNA
[0520] SEQ ID NO: 24: Synthetic DNA
[0521] SEQ ID NO: 25: Synthetic DNA
[0522] SEQ ID NO: 26: Synthetic DNA
[0523] SEQ ID NO: 27: Synthetic DNA
[0524] SEQ ID NO: 28: Synthetic peptide
[0525] SEQ ID NO: 29: Synthetic peptide
[0526] SEQ ID NO: 30: Synthetic DNA
[0527] SEQ ID NO: 31: Synthetic DNA
[0528] SEQ ID NO: 32: Synthetic DNA
[0529] SEQ ID NO: 33: Synthetic DNA
[0530] SEQ ID NO: 34: Synthetic DNA
[0531] SEQ ID NO: 35: Synthetic DNA
[0532] SEQ ID NO: 36: Synthetic DNA
[0533] SEQ ID NO: 37: Synthetic DNA
[0534] SEQ ID NO: 38: Synthetic DNA
[0535] SEQ ID NO: 39: Synthetic DNA
[0536] SEQ ID NO: 40: Synthetic DNA
[0537] SEQ ID NO: 41: Synthetic DNA
[0538] SEQ ID NO: 42: Synthetic DNA
[0539] SEQ ID NO: 43: Synthetic DNA
[0540] SEQ ID NO: 44: Synthetic DNA
[0541] SEQ ID NO: 45: Synthetic DNA
[0542] SEQ ID NO: 46: Synthetic DNA
[0543] SEQ ID NO: 47: Synthetic DNA
[0544] SEQ ID NO: 48: Synthetic DNA
[0545] SEQ ID NO: 49: Synthetic peptide
[0546] SEQ ID NO: 50: Synthetic peptide
[0547] SEQ ID NO: 51: Synthetic peptide
[0548] SEQ ID NO: 52: Synthetic peptide
[0549] SEQ ID NO: 53: Synthetic DNA
[0550] SEQ ID NO: 54: Synthetic DNA
[0551] SEQ ID NO: 55: Synthetic DNA
[0552] SEQ ID NO: 56: Synthetic DNA
[0553] SEQ ID NO: 57: Synthetic DNA
[0554] SEQ ID NO: 58: Synthetic DNA
[0555] SEQ ID NO: 59: Synthetic DNA
[0556] SEQ ID NO: 60: Synthetic DNA
[0557] SEQ ID NO: 61: Synthetic DNA
[0558] SEQ ID NO: 62: Synthetic DNA
[0559] SEQ ID NO: 63: Synthetic DNA
[0560] SEQ ID NO: 64: Synthetic DNA
[0561] SEQ ID NO: 65: Synthetic DNA
[0562] SEQ ID NO: 66: Synthetic DNA
[0563] SEQ ID NO: 67: Synthetic DNA
[0564] SEQ ID NO: 68: Synthetic DNA
[0565] SEQ ID NO: 69: Synthetic DNA
[0566] SEQ ID NO: 70: Synthetic DNA
[0567] SEQ ID NO: 71: Synthetic DNA
[0568] SEQ ID NO: 72: Synthetic DNA
[0569] SEQ ID NO: 73: Synthetic DNA
[0570] SEQ ID NO: 74: Synthetic DNA
[0571] SEQ ID NO: 75: Synthetic DNA
[0572] SEQ ID NO: 76: Synthetic DNA
[0573] SEQ ID NO: 77: Synthetic DNA
[0574] SEQ ID NO: 78: Synthetic DNA
[0575] SEQ ID NO: 79: Synthetic DNA
[0576] SEQ ID NO: 80: Synthetic DNA
[0577] SEQ ID NO: 81: Synthetic DNA
[0578] SEQ ID NO: 82: Synthetic DNA
[0579] SEQ ID NO: 83: Synthetic DNA
[0580] SEQ ID NO: 84: Synthetic DNA
[0581] SEQ ID NO: 85: Synthetic DNA
[0582] SEQ ID NO: 86: Synthetic DNA
[0583] SEQ ID NO: 87: Synthetic DNA
Sequence CWU 1
1
87 1 2770 DNA Homo sapiens CDS (133)..(885) 1 gaatccagtc taggatcctc
acaccagcta cttgcaaggg agaaggaaaa ggccagtaag 60 gcctgggcca
ggagagtccc gacaggagtg tcaggtttca atctcagcac cagccactca 120
gagcagggca cg atg ttg ggg gcc cgc ctc agg ctc tgg gtc tgt gcc ttg
171 Met Leu Gly Ala Arg Leu Arg Leu Trp Val Cys Ala Leu 1 5 10 tgc
agc gtc tgc agc atg agc gtc ctc aga gcc tat ccc aat gcc tcc 219 Cys
Ser Val Cys Ser Met Ser Val Leu Arg Ala Tyr Pro Asn Ala Ser 15 20
25 cca ctg ctc ggc tcc agc tgg ggt ggc ctg atc cac ctg tac aca gcc
267 Pro Leu Leu Gly Ser Ser Trp Gly Gly Leu Ile His Leu Tyr Thr Ala
30 35 40 45 aca gcc agg aac agc tac cac ctg cag atc cac aag aat ggc
cat gtg 315 Thr Ala Arg Asn Ser Tyr His Leu Gln Ile His Lys Asn Gly
His Val 50 55 60 gat ggc gca ccc cat cag acc atc tac agt gcc ctg
atg atc aga tca 363 Asp Gly Ala Pro His Gln Thr Ile Tyr Ser Ala Leu
Met Ile Arg Ser 65 70 75 gag gat gct ggc ttt gtg gtg att aca ggt
gtg atg agc aga aga tac 411 Glu Asp Ala Gly Phe Val Val Ile Thr Gly
Val Met Ser Arg Arg Tyr 80 85 90 ctc tgc atg gat ttc aga ggc aac
att ttt gga tca cac tat ttc gac 459 Leu Cys Met Asp Phe Arg Gly Asn
Ile Phe Gly Ser His Tyr Phe Asp 95 100 105 ccg gag aac tgc agg ttc
caa cac cag acg ctg gaa aac ggg tac gac 507 Pro Glu Asn Cys Arg Phe
Gln His Gln Thr Leu Glu Asn Gly Tyr Asp 110 115 120 125 gtc tac cac
tct cct cag tat cac ttc ctg gtc agt ctg ggc cgg gcg 555 Val Tyr His
Ser Pro Gln Tyr His Phe Leu Val Ser Leu Gly Arg Ala 130 135 140 aag
aga gcc ttc ctg cca ggc atg aac cca ccc ccg tac tcc cag ttc 603 Lys
Arg Ala Phe Leu Pro Gly Met Asn Pro Pro Pro Tyr Ser Gln Phe 145 150
155 ctg tcc cgg agg aac gag atc ccc cta att cac ttc aac acc ccc ata
651 Leu Ser Arg Arg Asn Glu Ile Pro Leu Ile His Phe Asn Thr Pro Ile
160 165 170 cca cgg cgg cac acc cgg agc gcc gag gac gac tcg gag cgg
gac ccc 699 Pro Arg Arg His Thr Arg Ser Ala Glu Asp Asp Ser Glu Arg
Asp Pro 175 180 185 ctg aac gtg ctg aag ccc cgg gcc cgg atg acc ccg
gcc ccg gcc tcc 747 Leu Asn Val Leu Lys Pro Arg Ala Arg Met Thr Pro
Ala Pro Ala Ser 190 195 200 205 tgt tca cag gag ctc ccg agc gcc gag
gac aac agc ccg atg gcc agt 795 Cys Ser Gln Glu Leu Pro Ser Ala Glu
Asp Asn Ser Pro Met Ala Ser 210 215 220 gac cca tta ggg gtg gtc agg
ggc ggt cga gtg aac acg cac gct ggg 843 Asp Pro Leu Gly Val Val Arg
Gly Gly Arg Val Asn Thr His Ala Gly 225 230 235 gga acg ggc ccg gaa
ggc tgc cgc ccc ttc gcc aag ttc atc 885 Gly Thr Gly Pro Glu Gly Cys
Arg Pro Phe Ala Lys Phe Ile 240 245 250 tagggtcgct ggaagggcac
cctctttaac ccatccctca gcaaacgcag ctcttcccaa 945 ggaccaggtc
ccttgacgtt ccgaggatgg gaaaggtgac aggggcatgt atggaatttg 1005
ctgcttctct ggggtccctt ccacaggagg tcctgtgaga accaaccttt gaggcccaag
1065 tcatggggtt tcaccgcctt cctcactcca tatagaacac ctttcccaat
aggaaacccc 1125 aacaggtaaa ctagaaattt ccccttcatg aaggtagaga
gaaggggtct ctcccaacat 1185 atttctcttc cttgtgcctc tcctctttat
cacttttaag cataaaaaaa aaaaaaaaaa 1245 aaaaaaaaaa aaaaagcagt
gggttcctga gctcaagact ttgaaggtgt agggaagagg 1305 aaatcggaga
tcccagaagc ttctccactg ccctatgcat ttatgttaga tgccccgatc 1365
ccactggcat ttgagtgtgc aaaccttgac attaacagct gaatggggca agttgatgaa
1425 aacactactt tcaagccttc gttcttcctt gagcatctct ggggaagagc
tgtcaaaaga 1485 ctggtggtag gctggtgaaa acttgacagc tagacttgat
gcttgctgaa atgaggcagg 1545 aatcataata gaaaactcag cctccctaca
gggtgagcac cttctgtctc gctgtctccc 1605 tctgtgcagc cacagccaga
gggcccagaa tggccccact ctgttcccaa gcagttcatg 1665 atacagcctc
accttttggc cccatctctg gtttttgaaa atttggtcta aggaataaat 1725
agcttttaca ctggctcacg aaaatctgcc ctgctagaat ttgcttttca aaatggaaat
1785 aaattccaac tctcctaaga ggcatttaat taaggctcta cttccaggtt
gagtaggaat 1845 ccattctgaa caaactacaa aaatgtgact gggaaggggg
ctttgagaga ctgggactgc 1905 tctgggttag gttttctgtg gactgaaaaa
tcgtgtcctt ttctctaaat gaagtggcat 1965 caaggactca gggggaaaga
aatcagggga catgttatag aagttatgaa aagacaacca 2025 catggtcagg
ctcttgtctg tggtctctag ggctctgcag cagcagtggc tcttcgatta 2085
gttaaaactc tcctaggctg acacatctgg gtctcaatcc ccttggaaat tcttggtgca
2145 ttaaatgaag ccttacccca ttactgcggt tcttcctgta agggggctcc
attttcctcc 2205 ctctctttaa atgaccacct aaaggacagt atattaacaa
gcaaagtcga ttcaacaaca 2265 gcttcttccc agtcactttt ttttttctca
ctgccatcac atactaacct tatactttga 2325 tctattcttt ttggttatga
gagaaatgtt gggcaactgt ttttacctga tggttttaag 2385 ctgaacttga
aggactggtt cctattctga aacagtaaaa ctatgtataa tagtatatag 2445
ccatgcatgg caaatatttt aatatttctg ttttcatttc ctgttggaaa tattatcctg
2505 cataatagct attggaggct cctcagtgaa agatcccaaa aggattttgg
tggaaaacta 2565 gttgtaatct cacaaactca acactaccat caggggtttt
ctttatggca aagccaaaat 2625 agctcctaca atttcttata tccctcgtca
tgtggcagta tttatttatt tatttggaag 2685 tttgcctatc cttctatatt
tatagatatt tataaaaatg taaccccttt ttcctttctt 2745 ctgtttaaaa
taaaaataaa attta 2770 2 251 PRT Homo sapiens 2 Met Leu Gly Ala Arg
Leu Arg Leu Trp Val Cys Ala Leu Cys Ser Val 1 5 10 15 Cys Ser Met
Ser Val Leu Arg Ala Tyr Pro Asn Ala Ser Pro Leu Leu 20 25 30 Gly
Ser Ser Trp Gly Gly Leu Ile His Leu Tyr Thr Ala Thr Ala Arg 35 40
45 Asn Ser Tyr His Leu Gln Ile His Lys Asn Gly His Val Asp Gly Ala
50 55 60 Pro His Gln Thr Ile Tyr Ser Ala Leu Met Ile Arg Ser Glu
Asp Ala 65 70 75 80 Gly Phe Val Val Ile Thr Gly Val Met Ser Arg Arg
Tyr Leu Cys Met 85 90 95 Asp Phe Arg Gly Asn Ile Phe Gly Ser His
Tyr Phe Asp Pro Glu Asn 100 105 110 Cys Arg Phe Gln His Gln Thr Leu
Glu Asn Gly Tyr Asp Val Tyr His 115 120 125 Ser Pro Gln Tyr His Phe
Leu Val Ser Leu Gly Arg Ala Lys Arg Ala 130 135 140 Phe Leu Pro Gly
Met Asn Pro Pro Pro Tyr Ser Gln Phe Leu Ser Arg 145 150 155 160 Arg
Asn Glu Ile Pro Leu Ile His Phe Asn Thr Pro Ile Pro Arg Arg 165 170
175 His Thr Arg Ser Ala Glu Asp Asp Ser Glu Arg Asp Pro Leu Asn Val
180 185 190 Leu Lys Pro Arg Ala Arg Met Thr Pro Ala Pro Ala Ser Cys
Ser Gln 195 200 205 Glu Leu Pro Ser Ala Glu Asp Asn Ser Pro Met Ala
Ser Asp Pro Leu 210 215 220 Gly Val Val Arg Gly Gly Arg Val Asn Thr
His Ala Gly Gly Thr Gly 225 230 235 240 Pro Glu Gly Cys Arg Pro Phe
Ala Lys Phe Ile 245 250 3 684 DNA Homo sapiens CDS (1)..(684) 3 tat
ccc aat gcc tcc cca ctg ctc ggc tcc agc tgg ggt ggc ctg atc 48 Tyr
Pro Asn Ala Ser Pro Leu Leu Gly Ser Ser Trp Gly Gly Leu Ile 1 5 10
15 cac ctg tac aca gcc aca gcc agg aac agc tac cac ctg cag atc cac
96 His Leu Tyr Thr Ala Thr Ala Arg Asn Ser Tyr His Leu Gln Ile His
20 25 30 aag aat ggc cat gtg gat ggc gca ccc cat cag acc atc tac
agt gcc 144 Lys Asn Gly His Val Asp Gly Ala Pro His Gln Thr Ile Tyr
Ser Ala 35 40 45 ctg atg atc aga tca gag gat gct ggc ttt gtg gtg
att aca ggt gtg 192 Leu Met Ile Arg Ser Glu Asp Ala Gly Phe Val Val
Ile Thr Gly Val 50 55 60 atg agc aga aga tac ctc tgc atg gat ttc
aga ggc aac att ttt gga 240 Met Ser Arg Arg Tyr Leu Cys Met Asp Phe
Arg Gly Asn Ile Phe Gly 65 70 75 80 tca cac tat ttc gac ccg gag aac
tgc agg ttc caa cac cag acg ctg 288 Ser His Tyr Phe Asp Pro Glu Asn
Cys Arg Phe Gln His Gln Thr Leu 85 90 95 gaa aac ggg tac gac gtc
tac cac tct cct cag tat cac ttc ctg gtc 336 Glu Asn Gly Tyr Asp Val
Tyr His Ser Pro Gln Tyr His Phe Leu Val 100 105 110 agt ctg ggc cgg
gcg aag aga gcc ttc ctg cca ggc atg aac cca ccc 384 Ser Leu Gly Arg
Ala Lys Arg Ala Phe Leu Pro Gly Met Asn Pro Pro 115 120 125 ccg tac
tcc cag ttc ctg tcc cgg agg aac gag atc ccc cta att cac 432 Pro Tyr
Ser Gln Phe Leu Ser Arg Arg Asn Glu Ile Pro Leu Ile His 130 135 140
ttc aac acc ccc ata cca cgg cgg cac acc cgg agc gcc gag gac gac 480
Phe Asn Thr Pro Ile Pro Arg Arg His Thr Arg Ser Ala Glu Asp Asp 145
150 155 160 tcg gag cgg gac ccc ctg aac gtg ctg aag ccc cgg gcc cgg
atg acc 528 Ser Glu Arg Asp Pro Leu Asn Val Leu Lys Pro Arg Ala Arg
Met Thr 165 170 175 ccg gcc ccg gcc tcc tgt tca cag gag ctc ccg agc
gcc gag gac aac 576 Pro Ala Pro Ala Ser Cys Ser Gln Glu Leu Pro Ser
Ala Glu Asp Asn 180 185 190 agc ccg atg gcc agt gac cca tta ggg gtg
gtc agg ggc ggt cga gtg 624 Ser Pro Met Ala Ser Asp Pro Leu Gly Val
Val Arg Gly Gly Arg Val 195 200 205 aac acg cac gct ggg gga acg ggc
ccg gaa ggc tgc cgc ccc ttc gcc 672 Asn Thr His Ala Gly Gly Thr Gly
Pro Glu Gly Cys Arg Pro Phe Ala 210 215 220 aag ttc atc tag 684 Lys
Phe Ile 225 4 227 PRT Homo sapiens 4 Tyr Pro Asn Ala Ser Pro Leu
Leu Gly Ser Ser Trp Gly Gly Leu Ile 1 5 10 15 His Leu Tyr Thr Ala
Thr Ala Arg Asn Ser Tyr His Leu Gln Ile His 20 25 30 Lys Asn Gly
His Val Asp Gly Ala Pro His Gln Thr Ile Tyr Ser Ala 35 40 45 Leu
Met Ile Arg Ser Glu Asp Ala Gly Phe Val Val Ile Thr Gly Val 50 55
60 Met Ser Arg Arg Tyr Leu Cys Met Asp Phe Arg Gly Asn Ile Phe Gly
65 70 75 80 Ser His Tyr Phe Asp Pro Glu Asn Cys Arg Phe Gln His Gln
Thr Leu 85 90 95 Glu Asn Gly Tyr Asp Val Tyr His Ser Pro Gln Tyr
His Phe Leu Val 100 105 110 Ser Leu Gly Arg Ala Lys Arg Ala Phe Leu
Pro Gly Met Asn Pro Pro 115 120 125 Pro Tyr Ser Gln Phe Leu Ser Arg
Arg Asn Glu Ile Pro Leu Ile His 130 135 140 Phe Asn Thr Pro Ile Pro
Arg Arg His Thr Arg Ser Ala Glu Asp Asp 145 150 155 160 Ser Glu Arg
Asp Pro Leu Asn Val Leu Lys Pro Arg Ala Arg Met Thr 165 170 175 Pro
Ala Pro Ala Ser Cys Ser Gln Glu Leu Pro Ser Ala Glu Asp Asn 180 185
190 Ser Pro Met Ala Ser Asp Pro Leu Gly Val Val Arg Gly Gly Arg Val
195 200 205 Asn Thr His Ala Gly Gly Thr Gly Pro Glu Gly Cys Arg Pro
Phe Ala 210 215 220 Lys Phe Ile 225 5 465 DNA Homo sapiens CDS
(1)..(465) 5 tat ccc aat gcc tcc cca ctg ctc ggc tcc agc tgg ggt
ggc ctg atc 48 Tyr Pro Asn Ala Ser Pro Leu Leu Gly Ser Ser Trp Gly
Gly Leu Ile 1 5 10 15 cac ctg tac aca gcc aca gcc agg aac agc tac
cac ctg cag atc cac 96 His Leu Tyr Thr Ala Thr Ala Arg Asn Ser Tyr
His Leu Gln Ile His 20 25 30 aag aat ggc cat gtg gat ggc gca ccc
cat cag acc atc tac agt gcc 144 Lys Asn Gly His Val Asp Gly Ala Pro
His Gln Thr Ile Tyr Ser Ala 35 40 45 ctg atg atc aga tca gag gat
gct ggc ttt gtg gtg att aca ggt gtg 192 Leu Met Ile Arg Ser Glu Asp
Ala Gly Phe Val Val Ile Thr Gly Val 50 55 60 atg agc aga aga tac
ctc tgc atg gat ttc aga ggc aac att ttt gga 240 Met Ser Arg Arg Tyr
Leu Cys Met Asp Phe Arg Gly Asn Ile Phe Gly 65 70 75 80 tca cac tat
ttc gac ccg gag aac tgc agg ttc caa cac cag acg ctg 288 Ser His Tyr
Phe Asp Pro Glu Asn Cys Arg Phe Gln His Gln Thr Leu 85 90 95 gaa
aac ggg tac gac gtc tac cac tct cct cag tat cac ttc ctg gtc 336 Glu
Asn Gly Tyr Asp Val Tyr His Ser Pro Gln Tyr His Phe Leu Val 100 105
110 agt ctg ggc cgg gcg aag aga gcc ttc ctg cca ggc atg aac cca ccc
384 Ser Leu Gly Arg Ala Lys Arg Ala Phe Leu Pro Gly Met Asn Pro Pro
115 120 125 ccg tac tcc cag ttc ctg tcc cgg agg aac gag atc ccc cta
att cac 432 Pro Tyr Ser Gln Phe Leu Ser Arg Arg Asn Glu Ile Pro Leu
Ile His 130 135 140 ttc aac acc ccc ata cca cgg cgg cac acc cgg 465
Phe Asn Thr Pro Ile Pro Arg Arg His Thr Arg 145 150 155 6 155 PRT
Homo sapiens 6 Tyr Pro Asn Ala Ser Pro Leu Leu Gly Ser Ser Trp Gly
Gly Leu Ile 1 5 10 15 His Leu Tyr Thr Ala Thr Ala Arg Asn Ser Tyr
His Leu Gln Ile His 20 25 30 Lys Asn Gly His Val Asp Gly Ala Pro
His Gln Thr Ile Tyr Ser Ala 35 40 45 Leu Met Ile Arg Ser Glu Asp
Ala Gly Phe Val Val Ile Thr Gly Val 50 55 60 Met Ser Arg Arg Tyr
Leu Cys Met Asp Phe Arg Gly Asn Ile Phe Gly 65 70 75 80 Ser His Tyr
Phe Asp Pro Glu Asn Cys Arg Phe Gln His Gln Thr Leu 85 90 95 Glu
Asn Gly Tyr Asp Val Tyr His Ser Pro Gln Tyr His Phe Leu Val 100 105
110 Ser Leu Gly Arg Ala Lys Arg Ala Phe Leu Pro Gly Met Asn Pro Pro
115 120 125 Pro Tyr Ser Gln Phe Leu Ser Arg Arg Asn Glu Ile Pro Leu
Ile His 130 135 140 Phe Asn Thr Pro Ile Pro Arg Arg His Thr Arg 145
150 155 7 219 DNA Homo sapiens CDS (1)..(216) 7 agc gcc gag gac gac
tcg gag cgg gac ccc ctg aac gtg ctg aag ccc 48 Ser Ala Glu Asp Asp
Ser Glu Arg Asp Pro Leu Asn Val Leu Lys Pro 1 5 10 15 cgg gcc cgg
atg acc ccg gcc ccg gcc tcc tgt tca cag gag ctc ccg 96 Arg Ala Arg
Met Thr Pro Ala Pro Ala Ser Cys Ser Gln Glu Leu Pro 20 25 30 agc
gcc gag gac aac agc ccg atg gcc agt gac cca tta ggg gtg gtc 144 Ser
Ala Glu Asp Asn Ser Pro Met Ala Ser Asp Pro Leu Gly Val Val 35 40
45 agg ggc ggt cga gtg aac acg cac gct ggg gga acg ggc ccg gaa ggc
192 Arg Gly Gly Arg Val Asn Thr His Ala Gly Gly Thr Gly Pro Glu Gly
50 55 60 tgc cgc ccc ttc gcc aag ttc atc tag 219 Cys Arg Pro Phe
Ala Lys Phe Ile 65 70 8 72 PRT Homo sapiens 8 Ser Ala Glu Asp Asp
Ser Glu Arg Asp Pro Leu Asn Val Leu Lys Pro 1 5 10 15 Arg Ala Arg
Met Thr Pro Ala Pro Ala Ser Cys Ser Gln Glu Leu Pro 20 25 30 Ser
Ala Glu Asp Asn Ser Pro Met Ala Ser Asp Pro Leu Gly Val Val 35 40
45 Arg Gly Gly Arg Val Asn Thr His Ala Gly Gly Thr Gly Pro Glu Gly
50 55 60 Cys Arg Pro Phe Ala Lys Phe Ile 65 70 9 543 DNA Mus sp.
CDS (1)..(540) 9 gcc ctg atg att aca tca gag gac gcc ggc tct gtg
gtg ata aca gga 48 Ala Leu Met Ile Thr Ser Glu Asp Ala Gly Ser Val
Val Ile Thr Gly 1 5 10 15 gcc atg act cga agg ttc ctt tgt atg gat
ctc cac ggc aac att ttt 96 Ala Met Thr Arg Arg Phe Leu Cys Met Asp
Leu His Gly Asn Ile Phe 20 25 30 gga tcg ctt cac ttc agc cca gag
aat tgc aag ttc cgc cag tgg acg 144 Gly Ser Leu His Phe Ser Pro Glu
Asn Cys Lys Phe Arg Gln Trp Thr 35 40 45 ctg gag aat ggc tat gac
gtc tac ttg tcg cag aag cat cac tac ctg 192 Leu Glu Asn Gly Tyr Asp
Val Tyr Leu Ser Gln Lys His His Tyr Leu 50 55 60 gtg agc ctg ggc
cgc gcc aag cgc atc ttc cag ccg ggc acc aac ccg 240 Val Ser Leu Gly
Arg Ala Lys Arg Ile Phe Gln Pro Gly Thr Asn Pro 65 70 75 80 ccg ccc
ttc tcc cag ttc ctg gct cgc agg aac gag gtc ccg ctg ctg 288 Pro Pro
Phe Ser Gln Phe Leu Ala Arg Arg Asn Glu Val Pro Leu Leu 85 90 95
cat ttc tac act gtt cgc cca cgg cgc cac acg cgc agc gcc gag gac 336
His Phe Tyr Thr Val Arg Pro Arg Arg His Thr Arg Ser Ala Glu Asp 100
105 110 cca ccg gag cgc gac cca ctg aac gtg ctc aag ccg cgg ccc cgc
gcc 384 Pro Pro Glu Arg Asp Pro Leu Asn Val Leu Lys Pro Arg Pro Arg
Ala 115 120 125 acg cct gtg cct gta tcc tgc tct cgc gag ctg ccg agc
gca gag gaa 432 Thr Pro Val Pro Val Ser Cys
Ser Arg Glu Leu Pro Ser Ala Glu Glu 130 135 140 ggt ggc ccc gca gcc
agc gat cct ctg ggg gtg ctg cgc aga ggc cgt 480 Gly Gly Pro Ala Ala
Ser Asp Pro Leu Gly Val Leu Arg Arg Gly Arg 145 150 155 160 gga gat
gct cgc ggg ggc gcg gga ggc gcg gat agg tgt cgc ccc ttt 528 Gly Asp
Ala Arg Gly Gly Ala Gly Gly Ala Asp Arg Cys Arg Pro Phe 165 170 175
ccc agg ttc gtc tag 543 Pro Arg Phe Val 180 10 180 PRT Mus sp. 10
Ala Leu Met Ile Thr Ser Glu Asp Ala Gly Ser Val Val Ile Thr Gly 1 5
10 15 Ala Met Thr Arg Arg Phe Leu Cys Met Asp Leu His Gly Asn Ile
Phe 20 25 30 Gly Ser Leu His Phe Ser Pro Glu Asn Cys Lys Phe Arg
Gln Trp Thr 35 40 45 Leu Glu Asn Gly Tyr Asp Val Tyr Leu Ser Gln
Lys His His Tyr Leu 50 55 60 Val Ser Leu Gly Arg Ala Lys Arg Ile
Phe Gln Pro Gly Thr Asn Pro 65 70 75 80 Pro Pro Phe Ser Gln Phe Leu
Ala Arg Arg Asn Glu Val Pro Leu Leu 85 90 95 His Phe Tyr Thr Val
Arg Pro Arg Arg His Thr Arg Ser Ala Glu Asp 100 105 110 Pro Pro Glu
Arg Asp Pro Leu Asn Val Leu Lys Pro Arg Pro Arg Ala 115 120 125 Thr
Pro Val Pro Val Ser Cys Ser Arg Glu Leu Pro Ser Ala Glu Glu 130 135
140 Gly Gly Pro Ala Ala Ser Asp Pro Leu Gly Val Leu Arg Arg Gly Arg
145 150 155 160 Gly Asp Ala Arg Gly Gly Ala Gly Gly Ala Asp Arg Cys
Arg Pro Phe 165 170 175 Pro Arg Phe Val 180 11 13200 DNA Homo
sapiens 11 ggctctcatg gctttagctc taacatgttg tatgggtttg gacaccttga
aaagtgctta 60 ggaggtcttg gggccagacc tggattcaaa tccaacctct
tccacatgtt acctttctat 120 ctctaggcaa gttacttaaa ctgtgtgcat
agatcagttt cctgatctat ataaagaaaa 180 taacagcatc tcctcaaaga
gttattctga agatgaaatg ggttactaca agtaaagcac 240 ttagagcagt
aagtggaaca gtaagctctc catgagcgtt agttcttgct gtgattcttt 300
ggagaagcag cctagggaag gagaagacct tgtcctggct ctaccattta tttgctttgt
360 gcctttggac atggaaacac taagatttcc atttcttcat gtaaagtatt
aaaatcttga 420 taatgcatgg agtgcctatc tcacagagtg gcagtgaggt
tcaaataagc taatagatgt 480 gaaaatgctt tgtaaactat aaaaaaaaac
tgtacaaatg tagggtaaca aatgccatct 540 ctttctgtct atacctgtaa
gcttgcactc attttgtatt atagttactt atttttatct 600 gcctcctgca
ttagatttga gctcctcaag ataggaatca catcttgctg tctcctatat 660
caacctgtac atgagtctag cttgatgcct gtacatggca tatactcagt acagggaaac
720 tggaagaata gcaaactcct tgtgtgtttt ttcgggtgtg tgttccagta
gcttgctccc 780 ctttagactg tatgtgccag gactattcca cccgacatca
ggtgtagctt cccagagggc 840 tcactgtgaa cagcatctgc aggcaggcat
ccaacaccaa gctgagcact catgtacaga 900 cactaaatgt ggggacaccc
tgtcctgagg ctcggatccc cagtgctcag ccagcagctg 960 ttccaatccc
catgaggtct ctgaatgagt ctccttacta ctggagagac tactagttta 1020
gttgccctcg atagtccaaa ctagggaaat tgagaaattg aaccttggca tattcagtga
1080 agtccagact aaggaagctg agaaactgaa ccttggcatg ttcactgacc
tcaaggcctt 1140 acttcttttt cttccatttc aaaatcttcc caaaatggca
actttggctt ttgtgcccct 1200 gctcctagct cccatctttc atgaagtggg
tgttcttaga ggatgccatc tgcctgatgg 1260 tgtcatgtat ctctatatcc
tgcaagctac ccaccaaatc ctgctcacag attaagcact 1320 ggatacatac
ttgctgattg gaatttaaag aaaaccaaaa taagtaaact cgacaggaga 1380
ttaattgcct aggagtcggt tgactgcttg actgaagctg gatttttttt gggaaccgct
1440 ggctgccttc acatttcctg atggaagtgg gacaggtcaa cagacagccc
agagtggcag 1500 ataacttttg cccacacgtc attcattttt tggagcgttg
gcttgaaatt gaggggtgtg 1560 tgtgtctgga accgacgtgc cttccgtgcg
cctccggggt ctttgcactt tctttcaatg 1620 ggctgattac aacacagagg
atgtggacag tggagttttt cctgtttgat gtcacactgc 1680 taccctttaa
aagtctgacg gcaaaaagga gggaatccag tctaggatcc tcacaccagc 1740
tacttgcaag ggagaaggaa aaggccagta aggcctgggc caggagagtc ccgacaggag
1800 tgtcaggttt caatctcagc accagccact cagagcaggg cacgatgttg
ggggcccgcc 1860 tcaggctctg ggtctgtgcc ttgtgcagcg tctgcagcat
gagcgtcctc agagcctatc 1920 ccaatgcctc cccactgctc ggctccagct
ggggtggcct gatccacctg tacacagcca 1980 cagccaggaa cagctaccac
ctgcagatcc acaagaatgg ccatgtggat ggcgcacccc 2040 atcagaccat
ctacagtgag tagggcttca ggctgggaag aaggggagca cccttgttgt 2100
ccatctacag gaggcttggg gaggttgggg actagactgg agggctaatc caaccctcct
2160 agctttctgc ccaggaacca cttattgtct ttgtgtgtgt gtgtgtttgt
gtgtgtgtgt 2220 gtgtgcgtgt gtatttaaaa cttaggggaa gattctgtca
ctctcctaat tagcatttgc 2280 tggtttttct caatatgaat aatttttatt
tcaactaaaa cccttcccac agtaggagcc 2340 atgttccctt tgccccgtca
aaagattgaa aaaatgtaca gaaagaaagg caaggagtct 2400 aagagaaaaa
gaagccaggc agatcaacag acactaagtt tcagctcatg gactttggac 2460
tgggttaact agggaggttg taaagattcc tgggtcatac ccagattgtt atagataaat
2520 ctgggggctc tttccaactc tggccttata aaaattcttg gaaaaatgta
ttaaagacag 2580 actgtccatg tatgttgtct tggtgagaat ggccaagtat
acaacccatc cacccattca 2640 tttgtccatg catccatcca tccatccatc
catccatcca tccatccatc catccatcca 2700 tccaacaggt ttaatgggtg
tttcagacac ccaggtaccc aggtacccag tatgagttgg 2760 tgattcttct
ctgtggaact gaaaggtgtc cagtcagtag caagtcaagc tagttagaaa 2820
cttattggca acatgagaca actggaatgt ttttaaagat caggtttgtg gagaattgga
2880 tcacaatcca caataatcaa tccattagca atgattcaaa tgagggtccc
tttgtgtgca 2940 cctatataag gggtaatgtg gtgaaagcag ggatagatat
tgaaaaagac tggatcttct 3000 attttaagaa aacgtgagaa aacacctcaa
agcatacaga aaaatgcaaa ctgatccaat 3060 atagctcaaa ttaaaggaaa
gaaaaattag ttagacaatc tctaactaaa gaaaacatag 3120 gaattatgat
ttgcccttta ttgttgtaat aaataaatta attatttttg ctgtgacctt 3180
gctgtgtgtg aggcatttat tcttctaggc tccaaaggtc cttaaacctg gttgcaagat
3240 atacaacatg caaaattaag ttgcaagata tataacatgc gaaattgaca
gtttaacctc 3300 ttcaagtact aaatgcatat tgacaggaga taaaaggaga
gaggaaagtt ctctccgaat 3360 accaaacagg ttccagaact ccagagaata
tagtaagact caggagtcaa catcttggaa 3420 accaagtttg agtctcatgg
caaaaatttc aattaaatct ggatacactt gatgcacccg 3480 aagtgttgtt
cattttattc aatggatatt taataggatc taccatgtgc ctggcattct 3540
accaagcgct gtggctgaaa actaagacac aacccttcaa ggacctcatg gtctgtcgtc
3600 ctaccttgtc agccagctca ctaccagact ttcaggaaat gcaattttgc
atgtctcatg 3660 gaggggacac ccttactcta attcaagact atatgtgggc
caggtgtggt ggctcacgcc 3720 tgtaatccca gcactctggg aggccgaggc
aggcagatca cgaggtcagg agattgagac 3780 catcctggct aacacagtga
aaccccatct ctactaaaaa tacaaaaaat tagccaggcg 3840 tggtggcagg
cgcctgtagt cccagctact tgggaggctg aggcaggaga atggcgtgaa 3900
cccgggaggc agagcttgca gtgagccgag attgcgccac tgcactccag cctgggtgac
3960 agagcgagac tccatctcaa aaaaaaaaaa aaaaaaaaaa aaaagactat
atgtgattta 4020 aaatgcagaa tagtagatta tggtaaatta ttttgattct
cttagatgga aagggctgca 4080 tccaactaga gaatgtttat acaacttgtc
tcgaatcctg gaatcccgtt gctgaaagga 4140 actccttaaa gacgtttctt
cctgaacaag aattagggta gaacagaaca ggccggctac 4200 ggtggggagt
gagtgtgaag agtcaacctc ctgggctggc agtctgaact tagacctttt 4260
ccttgaaagc ccacctcgta tcaggcccca agggatcact gagtgctagt tagagtgaat
4320 taaaatgact gagaagcagc aaaataaatt gaactgactt cagattttta
aaaatagaaa 4380 tgtgattttg tttccttaga cataggcact agctaaacat
tgcatcttta aagagttaaa 4440 catgaatgcg gggaggggaa cttcgggtca
gggtagtggg agggagatct acttttccta 4500 gtctatgtag ataacttttt
gtactgttca atgagttgtc atgtgtatta tgggaaaaaa 4560 aaagtgcagg
aaaaaattac acactacaat taaaagttac ccaaaagagg ccagatgcgg 4620
tggctcacgc gtataatctc agcactttgg gaggccgagg tgggaggatc acttgaagtc
4680 aggggttcga gaccagtctg gccaacatgg ggaaaccctg tctctactaa
aaatacaaaa 4740 attagtgggg tgtggtgcct gcctgtagtc acagctactt
gggaggctga ggcagaagaa 4800 tcgcttgtac cccagaggtg gagcttgcag
tgagctgaga tcgcaccact gtgctccagc 4860 ctgggtgaca gagtgagact
ccgtctcaaa aaaaaaacgt tacccaaaag aaagaggaaa 4920 gattaatgca
tgtagataag aagcacaact aattaaatgt ctggtgaagg atttgtaatg 4980
acctcatcag tgactgttaa gcaatgtttt tacactgatg gaaacctaaa atgtgagggg
5040 tattttttcc cctcctaatt atccatttct attgaattct ggtttatctt
attactttgc 5100 tgtaagaaat tcttaagagt tgagtgcaca acccatcttt
gggtcactgg acttgacaaa 5160 atgaagttat ctcctgccta ccagattctt
caagctccct taggactcac aagcgctgct 5220 gccaggtacc cctctggtgt
ccttatcacc cgttccatga acaaggccat cctctgactc 5280 ccctttatcc
ttattatatg gaaacagcaa gagagagatg cttattgtcc ctggtaatat 5340
catttcctag atcctgctat tttcacttcc tccatcttcc ccataggaac tatctttatt
5400 gaagctgaat taccgctgag ctccttcagc ctttttcata cgtttttctt
ttgaggtgct 5460 caaagcactt cactgctatc atcttattta tcacttactg
gccacaagag tagaatggag 5520 tagataattt tctacttcta taagcagagg
ttgaagcaaa gtggtaatag taaaaattac 5580 tagcacttag gaagtgcttt
ctaaaggata aaaacattat ctcattatgt cttcaagatg 5640 accaaatgag
gtaaatgttt ctgctgtcct cattttatat gtgaaaaaac aagagttaaa 5700
gaacttaaat aactggctga aggtcagaca ggtagtaaaa gacctagagg acaccttgac
5760 cccaaatccc aagctgttcc aaggtcacac gcacggagac acctccgtca
ttgaaggcaa 5820 agtccattaa gcctgctcag ctccaacagg cggggctggt
tgctccggca gtccatggtt 5880 tgttccttcc ttctgcaaaa ttctcccttg
aatctgtgca gactgcgaaa agatgccttt 5940 tgaaagcaca aaggaaagaa
actctgactc tctcacattt tctaaacttt cacattggct 6000 cacactgttt
gatggaagag tctgtgtgcc ctccgtagca gcttctcaca gttcctcaac 6060
caccgccgga tgttttctag ggggactggc tctggaaacc aggagcgtgt gtgccataca
6120 cactgcccac actgacccca agcatcaagc cagcacacct gtcaaggcat
gggcccggca 6180 taaacagcag gtcagaacac gccacgtgat gtcctctgtt
tgctcgacac tttcgagtca 6240 cttttaccac cattatcccc ttcgatcatc
accctatgaa gtaggcagga cagccattgt 6300 tctatttcac agctgaggaa
actgaggcta gtgcaaggtc agcaagtggc acaattggga 6360 ccaaaatcca
agtcccctgg ttcttggcac ctggctcagt gccccttccc cgagccctta 6420
gtgctgctgt gactccttgt cctcactgcc tgcagcatgt attttagcat ttgatattgt
6480 cttctcagac tcttagtgat ttctgctgga ggcttgttac cgggtggtgg
gaagatgtct 6540 ctgacctgag tttcaatcct gtcttagttt ccttatcact
taaattagga tattgctgct 6600 actactacta ctaataataa taataatcag
tactaacaat ataaaatttg ttttgagaat 6660 ttaaaatatg taaagtgctt
agagtgcaac cgctcaaaga ccattactaa atgtaatttt 6720 ttcccaatta
gactgaaaag tcaccagaac agaaaccatt tctttttttt tttttttgag 6780
acggagtctc gctctgtcgc ccaggctgga gtgcagtggt gcaatctcgg cttactgcta
6840 gctccgcctc ccaggttcat gccattctcc tgcctcagcc tcccgagtag
ctgggactac 6900 aggtgcccac caacacacct ggctaatttt ttttgtattt
tagtagagaa gaggtttcac 6960 cgtgttagcc aggatggtct tgatctcctg
acctcatgat ccacccgcct cagcctccca 7020 gagtgctggg attacaggca
tgagccaccg cacccagtcg aaaccatttc ttacacgttc 7080 cttatatttc
tccaaaggtt taccacaaaa ctagccatac atttgagaca cataggcaga 7140
caccactgtg gaggaattta agcatccttt gcgcctgtca taccccagtc cactttctgg
7200 gaagctttcc tcaaggttcc cctcagccaa actttgaaac aaattaactt
ctctctttgg 7260 ccctcttcct ttctcacctt tctggggctt gtctccaccc
tcattccctc tgttccgcgt 7320 cagtccaatt tcagtcccta tcatgatttc
tactcagcta gtacctaatt attgctgctt 7380 attttcctca aaggtccttg
tattgagcct gcatgggata ggagaagagc tttggccttt 7440 aaatcacccc
cacaaggctc catcacttgc taggcacaaa cattgggcat ttacaacctc 7500
tccaagctac agttgcagaa gaggaattag aatccctgca tgggattcta gctgcctccc
7560 tggaggctat gaggtcagat gagatgcacc tagaaattct gtgataggca
actggaagta 7620 gccccaaaga cctaaagtaa tgatttttaa cagaaaatgt
caggtaatta aataacatgt 7680 ggggaagaaa tctcgctgaa ttatcacgca
tgttacacca gtatatgatc taattgtgcc 7740 tttgccacaa aacagtaatt
taaagccatt atcaattact taagaggtag gtcgtgtgaa 7800 tgggtttcag
gcccttgtcg gagactagtt tttgagaggg gacactgaaa gtccatgagg 7860
ggctgcacct ggagaggtca ccaccaagtg agaaaatgac aaagaaccaa cccaagaaga
7920 gccaagaaga aaattccatc cgtcacttat attgattcaa cataaacagt
tataccctct 7980 gctcctaagc agctcactct aaggaacgca ctggataggt
aaactcagct aaagcaagtt 8040 aaatggaata catgctgtaa tagaggtgaa
ggcattgtcc tgaggagctg agaaggaaga 8100 acaactgatt ttgaatggaa
agatgaggaa agtcttcata gagatggtga cgcctgagcc 8160 tggtcttgaa
gagtgagtga cttcaataag tagagaagga agagggagat caactctact 8220
accattctgt acacatactg ggtgttgact gatgtattag acaattacac agacatccag
8280 gaggagaatc agactctatg gcaagctgga tccttgaaag acatctcagc
atagatttaa 8340 aaatcacaaa gtagaaggca tggaagaatg tgactatcac
cacaaacatt caaaggtatt 8400 agtaaggcaa aagggaaaat aaagacggtc
caggtagaga gagagaaaca tgtgttcagc 8460 acaggtagaa gaattccagg
agctcagagt gccccataca ggcaacaaga tgaagcagga 8520 ggtgaatgac
tgtatgtgtg ttgggggcaa gagaggatgt cagaagaaac gctgaatatg 8580
cagaaatgag gctgaattta agagtgctga agttatcacc acccttaaaa tcaatccagg
8640 gaggtttcat gaaggtaggt tttcaggagg tgcttgaagg tgggaattgg
atggcaatga 8700 gtctttgccc tgcctgtttt tctccatagg tgccctgatg
atcagatcag aggatgctgg 8760 ctttgtggtg attacaggtg tgatgagcag
aagatacctc tgcatggatt tcagaggcaa 8820 catttttgga tcagtgagtt
tcttttttgt gttggtcacc atttgcaaac aattaaccta 8880 atttctttga
cacgacatag acttttctag cttaacaatt ccatttgcag tgtaccctgg 8940
tgacctgttt ccactgatac ttcatgcagt ctaagtatga attaaacgtg aatgctcacg
9000 tttaatagct ggggtttgaa ctcaggcaat ctggctccag atcccaggct
ctcagcccct 9060 agtctgcact gcccattgga acccactttt tttttttatt
attatacttt aagttttagg 9120 gtacatgtgc acattgtgca ggttagttac
atacgtatac atgtgccatg ctggtgtgct 9180 gcacccacta actcatcatc
tagcattagg tatatctccc aatgctatcc ctcccccctc 9240 cccccacccc
acaacagtcc ccagagtgtg atgttcccct tcctgtgtcc atgtgatctc 9300
attgttcaat tcccacctac gagtgagaat atgcggtgtt tggttttttg ttcttgcgat
9360 agtttactga gaatgatggt ttccaatttc atccatgtcc ctacaaagga
catgaactca 9420 tcatttttta tggctgcata gtattccatg gtgtatatgt
gccacatttt cttaatccag 9480 tctatcattg ttggacattt gggttgtgga
acccactttt gaatctagca caggccctgg 9540 tatgtagtcg taggtcctca
agtcatgtca attcacgttg taaagtacat tagaaagggt 9600 gaaaagccat
tggtgtcttc ttgattctgg gactagctgt gactttgggt aaatctcctc 9660
acttctccaa acttcagcat tttcacttgg aaaaagcgaa gtggaataga ccacgtgacc
9720 tgtggagctc cctccagtta aagattttta attaataacc cctgccccaa
ttgtgatagc 9780 tattcattcg gattggtaag caaaggattt cccaaactaa
aggctgctgg cctcttttgg 9840 aggattttga gatagtaaaa tagtaggact
gcttatctca ggagtctctg accaccacac 9900 atgcccacta gaaactccac
aagaacagag actttctgtt ttgctttctg ctgcatccca 9960 gcccctacaa
tagtgtctgg ctagagtagg taaacaaaca aacaaaaaat ctgttgaacc 10020
actattgaaa tatagataac taactaaaca tccgttaacc cctctaggca tgtagatagt
10080 cctgatctgt aaactgctta ctttgtggag cttgaggatt aaaagaaata
acaggcaaag 10140 gccttcgtgg ggcactcagt aaaacccagt actagtggta
gaggattcaa acccagctca 10200 tctgtcagca aagttcatgt ctccaatccc
gtcagtgctc tcattcatgt ttgaccctat 10260 aaactccatc ccctctcctt
tctccagtaa agagacaaac ccaagccaat tttcagccag 10320 cagaggcttg
gaaaagatag agggcaggaa ggacaaggtg gtgcctactc caggaaaacc 10380
acaggccagg ccagcccggg cctccaggca gtaagcggag gccccagtag tcgtagtctc
10440 tgaaagggcg aactatatag tcagggcttg agcattaatc aaaaccactc
taccccagca 10500 gggaacaaag gggtgaaggc tcaacgccct aagaactgca
gagcttcagg ccggctggca 10560 caagcatgtg gccccaggag gagctgggga
gtgggtgggg cccccactgc cagccttcac 10620 gtggttcgct cttgtccttc
cagcactatt tcgacccgga gaactgcagg ttccaacacc 10680 agacgctgga
aaacgggtac gacgtctacc actctcctca gtatcacttc ctggtcagtc 10740
tgggccgggc gaagagagcc ttcctgccag gcatgaaccc acccccgtac tcccagttcc
10800 tgtcccggag gaacgagatc cccctaattc acttcaacac ccccatacca
cggcggcaca 10860 cccggagcgc cgaggacgac tcggagcggg accccctgaa
cgtgctgaag ccccgggccc 10920 ggatgacccc ggccccggcc tcctgttcac
aggagctccc gagcgccgag gacaacagcc 10980 cgatggccag tgacccatta
ggggtggtca ggggcggtcg agtgaacacg cacgctgggg 11040 gaacgggccc
ggaaggctgc cgccccttcg ccaagttcat ctagggtcgc tggaagggca 11100
ccctctttaa cccatccctc agcaaacgca gctcttccca aggaccaggt cccttgacgt
11160 tccgaggatg ggaaaggtga caggggcatg tatggaattt gctgcttctc
tggggtccct 11220 tccacaggag gtcctgtgag aaccaacctt tgaggcccaa
gtcatggggt ttcaccgcct 11280 tcctcactcc atatagaaca cctttcccaa
taggaaaccc caacaggtaa actagaaatt 11340 tccccttcat gaaggtagag
agaaggggtc tctcccaaca tatttctctt ccttgtgcct 11400 ctcctcttta
tcacttttaa gcataaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaagcag 11460
tgggttcctg agctcaagac tttgaaggtg tagggaagag gaaatcggag atcccagaag
11520 cttctccact gccctatgca tttatgttag atgccccgat cccactggca
tttgagtgtg 11580 caaaccttga cattaacagc tgaatggggc aagttgatga
aaacactact ttcaagcctt 11640 cgttcttcct tgagcatctc tggggaagag
ctgtcaaaag actggtggta ggctggtgaa 11700 aacttgacag ctagacttga
tgcttgctga aatgaggcag gaatcataat agaaaactca 11760 gcctccctac
agggtgagca ccttctgtct cgctgtctcc ctctgtgcag ccacagccag 11820
agggcccaga atggccccac tctgttccca agcagttcat gatacagcct caccttttgg
11880 ccccatctct ggtttttgaa aatttggtct aaggaataaa tagcttttac
actggctcac 11940 gaaaatctgc cctgctagaa tttgcttttc aaaatggaaa
taaattccaa ctctcctaag 12000 aggcatttaa ttaaggctct acttccaggt
tgagtaggaa tccattctga acaaactaca 12060 aaaatgtgac tgggaagggg
gctttgagag actgggactg ctctgggtta ggttttctgt 12120 ggactgaaaa
atcgtgtcct tttctctaaa tgaagtggca tcaaggactc agggggaaag 12180
aaatcagggg acatgttata gaagttatga aaagacaacc acatggtcag gctcttgtct
12240 gtggtctcta gggctctgca gcagcagtgg ctcttcgatt agttaaaact
ctcctaggct 12300 gacacatctg ggtctcaatc cccttggaaa ttcttggtgc
attaaatgaa gccttacccc 12360 attactgcgg ttcttcctgt aagggggctc
cattttcctc cctctcttta aatgaccacc 12420 taaaggacag tatattaaca
agcaaagtcg attcaacaac agcttcttcc cagtcacttt 12480 tttttttctc
actgccatca catactaacc ttatactttg atctattctt tttggttatg 12540
agagaaatgt tgggcaactg tttttacctg atggttttaa gctgaacttg aaggactggt
12600 tcctattctg aaacagtaaa actatgtata atagtatata gccatgcatg
gcaaatattt 12660 taatatttct gttttcattt cctgttggaa atattatcct
gcataatagc tattggaggc 12720 tcctcagtga aagatcccaa aaggattttg
gtggaaaact agttgtaatc tcacaaactc 12780 aacactacca tcaggggttt
tctttatggc aaagccaaaa tagctcctac aatttcttat 12840 atccctcgtc
atgtggcagt atttatttat ttatttggaa gtttgcctat ccttctatat 12900
ttatagatat ttataaaaat gtaacccctt tttcctttct tctgtttaaa ataaaaataa
12960 aatttatctc agcttctgtt agcttatcct ctttgtagta ctacttaaaa
gcatgtcgga 13020 atataagaat aaaaaggatt atgggagggg aacattaggg
aaatccagag aaggcaaaat 13080 tgaaaaaaag attttagaat tttaaaattt
tcaaagattt cttccattca taaggagact 13140 caatgatttt aattgatcta
gacagaatta tttaagtttt atcaatattg gatttctggt 13200 12 19 DNA
Artificial Sequence synthetic DNA 12 ttctgtctcg ctgtctccc 19 13 19
DNA Artificial Sequence synthetic DNA 13 ccccttccca gtcacattt
19
14 20 DNA Artificial Sequence synthetic DNA 14 ggggcatcta
acataaatgc 20 15 20 DNA Artificial Sequence synthetic DNA 15
agccactcag agcagggcac 20 16 21 DNA Artificial Sequence synthetic
DNA 16 ggtggcggcc gtctagaact a 21 17 21 DNA Artificial Sequence
synthetic DNA 17 tcagtctggg ccgggcgaag a 21 18 20 DNA Artificial
Sequence synthetic DNA 18 cacgttcaag gggtcccgct 20 19 20 DNA
Artificial Sequence synthetic DNA 19 tctgaaatcc atgcagaggt 20 20 21
DNA Artificial Sequence synthetic DNA 20 gggaggcatt gggataggct c 21
21 21 DNA Artificial Sequence synthetic DNA 21 ctagatgaac
ttggcgaagg g 21 22 30 DNA Artificial Sequence synthetic DNA 22
ccggaattca gccactcaga gcagggcacg 30 23 52 DNA Artificial Sequence
synthetic DNA 23 ataagaatgc ggccgctcaa tggtgatggt gatgatggat
gaacttggcg aa 52 24 20 DNA Artificial Sequence synthetic DNA 24
taatacgact cactataggg 20 25 20 DNA Artificial Sequence synthetic
DNA 25 attaaccctc actaaaggga 20 26 20 DNA Artificial Sequence
synthetic DNA 26 accacagtcc atgccatcac 20 27 20 DNA Artificial
Sequence synthetic DNA 27 tccaccaccc tgttgctgta 20 28 21 PRT
Artificial Sequence synthetic peptide 28 Arg Asn Ser Tyr His Leu
Gln Ile His Lys Asn Gly His Val Asp Gly 1 5 10 15 Ala Pro His Gln
Cys 20 29 21 PRT Artificial Sequence synthetic peptide 29 Arg Phe
Gln His Gln Thr Leu Glu Asn Gly Tyr Asp Val Tyr His Ser 1 5 10 15
Pro Gln Tyr His Cys 20 30 26 DNA Artificial Sequence synthetic DNA
30 tgaaggtcgg tgtgaacgga tttggc 26 31 24 DNA Artificial Sequence
synthetic DNA 31 catgtaggcc atgaggtcca ccac 24 32 21 DNA Artificial
Sequence synthetic DNA 32 gtaaagaacc ctgtgtattc c 21 33 21 DNA
Artificial Sequence synthetic DNA 33 ctgccttaag aaatccataa t 21 34
20 DNA Artificial Sequence synthetic DNA 34 gaggaatcac agtctcattc
20 35 20 DNA Artificial Sequence synthetic DNA 35 cttggggagg
tgcccgggac 20 36 21 DNA Artificial Sequence synthetic DNA 36
tccctcttag aagacaatac a 21 37 21 DNA Artificial Sequence synthetic
DNA 37 gtgtttaaag gcagtattac a 21 38 21 DNA Artificial Sequence
synthetic DNA 38 cagacagaga catccgtgta g 21 39 21 DNA Artificial
Sequence synthetic DNA 39 ccacatggtc caggttcagt c 21 40 20 DNA
Artificial Sequence synthetic DNA 40 gacggtgaga ctcggaacgt 20 41 20
DNA Artificial Sequence synthetic DNA 41 tccggaaaat ctggccatac 20
42 20 DNA Artificial Sequence synthetic DNA 42 taatacgact
cactataggg 20 43 19 DNA Artificial Sequence synthetic DNA 43
gatttaggtg acactatag 19 44 34 DNA Artificial Sequence synthetic DNA
44 ataagaatgc ggccgctcag atgaacttgg cgaa 34 45 33 DNA Artificial
Sequence synthetic DNA 45 atgaattcca ccatgttggg ggcccgcctc agg 33
46 49 DNA Artificial Sequence synthetic DNA 46 atgcggccgc
ctaatgatga tgatgatgat ggatgaactt ggcgaaggg 49 47 30 DNA Artificial
Sequence synthetic DNA 47 ataccacggc agcacaccca gagcgccgag 30 48 30
DNA Artificial Sequence synthetic DNA 48 ataccacggc agcacaccca
gagcgccgag 30 49 17 PRT Artificial Sequence synthetic peptide 49
Gly Met Asn Pro Pro Pro Tyr Ser Gln Phe Leu Ser Arg Arg Asn Glu 1 5
10 15 Cys 50 11 PRT Artificial Sequence synthetic peptide 50 Cys
Asn Thr Pro Ile Pro Arg Arg His Thr Arg 1 5 10 51 16 PRT Artificial
Sequence synthetic peptide 51 Ser Ala Glu Asp Asp Ser Glu Arg Asp
Pro Leu Asn Val Leu Lys Cys 1 5 10 15 52 14 PRT Artificial Sequence
synthetic peptide 52 Leu Pro Ser Ala Glu Asp Asn Ser Pro Met Ala
Ser Asp Cys 1 5 10 53 33 DNA Artificial Sequence synthetic DNA 53
atgaattcca ccatgttggg ggcccgcctc agg 33 54 33 DNA Artificial
Sequence synthetic DNA 54 atgcggccgc tatcgaccgc ccctgaccac ccc 33
55 33 DNA Artificial Sequence synthetic DNA 55 atgcggccgc
tacgggagct cctgtgaaca gga 33 56 34 DNA Artificial Sequence
synthetic DNA 56 atgcggccgc tcacggggtc atccgggccc gggg 34 57 79 DNA
Artificial Sequence synthetic DNA 57 aattccacca tgttgggggc
ccgcctcagg ctctgggtct gtgcttgtgc agcgtctgca 60 gcatgagcgt cctgcatgc
79 58 80 DNA Artificial Sequence synthetic DNA 58 aattgcatgc
aggacgctca tgctgcagac gctgcacaag gcacagaccc agagcctgag 60
gcgggccccc aacatggtgg 80 59 35 DNA Artificial Sequence synthetic
DNA 59 atatgcatgc ctccagctgg ggtggcctga tccac 35 60 23 DNA
Artificial Sequence synthetic DNA 60 tgtatcccaa tgcctcccca ctg 23
61 29 DNA Artificial Sequence synthetic DNA 61 atggatccct
agatgaactt ggcgaaggg 29 62 29 DNA Artificial Sequence synthetic DNA
62 atcatatgta tcccaatgcc tccccactg 29 63 31 DNA Artificial Sequence
synthetic DNA 63 atgcggccgc ctagatgaac ttggcgaagg g 31 64 49 DNA
Artificial Sequence synthetic DNA 64 gaattcatat gaaatacccg
aacgcttccc cgctgctggg ctccagctg 49 65 32 DNA Artificial Sequence
synthetic DNA 65 cccaagcttg cggccgccta gatgaacttg gc 32 66 25 DNA
Artificial Sequence synthetic DNA 66 aacaccccca tagcacggcg gcaca 25
67 25 DNA Artificial Sequence synthetic DNA 67 tgtgccgccg
tgctatgggg gtgtt 25 68 26 DNA Artificial Sequence synthetic DNA 68
acccccatac cagcgcggca cacccg 26 69 26 DNA Artificial Sequence
synthetic DNA 69 cgggtgtgcc gcgctggtat gggggt 26 70 26 DNA
Artificial Sequence synthetic DNA 70 cccataccac gggcgcacac ccggag
26 71 26 DNA Artificial Sequence synthetic DNA 71 ctccgggtgt
gcgcccgtgg tatggg 26 72 26 DNA Artificial Sequence synthetic DNA 72
ataccacggc gggccacccg gagcgc 26 73 26 DNA Artificial Sequence
synthetic DNA 73 gcgctccggg tggcccgccg tggtat 26 74 25 DNA
Artificial Sequence synthetic DNA 74 ccacggcggc acgcccggag cgccg 25
75 25 DNA Artificial Sequence synthetic DNA 75 cggcgctccg
ggcgtgccgc cgtgg 25 76 26 DNA Artificial Sequence synthetic DNA 76
cggcggcaca ccgcgagcgc cgagga 26 77 26 DNA Artificial Sequence
synthetic DNA 77 tcctcggcgc tcgcggtgtg ccgccg 26 78 26 DNA
Artificial Sequence synthetic DNA 78 cggcacaccc gggccgccga ggacga
26 79 26 DNA Artificial Sequence synthetic DNA 79 tcgtcctcgg
cggcccgggt gtgccg 26 80 26 DNA Artificial Sequence synthetic DNA 80
acccccatac cacagcggca cacccg 26 81 26 DNA Artificial Sequence
synthetic DNA 81 cgggtgtgcc gctgtggtat gggggt 26 82 26 DNA
Artificial Sequence synthetic DNA 82 cccataccac ggcagcacac ccggag
26 83 26 DNA Artificial Sequence synthetic DNA 83 ctccgggtgt
gctgccgtgg tatggg 26 84 26 DNA Artificial Sequence synthetic DNA 84
cggcggcaca cccagagcgc cgagga 26 85 26 DNA Artificial Sequence
synthetic DNA 85 tcctcggcgc tctgggtgtg ccgccg 26 86 25 DNA
Artificial Sequence synthetic DNA 86 cggcggcaca cctggagcgc cgagg 25
87 25 DNA Artificial Sequence synthetic DNA 87 cctcggcgct
ccaggtgtgc cgccg 25
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