U.S. patent application number 10/327514 was filed with the patent office on 2003-07-03 for bone polypeptide-1.
Invention is credited to Lanctot, Christian, Moffatt, Pierre, Salois, Patrick.
Application Number | 20030125258 10/327514 |
Document ID | / |
Family ID | 23336705 |
Filed Date | 2003-07-03 |
United States Patent
Application |
20030125258 |
Kind Code |
A1 |
Lanctot, Christian ; et
al. |
July 3, 2003 |
Bone polypeptide-1
Abstract
The present invention relates to a bone polypeptide,
particularly bone polypeptide-1 and nucleic acid molecules encoding
the same. The present invention provides the human primary sequence
of bone polypeptide-1 as well as sequences of vertebrate homologs.
The invention also provides cell lines engineered to express the
cDNA, antibodies to detect its translation products, and
recombinant adenoviruses to deliver an expressible cDNA into a
host. Bone polypeptide-1 can be utilized to treat diseases
affecting renal and bone functions.
Inventors: |
Lanctot, Christian;
(Montreal, CA) ; Salois, Patrick; (Montreal,
CA) ; Moffatt, Pierre; (Lachine, CA) |
Correspondence
Address: |
Michael R. Ward
Morrison & Foerster LLP
425 Market Street
San Francisco
CA
94105-2482
US
|
Family ID: |
23336705 |
Appl. No.: |
10/327514 |
Filed: |
December 20, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60341224 |
Dec 20, 2001 |
|
|
|
Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 514/16.9; 514/8.8; 530/350; 530/388.1;
536/23.5 |
Current CPC
Class: |
C07K 14/51 20130101;
A61K 48/00 20130101; A61K 38/00 20130101 |
Class at
Publication: |
514/12 ;
435/69.1; 435/320.1; 435/325; 530/350; 530/388.1; 536/23.5 |
International
Class: |
A61K 038/17; C12P
021/02; C12N 005/06; C07K 014/47; C07K 016/40; C07H 021/04 |
Claims
We claim:
1. An isolated nucleic acid encoding bone polypeptide-1 wherein
said bone polypeptide-1 comprises a polypeptide sequence selected
from the group consisting of SEQ ID NO:9, SEQ ID NO:10; SEQ ID
NO:11, SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14.
2. A vector comprising the nucleic acid of claim 1.
3. The vector of claim 2 comprising an expression control sequence
operably linked to said nucleic acid.
4. The vector of claim 3 wherein said expression control sequence
is a promoter.
5. The vector of claim 4 wherein the promoter is a prokaryotic
promoter.
6. The vector of claim 4 wherein the promoter is a eukaryotic
promoter.
7. An isolated cell comprising the vector of claim 3.
8. The cell of claim 7 wherein the isolated cell is a prokaryotic
cell.
9. The cell of claim 7 wherein the isolated cell is a eukaryotic
cell.
10. A method of producing bone polypeptide-1, comprising culturing
the cell of claim 7 under conditions permitting expression of the
polypeptide and purifying the polypeptide from the cell or culture
medium of the cell.
11. The method of claim 10 wherein the cell is a prokaryotic
cell.
12. The method of claim 10 wherein the cell is a eukaryotic
cell.
13. An isolated nucleic acid wherein said nucleic acid hybridizes
under high stringency conditions to the full length nucleic acid
sequence of a nucleic acid selected from the group consisting of
SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO: 6
and SEQ ID NO:7 or the complements thereof wherein said high
stringency conditions includes hybrizing in 0.15 M NaCl at
72.degree. C. for about 15 minutes and washing said hybridized DNA
in 0.2.times.SSC at 65.degree. C. for 15 minutes.
14. The nucleic acid of claim 13 wherein said nucleic acid
comprises the nucleotide sequence of SEQ ID NO:2.
15. A vector comprising the nucleic acid of claim 13.
16. The vector of claim 15 comprising an expression control
sequence operably linked to said nucleic acid.
17. The vector of claim 15 wherein said expression control sequence
is a promoter.
18. An isolated cell comprising the vector of claim 15.
19. A method of producing bone polypeptide-1, comprising culturing
the cell of claim 18 under conditions permitting expression of bone
polypeptide-1 and purifying bone polypeptide-1 from the cell or
culture medium of the cell.
20. An isolated or recombinant bone polypeptide-1 wherein said bone
polypeptide-1 comprises the amino acid sequence of SEQ ID NO:
9.
21. An isolated or recombinant bone polypeptide-1 fragment wherein
said bone polypeptide-1 fragment comprises one or more peptides
selected from the group consisting of SEQ ID NO:20, SEQ ID NO: 21,
SEQ ID NO:22, SEQ ID NO:22; SEQ ID NO:23, SEQ ID NO:24 and SEQ ID
NO:25.
22. A pharmaceutical composition comprising the bone polypeptide-1
of claim 20.
23. A pharmaceutical composition comprising the bone polypeptide-1
fragment of claim 21.
24. An antibody that binds to an epitope in the polypeptide encoded
by the nucleic acid of SEQ ID NO:2.
25. The antibody of claim 24 wherein the epitope comprises a
peptide selected from the group consisting of SEQ ID NO:26, SEQ ID
NO: 27 and SEQ ID NO:28.
26. An expression vector comprising a nucleic acid sequence
encoding the bone polypeptide-1 of claim 20, the vector being
suitable for genetic therapy.
27. An expression vector comprising a nucleic acid sequence
encoding the bone polypeptide-1 fragment of claim 21, the vector
being suitable for genetic therapy.
28. A method of treating a bone disorder or osteoporosis of a
patient comprising administering an effective amount of the bone
polypeptide-1 of claim 20 to said patient.
29. A method of treating a bone disorder or osteoporosis of a
patient comprising administering an effective amount of the bone
polypeptide-1 fragment of claim 21 to said patient.
Description
RELATED APPLICATION
[0001] This application claims the benefit under 35 U.S.C.
.sctn.119(e) of United States provisional patent application No.
60/341,224, filed Dec. 20, 2001 which is hereby incorporated by
reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] a) Field of the Invention
[0003] The present invention relates to a bone polypeptide and
active fragments thereof. In particular, the present invention
relates to bone polypeptide-1, active fragments thereof and nucleic
acids encoding the same.
[0004] b) Brief Description of the Prior Art
[0005] Bone is a specialized connective tissue that is constantly
remodelled by the reciprocal action of bone-forming cells
(osteoblasts) and bone-resorbing cells (osteoclasts) (Manolagas,
2000). Full citations for the references cited herein are provided
before the claims. In the developing organism, the skeletal system
is formed either through endochondral or intramembranous
ossification (Baron, 1999). Endochondral bone formation,
exemplified by the development of the long bones of the limbs,
involves replacement of a preformed cartilage template whereas
intramembranous ossification, a process typical-of flat bones of
the skull, does not rely on an intermediate cartilaginous step. In
both cases, osteoblasts originate from the mesenchyme and initially
differentiate from the inner layer of the periosteum. Periosteal
osteoblasts deposit lamellar bone, progressively becoming enthumbed
in the matrix they deposit and finally differentiating into
osteocytes. Osteocytes elaborate cytoplasmic extensions through
canaliculi, thereby constituting a network of interconnected cells
within bone tissue (Aarder et al., 1994). It is thought that this
network senses load on the bones and alters bone activity according
to the demands of that load.
[0006] Osteoporosis is characterized by a loss of bone mass due to
an imbalance between bone resorption and bone formation. This
degenerative disease affects 20 million women in the United States.
Current treatment mainly involves the inhibition of the activity of
osteoclasts by inhibitors such as bisphosphonates (Russell, 1999).
Such anti-resorptive therapies slow down the progression of the
disease but do not really help in rebuilding lost bone. Effective
bone anabolics are thus needed. Unfortunately, with the possible
exception of a fragment of parathyroid hormone (PTH.sub.1-34; Neer
et al., 2001), very few molecules have been shown to notably
increase bone mass in osteoporotic patients. A number of growth
factors and related molecules are now being considered as
therapeutic agents. Insulin-like growth factor 1 (IGF1) is a
protein that displays bone-sparing activity in the ovariectomized
rat, a model of postmenopausal osteoporosis (Bagi et al., 1995).
Basic fibroblast growth factor (bFGF) is another protein that was
shown to enhance bone formation in vivo (Mayahara et al., 1993).
However IGF1, as the name implies, is also an hypoglycemic factor
and bFGF severely disrupts hematopoiesis. Hence, the in vivo
specificity of these growth factors is difficult to ascertain and
their clinical potential is unproven. There is thus a need to
identify molecules that can efficiently and specifically increase
bone formation.
[0007] Regulation of bone mass by the central nervous system has
recently been reported by Ducy and colleagues (Ducy et al., 2000).
These authors have shown that the ob/ob mice lacking a functional
leptin gene have a higher bone mass. Leptin is a 16 kD hormone
synthesized by the adipocytes and acting on hypothalamic neurons to
regulate caloric intake (Unger, 2000). When injected
intracerebroventricularly, leptin caused a loss of bone. It has
been hypothesized that, in addition to adipocytes, osteoblasts per
se might secrete a humoral factor that regulates bone mass through
an hypothalamic relay. Thus, identification of such a factor might
be useful to devise new therapies for the treatment of
osteoporosis.
[0008] The inorganic component of bone is made up of hydroxyapatite
crystals. Hence, in addition to its role as a supporting mechanical
structure, the skeleton is a reservoir of calcium. Bone cells play
a crucial role in the homeostasis of calcium and other ions such as
phosphate. In particular, it has been proposed that bone cells
secrete a hormone, tentatively called phosphatonin, that regulates
phosphate retention by kidney tubules. It has been reported that
phosphatonin levels are elevated in conditioned medium of tumor
cells derived from a rare disease called oncogenic hypophosphatemia
osteomalacia (Kumar, 2000). Furthermore, it has been postulated
that phosphatonin is a substrate for Phex, a metallopeptidase found
at the cell surface of osteoblasts and osteocytes (Frota Ruchon et
al., 2000). This hypothesis is supported by the fact that patients
with X-linked hypophosphatemia harbor deleterious mutations in the
Phex coding sequence (The Hyp consortium, 1995) and presumably have
elevated levels of active phosphatonin. Low serum phosphate levels
are associated with impaired bone quality. Hypophosphatemia could
be remedied by injection of phosphatonin antagonists. In view of
the above, it is clear that there is a need to identify the
molecular nature of phosphatonin or other molecules that regulate
phosphate metabolism. In particular, there is a tremendous need to
identify bone polypeptides that may be useful as therapeutic
agents.
SUMMARY OF THE INVENTION
[0009] In order to meet these needs, the present invention is
directed to recombinant bone polypeptide-1 (BP-1), proteins sharing
substantial homology to BP-1 and active fragments thereof. Bone
polypeptide-1 is polypeptide expressed in bone. Bone polypeptide 1
can be synthesized chemically, recombinantly produced, isolated
and/or purified from a recombinant host or it and it can be
isolated and/or purified from its natural source. Sources of bone
polypeptide-1 include all organisms containing bone since bone
polypeptide-1 is predominantly expressed in bone cells. Preferred
sources of bone polypeptide 1 include rat, mouse, python, cow and
chicken. An especially preferred source of bone polypeptide-1 is a
human.
[0010] The present invention is further directed to nucleic acids
encoding bone polypeptide-1 and active fragments thereof; a vector
containing the nucleic acids and a host cell carrying the vector.
The present invention is further directed to processes to produce
bone polypeptide-1 and active fragments thereof, processes to
produce cells capable of producing the bone polypeptide-1 and
active fragments thereof and to produce a vector containing DNA or
RNA encoding bone polypeptide-1 and active fragments thereof. The
present invention is further directed to methods for treating bone
and renal diseases and methods for using pharmacologic compositions
comprising an effective amount of bone polypeptide-1 and active
fragments thereof. The invention also encompasses monoclonal and
polyclonal antibodies specifically recognizing bone polypeptide-1
and active fragments thereof.
[0011] The present invention is further directed to an isolated
nucleic acid encoding bone polypeptide-1 and active fragments
thereof. The nucleic acid may be isolated from an animal,
preferably a human. Other sources of the nucleic acid include rats,
cows, snakes, mice and chickens.
[0012] Bone polypeptide-1 may regulate bone cell proliferation
and/or bone cell differentiation and/or osteoblast activity via an
autocrine, paracrine or endocrine pathway. Active fragments of bone
polypeptide-1 are fragments that have similar activity as the full
length protein and therefore may regulate bone cell proliferation
and/or bone cell differentiation and/or osteoblast activity via an
autocrine, paracrine or endocrine pathway.
[0013] The present invention is further directed to nucleic acids
having sequences selected from: SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID
NO: 4; SEQ ID NO: 5; SEQ ID NO: 6, SEQ ID NO: 7 and homologous
sequences.
[0014] The present invention is further directed to vector
including a nucleic acid encoding bone polypeptide-1 and active
fragments thereof. In one format, the vector includes an expression
control sequence operably linked to the nucleic acid. The
expression control sequence may be a promoter such as a prokaryotic
promoter or a eukaryotic promoter. The vector may be present in an
isolated cell such as a prokaryotic cell or a eukaryotic cell.
[0015] The present invention is further directed to a method of
producing bone polypeptide-1 or active fragments thereof by
culturing a cell containing an vector containing a nucleic acid
encoding bone polypeptide-1 under conditions permitting expression
of the polypeptide and purifying the polypeptide from the cell or
culture medium of the cell. The cell may be a prokaryotic cell or a
eukaryotic cell.
[0016] The present invention is further directed to an isolated
nucleic acid that hybridizes under high stringency conditions to a
nucleic acid including nucleotide sequences such as SEQ ID NO: 2;
SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6, SEQ ID NO:
7, SEQ ID NO: 8, and the complements thereof wherein the high
stringency conditions includes hybridizing in 0.15 M NaCl at
72.degree. C. for about 15 minutes and washing the hybridized DNA
in 0.2.times.SSC at 65.degree. C. for 15 minutes. The nucleic acid
may be isolated from an animal, preferably a human. Other sources
of the nucleic acid include rats, cows, snakes, mice and
chickens.
[0017] The nucleic acids of the invention may be cloned into an
vector. The vector may further include an expression control
sequence operably linked to the nucleic acid. The expression
control sequence may be a promoter such as a prokaryotic promoter
or a eukaryotic promoter.
[0018] The present invention is further directed to isolated and/or
purified and/or recombinant bone polypeptide-1 or an active
fragment thereof in a pharmaceutical composition. The polypeptide
may be isolated and/or purified from a human. Alternatively, the
polypeptide may be recombinantly produced from the nucleic acid
encoding the human polypeptide. The polypeptide may also be
isolated from rat, cow, snake, mouse or chicken. Alternatively, the
polypeptide may be recombinantly produced from the nucleic acid
encoding the rat, cow, snake, mouse or chicken polypeptide.
[0019] The present invention is further directed to polypeptides
having amino acid sequences selected from of SEQ ID NO: 9; SEQ ID
NO: 10; SEQ ID NO: 11; SEQ ID NO: 12; SEQ ID NO: 13; SEQ ID NO: 14,
SEQ ID NO: 20, SEQ ID NO: 21 SEQ ID NO: 22; SEQ ID NO: 23; SEQ ID
NO: 24; SEQ ID NO: 25; SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO:
28.
[0020] The present invention is further directed to an expression
vector containing a coding sequence the sequence of which encodes
bone polypeptide-1 or an active fragment thereof, the vector being
suitable for genetic therapy.
[0021] The present invention is further directed to a method to
deliver a nucleic acid, the sequence of which encodes bone
polypeptide-1 or a fragment thereof, into a host for therapy. In
one format of the method of the invention, the delivery is by
adenovirus.
[0022] The present invention is further directed to a method of
treating a bone disorder or osteoporosis comprising administering
an effective amount of bone polypeptide-1 or a fragment
thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIGS. 1A, B show the results of a Northern analysis
comparing the expression of a mouse cDNA encoding bone
polypeptide-1 (BP-1) in a variety of embryonic and adult mouse
tissues. A. Five micrograms of total RNA was loaded in lanes 1 to
16.1, brain e1.5; 2, adult brain; 3, gonads e14.5; 4, adult testis;
5, heart e12.5; 6, adult heart; 7, intestine e14.5; 8, adult
intestine; 9, kidney e13.5; 10, adult kidney; 11, liver e11.5; 12,
adult liver; 13, lung e12.5; 14, adult lung; 15, spleen e15.5; 16,
adult spleen. B. One and a half microgram of polyA(+) RNA was
loaded in lanes 21 to 23.21, calvaria e15.5; 22, adult kidney; 23,
adult liver. Bars indicate migration of 28S and 18S ribosomal
RNA.
[0024] FIG. 2 shows the nucleotide sequence of a full length mouse
cDNA encoding bone protein-1 (SEQ ID NO:1). The open reading frame
is written in capital letters, the polyadenylation signal is in
bold characters, the sequence corresponding to the original cal
55-cap1-c2-neg306 clone is underlined.
[0025] FIGS. 3A-F show brightfield (A,D) and darkfield (B,C,E,F)
images of sections through the limb of a mouse embryo at 16 days of
gestation (A-C) or through the head of a mouse embryo at 13 days of
gestation (D-F) hybridized with a probe complementary to a novel
cDNA encoding bone polypeptide-1 (B,E) or to Cbfa1, a transcription
factor expressed mainly in the osteoblast lineage (C,F).
[0026] FIG. 4 shows an alignment of the coding sequences from human
(Homo sapiens) (SEQ ID NO:2), mouse (Mus musculus) (SEQ ID
NO:3),rat (Rattus norvegicus) (SEQ ID NO:4), bovine (Bos taurus)
(SEQ ID NO:5), chicken (Gallus gallus) (SEQ ID NO:6), and python
(Python molurus bivittatus) (SEQ ID NO: 7) BP-1 cDNAs. The
consensus DNA sequence for BP-1 is provided as SEQ ID NO: 8.
[0027] FIG. 5A shows an alignment of the primary sequences of BP
deduced from cDNAs isolated from tissues of the human (Homo
sapiens) (SEQ ID NO:9), mouse (Mus musculus) (SEQ ID NO:10), rat
(Rattus norvegicus) (SEQ ID NO:11), bovine (Bos taurus) (SEQ ID
NO:12), chicken (Gallus gallus) (SEQ ID NO:13) and python (Python
molurus bivittatus) (SEQ ID NO:14). The consensus polypeptide
sequence for BP-1 is provided as SEQ ID NO: 15. Sequence of the
putative signal peptides are underlined. Putative processing sites
are boxed.
[0028] FIG. 5B shows an alignment of a region of homology between
BP-1 and members of the natriuretic peptides family. Numbering of
amino acids is according to the full length precursor protein (SEQ
ID NO:9 for BP-1). The human sequence is SEQ ID NO: 16. The rat
atrial natriuretic factor protein region is SEQ ID NO: 17. The rat
brain natriuretic factor protein region is SEQ ID NO: 18. The rat
C-type natriuretic factor protein region is SEQ ID NO: 19.
[0029] FIG. 6A depicts the full length protein derived from the
human BP-1 cDNA (SEQ ID NO: 2) and is presented as SEQ ID NO: 9.
The signal peptide and corresponding signal peptidase cleavage site
are shown. Also shown are the conserved dibasic cleavage sites, the
putative products generated by processing of the protein (PROT 1
(SEQ ID NO: 20), PEPT 1 (SEQ ID NO: 21), PEPT 2A (SEQ ID NO: 22),
PEPT 3A (SEQ ID NO: 23), PEPT 2B (SEQ ID NO: 24), PEPT 3B (SEQ ID
NO: 25)), and the position of synthetic peptides used to raise
antibodies (AG1 (SEQ ID NO: 26), AG2 (SEQ ID NO: 27), AG3 (SEQ ID
NO: 28)). The residues at the N and C termini of peptides are
indicated and numbered according to the initiator methionine of SEQ
ID NO:9.
[0030] FIG. 6B shows a picture of Western analysis on cell extracts
(lanes 620) or precipitated medium (lanes 621) from cells
transfected with a vector expressing wild type BP-1 mouse cDNA
(lanes 620, 621, 622), or a form of BP-1 mouse cDNA where one of
the dibasic cleavage site was mutated (lanes 623). Migration of
molecular weight markers is indicated.
[0031] FIG. 7A depicts an expression vector for BP-1 (plasmid GD46)
that can be used to generate cell lines secreting the BP-1
polypeptide or fragments thereof in the culture medium. The vector
comprises components of a transcription unit for mouse BP-1 (700,
701, 702) as well as a transcription unit to confer resistance to
puromycin (703).
[0032] FIG. 7B shows the standard curve of an enzyme-linked
immunoadsorbent assay (ELISA) performed on increasing quantities of
synthetic peptide derived from the C terminal region of BP-1.
[0033] FIG. 8A shows the levels of BP-1 immunoreactivity in
fractions collected from a cation-exchange chromatography column
after passage of conditioned medium from 293-GD46-7 cells
overexpressing the mouse BP-1 coding sequence.
[0034] FIG. 8B is a picture of a silver-stained polyacrylamide gel
showing the proteins found in the conditioned medium from
293-GD46-7 cells (lane 810), in the flow through from a
cation-exchange chromatography column (lane 811) and in
BP-1-immunoreactive fractions from the same column (lane 812).
Arrow 813 points to a BP-1 product. Arrowhead 814 points to
aprotinin, a contaminating protease inhibitor included in the
conditioned medium before the chromatographic step.
[0035] FIG. 8C shows the result of a Western analysis performed on
conditioned medium from 293-GD46-7 cells (lane 820), on the flow
through from a cation-exchange chromatography column (lane 821) and
on BP-1-immunoreactive fractions from the same column (lane
822).
[0036] FIG. 9A depicts a vector (plasmid GD28b) that can be used to
generate recombinant adenoviral particles expressing BP-1 coding
sequence. The vector comprises a transcription unit for mouse BP-1
(901) flanked by two regions of the adenovirus serotype 5 genome
(902, 903).
[0037] FIG. 9B shows the result of an ELISA to detect BP-1 products
in the medium of primary cultures of rat osteoblasts infected with
recombinant adenoviral particles at day 11 of culture.
[0038] FIG. 10 shows the result of a Northern analysis to detect
osteocalcin (OCN) in total RNA extracted from primary cultures of
osteoblasts treated chronically with control medium (1001) or
medium containing BP-1 products (1002). The levels of a control
messenger (GAPDH) are also shown.
DETAILED DESCRIPTION OF THE INVENTION
[0039] A) Definitions
[0040] Throughout the text, the word "kilobase" is generally
abbreviated as "kb", the words "deoxyribonucleic acid" as "DNA",
the words "ribonucleic acid" as "RNA", the words "complementary
DNA" as "cDNA", the words "polymerase chain reaction" as "PCR", the
words "expressed sequenced tag" as "EST", and the words "reverse
transcription" as "RT". Nucleotide sequences are written in the 5'
to 3' orientation unless stated otherwise. Amino acid sequences are
written from the N terminus unless stated otherwise.
[0041] In order to provide an even clearer and more consistent
understanding of the specification and the claims, including the
scope given herein to such terms, the following definitions are
provided:
[0042] Exogenous nucleic acid: A nucleic acid (such as cDNA, cDNA
fragments, genomic DNA fragments, mRNA fragments, antisense RNA,
oligonucleotide) which is not naturally part of another nucleic
acid molecule. The "exogenous nucleic acid" may be from any
organism, purely synthetic, or any combination thereof.
[0043] Expressed sequenced tag: sequence information on a small
nucleic acid (typically 200-500 bp) derived from a gene
transcript.
[0044] Hormone: a molecule, generally polypeptidic in nature,
secreted extracellularly by one cell type to modulate the activity
of target cells.
[0045] Hormone precursor. Refers to a secreted protein that is
processed in the secretory pathway such that one or more fragments
are released extracellularly. Fragments can be further modified
(e.g. amidation at the C terminus) before or after release in the
extracellular space.
[0046] Host: A cell, tissue, organ or organism capable of providing
cellular components for allowing the expression of an exogenous
nucleic acid. The exogenous nucleic acid maybe cloned into a
vector. This term is intended to also include hosts which have been
modified in order to accomplish these functions. Bacteria, fungi,
animal (cells, tissues, or organisms) and plant (cells, tissues, or
organisms) are examples of a host.
[0047] Insertion: The process by which a nucleic acid is introduced
into another nucleic acid. Methods for inserting a nucleic acid
into another normally requires the use of restriction enzymes and
such methods of insertion are well known in the art.
[0048] In silico: using computer and bioinformatics software and
hardware.
[0049] Nucleic acid: Any DNA, RNA sequence or molecule having one
nucleotide or more, including nucleotide sequences encoding a
complete gene. The term is intended to encompass all nucleic acids
whether occurring naturally or non-naturally in a particular cell,
tissue or organism. This includes DNA and fragments thereof, RNA
and fragments thereof, cDNAs and fragments thereof, expressed
sequence tags, artificial sequences including randomized artificial
sequences.
[0050] Open reading frame ("ORF"). The portion of a cDNA that is
translated into a protein. Typically, an open reading frame starts
with an initiator ATG codon and ends with a termination codon (TM,
TAG or TGA).
[0051] Protein products: refers to the various peptides or
polypeptides generated after translation of a single mRNA.
Polypeptides refer to amino acids linked to form a peptide or a
full length protein.
[0052] Recombinant: The term "recombinant" in association with
"vector" refers to a vector which has been modified to contain a
non-native exogenous nucleic acid.
[0053] Secreted protein: any protein that enters the cellular
secretory pathway. Typically, secreted proteins are synthesized
bearing a signal peptide which is removed shortly after its
synthesis.
[0054] Signal peptide: a peptide capable of directing a nascent
protein to the cell secretory pathway. It is generally accepted
that a signal peptide is composed of an initiating methionine, a
highly hydrophobic stretch, typically 10 to 15 residues long, and a
signal peptidase cleavage site.
[0055] Transcription unit: As used herein, a "transcription unit"
refers to a nucleic acid which comprises an enhancer sequence, a
promoter sequence, and a transcription termination sequence, all
operably linked together. Preferably, the enhancer and promoter
sequences are constitutively active in various hosts. Enhancer and
promoter sequences can be derived for example from the
cytomegalovirus (CMV) immediate-early genes or from the Rous
sarcoma virus (RSV) long terminal repeat. Preferably, the
transcription unit is comprised within a vector.
[0056] Transfection: the process of introducing nucleic acids in
eukaryotic cells by any means such as electroporation, lipofection,
precipitate uptake, micro-injection. A cell having incorporated an
exogenous nucleic acid is said to be transfected.
[0057] Vector: A RNA or DNA molecule which can be used to transfer
an RNA or DNA segment from one organism to another. Vectors are
particularly useful for manipulating genetic constructs and
different vectors may have properties particularly appropriate to
express protein(s) in a recipient during cloning procedures and may
comprise different selectable markers. Bacterial plasmids are
ucommonly used vectors.
[0058] B) General Overview of the Invention
[0059] The invention is based on isolated nucleic acids encoding
bone polypeptide-1. The invention encompasses isolated or
substantially purified nucleic acid or protein compositions. In the
context of the present invention, an "isolated" or "substantially
purified" DNA molecule or an "isolated" or "substantially purified"
polypeptide is a DNA molecule or polypeptide that, by the hand of
man, exists apart from its native environment and is therefore not
a product of nature. An isolated DNA molecule or polypeptide may
exist in a purified form or may exist in a non-native environment
such as, for example, a transgenic host cell. An isolated or
purified DNA or polypeptide may be synthesized chemically, may be
produced using recombinant DNA techniques and then isolated or
purified or may be isolated or purified from its natural host. An
"isolated" or "substantially purified" nucleic acid molecule or
protein, or biologically active portion thereof, is substantially
free of other cellular material, or culture medium when produced by
recombinant techniques and, in some circumstances, further purifed,
or substantially free of chemical precursors or other chemicals
when chemically synthesized. Preferably, an "isolated" nucleic acid
is free of sequences (preferably protein encoding sequences) that
naturally flank the nucleic acid (i.e., sequences located at the 5'
and 3' ends of the nucleic acid) in the genomic DNA of the organism
from which the nucleic acid is derived. For example, in various
embodiments, the isolated nucleic acid molecule can contain less
than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of
nucleotide sequences that naturally flank the nucleic acid molecule
in genomic DNA of the cell from which the nucleic acid is derived.
A protein that is substantially free of cellular material includes
preparations of protein or polypeptide having less than about 30%,
20%, 10%, 5%, (by dry weight) of contaminating protein. When the
protein of the invention, or biologically active portion thereof,
is recombinantly produced, preferably culture medium represents
less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical
precursors or non-protein of interest chemicals.
[0060] i) Cloning of a cDNA Fragment Encoding Bone polypetide-1
[0061] We have previously developed a screening system that allows
the rapid identification of nucleic acids encoding signal peptides
from complex libraries of cDNA fragments (International patent
application No. PCT/CA01/01169; U.S. patent application Ser. No.
09/641,931) both of which are hereby incorporated by reference in
their entirety. A library enriched in 5' fragments of cDNAs derived
from developing calvaria was obtained using the so-called
"oligo-capping" method (Maruyama and Sugano, 1994). Briefly, the
top halves of skulls of e15.5 mouse embryos were dissected free
from nervous or skin tissue. Total RNA was extracted using the
guanidium/phenol method and mRNA was isolated using Oligotex mRNA
kit (Qiagen, Mississauga, Canada). A synthetic oligoribonucleotide
(RNA30-1; 5'-agcaucgagucggccuuguuggccuacugg-3') SEQ ID NO:29 was
specifically ligated to the 5' extremities of mRNAs whose CAP
moiety had been converted to a free phosphate group by the action
of tobacco acid pyrophosphatase. The first strand of corresponding
cDNA was synthesized using a primer-linker comprising a random
sequence of 9 nucleotides (36-2V;
5'-gagagagagagagcgactcggatccannnnnnnnnc-3') SEQ ID NO 30. The
resulting first strand cDNA was amplified by 30 cycles of PCR using
primers complementary to both extremities (18-114V;
5'-agcatcgagtcggccttg-3') SEQ ID NO: 31 and (18-99V;
5'-gagagcgactcggatcca-3') SEQ ID NO: 32. Amplicons were purified
and subjected to a further 10 cycles of semi-nested PCR using
primers 18-99V and 32-4V (5'-ggacgagcggccgcgccttgttggcctactgg-3')
SEQ ID NO:33. PCR products were digested with NotI and BamHI,
size-selected by electrophoresis (500-1200 bp) and directionally
cloned in an expression/screening vector digested with NotI and
BamHI. The library numbered approximately 3,000,000 primary clones.
Sequencing of randomly chosen inserts revealed that the average
insert size was 575 bp and that 60% of inserts corresponded to 5'
fragments of cDNAs. The library was expression screened in BHK-21
fibroblasts. Selected fragments were amplified, cloned in the
pBluescript KS II+ vector and sequenced using a CEQ2000.TM.
automated sequencer.
[0062] One of the retrieved fragments (ca155-cap1-c2-neg306, 433 bp
in length) encoded a putative translation product of 124 residues
containing a signal peptide. Interestingly, this translation
product harbored sequences reminiscent of dibasic cleavage sites
found in peptide hormone precursor (KKKR at position 76-79 and KKR
at position 110-112). This observation prompted us to determine the
expression profile of the corresponding gene by Northern analysis
of RNA extracted from 52 different tissues and cell lines. No
signal was detected after an exposure of 7 days; a representative
autoradiograph is shown in FIG. 1A. In order to verify that the
retrieved fragment derived from a cellular transcript, Northern
analysis was performed with 1.5 .mu.g of polyA+ RNA extracted from
e15.5 mouse calvaria, adult liver and adult kidney (FIG. 1B). A
clear signal (arrow; 25), corresponding to a mRNA of approximately
1.3 kb, was specifically detected in the embryonic calvaria. Taken
together, these results indicate that ca155-cap1-c2-neg306 is
derived from a mRNA that encodes a bone protein that is
specifically expressed in bone tissue.
[0063] ii) Results from Database Mining; Cloning of Full Length
Mouse cDNA Containing c155-cap1-c2-neg306
[0064] Because of the interesting features of clone
ca155-cap1-c2-neg306, we carried out extensive database searches to
compare its sequence with those deposited in the public domain. We
used the standard Blastn tool (v2.2.4, Aug. 26, 2002) with the
following parameter set: expect value of 10, low complexity filter.
There was only one significant match to an entry (accession
XM.sub.--155941.1) in the non redundant (nr) set of the Genbank.TM.
database. This clone (designated LOC239790) encodes a putative
protein whose N terminal sequence is identical to that of
ca155-cap1-c2-neg306 but diverges at residues Gln.sup.102. Searches
in dbEST revealed that cal 55-cap1-c2-neg306 shows high homology to
a bovine EST (77.5% homology, accession number BF045261), to an
uncharacterized mouse EST (accession number BB638598.1), to 3 human
ESTs from metastatic chondrosarcoma (accession numbers BQ021661,
BQ001512, BQ000995) and that the last 102 nucleotides of cal
55-cap1-c2-neg306 shows high homology (85.8%) to the last 105
nucleotides of a rat EST (accession number Al178209). It should be
noted that a valid ORF can not be deduced from the sequences of
either mouse, rat or human ESTs. The 526 bp bovine EST contained an
ORF of 132 residues. In silico assembly of cal 55-cap1-c2-neg306
and the rat EST yielded a 0.99 kb chimeric sequence, roughly
corresponding to the size of the expected full length transcript
minus polyA tail (see Northern analysis, FIG. 1B).
[0065] Since we had initially cloned the 5' end of
ca155-cap1-c2-neg306, we used a modified 3' RACE strategy to obtain
a full length cDNA (see Materials and Methods in the Examples
section). Sequences from at least five different clones were
aligned together with cal 55-cap1-c2-neg306 to reconstitute the
full length consensus cDNA sequence shown on FIG. 2. The 1280 bp
cDNA (SEQ ID NO:1) contains an ORF of 393 bp (uppercase) flanked by
61 bp and 811 bp of untranslated sequences at the 5' and 3' ends,
respectively (lowercase). A polyadenylation signal is found 14 bp
upstream of a polyA stretch. The putative initiator ATG codon is
found in an adequate Kozak context (MGATGC, SEQ ID NO: 25).
[0066] iii) Expression Profiling on Histological Sections of Mouse
Embryos
[0067] To determine which bone cells express the cDNA from which
ca155-cap1-c2-neg306 derives, in situ hybridization was performed
on sections of e13.5 and e16.5 mouse embryos using standard
procedures. Consecutive sections were hybridized with a probe
partially complementary to Cbfa1 transcripts. Cbfa1 is a
transcription factor expressed in cells of the osteoblast lineage
and whose activity is essential for proper development of the
skeleton (Ducy, 2000). As shown in FIG. 3B, the
ca155-cap1-c2-neg306 probe specifically labels cells surrounding
the bone collar in e16.5 limbs. These cells are located in the
periosteum and are cells of the osteoblast lineage. This is
revealed by the fact that they express Cbfa1 (FIG. 3C). At e13.5,
expression of ca155-cap1-c2-neg306 is seen in a few cells in the
vicinity of cartilaginous formations in the skull (FIG. 3E). These
cells are also labeled by the Cbfa1 probe (FIG. 3F). Thus, the cDNA
from which ca155-cap1-c2-neg306 derives is expressed in cells of
the osteoblast lineage.
[0068] iv) Cloning of Vertebrate Homologs
[0069] Because of its features, the cDNA from which cal
55-cap1-c2-neg306 derives will be referred hereafter as mouse BP-1,
mouse bone polypeptide-1. As discussed in more detail below,
various homologs, including at least one human homolog to BP-1
exist. Bone polypeptide-1, as defined herein, refers to a
polypeptide expressed in bone. As discussed in more detail below,
bone polypeptide-1 may regulate bone cell proliferation and/or bone
cell differentiation and/or osteoblast activity via an autocrine,
paracrine or endocrine pathway.
[0070] BP-1 DNA Sequences
[0071] The BP-1 DNA used in any embodiment of this invention can be
BP-1 cDNA obtained as described herein, or alternatively, can be
any oligonucleotide sequence having all or a portion of a sequence
represented herein, or their functional equivalents. Such
oligodeoxynucleotide sequences can be produced chemically or
mechanically, using known techniques.
[0072] The following terms are used to describe the sequence
relationships between two or more nucleic acids or polynucleotides:
(a) "reference sequence", (b)"comparison window", (c) "sequence
identity", (d) "percentage of sequence identity", and (e)
"substantial identity".
[0073] (a) As used herein, "reference sequence" is a defined
sequence used as a basis for sequence comparison. A reference
sequence may be a subset or the entirety of a specified sequence;
for example, as a segment of a full length BP-1 cDNA or gene
sequence, or the complete cDNA or gene sequence.
[0074] (b) As used herein, "comparison window" makes reference to a
contiguous and specified segment of a polynucleotide sequence,
wherein the polynucleotide sequence in the comparison window may
comprise additions or deletions (i.e., gaps) compared to the
reference sequence (which does not comprise additions or deletions)
for optimal alignment of the two sequences. Generally, the
comparison window is at least 20 contiguous nucleotides in length,
and optionally can be 30, 40, 50, 100, or longer. Those of skill in
the art understand that to avoid a high similarity to a reference
sequence due to inclusion of gaps in the polynucleotide sequence a
gap penalty is typically introduced and is subtracted from the
number of matches.
[0075] Methods of alignment of sequences for comparison are well
known in the art. Thus, the determination of percent identity
between any two sequences can be accomplished using a mathematical
algorithm. Preferred, non-limiting examples of such mathematical
algorithms are the algorithm of Myers and Miller, 1988; the local
homology algorithm of Smith et al. 1981; the homology alignment
algorithm of Needleman and Wunsch 1970; the
search-for-similarity-method of Pearson and Lipman 1988; the
algorithm of Karlin and Altschul, 1990, modified as in Karlin and
Altschul, 1993.
[0076] Computer implementations of these mathematical algorithms
can be utilized for comparison of sequences to determine sequence
identity. Such implementations include, but are not limited to:
CLUSTAL in the PC/Gene program (available from Intelligenetics,
Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP,
BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics
Software Package, Version 8 (available from Genetics Computer Group
(GCG), 575 Science Drive, Madison, Wis., USA). Alignments using
these programs can be performed using the default parameters. The
CLUSTAL program is well described by Higgins et al. 1988; Higgins
et al. 1989; Corpet et al. 1988; Huang et al. 1992; and Pearson et
al. 1994. The ALIGN program is based on the algorithm of Myers and
Miller, supra. The BLAST programs of Altschul et al., 1990, are
based on the algorithm of Karlin and Altschul supra.
[0077] Software for performing BLAST analyses is publicly available
through the web site for National Center for Biotechnology
Information (NCBI). This algorithm involves first identifying high
scoring sequence pairs (HSPs) by identifying short words of length
W in the query sequence, which either match or satisfy some
positive-valued threshold score T when aligned with a word of the
same length in a database sequence. T is referred to as the
neighborhood word score threshold (Altschul et al., 1990). These
initial neighborhood word hits act as seeds for initiating searches
to find longer HSPs containing them. The word hits are then
extended in both directions along each sequence for as far as the
cumulative alignment score can be increased. Cumulative scores are
calculated using, for nucleotide sequences, the parameters M
(reward score for a pair of matching residues; always >0) and N
(penalty score for mismatching residues; always <0). For amino
acid sequences, a scoring matrix is used to calculate the
cumulative score. Extension of the word hits in each direction are
halted when the cumulative alignment score falls off by the
quantity X from its maximum achieved value, the cumulative score
goes to zero or below due to the accumulation of one or more
negative-scoring residue alignments, or the end of either sequence
is reached.
[0078] In addition to calculating percent sequence identity, the
BLAST algorithm also performs a statistical analysis of the
similarity between two sequences (see, e.g., Karlin & Altschul
(1993). One measure of similarity provided by the BLAST algorithm
is the smallest sum probability (P(N)), which provides an
indication of the probability by which a match between two
nucleotide or amino acid sequences would occur by chance. For
example, a test nucleic acid sequence is considered similar to a
reference sequence if the smallest sum probability in a comparison
of the test nucleic acid sequence to the reference nucleic acid
sequence is less than about 0.1, more preferably less than about
0.01, and most preferably less than about 0.001.
[0079] To obtain gapped alignments for comparison purposes, Gapped
BLAST (in BLAST 2.0) can be utilized as described in Altschul et
al. 1997. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to
perform an iterated search that detects distant relationships
between molecules. See Altschul et al., supra. When utilizing
BLAST, Gapped BLAST, PSI-BLAST, the default parameters of the
respective programs (e.g. BLASTN for nucleotide sequences, BLASTX
for proteins) can be used. The BLASTN program (for nucleotide
sequences) uses as defaults a wordlength (W) of 11, an expectation
(E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both
strands. For amino acid sequences, the BLASTP program uses as
defaults a wordlength (W) of 3, an expectation (E) of 10, and the
BLOSUM62 scoring matrix (see Henikoff & Henikoff, 1989). See
the NCBI web site. Alignment may also be performed manually by
inspection.
[0080] For purposes of the present invention, comparison of BP-1
nucleotide sequences for determination of percent sequence identity
to the BP-1 sequences disclosed herein is preferably made using the
BlastN program (version 1.4.7 or later) with its default parameters
or any equivalent program. By "equivalent program" is intended any
sequence comparison program that, for any two sequences in
question, generates an alignment having identical nucleotide or
amino acid residue matches and an identical percent sequence
identity when compared to the corresponding alignment generated by
the preferred program.
[0081] .COPYRGT. As used herein, "sequence identity" or "identity"
in the context of two BP-1 nucleic acid or polypeptide sequences
makes reference to the residues in the two sequences that are the
same when aligned for maximum correspondence over a specified
comparison window. When percentage of sequence identity is used in
reference to proteins it is recognized that residue positions which
are not identical often differ by conservative amino acid
substitutions, where amino acid residues are substituted for other
amino acid residues with similar chemical properties (e.g., charge
or hydrophobicity) and therefore do not change the functional
properties of the molecule. When sequences differ in conservative
substitutions, the percent sequence identity may be adjusted
upwards to correct for the conservative nature of the substitution.
Sequences that differ by such conservative substitutions are said
to have "sequence similarity" or "similarity." Means for making
this adjustment are well known to those of skill in the art.
Typically this involves scoring a conservative substitution as a
partial rather than a full mismatch, thereby increasing the
percentage sequence identity. Thus, for example, where an identical
amino acid is given a score of 1 and a non-conservative
substitution is given a score of zero, a conservative substitution
is given a score between zero and 1. The scoring of conservative
substitutions is calculated, e.g., as implemented in the program
PC/GENE (Intelligenetics, Mountain View, Calif.).
[0082] (d) As used herein, "percentage of sequence identity" means
the value determined by comparing two optimally aligned sequences
over a comparison window, wherein the portion of the polynucleotide
sequence in the comparison window may comprise additions or
deletions (i.e., gaps) as compared to the reference sequence (which
does not comprise additions or deletions) for optimal alignment of
the two sequences. The percentage is calculated by determining the
number of positions at which the identical nucleic acid base or
amino acid residue occurs in both sequences to yield the number of
matched positions, dividing the number of matched positions by the
total number of positions in the window of comparison, and
multiplying the result by 100 to yield the percentage of sequence
identity.
[0083] (e)(I) The term "substantial identity" of polynucleotide
sequences means that a BP-1 polynucleotide comprises a sequence
that has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,
70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, preferably at
least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more
preferably at least 90%, 91%, 92%, 93%, or 94%, and most preferably
at least 95%, 96%, 97%, 98%, or 99% sequence identity, compared to
a reference sequence using one of the alignment programs described
using standard parameters. One of skill in the art will recognize
that these values can be appropriately adjusted to determine
corresponding identity of proteins encoded by two nucleotide
sequences by taking into account codon degeneracy, amino acid
similarity, reading frame positioning, and the like. Substantial
identity of amino acid sequences for these purposes normally means
sequence identity of at least 70%, more preferably at least 80%,
90%, and most preferably at least 95%.
[0084] Another indication that nucleotide sequences are
substantially identical is if two molecules hybridize to each other
under stringent conditions (see below). Generally, stringent
conditions are selected to be about 5.degree. C. lower than the
thermal melting point (T.sub.m) for the specific sequence at a
defined ionic strength and pH. However, stringent conditions
encompass temperatures in the range of about 1.degree. C. to about
20.degree. C., depending upon the desired degree of stringency as
otherwise qualified herein. Nucleic acids that do not hybridize to
each other under stringent conditions are still substantially
identical if the polypeptides they encode are substantially
identical. This may occur, e.g., when a copy of a nucleic acid is
created using the maximum codon degeneracy permitted by the genetic
code. One indication that two nucleic acid sequences are
substantially identical is when the polypeptide encoded by the
first nucleic acid is immunologically cross reactive with the
polypeptide encoded by the second nucleic acid.
[0085] (e)(ii) The term "substantial identity" in the context of a
BP-1 peptide indicates that a peptide comprises a sequence with at
least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,
72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, preferably 80%, 81%,
82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more preferably at least
90%, 91%, 92%, 93%, or 94%, or even more preferably, 95%, 96%, 97%,
98% or 99%, sequence identity to the reference sequence over a
specified comparison window. Preferably, optimal alignment is
conducted using the homology alignment algorithm of Needleman and
Wunsch (1970). An indication that two peptide sequences are
substantially identical is that one peptide is immunologically
reactive with antibodies raised against the second peptide. Thus, a
peptide is substantially identical to a second peptide, for
example, where the two peptides differ only by a conservative
substitution.
[0086] For sequence comparison, typically one sequence acts as a
reference sequence to which test sequences are compared. When using
a sequence comparison algorithm, test and reference sequences are
input into a computer, subsequence coordinates are designated if
necessary, and sequence algorithm program parameters are
designated. The sequence comparison algorithm then calculates the
percent sequence identity for the test sequence(s) relative to the
reference sequence, based on the designated program parameters.
[0087] As noted above, another indication that two BP-1 nucleic
acid sequences are substantially identical is that the two
molecules hybridize to each other under stringent conditions. The
phrase "hybridizing specifically to" refers to the binding,
duplexing, or hybridizing of a molecule only to a particular
nucleotide sequence under stringent conditions when that sequence
is present in a complex mixture (e.g., total cellular) DNA or RNA.
"Bind(s) substantially" refers to complementary hybridization
between a probe nucleic acid and a target nucleic acid and embraces
minor mismatches that can be accommodated by reducing the
stringency of the hybridization media to achieve the desired
detection of the target nucleic acid sequence.
[0088] "Stringent hybridization conditions" and "stringent
hybridization wash conditions" in the context of nucleic acid
hybridization experiments such as Southern and Northern
hybridization are sequence dependent, and are different under
different environmental parameters. The T.sub.m is the temperature
(under defined ionic strength and pH) at which 50% of the target
sequence hybridizes to a perfectly matched probe. Specificity is
typically the function of post-hybridization washes, the critical
factors being the ionic strength and temperature of the final wash
solution. For DNA-DNA hybrids, the T.sub.m can be approximated from
the equation of Meinkoth and Wahl, 1984; T.sub.m 81.5.degree.
C.+16.6 (log M)+0.41 (% GC)-0.61 (% form) -500/L; where M is the
molarity of monovalent cations, % GC is the percentage of guanosine
and cytosine nucleotides in the DNA, % form is the percentage of
formamide in the hybridization solution, and L is the length of the
hybrid in base pairs. T.sub.m is reduced by about 1.degree. C. for
each 1% of mismatching; thus, T.sub.m, hybridization, and/or wash
conditions can be adjusted to hybridize to sequences of the desired
identity. For example, if sequences with >90% identity are
sought, the T.sub.m can be decreased 10.degree. C. Generally,
stringent conditions are selected to be about 5.degree. C. lower
than the thermal melting point I for the specific sequence and its
complement at a defined ionic strength and pH. However, severely
stringent conditions can utilize a hybridization and/or wash at 1,
2, 3, or 4.degree. C. lower than the thermal melting point I;
moderately stringent conditions can utilize a hybridization and/or
wash at 6, 7, 8, 9, or 10.degree. C. lower than the thermal melting
point I; low stringency conditions can utilize a hybridization
and/or wash at 11, 12, 13, 14, 15, or 20.degree. C. lower than the
thermal melting point I. Using the equation, hybridization and wash
compositions, and desired T, those of ordinary skill will
understand that variations in the stringency of hybridization
and/or wash solutions are inherently described. If the desired
degree of mismatching results in a T of less than 45.degree. C.
(aqueous solution) or 32.degree. C. (formamide solution), it is
preferred to increase the SSC concentration so that a higher
temperature can be used. An extensive guide to the hybridization of
nucleic acids is found in Tijssen, 1993. Generally, highly
stringent hybridization and wash conditions are selected to be
about 5.degree. C. lower than the thermal melting point T.sub.m for
the specific sequence at a defined ionic strength and pH.
[0089] An example of highly stringent wash conditions is 0.15 M
NaCl at 72.degree. C. for about 15 minutes. An example of stringent
wash conditions is a 0.2.times.SSC wash at 65.degree. C. for 15
minutes (see, Sambrook, infra, for a description of SSC buffer).
Often, a high stringency wash is preceded by a low stringency wash
to remove background probe signal. An example medium stringency
wash for a duplex of, e.g., more than 100 nucleotides, is
1.times.SSC at 45.degree. C. for 15 minutes. An example low
stringency wash for a duplex of, e.g., more than 100 nucleotides,
is 4-6.times.SSC at 40.degree. C. for 15 minutes. For short probes
(e.g., about 10 to 50 nucleotides), stringent conditions typically
involve salt concentrations of less than about 1.5 M, more
preferably about 0.01 to 1.0 M, Na ion concentration (or other
salts) at pH 7.0 to 8.3, and the temperature is typically at least
about 30.degree. C. and at least about 60.degree. C. for long robes
(e.g., >50 nucleotides). Stringent conditions may also be
achieved with the addition of destabilizing agents such as
formamide. In general, a signal to noise ratio of 2.times. (or
higher) than that observed for an unrelated probe in the particular
hybridization assay indicates detection of a specific
hybridization. Nucleic acids that do not hybridize to each other
under stringent conditions are still substantially identical if the
proteins that they encode are substantially identical. This occurs,
e.g., when a copy of a nucleic acid is created using the maximum
codon degeneracy permitted by the genetic code.
[0090] Very stringent conditions are selected to be equal to the
T.sub.m for a particular probe. An example of stringent conditions
for hybridization of complementary nucleic acids which have more
than 100 complementary residues on a filter in a Southern or
Northern blot is 50% formamide, e.g., hybridization in 50%
formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in
0.1.times.SSC at 60 to 65.degree. C. Exemplary low stringency
conditions include hybridization with a buffer solution of 30 to
35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at
37.degree. C., and a wash in 1.times. to 2.times.SSC
(20.times.SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to
55.degree. C. Exemplary moderate stringency conditions include
hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at
37.degree. C., and a wash in 0.5.times. to 1.times.SSC at 55 to
60.degree. C.
[0091] The following are examples of sets of hybridization/wash
conditions that may be used to clone orthologous nucleotide
sequences that are substantially identical to reference nucleotide
sequences of the present invention: a reference nucleotide sequence
preferably hybridizes to the reference nucleotide sequence in 7%
sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at
50.degree. C. with washing in 2.times.SSC, 0.1% SDS at 50.degree.
C., more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M
NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in 1.times.SSC,
0.1% SDS at 50.degree. C., more desirably still in 7% sodium
dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C.
with washing in 0.5.times.SSC, 0.1% SDS at 50.degree. C.,
preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO.sub.4, 1
mM EDTA at 50.degree. C. with washing in 0.1.times.SSC, 0.1% SDS at
50.degree. C., more preferably in 7% sodium dodecyl sulfate (SDS),
0.5 M NaPO.sub.4, 1 mM EDTA at 50.degree. C. with washing in
0.1.times.SSC, 0.1% SDS at 65.degree. C.
[0092] Protein Sequences
[0093] The present invention includes peptides which are derivable
from BP-1. A peptide is said to be "derivable from a naturally
occurring BP-1 amino acid sequence" if it can be obtained by
fragmenting a naturally occurring sequence, or if it can be
synthesized based upon a knowledge of the sequence of the naturally
occurring BP-1 amino acid sequence or of the genetic material (DNA
or RNA) which encodes this sequence.
[0094] Included within the scope of the present invention are those
molecules which can be said to be "derivatives" or "active
fragments" of BP-1 referred herein as "BP-1 products". Such a
"derivative" or "active fragment" has one or more of the following
characteristics: (1) it shares substantial homology with BP-1 or a
similarly sized fragment of BP-1; (2) it is capable of functioning
as a bone hormone and (3) using at least one of the assays provided
herein, the derivative has either (I) a bone hormone activity, or,
(ii) an activity on the kidney. Bone hormone activity refers to an
ability to regulate bone cell proliferation and/or bone cell
differentiation and/or of osteoblast activity, via an autocrine,
paracrine or endocrine pathways.
[0095] A derivative of BP-1 is said to share "substantial homology"
with BP-1 if the amino acid sequences of the derivative is at least
60%, and more preferably at least 80%, and most preferably at least
90%, the same as that of BP-1.
[0096] The derivatives of the present invention include BP-1
fragments which, in addition to containing a sequence that is
substantially homologous to that of a naturally occurring BP-1
peptide may contain one or more additional amino acids at their
amino and/or their carboxy termini. Thus, the invention pertains to
polypeptide fragments of BP-1 that may contain one or more amino
acids that may not be present in a naturally occurring BP-1
sequence. The additional amino acids may be D-amino acids or
L-amino acids or combinations thereof. Furthermore, the additional
amino acids may be naturally occurring amino acids or non-naturally
occurring amino acids such as L-tert-leucine; L-homophenylalanine;
D-homophenylalanine; D-methionine; Halogenated D and
L-phenylalanines, tyrosines, and tryptophans; D-2-aminopimelic acid
and L-2-aminopimelic acid
[0097] The invention also includes BP-1 fragments which, although
containing a sequence that is substantially homologous to that of a
naturally occurring BP-1 peptide may lack one or more additional
amino acids at their amino and/or their carboxy termini that are
naturally found on a BP-1 peptide. Thus, the invention pertains to
polypeptide fragments of BP-1 that may lack one or more amino acids
that are normally present in a naturally occurring BP-1.
[0098] The invention also encompasses the obvious or trivial
variants of the above-described fragments which have
inconsequential amino acid substitutions (and thus have amino acid
sequences which differ from that of the natural sequence) provided
that such variants have a bone hormone activity which is
substantially identical to that of the above-described BP-1
derivatives. Examples of obvious or trivial substitutions include
the substitution of one basic residue for another (i.e. Arg for
Lys), the substitution of one hydrophobic residue for another (i.e.
Leu for lie), or the substitution of one aromatic residue for
another (i.e. Phe for Tyr), etc.
[0099] Bone Polypeptide-1 Homologs
[0100] A cDNA encoding BP-1, or a portion thereof can be used to
identify similar sequences in other vertebrate species and thus, to
identify or "pull out" sequences which have sufficient homology to
hybridize to BP-1 cDNA or mRNA or a portion thereof under
conditions of low stringency. Those sequences which have sufficient
homology (generally greater than 40%) can be selected for further
assessment using the method described herein. Alternatively, high
stringency conditions can be used as discussed above. In this
manner, DNA of the present invention can be used to identify, in
other species, sequences encoding polypeptides having amino acid
sequences similar to that of BP-1 and thus to identify bone
polypeptides in other species. Thus, the present invention includes
not only BP-1, but also related proteins encoded by DNA which
hybridizes, preferably under high stringency conditions, to DNA of
the present invention.
[0101] BLAST searches of the mouse BP-1 sequence using the Ensembl
web site (containing the sequence of the human genome, v8.3.0a.1,
August 2002 data) revealed that a human homolog of BP-1 might
exist. Indeed, homologous sequences are found on human chromosome
3q28 in contigs AC019234.5.48926.64909 (nt 12659-12937, 75%
homology), AC019234.5.130697.209258 (nt 75405-75529, 79% homology),
AC019234.5.89412.130596 (nt 39498-39560, 92% homology). The
Genescan algorithm predicts 4 exons but those are disordered and
not annotated as part of a single gene and, furthermore, the
predicted translation product do not match the primary sequence of
BP-1, presumably because Genescan has not identified the correct
ORF. Thus, the ORF predicted from the BP-1 cDNA (FIG. 2) can not be
determined solely by in silico analysis of the sequence of the
human genome. Therefore, in order to clone a human homolog of BP-1,
we performed RT-PCR on human bone marrow mRNA using two primers
encompassing the putative initiator and termination codons found in
the human genome sequence. In addition, the rat homolog of BP was
cloned by low-stringency RT-PCR using one primer encompassing the
putative initiator codon and another encompassing a region in the
3' untranslated region of the mouse cDNA. To identify
evolutionarily conserved domains within the BP-1 protein, we
obtained sequence information on BP-1 from non mammalian species.
The cDNA encoding chicken (Gallus gallus) BP-1 was retrieved by
`BLASTing` the human ORF against the BBSRC Chicken EST Project
database (Blastn tool, all tissues, matrix BLOSUM62, expectation
10-2). A portion of the cDNA encoding snake (Python molurus
bivittatus) BP-1 was cloned by RT-PCR using degenerate
oligonucleotide primers and starting from RNA extracted from the
vertebrae of a young python (See Materials and Methods in the
Examples section.) FIG. 4 shows the alignment of the nucleic acid
sequences encoding human, bovine, mouse, rat, chicken, and python
BP-1. Allelic variations of the nucleic acid sequences described
herein are also encompassed in the invention.
[0102] FIG. 5A shows the alignment of the ORFs derived from the
BP-1 cDNAs of mouse, human, rat, cow, chicken. The partial ORF
deduced from the python sequence is also aligned. The presence of a
cleavable signal peptide as well as the position of the putative
processing sites are conserved across species (respectively
underlined and boxed in FIG. 5A). In silico analysis indicates that
a) the region of the protein that lies between the 2 dibasic
cleavage sites (residues 83-112 in the human sequence) is well
conserved (overall identity of 60%, overall similarity of 76%); b)
the C terminal region of the protein (residues 116-133 in the human
sequence) is also well conserved (human and mouse share 94.4%
similarity and identity; human and chicken share 83.3% similarity
and 61.1% identity). The observation that the dibasic cleavage
sites are found in various species suggests that BP-1 is a
prohormone precursor conserved in terrestrial vertebrates.
Furthermore, analysis of the conserved domains suggests that the
bioactive protein products derive from the C terminal half of
BP-1.
[0103] In silico analysis indicates that full length human BP-1 has
the following feature: it has a molecular weight of 14,832 g/mol
and an isoelectric point of 9.62; it contains neither
N-glycosylation sites nor disulfide bridges. Despite extensive
searches in public databases (e.g. InterPro, ProDom, STN DGENE),
BP-1 shows no significant homology to any known protein, protein
motif or protein domain. We have found however that residues
116-124 of BP-1 (numbering from the human sequence) show some
homology to members of the natriuretic peptides family. FIG. 5B
shows the alignment of the homologous regions between BP-1 and
natriuretic peptides. The 2 cysteine residues conserved in all
natriuretic peptides form an intramolecular disulfide bridge
essential for their bioactivity(Inagami et al., 1985). Cysteine
residues involved in cyclization are not found in BP-1.
Interestingly however, it has been reported that a synthetic
`linear` analog of the atrial natriuretic peptide could displace
the binding of atrial natriuretic factor to its receptor but failed
to activate synthesis of second messengers (Olins et al., 1988).
Thus, it is possible that a BP-1 protein product could be related
both biochemically and functionally to the natriuretic
peptides.
[0104] v) Production of Antibodies Against Portions of BP-1.
[0105] In order to detect the BP-1 protein products, antibodies
were raised against synthetic peptides derived from 3 regions of
the full length protein AG-1: (DELVSLENDVIETK (SEQ ID NO:26), AG-2:
(RLSAGSVDHKGKQR) SEQ ID NO: 27, and AG-3: (MDRIGRNRLSNSRG) SEQ ID
NO: 28). The chosen regions have a high antigenicity index as
predicted by the algorithm of Hopp and Woods (Hopp and Woods,
1981). The positions of the antigenic peptides are indicated on the
model of human BP-1 shown on FIG. 6A. To increase immunogenicity,
antigenic peptides comprise a N- or C-terminal cysteine to allow
covalent coupling to a carrier protein (e.g. keyhole limpet
hemocyanin, bovine serum albumin). The peptide/carrier complex are
injected subcutaneously into animals (e.g. rabbits) and antisera
are obtained using standard protocols. (See Materials and Methods
in Examples section.) Affinity chromatography was used to purifiy
the fraction of immunoglobulins specific to the peptide used to
elicit an immune response.
[0106] Both monoclonal and polyclonal antibodies are included
within the scope of this invention as they can be produced by well
established procedures known to those of skill in the art.
Additionally, any secondary antibodies, either monoclonal or
polyclonal, directed to the first antibodies would also be included
within the scope of this invention.
[0107] BP-1 is Secreted Extracellularly and can be Processed
[0108] To begin to assess the biochemical and biological properties
of BP-1, a cDNA encoding the mouse protein was inserted into a
mammalian expression vector and this vector was transfected in
HEK293A cells. Immunofluorescence analysis after cell
permeabilization showed that BP-1 is mainly localized to the Golgi
apparatus and in small cytoplasmic vesicles of transfected cells.
No signal was detected at the cell surface. Western analysis of
cell lysates and culture medium using antibodies raised against the
C terminal region of human BP-1 (AG3) (SEQ ID NO: 28) revealed that
the bulk of the protein products is secreted in the culture medium
(FIG. 6B, compare lanes 620 and 621). The major secreted form has
an apparent molecular weight of 13 kD, presumably corresponding to
the BP-1 precursor after removal of the signal peptide. Upon longer
exposure (FIG. 6B, lane 622), a smaller product with an apparent
molecular weight of .about.6 kD is reproducibly found in the
culture medium of transfected HEK293A cells. This .about.6 kD band
is absent when cells are transfected with a vector expressing a
form of the mouse cDNA where the dibasic cleavage site at position
76-79 is mutated (KKKR->AS, FIG. 6B, lane 623).
[0109] Given these results, we predict that BP-1 is processed
according to the following scheme. After translocation in the
endoplasmic reticulum, the signal peptide is cleaved and the
protein (PROT 1, SEQ ID NO: 20) may be further processed in the
secretory pathway or extracellular space to yield bioactive
products. Candidate processing enzymes include furin (Denault and
Leduc, 1996; GenBank.TM. PID accession number g31478), a related
convertase or, as discussed below, corin (GenBank.TM. PID accession
number g5729989). Our results indicate that the BP-1 precursor may
be cleaved at arginine.sup.82 (numbering according to human
sequence) in an heterologous expression system (e.g. HEK293A
fibroblasts) to generate PEPT 1 (SEQ ID NO: 21). Additional
processing may occur C terminal to arginine.sup.115 at a typical
dibasic site to generate PEPT 2A (SEQ ID NO: 22) and PEPT 3A (SEQ
ID NO: 23). Alternative processing may occur at a basic residue
(lysine.sup.104) upstream of the second dibasic site by an a
typical convertase, similar to what has been reported for the
processing of a natriuretic peptide precursor by the corin protease
(Yan et al., 2000; Yan et al., 1999). This would generate PEPT 2B
(SEQ ID NO: 24) and PEPT 3B (SEQ ID NO: 25). Interestingly in this
latter case, both cleavage products of BP-1 end with a glycine
residue. This observation raises the possibility that BP-1 products
are amidated by peptidylglycine alpha-amidating monooxygenase
(GenBank.TM. accession number no. BAC22594), an enzyme whose
activity has been detected in mouse calvaria, a tissue that
produces BP-1 (Birnbaum et al., 1989). FIG. 6A illustrates the
processing scenario for human BP-1 and depicts its putative protein
products.
[0110] vi) Methods of Production of Recombinant or Synthetic BP-1
Protein Products.
[0111] The present invention provides expression vectors and host
cells transformed to express the nucleic acid sequences encoding
BP-1 of the invention. Expression vectors of the invention comprise
a nucleic acid sequence coding for at least one BP-1, or at least
one antigenic fragment thereof, or derivative or homologue thereof,
or the functional equivalent of such nucleic acid sequence. Nucleic
acid sequences coding for BP-1, or at least one fragment thereof
may be expressed in prokaryotic or eukaryotic host cells. Suitable
host cells include bacterial cells such as E. coli, insect cells,
yeast, or mammalian cells such as Chinese hamster ovary cells
(CHO). Suitable expression vectors, promoters, enhancers, and other
expression control elements may be found in Sambrook et al.
Molecular Cloning: A Laboratory Manual, second edition, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
[0112] Whatever the producing host, the BP-1 portion of the
transcription unit may be engineered to increase or control the
stability or translatability of the expressed mRNA, e.g. through
the insertion of exogenous cis-acting nucleic acid sequences, the
replacement of poorly translated codons, the removal of
untranslated sequences and/or the engineering of a proper context
for the initiation of translation (Kozak, 1986). The BP-1 cDNA may
also be engineered to increase the yield of secreted products,
typically by replacing its signal peptide coding sequence by an
exogenous nucleic acid encoding a more active signal peptide in the
chosen host. Furthermore, minor modifications or variations may be
introduced in the BP-1 coding sequence to enhance the stability
and/or the activity of BP-1 product(s). These modifications may be
made by site-directed mutagenesis or may be the result of
spontaneous mutations.
[0113] Vertebrate Cell Lines
[0114] An aspect of this invention relates to modified cell lines
and transgenic organisms used to produce BP-1 protein products.
According to a preferred embodiment, there is provided an
eukaryotic cell line having incorporated in its genome a DNA
segment comprising a BP-1 coding sequence operably linked within a
transcription unit. Enhancer and promoter sequences driving robust
expression in a wide variety of cells are generally preferred.
These include but are not limited to sequences derived from
cytomegalovirus immediate-early genes (CMV; e.g. GenBank.TM.
accession no. X03922) and Rous sarcoma virus long terminal repeat
(RSV; e.g. GenBank.TM. accession no M83237) as well as sequences
derived from widely expressed cellular genes such as chicken
.beta.-actin, human elongation factor 1.alpha., phosphoglycerate
kinase, and metallothionein. Signals for the termination and
polyadenylation of transcripts are well known in the art. Examples
include part of the 3' untranslated region of the bovine growth
hormone gene or of the SV40 virus.
[0115] Methods to incorporate DNA segments into the genome of a
cell are well known in the art. According to a preferred embodiment
of the invention, the transcription unit is inserted into a plasmid
containing a gene conferring resistance to a selective agent (e.g.
puromycin-N-acetyltransferase conferring resistance to puromycin).
The resulting construct is electroporated into cells using standard
protocols. Transfection by lipofection, calcium phosphate
precipitate, and micro-injection are other techniques available to
introduce nucleic acids into eukaryotic cells. Selection is applied
on the pool of transfected cells. Surviving and growing cells are
thought to have incorporated the plasmid and are cloned. Individual
clones are analyzed for the production of BP-1 protein products in
the culture medium. Typically, the levels of BP-1 products are
determined by Western analysis or ELISA. Preferred cellular hosts
include, but are not limited to, human embryonic kidney 293 cells
(HEK293; American Type Culture Collection no. CRL-1573) and chinese
hamster ovary cells (CHO; American Type Culture Collection no.
CCL-61). It is known in the prior art how to cultivate large
quantities of these cells in order to obtain large amounts of
recombinant proteins. It is understood that a BP-1-producing cell
line can be further engineered to express a convertase involved in
BP-1 processing, thereby allowing the release of bioactive BP-1
products into the culture medium.
[0116] Transgenic Organisms
[0117] Alternatively, the transcription unit comprising the BP-1
coding sequence can be inserted into a fertilized egg (e.g. of a
mouse), which is re-implanted into a pseudo-pregnant mother. DNA
extracted from resulting organisms (e.g. embryos, pups or adults)
is analyzed by Southern blotting to determine whether the organism
is transgenic. Positive animals are bred and used to produce large
quantities of BP-1 products. Ideally, the recombinant protein
products should be produced in an easily collectable tissue or
biological fluid (e.g. hair, milk). Furthermore, expression in
tissues that are targets of BP-1 action (e.g. bone, kidney) should
be limited. In order to achieve these goals, the enhancer and
promoter elements of the transcription unit are chosen so that the
BP-1 coding sequence is mainly expressed in the appropriate tissue
(e.g. keratinocytes, mammary epithelium).
[0118] Fusion Protein in Bacteria
[0119] For some aspects of the present invention, it is desirable
to produce a fusion protein comprising a BP-1 polypeptide or at
least one fragment thereof or their derivatives and an amino acid
sequence from another peptide or protein, examples of the latter
being enzymes such as beta-galactosidase, phosphatase, urease and
fusion proteins incorporating purification moieties such as
His-tags, FLAG-tags, myc-epitope tags and the like. Most fusion
proteins are formed by the expression of a recombinant gene in
which two coding sequences have been joined together such that
their reading frames are in phase.
[0120] For expression in E. coli, suitable expression vectors
include pTRC (Amann et al. (1988) Gene 69: 301-315); pET-11d
(Novagen, Madison, Wis.); pGEX (Amrad Corp., Melbourne, Australia);
pMAL (N.E. Biolabs, Beverly, Mass.); pRIT5 (Pharmacia, Piscataway,
N.J.); PSEM (Knapp et al. (1990) BioTechniques 8: 280-281); pQE30
(Qiagen, Germany); and pTrxFus (Invitrogen, Carlsbad, Calif.). The
use of pTRC and pET-11d will lead to the expression of unfused
protein. The use of PGEX, pMAL, pRIT5, pSEM, pQE30, and pTrxFus
will lead to the expression of BP-1 fused to glutathione
S-transferase (pGEX), maltose E binding protein (pMAL), protein A
(pRIT5), truncated .beta.-galactosidase (PSEM), hexahistidine
(His6), or thioredoxin. When a BP-1, fragment, or fragments thereof
is expressed as a fusion protein, it is particularly advantageous
to introduce an enzymatic cleavage site at the fusion junction
between the carrier protein and the BP-1 or fragment thereof. A
BP-1 or fragment thereof may then be recovered from the fusion
protein through enzymatic cleavage at the enzymatic site and
biochemical purification using conventional techniques for
purification of proteins and peptides. Suitable enzymatic cleavage
sites include those for blood clotting Factor Xa or thrombin for
which the appropriate enzymes and protocols for cleavage are
commercially available from for example Sigma Chemical Company, St.
Louis, Mo. and N.E. Biolabs, Beverly, Mass.
[0121] Host cells can be transformed to express the nucleic acid
sequences encoding the BP-1 of the invention using conventional
techniques such as calcium phosphate or calcium chloride
co-precipitation, DEAE-dextran-mediated transfection, or
electroporation. Suitable methods for transforming the host cells
may be found in Sambrook et al. supra, and other laboratory
textbooks. The nucleic acid sequences of the invention may also be
synthesized using standard techniques.
[0122] Chemical Synthesis
[0123] Homologues and derivatives of BP-1 is meant to include
synthetic derivatives thereof. The nucleotide sequences encoding
BP-1 can be used to chemically synthesize the entire protein or
generate any number of fragments (peptides) by chemical synthesis
by well known methods (e.g., solid phase synthesis). All such
chemically synthesized peptides are encompassed by the present
invention. Alternatively, proteins or peptides can be linked in
vitro by chemical means. All such fusion protein or hybrid genetic
derivatives of BP-1 or its encoding nucleotide sequences are
encompassed by the present invention. Accordingly, the present
invention extends to isolated BP-1, fragments thereof and their
derivatives, homologues and immunological relatives made by
recombinant means or by chemical synthesis.
[0124] vii) Methods of Purification of Recombinant BP-1 Protein
Products.
[0125] BP-1 and fragments (peptides) thereof can be purified from
cell culture medium, host cells, or both using techniques known in
the art for purifying peptides and proteins, including ion-exchange
chromatography, hydrophobic chromatography, gel filtration
chromatography, ultrafiltration, electrophoresis and
immunopurification with antibodies specific for BP-1 or fragments
thereof. The terms isolated and purified are used interchangeably
herein and refer to peptides, protein, protein fragments, and
nucleic acid sequences substantially free of cellular material or
culture medium when produced by recombinant DNA techniques, or
chemical precursors or other chemicals when synthesized
chemically.
[0126] Typically, recombinant proteins found in culture medium or
biological fluids are purified by successive steps of
chromatography according to methods known to an experimentator
skilled in the art. According to a preferred embodiment of the
invention, accumulation of secreted recombinant BP-1 is done by
incubating engineered mammalian cells in serum-free medium, in
order to limit the amount of contaminating proteins found in the
medium. This medium is then diluted with column buffer (neutral
HEPES-buffered saline) and passed on a cation-exchange column (e.g.
Sepharose.TM. SP, Amersham Pharmacia Biosciences). Since the
isoelectric point of human BP-1 is 9.62, it binds strongly to the
negatively-charged resin in column buffer. After extensive washes
with column buffer, bound proteins are eluted with a salt gradient.
Fractions are collected and assayed for BP-1 immunoreactivity by
ELISA. Under these conditions, the BP-1 products elute at a salt
concentration of 250-300 mM. Further purification is achieved by
affinity chromatography. Monoclonal or polyclonal antibodies
specific to BP-1 (e.g. raised against AG2, see section v) are
covalently attached to a resin, either directly (e.g.
CNBR-Sepharose.TM.) or through binding and crosslinking to
immobilized protein A. In the latter case, protein A specifically
binds the Fc portion of the antibodies, thereby avoiding
crosslinking/inactivation of their F(ab).sub.2 portion to the resin
and increasing the binding capacity of the affinity resin.
BP-1-containing fractions from the ion-exchange column are pooled,
diluted to a salt concentration of approximately 100 mM and passed
over the affinity column at neutral pH. After washes, bound
proteins are eluted under basic (pH 10) and acidic (pH 2.9)
conditions. Fractions are collected, neutralized and assayed for
BP-1 immunoreactivity by ELISA. BP-1-containing fractions are
pooled and, if necessary, dialyzed against physiological saline and
concentrated (e.g. by lyophilization).
[0127] viii) Recombinant Adenoviruses Expressing BP-1 cDNA for Gene
Delivery
[0128] Since processing of BP-1 to bioactive products may occur
uniquely in osteoblasts or within the bone microenvironment, it may
be advantageous to express a BP-1 coding sequence in cultured
osteoblasts or directly in vivo. Recombinant adenovirus particles
are particularly useful to deliver a transcription unit into a
whole organism or into cells that are difficult to transfect by
conventional methods such as differentiated osteoblasts (Ragot et
al., 1998). Standard methodology may be used to insert a
transcription unit in an adenoviral genome and package the
resulting recombinant adenoviral genome within an infectious viral
particle. Briefly, the transcription unit comprising a BP-1 coding
sequence is cloned in a plasmid between regions of the adenovirus
serotype 5 genome. This plasmid is transfected, along with a
replication-defective viral genome, in a cell line that can
complement the replication defect, usually HEK293 cells. Homologous
recombination between a plasmid and a viral genome generates a
recombinant viral genome having inserted a BP-1 transcription unit.
This recombinant viral genome is subsequently packaged into
infectious particles. Typically, the replication-defective viral
genome minimally lacks the E1 protein such that it can not
replicate in infected cells unless these are HEK293 cells that
endogenously synthesize the E1 protein (Louis et al., 1997). The
recombinant adenovirus can be propagated in HEK293 cells to very
high titers (>10.sup.10 pfu/ml). Stocks of recombinant
adenoviral particles are concentrated and purified by
centrifugation on cesium chloride gradient according to standard
procedures.
[0129] The resulting recombinant adenovirus can be used to infect
large numbers of various cell types, including primary osteoblasts,
to produce recombinant BP-1. It can also be used as a delivery
system for systemic or local gene therapy or other protocols that
may require overexpression of BP-1 in vivo.
[0130] ix) Therapeutic Utility of BP-1 Products
[0131] Considering the fact that BP-1 is specifically expressed in
the osteoblast lineage, BP-1 products may regulate bone cell
proliferation and/or bone cell differentiation and/or of osteoblast
activity, via an autocrine, paracrine or endocrine pathway. BP-1
products may also control osteoclast activity via a paracrine or
endocrine pathway. In either cases, pharmaceutical compositions
containing a therapeutically effective amount of BP-1 products may
be useful in the treatment of bone diseases, particularly in those
characterized by bone loss such as osteoporosis. The BP-1
compositions may be further employed in methods for treating bone
fractures or defects. Drugs that augment, mimick, antagonize or
blunt the activity of BP-1 products may also be beneficial for the
treatment of bone diseases. Considering the in vitro effect of our
BP-1 preparations (see Example 4), drugs that antagonize or blunt
the activity of BP-1 products could be particularly useful to treat
osteopenic or osteoporotic conditions.
[0132] Examples of drugs that augment the activity of BP-1 products
include chemicals that activate proteins involved in the
transcription of the BP-1 gene, thereby upregulating its
expression. Drugs that inhibit the degradation of a given BP-1
product should also lead to increased activity of this BP-1
product. Examples of drugs that mimick the activity of BP-1
products include peptidomimetics or peptides, modified or not,
corresponding to fragments of BP-1 that regulate bone or kidney
functions. In most cases, such drugs are agonists of the receptor
that binds a given BP-1 product.
[0133] Examples of drugs that antagonize or blunt the activity of
BP-1 products include a) one or a mixture of antisense
oligonucleotides blocking the expression or translation of the BP-1
mRNA; b) one or a mixture of antibodies raised against a given BP-1
product quenching the activity of this product; c) an antagonist
binding the cognate receptor of a given BP-1 product; d) a molecule
inhibiting the enzymes responsible for the processing of the BP-1
precursor to a given product.
[0134] Phosphatonin is a putative hormone which regulates phosphate
retention by kidney tubules. It has been hypothesized that
phosphatonin is produced by cells of the osteoblast lineage and is
a substrate for Phex, a metallopeptidase found at the cell surface
of osteoblasts and osteocytes (Frota Ruchon et al., 2000; Ruchon et
al., 1998; GenBank.TM. PID accession number g2499917). Our
observations are consistent with the possibility that a given BP-1
product could have phosphatonin activity. If this is the case, then
hypophosphatemia could be remedied by injection of a molecule that
antagonize this activity. On the other hand, hyperphosphatemia
could be remedied by injection of a molecule that stimulate or
mimick phosphatonin activity. Alternatively, phosphatonin activity
may be prolonged by using a drug that inhibits its degradation
(e.g. inhibitors of the Phex endopeptidase activity). Such
proteolysis inhibitors could be useful to treat hyperphosphatemic
conditions. Thus, drugs that augment, mimick, antagonize or blunt
the activity of BP-1 products may also be beneficial for the
treatment of diseases characterized by abnormal serum phosphate
levels.
[0135] Pharmaceutical Compositions
[0136] The present invention, therefore, provides a pharmaceutical
composition comprising a therapeutically effective amount of BP-1
or derivatives, homologues or immunological relatives thereof and
one or more pharmaceutically acceptable carriers and/or diluents.
The active ingredients of a pharmaceutical composition comprising
BP-1 is contemplated to exhibit therapeutic activity when
administered in amount which depends on the particular case. Dosage
regime may be adjusted to provide the optimum therapeutic response.
For example, several divided doses may be administered daily or the
dose may be proportionally reduced as indicated by the exigencies
of the therapeutic situation. The active compound may be
administered in a convenient manner such as by the oral,
intravenous (where water soluble), intramuscular, subcutaneous,
intranasal, intradermal or suppository routes or implanting (e.g.,
using slow release molecules). Depending on the route of
administration, the active ingredients which comprise the
pharmaceutical composition of the invention may be required to be
coated in a material to protect the ingredients from the action of
enzymes, acids and other natural conditions which may inactivate
said ingredients
[0137] The active compounds may also be administered parenterally
or intraperitoneally. Dispersions can also be prepared in glycerol,
liquid polyethylene glycols, and mixtures thereof and in oils.
Under ordinary conditions of storage and use, these preparations
contain a preservative to prevent the growth of microorganisms.
[0138] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions (where water soluble) or dispersions and
sterile powders of the extemporaneous dispersion. In all cases the
form must be sterile and must be fluid to the extent that easy
syringability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyethylene glycol, and the like), suitable
mixtures thereof, and vegetable oils. The proper fluidity can be
maintained, for example, by the use of a coating such as lecithin,
by the maintenance of the required particle size in the case of
dispersion and by the use of superfactants. The preventions of the
action of microorganisms can be brought about by various
antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In
many cases, it will be preferable to include isotonic agents, for
example, sugars or sodium chloride. Prolonged absorption of the
injectable compositions can be brought about by the use in the
compositions of agents delaying absorption, for example, aluminum
monostearate and gelatin.
[0139] Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum drying and the freeze-drying technique
which yield a powder of the active ingredient plus any additional
desired ingredient from previously sterile-filtered solution
thereof.
[0140] When at least BP-1, or at least one fragment thereof is
suitably protected as described above, the active compound may be
orally administered, for example, with an inert diluent or with an
assimilable edible carrier, or it may be enclosed in hard or soft
shell gelatin capsule, or it may be compressed into tablets, or it
may be incorporated directly with food of the diet. For oral
therapeutic administration, the active compound may be incorporated
with excipients and used in the form of ingestible tablets, buccal
tablets, troches, capsules, elixirs, suspensions, syrups, wafers,
and the like. Such compositions and preparations should contain at
least 1% by weight of active compound. The percentage of the
compositions and preparations may, of course, be carried and may
conveniently be between about 5 to 80% of the weight of the unit.
The amount of active compound in such therapeutically useful
compositions is such that a suitable dosage will be obtained.
[0141] The tablets, troches, pills, capsules and the like may also
contain the following: A binder such as gum tragacanth, acacia,
corn starch or gelatin; excipients such as dicalcium phosphate; a
disintegrating agent such as corn starch, potato starch, alginic
acid and the like; a lubricant such as magnesium stearate; and a
sweetening agent such as sucrose, lactose or saccharin may be added
or a flavoring agent such as peppermint, oil of wintergreen, or
cherry flavoring. When the dosage unit form is a capsule, it may
contain, in addition to materials of the above type, a liquid
carrier. Various other materials may be present as coatings or to
otherwise modify the physical form of the dosage unit. For
instance, tablets, pills, or capsules may be coated with shellac,
sugar or both. A syrup or elixir may contain the active compound,
sucrose as a sweetening agent, methyl and propylparabens as
preservatives, a dye and flavoring such as cherry or orange flavor
of course, any material used in preparing any dosage unit form
should be pharmaceutically pure and substantially non-toxic in the
amounts employed. In addition, the active compound may be
incorporated into sustained-release preparations and
formulations.
[0142] As used herein "pharmaceutically acceptable carrier and/or
diluent" includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents and the like. The use of such media and agents for
pharmaceutical active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active ingredient, use thereof in the therapeutic compositions is
contemplated. Supplementary active ingredients can also be
incorporated into the compositions.
[0143] It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the
mammalian subjects to be treated; each unit containing a
predetermined quantity of active material calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the novel dosage unit
forms of the invention are dictated by and directly dependent on
(1) the unique characteristics of the active material and the
particular therapeutic effect to be achieved, and (b) the
limitations inherent in the art of compounding such an active
material for the treatment of bone disease.
[0144] x) Screening Methods Using BP-1 Products
[0145] It is known in the prior art that proteins or peptides such
as those derived from BP-1 act through binding to cognate
receptors. It is possible to use labelled BP-1 products to identify
such receptors. This could be done as follows. BP-1 (PROT 1, SEQ ID
NO: 20) can be synthesized in the presence of labeled amino acids
(e.g. tritiated L-leucine). The protein can be synthesized and
labeled in vivo after transfection of a suitable host with a vector
capable of expressing the BP-1 cDNA. Alternatively, transcripts can
be synthesized, translated and labeled in vitro starting with a
plasmid harboring the BP-1 cDNA operably linked to a DNA-dependent
RNA polymerase promoter. Ideally, the in vitro translation step
should be performed using rabbit reticulocyte lysates containing
microsomal membranes to allow for co-translational and
post-translational modifications to occur (Lingappa et al., 1979).
In the case of peptides derived from BP-1 (PEPT 2A (SEQ ID NO: 22),
PEPT 3A (SEQ ID NO: 23), PEPT 2B (SEQ ID NO: 24), PEPT 3B (SEQ ID
NO: 25)), these can be labeled during chemical synthesis (e.g. by
incorporating a biotinylated derivative of amino acids) or by
adding a N-terminal tyrosine residue that can be iodinated
according to standard methods (Tsomides and Eisen, 1993). The
labeled BP-1 product(s) can be purified (e.g. by affinity
chromatography). To screen for cognate receptors, a cDNA library is
constructed according to standard protocols starting with mRNA
extracted from a target organ (e.g. bone, kidney). cDNAs are cloned
in an expression vector downstream of strongly active
enhancer/promoter elements. The library of expression vectors is
screened for BP-1, product(s) binding activity as follows. The
initial step is to find a suitable host, i.e. one that does not
bind the labeled BP-1 product(s) under basal conditions and in the
absence of exogenous expression vectors, preferably a mammalian
cell line. Examples of hosts include, but are not limited to,
HEK293 cells, CHO cells, COS cells (American Type Culture
Collection no. CRL-1650) and CV-1 cells (American Type Culture
Collection no. CCL-70). The library is then divided into pools
(e.g. 500 clones per pool), each pool is transfected into the host
and incubated with the labeled BP-1 product(s). Expression vectors
are extracted from cells transfected with a pool of clones that
confers specific binding of the labeled BP-1 product(s). Rounds of
selection are repeated until a single clone is identified. This
clone encodes a putative receptor for BP-1 product(s). The receptor
can be used to screen for molecules that bind to it and for
molecules that modulate the binding of BP-1 product(s). Such assays
can be performed on cells transfected with a vector expressing the
receptor cDNA.
[0146] vi) Diagnostic Assays
[0147] Based on the features described in the invention, it can be
expected that methods to assay BP-1 products or nucleic acid
sequences encoding BP-1 products will be useful as diagnostic
reagents i.e to diagnose or monitor diseases such as osteoporosis
and disorders of phosphate metabolism. Levels of BP-1 products or
expression of the BP-1 gene may correlate with the occurrence of a
bone disease. For example, the amount of BP-1 products may be
elevated in bones of osteoporotic patients. Various immunological
methods to assay soluble extracellular proteins are well known in
the prior art (e.g. radioimmunoassays, ELISA, `sandwich` ELISA).
Such methods generally uses monoclonal or polyclonal antibodies
specific to the protein of interest. In the case of the present
invention, these antibodies could be raised against antigenic
peptides and purified as described in section v. Example 1
describes an enzyme-linked immunoadsorbent assay to quantify BP-1
products found in cell culture medium. Other immunological methods
to assay BP-1 products in tissues or biological fluids may be
developed by an experimentator skilled in the art. Various
hybridization-based methods to assay for specific nucleic acids are
well known in the prior art. In the case of BP-1 for example, RNA
extracted from cells of the bone marrow can be labeled during
reverse transcription (e.g. radioactively or fluorescently) and
single stranded cDNA can be hybridized to an immobilized nucleic
acid probe comprising part of the BP-1 sequence (e.g.
oligonucleotide, cDNA).
[0148] Proteins, peptides, or antibodies of the present invention
can also be used for detecting and diagnosing bone or renal
conditions that involve secretion of BP-1. For example, diagnosis
can be accomplished by combining blood obtained from an individual
to be tested with antibodies to BP-1 and determining the extent to
which antibody is bound to the sample.
[0149] Futhermore, it is expected that there are sequence
polymorphisms in the nucleic acid sequence coding for BP-1, and it
will be appreciated by one skilled in the art that one or more
nucleotides in the nucleic acid sequence coding for BP-1 may vary
due to natural allelic variation. Any and all such nucleotide
variations and resulting amino acid polymorphisms are within the
scope of the invention. It may also be appreciated by one skilled
in the art that BP-1 maybe a member of a family of highly related
genes. Nucleotide sequences and corresponding deduced amino acid
sequences of any and all such related family members including BP-1
are within the scope of the invention.
EXAMPLES
[0150] As it will now be demonstrated by way of examples
hereinafter, the invention provides methods to produce, partially
purify and characterize the BP-1 protein products. Example 1 gives
an example of a cell line secreting BP-1 protein products in the
culture medium. Example 2 gives an example of a method to partially
purify the BP-1 protein products from cell culture medium. Example
3 gives an example of recombinant adenovirus particles expressing a
BP-1 coding sequence and uses thereof. Example 4 gives an example
of treatment of primary cultures of osteoblasts with medium
containing BP-1 products.
[0151] Although any methods and materials similar or equivalent to
those described herein can be used in the practice or testing of
the present invention, the preferred methods and materials are
described.
[0152] A) Materials and Methods
[0153] The following are experimental procedures and materials that
were used to describe the present invention.
[0154] Enzymes and Reagents
[0155] Restriction enzymes and DNA-modifying enzymes were purchased
from New England Biolabs (Cambridge, Ma.) unless otherwise stated.
Synthetic oligonucleotides were obtained either from Hukabel Ltd.
(Montreal, Quebec, Canada) or MWG Biotech Inc. (High Point, N.C.).
Cell culture reagents were from Life Technologies (subsidiary of
Invitrogen, Carlsbad, Calif.) unless otherwise stated. Chemicals
were obtained either from Roche Molecular Biochemicals (Laval,
Canada), Calbiochem (San Diego, Calif.) or Sigma (St.Louis, Mo.).
Radiochemicals were purchased from Amersham Pharmacia Biotech (Baie
d'Urfe, Canada). Sequencing of DNA was done using the Dye
terminator cycle sequencing DTCS.TM. sequencing kit (Beckman
Coulter, Fullerton, Calif.) and a Beckman CEQ 2000 automated
sequencer.
[0156] RNA Extraction and Analysis
[0157] Total RNA was purified either by the guanidium
isothiocyanate/acid phenol method (Chomczynski and Sacchi, 1987) or
using the RNEASY.TM. kit according to the manufacturer's
instructions (Qiagen, Mississauga, Canada). PolyA+RNA was purified
using the Oligotex.TM. kit according to the manufacturer's
instructions (Qiagen). Northern analysis were performed as follows.
RNA was electrophoresed on 1.2% agarose/1.2% formaldehyde gels in
1.times.MOPS buffer (10 mM 3-(N-morpholino)propanesu- lfonic acid,
4 mM sodium acetate, 0.5 mM EDTA, pH 7) and transferred onto a
nylon membrane (Osmonics, Westborough, Ma.) by capillarity in
20.times.SSC. After UV crosslinking, the blot was incubated in
Church buffer (Church and Gilbert, 1984) for 4-8 hours at
63.degree. C. and hybridized overnight in Church buffer containing
approximately 1 ng/ml of ca155-cap1-c2-neg306 fragment that had
been labelled with [.alpha..sup.32P]dCTP by random priming.
[0158] Cloning of Full Length Mouse cDNA Corresponding to
ca155-cap1-c2-neg306
[0159] First, an expression vector was generated as follows. A 5498
bp SacII-ApaI fragment of pQBI25fc3 (qBiogene, Montreal, Canada)
was blunted and ligated unto itself to generate pCMVneo.
Complementary oligonucleotides 30-11 (SEQ ID NO. 35) and 30-12 (SEQ
ID NO. 36) were annealed and cloned in pCMVneo previously digested
with BamHI and EcoRI and blunted using the Klenow fragment of DNA
polymerase 1. The resulting plasmid is pCMVneoXN. The first strand
of cDNAs was reverse transcribed at 42.degree. C. from 5 .mu.g of
e15.5 mouse calvaria total RNA using a dT.sub.15 primer-linker
(33-1V, SEQ ID NO: 37) and Superscript II.TM. (Invitrogen,
Carlsbad, Calif.). After RNAse H treatment (Roche Molecular
Biochemicals, Laval, Canada), {fraction (3/10)} of the first strand
cDNA preparation was subjected to 25 cycles of PCR using DNA
polymerases from the Titan.TM. RT-PCR kit, forward primer 25-10G
(25-10G, 5'-aaacgctctgacttctcacaagatg-3', SEQ ID NO. 38) and
reverse primer 18-30V (18-30V, 5'-gagatgaattcctcgagc-3', SEQ ID NO.
39). Cycling conditions were as follows: 94.degree. C. for 45
seconds, 54.degree. C. for 30 seconds, 68.degree. C. for 3 minutes.
PCR products were cloned in pCMVneoXN to generate GD25. Bacterial
colonies were hybridized with a ca155-cap1-c2-neg306 radioactive
probe. Inserts from positive colonies were sequenced.
[0160] Cloning of Vertebrate Homologs of BP-1
[0161] PolyA+ RNA from human bone marrow was purchased from
Clontech Inc. (Palo Alto, Calif.). RT-PCR was performed on 50 ng of
polyA+ RNA using forward primer 18-131G (SEQ ID NO. 40) and reverse
primer 19-9G (SEQ ID NO. 41) and Titan.TM. one tube RT-PCR system
according to the manufacturer's instructions (Roche Molecular
Biochemicals). Cycling conditions (40 cycles) were as follows:
94.degree. C. for 1 minute, 54.degree. C. for 1 minute, 68.degree.
C. for 1 minute. PCR products (predominant band at approximately
400 bp) were cloned in pBluescript II KS (Stratagene; La Jolla,
Calif.) and inserts from randomly chosen colonies were
sequenced.
[0162] Total RNA was extracted from UMR-106 cells (CRL-1661,
American Type Culture Collection, Manassas, Va.). This cell line is
derived from a rat osteosarcoma and possesses characteristics of
the osteoblast lineage (Partridge et al., 1983). The first strand
of cDNAs was reverse transcribed at 42.degree. C. from 5 .mu.g of
total RNA using 33-1V and Superscript II.TM. (Invitrogen). After
RNAse H treatment (Roche Molecular Biochemicals) and purification
using the minElute kit (Qiagen), 1/4 of the first strand cDNA
preparation was subjected to 35 cycles of PCR using the DNA
polymerases from the Titan.TM. RT-PCR kit, forward primer 18-131G
and reverse primer 18-30V. Cycling conditions were as follows:
94.degree. C. for 1 minute, 48.degree. C. for 1 minute, 68.degree.
C. for 1.5 minute. RT-PCR products were purified using the minElute
kit. 1/6 of the initial PCR reaction was subjected to 35 cycles of
semi-nested PCR using the DNA polymerases from the Titan.TM. RT-PCR
kit, forward primer 18-131G and reverse primer 21-9G (SEQ ID NO.
42). PCR products (predominant band at approximately 500 bp) were
cloned in pBluescript II KS (Stratagene, La Jolla, Calif.) and
inserts from randomly chosen colonies were sequenced.
[0163] Total RNA was extracted from the vertebrae and surrounding
muscles of a 2-month old birman python (.about.130 g) using the
Trizol.TM. reagent (Invitrogen). The first strand of cDNAs was
synthesized at 42.degree. C. for 1 hour from 4 .mu.g of total RNA
using 0.5 .mu.g of oligo-dT.sub.18 and 200U Superscript II.TM.
(Invitrogen) in a total volume of 20 .mu.l. After RNAse H treatment
(2U added, Roche Molecular Biochemicals) and purification using the
QIAQuick kit (Qiagen) with 30 .mu.l of elution buffer, the first
strand cDNA preparation (2 .mu.l) was subjected to 33 cycles of PCR
using Taq DNA polymerase, forward primer 23-4V (SEQ ID NO. 44) and
reverse primer 23-6V (SEQ ID NO. 45). Cycling conditions were as
follows: 94.degree. C. for 30 seconds, 56.degree. C. for 30
seconds, 72.degree. C. for 40 seconds. RT-PCR products were
purified using the minElute kit. PCR products (predominant band at
approximately 130 bp) were cloned in pBluescript II KS (Stratagene,
La Jolla, Calif.) and inserts from randomly chosen colonies were
sequenced.
[0164] In situ Hybridization
[0165] In situ hybridization on paraformaldehyde-fixed
paraffin-embedded tissue sections was performed according to
standard procedures (for example, see Wilkinson and Nieto, 1993).
Briefly, [.alpha..sup.3.sup.5S]U- TP-labeled cRNA probe were
synthesized in vitro from 0.5 .mu.g of purified linearized template
DNA using DNA-dependent RNA polymerase (SP6, T7 or T3 RNA
polymerase). After phenol/chloroform extraction and ethanol
precipitation, quality of the probe was assessed by denaturing 4%
polyacrylamide gel electrophoresis. Tissue sections were
deparaffinized, rehydrated, treated with 5 .mu.g/ml of proteinase K
(Roche Molecular Biochemicals) for 15 minutes in 50 mM Tris-HCl pH
8/5 mM EDTA, refixed in 4% paraformaldehyde, acetylated with 0.25%
(v/v) acetic anhydride in 0.1M triethanolamine pH 8, dehydrated and
air-dried. Prehybridization was carried out for 2-4 hours at room
temperature in 4.times. SET, 1.times. Denhardt's solution, 0.5
mg/ml ssDNA, 0.6 mg/ml yeast RNA, and 50% deionized formamide.
1.times. SET is 0.15M NaCl, 0.03M Tris-HCl pH 8 and 2 mM EDTA.
1.times. Denhardt's solution is 0.02% (w/v) Ficoll, 0.02% (w/v)
polyvinylpyrrolidone, 0.1% (w/v) BSA fraction V. ssDNA is salmon
sperm DNA that has been sonicated to obtain fragments averaging 2
kb, purified by phenol/chloroform extraction/ethanol
precipitation/dialysis and denatured by boiling. Hybridization was
carried out for 12-16 hours at 54.degree. C. with 40,000 cpm/.mu.l
of cRNA probe in 4.times. SET, 1.times. Denhardt's solution, 0.1
mg/ml ssDNA, 0.1 mg/ml yeast RNA, 10% dextran sulfate, 10 mM DTT,
0.1% SDS, and 50% deionized formamide. High stringency washes were
performed as follows: formamide wash for 30 minutes at 55.degree.
C. and 30 minutes at 62.degree. C.; 20 .mu.g/ml RNAse A treatment
in 3.5.times. SSC, 30 minutes, 37.degree. C.; formamide wash for 30
minutes at 62.degree. C. Formamide wash is 50% deionized
formamide/0.15M NaCl/0.15M sodium citrate/10 mM DTT. After rinses,
slides were dehydrated, dipped in Kodak NTB-2 liquid emulsion
(Intersciences, Markham, Canada), developed after 3-4 weeks of
exposition and counterstained by hematoxylin-eosin.
[0166] Cloning of Mouse BP-1 into a Mammalian Expression Vector
[0167] To optimize expression of BP-1 in heterologous systems, a
vector expressing the coding sequence of mouse BP-1 was obtained as
follows. A 411 bp fragment was amplified from 100 ng of plasmid
GD25 using forward primer 25-10G (SEQ ID NO: 38) and reverse primer
24-10G (SEQ ID NO: 43). The PCR reaction contained 25 pmoles/.mu.l
of each primer, deoxynucleotides at 200 .mu.M, dithiotreitol at 5
mM, the buffer and the enzyme mix from the Titan.TM. one-step
RT-PCR kit (Roche Molecular Biochemicals). Cycling conditions were
as follows: 94.degree. C. for 30 seconds, 56.degree. C. for 30
seconds, 68.degree. C. for 30 seconds. The PCR product was cloned
in the unique PmeI site of pCMVneoXN to generate GD23. The identity
of the insert was verified by DNA sequencing.
[0168] Production and Purification of Antisera
[0169] Peptides were synthesized, purified and coupled to activated
keyhole lympet hemocyanin via sulfhydryl groups. The
peptide/carrier complex was mixed with complete Freund's adjuvant
and injected subcutaneously to rabbits on day 1. On days 21, 35 and
49, the peptide/carrier complex mixed with incomplete Freund's
adjuvant was again injected. Blood samples were collected on days
44 and 59 to determine the antiserum titer by ELISA. Animals were
exsanguinated on day 63. Immunoglobulins were precipitated from
pooled antisera and purified by peptide affinity chromatography
according to standard protocols (Affinity Bioreagents, Golden,
Co.).
[0170] Cell Culture and Transfection
[0171] HEK293A cells (purchased from Quantum Biotechnologies,
Montreal, Canada) are grown in Dulbecco modified essential medium
supplemented with 10% (v/v) fetal bovine serum, 100 U/ml
penicillin, 100 mg/ml streptomycin (referred hereafter as complete
medium). Cells are passaged when reaching 80-95% confluence by
incubating with 0.05% (v/v) trypsin/0.5 mM EDTA (Wisent Inc.,
St-Bruno, Canada). Lipofection is performed as follows. Lipid-DNA
complexes (containing typically 1 .mu.g of DNA) are formed using
the Effectene.TM. reagent (Qiagen) according to the manufacturer's
instructions. Cells are transfected the day after plating
(typically 18,700 cells/cm.sup.2) by adding the lipid-DNA complex
to the culture medium. After a 6 hour incubation, the medium is
changed and cells are usually processed after 48 hours.
[0172] Immunofluorescence
[0173] All incubations and washes are done at room temperature.
Cells are rinsed with PBS and fixed with 2% (w/v) paraformaldehyde
in PBS. Cells are washed with PBS. For detection of intracellular
protein, cells are permeabilized by incubation in 0.1% TRITON
X-100.TM. in PBS for 4 minutes and washed twice with PBS. Cells are
then incubated in 50 mM NH.sub.4Cl in PBS for 10 minutes at room
temperature and washed with PBS. Cells are incubated in PBS
supplemented with 0.1% (w/v) bovine serum albumin fraction V and 2%
(w/v) dried milk for 1 hour. Cells are then incubated for 1 hour
with a {fraction (1/100)} dilution (v/v) of antiserum raised
against BP-1 in PBS supplemented with 0.1% (w/v) bovine serum
albumin and 0.5% (w/v) dried milk. Cells are washed twice with PBS
and incubated for 1 hour in {fraction (1/200)} dilution of goat
anti-mouse coupled to fluorescein isothiocyanate (Sigma) in PBS
supplemented with 0.1% (w/v) bovine serum albumin. Cells are washed
twice and observed by fluorescence microscopy.
[0174] Western Analysis
[0175] Cell extracts. Cells are rinsed with PBS. Membrane proteins
are solubilized in 0.1 ml of Lysis buffer (50 mM Tris-Cl pH 8.0,
150 mM NaCl, 2 mM EDTA, 1% IGEPAL-630.TM. and 1% (v/v) protease
inhibitor cocktail (Sigma)). Cell debris and insoluble material are
pelleted by centrifugation at 12,000 g for 5 minutes at 4.degree.
C. Protein concentration in the supernatant is determined using the
Bradford assay according to the manufacturer's instructions
(Bio-Rad, Hercules, Calif.).
[0176] Medium. 24 hours post-transfection, medium is replaced by
medium containing 0.5% (v/v) fetal bovine serum. The following day,
this medium is collected and centrifuged at 12,000 g for 5 minutes
at 4.degree. C. to remove cell debris. Proteins in the supernatant
are precipitated by adding trichloroacetic acid at a final
concentration of 10% (v/v) and incubating on ice for 1 hour. After
centrifugation at 12,000 g for 15 minutes at 4.degree. C., the
pellet is washed once with cold acetone, dried briefly and
resuspended in 24 .mu.l of Lysis buffer.
[0177] Proteins are boiled for 5 minutes in the following Laemmli
1.times. solution: 50 mM Tris-HCl, pH 6.8, 100 mM dithiothreitol,
2% sodium dodecyl sulfate, 0.1% bromophenol blue, 10% glycerol.
Proteins are electrophoresed on denaturing polyacrylamide gel and
transferred to 0.2 .mu.m nitrocellulose (Protran.RTM., Schleicher
& Schnell, Keene, N H) according to standard Western protocols.
The nitrocellulose membrane is incubated overnight in Tris-buffered
saline (TBS; 25 mM Tris-HCl, pH 7.4, 137 mM NaCl, 2.7 mM KCl)
supplemented with 5% (w/v) dried milk and 0.1% (v/v) TWEEN-20.TM..
It is then incubated for 1 hour at room temperature with a
{fraction (1/800)} dilution (v/v) of antiserum raised against BP-1
in TBS supplemented with 2.5% (w/v) dried milk and 0.1% (v/v)
TWEEN-20.TM.. The membrane is washed twice with TBS supplemented
with 0.1% (v/v) TWEEN-20.TM.. It is then incubated for 1 hour at
room temperature with goat anti-mouse coupled to horseradish
peroxidase (Sigma) diluted {fraction (1/30,000)} in TBS
supplemented with 2.5% (w/v) dried milk and 0.1% (v/v)
TWEEN-20.TM.. The membrane is washed twice with TBS supplemented
with 0.1% (v/v) TWEEN-20.TM.. Detection of the protein bound to the
antibody complex is performed with the ECL.TM. reagent according to
the manufacturer's instructions (Amersham Pharmacia Biotech, Baie
d'Urf, Canada).
[0178] Primary Culture of Rat Calvarial Osteoblasts
[0179] Calvariae were dissected from embryonic rats from days 19-21
of gestation. Osteoblasts were isolated by 5 successive digestions
of the tissues with a mix of collagenase (2 mg/ml, Worthington,
N.J.), trypsin (0.05% w/v, Roche Molecular Biochemicals, IN.) and
EDTA (0.02% w/v) for 20 minutes with agitation at 37.degree. C.
Cells from digests 2 to 5 were seeded at 10,000 cells/cm.sup.2 and
cultured in .alpha.MEM (Invitrogen, Carlsbad, Calif.) supplemented
with 10% (v/v) FBS, 20 mM HEPES pH 7.4, 2 mM L-glutamine, 5 mM
.beta.-glycerophosphate, 50 .mu.g/ml ascorbic acid, 100U/ml
penicillin, 100 .mu.g/ml streptomycin. Medium was changed every 2-3
days.
[0180] Incorporation of .sup.45Ca into Primary Cultures of Rat
Calvarial Osteoblasts
[0181] Cells were incubated in medium containing 1 .mu.Ci/Ml
.sup.45CaCl.sub.2 to assay calcium uptake into mineralized matrix.
Medium was collected 48 hours later from each well and added to 4
ml of scintillation cocktail, and counted on a scintillation
counter. The cells were washed twice in PBS then scraped into 500
.mu.l of lysis buffer (20 mM Tris, 0.5 mM MgCl.sub.2, 0.1% (v/v)
Triton X-100.TM.). Cellular debris was spun out, the lysis buffer
removed and stored and the pellet resuspended in 1 ml 0.5M EDTA.
The pellet in EDTA was placed overnight on a rocking platform then
the debris spun out and the EDTA solution removed and stored. The
pellet was then resuspended in 1 ml 0.5M NaOH and placed overnight
on a rocking platform. The remaining cell debris was spun out and
the NaOH removed and stored. To ascertain .sup.45Ca incorporation,
100 .mu.l of the lysis buffer and 500 .mu.l of the EDTA and NaOH
fractions were counted in 4 ml of scintillation fluid. The
proportion of .sup.45Ca taken up into the cells in each well was
then calculated as a percentage of the total counts in each
well.
B) Example 1
Cell Lines Secreting BP-1 Protein Products in the Culture
Medium
[0182] The expression vector used to obtain stable cell lines
producing BP-1 is schematized on FIG. 7A. Plasmid RC33 (see
provisional patent application docket number 29313-0002 and
international patent application PCT/CA02/00997) was digested with
KpnI and EcoRI and plasmid GD23 (see Materials and Methods) was
digested with KpnI and MluI. Both digests were treated using the
Klenow fragment of DNA polymerase I to blunt the 5' protruding
extremities generated by EcoRI and MluI. The 5194 bp fragment of
the RC33 digest and the 473 bp fragment of the GD23 digest were
purified and ligated. The resulting vector is designated GD46. A
similar vector comprising the coding sequence for human BP-1 was
obtained and is designated GD45. One of the transcription unit of
GD46 comprises the cytomegalovirus immediate early gene
enhancer/promoter regions (700) followed by the coding sequence for
mouse BP-1 (701) and a bovine growth hormone polyadenylation signal
(702). GD46 also comprises a transcription unit to confer
resistance to puromycin (703).
[0183] To obtain cell lines having integrated one or more copies of
GD46 in its genome, 3.5 million of HEK293 cells were electroporated
at 600V/cm with a mixture of 3.5 .mu.g of ScaI-linearized
expression vector and 15 .mu.g of denatured salmon sperm DNA. Cells
were plated and selection (2.5 .mu.g/ml puromycin) was applied 24
hours later. The concentration of puromycin was reduced to 0.2
.mu.g/ml on day 4 and colonies were picked on day 11. Clones were
grown until confluence in 24-well plates. At this time, cells were
incubated in 0.5 ml of medium without serum for 48 hours. One-fifth
(100 .mu.l) was analyzed by ELISA as follows. One volume (100
.mu.l) of 2.times. carbonate buffer (13 mM Na.sub.2CO.sub.3/35 mM
NaHCO.sub.3) was added to the sample. Samples were transferred in
wells of a high-binding capacity polystyrene plate (Corning, USA)
and incubated 1 hour at 37.degree. C. The medium was then aspirated
and the well was washed once with TBST (Tris-buffered saline pH 7.4
[TBS; 100 mM Tris], 0.1% (v/v) Tween-20.TM.). Non-specific binding
sites were blocked for 1 hour at 37.degree. C. with 300 .mu.l of
TBS supplemented with 5% (w/v) of non fat milk. After 5 washes with
TBST, incubation proceeded for 1 hour at 37.degree. C. with 200
.mu.l of a {fraction (1/2500)} dilution of the affinity-purified
antibody against the C-terminal portion of BP-1 in TBST
supplemented with 2.5% (w/v) of non fat milk. The plate was then
washed 5 times with TBST. Samples were incubated for 1 hour at
37.degree. C. with 200 .mu.l of a {fraction (1/30,000)} dilution of
anti-rabbit IgG coupled to horseradish peroxydase (Sigma, St.Louis,
Mo.). After 5 washes with TBST, the antibody complex was revealed
with Sigma Fast.TM. o-phenylenediamine dihydrochloride according to
the manufacturer's recommendations. Reactions were stopped by
adding 25 .mu.l of 5N sulfuric acid per well. Absorbance at 490 nm
was determined using a plate reader. Known quantities of antigenic
peptide are also assayed to determine a standard curve (FIG. 7B).
The clones of GD46 transfectants secreting immunoreactive BP-1
products in the culture medium were expanded. The amount of BP-1
products released by each clone was calculated by regression
analysis after assaying varing volumes of culture medium. The
highest expressor (clone 293-GD46-7) was found to secrete 8 pg of
BP-1 products per cell in the culture medium in 48 hours. This
corresponds to a production of approximately 10 mg/l for a
confluent monolayer in a 175 cm.sup.2 flask. We also obtained a
cell line overexpressing the human BP-1 coding sequence
(293-GD45-58B). Confluent 175 cm.sup.2 monolayers of these cells
secrete approximately 0.6 mg of BP-1 products per liter of culture
medium in 48 hours.
[0184] To ascertain that the cell lines we derived were of
monoclonal origin and would not be diluted by non BP-1-expressing
cells upon passaging, immunofluorescence analysis was performed on
confluent monolayers of 293A-GD46-7 using an antibody directed
against the C terminal portion of BP-1. BP-1 immunoreactivity was
seen in the secretory apparatus in over 98% of the cells.
C) Example 2
Method to Partially Purifiy the BP-1 Protein Products from Cell
Culture Medium
[0185] Six million cells stably overexpressing mouse BP-1 (clone
293-GD46-7) were seeded in a 175 cm.sup.2 flask and grown for 48
hours in DMEM supplemented with 10% FBS, at which stage the
monolayer was approximately 90% confluent. The cells were then
washed 2 times with pre-warmed phosphate buffered saline and once
with serum-free DMEM. The cells were incubated for 48 hours in 20
ml of serum-free DMEM. The medium was then collected and spun down
at 800 g for 2 minutes to remove floating cells and debris. All
subsequent procedures were performed at 4.degree. C. using a BioRad
BioLogic.TM. LP Chromatography system. The conditioned medium
containing BP-1 products was first diluted 2-fold with
equilibration buffer (Buffer A: 25 mM HEPES pH 7.8 and 100 mM NaCl)
and filtered through a 0.45 .mu.m Filtropur.TM. S membrane. The
diluted medium was then loaded at a flow rate of 2 ml/min on a
column (1.5 cm.times.20 cm) packed with 3.5 ml gel bed of a
Sepharose.TM. SP cation exchange resin (Amersham-Pharmacia
Biosciences). The flow-through was collected until the absorbance
at 280 nm returned to baseline. The resin was washed with 5 bed
volume of Buffer A at 2 ml/min. The bound-proteins were eluted at 1
ml/min with 50 ml of a linear gradient from 0-100% of Buffer B (25
mM HEPES pH 7.8 and 1 M NaCl). Fractions of 1 ml were collected and
monitored for the presence of BP-1 by ELISA (see Example 1). The
fractions containing BP-1 were pooled and concentrated 10-fold on
an Amicon Centriplus.TM. YM-3 (3 kDa cut-off membrane) by
centrifugation at 3400 g at 4.degree. C. No significant loss of
BP-1 immunoreactivity occurred during this concentration
procedure.
[0186] FIG. 8 shows the results of the initial steps of the
purification scheme described above. The fractions collected from
the cation-exchange column were individually assayed for BP-1
immunoreactivity by ELISA (FIG. 8A). The input (conditioned medium
0.9% of total), the flow through (0.9% of total) and
BP-1-containing fractions (0.8% of total) were analyzed by SDS-PAGE
10-20% (FIG. 8B, lanes 810, 811, 812, respectively). After
electrophoresis, the gel was stained by PlusOne.TM. silver staining
kit (Amersham Pharmacia Biosciences). Lane 800 shows the migration
of molecular weight markers. Arrow 813 points to a BP-1 product.
Arrowhead 814 points to aprotinin, a contaminating protease
inhibitor included in the conditioned medium before the
chromatographic step. A duplicate gel was transferred onto
nitrocellulose membrane and BP-1 products were revealed by
immunostaining (FIG. 8C, lane 820:input, lane 821:flow through,
lane 822: BP-1-containing fractions). Results indicate that the
bulk of the proteins in the culture medium do not bind the cation
exchange column (compare lanes 810 and 811). By Coomassie and
silver staining, we estimate that these initial steps of
purification allowed a 25-fold enrichment in BP-1 products. As can
be seen on the Sepharose.TM. SP elution profile (FIG. 8A), BP-1
products eluted at a salt concentration of approximately 300 mM
NaCl. Importantly, the immunoreactive fractions contain products of
lower apparent molecular weight (lane 822, band at .about.6 kD) in
addition to the .about.13 kD form of BP-1. This indicates that the
partial purification scheme described above can enrich for all BP-1
products secreted by an overexpressing cell line.
D) Example 3
Recombinant Adenovirus Particles Expressing the BP-1 Coding
Sequence and Uses Thereof
[0187] This example illustrates the various functionalities of an
adenovirus-based expression vector designed according to the
present invention. To obtain a `shuttle` vector for inserting a
transcription unit into an adenoviral genome, a 1689 bp StuI-NaeI
fragment from GD23 (see Materials and Methods) was blunted and
cloned in the EcoRV site of pQBI-AdBN (qBiogene, Montreal, Canada)
which had been previously engineered to replace the unique ClaI
site with a unique PmeI site. The resulting vector is designated
GD28b and comprises a transcription unit for mouse BP-1 coding
sequence (901) flanked by nucleotides 1-102 (902) and nucleotides
3334-5779 (903) of the Adenovirus serotype 5 genome (GenBank.TM.
accession number 9626187). A map of GD28b is given in FIG. 9A.
[0188] The transcription unit for BP-1 comprised in GD28b was
incorporated in an adenoviral genome by in vivo homologous
recombination. This was done by co-transfecting 5 .mu.g of
PmeI-linearized GD28b with 5 .mu.g of AdCMVIacZ.DELTA.E1/.DELTA.E3,
a replication-defective genome obtained commercially (qBiogene,
Montreal, Canada). Co-transfection of DNA molecules in HEK293 cells
was carried out by means of a calcium phosphate precipitate using
standard protocols. Two days post-transfection, cells were overlaid
with medium containing 1.25% (w/v) low melting agarose. Recombinant
viral genome resulting from homologous recombination between GD28b
and the replication-defective adenoviral genome can be propagated
in HEK293 cells, as indicated by the appearance of viral plaques
starting at day 8 post-infection. Plaques were picked at day 14
post-transfection. After elution, viral particles ({fraction
(1/10)} of the elutate) were used to infect 3.times.10.sup.5 HEK293
cells to assay for BP-1 expression. This was done by measuring BP-1
immunoreactivity by ELISA in {fraction (1/10)} of the culture
medium 2 days post-infection. Expression of BP-1 was also confirmed
by Northern and Western analysis. A stock of recombinant adenoviral
particles (Ad5GD28b) was obtained after 2 successive rounds of
plaque-purification according to standard protocols. This stock was
amplified on HEK293 cells. At the time of full cytophathic effect,
the infected cells were harvested and the viral particles were
purified on a discontinuous cesium chloride gradient and on a
continuous cesium chloride gradient according to standard protocols
(O.D.260 Inc., Boise, Id., USA).
[0189] The following experiment was performed to assess the
efficiency of BP-1 gene transfer in osteoblasts by recombinant
adenoviruses. Primary cultures of osteoblasts were grown in vitro
(see Materials and Methods). Cells reached confluence 5 days after
seeding, at which time they started depositing an extracellular
collagen matrix. Mineralization of this matrix was routinely
visible around days 13-15. At day 11, cells were infected with
CsCl-purified Ad5GD28b at a multiplicity of infection of 100
pfu/cell for 3 hours. Relative levels of BP-1 products were
determined by ELISA on aliquots ({fraction (1/40)}) of culture
medium taken 2, 4, 7 and 10 days post-infection (FIG. 9B). Results
show that BP-1 immunoreactivity is detected as early as 2 days
post-infection, peaks at day 4 and is still detectable 10 days
post-infection. These results indicate that a preparation of
Ad5GD28b can be used to express high levels of BP-1 in
osteoblasts.
E) Example 4
Treatment of Osteoblasts with Medium Containing BP-1 Products
[0190] This example demonstrates the effects of treating primary
cultures of osteoblasts with medium containing BP-1 products. The
BP-1-containing medium was obtained by transient transfection of
1.5 million HEK293A cells with 2 .mu.g of GD23, an expression
vector for mouse BP-1 cDNA. Control medium was obtained by
similarly transfecting pCMVneo, a control expression vector. At 24
hours post-transfection, cells were washed twice with serum-free
.alpha.MEM. The transfected cells were then incubated for 48 hours
in primary culture medium. BP-1 products were characterized by
Western analysis using an antiserum raised against a C-terminal
portion of BP-1 (SEQ AG3). The concentration of BP-1 products was
estimated at 5-10 .mu.g/ml with the bulk of immunoreactive protein
having an apparent molecular weight of .about.13 kD and a minor
fraction having an apparent molecular weight of .about.6 kD after
SDS-PAGE (e.g. FIG. 6B).
[0191] Primary cultures of osteoblasts prepared as described in
Materials and Methods were cultivated in a 1/6 (v/v) dilution of
either BP-1-containing or control medium. .beta.-glycerophosphate
(10 mM) was added to the culture medium from day 12. The effect of
the treatment was determined by measuring osteocalcin expression
and matrix mineralization, both markers of the mature osteoblastic
phenotype. As shown in FIG. 10, treatment with BP-1-containing
medium (1002) for 18 days completely suppressed osteocalcin (OCN)
expression compared to treatment of cells with control medium
(1001). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression
in the two cell populations was similar, demonstrating no general
effects on the cell populations. Incorporation of .sup.45Ca was
also markedly reduced in the cells treated with BP-1-containing
medium compared to cells treated with control medium (-60%,
p<0.01). These results suggest that BP-1 products restrict the
progression from early to late stage osteoblasts.
REFERENCES
[0192] Throughout this paper, reference is made to a number of
articles of scientific literature which are listed below and are
hereby incorporated by reference in their entirety.
[0193] Aarder, E. M., Burger, E. H., Nijweide, P. J. (1994)
Function of osteocytes in bone, J. Cell Biochem., 55, 287-299
[0194] Bagi, C. M., DeLeon, E., Brommage, R., Adams, S., Rosen, D.,
Sommer, A. (1995) Systemic administration of rhIGF-I or
rhIGF-I/IGFBP-3 increases cortical bone and lean body mass in
ovariectomized rats, Bone, 16(Suppl.), 263S-269S
[0195] Baron, R. (1999) Anatomy and ultrastructure of bone, in
Primer on the Metabolic Bone Diseases and Disorders of Mineral
Metabolism, ed. Lippincott Williams & Wilkins, Philadelphia,
pp.3-10
[0196] Birnbaum, R. S., Howard, G. A., Roos, B. A. (1989) Ontogeny
of peptidylglycine alpha-amidating monooxygenase activity in
rapidly mineralizing bone from neonatal mouse, Endocrinology, 124,
3134-3136
[0197] Chomczynski P, Sacchi N. (1987) Single-step method of RNA
isolation by acid guanidinium thiocyanate-phenol-chloroform
extraction, Anal Biochem., 162, 156-9.
[0198] Church G M, Gilbert W. (1984) Genomic Sequencing, Proc NatI
Acad Sci U S A, 81,1991-5
[0199] Denault, J.-B., Leduc, R. (1996) Furin/PACE/SPC1: a
convertase involved in exocytic and encodytic processing of
precursor proteins, FEBS Lett., 379, 113-116
[0200] Ducy, P. (2000) Cbfa1: a molecular switch in osteoblast
biology, Dev. Dyn. 219, 461-471
[0201] Ducy, P., Amling, M., Takeda, S., Priemel, M., Schilling, A.
F., Biel, F. T., Shen, J., Vinson, C., Rueger, J. M., Karsenty, G.
(2000) Leptin inhibits bone formation through a hypothalamic relay:
a central control bone mass, Cell, 100, 197-207
[0202] Frota Ruchon, A., Tenenhouse, T. S., Marcinkiewicz, M.,
Siegfried, G., Aubin, J. E., Desgroseillers, L., Crine, P.,
Boileau, G. (2000) Developmental expression and tissue distribution
of phex protein: effect of the hyp mutation and relationship to
bone markers, J. Bone Miner. Res., 15, 1440-1450.
[0203] Henikoff S., Henikoff J. G.; RT "Automated assembly of
protein blocks for database searching."; RL Nucleic Acids Res.
19:6565-6572(1991).
[0204] Hopp, T. P., Woods, K. R. (1981) Prediction of protein
antigenic determinants from amino acid sequences, Proc Natl Acad
Sci USA, 86, 152-156
[0205] The Hyp consortium (1995) A gene (PEX) with homologies to
endopeptidases is mutated in patients with X-linked
hypophosphatemic rickets, Nat. Genet. 11, 130-6
[0206] Inagami T, Misono K S, Grammer R T, Fukumi H, Maki M, Tanaka
I, McKenzie J C, Takayanagi R, Pandey K N, Parmentier M. (1985)
Biochemical studies of rat atrial natriuretic factor, Clin Exp
Hypertens A, 7, 851-867
[0207] Karlin S, Altschul S F, Proc Natl Acad Sci U S A 1990 March,
87:2264-8;
[0208] Karlin S, Altschul S F, Proc Natl Acad Sci U S A Jun. 15,
1993;90(12):5873-7; Altschul, S F (1993),J. Mol. Evol.
36:290-300
[0209] Kozak M (1986) Point mutations define a sequence flanking
the AUG initiator codon that modulates translation by eukaryotic
ribosomes, Cell, 44, 283-292
[0210] Kumar, R. (2000) Tumor-induced osteomalacia and the
regulation of phosphate homeostasis, Bone, 27,:333-8
[0211] Lingappa V. R., Cunningham B. A., Jazwinski S. M., Hopp T.
P., Blobel G., Edelman G. M. (1979) Cell-free synthesis and
segregation of beta 2-microglobulin, Proc Natl Acad Sci U S A, 76,
3651-3655
[0212] Louis, N., Evelegh, C., Graham, F. L. (1997) Cloning and
sequencing of the cellular-viral junctions from the human
adenovirus type 5 transformed 293 cell line, Virology, 233,
423-429
[0213] Mayahara H, Ito T, Nagal H, Miyajima H, Tsukuda R, Taketomi
S, Mizoguchi J, kato K (1993) In vivo stimulation of endosteal bone
formation by basic fibroblast growth factor in rats, Growth
Factors, 9, 73-80
[0214] Manolagas, S. C. (2000) Birth and death of bone cells: basic
regulatory mechanisms and implications for the pathogenesis and
treatment of osteoporosis, Endocr. Rev., 21, 115-137
[0215] Maruyama K, Sugano S (1994) Oligo-capping: a simple method
to replace cap structure of eukaryotic mRNAs with
oligoribonucleotides, Gene, 138, 171-174
[0216] Myers., W. Technical Report 29, Department of Computer
Science, University of Arizona, Tucson, 1991.
[0217] Needleman, S. B. & Wunsch, C. D. (1970). A general
method applicable to the search for similarities in the amino acid
sequence of two proteins. J. Mol. Biol. 48, 443-453.
[0218] Neer R. M., Arnaud C. D., Zanchetta J. R., Prince R., Gaich
G. A., Reginster J. Y., Hodsman A. B., Eriksen E. F., Ish-Shalom
S., Genant H. K., Wang O., Mitlak B. H. (2001) Effect of
parathyroid hormone (1-34) on fractures and bone mineral density in
postmenopausal women with osteoporosis, N Engl J Med, 344,
1434-41
[0219] Olins, G. M., Patton, D. R., Bovy, P. R., Mehta, P. P.
(1988) A linear analog of atrial natriuretic peptide (ANP)
discriminates guanylate cyclase-coupled ANP receptors from
non-coupled receptors, J. Biol. Chem., 263, 10989-10993
[0220] Partridge, N. C., Alcorn, D., Michelangeli, V. P., Ryan, G.,
Martin, T. J. (1983) Morphological and biochemical characterization
of four clonal osteogenic sarcoma cell lines of rat origin, Cancer
Res., 43, 4308-4314
[0221] Pearson, W. R. & D. J. Lipman., Improved Tools for
Biological Sequence Analysis., 1988, Proc. Natl. Acad. Sci.,
85,2444-2448
[0222] Ragot, T., et al. (1998), Meth. Cell Biol., 52, 229-260.
[0223] Ruchon A. F., Marcinkiewicz M., Siegfried G., Tenenhouse H.
S., DesGroseillers L., Crine P., Boileau G. (1998) Pex mRNA is
localized in developing mouse osteoblasts and odontoblasts, J
Histochem Cytochem, 46, 459-68
[0224] Russell, R. G. G, Rogers, M. J. (1999) Bisphosphonates: from
the laboratory to the clinic and back again, Bone, 25, 97-106
[0225] Smith, T. F. & Waterman, M. S. (1981). Identification of
common molecular subsequences. J. Mol. Biol. 147, 195-197.
[0226] Tsomides, T. J., Eisen, H. N. (1993) Stoichiometric labeling
of peptides by iodination on tyrosyl or histidyl residues, Anal.
Biochem., 210, 129-135
[0227] Unger, R. H. (2000) Leptin physiology: a seond look, Reg.
Pept., 92, 87-95
[0228] Wilkinson D G, Nieto M A. (1993) Detection of messenger RNA
by in situ hybridization to tissue sections and whole-mounts,
Methods Enzymol, 225, 361-73
[0229] Yan, W., Sheng, N., Seto, M., Morser, J., Wu, Q. (1999)
Corin, a mosaic transmembrane serine protease encoded by a novel
CDNA from human heart, J. Biol. Chem., 274, 14926-14935
[0230] Yan, W., Wu, F., Morser, J., Wu, Q. (2000) Corin, a
transmembrane cardiac serine protease, acts as a
pro-atrial-natriuretic peptide-converting enzyme, Proc Natl Acad
Sci U S A, 97, 8525-8529
[0231] While several embodiments of the invention have been
described, it will be understood that the present invention is
capable of further modifications, and this application is intended
to cover any variations, uses, or adaptations of the invention,
following in general the principles of the invention and including
such departures from the present disclosure as to come within
knowledge or customary practice in the art to which the invention
pertains, and as may be applied to the essential features
hereinbefore set forth and falling within the scope of the
invention.
Sequence CWU 1
1
45 1 1280 DNA Mus musculus 1 gctaagtttg ggataagctg caggcgggac
tccaaagtta ggagctctga cttctcacaa 60 gatgctggac tggagattgg
caagtacaca cttcatcctg gctatgattg tgatgctgtg 120 gggctcagga
aaggcattct ctgtggactt agcatcacag gagtttggaa cagcaagctt 180
gcagtctcca cccacagcca gagaagagaa gtcagccact gagctttcgg ctaagctcct
240 gcgtcttgat gatctggtgt ccttagagaa tgacgtattt gagaccaaga
aaaagagaag 300 cttctctggc tttgggtctc cccttgacag actctcagct
gggtctgtag agcatagagg 360 gaaacaaagg aaagcagtag atcattcaaa
aaagcggttt ggtattccca tggatcggat 420 tggtagaaac cggctctcca
gttccagagg ctgatggatt cttattgtgc gacttacttg 480 tgtgagatgg
cacagaacta tagaagacac ttcagtgaag ttcactaccc cttttgtcaa 540
ggaattggcc tttcgcaaac cttcccaaag cttgatcctc cccagaccat cacgtcatag
600 tgttgctgtg gttttagttg agttgtgcag atcatttcag tgcatggata
tctctgaaag 660 tatttttcaa tgattcccaa attgtaacgt ggcccctgaa
cctacttttt ttaaacagca 720 gaccaatata atgcattctc ttgccattaa
tattttcaca tttcagttaa tcaatgtgct 780 ttctagaaac ctagtgtctg
aagatctgat gatctaaaga aatcagaaat gagcacatgg 840 tgatttatat
aggtttcttt agtttttctg aggtttgtcg aattgttgta aacttcaact 900
tcaagcttag aaaaaagaca ttacatgagt gtttgcttca actgtgtcag agggcaaata
960 aattttgaga aacctgagca attgtgttct ttaggaacta ataaaggata
gtataattgg 1020 cccatatgta atattctgac aaactctgaa tgtaaaagac
tcatttgaaa agaagttact 1080 gcctggcttg tttacttcta ccagcctagg
ggtgaattgt tcaaatgttt cctatgttag 1140 cagcttttct tcttcttttt
tttctttcta ttttactttt tttcttcatt caatgtttat 1200 aagctaaaaa
tccaaccaaa tagtgctttg tgctttaaaa gggggtatta aaatcaacat 1260
taatctaaaa aaaaaaaaaa 1280 2 402 DNA Homo sapiens 2 atgctggact
ggagattggc aagtgcacat ttcatcctgg ctgtgacact gacactgtgg 60
agctcaggaa aagtcctctc agtagatgta acaacaacag aggcctttga ttctggagtc
120 atagatgtgc agtcaacacc cacagtcagg gaagagaaat cagccactga
cctgacagca 180 aaactcttgc ttcttgatga attggtgtcc ctagaaaatg
atgtgattga gacaaagaag 240 aaaaggagtt tctctggttt tgggtctccc
cttgacagac tctcagctgg ctctgtagat 300 cacaaaggta aacagaggaa
agtagtagat catccaaaaa ggcgatttgg tatccccatg 360 gatcggattg
gtagaaaccg gctttcaaat tccagaggct aa 402 3 393 DNA Mus musculus 3
atgctggact ggagattggc aagtacacac ttcatcctgg ctatgattgt gatgctgtgg
60 ggctcaggaa aggcattctc tgtggactta gcatcacagg agtttggaac
agcaagcttg 120 cagtctccac ccacagccag agaagagaag tcagccactg
agctttcggc taagctcctg 180 cgtcttgatg atctggtgtc cttagagaat
gacgtatttg agaccaagaa aaagagaagc 240 ttctctggct ttgggtctcc
ccttgacaga ctctcagctg ggtctgtaga gcatagaggg 300 aaacaaagga
aagcagtaga tcattcaaaa aagcggtttg gtattcccat ggatcggatt 360
ggtagaaacc ggctctccag ttccagaggc tga 393 4 399 DNA Rattus
norvegicus 4 atgctggact ggagattggc aagtgcacac ttcctcctgg ctatgatcct
gatgctgtgg 60 ggctcaggaa aggcattctc cgtggactta gcatcagagg
cctccgagtt tggagcagaa 120 agcttgcagt ccccacccac aaccagagaa
gagaagtcag ccacggagct tgcagctaag 180 ctcctgcttc ttgatgatct
ggtgtccttg gagaatgatg tgtttgagac caagaagaag 240 agaagcttct
ctggcttcgg gtctcccctt gacagactct cggctgggtc tgtagagcat 300
agagggaaac aaaggagagt agttgatcat tcaaaaaagc gatttggtat tcccatggat
360 cgaattggta gaaaccgtct ctccagttcc aggggctga 399 5 399 DNA Bos
taurus 5 atgctggact ggagattagc aagtgcacat tttatcctgg ctatgacact
gatgctctgg 60 agctcaggaa aagtgttctc agtgggtgtc acaacagagg
cctttgattc tggagtctta 120 ggtgttcagt catcacccac agtcagagaa
gcgaagtcgg ccactgacct ggcagcaaaa 180 ctcttacttc ttgatgaact
tgtgtctctg gagaatgacg tgattgaaac aaagaagaaa 240 agaagcttct
ctgggtttgg ttctcccctg gacagactct cagctggctc tgtaagtcat 300
aaaggtaaac agaggaaagt agtagatcat ccaaaaaggc gatttggtat ccctatggat
360 cggattggaa gaaaccggct ttcaaattcc agaggctaa 399 6 402 DNA Gallus
gallus 6 atgctgcagt tccagcttgt tgtggtccat ctggcccttg tgatcaccct
gctgcagtgg 60 cattctagtt cagtgctcct tgcagaggca gctccagagc
ctttggagcc ttctgctgct 120 ctgggcatgg cagcacatcc tactgccagc
gaggagaagt cagcctccag cctggcagcc 180 aaactgctcc ttcttgatga
gttggtgtct ctggagaatg aggtaactga gacaaagaag 240 aaaagaagtt
ttccaggatt tggctccccg atcgacagaa tttctgcgac atctgtggat 300
gctaaaggca aacagaggaa agtggttgag ctgcctaaga gacggtttgg agttcctctt
360 gaccggatcg gagtgagtcg tcttggcaac accaagggtt ag 402 7 253 DNA
Python molurus bivittatus 7 tacggcgtcg gaggagaagt cggctactga
cctggtggcc aaaattttgc tcctcaacga 60 attggtgtcc cttgaaaacg
atgtctttga gaccaagaag aagaggagct tctccgggtt 120 tggctcccca
cttgacagac tttcggtggg cctgaaagcc aagcagagga aagctgtgga 180
gctgccaaag aagcggtttg ggattcctct agatcggatt ggcgtgaatc gtttgagcgg
240 ctccagaggt tag 253 8 405 DNA Artificial Sequence Consensus DNA
sequence for BP-1 8 atgctggact ggagattggc aagtgcacat ttcatcctgg
ctatgacnct gatgctgtgg 60 ngctcaggaa aagtnttctc ngtggangta
gcancannng aggccttnga gnnntctgga 120 gcnntaggcn tgcagtcacc
acccacagcc agagaagaga agtcagccac tgacctggca 180 gcnaaactct
tgcttcttga tgaattggtg tccctggaga atgatgtgtt tgagaccaag 240
aagaagagaa gcttctctgg ntttgggtct ccccttgaca gactctcagc tgggtctgta
300 gatcataaag gnaaacagag gaaagtagta gatcatccaa aaaggcggtt
tggtattcct 360 atggatcgga ttggtagaaa ccgtctttcc agttccagag gctaa
405 9 133 PRT Homo sapiens 9 Met Leu Asp Trp Arg Leu Ala Ser Ala
His Phe Ile Leu Ala Val Thr 1 5 10 15 Leu Thr Leu Trp Ser Ser Gly
Lys Val Leu Ser Val Asp Val Thr Thr 20 25 30 Thr Glu Ala Phe Asp
Ser Gly Val Ile Asp Val Gln Ser Thr Pro Thr 35 40 45 Val Arg Glu
Glu Lys Ser Ala Thr Asp Leu Thr Ala Lys Leu Leu Leu 50 55 60 Leu
Asp Glu Leu Val Ser Leu Glu Asn Asp Val Ile Glu Thr Lys Lys 65 70
75 80 Lys Arg Ser Phe Ser Gly Phe Gly Ser Pro Leu Asp Arg Leu Ser
Ala 85 90 95 Gly Ser Val Asp His Lys Gly Lys Gln Arg Lys Val Val
Asp His Pro 100 105 110 Lys Arg Arg Phe Gly Ile Pro Met Asp Arg Ile
Gly Arg Asn Arg Leu 115 120 125 Ser Asn Ser Arg Gly 130 10 130 PRT
Mus musculus 10 Met Leu Asp Trp Arg Leu Ala Ser Thr His Phe Ile Leu
Ala Met Ile 1 5 10 15 Val Met Leu Trp Gly Ser Gly Lys Ala Phe Ser
Val Asp Leu Ala Ser 20 25 30 Gln Glu Phe Gly Thr Ala Ser Leu Gln
Ser Pro Pro Thr Ala Arg Glu 35 40 45 Glu Lys Ser Ala Thr Glu Leu
Ser Ala Lys Leu Leu Arg Leu Asp Asp 50 55 60 Leu Val Ser Leu Glu
Asn Asp Val Phe Glu Thr Lys Lys Lys Arg Ser 65 70 75 80 Phe Ser Gly
Phe Gly Ser Pro Leu Asp Arg Leu Ser Ala Gly Ser Val 85 90 95 Glu
His Arg Gly Lys Gln Arg Lys Ala Val Asp His Ser Lys Lys Arg 100 105
110 Phe Gly Ile Pro Met Asp Arg Ile Gly Arg Asn Arg Leu Ser Ser Ser
115 120 125 Arg Gly 130 11 132 PRT Rattus norvegicus 11 Met Leu Asp
Trp Arg Leu Ala Ser Ala His Phe Leu Leu Ala Met Ile 1 5 10 15 Leu
Met Leu Trp Gly Ser Gly Lys Ala Phe Ser Val Asp Leu Ala Ser 20 25
30 Glu Ala Ser Glu Phe Gly Ala Glu Ser Leu Gln Ser Pro Pro Thr Thr
35 40 45 Arg Glu Glu Lys Ser Ala Thr Glu Leu Ala Ala Lys Leu Leu
Leu Leu 50 55 60 Asp Asp Leu Val Ser Leu Glu Asn Asp Val Phe Glu
Thr Lys Lys Lys 65 70 75 80 Arg Ser Phe Ser Gly Phe Gly Ser Pro Leu
Asp Arg Leu Ser Ala Gly 85 90 95 Ser Val Glu His Arg Gly Lys Gln
Arg Arg Val Val Asp His Ser Lys 100 105 110 Lys Arg Phe Gly Ile Pro
Met Asp Arg Ile Gly Arg Asn Arg Leu Ser 115 120 125 Ser Ser Arg Gly
130 12 132 PRT Bos taurus 12 Met Leu Asp Trp Arg Leu Ala Ser Ala
His Phe Ile Leu Ala Met Thr 1 5 10 15 Leu Met Leu Trp Ser Ser Gly
Lys Val Phe Ser Val Gly Val Thr Thr 20 25 30 Glu Ala Phe Asp Ser
Gly Val Leu Gly Val Gln Ser Ser Pro Thr Val 35 40 45 Arg Glu Ala
Lys Ser Ala Thr Asp Leu Ala Ala Lys Leu Leu Leu Leu 50 55 60 Asp
Glu Leu Val Ser Leu Glu Asn Asp Val Ile Glu Thr Lys Lys Lys 65 70
75 80 Arg Ser Phe Ser Gly Phe Gly Ser Pro Leu Asp Arg Leu Ser Ala
Gly 85 90 95 Ser Val Ser His Lys Gly Lys Gln Arg Lys Val Val Asp
His Pro Lys 100 105 110 Arg Arg Phe Gly Ile Pro Met Asp Arg Ile Gly
Arg Asn Arg Leu Ser 115 120 125 Asn Ser Arg Gly 130 13 133 PRT
Gallus gallus 13 Met Leu Gln Phe Gln Leu Val Val Val His Leu Ala
Leu Val Ile Thr 1 5 10 15 Leu Leu Gln Trp His Ser Ser Ser Val Leu
Leu Ala Glu Ala Ala Pro 20 25 30 Glu Pro Leu Glu Pro Ser Ala Ala
Leu Gly Met Ala Ala His Pro Thr 35 40 45 Ala Ser Glu Glu Lys Ser
Ala Ser Ser Leu Ala Ala Lys Leu Leu Leu 50 55 60 Leu Asp Glu Leu
Val Ser Leu Glu Asn Glu Val Thr Glu Thr Lys Lys 65 70 75 80 Lys Arg
Ser Phe Pro Gly Phe Gly Ser Pro Ile Asp Arg Ile Ser Ala 85 90 95
Thr Ser Val Asp Ala Lys Gly Lys Gln Arg Lys Val Val Glu Leu Pro 100
105 110 Lys Arg Arg Phe Gly Val Pro Leu Asp Arg Ile Gly Val Ser Arg
Leu 115 120 125 Gly Asn Thr Lys Gly 130 14 75 PRT Python molurus
bivittatus 14 Thr Asp Leu Val Ala Lys Ile Leu Leu Leu Asn Glu Leu
Val Ser Leu 1 5 10 15 Glu Asn Asp Val Phe Glu Thr Lys Lys Lys Arg
Ser Phe Ser Gly Phe 20 25 30 Gly Ser Pro Leu Asp Arg Leu Ser Val
Gly Leu Lys Ala Lys Gln Arg 35 40 45 Lys Ala Val Glu Leu Pro Lys
Lys Arg Phe Gly Ile Pro Leu Asp Arg 50 55 60 Ile Gly Val Asn Arg
Leu Ser Gly Ser Arg Gly 65 70 75 15 132 PRT Artificial Sequence
Consensus polypeptide sequence for BP-1 15 Met Leu Asp Trp Arg Leu
Ala Ser Ala His Phe Ile Leu Ala Met Thr 1 5 10 15 Leu Met Leu Trp
Xaa Ser Gly Lys Val Phe Ser Val Asp Leu Ala Ser 20 25 30 Glu Xaa
Xaa Xaa Asp Ser Gly Xaa Leu Xaa Leu Gln Ser Xaa Pro Thr 35 40 45
Xaa Glu Glu Lys Ser Ala Thr Asp Leu Ala Ala Lys Leu Leu Leu Leu 50
55 60 Asp Glu Leu Val Ser Leu Glu Asn Asp Val Phe Glu Thr Lys Lys
Lys 65 70 75 80 Arg Ser Phe Ser Gly Phe Gly Ser Pro Leu Asp Arg Leu
Ser Ala Gly 85 90 95 Ser Val Asp His Lys Gly Lys Gln Arg Lys Val
Val Asp His Pro Lys 100 105 110 Lys Arg Phe Gly Ile Pro Met Asp Arg
Ile Gly Arg Asn Arg Leu Ser 115 120 125 Asn Ser Arg Gly 130 16 9
PRT Homo sapiens 16 Phe Gly Ile Pro Met Asp Arg Ile Gly 1 5 17 17
PRT Rattus norvegicus 17 Cys Phe Gly Gly Arg Ile Asp Arg Ile Gly
Ala Gln Ser Gly Leu Gly 1 5 10 15 Cys 18 17 PRT Rattus norvegicus
18 Cys Phe Gly Gln Lys Ile Asp Arg Ile Gly Ala Val Ser Arg Leu Gly
1 5 10 15 Cys 19 17 PRT Rattus norvegicus 19 Cys Phe Gly Leu Lys
Leu Asp Arg Ile Gly Ser Met Ser Gly Leu Gly 1 5 10 15 Cys 20 106
PRT Homo sapiens 20 Val Asp Val Thr Thr Thr Glu Ala Phe Asp Ser Gly
Val Ile Asp Val 1 5 10 15 Gln Ser Thr Pro Thr Val Arg Glu Glu Lys
Ser Ala Thr Asp Leu Thr 20 25 30 Ala Lys Leu Leu Leu Leu Asp Glu
Leu Val Ser Leu Glu Asn Asp Val 35 40 45 Ile Glu Thr Lys Lys Lys
Arg Ser Phe Ser Gly Phe Gly Ser Pro Leu 50 55 60 Asp Arg Leu Ser
Ala Gly Ser Val Asp His Lys Gly Lys Gln Arg Lys 65 70 75 80 Val Val
Asp His Pro Lys Arg Arg Phe Gly Ile Pro Met Asp Arg Ile 85 90 95
Gly Arg Asn Arg Leu Ser Asn Ser Arg Gly 100 105 21 51 PRT Homo
sapiens 21 Ser Phe Ser Gly Phe Gly Ser Pro Leu Asp Arg Leu Ser Ala
Gly Ser 1 5 10 15 Val Asp His Lys Gly Lys Gln Arg Lys Val Val Asp
His Pro Lys Arg 20 25 30 Arg Phe Gly Ile Pro Met Asp Arg Ile Gly
Arg Asn Arg Leu Ser Asn 35 40 45 Ser Arg Gly 50 22 30 PRT Homo
sapiens 22 Ser Phe Ser Gly Phe Gly Ser Pro Leu Asp Arg Leu Ser Ala
Gly Ser 1 5 10 15 Val Asp His Lys Gly Lys Gln Arg Lys Val Val Asp
His Pro 20 25 30 23 18 PRT Homo sapiens 23 Phe Gly Ile Pro Met Asp
Arg Ile Gly Arg Asn Arg Leu Ser Asn Ser 1 5 10 15 Arg Gly 24 21 PRT
Homo sapiens 24 Ser Phe Ser Gly Phe Gly Ser Pro Leu Asp Arg Leu Ser
Ala Gly Ser 1 5 10 15 Val Asp His Lys Gly 20 25 26 PRT Homo sapiens
25 Val Val Asp His Pro Lys Arg Arg Phe Gly Ile Pro Met Asp Arg Ile
1 5 10 15 Gly Arg Asn Arg Leu Ser Asn Ser Arg Gly 20 25 26 14 PRT
Homo sapiens 26 Asp Glu Leu Val Ser Leu Glu Asn Asp Val Ile Glu Thr
Lys 1 5 10 27 14 PRT Homo sapiens 27 Arg Leu Ser Ala Gly Ser Val
Asp His Lys Gly Lys Gln Arg 1 5 10 28 14 PRT Homo sapiens 28 Met
Asp Arg Ile Gly Arg Asn Arg Leu Ser Asn Ser Arg Gly 1 5 10 29 30
RNA Artificial Sequence Synthetic oligoribonucleotide 29 agcaucgagu
cggccuuguu ggccuacugg 30 30 36 DNA Artificial Sequence Synthetic
primer-linker 30 gagagagaga gagcgactcg gatccannnn nnnnnc 36 31 18
DNA Artificial Sequence Synthetic primer 31 agcatcgagt cggccttg 18
32 18 DNA Artificial Sequence Synthetic primer 32 gagagcgact
cggatcca 18 33 32 DNA Artificial Sequence Synthetic primer 33
ggacgagcgg ccgcgccttg ttggcctact gg 32 34 7 DNA Artificial Sequence
Synthetic construct 34 aagatgc 7 35 30 DNA Artificial Sequence
Synthetic oligonucleotide 35 gtgcggcggc gtttaaacgg tcacctcgag 30 36
30 DNA Artificial Sequence Synthetic oligonucleotide 36 ctcgaggtga
ccgtttaaac gcggccgcac 30 37 33 DNA Artificial Sequence Synthetic
primer-linker 37 gagatgaatt cctcgagctt tttttttttt ttt 33 38 25 DNA
Artificial Sequence Synthetic primer 38 aaacgctctg acttctcaca agatg
25 39 18 DNA Artificial Sequence Synthetic primer 39 gagatgaatt
cctcgagc 18 40 18 DNA Artificial Sequence Synthetic primer 40
agatgctgga ctggagat 18 41 19 DNA Artificial Sequence Synthetic
primer 41 gaatcaatta gcctctgga 19 42 21 DNA Artificial Sequence
Synthetic primer 42 aact tcactgaagt g 21 43 24 DNA Artificial
Sequence Synthetic primer 43 gcct ctggaactgg agag 24 44 23 DNA
Artificial Sequence Synthetic primer 44 acnrynhsng argmgaartc rgc
23 45 23 DNA Artificial Sequence Synthetic primer 45 ctrtcnadsg
grswnccraa ncc 23
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