U.S. patent application number 13/084267 was filed with the patent office on 2012-10-04 for novel neurotrophic factors.
This patent application is currently assigned to NSGENE A/S. Invention is credited to Nikolaj Blom, Claus Hansen, Teit E. Johansen.
Application Number | 20120252726 13/084267 |
Document ID | / |
Family ID | 27561795 |
Filed Date | 2012-10-04 |
United States Patent
Application |
20120252726 |
Kind Code |
A1 |
Johansen; Teit E. ; et
al. |
October 4, 2012 |
NOVEL NEUROTROPHIC FACTORS
Abstract
The invention relates to neublastin neurotrophic factor
polypeptides, nucleic acids encoding neublastin polypeptides, and
antibodies that bind specifically to neublastin polypeptides, as
well as methods of making and methods of using the same.
Inventors: |
Johansen; Teit E.;
(Horsholm, DK) ; Blom; Nikolaj; (Copenhagen,
DK) ; Hansen; Claus; (Holbaek, DK) |
Assignee: |
NSGENE A/S
Ballerup
DK
|
Family ID: |
27561795 |
Appl. No.: |
13/084267 |
Filed: |
April 11, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12606602 |
Oct 27, 2009 |
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13084267 |
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10806793 |
Mar 22, 2004 |
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12606602 |
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09662183 |
Sep 15, 2000 |
6734284 |
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10806793 |
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09347613 |
Jul 2, 1999 |
6593133 |
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09662183 |
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60092229 |
Jul 9, 1998 |
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60103908 |
Oct 13, 1998 |
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60097774 |
Aug 25, 1998 |
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Current U.S.
Class: |
514/8.4 ;
435/252.3; 435/254.11; 435/254.2; 435/320.1; 435/325; 435/348;
435/360; 435/69.4; 514/44R; 530/350; 530/395; 536/23.5 |
Current CPC
Class: |
A61K 48/00 20130101;
A61P 35/00 20180101; A61P 43/00 20180101; A61K 38/00 20130101; A61P
27/06 20180101; A61P 13/12 20180101; A61P 25/02 20180101; A61P
39/02 20180101; A61P 25/16 20180101; A61P 27/02 20180101; A61P
21/06 20180101; A61P 25/00 20180101; A61P 9/10 20180101; A61P 37/02
20180101; A61P 3/00 20180101; A61P 3/10 20180101; A61P 25/28
20180101; C07K 14/4756 20130101; A61P 21/02 20180101; A61P 25/14
20180101; A61P 17/00 20180101 |
Class at
Publication: |
514/8.4 ;
435/320.1; 435/254.11; 435/254.2; 435/348; 435/252.3; 435/360;
435/325; 435/69.4; 536/23.5; 514/44.R; 530/350; 530/395 |
International
Class: |
A61K 38/18 20060101
A61K038/18; C12N 1/15 20060101 C12N001/15; C12N 1/19 20060101
C12N001/19; C12N 5/10 20060101 C12N005/10; A61P 25/28 20060101
A61P025/28; C12P 21/02 20060101 C12P021/02; C12N 15/12 20060101
C12N015/12; A61K 31/7088 20060101 A61K031/7088; C07K 14/575
20060101 C07K014/575; C12N 15/63 20060101 C12N015/63; C12N 1/21
20060101 C12N001/21 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 6, 1998 |
DK |
199800904 |
Aug 19, 1998 |
DK |
199801048 |
Oct 6, 1998 |
DK |
199801265 |
Claims
1. An isolated neublastin nucleic acid comprising the sequence of
any one of SEQ. ID. NOS. 1, 3, 8, 13, 14, 15, 29 and 30.
2. A nucleic acid sequence comprising an open reading frame which
encodes neublastin neurotrophic factor or a unique subregion
thereof, and which codes on expression for aneublastin polypeptide
which comprises at least 70% homology to SEQ. ID. NOS. 2, 4, 5, 6,
7, 9, 10, 11, 12, and 16.
3. A nucleic acid that hybridizes specifically under high
stringency solution hybridization conditions to the nucleic acid of
claim 1.
4. A nucleic acid that comprises a nucleic acid sequence that is
complementary to the nucleic acid of claim 3.
5. A method of using a nucleic acid of claim 1, comprising the step
of causing a polypeptide encoded by said nucleic acid to be
expressed in a cell.
6. The method of claim 5, further comprising the step of
administering said nucleic acid to an animal, and causing said
polypeptide to be expressed in said animal.
7. A vector comprising the nucleic acid of claim 18
8. The vector of claim 7, wherein said vector is an expression
vector.
9. A method of using the vector of claim 8, comprising the step of
causing a polypeptide encoded by said nucleic acid to be expressed
from said nucleic acid.
10. A cell transformed with the nucleic acid of claim 1.
11. The cell of claim 10, wherein said cell is selected from the
group consisting of mammalian cells, fungal cells, yeast cells,
insect cells and bacterial cells.
12. The method of claim 11, wherein said cell is a Chinese hamster
ovary cell.
13. The method of claim 11, wherein said cell is a cell derived
from the mammalian central nervous system.
14. A neublastin neurotrophic factor polypeptide comprising any one
of the amino acid sequences set forth in SEQ. ID. NOS. 2, 4, 5, 6,
7, 9, 10, 11, 12 and 16.
15. The polypeptide of claim 14, wherein said polypeptide is
glycosylated.
16. The polypeptide of claim 14, wherein said polypeptide is coded
for by a nucleic acid of claim 1.
17. A method of making the polypeptide of claim 14, said method
comprising the step of expressing said polypeptide from a
neublastin neurotrophic factor nucleic acid.
18. The method of claim 17, comprising the step of culturing a cell
comprising said neublastin neurotrophic factor nucleic acid in a
culture medium which permits the production of said
polypeptide.
19. The method of claim 18, further comprising the step of
recovering said polypeptide from said culture medium.
20. A method of treating a neurodegenerative disease or disorder in
an animal, comprising administering to said animal a neublastin
polypeptide one or more of the neublastin polypepetides set forth
in SEQ. ID. NOs. 2, 4, 5, 6, 7, 9, 10, 11, 12 and 16.
Description
FIELD OF THE INVENTION
[0001] The invention relates to neurotrophic factor polypeptides,
nucleic acids encoding neurotrophic factor polypeptides, and
antibodies that bind specifically to neurotrophic factors.
BACKGROUND
[0002] Neurotrophic factors are naturally-occurring proteins which
promote survival, maintain phenotypic differentiation, prevent
degeneration, and enhance the activity of neuronal cells and
tissues. Neurotrophic factors are isolated from neural tissue and
from non-neural tissue that is innervated by the nervous system,
and have been classified into functionally and structurally related
groups, also referred to as families, superfamilies, or
subfamilies. Among the neurotrophic factor superfamilies are the
fibroblast growth factor, neurotrophin, and transforming growth
factor-.beta. (TGF-.beta.) superfamilies. Individual species of
neurotrophic factors are distinguished by their physical structure,
their interaction with their composite receptors, and their affects
on various types of nerve cells. Classified within the TGF-.beta.
superfamily (Massague, et al., 1994, Trends in Cell Biology,
4:172-178) are the glial cell line-derived neurotrophic factor
ligands ("GDNF"; WO 93/06116, incorporated herein by reference),
which include GDNF, persephin ("PSP"; Milbrandt et al., 1998,
Neuron 20:245-253, incorporated herein by reference) and neurturin
("NTN"; WO 97/08196, incorporated herein by reference). The ligands
of the GDNF subfamily have in common their ability to induce
signalling through the RET receptor tyrosine kinase. These three
ligands of the GDNF subfamily differ in their relative affinities
for a family of neurotrophic receptors, the GFR.alpha.
receptors.
[0003] Due to the affects of neurotrophic factors on neuronal
tissue, there remains a need to identify and characterise
additional neurotrophic factors for diagnosing and treating
disorders of the nervous system.
SUMMARY OF THE INVENTION
[0004] This invention relates to a novel neurotrophic factor herein
called "neublastin," or "NBN." Neublastin is classified within the
GDNF subfamily because it shares regions of homology with other
GDNF ligands (see Tables 3 and 4, infra) and because of its ability
to interact with RET (see, e.g., Airaksinen et al., Mol. Cell.
Neuroscience, 13, pp. 313-325 (1999)), neublastin is a novel and
unique neurotrophic factor. Unlike other GDNF ligands, neublastin
exhibits high affinity for the GFR.alpha.3-RET receptor complex and
unique subregions in its amino acid sequence.
[0005] A "neublastin polypeptide," as used herein, is a polypeptide
which possesses neurotrophic activity (e.g., as described in
Examples 6, 7, 8, and 9) and includes those polypeptides which have
an amino acid sequence that has at least 70% homology to the human
"neublastin" polypeptides set forth in AA.sub.-95-AA.sub.105 of
SEQ. ID. NO. 2, AA.sub.1-AA.sub.105 of SEQ. ID. NO. 2,
AA.sub.-97-AA.sub.140 of SEQ. ID. NO. 4, AA.sub.-41-AA.sub.140 of
SEQ. ID. NO. 4 ("pro"), AA.sub.1-AA.sub.140 of SEQ. ID. NO. 4,
AA.sub.-80-AA.sub.140 of SEQ. ID. NO. 9 ("wild type" prepro),
AA.sub.-41-AA.sub.140 of SEQ. ID. NO. 9 (pro), AA.sub.1-AA.sub.140
of SEQ. ID. NO. 5 (mature 140AA), AA.sub.1-AA.sub.116 of SEQ. ID.
NO. 6 (mature 116AA), AA.sub.1-AA.sub.113 of SEQ. ID. NO. 7 (mature
113AA), AA.sub.1-AA.sub.140 of SEQ. D. NO. 10 (mature 140AA),
AA.sub.1-AA.sub.116 of SEQ. ID. NO. 11 (mature 116AA),
AA.sub.1-AA.sub.113 of SEQ. ID. NO. 12 (mature 113AA), and variants
and derivatives thereof. In addition, this invention contemplates
those polypeptides which have an amino acid sequence that has at
least 70% homology to the murine "neublastin" polypeptides set
forth in AA.sub.1-AA.sub.224 of SEQ. ID. NO. 16.
[0006] Preferably, the C-terminal sequence of the above identified
neublastin polypeptides has an amino acid sequence as set forth in
AA.sub.72-AA.sub.103 of SEQ. ID. NO. 2 (i.e., AA.sub.107-AA.sub.140
of SEQ. ID. NO. 9), more preferably AA.sub.41-AA.sub.105 of SEQ.
ID. NO. 2 (i.e., AA.sub.76-AA.sub.140 of SEQ. ID. NO. 9), or the
amino acid sequence set forth in AA.sub.191-AA.sub.224 of SEQ. ID.
NO. 16.
[0007] Also, it is preferable that the neublastin polypeptide
retain the 7 conserved Cys residues that are characteristic of the
GDNF family and of the TGF-beta super family.
[0008] Preferably, the neublastin polypeptide has an amino acid
sequence greater than 85% homology, most preferably greater than
95% homology, to the foregoing sequences (i.e.,
AA.sub.-95-AA.sub.105 of SEQ. ID. NO. 2, AA.sub.1-AA.sub.105 of
SEQ. ID. NO. 2, AA.sub.-97-AA.sub.140 of SEQ. ID. NO. 4,
AA.sub.1-AA.sub.140 of SEQ. ID. NO. 4, AA.sub.-41-AA.sub.140 of
SEQ. ID. NO. 4, AA.sub.-80-AA.sub.140 of SEQ. ID. NO. 9 ("wild
type" prepro), AA.sub.-41-AA.sub.140 of SEQ. ID. NO. 9 (pro),
AA.sub.1-AA.sub.140 of SEQ. ID. NO. 5 (mature 140AA),
AA.sub.1AA.sub.116 of SEQ. ID. NO. 6 (mature 116AA),
AA.sub.1-AA.sub.113 of SEQ. ID. NO. 7 (mature 113AA),
AA.sub.1-AA.sub.140 of SEQ. ID. NO. 10 (mature 140AA),
AA.sub.1-AA.sub.116 of SEQ. ID. NO. 11 (mature 116AA),
AA.sub.1-AA.sub.113 of SEQ. ID. NO. 12 (mature 113AA)), and
AA.sub.1-AA.sub.224 of SEQ. ID. NO. 16.
[0009] A "neublastin nucleic acid," as used herein, is a
polynucleotide which codes for a neublastin polypeptide.
Accordingly, an isolated neublastin nucleic acid is a
polynucleotide molecule having an open reading frame of nucleotide
codons that, were it to be exposed to the appropriate components
required for translation, would encode, or code for, a neublastin
polypeptide. Neublastin nucleic acids of the invention may be RNA
or DNA, e.g., genomic DNA, or DNA which is complementary to and/or
transcribed from, a neublastin mRNA ("cDNA"). Thus, a neublastin
nucleic acid of the invention further includes polynucleotide
molecules which hybridize with specificity, under high stringency
hybridization conditions, to a polynucleotide that codes for a
neublastin polypeptide. This invention also relates to nucleic acid
primers that are useful in identifying, isolating and amplifying
polynucleotides that encode neublastin polypeptides, or fragments
thereof. In certain embodiments of the invention, certain of these
primers are neublastin-specific probes useful for hybridization to
a neublastin nucleic acid, but not to nucleic acids coding for the
other members of the GDNF family. By "specific", "specificity", or
"specifically", is meant an ability to hybridize with neublastin
nucleic acid and inability to hybridize with non-neublastin nucleic
acids, including an inability to hybridize to nucleic acids that
code uniquely for the GDNF ligands (e.g., GDNF, persephin, and
neurturin).
[0010] In another embodiment, a neublastin nucleic acid of the
invention is one that is identified as being complementary to a
polynucleotide that codes for a neublastin polypeptide, either by
having a complementary nucleic acid sequence or demonstrating that
it hybridizes with specificity at high stringency hybridization
conditions to a polynucleotide that codes for neublastin.
Particular neublastin nucleic acids include, without limitation,
the nucleic acid sequences shown herein and designated SEQ ID NO:
1, SEQ ID NO: 3, SEQ ID NO: 8, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID
NO: 15, SEQ ID NO: 29 and SEQ ID NO: 30 as well as primers SEQ ID
NOS: 17-28, 31 and 32. A neublastin nucleic acid of the invention
further includes a unique subregion, or fragment, of a neublastin
nucleic acid, including without limitation the nucleic acid
fragments shown in FIG. 8.
[0011] The neublastin nucleic acids of the invention may be used to
express a neublastin polypeptide, e.g., by expressing a neublastin
polypeptide in vitro, or by administering a neublastin nucleic acid
to an animal for in vivo expression. Neublastin nucleic acids may
be included within a nucleic acid vector, e.g., an expression
vector or a cloning vector. A neublastin nucleic acid may, but need
not of necessity, be maintained, reproduced, transferred, or
expressed as part of a nucleic acid vector. A recombinant
expression vector containing a neublastin polynucleotide sequence
can be introduced into and/or maintained within a cell. Cells
hosting a neublastin vector may be prokaryotic. Alternatively, a
neublastin nucleic acid can be introduced into a eukaryotic cell,
e.g., a eukaryotic cell that contains the appropriate apparati for
post-translational processing of a polypeptide into a mature
protein, and/or the appropriate apparati for secreting a
polypeptide into the extracellular environment of the cell.
[0012] The invention further features a neublastin neurotrophic
factor, "neublastin." Neublastin may be in the form of a
polypeptide, or may be a multimer of two or more neublastin
polypeptides, e.g., a neublastin dimer. Neublastin polypeptides are
associated as multimers by intermolecular structural associations
known to those skilled in the art, including without limitation
cysteine-cysteine interaction, sulfhydryl bonds, and noncovalent
interactions. Particular neublastin polypeptides include, without
limitation, an amino acid sequence disclosed herein and designated
SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; SEQ ID NO:
7; SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 11, SEQ ID NO: 12 and
SEQ ID NO: 16.
[0013] A neublastin polypeptide of the invention is useful for
treating a defect in a neuron, including without limitation
lesioned neurons and traumatized neurons. Peripheral nerves that
experience trauma include, but are not limited to, nerves of the
medulla or of the spinal cord. Neublastin polypeptides are useful
in the treatment of neurodegenerative disease, e.g., cerebral
ischemic neuronal damage; neuropathy, e.g., peripheral neuropathy,
Alzheimer's disease, Huntington's disease, Parkinson's disease,
amyotrophic lateral sclerosis (ALS). Neublastin polypeptides are
further contemplated for use in the treatment of impaired memory,
e.g., memory impairment associated with dementia.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 is a photographic image of two northern blots probed
with .sup.32P-labelled neublastin cDNA, comparing relative levels
of expression of the neublastin gene in various human adult tissue
types (panel A) and in various regions of the adult human brain
(panel B).
[0015] FIG. 2 is a photographic image of a northern blot probed
with .sup.32P-labelled neublastin cDNA, comparing the amount of
neublastin cDNA expressed in a non-transfected cell-line, HiB5,
with the amount of neublastin cDNA expressed in a cell-line
transfected with neublastin cDNA, and with a cell-line transfected
with GDNF-cDNA.
[0016] FIG. 3 is a photographic image of two western blots which
compare the degrees to which neublastin protein is expressed in
non-transfected HiB5 cells (lane 1) relative to an HiB5 cell-line
stably-transfected with neublastin cDNA (lane 2) was probed with
either the neublastin-specific antibody Ab-2 (left blot; Panel A)
or the neublastin-specific antibody Ab-1 (right blot; Panel B).
[0017] FIG. 4 is a graphical illustration of the effect of
neublastin on the survival of cultured rat embryonic, dopaminergic,
ventral mesencephalic neurons and ChAT activity in cholinergic
cranial nerve motor neurons in serum-free medium. In particular,
FIG. 4A is an illustration of the dose-response curve for
recombinant GDNF on ChAT activity (dpm/hour). FIG. 4B is an
illustration of ChAT activity (dpm/hour) using diluted conditioned
medium from either neublastin producing or GDNF-producing cells.
FIG. 4C is an illustration of the number of tyrosine hydroxylase
immunoreactive cells per well.
[0018] FIG. 5 is an illustration of the effect of neublastin
secreted from HiB5pUbi1zNBN22 cells on the function and survival of
slice cultures of pig embryonic dopaminergic ventral mesencephalic
neurons co-cultured with either HiB5pUbi1zNBN22 cells (neublastin)
or HiB5 cells (control). FIG. 5A and FIG. 5B illustrate dopamine
released to the medium at DIV12 [Dopamine (pmol/ml)--day 12] and
DIV21 [Dopamine (pmol/ml)--day 21], respectively. FIG. 5C is an
illustration of the number of tyrosine hydroxylase immunoreactive
cells per culture [TH-ir cells per culture] at DIV21.
[0019] FIG. 6 is an illustration of the in vivo effect of
lentiviral-produced neublastin on nigral dopamine neurons.
[0020] FIG. 7 is a schematic diagram of the genomic structure of
the neublastin gene, including the nucleic acid primers which can
be used to identify the full length neublastin gene, and their
spatial orientation in relation to the genomic Neublastin-encoding
sequence (i.e., gene).
[0021] FIG. 8 is an illustration of neublastin specific primers
used to identify the cDNA clone encoding the human neublastin
polypeptide that hybridize to nucleic acids that encode neublastin
polypeptides, but do not hybridize to nucleic acids encoding the
other known GDNF family members (i.e., GDNF, Persephin and
neurturin).
[0022] FIG. 9 illustrates the neurotrophic activity on cultures of
dissociated rat dorsal root ganglion cells from different
development stages of a polypeptide disclosed in the present
invention in comparison to those obtained with known neurotrophic
factors [0 control experiment (in absence of factors); 1 in the
presence of GDNF; 2 in the presence of Neurturin; 3 in the presence
of Neublastin of the invention; E12 embryonic day 12; E16 embryonic
day 16; P0 the day of birth; P7 day 7 after birth; and P15 day 15
after birth].
[0023] FIG. 10 illustrates neublastin production from CHO cell
lines.
[0024] FIG. 11 illustrates a comparison of neublastin and GDNF
binding to GFR.alpha.-1 and GFR.alpha.-3 receptors.
[0025] FIG. 12 is a photographic image of a western blot which
illustrates R30 anti-peptide antibody and R31 anti-peptide antibody
binding to neublastin.
[0026] FIG. 13 is a picture of a gel showing extraction of
neublastin by affinity binding on RETL3-Ig.
[0027] FIG. 14 is a plasmid map of pET19b-Neublastin, along with
the sequence of the synthetic gene for Neublastin.
[0028] FIG. 15 is a plasmid map of pMJB164-HisNeublastin, along
with the sequence of the synthetic gene for HisNeublastin.
DETAILED DISCLOSURE OF THE INVENTION
[0029] Applicant have identified a nucleic acid that encodes a
novel neurotrophic factor which is referred to herein as
"neublastin," or "NBN." Neublastin is a member of the glial cell
line-derived neurotrophic factor (GDNF) sub-class of the
transforming growth factor-.beta. (TGF-.beta.) super-family of
neurotrophic factors.
[0030] The cDNA encoding neublastin was originally identified as
follows. Using the TBLASTN 1.4.11 algorithm (Atschul et al., Nucl.
Acids Res., 25, pp. 3389-3402 (1997)) and human persephin as query
(GenBank Acc. No. AF040962), a 290 bp fragment was initially
identified in High-Throughput Genomic Sequence (HGTS) of two human
bacterial artificial chromosomes (BAC) with GenBank entries
AC005038 and AC005051. AC005038 consists of approximately 190,000
bp of 5 contigs of unordered sequences and AC005051 consists of
approximately 132,000 bp of 12 contigs of unordered sequences. The
290 bp fragment identified in the two BAC clones proved to have
regions that were homologous, but not identical, to a coding region
of the cDNA of the neurotrophic factor, human persephin.
[0031] From this 290 bp sequence two Neublastin-specific PCR
primers were synthesised (Top Stand Primer [SEQ ID NO. 17] and
Bottom Strand Primer [SEQ ID NO. 18]). Screening of human fetal
brain cDNA library was performed. The initial screening comprised
96-well PCR-based screening with the two PCR primers [SEQ ID NOS.
17 and 18] of a cDNA library "Master Plate" from 500,000 cDNA
clones containing approximately 5,000 clones/well. A second
PCR-based screen was performed on a human fetal brain cDNA library
"Sub-Plate" containing E. coli glycerol stock with approximately
5,000 clones/well.
[0032] A 102 bp fragment [SEQ ID NO. 13] was identified in the
PCR-based screenings of both the Master Plate and Sub Plate. A
positive cDNA clone (possessing the 102 bp fragment) was selected,
plated on two LB/antibiotic-containing plates, and grown overnight.
From these plates, a total of 96 bacterial colonies were selected
and individually placed in the wells of a new, 96-well PCR plate
containing both PCR primers [SEQ ID NOS. 17 and 18] and the
requisite PCR amplification reagents. PCR amplification was then
performed and the 96 individual PCR reactions were analyzed by 2%
agarose gel electrophoresis. The positive colony with the clone
containing the 102 bp fragment was then identified. Plasmid DNA was
obtained from the positive colony containing the 102 bp fragment
and sequenced. Subsequent sequencing analysis revealed the presence
of a full-length cDNA of 861 bp [SEQ ID NO. 8]. The Open Reading
Frame (ORF) of 663 bp, or coding region (CDS), identified in SEQ ID
NO. 8, encodes the pre-pro-polypeptide (designated
"pre-pro-Neublastin") and is shown in SEQ ID NO: 9. Based on SEQ ID
NO: 9, three variants of Neublastin polypeptides were identified.
These variants include: [0033] (i) the 140 AA polypeptide
designated herein as NBN140, which possesses the amino acid
sequence designated as SEQ ID NO: 10; [0034] (ii) the 116 AA
polypeptide designated herein as NBN116, which possesses the amino
acid sequence designated as SEQ ID NO: 11; and [0035] (iii) the 113
AA polypeptide designated herein as NBN113, which possesses the
amino acid sequence designated as SEQ ID NO: 12.
[0036] The entire cDNA sequence containing 782 bp 5' untranslated
DNA, 663 bp encoding DNA, and 447 3' untranslated (totaling 1992
bp) has been submitted to GenBank under the Accession Number AF
120274.
[0037] The genomic Neublastin-encoding sequence was identified as
follows:
[0038] With the goal of cloning the genomic neublastin-encoding
sequence, an additional set of primers were prepared. In
particular, Primer Pair No. 1 comprised [sense=SEQ ID NO:23 and
antisense=SEQ ID NO:24] and Primer Pair No. 2 comprised [sense=SEQ
ID NO:25 and antisense=SEQ ID NO:26].
[0039] Using Primer Pair No. 2, a 887 bp DNA fragment was amplified
by PCR from a preparation of human genomic DNA, and cloned into the
pCR11 vector (Invitrogen) and transformed into E. coli. The
resulting plasmid was sequenced and a 861 bp putative cDNA sequence
(encoding a protein named neublastin herein) was predicted (as set
forth in SEQ.ID.NO.3). Similarly, using Primer Pair No. 1, an 870
bp DNA fragment was obtained by PCR of human genomic DNA. An
additional 42 bp region at the 3'-terminus of the Open Reading
Frame (ORF) was found in this fragment, in comparison to the 887 bp
sequence. The genomic structure of the neublastin gene was
predicted by comparing it to the sequences of nucleic acids of
other neurotrophic factors, by mapping exon-intron boundaries. This
analysis demonstrated that the neublastin gene has at least two
exons separated by a 70 bp intron.
[0040] This sequence was also used to screen GenBank for neublastin
EST sequences. Three were identified with GenBank entries AA844072,
AA931637 and AA533512, indicating that neublastin nucleic acids are
transcribed into mRNA.
[0041] Comparison of the entire cDNA sequence obtained (AF 120274)
and the genomic sequence present in GenBank entries AC005038 and
AC005051 revealed that the neublastin gene consists of at least
five exons (including three coding) separated by four introns (see,
e.g., FIG. 8). Together, the exons have a predicted amino acid
sequence of a full-length Neublastin polypeptide. It should also be
noted that the 887 bp fragment was found to contain the complete
coding region of pro-neublastin. The predicted cDNA [SEQ ID NO: 3]
contains an Open Reading Frame (ORF) encoding pro-neublastin (181
amino acid residues) which showed homology to the three known human
proteins--Persephin, Neurturin, and GDNF.
Neublastin Nucleic Acids of the Invention
[0042] In another aspect the invention provides polynucleotides
capable of expressing the polypeptides of the invention. The
polynucleotides of the invention include DNA, cDNA and RNA
sequences, as well as anti-sense sequences, and include naturally
occurring, synthetic, and intentionally manipulated
polynucleotides. The polynucleotides of the invention also include
sequences that are degenerate as a result of the genetic code, but
which code on expression for a neublastin polypeptide.
[0043] As defined herein, the term "polynucleotide" refers to a
polymeric form of nucleotides of at least 10 bases in length,
preferably at least 15 bases in length. By "isolated
polynucleotide" is meant a polynucleotide that is not immediately
contiguous with both of the coding sequences with which it is
immediately contiguous (one on the 5' end and one on the 3' end) in
the naturally occurring genome of the organism from which it is
derived. The term therefore includes recombinant DNA which is
incorporated into an expression vector, into an autonomously
replicating plasmid or virus, or into the genomic DNA of a
prokaryote or eukaryote, or which exists as a separate molecule,
e.g. a cDNA, independent from other sequences.
[0044] The polynucleotides of the invention also include allelic
variants and "mutated polynucleotides" having a nucleotide sequence
that differs from the nucleotide sequences presented herein at one
or more nucleotide positions.
[0045] In a preferred embodiment, the polynucleotide of the
invention has a nucleic acid (DNA) sequence capable of hybridizing
with the polynucleotide sequence presented as SEQ ID NO: 1, the
polynucleotide sequence presented as SEQ ID NO: 3, the
polynucleotide sequence presented as SEQ ID NO: 8, or the
polynucleotide sequence presented as SEQ ID NO: 15, its
complementary strand, or a sub-sequence hereof under at least
medium, medium/high, or high stringency conditions, as described in
more detail below.
[0046] In another preferred embodiment, the isolated polynucleotide
of the invention has a nucleic acid (DNA) sequence that is at least
70%, preferably at least 80%, more preferred at least 90%, most
preferred at least 95% homologous to the polynucleotide sequence
presented as SEQ ID NO: 1, the polynucleotide sequence presented as
SEQ ID NO: 3, the polynucleotide sequence presented as SEQ ID NO:
8, or the polynucleotide sequence presented as SEQ ID NO: 15.
[0047] In its most preferred embodiment, the polynucleotide has the
DNA sequence presented as SEQ ID NO: 1, the DNA sequence presented
as SEQ ID NO: 3, the DNA sequence presented as SEQ ID NO: 8, or the
polynucleotide sequence presented as SEQ ID NO: 15.
[0048] This invention also provides novel primers and DNA sequences
for identifying, isolating and amplifying neublastin
polynucleotides which code on expression for neublastin
polypeptides or fragments thereof. Such primers include the
polynucleotides set forth in SEQ.ID.NOS. 17-28, and 31-32. In
addition, this invention provides neublastin DNA sequences
generated from those primers, including those set forth in
SEQ.ID.NOS. 13 and 14: Further, this invention provides DNA
sequences from 3' or 5' untranslated regions ("UTR") in genomic DNA
that flank neublastin exons; such sequences are useful in
identifying, isolating and amplifying neublastin polynucleotides
which code on expression for neublastin polypeptides or fragments
thereof.
[0049] 3' UTR sequences of this invention include the sequences set
forth in: [0050] nucleotides 721-865 of SEQ.ID.NO. 1, [0051]
nucleotides 718-861 of SEQ.ID.NO. 3, [0052] nucleotides 718-861 of
SEQ.ID.NO. 8, [0053] nucleotides 1647-2136 of SEQ.ID.NO. 15, and
[0054] contiguous sequences of between 10-25 nucleotides derived
from (i.e., falling within) the foregoing sequences (which are
useful, e.g., as primers).
[0055] 5' UTR sequences of this invention include the sequences set
forth in: [0056] nucleotides 1-10 of SEQ.ID.NO. 1, [0057]
nucleotides 1-57 of SEQ.ID.NO. 8, [0058] nucleotides 1-974 of
SEQ.ID.NO. 15, and [0059] contiguous sequences of between 10-25
nucleotides derived from (i.e., falling within) the foregoing
sequences (which are useful, e.g., as primers).
[0060] The polynucleotides of the invention may preferably be
obtained by cloning procedures, e.g. as described in "Current
Protocols in Molecular Biology" [John Wiley & Sons, Inc.]. In a
preferred embodiment, the polynucleotide is cloned from, or
produced on the basis of human genomic DNA or a cDNA library of the
human brain.
Homology of DNA Sequences
[0061] The DNA sequence homology referred to above may be
determined as the degree of identity between two sequences
indicating a derivation of the first sequence from the second. The
homology may suitably be determined by means of computer programs
known in the art, such as GAP provided in the GCG program package
[Needleman, S. B. and Wunsch C. D., Journal of Molecular Biology
1970 48 443-453]. Using GAP with the following settings for DNA
sequence comparison: GAP creation penalty of 5.0 and GAP extension
penalty of 0.3, the coding region of the analogous DNA sequences
referred to above exhibits a degree of identity preferably of at
least 70%, more preferably at least 80%, more preferably at least
90%, more preferably at least 95%, with the CDS (encoding) part of
the DNA sequence shown in SEQ ID No. 1, or the CDS (encoding) part
of the DNA sequence shown in SEQ ID No. 3, or the CDS (encoding)
part of the DNA sequence shown in SEQ ID No. 8, the CDS (encoding)
part of the DNA sequence shown in SEQ.ID.NO. 15.
[0062] The term "sequence identity" refers to the degree to which
two polynucleotide sequences are identical on a
nucleotide-by-nucleotide basis over a particular region of
comparison. The term "percentage of sequence identity" is
calculated by comparing two optimally aligned sequences over that
region of comparison, determining the number of positions at which
the identical nucleic acid base (e.g., A, T, C, G, U, or I) 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 region of comparison (i.e., the window size), and
multiplying the result by 100 to yield the percentage of sequence
identity. The term "substantial identity" as used herein denotes a
characteristic of a polynucleotide sequence, wherein the
polynucleotide comprises a sequence that has at least 80 percent
sequence identity, preferably at least 85 percent identity and
often 90 to 95 percent sequence identity, more usually at least 99
percent sequence identity as compared to a reference sequence over
a comparison region.
Hybridization Protocol
[0063] The polynucleotides of the invention are such which have a
nucleic acid sequence capable of hybridizing with the
polynucleotide sequence presented as SEQ ID NO: 1, the
polynucleotide sequence presented as SEQ ID NO: 3, or the
polynucleotide sequence presented as SEQ ID NO: 8, or the
polynucleotide sequence presented as SEQ ID NO: 15, or their
complementary strand, or a sub-sequence hereof under at least
medium, medium/high, or high stringency conditions, as described in
more detail below.
[0064] Suitable experimental conditions for determining
hybridization between a nucleotide probe and a homologous DNA or
RNA sequence, involves pre-soaking of the filter containing the DNA
fragments or RNA to hybridize in 5.times.SSC [Sodium
chloride/Sodium citrate; cf. Sambrook et al.; Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Lab., Cold Spring Harbor,
N.Y. 1989] for 10 minutes, and pre-hybridization of the filter in a
solution of 5.times.SSC, 5.times.Denhardt's solution [cf. Sambrook
et al.; Op cit.], 0.5% SDS and 100 .mu.g/ml of denatured sonicated
salmon sperm DNA [cf. Sambrook et al.; Op cit.], followed by
hybridization in the same solution containing a concentration of 10
ng/ml of a random-primed [Feinberg A P & Vogelstein B; Anal.
Biochem. 1983 132 6-13], .sup.32P-dCTP-labeled (specific activity
>1.times.10.sup.9 cpm/.mu.g) probe for 12 hours at approximately
45.degree. C. The filter is then washed twice for 30 minutes in
0.1.times.SSC, 0.5% SDS at a temperature of at least at least
60.degree. C. (medium stringency conditions), preferably of at
least 65.degree. C. (medium/high stringency conditions), more
preferred of at least 70.degree. C. (high stringency conditions),
and even more preferred of at least 75.degree. C. (very high
stringency conditions). Molecules to which the oligonucleotide
probe hybridizes under these conditions may be detected using a
x-ray film.
Cloned Polynucleotides
[0065] The isolated polynucleotide of the invention may in
particular be a cloned polynucleotide. As defined herein, the term
"cloned polynucleotide", refers to a polynucleotide or DNA sequence
cloned in accordance with standard cloning procedures currently
used in genetic engineering to relocate a segment of DNA, which may
in particular be cDNA, i.e. enzymatically derived from RNA, from
its natural location to a different site where it will be
reproduced.
[0066] Cloning may be accomplished by any suitable route and may
involve techniques such as reverse transcriptase technology, PCR
technology, and the like, as well as excision and isolation of the
desired DNA segment.
[0067] The cloned polynucleotide of the invention may alternatively
be termed "DNA construct" or "isolated DNA sequence", and may in
particular be a complementary DNA (cDNA).
Biological Sources
[0068] The isolated polynucleotide of the invention may be obtained
from any suitable source.
[0069] In a preferred embodiment, which the polynucleotide of the
invention is cloned from, or produced on the basis of a cDNA
library, e.g. of a cDNA library of the fetal or adult brain, in
particular of the forebrain, the hindbrain, the cortex, the
striatum, the amygdala, the cerebellum, the caudate nucleus, the
corpus callosum, the hippocampus, the thalamic nucleus, the
subthalamic nucleus, the olfactory nucleus, the putamen, the
substantia nigra, the dorsal root ganglia, the trigeminal ganglion,
the superior mesenteric artery, or the thalamus; of the spinal
cord; of the heart; the placenta; of the lung; of the liver; of the
skeletal muscle; of the kidney; of the liver; of the pancreas; of
the intestines; of the eye; of the retina; of the tooth pulp; of
the hair follicle; of the prostate; of the pituitary; or of the
trachea.
[0070] Commercial cDNA libraries from a variety of tissues, both
human and non-human, are available from e.g. Stratagene and
Clontech. The isolated polynucleotide of the invention may be
obtained by standard methods, e.g. those described in the working
examples.
Neublastin Polypeptides of the Invention
[0071] As noted above, a "neublastin polypeptide," as used herein,
is a polypeptide which possesses neurotrophic activity (e.g., as
described in Examples 6, 7, 8, and 9) and includes those
polypeptides which have an amino acid sequence that has at least
70% homology to the "neublastin" polypeptides set forth in
AA.sub.-95-AA.sub.105 of SEQ. ID. NO. 2, AA.sub.1-AA.sub.105 of
SEQ. ID. NO. 2, AA.sub.-97-AA.sub.140 of SEQ. ID. NO. 4,
AA.sub.-41-AA.sub.140 of SEQ. ID. NO. 4, AA.sub.1-AA.sub.140 of
SEQ. ID. NO. 4, AA.sub.-80-AA.sub.140 of SEQ. ID. NO. 9 ("wild
type" prepro), AA.sub.-41-AA.sub.140 of SEQ. ID. NO. 9 (pro),
AA.sub.1-AA.sub.140 of SEQ. ID. NO. 5 (mature 140AA),
AA.sub.1-AA.sub.116 of SEQ. ID. NO. 6 (mature 116AA),
AA.sub.1-AA.sub.113 of SEQ. ID. NO. 7 (mature 113AA),
AA.sub.1-AA.sub.140 of SEQ. ID. NO. 10 (mature 140AA),
AA.sub.1-AA.sub.116 of SEQ. ID. NO. 11 (mature 116AA),
AA.sub.1-AA.sub.113 of SEQ. ID. NO. 12 (mature 113AA),
AA.sub.1-AA.sub.224 of SEQ. ID. NO. 16 (murine prepro), and
variants and derivatives of each of the foregoing.
[0072] Preferably, the C-terminal sequence of the above identified
neublastin polypeptides has an amino acid sequence as set forth in
AA.sub.72-AA.sub.105 of SEQ. ID. NO. 2 (i.e., AA.sub.107-AA.sub.140
of SEQ. ID. NO. 9), more preferably AA.sub.41-AA.sub.105 of SEQ.
ID. NO. 2 (i.e., AA.sub.76-AA.sub.140 of SEQ. ID. NO. 9).
[0073] Also, it is preferable that the neublastin polypeptide
retain the 7 conserved Cys residues that are characteristic of the
GDNF family and of the TGF-beta super family.
[0074] Preferably the neublastin polypeptide has an amino acid
sequence greater than 85% homology, most preferably greater than
95% homology, to the foregoing sequences (i.e.,
AA.sub.-95-AA.sub.105 of SEQ. ID. NO. 2, AA.sub.1-AA.sub.105 of
SEQ. ID. NO. 2, AA.sub.-97-AA.sub.140 of SEQ. ID. NO. 4,
AA.sub.-41-AA.sub.140 of SEQ. ID. NO. 4, AA.sub.1-AA.sub.140 of
SEQ. ID. NO. 4, AA.sub.-80-AA.sub.140 of SEQ. ID. NO. 9 ("wild
type" prepro), AA.sub.-41-AA.sub.140 of SEQ. ID. NO. 9 (pro),
AA.sub.1-AA.sub.140 of SEQ. ID. NO. 5 (mature 140AA),
AA.sub.1-AA.sub.116 of SEQ. ID. NO. 6 (mature 116AA),
AA.sub.1-AA.sub.113 of SEQ. ID. NO. 7 (mature 113AA),
AA.sub.1-AA.sub.140 of SEQ. ID. NO. 10 (mature 140AA),
AA.sub.1-AA.sub.116 of SEQ. ID. NO. 11 (mature 116AA),
AA.sub.1-AA.sub.113 of SEQ. ID. NO. 12 (mature 113AA),
AA.sub.1-AA.sub.224 of SEQ. ID. NO. 16 (murine prepro), and
preferably any of the foregoing polypeptides with a C-terminal
sequence of the above identified neublastin polypeptides has an
amino acid sequence as set forth in AA.sub.72-AA.sub.105 of SEQ.
ID. NO. 2 (i.e., AA.sub.107-AA.sub.140 of SEQ. ID. NO. 9), more
preferably AA.sub.41-AA.sub.105 of SEQ. ID. NO. 2 (i.e.,
AA.sub.76-AA.sub.140 of SEQ. ID. NO. 9) or AA.sub.191-AA.sub.224 of
SEQ. ID. NO. 16.
[0075] In addition, this invention contemplates those polypeptides
which have an amino acid sequence that has at least 70% homology to
the murine "neublastin" polypeptides set forth in
AA.sub.1-AA.sub.224 of SEQ. ID. NO. 16.
[0076] Among the preferred polypeptides of the invention in one
embodiment represent the preprosequence (as set forth in SEQ. ID.
NOS. 2, 4, 9, and 16, respectively), the pro sequence (as set forth
in AA.sub.-75-AA.sub.105 of SEQ. ID. NO. 2, or
AA.sub.-41-AA.sub.140 of SEQ.ID.NOS. 4 and 9, respectively) and the
mature sequence of neublastin (as set forth in SEQ. ID. NOS. 5, 6,
7, 10, 11, or 12, preferably SEQ. ID. NOS. 10, 11, 12).
[0077] The polypeptides of the invention include variant
polypeptides. In the context of this invention, the term "variant
polypeptide" means a polypeptide (or protein) having an amino acid
sequence that differs from the sequence presented as SEQ ID NO: 2,
SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO:
9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 16,
at one or more amino acid positions. Such variant polypeptides
include the modified polypeptides described above, as well as
conservative substitutions, splice variants, isoforms, homologues
from other species, and polymorphisms.
[0078] As defined herein, the term "conservative substitutions"
denotes the replacement of an amino acid residue by another,
biologically similar residue. For example, one would expect
conservative amino acid substitutions to have little or no effect
on the biological activity, particularly if they represent less
than 10% of the total number of residues in the polypeptide or
protein. Preferably, conservative amino acids substitutions
represent changes in less than 5% of the polypeptide or protein,
most preferably less than 2% of the polypeptide or protein (e.g.,
when calculated in accordance with NBN113, most preferred
conservative substitutions would represent fewer than 3 amino acid
substitutions in the wild type mature amino acid sequence). In a
particularly preferred embodiment, there is a single amino acid
substitution in the mature sequence, wherein the both the
substituted and replacement amino acid are non-cyclic.
[0079] Other examples of particularly conservative substitutions
include the substitution of one hydrophobic residue such as
isoleucine, valine, leucine or methionine for another, or the
substitution of one polar residue for another, such as the
substitution of arginine for lysine, glutamic for aspartic acid, or
glutamine for asparagine, and the like.
[0080] The term conservative substitution also include the use of a
substituted amino acid residue in place of an un-substituted parent
amino acid residue provided that antibodies raised to the
substituted polypeptide also immunoreact with the un-substituted
polypeptide.
[0081] Modifications of this primary amino acid sequence may result
in proteins which have substantially equivalent activity as
compared to the unmodified counterpart polypeptide, and thus may be
considered functional analogous of the parent proteins. Such
modifications may be deliberate, e.g. as by site-directed
mutagenesis, or they may occur spontaneous, and include splice
variants, isoforms, homologues from other species, and
polymorphisms. Such functional analogous are also contemplated
according to the invention.
[0082] Moreover, modifications of the primary amino acid sequence
may result in proteins which do not retain the biological activity
of the parent protein, including dominant negative forms, etc. A
dominant negative protein may interfere with the wild-type protein
by binding to, or otherwise sequestering regulating agents, such as
upstream or downstream components, that normally interact
functionally with the polypeptide. Such dominant negative forms are
also contemplated according to the invention.
[0083] A "signal peptide" is a peptide sequence that directs a
newly synthesized polypeptide to which the signal peptide is
attached to the endoplasmic reticulum (ER) for further
post-translational processing and distribution.
[0084] An "heterologous signal peptide," as used herein in the
context of neublastin, means a signal peptide that is not the human
neublastin signal peptide, typically the signal peptide of some
mammalian protein other than neublastin.
Skilled artisans will recognize that the human neublastin DNA
sequence (either cDNA or genomic DNA), or sequences that differ
from human neublastin DNA due to either silent codon changes or to
codon changes that produce conservative amino acid substitutions,
can be used to genetically modify cultured human cells so that they
will overexpress and secrete the enzyme.
[0085] Polypeptides of the present invention also include chimeric
polypeptides or cleavable fusion polypeptides in which another
polypeptide is fused at the N-terminus or the C-terminus of the
polypeptide or fragment thereof. A chimeric polypeptide may be
produced by fusing a nucleic acid sequence (or a portion thereof)
encoding another polypeptide to a nucleic acid sequence (or a
portion thereof) of the present invention.
[0086] Techniques for producing chimeric polypeptides are standard
techniques. Such techniques usually requires joining the sequences
in a way so that they are in both in the same reading frame, and
expression of the fused polypeptide under the control of the same
promoter(s) and terminator.
[0087] Polypeptides of the present invention also include truncated
forms of the full length neublastin molecule. In such truncated
molecules, one or more amino acids have been deleted from the
N-terminus or the C-terminus, preferably the N-terminus.
Amino Acid Sequence Homology
[0088] The degree to which a candidate polypeptide shares homology
with a neublastin polypeptide of the invention is determined as the
degree of identity between two amino acid sequences. A high level
of sequence identity indicates a likelihood that the first sequence
is derived from the second
[0089] Homology is determined by computer analysis, such as,
without limitations, the ClustaiX computer alignment program
[Thompson J D, Gibson T J, Plewniak F, Jeanmougin F, & Higgins
D G: The ClustaiX windows interface: flexible strategies for
multiple sequence alignment aided by quality analysis tools;
Nucleic Acids Res. 1997, 25 (24): 4876-82], and the default
parameters suggested herein. Using this program, the mature part of
a polypeptide encoded by an analogous DNA sequence of the invention
exhibits a degree of identity of at least 90%, more preferred of at
least 95%, most preferred of at least 98% with the amino acid
sequence presented herein as SEQ ID NO: 2, SEQ. ID. NO: 4; SEQ. ID.
NO.: 5; SEQ. ID. NO.: 6; SEQ. ID. NO.: 7; SEQ. ID. NO.: 9; SEQ. ID.
NO.: 10; SEQ. ID. NO.: 11; SEQ. ID. NO.: 12, or SEQ. ID. NO.:
16.
[0090] Based on the homology determination it is confirmed that the
polypeptide of the invention, belonging to the TGF-.beta.
superfamily, is related to the GDNF subfamily, but represents a
distinct member of this subfamily.
Bioactive Polypeptides
[0091] The polypeptide of the invention may be provided on any
bioactive form, including the form of pre-pro-proteins,
pro-proteins, mature proteins, glycosylated proteins,
phosphorylated proteins, or any other posttranslational modified
protein.
[0092] The polypeptide of the invention may in particular be a
N-glycosylated polypeptide, which polypeptide preferably is
glycosylated at the N-residues indicated in the sequence
listings.
[0093] In a preferred embodiment, the polypeptide of the invention
has the amino acid sequence presented as SEQ ID NO: 9, holding a
glycosylated asparagine residue at position 122; the amino acid
sequence presented as SEQ ID NO: 10, holding a glycosylated
asparagine residue at position 122; the amino acid sequence
presented as SEQ ID NO: 11, holding a glycosylated asparagine
residue at position 98; or the amino acid sequence presented as SEQ
ID NO: 12, holding a glycosylated asparagine residue at position
95.
[0094] This invention also contemplates neublastin fusion proteins,
such as Ig-fusions, as described, e.g., in U.S. Pat. No. 5,434,131,
herein incorporated by reference.
[0095] In one embodiment, the invention provides a polypeptide
having the amino acid sequence shown as SEQ ID NO: 2, or an amino
acid sequence which is at least about 85%, preferably at least
about 90%, more preferably at least about 98%, and most preferably
at least about 99% homologous to the sequence presented as SEQ ID
NO: 2.
[0096] In another embodiment, the invention provides a polypeptide
having the amino acid sequence of SEQ ID NO: 4, or an amino acid
sequence which is at least 90%, more preferred at least 95%, yet
more preferred at least 98%, most preferred at least 99% homologous
to the sequence presented as SEQ ID NO: 4.
[0097] In a third embodiment, the invention provides a polypeptide
having the amino acid sequence of SEQ ID NO: 5, or an amino acid
sequence which is at least 90%, more preferred at least 95%, most
preferred at least 98% homologous to the sequence presented as SEQ
ID NO: 5.
[0098] In a fourth embodiment, the invention provides a
polypeptides having the amino acid sequence of SEQ ID NO: 6, or an
amino acid sequence which is at least 90%, more preferred at least
95%, most preferred at least 98% homologous to the sequence
presented as SEQ ID NO: 6.
[0099] In a fifth embodiment, the invention provides a polypeptides
having the amino acid sequence of SEQ ID NO: 7, or an amino acid
sequence which is at least 90%, more preferred at least 95%, most
preferred at least 98% homologous to the sequence presented as SEQ
ID NO: 7.
[0100] The neublastin polypeptide of the invention includes allelic
variants, e.g., the polypeptide amino acid sequences of SEQ ID NOS.
5-7, in which Xaa designates Asn or Thr, and Yaa designates Ala or
Pro.
[0101] In a sixth embodiment, the invention provides a polypeptide
shaving the amino acid sequence of SEQ ID NO: 9, or an amino acid
sequence which is at least 90%, more preferred at least 95%, most
preferred at least 98% homologous to the sequence presented as SEQ
ID NO: 9.
[0102] In a seventh embodiment, the invention provides a
polypeptide having the amino acid sequence of SEQ ID NO: 10, or an
amino acid sequence at least 90%, more preferred at least 95%, most
preferred at least 98%, homologous to the sequence presented as SEQ
ID NO: 10.
[0103] In a eight embodiment, the invention provides a polypeptide
having the amino acid sequence of SEQ ID NO: 11, or an amino acid
sequence at least 90%, more preferred at least 95%, most preferred
at least 98% homologous to the sequence presented as SEQ ID NO:
11.
[0104] In a ninth embodiment, the invention provides a polypeptide
having the amino acid sequence of SEQ ID NO: 12, or an amino acid
sequence at least 90%, more preferred at least 95%, most preferred
at least 98% homologous to the sequence presented as SEQ ID NO:
12.
[0105] In a tenth embodiment, the invention provides a polypeptide
having the amino acid sequence of SEQ ID NO: 16, or an amino acid
sequence at least 90%, more preferred at least 95%, most preferred
at least 98% homologous to the sequence presented as SEQ ID NO: 16,
which is a pre-pro-neublastin of murine origin.
[0106] In another embodiment, the polypeptide of the invention
holds the GDNF subfamily fingerprint, i.e. the amino acid residues
underlined in Table 3.
[0107] In a further embodiment, the invention provides a
polypeptide encoded by a polynucleotide sequence capable of
hybridizing under high stringency conditions with the
polynucleotide sequence presented as SEQ ID NO: 1, its
complementary strand, or a sub-sequence thereof. In a preferred
embodiment, the polypeptide of the invention is encoded by a
polynucleotide sequence being at least 70% homologous to the
polynucleotide sequence presented as SEQ ID NO: 1. In its most
preferred embodiment, the polypeptide of the invention is encoded
by the polynucleotide sequence presented as SEQ ID NO: 1.
[0108] In a yet further embodiment, the invention provides novel
polypeptides encoded by a polynucleotide sequence capable of
hybridizing under high stringency conditions with the
polynucleotide sequence presented as SEQ ID NO: 3, its
complementary strand, or a sub-sequence thereof. In a preferred
embodiment, the polypeptide of the invention is encoded by a
polynucleotide sequence being at least 70% homologous to the
polynucleotide sequence presented as SEQ ID NO: 3. In its most
preferred embodiment, the polypeptide of the invention is encoded
by the polynucleotide sequence presented as SEQ ID NO: 3.
[0109] In a still further embodiment, the invention provides novel
polypeptides encoded by a polynucleotide sequence capable of
hybridizing under high stringency conditions with the
polynucleotide sequence presented as SEQ ID NO: 8, its
complementary strand, or a sub-sequence thereof. In a preferred
embodiment, the polypeptide of the invention is encoded by a
polynucleotide sequence being at least 70% homologous to the
polynucleotide sequence presented as SEQ ID NO: 8. In its most
preferred embodiment, the polypeptide of the invention is encoded
by the polynucleotide sequence presented as SEQ ID NO: 8.
[0110] In a still further embodiment, the invention provides novel
polypeptides encoded by a polynucleotide sequence capable of
hybridizing under high stringency conditions with the
polynucleotide sequence presented as SEQ ID NO: 15, its
complementary strand, or a sub-sequence thereof. In a preferred
embodiment, the polypeptide of the invention is encoded by a
polynucleotide sequence being at least 70% homologous to the
polynucleotide sequence presented as SEQ ID NO: 15. In its most
preferred embodiment, the polypeptide of the invention is encoded
by the polynucleotide sequence presented as SEQ ID NO: 15.
Biological Origin
[0111] The polypeptide of the invention may be isolated from
mammalian cells, preferably from a human cell or from a cell of
murine origin.
[0112] In a most preferred embodiment, the polypeptide of the
invention may be isolated from human heart tissue, from human
skeletal muscle, from human pancreas, or from human brain tissue,
in particular from caudate nucleus or from thalamus, or it may be
obtained from DNA of mammalian origin, as discussed in more detail
below.
Neurotrophic Activity
[0113] Neublastin polypeptides of the invention are useful for
moderating metabolism, growth, differentiation, or survival of a
nerve or neuronal cell. In particular, neublastin polypeptides are
used to treating or to alleviate a disorder or disease of a living
animal, e.g., a human, which disorder or disease is responsive to
the activity of a neurotrophic agents. Such treatments and methods
are described in more details below.
Antibodies
[0114] Neublastin polypeptides or polypeptide fragments of the
invention are used to produce neublastin-specific antibodies. As
used herein, a "neublastin-specific antibody is an antibody, e.g.,
a polyclonal antibody or a monoclonal antibody, that is
immunoreactive to a neublastin polypeptide or polypeptide fragment,
or that binds with specificity to an epitopes of a neublastin
polypeptides.
[0115] The preparation of polyclonal and monoclonal antibodies is
well known in the art. Polyclonal antibodies may in particular be
obtained as described by, e.g., Green et al.: "Production of
Polyclonal Antisera" in Immunochemical Protocols (Manson, Ed.);
Humana Press, 1992, pages 1-5; by Coligan et al.: "Production of
Polyclonal Antisera in Rabbits, Rats, Mice and Hamsters" in Current
Protocols in Immunology, 1992, Section 2.4.1, and by Ed Harlow and
David Lane (Eds.) in "Antibodies; A laboratory manual" Cold Spring
Harbor Lab. Press 1988. These protocols are hereby incorporated by
reference. Monoclonal antibodies may in particular be obtained as
described by, e.g., Kohler & Milstein, Nature, 1975, 256:495;
Coligan et al., in Current Protocols in Immunology, 1992, Sections
2.5.1-2.6.7; and Harlow et al., in Antibodies: A Laboratory Manual;
Cold Spring Harbor, Pub., 1988, page 726; which protocols are
hereby incorporated by reference.
[0116] Briefly, monoclonal antibodies may be obtained by injecting,
e.g., mice with a composition comprising an antigen, verifying the
presence of antibody production by removing a serum sample,
removing the spleen to obtain B lymphocytes, fusing the B
lymphocytes with myeloma cells to produce hybridomas, cloning the
hybridomas, selecting positive clones that produce the antibodies
to the antigen, and isolating the antibodies from the hybridoma
cultures.
[0117] Monoclonal antibodies can be isolated and purified from
hybridoma cultures by a variety of well-established techniques,
including affinity chromatography with protein A Sepharose,
size-exclusion chromatography, and ion-exchange chromatography,
see. e.g. Coligan et al. in Current Protocols in Immunology, 1992,
Sections 2.7.1-2.7.12, and Sections 2.9.1-2.9.3; and Barnes et al.:
"Purification of Immunoglobulin G (IgG)" in Methods in Molecular
Biology; Humana Press, 1992, Vol. 10, Pages 79-104. Polyclonal or
monoclonal antibodies may optionally be further purified, e.g. by
binding to and elution from a matrix to which the polypeptide, to
which the antibodies were raised, is bound.
[0118] Antibodies which bind to the neublastin polypeptide of the
invention can be prepared using an intact polypeptide or fragments
containing small peptides of interest as the immunising antigen.
The polypeptide used to immunise an animal may be obtained by
recombinant DNA techniques or by chemical synthesis, and may
optionally be conjugated to a carrier protein. Commonly used
carrier proteins which are chemically coupled to the peptide
include keyhole limpet hemocyanin (KLH), thyroglobulin, bovine
serum albumin (BSA), and tetanus toxoid. The coupled peptide may
then be used to immunise the animal, which may in particular be a
mouse, a rat, a hamster or a rabbit.
[0119] In one embodiment, antibodies are produced using the
following peptides: Peptide 1: CRPTRYEAVSFMDVNST (amino acids
108-124 of SEQ ID NO: 9); or Peptide 2: ALRPPPGSRPVSQPC (amino
acids 93-107 of SEQ ID NO: 9). Methods for producing antibodies
using these polypeptides are described in Example 10.
[0120] We also generated rabbit polyclonal antibodies to the
following peptides:
TABLE-US-00001 (amino acids 30-43 of SEQ ID NO: 9) Peptide R27:
GPGSRARAAGARGC; (amino acids 57-70 of SEQ ID NO: 9) Peptide R28:
LGHRSDELVRFRFC; (amino acids 74-85 of SEQ ID NO: 9) Peptide R29:
CRRARSPHDLSL; (amino acids 94-107 of SEQ ID NO: 9) Peptide R30:
LRPPPGSRPVSQPC; and (amino acids 123-136 of SEQ ID NO: 9) Peptide
R31: STWRTVDRLSATAC.
[0121] Of this group, only peptides R30 and R31, relatively close
to the C-terminus, recognized the denatured protein under reducing
conditions on a Western blot.
[0122] We have also identified additional neublastin-derived
peptides derived from the mature protein, as detailed below, which
are predicted surface exposed loops based on the known GDNF
structure (Eigenbrot and Gerber, Nat. Struct. Biol., 4, pp. 435-438
(1997)), and are thus useful for antibody generation:
TABLE-US-00002 (AA 43-70 of SEQ. ID. NO: 9) Region 1:
CRLRSQLVPVRALGLGHRSDELVRFRFC (AA 74-107 of SEQ. ID. NO: 9) Region
2: CRRARSPHDLSLASLLGAGALRPPPGSRPVSQPC (AA 108-136 of SEQ. ID. NO:
9) Region 3: CRPTRYEAVSFMDVNSTWRTVDRLSATAC
[0123] In another aspect of the invention, antibodies which
specifically bind neublastin or neublastin-derived peptides may be
used for detecting the presence of such neublastin neurotrophic
factors in various media, and in particular for the diagnosis of
conditions or diseases associated with the neublastin molecules of
the invention. A variety of protocols for such detection, including
ELISA, RIA and FACS, are known in the art.
[0124] The antibodies of this invention may also be used for
blocking the effect of the neurotrophic factor, and may in
particular be neutralizing antibodies.
Methods of Producing the Polypeptides of the Invention
[0125] A cell comprising a DNA sequence encoding a neublastin
polypeptide of the invention is cultured under conditions
permitting the production of the polypeptide, followed by recovery
of the polypeptide from the culture medium, as detailed below. When
cells are to be genetically modified for the purposes of producing
a neublastin polypeptide, the cells may be modified by conventional
methods or by gene activation.
[0126] According to conventional methods, a DNA molecule that
contains a neublastin cDNA or genomic DNA sequence may be contained
within an expression construct and transfected into cells by
standard methods including, but not limited to, liposome-,
polybrene-, or DEAE dextran-mediated transfection, electroporation,
calcium phosphate precipitation, microinjection, or velocity driven
microprojectiles ("biolistics"). Alternatively, one could use a
system that delivers DNA by viral vector. Viruses known to be
useful for gene transfer include adenoviruses, adeno-associated
virus, lentivirus, herpes virus, mumps virus, poliovirus,
retroviruses, Sindbis virus, and vaccinia virus such as canary pox
virus, as well as Baculovirus infection of insect cells, in
particular SfP9 insect cells.
[0127] Alternatively, the cells may be modified using a gene
activation ("GA") approach, such as described in U.S. Pat. Nos.
5,733,761 and 5,750,376, each incorporated herein by reference.
[0128] Accordingly, the term "genetically modified," as used herein
in reference to cells, is meant to encompass cells that express a
particular gene product following introduction of a DNA molecule
encoding the gene product and/or regulatory elements that control
expression of a coding sequence for the gene product. The DNA
molecule may be introduced by gene targeting, allowing
incorporation of the DNA molecule at a particular genomic site.
Recombinant Expression Vectors
[0129] In a further aspect the invention provides a recombinant
expression vector comprising the polynucleotide of the invention.
The recombinant expression vector of the invention may be any
suitable eukaryotic expression vector. Preferred recombinant
expression vectors are the ubiquitin promoter containing vector
pTEJ-8 (FEBS Lett. 1990 267 289-294), and derivatives hereof, e.g.
pUbi1Z. A preferred commercially available eukaryotic expression
vectors is e.g. the virus promoter containing vector pcDNA-3
(available from Invitrogen). Another preferred expression vector
uses SV40 early and adenovirus major late promoters (derived from
plasmid pAD2beta; Norton and Coffin, Mol. Cell. Biol. 5: 281
(1985)).
[0130] This invention also provides prokaryotic expression vectors
and synthetic genes (syngenes) with codon optimization for
prokaryotic expression. Syngenes were constructed with lower GC
content and preferred bacterial (e.g., E. coli) codons. The syngene
is being cloned into two vectors, pET19b and pMJB164, a derivative
of pET19b. The construction with pET19b is shown in FIG. 14. In
this construct, the sequence encoding the mature domain of
neublastin is directly fused to an initiating methionine. The
construction with pMJB164 is shown in FIG. 15.
Production Cells
[0131] In a yet further aspect the invention provides a production
cell genetically manipulated to comprise the isolated
polynucleotide sequence of the invention, and/or or a recombinant
expression vector of the invention. The cell of the invention may
in particular be genetically manipulated to transiently or stably
express, over-express or co-express polypeptide of the invention.
Methods for generating transient and stable expression are known in
the art.
[0132] The polynucleotide of the invention may be inserted into an
expression vector, e.g. a plasmid, virus or other expression
vehicle, and operatively linked to expression control sequences by
ligation in a way that expression of the coding sequence is
achieved under conditions compatible with the expression control
sequences. Suitable expression control sequences include promoters,
enhancers, transcription terminators, start codons, splicing
signals for introns, and stop codons, all maintained in the correct
reading frame of the polynucleotide of the invention so as to
permit proper translation of mRNA. Expression control sequences may
also include additional components such as leader sequences and
fusion partner sequences.
[0133] The promoter may in particular be a constitutive or an
inducible promoter. When cloning in bacterial systems, inducible
promoters such as pL of bacteriophage .lamda., plac, ptrp, ptac
(ptrp-lac hybrid promoter), may be used. When cloning in mammalian
systems, promoters derived from the genome of mammalian cells, e.g.
the ubiquitin promoter, the TK promoter, or the metallothionein
promoter, or from mammalian viruses, e.g. the retrovirus long
terminal repeat, the adenovirus late promoter or the vaccinia virus
7.5K promoter, may be used. Promoters obtained by recombinant DNA
or synthetic techniques may also be used to provide for
transcription of the polynucleotide of the invention.
[0134] Suitable expression vectors typically comprise an origin of
expression, a promoter as well as specific genes which allow for
phenotypic selection of the transformed cells, and include vectors
like the T7-based expression vector for expression in bacteria
[Rosenberg et al; Gene 1987 56 125], the pTEJ-8, pUbilZ, pcDNA-3
and pMSXND expression vectors for expression in mammalian cells
[Lee and Nathans, J. Biol. Chem. 1988 263 3521], baculovirus
derived vectors for expression in insect cells, and the oocyte
expression vector PTLN [Lorenz C, Pusch M & Jentsch T J:
Heteromultimeric CLC chloride channels with novel properties; Proc.
Natl. Acad. Sci. USA 1996 93 13362-13366].
[0135] In a preferred embodiment, the cell of the invention is an
eukaryotic cell, e.g., a mammalian cell, e.g., a human cell, an
oocyte, or a yeast cell. The cell of the invention may be without
limitation a human embryonic kidney (HEK) cell, e.g., a HEK 293
cell, a BHK21 cell, a Chinese hamster ovary (CHO) cell, a Xenopus
laevis oocyte (XLO) cell. In another embodiment, the cell of the
invention is a fungal cell, e.g., a filamentous fungal cell. In
another preferred embodiment, the cell is an insect cell, most
preferably the Sf9 cell. Additional preferred mammalian cells of
the invention are PC12, HiB5, RN33b cell lines and human neural
progenitor cells. Most preferred are human cells.
[0136] Examples of primary or secondary cells include fibroblasts,
epithelial cells including mammary and intestinal epithelial cells,
endothelial cells, formed elements of the blood including
lymphocytes and bone marrow cells, glial cells, hepatocytes,
keratinocytes, muscle cells, neural cells, or the precursors of
these cell types. Examples of immortalized human cell lines useful
in the present methods include, but are not limited to, Bowes
Melanoma cells (ATCC Accession No. CRL 9607), Daudi cells (ATCC
Accession No. CCL 213), HeLa cells and derivatives of HeLa cells
(ATCC Accession Nos. CCL 2, CCL 2.1, and CCL 2.2), HL-60 cells
(ATCC Accession No. CCL 240), HT-1080 cells (ATCC Accession No. CCL
121), Jurkat cells (ATCC Accession No. TIB 152), KB carcinoma cells
(ATCC Accession No. CCL 17), K-562 leukemia cells (ATCC Accession
No. CCL 243), MCF-7 breast cancer cells (ATCC Accession No. BTH
22), MOLT-4 cells (ATCC Accession No. 1582), Namalwa cells (ATCC
Accession No. CRL 1432), Raji cells (ATCC Accession No. CCL 86),
RPMI 8226 cells (ATCC Accession No. CCL 155), U-937 cells (ATCC
Accession No. CRL 1593), WI-38VA13 sub line 2R4 cells (ATCC
Accession No. CLL 75.1), and 2780AD ovarian carcinoma cells (Van
der Buick et al., Cancer Res. 48: 5927-5932, 1988), as well as
heterohybridoma cells produced by fusion of human cells and cells
of another species. Secondary human fibroblast strains, such as
WI-38 (ATCC Accession No. CCL 75) and MRC-5 (ATCC Accession No. CCL
171), may also be used.
[0137] When the cell of the invention is an eukaryotic cell,
incorporation of the heterologous polynucleotide of the invention
may be in particular be carried out by infection (employing a virus
vector), by transfection (employing a plasmid vector), using
calcium phosphate precipitation, microinjection, electroporation,
lipofection, or other physical-chemical methods known in the
art.
[0138] In a more preferred embodiment the isolated polynucleotide
sequence of the invention, and/or or a recombinant expression
vector of the invention are transfected in a mammalian host cell, a
neural progenitor cell, an astrocyte cell, a T-cell, a hematopoitic
stem cell, a non-dividing cell, or a cerebral endothelial cell,
comprising at least one DNA molecule capable of mediating cellular
immortalization and/or transformation.
[0139] Activation of an endogenous gene in a host cell may be
accomplished by the introducing regulatory elements, in particular
by the introducing a promoter capable of effecting transcription of
an endogenous gene encoding the neublastin polypeptide of the
invention.
Pharmaceutical Compositions
[0140] In another aspect the invention provides novel
pharmaceutical compositions comprising a therapeutically effective
amount of the polypeptide of the invention.
[0141] For use in therapy the polypeptide of the invention may be
administered in any convenient form. In a preferred embodiment, the
polypeptide of the invention is incorporated into a pharmaceutical
composition together with one or more adjuvants, excipients,
carriers and/or diluents, and the pharmaceutical composition
prepared by the skilled person using conventional methods known in
the art.
[0142] Such pharmaceutical compositions may comprise the
polypeptide of the invention, or antibodies hereof. The composition
may be administered alone or in combination with at one or more
other agents, drugs or hormones.
[0143] The pharmaceutical composition of this invention may be
administered by any suitable route, including, but not limited to
oral, intravenous, intramuscular, inter-arterial, intramedullary,
intrathecal, intraventricular, transdermal, subcultaneous,
intraperitoneal, intranasal, anteral, topical, sublingual or rectal
application, buccal, vaginal, intraorbital, intracerebral,
intracranial, intraspinal, intraventricular, intracisternal,
intracapsular, intrapulmonary, transmucosal, or via inhalation.
[0144] Intrapulmonary delivery methods, apparatus and drug
preparation are described, for example, in U.S. Pat. Nos.
5,785,049, 5,780,019, and 5,775,320, each incorporated herein by
reference. Administration may be by periodic injections of a bolus
of the preparation, or may be made more continuous by intravenous
or intraperitoneal administration from a reservoir which is
external (e.g., an IV bag) or internal (e.g., a bioerodable
implant, a bioartificial organ, or a colony of implanted neublastin
production cells). See, e.g., U.S. Pat. Nos. 4,407,957, 5,798,113,
and 5,800,828, each incorporated herein by reference.
Intrapulmonary delivery methods and apparatus are described, for
example, in U.S. Pat. Nos. 5,654,007, 5,780,014, and 5,814,607,
each incorporated herein by reference.
[0145] In particular, administration of a neublastin according to
this invention may be achieved using any suitable delivery means,
including: [0146] (a) pump (see, e.g., Annals of Pharmacotherapy,
27:912 (1993); Cancer, 41:1270 (1993); Cancer Research, 44:1698
(1984), incorporated herein by reference), [0147] (b),
microencapsulation (see, e.g., U.S. Pat. Nos. 4,352,883; 4,353,888;
and 5,084,350, herein incorporated by reference), [0148] (c)
continuous release polymer implants (see, e.g., Sabel, U.S. Pat.
No. 4,883,666, incorporated herein by reference), [0149] (d)
macroencapsulation (see, e.g., U.S. Pat. Nos. 5,284,761, 5,158,881,
4,976,859 and 4,968,733 and published PCT patent applications
WO92/19195, WO 95/05452, each incorporated herein by reference);
[0150] (e) naked or unencapsulated cell grafts to the CNS (see,
e.g., U.S. Pat. Nos. 5,082,670 and 5,618,531, each incorporated
herein by reference); or [0151] (f) injection, either
subcutaneously, intravenously, intra-arterially, intramuscularly,
or to other suitable site; [0152] (g) oral administration, in
capsule, liquid, tablet, pill, or prolonged release
formulation.
[0153] In one embodiment of this invention, a neublastin is
delivered directly into the CNS, preferably to the brain
ventricles, brain parenchyma, the intrathecal space or other
suitable CMS location, most preferably intrathecally.
[0154] In another preferred embodiment, we contemplate systemic
delivery by subcutaneous injection, intravenous administration, or
intravenous infusion.
[0155] Other useful parenteral delivery systems include
ethylene-vinyl acetate copolymer particles, osmotic pumps,
implantable infusion systems, pump delivery, encapsulated cell
delivery, liposomal delivery, needle-delivered injection,
needle-less injection, nebulizer, aeorosolizer, electroporation,
and transdermal patch.
[0156] Further details on techniques for formulation and
administration may be found in the latest edition of Remington's
Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa.).
[0157] The active ingredient may be administered in one or several
doses per day. Currently contemplated appropriate dosages are
between 0.5 ng neublastin/kg body weight to about 50 .mu.g/kg per
administration, and from about 1.0 ng/kg to about 100 .mu.g/kg
daily. The neublastin pharmaceutical composition should provide a
local concentration of neurotrophic factor of from about 5 ng/ml
cerebrospinal fluid ("CSF") to 25 ng/ml CSF.
[0158] The dose administered must of course be carefully adjusted
to the age, weight and condition of the individual being treated,
as well as the route of administration, dosage form and regimen,
and the result desired, and the exact dosage should of course be
determined by the practitioner.
[0159] In further embodiments, the Neublastin polypeptide of the
invention may be administered by genetic delivery, using cell lines
and vectors as described below under methods of treatment. To
generate such therapeutic cell lines, the polynucleotide of the
invention may be inserted into an expression vector, e.g. a
plasmid, virus or other expression vehicle, and operatively linked
to expression control sequences by ligation in a way that
expression of the coding sequence is achieved under conditions
compatible with the expression control sequences. Suitable
expression control sequences include promoters, enhancers,
transcription terminators, start codons, splicing signals for
introns, and stop codons, all maintained in the correct reading
frame of the polynucleotide of the invention so as to permit proper
translation of mRNA. Expression control sequences may also include
additional components such as leader sequences and fusion partner
sequences.
[0160] The promoter may in particular be a constitutive or an
inducible promoter. Constitutive promoters could be synthetic,
viral or derived from the genome of mammalian cells, e.g. the human
ubiquitin promoter. In a preferred embodiment the therapeutic cell
line will be a human immortalised neural cell line expressing the
polypeptide of the invention. For implantation, we contemplate
implanting between about 10.sup.5 to 10.sup.10 cells, more
preferably 10.sup.6 to about 10.sup.8 cells.
Methods of Treatment
[0161] The present invention, which relates to polynucleotides and
proteins, polypeptides, peptide fragments or derivatives produced
therefrom, as well as to antibodies directed against such proteins,
peptides or derivatives, may be used for treating or alleviating a
disorder or disease of a living animal body, including a human,
which disorder or disease is responsive to the activity of
neurotrophic agents.
[0162] The polypeptides of the present invention may be used
directly via, e.g., injected, implanted or ingested pharmaceutical
compositions to treat a pathological process responsive to the
neublastin polypeptides.
[0163] The polynucleotide of the invention, including the
complementary sequences thereof, may be used for the expression of
the neurotrophic factor of the invention. This may be achieved by
cell lines expressing such proteins, peptides or derivatives of the
invention, or by virus vectors encoding such proteins, peptides or
derivatives of the invention, or by host cells expressing such
proteins, peptides or derivatives. These cells, vectors and
compositions may be administered to treatment target areas to
affect a disease process responsive to the neublastin
polypeptides.
[0164] Suitable expression vectors may be derived from
lentiviruses, retroviruses, adenoviruses, herpes or vaccinia
viruses, or from various bacterially produced plasmids may be used
for in vivo delivery of nucleotide sequences to a whole organism or
a target organ, tissue or cell population. Other methods include,
but are not limited to, liposome transfection, electroporation,
transfection with carrier peptides containing nuclear or other
localizing signals, and gene delivery via slow-release systems. In
still another aspect of the invention, "antisense" nucleotide
sequences complementary to the neublastin gene or portions thereof,
may be used to inhibit or enhance neublastin expression.
[0165] In yet another aspect the invention relates to a method of
treating or alleviating a disorder or disease of a living animal
body, including a human, which disorder or disease is responsive to
the activity of neurotrophic agents.
[0166] The disorder or disease may in particular be damages of the
nervous system caused by trauma, surgery, ischemia, infection,
metabolic diseases, nutritional deficiency, malignancy or toxic
agents, and genetic or idiopathic processes.
[0167] The damage may in particular have occurred to sensory
neurons or retinal ganglion cells, including neurons in the dorsal
root ganglion or in any of the following tissues: The geniculate,
petrosal and nodose ganglia; the vestibuloacoustic complex of the
VIIIth cranial nerve; the ventrolateral pole of the
maxillomandribular lobe of the trigeminal ganglion; and the
mesencephalic trigeminal nucleus.
[0168] In a preferred embodiment of the method of the invention,
the disease or disorder is a neurodegenerative disease involving
lesioned and traumatic neurons, such as traumatic lesions of
peripheral nerves, the medulla, and/or the spinal cord, cerebral
ischaemic neuronal damage, neuropathy and especially peripheral
neuropathy, peripheral nerve trauma or injury, ischemic stroke,
acute brain injury, acute spinal cord injury, nervous system
tumors, multiple sclerosis, exposure to neurotoxins, metabolic
diseases such as diabetes or renal dysfunctions and damage caused
by infectious agents, neurodegenerative disorders including
Alzheimer's disease, Huntington's disease, Parkinson's disease,
Parkinson-Plus syndromes, progressive Supranuclear Palsy
(Steele-Richardson-Olszewski Syndrome), Olivopontocerebellar
Atrophy (OPCA), Shy-Drager Syndrome (multiple systems atrophy),
Guamanian parkinsonism dementia complex, amyotrophic lateral
sclerosis, or any other congenital or neurodegenerative disease,
and memory impairment connected to dementia.
[0169] In a preferred embodiment, we contemplate treatment of
sensory and/or autonomic system neurons. In another preferred
embodiment, we contemplate treatment of motor neuron diseases such
as amyotrophic lateral sclerosis ("ALS") and spinal muscular
atrophy. In yet another preferred embodiment, we contemplate use of
the neublastin molecules of this invention to enhance nerve
recovery following traumatic injury. In one embodiment we
contemplate use of a nerve guidance channel with a matrix
containing neublastin polypeptides. Such nerve guidance channels
are disclosed, e.g., U.S. Pat. No. 5,834,029, incorporated herein
by reference.
[0170] In a preferred embodiment, the polypeptides and nucleic
acids of this invention (and pharmaceutical compositions containing
same) are used in the treatment of peripheral neuropathies. Among
the peripheral neuropathies contemplated for treatment with the
molecules of this invention are trauma-induced neuropathies, e.g.,
those caused by physical injury or disease state, physical damage
to the brain, physical damage to the spinal cord, stroke associated
with brain damage, and neurological disorders related to
neurodegeneration.
[0171] We also contemplate treatment of chemotherapy-induced
neuropathies (such as those caused by delivery of chemotherapeutic
agents, e.g., taxol or cisplatin); toxin-induced neuropathies,
drug-induced neuropathies, vitamin-deficiency-induced neuropathies;
idiopathic neuropathies; and diabetic neuropathies. See, e.g., U.S.
Pat. Nos. 5,496,804 and 5,916,555, each herein incorporated by
reference.
[0172] We also contemplate treatment of mon-neuropathies,
mono-multiplex neuropathies, and poly-neuropathies, including
axonal and demyelinating neuropathies, using the neublastin
nucleotides and polypeptides of this invention.
[0173] In another preferred embodiment, the polypeptides and
nucleic acids of this invention (and pharmaceutical compositions
containing same) are used in the treatment of various disorders in
the eye, including photoreceptor loss in the retina in patients
afflicted with macular degeneration, retinitis pigmentosa,
glaucoma, and similar diseases.
[0174] Another object of the present invention is to provide a
method for the prevention of the degenerative changes connected
with the above diseases and disorders, by implanting into mammalian
brain including human vectors or cells capable of producing a
biologically active form of neublastin or a precursor of
neublastin, i.e. a molecule that can readily be converted to a
biologically active form of neublastin by the body, or additionally
cells that secrete neublastin may be encapsulated, e.g. into
semipermeable membranes.
[0175] Cells can be grown in vitro for use in transplantation or
engraftment into mammalian brain including human.
[0176] In a preferred embodiment, the gene encoding the polypeptide
of the invention is transfected into a suitable cell line, e.g.
into an immortalised rat neural stem cell line like HiB5 and RN33b,
or into a human immortalised neural progenitor cell line, and the
resulting cell line is implanted in the brain of a living body,
including a human, to secrete the therapeutic polypeptide of the
invention in the CNS, e.g. using the expression vectors described
in International Patent Application WO 98/32869.
Methods of Diagnosis and Screening
[0177] A neublastin nucleic acid can be used to determine whether
an individual is predisposed to developing a neurological disorder
resulting from a defect in the neublastin gene, e.g., an defect in
a neublastin allele, which has been acquired by, e.g., genetic
inheritence, by abnormal embryonic development, or by acquired DNA
damage. The analysis can be by, e.g., detecting a deletion(s) or a
point-mutation(s) within the neublastin gene, or by detecting the
inheritance of such predisposition of such genetic defects with
specific restriction fragment length polymorphisms (RFLPs), by
detecting the presence or absence of a normal neublastin gene by
hybridizing a nucleic acid sample from the patient with a nucleic
acid probe(s) specific for the neublastin gene, and determining the
ability of the probe to hybridize to the nucleic acid.
[0178] In particular, a neublastin nucleic acid can be used as a
hybridization probe. Such hybridization assays may be used to
detect, prognose, diagnose, or monitor the various conditions,
disorders, or disease states associated with aberrant levels of the
mRNAs encoding the Neublastin protein. A neublastin nucleic acid
can be construed as a "marker" for neublastin neurotrophic
factor-dependant physiological processes. These processes include,
but are not limited to, "normal" physiological processes (e.g.,
neuronal function) and pathological processes (e.g.,
neurodegenerative disease). The characterization of a particular
patient sub-population(s) with aberrant (i.e., elevated or
deficient) levels of the neublastin protein and or
neublastin-encoding mRNA may lead to new disease classifications.
By "aberrant levels," as defined herein, is meant an increased or
decreased level relative to that in a control sample or individual
not having the disorder determined by quantitative or qualitative
means.
[0179] The neublastin nucleic acids and polypeptides of this
invention may also be used to screen for and identify neublastin
analogs, including small molecule mimetics of neublastin. In one
contemplated embodiment, the invention provides a method for
identifying a candidate compound that induces a
neuroblastin-mediated biological effect, the method comprising the
steps of providing a test cell which when contacted with neublastin
is induced to express a detectable product, exposing the cell to
the candidate compound, and detecting the detectable product. The
expression of the detectable product is indicative of the ability
of the candidate compound to induce the neuroblastin-mediated
biological effect.
[0180] Further, the neublastin nucleic acids and polypeptides of
this invention may be used on DNA chip or protein chips, or in
computer programs to identify related novel gene sequences and
proteins encoded by them, including allelic variants and single
nucleotide polymorphisms ("SNPs"). Such methods are described,
e.g., in U.S. Pat. Nos. 5,795,716; 5,754,524; 5,733,729; 5,800,992;
5,445,934; 5,525,464, each herein incorporated by reference.
EXAMPLES
Example 1
Methods for Isolating Neublastin Nucleic Acids
[0181] Method 1: Rapid-Screening of Human Fetal Brain cDNA for the
Neublastin Gene
[0182] A 290 bp fragment was identified in two high throughput
genomic sequences (HGTS) submitted to GenBank (Accession No.
AC005038 and AC005051) by its homology to human persephin. From the
nucleic acid sequence of the 290 bp fragment, two neublastin
specific primers were synthesized. The neublastin top strand primer
("NBNint.sence") had the sequence 5'-CCT GGC CAG CCT ACT GGG-3'
(SEQ. ID. NO.: 17). The neublastin bottom strand primer
("NBNint.antisence") had the sequence 5'-AAG GAG ACC GCT TCG TAG
CG-3' (SEQ. ID. NO.: 18). With these primers, 96-well PCR reactions
were performed.
[0183] A 96-well master plate, containing plasmid DNA from 500,000
cDNA clones, was loaded with approximately 5000 clones per well. A
96-well sub-plate was utilized with E. coli DH10B glycerol stock
containing 50 clones per well.
[0184] A neublastin nucleic acid was identified by three rounds of
amplification using polymerase chain reaction ("PCR") techniques;
amplification increases the number of copies of the nucleic acid in
the sample.
[0185] Master Plate Screening:
[0186] Using the 96-well PCR screening technique described above, a
human fetal brain cDNA master plate was screened with the
gene-specific primers to isolate the human neublastin cDNA.
[0187] Thirty nanograms (30 ng) of human fetal brain cDNA (6
ng/.mu.l; Origene Technologies) was obtained from the corresponding
well of the master plate and placed in a total volume of 25 .mu.l
which contained the following reagents: 0.2 mM of each of the two
aforementioned gene-specific primers (i.e., NBNint.sence and
NBNint.antisence), 1.times. standard PCR buffer (Buffer V, Advanced
Biotechnologies, UK), 0.2 mM dNTPs (Amersham-Pharmacia), 0.1 M
GC-Melt (Clontech Laboratories, USA); and 0.5 units of Taq DNA
polymerase (5 U/.mu.l; Advanced Biotechnologies, UK).
[0188] PCR thermocycling reactions were performed using the
following procedure and conditions. DNA was initially denatured at
94.degree. C. for 3 minutes, and then followed by 35 cycles of
denaturation at 94.degree. C. for 1 minute each, annealing at
55.degree. C. for 1 minute, a first extension at 72.degree. C. for
90 seconds; and a final extension at 72.degree. C. for 5 minutes.
The products of 96 individual PCR reactions were analysed by gel
electrophoresis using a 2% agarose gel containing ethidium bromide
stain. The 102 bp, positive PCR product seen from a well was found
to correspond to a unique 96-well sub-plate.
[0189] The 102 bp nucleic acid fragment had the following sequence
[SEQ ID NO. 13]:
TABLE-US-00003 5'-CCTGGCCAGCCTACTGGGCGCCGGGGCCCTGCGACCGCCCCCGGGC
TCCCGGCCCGTCAGCCAGCCCTGCTGCCGACCCACGCGCTACGAAGCG GTCTCCTT-3'
[0190] Sub-Plate Screening:
[0191] The 96-well human fetal brain sub-plate was screened by
PCR-mediated amplification by placing 1 .mu.l of the glycerol stock
from the corresponding sub-plate well in a total volume of 25 .mu.l
which contained: 0.2 mM of each of the two gene-specific primers;
1.times. standard PCR buffer (Buffer V; Advanced Biotechnologies,
UK); 0.2 mM dNTPs (Amersham-Pharmacia); 0.1 M GC-Melt (Clontech
Laboratories, USA); and 0.5 units of Taq DNA polymerase (5 U/.mu.l;
Advanced Biotechnologies, UK).
[0192] The same PCR thermocycling conditions as described for the
masterplate screening were utilized. The 96 individual PCR
reactions were analysed on a 2% agarose gel containing ethidium
bromide and a positive well was identified which gave the 102 bp
PCR fragment.
[0193] Colony PCR:
[0194] One ml of the glycerol stock from the positive sub-plate
well was diluted 1:100 in Luria broth (LB). One ml and 10 ml of the
aforementioned dilution were then plated on two separate agar
plates containing Luria broth ("LB"), and 100 .mu.g/ml
carbenicillin. The LB plates were then incubated overnight at
30.degree. C. From these plates, 96 colonies were picked into a new
96-well PCR plate containing: 0.2 mM of each of the two
aforementioned gene-specific primers, 1.times. standard PCR buffer
(Buffer V; Advanced Biotechnologies, UK), 0.2 mM dNTPs
(Amersham-Pharmacia), 0.1 M GC-Melt (Clontech Laboratories, USA),
and 0.5 units of Taq DNA polymerase (5 U/.mu.l; Advanced
Biotechnologies, UK) in a final volume of 25 .mu.l.
[0195] The same PCR thermocycling conditions as described for the
masterplate screening were utilized. The 96 individual PCR
reactions were then analysed on a 2% agarose gel containing
ethidium bromide. A positive colony containing the 102 bp fragment
was subsequently identified.
[0196] Sequencing of the plasmid DNA prepared from this positive
colony revealed a full-length cDNA of 861 bp [SEQ ID NO: 8]. The
cDNA coded for a pre-pro-neublastin [SEQ ID NO: 9]. Automated DNA
sequencing was performed using the BigDye.RTM. terminator cycle
sequencing kit (PE Applied Biosystems, USA). The sequencing gels
were run on the ABI Prism 377 (PE Applied Biosystems, USA).
Method 2: Cloning Neublastin cDNA from Human Brain:
[0197] An additional method of amplifying the full-length
neublastin cDNA or cDNA fragment can be performed by RACE (Rapid
Amplification of cDNA ends) and the neublastin-specific primers
NBNint.sence and NBNint.antisence described above, combined with
vector-specific or adapter-specific primers, for example by using
the Marathon cDNA amplification kit (Clontech Laboratories, USA,
Cat. No. K1802-1).
[0198] Whole human brain Marathon-Ready cDNA (Clontech
Laboratories, USA, Catalogue. No. 7400-1) can be used to amplify
the full-length neublastin cDNA. Useful primers for amplification
include a neublastin top strand primer 5'-ATGGAACTTGGACTTGG-3' (SEQ
ID NO.: 19) ("NBNext.sence"), and a neublastin bottom strand primer
5'-TCCATCACCCACCGGC-3' (SEQ ID NO.: 20) ("NBNext.antisence"),
combined with the adaptor primer AP1 included with the
Marathon-Ready cDNA. An alternative top strand primer has also been
used, 5'-CTAGGAGCCCATGCCC-3' (SEQ ID NO.: 28). A further
alternative bottom strand primer, 5'-GAGCGAGCCCTCAGCC-3' (SEQ ID
NO.: 33) may also be used. Likewise, alternative bottom strand
primers SEQ ID NOS.: 24 and 26 may also be used.
Method 3: Cloning Neublastin cDNA from Human Brain:
[0199] Another method of cloning neublastin cDNA is by screening
human adult or fetal brain libraries with one or more neublastin
probes described herein (and as exemplified in FIG. 1). These
libraries include: .lamda.gt11 human brain (Clontech Laboratories,
USA, Cat. No. HL3002b); or .lamda.gt11 human fetal brain (Clontech
Laboratories, USA, Cat. No. HL3002b).
Method 4: Rapid-Screening of Mouse Fetal cDNA for the Neublastin
Gene
[0200] A rapid screening procedure for the neublastin gene was
performed in the following manner. A 96-well master plate,
containing plasmid DNA from 500,000 cDNA clones, was loaded with
approximately 5000 clones per well. A 96-well sub-plate was
utilized with E. Coli glycerol stock containing 50 clones per well.
Three rounds of PCR-mediated amplification was performed in order
to identify a gene of interest (i.e., neublastin).
[0201] Master Plate Screening:
[0202] A mouse fetal cDNA master plate was screened by 96-well PCR
using gene-specific primers to isolate the mouse neublastin cDNA.
The following two primers were synthesised:
[0203] (1) neublastin C2 primer (NBNint.sence):
5'-GGCCACCGCTCCGACGAG-3' (SEQ ID NO: 21); and (2) neublastin C2 as
primer (NBNint.antisence): 5'-GGCGGTCCACGGTTCTCCAG-3' (SEQ ID NO:
22). By using these two gene-specific primers a 220 bp positive PCR
product was identified. The 220 bp nucleic acid possessed the
following sequence [SEQ ID NO. 14]:
TABLE-US-00004 5'-GGCCACCGCTCCGACGAGCTGATACGTTTCCGCTIVTGCAGCGGCTC
GTGCCGCCGAGCACGCTCCCAGCACGATCTCAGTCTGGCCAGCCTACT
GGGCGCTGGGGCCCTACGGTCGCCTCCCGGGTCCCGGCCGATCAGCCA
GCCCTGCTGCCGGCCCACTCGCTATGAGGCCGTCTCCTTCATGGACGT
GAACAGCACCTGGAGAACCGTGGACCGCC-3'
[0204] 96-well PCR reactions were then performed in the following
manner. Thirty nanograms of mouse fetal brain cDNA (6 ng/.mu.l;
Origene Technologies) was obtained from the corresponding well of
the master plate and placed in a total volume of 25 .mu.l which
also contained: 0.2 mM of each of the two aforementioned
gene-specific primers (i.e., C2 primer (NBNint.sence) and
neublastin C2 as primer (NBNint.antisence)), 1.times. standard PCR
buffer (Buffer V; Advanced Biotechnologies, UK), 0.2 mM dNTPs
(Amersham-Pharmacia), 0.1 M GC-Melt (Clontech Laboratories, USA),
and 0.5 units of Taq DNA polymerase (5 U/.mu.l; Advanced
Biotechnologies, UK).
[0205] The following PCR thermocycling conditions were utilized: an
initial denaturation at 94.degree. C. for 3 minutes, followed by 35
cycles of denaturation at 94.degree. C. for 1 minute each,
annealing at 55.degree. C. for 1 minute, extension at 72.degree. C.
for 90 seconds; and a final extension at 72.degree. C. for 5
minutes. The 96 individual PCR reactions were analysed on a 2%
agarose gel containing ethidium bromide stain. The 220 bp, positive
PCR product seen from a well was found to correspond to a unique
96-well sub-plate. The 96 individual PCR reactions were then
analysed by gel electrophoresis on a 2% agarose gel containing
ethidium bromide stain. The 220 bp positive PCR product which had
been identified corresponded to a unique well of the 96-well
sub-plate.
[0206] Sub-Plate Screening:
[0207] The 96-well mouse fetal sub-plate was screened by
PCR-mediated amplification by placing 1 .mu.l of the glycerol stock
from the corresponding sub-plate well into a final, total volume of
25 .mu.l which contained: 0.2 mM of each of the two aforementioned
gene-specific primers; 1.times. standard PCR buffer (Buffer V;
Advanced Biotechnologies, UK); 0.2 mM dNTPs (Amersham-Pharmacia);
0.1 M GC-Melt (Clontech Laboratories, USA); and 0.5 units of Taq
DNA polymerase (5 U/.mu.l; Advanced Biotechnologies, UK). The PCR
thermocycling was performed according to the conditions described
above for the master plate screening.
[0208] The individual 96 PCR reactions were then analysed on a 2%
agarose gel containing ethidium bromide and a positive well was
identified which produced the 220 bp fragment.
[0209] Colony PCR:
[0210] One ml of the glycerol stock from the positive sub-plate
well was diluted 1:100 in Luria broth (LB). One ml and 10 ml of the
aforementioned dilution were then plated on two separate LB plates,
containing 100 .mu.g/ml carbenicillin, and incubated at 30.degree.
C. overnight. A total of 96 colonies were isolated and transferred
to a 96-well PCR plate containing: 0.2 mM of each of the two
aforementioned gene-specific primers, 1.times. standard PCR buffer
(Buffer V; Advanced Biotechnologies, UK), 0.2 mM dNTPs
(Amersham-Pharmacia); 0.1 M GC-Melt (Clontech Laboratories, USA),
and 0.5 units of Taq DNA polymerase (5 U/.mu.l; Advanced
Biotechnologies UK) in a final volume of 25 .mu.l.
[0211] PCR thermocycling was performed according to the conditions
described above (see, "master plate screening", infra). The 96
individual PCR reactions were analysed by gel electrophoresis on a
2% agarose gel containing ethidium bromide. A positive colony was
identified by the presence of the 220 bp fragment. Plasmid DNA was
prepared from this positive colony. The clone was sequenced by
automated DNA sequencing using the BigDye.RTM. terminator cycle
sequencing kit with AmpliTaq DNA polymerase. The sequencing gels
were run on the ABI Prism 377 (PE Applied Biosystems). The
resulting sequence of this clone revealed a full-length cDNA of
2136 bp (SEQ ID NO: 15). The cDNA includes an open reading frame
with the predicted amino acid sequence shown in SEQ ID NO: 16,
which codes for a mouse pre-pro-neublastin polypeptide.
Example 2
Cloning of Genomic Neublastin As discussed above, applicants
identified a 290 bp nucleic acid fragment in two human BAC clones
with entries in GenBank (with the Accession Nos. AC005038 and
AC005051) which had regions of homology to persephin and to the
flanking sequences of persephin. Applicants used the 861 bp
predicted sequence described above to design additional primers,
with the goal of cloning a nucleic acid encoding additional
neublastin nucleic acids using Lasergene Software (DNAStar, Inc.).
Two pairs of primers were used to clone the neublastin gene by
using PCR reactions on genomic DNA. The two pairs of primers are
illustrated below.
TABLE-US-00005 [0212] Primer Pair No. 1 (SEQ ID NO: 23) 5' CCA AgC
CCA CCT ggg TgC CCT CTT TCT CC 3' (sense). (SEQ ID NO: 24) 5' CAT
CAC CCA CCg gCA ggg gCC TCT CAg 3' (antisense). Primer Pair No. 2
(SEQ ID NO: 25) 5' gAgCCCAtgCCCggCCTgATCTCAgCCCgA ggACA 3' (sense).
(SEQ ID NO: 26) 5' CCCTggCTgAggCCgCTggCTAgTgggACTCTgC 3'
(antisense).
[0213] Using primer pair No. 1, a 887 bp DNA fragment was amplified
from a preparation of human genomic DNA purchased from Clontech
Laboratories, (Cat. No. 6550-1).
[0214] PCR Protocol:
[0215] PCR was performed using the Expand High Fidelity PCR system
(Boehringer Mannheim) with buffer 1. The PCR reaction mixture was
supplemented with 5% dimethylsulfoxide (DMSO) and 17.5 pmol of each
dNTP in a total volume of 50 .mu.l. Thermocycling was performed
with a pre-denaturation step at 94.degree. C. for 2 minutes,
followed by 35 two-step cycles at 94.degree. C. for 10 seconds, and
68.degree. C. for 1 minute, respectively. Thermocycling was
terminated by incubation at 68.degree. C. for 5 minutes.
Thermocycling was carried out in a PTC-225 DNA Engine Tetrad
thermocycler (MJ Research, MA). The PCR products were analysed by
gel electrophoresis on 2% agarose (FMC) and then photographed.
[0216] The 887 bp fragment amplified from human genomic DNA with
primer pair No. 1 was cloned into the pCRII vector (Invitrogen),
and transformed into XL 1-Blue competent E. coli cells
(Stratagene). The resulting plasmid, designated neublastin-2, was
sequenced using Thermosequenase (Amersham Pharmacia Biotech).
Sequencing products were analysed by electrophoreses on an
ALFExpress automated sequencer (Amersham Pharmacia Biotech).
[0217] Fragments obtained by PCR amplification of human genomic DNA
with the second pair of primers (Primer Pair No. 1, above), were
sequenced, revealing an additional 42 bp region at the 3' prime end
of the open reading frame. The full-length sequence was analysed by
comparing it to the sequences of nucleic acids of other
neurotrophic factors, as well as by mapping exon-intron boundaries
using gene-finding software programs which identify probable splice
junctions and regions of high coding potential using Netgene and
Gene Mark software (Brunak et al., J. Mol. Biol., 220, pp. 49-65
(1991); Borodovsky et al., Nucl. Acids Res., 23, pp. 3554-62
(1995)). The exon-intron boundaries were confirmed by the cDNA
obtained from the Rapid Screen described above.
[0218] As illustrated in FIG. 7, the resulting neublastin gene has
two exons separated by a 70 bp intron. Together, the exons have a
predicted amino acid sequence of a full-length Neublastin
polypeptide. The predicted cDNA (SEQ ID NO: 3) contains an open
reading frame (ORF) encoding 238 amino acid residues (SEQ ID NO:
4). The Neublastin-2 clone contained the complete coding sequence
of pro-neublastin. The amino acid sequence encoded by the gene
showed high homology to three proteins, persephin, neurturin, and
GDNF.
Example 3
Expression of Neublastin Nucleic Acids
[0219] Expression of neublastin RNA was detected in both nervous
and non-nervous tissue in rodents and in humans, and at various
developmental immature and adult stages, using the techniques
described below.
Method of Detecting Neublastin RNA Expression Using RT-PCR:
[0220] Based on the neublastin DNA sequence identified as SEQ ID
NO: 1, the following primers were synthesised: (1) a neublastin C2
primer 5'-GGCCACCGCTCCGACGAG-3' (SEQ ID NO. 21), and (2) a
neublastin. C2 as primer 5'-GGCGGTCCACGGTTCTCCAG-3' (SEQ ID NO.
22). This primer set was used to RT-PCR amplify a DNA fragment from
adult and fetal human whole-brain mRNA. Among the DNA fragments
produced by this reaction was one of 220 bp. Identification of this
220 bp DNA fragment confirmed that the neublastin gene is expressed
in adult and fetal brain tissue. A 220 bp DNA fragment was also
amplified from genomic DNA with using these primers.
Method of Detecting Neublastin RNA Expression by Northern Blot
Hybridization:
[0221] Northern blots with polyA.sup.+ RNA from adult human tissue
were purchased from a commercial supplier (Clontech Laboratories,
USA) and probed with a .sup.32P-labeled neublastin cDNA. The
labelled neublastin cDNA was prepared according to the methods
described in Example 1, above.
[0222] Preparation of Probes:
[0223] A neublastin nucleic acid DNA fragment (nucleotides 296-819
of SEQ ID NO: 8) was labelled by the Rediprime II labelling kit
(Amersham; Cat. No. RPN1633) for use as a hybridization probe, as
recommended by the manufacturer. Briefly, the DNA sample was
diluted to a concentration of 2.5-25 ng in 45 .mu.l of 10 mM TE
Buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). The DNA was then
denatured by heating the sample to 95-100.degree. C. for 5 minutes
in a boiling water bath, quick cooling the sample by placing it on
ice for 5 minutes, and then briefly centrifuging it to bring the
contents to the bottom of the reaction tube. The total amount of
denatured DNA was added together with 5 .mu.l of Redivue [.sup.32P]
dCTP (Amersham Pharmacia Biotech Ltd.) in the reaction tube
containing buffered solution of dATP, dGTP, dTTP, exonuclease free
Klenow enzyme and random primer in dried stabilised form. The
solution was mixed by pipetting up and down 2 times, moving the
pipette tip around in the solution, and the reaction mixture was
incubated at 37.degree. C. for 10 minutes. The labelling reaction
was stopped by adding 5 .mu.l of 0.2 M EDTA. For use as a
hybridization probe the labelled DNA was denatured to single
strands by heating the DNA sample to 95-100.degree. C. for 5
minutes, then snap cooling the DNA sample on ice for 5 minutes. The
tube was centrifuged and its contents mixed well. Finally the
single-stranded DNA probe was purified using the Nucleotide Removal
Kit (Qiagen).
[0224] Hybridization Techniques:
[0225] Prepared northern blots were purchased from a commercial
supplier ("Multiple Tissue Northern Blots, Clontech Laboratories,
USA, Catalogue Nos. 7760-1 and 7769-1) and were hybridized
according to the manufacturer's instructions using the neublastin
.sup.32P-labeled probe prepared above. For hybridization,
ExpressHyb Solution (Clontech Laboratories, USA) was used, and a
concentration of approximately 3 ng/ml of the labelled probe was
employed. The ExpressHyb solution was heated to 68.degree. C. and
then stirred to dissolve any precipitate. Each northern blot
membrane (10.times.10 cm) was pre-hybridized in at least 5 ml of
ExpressHyb Solution at 68.degree. C. for 30 minutes in a Hybaid
Hybridization Oven according to the manufacturer's instructions.
The neublastin .sup.32P-labeled probe was denatured at
95-100.degree. C. for 2 minutes and then chilled quickly on ice.
Fourteen microliters (14 .mu.l) of the labelled probe was added to
5 ml of fresh ExpressHyb, and thoroughly mixed. The ExpressHyb
Solution used in the pre-hybridization was replaced by evenly
distributing over the blots the 5 ml of fresh ExpressHyb Solution
containing labelled DNA probe. Blots were incubated at 68.degree.
C. for 1 hour in a Hybaid hybridization Oven. After incubation, the
blots were rinsed and washed several times at low stringency
(2.times.SSC buffer containing 0.05% SDS at room temperature)
followed by a high stringency wash (0.1.times.SSC containing 0.1%
SDS at 50.degree. C.) [20.times.SSC is 0.3 M NaCl/0.3 M Na citrate,
pH 7.0]. The blots were exposed to a Hyperfilm MP (Amersham
Pharmacia Biotech Ltd.) at -80.degree. C. using intensifying
screens.
[0226] The results of the northern blot hybridization experiments
are presented in FIG. 1. FIG. 1A (left) and FIG. 1B (right) are
northern blots of polyA.sup.+ RNA which were probed with
.sup.32P-labelled neublastin cDNA as described in Example 3. The
markers represent polynucleotides of 1.35 kilobase pairs ("kb"),
2.4 kb, 4.4 kb, 7.5 kb, and 9.5 kb in size. The membrane of FIG. 1A
was prepared with mRNA extracted from various adult human tissues:
From the results of the northern blot hybridization analysis,
applicants conclude that neublastin mRNA is expressed in many adult
human tissues. The highest level of neublastin expression is
detected in the heart, in skeletal muscle and in the pancreas. The
membrane of FIG. 1B was prepared with RNA extracted from various
regions of the adult human brain. Within the adult brain, the
highest level of expression is seen in the caudate nucleus and in
the thalamus. An mRNA transcript of approximately 5 kb was the
predominant form of neublastin mRNA expressed in the brain.
Method of detecting Neublastin RNA Expression Using by In Situ
Hybridization in Tissues:
[0227] The following techniques are used to measure the expression
of neublastin RNA in animal tissues, e.g., rodent tissues, with a
neublastin anti-sense probe.
Expression in Mice:
[0228] Preparation of Tissue Samples:
[0229] Time pregnant mice (B&K Universal, Stockholm, Sweden)
were killed by cervical dislocation on gestational day 13.5 or
18.5. Embryos were removed by dissection under sterile conditions,
and immediately immersed in a solution of 0.1M phosphate buffer
(PB) containing 4% paraformaldehyde ("PFA") for 24-30 hours, and
then removed from the PFA and stored in PBS. The tissue was
prepared for sectioning by immersing the tissue in a solution of
30% sucrose, and then embedding it in TissueTech (O.C.T. Compound,
Sakura Finetek USA, Torrance, Calif.). Six series of coronal or
sagittal sections (12 .mu.m each) were cut on a cryostat and thaw
mounted onto positively charged glass slides. Neonatal heads/brains
(P1, P7) were fixed following the same protocol as for the
embryonic stages, and adult brain tissue was dissected, immediately
frozen on dry ice, and cut on a cryostat without any prior
embedding.
[0230] Preparation of Neublastin Riboprobes:
[0231] An antisense neublastin RNA probe (hereafter a "neublastin
riboprobe") was made as follows. Nucleotides 1109-1863 of the mouse
neublastin cDNA sequence (SEQ ID NO: 15) were sub-cloned into the
BlueScript vector (Stratagene). The resulting plasmid was cut into
a linear DNA using EcoRI restriction endonuclease. The EcoRI DNA
fragment was in vitro transcribed with T3 RNA polymerase and the
digoxigenin ("DIG") RNA Labelling Kit according to the
manufacturer's instructions (Boehringer Mannheim).
[0232] Hybridization:
[0233] Cryostat sections were fixed for 10 minutes in 4% PFA,
treated for 5 minutes with 10 mg/ml of proteinase K, dehydrated
sequentially in 70% and 95% ethanol for 5 and 2 min, respectively,
and then allowed to air dry. Hybridization buffer (50% deionized
formamide, 10% of a 50% dextran sulphate solution, 1% Denhardt's
solution, 250 .mu.g/ml yeast tRNA, 0.3M NaCl, 20 mM Tris-HCl (pH8),
5 mM EDTA, 10 mM NaPO.sub.4, 1% sarcosyl) containing 1 .mu.g/ml of
the DIG-labelled probe was heated to 80.degree. C. for 2 minutes
and applied onto the sections. The sections was then covered with
parafilm and incubated at 55.degree. C. for 16-18 hours.
[0234] The next day the sections were washed at high stringency
(2.times.SSC containing 50% formamide) at 55.degree. C. for 30
minutes, and then washed in RNase buffer and incubated with 20
.mu.g/ml of RNaseA for 30 minutes at 37.degree. C. In order to
detect the DIG-labelled probe, sections were pre-incubated in
blocking solution (PBS containing 0.1% Tween-20 and 10%
heat-inactivated goat serum) for 1 hour and then incubated over
night at 4.degree. C. with a 1:5000 dilution of
alkaline-phosphatase-coupled anti-DIG antibody (Boehringer
Mannheim). The following day, each section was given four, two-hour
washes in PBS containing 0.1% Tween-20, and then given two
ten-minute washes in NTMT buffer (100 mM NaCl, 100 mM Tris-HCl
(pH9.5), 50 mM MgCl.sub.2, 0.1% Tween-20). The sections were then
incubated in BM-purple substrate containing 0.5 mg/ml of levamisole
for 48 hours. The color reaction was stopped by washing in PBS. The
sections were air dried and covered with cover-slip with DPX
(KEBO-lab, Sweden).
[0235] The results of the in situ hybridization reactions are
presented in Table 1.
TABLE-US-00006 TABLE 1 Expression of neublastin in Mice Structure
E13.5 E18.5 P1 P7 Adult Forebrain ++ Ventral Midbrain - Dorsal Root
ganglia ++ Spinal chord + Retina +++ +++ + Olfactory bulb ++ ++ ++
Tooth pulp ++ ++ + Trigeminal ganglia ++ ++ ++ Striatum + + ++
Cortex ++ ++ ++ + Dentate gyrus ++ +
[0236] As shown in Table 1, at embryonic day 13.5 ("E13.5"),
neublastin was expressed in the spinal chord and in the hindbrain,
and weakly in the forebrain. Neublastin expression was also
detected in the developing retina and in the sensory ganglia
(dorsal root ganglia and trigeminal ganglia (V)). Outside the
nervous system, a weak signal was found in the kidney, the lung and
the intestine, indicating that neublastin is also expressed in
those tissues.
[0237] At embryonic day 18.5 ("E18.5"), neublastin was expressed
most prominently in the trigeminal ganglion (V). Neublastin
expression was also detected in the retina, the striatum, and the
cortex. In addition, expression was seen in tooth anlage.
[0238] Again referring to Table 1, increased neublastin expression,
from the E18.5 time-point to postnatal days 1 and 7, was seen in
the cortex, the striatum and the trigeminal ganglion (V).
Neublastin expression was more prominent in the outer layers of the
cortex than in the inner layers of the cortex. On P7, expression
was found in the same structures as at day 1 but in addition
neublastin expression was found in the hippocampus, especially in
the dentate gyrus and in the cerebellum. In the adult murine brain,
neublastin was strongly expressed in dentate gyrus, with very low
or undetectable levels of neublastin expression detected other
tissues tested.
Expression in Rat:
[0239] The following experiment describes the hybridization of rat
tissues with a alkaline-phosphatase-labelled oligodeoxynucleotide
neublastin anti-sense probe.
[0240] Preparation of Tissue Samples:
[0241] Rat embryos (E14) were obtained from pregnant Wistar rats
(Mollegard, Denmark) following pentobarbital anaesthesia. Postnatal
rats (P0, P7, adult) were killed by decapitation. Dissected brains
and whole heads were immediately immersed in cold 0.9% NaCl, fresh
frozen and sectioned at 20 .mu.m on a cryostat (coronal and
sagittal sections, 10 series).
[0242] In Situ Hybridization:
[0243] Two series of sections were hybridized using an anti-sense
alkaline-phosphatase (AP) conjugated oligodeoxynucleotide probe
(5'-NCA GGT GGT CCG TGG GGG GCG CCA AGA CCG G-3' (SEQ ID NO:27),
Oligo. No. 164675, DNA Technology, Denmark,). This probe is
complementary to bases 1140 to 1169 of the mouse neublastin cDNA of
SEQ ID NO:15).
[0244] Prior to hybridization, the sections were air dried at room
temperature, heated at 55.degree. C. for 10 min., and then treated
with 96% ethanol at 4.degree. C. overnight. The sections were then
air dried and incubated in hybridization medium (5.0 pmol probe/ml)
overnight at 39.degree. C. (Finsen et al., 1992, Neurosci.
47:105-113; West et al., 1996, J. Comp. Neurol., 370:11-22).
[0245] Post-hybridization treatment consisted of four,
thirty-minute rinses in 1.times.SSC (0.15M NaCl, 0.015 M
Na-citrate) at 55.degree. C., followed by three ten-minute rinses
in Tris-HCl, pH 9.5 at room temperature prior to applying AP
developer. AP developer was prepared immediately before use and
contained nitroblue tetrazoleum (NBT, Sigma), 5-bromo, 4-chloro,
3-indolylphosphate (BCIP, Sigma), and Tris-HCl--MgCl.sub.2 buffer,
pH 9.5 (Finsen et al., Neurosci. 1992 47 105-113). AP development
took place in the dark at room temperature for 48 hours. The color
reaction was stopped by rinsing the sections in destilled water.
The sections were dehydrated in graded acetone, softened in
xylene-phenol creosote (Allchem, UK), cleared in xylene, and
coverslipped using Eukitt (Bie & Berntsen, Denmark).
[0246] Control reactions consisted of (1) pre-treating the sections
with RNase A (50 .mu.g/ml, Pharmacia, Sweden) prior to
hybridization; (2) hybridizing the sections with a hundred-fold
excess of unlabelled probe; and (3) hybridizing the sections with
hybridization buffer alone. The results of the hybridization
reactions are presented in Table 2.
TABLE-US-00007 TABLE 2 Expression of neublastin in rats Structure
E14 P0/P1 P7 Adult Forebrain ++ Ventral Midbrain - Dorsal root
ganglia ++ Spinal cord + Retina + Olfactory bulb (+) ++ ++
Cerebellum + ++ + Trigeminal ganglia ++ ++ Striatum + +(+) Cortex
(+) ++ ++ + Hippocampus (+) ++ ++
At embryonic day 14 (E14), neublastin was weakly expressed in rat
embryos in the forebrain, in the hindbrain, and in the spinal cord.
Neublastin mRNA was also detected in the eye (retina), dorsal root
ganglia, the trigeminal ganglia (V), and in the kidneys, lungs,
heart, liver, and intestines. In newborn (P0) rats there was marked
neublastin expression in the cortex and in the striatum. Neublastin
expression was also detected in the olfactory bulb and in the
hippocampus. In 7-day-old (P7) rats, neublastin was expressed in
the cortex, the striatum, the olfactory bulb, and in the
cerebellum. A marked signal was seen in the hippocampus. In adult
rats, very low or undetectable levels of neublastin expression were
detected in most areas of the brain. Weak signals were detected in
the thalamic nucleus, and marked neublastin expression was detected
in the hippocampus.
Example 4
Neublastin Polypeptides
[0247] The open reading frame, or coding region (CDS), identified
in SEQ ID NO: 8 encodes the pre-pro-polypeptide (designated
"pre-pro-neublastin"). The amino acid sequence predicted from this
open reading frame is shown in SEQ ID NO: 9. Based on SEQ ID NO: 9,
three variants of neublastin polypeptides were identified. These
variants include: (i) the polypeptide designated herein as NBN140,
which possesses the amino acid sequence designated as SEQ ID NO:
10; (ii) the polypeptide designated herein as NBN116, which
possesses the amino acid sequence designated as SEQ ID NO: 11; and
(iii) the polypeptide designated herein as NBN113, which possesses
the amino acid sequence designated as SEQ ID NO: 12.
[0248] Similarly, based on the coding region (CDS) as identified in
SEQ ID NO: 3, which encodes the pre-pro-polypeptide possessing the
amino acid sequence (designated as SEQ ID NO: 4), three variants of
neublastin were identified. These variants include: (i) the
polypeptide which possesses the amino acid sequence designated as
SEQ ID NO: 5; (ii) the polypeptide which possesses the amino acid
sequence designated as SEQ ID NO: 6; and (iii) the polypeptide
which possesses the amino acid sequence designated as SEQ ID NO:
7.
[0249] Based on a Clustal W (1.75)-based multiple sequence
alignment, SEQ ID NO: 9 was aligned with the amino acid sequences
of GDNF, persephin and neurturin. This alignment is illustrated in
Table 3.
TABLE-US-00008 TABLE 3 Amino Acid Sequence Comparison of Neublastin
to Persephin, Neurturin, and GDNF Neurturin-full
--------------------MQRWKAAALASVLCSSVLSIWMCREGLLLSHRLGPA Neublastin
MELGLGGLSTLSHCPWPRRQPALWPTLAALALLSSVAEASLGSAPRSPAPREGPPP
Persephin-full
--------------------------------------------------------
GDNF_HUMAN-full
-----MKLWDVVAVCLVLLHTASAFPLPAGKRPPEAPAEDRSLGRREAPFALSSDS
Neurturin-full
LVPLHRLPRTLDARIARLAQYRALLQGAPDAMELRELTPWAGRPPGPRRRAGPRRR Neublastin
VLASPAGHLPGGRTARWCSGRARRPPPQPSRPAPPPPAPPSALPRGGRAARAGGPG
Persephin-full
-MAVGKFLLGSLLLLSLQLGQGWGPDARGVPVADGEFSSEQVAKAGGTWLGTHRPL
GDNF_HUMAN-full
NMPEDYPDQFDDVMDFIQATIKRLKRSPDKQMAVLPRRERNRQAAAANPENSRGKG
Neurturin-full
RARARLGARPCGLRELEVRVSELGLGYASDETVLFRYCAGACEA-AARVYDLGLRR Neublastin
SRARAAGARGCRLRSQLVPVRALGLGHRSDELVRFRFCSGSCRR-ARSPHDLSLAS
Persephin-full
ARLRRALSGPCQLWSLTLSVAELGLGYASEEKVIFRYCAGSCPRGARTQHGLALAR
GDNF_HUMAN-full
RRGQRGKNRGCVLTAIHLNVTDLGLGYETKEELIFRYCSGSCDA-AETTYDKILKN * * : *
****: :.* : **:*:*:* * :. * Neurturin-full
LRQRRRLRRE---RVRAQPCCRPTAYEDEVSFLDAHSRYHTVHELSARECACV- Neublastin
LLGAGALRPPPGSRPVSQPCCRPTRYE-AVSFMDVNSTWRTVDRLSATACGCLG
Persephin-full
LQGQGRAHGG--------PCCRPTRYT-DVAFLDDRHRWQRLPQLSAAACGCGG
GDNF_HUMAN-full
LSRNRRLVSD----KVGQACCRPIAFDDDLSFLDDNLVYHILRKHSAKRCGCI- * .**** :
::*:* . :: : . ** *.* * indicates positions which have a single,
fully conserved residue. : indicates that one of the following
'strong' groups is fully conserved: -STA, NEQK, NHQK, NDEQ, QHRK,
MILV, MILF, HY, FYW. . indicates that one of the following 'weaker'
groups is fully conserved: -CSA, ATV, SAG, STNK, STPA, SGND,
SNDEQK, NDEQHK, NEQHRK, VLIM, HFY.
[0250] From the amino acid sequence alignment shown in Table 3, it
can be seen that neublastin has seven conserved cysteine residues
at locations that are conserved within the TGF-.beta. superfamily.
In one embodiment, the preferred neuroblastin polypeptide contains
(seven) cysteines conserved as in SEQ ID NO: 2 at positions 8, 35,
39, 72, 73, 101 and 103, or as in SEQ ID NOS: 4 and 9 at positions
43, 70, 74, 107, 108, 136 and 138. These seven conserved cysteine
residues are known within the TGF-b superfamily to form three
intramonomeric disulfide bonds (contemplated, e.g., in SEQ ID NO: 2
between cysteine residues 8-73, 35-101, and 39-103, and, e.g., in
SEQ ID NOS: 4 and 9 between cysteine residues 43-108, 70-136, and
74-138) and one intermonomeric disulfide bond (contemplated, e.g.,
in SEQ ID NO: 2 between cysteine residues 72-72, and, e.g., in SEQ
ID NOS: 4 and 9 between cysteine residues 107-107), which together
with the extended beta strand region constitutes the conserved
structural motif for the TGF-b superfamily. See, e.g., Daopin et
al., Proteins 1993 17 176-192.
[0251] Based on this sequence alignment, neublastin was shown to be
a member of the GDNF subfamily of neurotrophic factors
(LGLG-FR(Y/F)CSGSC-QxCCRP-SAxxCGC, the GDNF subfamily fingerprint,
underlined in Table 3).
[0252] The homology of neublastin to other members of the GDNF
family was calculated, and the results are presented Table 4,
below.
TABLE-US-00009 TABLE 4 Homology of Neublastin Polypeptides to other
members of the GDNF Family Mature Protein NBN140 Mature Protein
NBN113 Homology of Homology of Homology full length Homology full
length Neurotrophic Overlap Strong peptides Overlap Strong peptides
Factor Identity (aa) Homology Identity Identity (aa) Homology
Identity GDNF 34% 137 48% 31.9% 36% 111 52% 29.5% (47/137) (67/137)
(41/111) (59/111) NTN 48% 127 56% 36.9% 49% 114 57% 44.7% (61/127)
(72/127) (56/114) (66/114) PSP 44% 125 56% 36.9 45% 111 57% 44.3%
(55/125) (71/125) (51/111) (65/111) IHA 31% 81 -- 25.2% 31% 81 --
22.5% (25/81) (25/81) TGF-.beta.2 23% 73 -- 18.5% 23% 73 -- 20.2%
(17/73) (17/73) GDNF = Glial cell line Derived Neurotrophic Factor
NTN = Neurturin PSP = Persephin IHA = Inhibin-.alpha. TF-.beta.2 =
Transforming Growth Factor-.beta.2 Strong homology indicates that
one of the following "strong" groups are conserved: STA, NEQK,
NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW.
Example 5
Production of Neublastin
[0253] We have produced neublastin in both eukaryotic and
prokaryotic cells, as described below.
Expression Vectors
[0254] The full length cDNA encoding neublastin was inserted into
the eukaryotic expression vector pUbi1Z. This vector was generated
by cloning the human UbC promoter into a modified version of
pcDNA3.1/Zeo. The unmodified pcDNA3.1/Zeo is commercially available
(Invitrogen). The modified pcDNA3.1/Zeo is smaller than the parent
vector, because the ampicillin gene (from position 3933 to 5015)
and a sequence from position 2838 to 3134 were removed. In this
modified version of pcDNA3.1/Zeo, the CMV promoter was replaced
with the UbC promoter from pTEJ-8 (Johansen et al., FEES Lett. 1990
267 289-294), resulting in pUbi1Z.
Mammalian Cell Expression
[0255] The pUbi1Z vector which contained neublastin coding
sequences was then transfected into the mammalian cell line HiB5,
which is an immortalised rat neural cell line (Renfranz et al.,
Cell, 66, pp. 713-729 (1991)). Several HiB5 cell lines stably
expressing neublastin (as determined by RT-PCR) have been
established. In one of these stable cell lines, HiB5pUbi1zNBN22
expression was confirmed by hybridizing total RNA on a Northern
blot with a .sup.32P-labelled neublastin probe. The results of
these studies are shown in FIG. 2. HiB5pUbi1zNBN22 was then used as
a source of neublastin for studies of neublastin neurotrophic
activity.
[0256] FIG. 2 shows the expression of neublastin cDNA in the
HiB5pUbi1zNBN22 clone (i.e., Northern blot probed with
.sup.32P-labelled neublastin cDNA of the present invention as
described infra). The blot was prepared by total RNA extracted from
untransfected HiB5 cells, HiB5pUbi1zNBN22 cells and
HiB5pUbi1zGDNF14, respectively, as indicated. The positions of the
28S and 18S rRNA bands corresponding to 4.1 kb and 1.9 kb,
respectively, are indicated on the blot.
[0257] As shown in FIG. 3, antibodies raised against
neublastin-derived polypeptides also recognised a protein of
approximately 13 kilodaltons ("kD") in conditioned medium from the
HiB5pUbi1zNBN22 clone but not from non-transfected HiB5 cells (cf.
Example 6).
[0258] The predicted molecular weights of the non-modified (i.e.
lacking post-translational modifications) neublastin polypeptides
NBN140 (SEQ ID NO:10), NBN116 (SEQ ID NO:11) and NBN113 (SEQ ID
NO:12) were determined to be 14.7 kilodaltons ("kD"), 12.4 kD, and
12.1 kD, respectively.
[0259] Methods:
[0260] A Northern blot with total RNA (10 .mu.g) from untransfected
HiB5 cells and the HiB5pUbi1zNBN22 clone was prepared by
electrophoresis on a 0.8% formaldehyde agarose gel and blotted onto
a nylon membrane (Duralone, Stratagene). The blot was hybridized
and washed as described in Example 3 with a 1.3 kb
.sup.32P-labelled probe prepared by random labelling covering SEQ
ID. NO: 8 and additional nucleotides from the 5'UTR and 3'UTR of
the neublastin cDNA. The blot was exposed to a Hyperfilm MP
(Amersham) at -80.degree. C. using intensifying screens.
[0261] Conditioned medium from Hib5pUbi1zNBN22, or untransfected
Hib5 cells incubated overnight in serum-free medium supplemented
with N2 supplement (Life Technologies; Cat. No. 17502-048) was
concentrated and separated on 15% polyacrylamide gels (Amersham
Pharmacia Biotech; Cat. No. 80-1262-01). Proteins were transferred
to PVDF-membranes (Amersham Pharmacia Biotech; Cat. No. RPN-303F)
and non-specific protein-binding sites were blocked with 5% non-fat
dry milk in PBS with 0.1% Tween-20. Membranes were incubated
overnight with a polyclonal neublastin antibody (1:1000), followed
by incubation with a secondary anti-rabbit IgG antibody (Amersham
Pharmacia Biotech; Cat. No. NA 934) conjugated to horseradish
peroxidase (1:2000). Immunostaining was visualised using enhanced
chemoluminiscence (ECL) (Amersham Pharmacia Biotech; Cat. No.
RPN2109) or ECL+ (Amersham Pharmacia Biotech; Cat. No. RPN2132)
according to the manufacturer's instructions (Amersham).
[0262] The results of these experiments are shown in FIG. 3. FIGS.
3A and 3B are illustrations of the expression of neublastin protein
in transfected HiB5 cells. Overnight medium from non-transfected
HiB5 cells [Lane 1], or from an HiB5 clone stable transfected with
neublastin cDNA [Lane 2], were concentrated as described infra. The
medium was then analyzed by Western blotting using two different
polyclonal antibodies, Ab-1 and Ab-2 described in Example 10,
specific for neublastin. In the medium derived from transfected
cells, both of the antibodies were found to recognize a protein
with a molecular weight of approximately 15 kDa. This protein was
not seen in non-transfected HiB5 cells.
[0263] The cloned cDNA encoding neublastin can also be inserted
into other eukaryotic expression vector, e.g., the eukaryotic
expression vector TEJ-8 (Johansen et al., FEBS Lett., 1990,
267:289-294) or pcDNA-3 (Invitrogen), and the resulting expression
plasmid transfected into an alternative mammalian cell line, e.g.,
Chinese Hamster Ovary ("CHO") cells, the HEK293, the COS, the PC12,
or the RN33b cell lines, or a human neural stem cell. Stable cell
lines expressing neublastin are used, e.g., to produce the
neublastin protein.
Expression in CHO Cells
[0264] Construction of Plasmid pJC070.14
[0265] In order to express the Neublastin cDNA in Chinese hamster
ovary cells, a cDNA fragment encoding the prepro form of human
Neublastin was inserted into the mammalian expression vector
pEAG347 to generate plasmid pJC070.14. pEAG347 contains tandem SV40
early and adenovirus major late promoters (derived from plasmid
pAD2beta; Norton and Coffin, Mol. Cell. Biol. 5: 281 (1985)), a
unique NotI cloning site, followed by SV40 late transcription
termination and polyA signals (derived from plasmid pCMVbeta;
MacGregor and Caskey, Nucl. Acids. Res. 17: 2365 (1989)). In
addition, pEAG347 contains a pUC19-derived plasmid backbone and a
pSV2dhfr-derived dhfr for MTX selection and amplification in
transfected CHO cells.
[0266] Plasmid pJC070.14 was generated in two steps. First, a
fragment encoding the prepro form of human Neublastin was isolated
from plasmid pUbi1Z-NBN using the polymerase chain reaction with
oligonucleotides KD2-824 5'AAGGAAAAAA GCGGCCGCCA TGGAACTTGG
ACTTGGAGG3' (SEQ. ID. NO. 31), KD2-825 5'TTTTTTCCTT GGCGGCCGCT
CAGCCCAGGC AGCCGCAGG3' (SEQ. ID. NO. 32) and PFU polymerase. The
fragment was cloned into the Srf-1 site of pPCR-Script Amp SK(+) to
generate the plasmid pJC069. In the second step, a partial Not-1
digest was performed on plasmid pJC069 to generate a 685 bp
fragment (containing the neublastin gene) which was cloned into the
Not-1 site of plasmid pEAG347 to generate plasmid pJC070.14.
Transcription of the neublastin gene in plasmid pJC070.14 is
controlled by the adenovirus major late promoter.
[0267] Generation of CHO Cell Lines Expressing Neublastin.
[0268] 200 .mu.g of pJC070.14 was linearized by digestion with the
restriction endonuclease Mlu-1. The DNA was extracted with
phenol:chloroform:isoamyl alchohol (25:24:1) and ethanol
precipitated. The linearized DNA was resuspended in 20 mM Hepes
pH7.05, 137 mM NaCl, 5 mM KCl, 0.7 mM Na.sub.2HPO.sub.4, 6 mM
dextrose (HEBS) and introduced into .about.4E7 CHO dukx B1(dhfr-)
cells (p23) by electroporation (280V and 960 .mu.F). Following
electroporation, the cells were returned to culture in .alpha.+
Modified Eagle's Medium (MEM) supplemented with 10% fetal bovine
serum (FBS) for two days. The cells were then trypsinized and
replated in 100 mm dishes (100,000 cells/plate) in .alpha.-MEM
(lacking ribo- and deoxyribonucleosides), supplemented with 10%
dialyzed FBS, for five days. The cells were subsequently split at a
density of 100,000 cells/100 mm plate, and selected in 200 nM
methotrexate. Resistant colonies were picked and scaled up to 6
well plates; conditioned media from each clone was screened using a
specific assay for neublastin described below. The twelve clones
expressing the highest level of neublastin were scaled up to T162
flasks and subsequently reassayed. As shown in FIG. 10, the CHO
cell lines produced neublastin in the range of 25 to 50 ng/ml.
[0269] Ternary Complex Assay for Neublastin.
[0270] We assayed for the presence of neublastin in the media of
CHO cell line supernatants using a modified form of a ternary
complex assay described by Sanicola et al. (Proc Natl Acad Sci USA
94: 6238 (1997).
[0271] In this assay, the ability of GDNF-like molecules can be
evaluated for their ability to mediate binding between the
extracellular domain of RET and the various co-receptors,
GFR.alpha.1, GFR.alpha.2, and GFR.alpha.3. Soluble forms of RET and
the co-receptors were generated as fusion proteins. A fusion
protein between the extracellular domain of rat RET and placental
alkaline phosphatase (RET-AP) and a fusion protein between the
extracellular domain of rat GFR.alpha.1 (disclosed in published
application WO9744356; Nov. 27, 1997, herein incorporated by
reference) and the Fc domain of human IgG1 (rGFR.alpha.1-Ig) have
been described (Sanicola et al. Proc Natl Acad Sci USA 94: 6238
(1997)).
[0272] To generate a fusion protein between the extracellular
domain of murine GFR.alpha.3 and the Fc domain of human IgG1
(mGFR.alpha.3-Ig), a DNA fragment encoding amino acids 1-359 of
murine RETL3 was ligated to a fragment containing the Fc domain of
human IgG1 and cloned into the expression vector pEAG347 to
generate plasmid pGJ144. Plasmid pGJ144 was transfected into
Chinese hamster ovary cells (CHO) to generate a stable cell line
producing the fusion protein, which was purified on a Protein A
Sepharose immunoaffinity column using standard methods. In summary,
if the GDNF-like molecule can mediate binding of the co-receptor to
RET in this assay, then the RET-AP fusion protein will be retained
on the plate and the amount that is retained can be measured using
a chemiluminescent substrate for alkaline phosphatase.
[0273] Dynex Microlite-1 ELISA plates (Dynex Technologies) were
coated with 1 .mu.g/ml goat antibody specific for human Fc in 50 mM
bicarbonate/carbonate, pH 9.6 for 16 hr. The plates were emptied
and filled with 300 .mu.l of 1% I-block (Tropix) in TBS/0.5%
Tween-20 (TBST), for 1 hr. After washing three times with TBST the
wells were filled with 100 .mu.l of 1 .mu.g/ml rGFR.alpha.1-Ig or
mGFR.alpha.3-Ig diluted in conditioned media from 293 EBNA cells
expressing the RET-AP fusion gene. 100 ul of conditioned media from
the CHO neublastin clones was then added to the top well of a
column of wells, and 2 fold serial dilutions were performed down
each row of wells, and incubated for 1.5 hr at room temperature.
The plates were then washed three times with TBST, and twice with
200 mMTris pH9.8, 10 mMMgCl.sub.2 (CSPD buffer). The wash solution
was then replaced with 425 .mu.M CSPD (Tropix) in CSPD buffer
containing 1 mg/ml Sapphire chemiluminescence enhancer (Tropix),
and incubated for 30' at room temperature. The chemiluminescent
output was measured using a Dynatech luminometer.
[0274] In the initial experiments, we investigated whether
neublastin produced by the CHO cell lines could mediate the binding
of GFR.alpha.1 or GFR.alpha.3 to the extracellular domain of RET.
As shown in FIG. 11, conditioned medium from CHO cell clone #53
produced a robust signal in the ternary complex assay when the
mGFR.alpha.3-Ig fusion protein was included, but no signal when the
rGFR.alpha.1-Ig fusion protein was included, indicating that
neublastin binds to GFR.alpha.3 but not to GFR.alpha.1. This
behavior clearly distinguishes neublastin from GDNF; as shown in
FIG. 11, GDNF binds to GFR.alpha.1 but not to GFR.alpha.3. No
signal was observed with either co-receptor fusion protein, when
conditioned medium from the parental CHO cell line or straight
medium was assayed.
[0275] In order to quantitate the expression levels of neublastin
in the CHO cell lines, a standard curve was prepared using
rGFR.alpha.1-Ig and GDNF starting at a concentration of 1 ng/ml.
Neublastin concentrations for the different CHO cell lines were
then calculated using this standard curve; the levels produced by
five CHO cell lines are shown in FIG. 10. Because this estimation
depends on the untested assumption that the binding affinity
between GDNF and GFR.alpha.1 is similar to the binding affinity
between neublastin and GFR.alpha.3, these levels are only
approximate.
[0276] Analysis of Neublastin from CHO Cell Line Supernatants.
[0277] In order to further analyze the neublastin produced by the
CHO cell lines, the protein was extracted from the medium using the
GFR.alpha.3-Ig fusion protein and analyzed by western blots with
polyclonal antibodies raised against neublastin peptides.
[0278] In the first experiment, the neublastin was extracted with
mGFR.alpha.3-Ig attached to Sepharose beads. mGFR.alpha.3-Ig was
attached to Sepharose beads using the conditions suggested by the
manufacturer, Pharmacia Inc. 100 .mu.L of mGFR.alpha.3-Ig-Sepharose
was added to 1.0 mL samples of conditioned medium from a negative
control CHO cell line or from the neublastin producing CHO cell
line #16. The suspensions were incubated for two hours on a rocking
platform. Each suspension was centrifuged and the supernatant
removed followed with three 1.0 mL washes with 10 mM HEPES, 100 mM
NaCl, pH 7.5. Each resin was resuspended in 100 .mu.L of 2.times.
reducing sample buffer and heated to 100.degree. C. for 5 minutes.
20 .mu.L of the sample buffer supernatant and 10 uL of a molecular
weight standard (FMC) were applied to each well of a 10-20% precast
SDS-PAGE gel (Owl Scientific). The gel was electrophoresed at 40 mA
constant current for 72 minutes.
[0279] For western blot analysis, the protein was electroblotted to
nitrocellulose (Schleicher and Schuell) in a Hofer Scientific
apparatus in 10 mM CAPS, 10% methanol, 0.05% SDS, pH 11.2 buffer
system (45 minutes at 400 mA constant current). After the transfer,
the nitrocellulose filter was removed from the cassette and the
molecular weight markers were visualized by staining with a
solution of 0.1% Ponceau S in 1% acetic acid for 60 seconds. The
membrane was cut into two sections and the excess stain was removed
by gentle agitation in distilled water. The membranes were blocked
in 2% nonfat dry milk in TBS overnight at 4.degree. C. The
membranes were incubated individually with two of the
affinity-purified anti-neublastin peptide antibodies (R30 and R31)
at a concentration of 1.0 .mu.g/mL in 2% nonfat dry milk in TBS).
The membranes were washed with three 10 minute washes in TBS-Tween
and incubated in a 1:5000 dilution of goat anti-rabbit IgG-HRP
conjugate (Biorad) for 30 minutes. The membranes were washed with
three 10 minute washes of TBS-Tween and developed with ECL
substrate (Amersham). As shown in FIG. 12, specific bands were
detected in the proteins extracted from the neublastin producing
CHO cell line with both antibodies (lanes 2 and 4), when compared
to the bands observed in the extracted proteins from the negative
control cell line (lanes 1 and 3).
[0280] The molecular weight of the lower species is about 13 kD and
probably represents the mature domain of neublastin, generated
after cleavage of the pro-domain. This cleavage could occur after
any one of the three Arg-_ (e.g., -RXXR.dwnarw.-) residues of the
prepro neublastin protein to generate either the 140 AA, 116 AA or
113 AA forms, as set forth in SEQ.ID.NOS. 10, 11, or 12,
respectively. The predicted molecular weights of the non-modified
(i.e., lacking post-translational modifications) neublastin
polypeptides NBN140 (SEQ. ID. NO. 10), NBN116 (SEQ. ID. NO. 11),
and NBN113 (SEQ. ID. NO. 12) were determined to be 14.7 kD, 12.4
kD, and 12.1 kD, respectively. Further analysis will be needed to
confirm the structure of this species as well as the other
neublastin specific bands.
[0281] In the second experiment, the neublastin was extracted with
hGFR.alpha.3-Ig captured on an ELISA plate. To generate a fusion
protein between the extracellular domain of human GFR.alpha.3
(disclosed in published application WO97/44356; Nov. 27, 1997,
herein incorporated by reference) and the Fc domain of human IgG1
(hGFR.alpha.3-Ig), a DNA fragment encoding amino acids 1-364 of
human GFR.alpha.3 was ligated to a fragment containing the Fc
domain of human IgG1 and cloned into the expression vector CH269
described by Sanicola et al. (Proc Natl Acad Sci USA 94: 6238
(1997)). The fusion protein encoded by this plasmid was transiently
expressed in 293-Epstein-Barr virus-encoded nuclear antigen (EBNA)
cells and purified on a Protein A Sepharose immunoaffinity column
using standard methods.
[0282] Six wells of a 96-well plate were coated overnight at
4.degree. C. with goat anti-human IgG (Fc.gamma. fragment specific;
Jackson Immunulogics) at a concentration of 25 .mu.g/ml in PBS (300
.mu.l/well). The wells were blocked for 1 h at room temperature
with 400 .mu.l of 1% BSA in PBS. After 3 washes with PBST
(PBS+0.05% Tween 20), 300 .mu.l hGFR.alpha.3-Ig (10 .mu.g/ml in PBS
containing 0.1% BSA) was added to each well. The plate was
incubated for 1 h at room temperature and shaken gently (200
strokes/min) to maximize the binding. The wells were then emptied
and washed again 3 times with PBST. 250 .mu.l of conditioned media
from a negative control CHO cell line or from the neublastin
producing CHO cell line #25 was added to each of 3 wells. The plate
was incubated for 3 h at room temperature and shaken gently (300
strokes/min). The wells were then washed twice with PBST. 25 .mu.l
of non-reducing Laemli loading buffer was added to the first well
and the plate was shaken rapidly for 5 min to elute the bound
proteins (1300 strokes/min). The content was transferred to the
next well and the procedure was repeated to elute the proteins
bound in the second and third wells. After adding
.beta.-mercaptoethanol (5% final), the samples were boiled for 5
minutes and analyzed by SDS-PAGE on a 10-20% polyacrylamide
gel.
[0283] For western blot analysis, the proteins were transferred to
nitrocellulose. The membrane was blocked and probed in 5% non fat
dry milk, PBST and washed in PBST. Neublastin was detected by
electrochemoluminescence after reaction with polyclonal antibodies
(R30 and R31) raised against two neublastin peptides (at 1 ug/ml)
followed by reaction with HRP-conjugated goat anti-rabbit
antibodies (BioRad). As shown in FIG. 13, five neublastin specific
bands were detected in the extracted proteins from the neublastin
producing CHO cell line (lane 2). The lower two bands are very
similar to the bands observed in FIG. 12; again, the lower band
probably represents the mature domain of neublastin generated after
cleavage of the pro-domain.
[0284] Subsequent analysis (data not shown) of the bands in FIG. 13
shows that deglycosylation with PGNase F of the approximately 18 kD
band reduces that band to a size equivalent to the lower-most band
in the gel of FIG. 13. This suggests that neublastin may be
produced as a glycosylated protein in mammalian cells.
Expression of Neublastin in E. coli
[0285] In order to express the neublastin gene in E. coli, syngenes
were constructed with lower GC content and preferred E. coli
codons. The syngene is being cloned into two vectors, pET19b and
pMJB164, a derivative of pET19b. The construction with pET19b is
shown in FIG. 14. In this construct, the sequence encoding the
mature domain of neublastin (NBN113) is directly fused to an
initiating methionine. The construction with pMJB164 is shown in
FIG. 15. In this construct, the mature domain of neublastin is
fused to a histidine tag (i.e. 10 histidines) separated by an
enterokinase cleavage site. The initiating methionine precedes the
histidine tag.
TABLE-US-00010 Nucleotide sequence encoding neublastin in FIG. 14
SEQ. ID. NO. 29 ATGGCTGGAGGACCGGGATCTCGTGCTCGTGCAGCAGGAGCACGTGGCTG
TCGTCTGCGTTCTCAACTAGTGCCGGTGCGTGCACTCGGACTGGGACACC
GTTCCGACGAACTAGTACGTTTTCGTTTTTGTTCAGGATCTTGTCGTCGT
GCACGTTCTCCGCATGATCTATCTCTAGCATCTCTACTAGGAGCCGGAGC
ACTAAGACCGCCGCCGGGATCTAGACCTGTATCTCAACCTTGTTGTAGAC
CTACTAGATACGAAGCAGTATCTTTCATGGACGTAAACTCTACATGGAGA
ACCGTAGATAGACTATCTGCAACCGCATGTGGCTGTCTAGGATGATAATA G Nucleotide
sequence encoding his-tagged neublastin in FIG. 15 SEQ. ID. NO. 30
ATGGGCCATCATCATCATCATCATCATCATCATCACTCGAGCGGCCATAT
CGACGACGACGACAAGGCTGGAGGACCGGGATCTCGTGCTCGTGCAGCAG
GAGCACGTGGCTGTCGTCTGCGTTCTCAACTAGTGCCGGTGCGTGCACTC
GGACTGGGACACCGTTCCGACGAACTAGTACGTTTTCGTTTTTGTTCAGG
ATCTTGTCGTCGTGCACGTTCTCCGCATGATCTATCTCTAGCATCTCTAC
TAGGAGCCGGAGCACTAAGACCGCCGCCGGGATCTAGACCTGTATCTCAA
CCTTGTTGTAGACCTACTAGATACGAAGCAGTATCTTTCATGGACGTAAA
CTCTACATGGAGAACCGTAGATAGACTATCTGCAACCGCATGTGGCTGTC
TAGGATGATAATAG.
Example 6
Effect of Neublastin on the Survival of Rat Embryonic Dopaminergic
Neurons and ChAT Activity
[0286] In this series of experiments the effect of conditioned
medium from neublastin-producing HiB5pUbi1zNBN22 cells described
above was assessed.
[0287] Preparation of Cultures:
[0288] The ventral mesencephalon or spinal chord was dissected out
from rat E14 embryos in cold Hanks Buffered Salt Solution (HBSS).
Tissue pieces were incubate in sterile filtered 0.1% trypsin
(Worthington) and 0.05% DNase (Sigma) in HBSS at 37.degree. C. for
20 min. Tissue pieces was then rinsed four times in HBSS+0.05%
DNase and dissociated using a 1 ml automatic pipette. The
suspension was then centrifuged at 600 rpm for 5 min and the pellet
was re-suspended in serum conditioned medium (SCM; DMEM with 10%
foetal calf serum). The total number of cells was assessed by
tryphan blue dye exclusion method and plated at a density of
100.000 cells/cm.sup.2 in poly-L-lysine coated eight-well chamber
slides (Nunc) for assessment of dopaminergic neuron survival or at
200 000 cells/cm.sup.2 in 48 well plates (Nunc) for ChAT activity
measurements. Cells were incubated in SCM at 5% CO.sub.2/95%
O.sub.2 and 95% humidity in 37.degree. C. for 24-48 h before
changing to serum free medium (SFM) with addition of neurotrophic
factors.
[0289] Cells for assessing dopaminergic neuron survival was left
for 5 days in SFM+ trophic factor additions and then fixed for 5
min in 4% PFA and stained for tyrosine hydroxylase by
immunohistochemistry.
[0290] Cells for ChAT activity were left for 3 days with SFM and
then lysed in HBSS+0.1% Triton X-100 and immediately frozen down on
dry ice until Chat activity measurement.
[0291] Trophic Factor Addition:
[0292] Conditioned medium was collected from non-transfected HiB5
control or HiB5 producing neublastin (HiB5pUbi1zNBN22) or GDNF
(HiB5pUbi1zGDNF-L17). HiB5pUbi1zNBN22 produces approximately 20 ng
GDNF/24 hours/10.sup.5 cells as determined by GDNF-ELISA on
conditioned medium, collected from the cells. The respective cell
lines were incubated overnight with DMEM+1% FCS and the supernatant
was taken off and stored at -20.degree. C. until use. The
supernatants were diluted in 1:50 in SFM when added to the cells.
Separate wells were treated with HiB5 control supernatant
(1:50)+purified recombinant rat GDNF (from 0.03-10 ng/ml).
[0293] The results of these experiments are shown in FIG. 4. FIGS.
4A-4C are illustrations of the effect of neublastin, secreted from
HiB5pUbi1zNBN22 cells, on the survival of cultured rat embryonic,
dopaminergic, ventral mesencephalic neurons and ChAT activity in
cholinergic cranial nerve motor neurons in serum-free medium as
described infra in Example 5.1.
[0294] FIG. 4A is an illustration of the dose-response curve for
recombinant GDNF on ChAT activity (dpm/hour) measured at DIV5 in
serum-free cultures which were initially established from E14
ventral mesencephali [i.e., HiB5; GDNF 0.03 ng/ml; GDNF 0.1 ng/ml;
GDNF 0.3 ng/ml; GDNF 1 ng/ml; GDNF 10 ng/ml; GDNF 100 ng/ml].
[0295] FIG. 4B is an illustration of ChAT activity (dpm/hour)
measured at DIV5 in serum-free cultures which were initially
established from E14 ventral mesencephali. Diluted conditioned
medium from either neublastin producing HiB5pUbi1zNBN22 cells
(neublastin) or GDNF-producing HiB5GDNFL-17 (GDNFL-17) cells were
added as indicated in the figure [i.e., neublastin 1:10; neublastin
1:50; GDNF L-17 1:50].
[0296] FIG. 4C is an illustration of the number of tyrosine
hydroxylase immunoreactive cells per well [No. TH+ cells/well] at
DIV7 in serum-free cultures which were initially established from
E14 rat ventral mesencephali. Diluted conditioned medium from
either non-transfected HiB5 cells (HiB5) or neublastin-producing
HiB5pUbi1zNBN22 cells (neublastin) or recombinant GDNF, in various
concentrations, were added as indicated in the figure [i.e., HiB5
1:10; HiB5 1:40; GDNF 0.1 ng/ml; GDNF 10 ng/ml; GDNF 100 ng/ml; and
neublastin 1:40].
[0297] Conditioned medium from neublastin transfected HiB5 cells
diluted 1:40 significantly increases the number of TH
immunoreactive cells pr. well compared to control (untransfected)
HiB5 cells at an equivalent and a lower dilution (1:10 and 1:40)
(see, e.g., FIG. 4B). The increase in TH-immunoreactive cells are
comparable to the increase seen at a maximal GDNF concentration (10
ng/ml). This indicates that neublastin secreted to the medium has
an effect on survival of the dopaminergic neuron population from
rat embryonic ventral mesencephalon. In contrast, unlike GDNF
secreted from transfected HiB5 cells, no effect of conditioned
medium from neublastin transfected HiB5 cells is seen on another
neuronal population in the same culture, the cholinergic neurons
(see, e.g., FIG. 4A).
Example 7
Effect of Neublastin on the Survival of Slice Cultures of Pie
Embryonic Dopaminergic Ventral Mesencephalic Neurons
[0298] In this experiment the effect of co-culturing
neublastin-producing HiB5pUbi1zNBN22 cells with slice cultures of
ventral mesencephali from porcine embryos.
[0299] Preparation of Cultures:
[0300] Ventral mesencephali (VM) were isolated from porcine embryos
(E28; n=12) under sterile conditions, chopped into 400 .mu.m slices
and placed in chilled Gey's balanced salt solution (GIBCO) with
glucose (6.5 mg/ml). The tissue slices were cultured by the
interface culture method, originally developed by Stoppini et al.
[L. Stoppini, P. A. Buchs, D. Muller, J. Neurosci. Methods 1991 37
173-182].
[0301] In brief, slices were placed on semi-porous membranes
(Millipore, 0.3 .mu.m; 8 slices/membrane corresponding to one VM)
placed as inserts in 6-well plates (Costar) with serum containing
medium (Gibco BRL). Each well contained 1 ml medium (50% Optimem,
25% horse serum, 25% Hank's balanced salt solution (all GIBCO))
supplemented with D-glucose to a final concentration of 25 mM. At
day 0, 7000 transfected HiB5pUbi1zNBN22 (neublastin) or 7000
non-transfected HiB5 cells (control) were seeded on each tissue
slice. The co-cultures were first grown in an incubator at
33.degree. C. for 48 hours allowing the HiB5 cells immortalized
with a temperature sensitive oncogene to proliferate, and then
placed in an incubator at 37.degree. C., where the HiB5 cells
differentiate. The medium was changed twice a week. Antimitotics
and antibiotics were not used at any stage.
[0302] Determination of Dopamine by HPLC:
[0303] At day 12 and 21 in vitro, the culture medium was collected
and analysed for dopamine using HPLC with electrochemical detection
(W. N. Slooth. J. B. P. Gramsbergen, J. Neurosci. Meth. 1995 60
141-49).
[0304] Tissue Processine and Immunohistochemistry:
[0305] At day 21, the cultures were fixed in 4% paraformaldehyde in
phosphate buffer for 60 min., dehydrated in a 20% sucrose solution
for 24 hours, frozen, cryostat sectioned at 20 .mu.m (4 series),
and mounted onto gelatine coated microscope slides. One series of
sections was immunostained for tyrosine hydroxylase (TH). Briefly,
sections were washed in 0.05M tris-buffered saline (TBS, pH 7.4)
containing 1% Triton X-100 for 3.times.15 min. and incubated with
10% fettle bovine serum (FBS, Life Technologies) in TBS for 30 min.
The tissue was then incubated for 24 hours at 4.degree. C. with
monoclonal mouse anti-TH antibody (Boehringer Mannheim) diluted
1:600 in TBS with 10% FBS. After rinsing in TBS with 1% Triton
X-100 for 3.times.15 min., sections were incubated for 60 min. with
bio-tinylated anti-mouse IgG antibody (Amersham) diluted 1:200 in
TBS with 10% FBS. The sections were then washed in TBS with 1%
Triton X-100 (3.times.15 min.) and incubated for 60 min. with
streptavidin-peroxidase (Dako) diluted 1:200 in TBS with 10% FBS.
After washing in TBS (3.times.15 min.), bound antibody was
visualised by treatment with 0.05% 3,3-diaminobenzidine (Sigma) in
TBS containing 0.01% H.sub.2O.sub.2. Finally, the sections were
dehydrated in alcohol, cleared in xylene, and cover-slipped in
Eukitt.
[0306] Cell Counts and Morphometric Analysis:
[0307] Quantification of immunoreactive TH-ir neurons was performed
using bright field microscopy (Olympus). Only cells displaying an
intense staining with a well preserved cellular structure and a
distinct nucleus were counted. The estimation was based on cell
counts in every fourth culture section using a .times.20 objective.
Cell numbers were corrected for double counting according to
Abercrombie's formula (M. Abercrombie, Anat. Rec. 1946 94 239-47),
using the average diameter of the nuclei in the TH-ir neurons
(6.6.+-.0.2 .mu.m, n=30). The size of the nuclei was estimated
using a neuron tracing system (Neurolucida, MicroBrightField,
Inc.).
[0308] The results of these experiments are shown in FIG. 5. FIGS.
5A-5C are illustrations of the effect of neublastin secreted from
HiB5pUbi1zNBN22 cells on the function and survival of slice
cultures of pig embryonic dopaminergic ventral mesencephalic
neurons co-cultured with either HiB5pUbi1zNBN22 cells (neublastin)
or HiB5 cells (control) as described infra. FIG. 5A and FIG. 5B:
illustrate dopamine released to the medium at DIV12 [Dopamine
(pmol/ml)--day 12] and DIV21 [Dopamine (pmol/ml)--day 21],
respectively. FIG. 5C is an illustration of the number of tyrosine
hydroxylase immunoreactive cells per culture [TH-ir cells per
culture] at DIV21.
[0309] At day 12 HPLC analysis revealed that medium from
HiB5-neublastin co-cultures contained 84% more dopamine than medium
from HiB5-C co-cultures (FIG. 5A). At day 21 the difference was 78%
(FIG. 5B), and cell counts showed that HiB5-neublastin co-cultures
contained 66% more tyrosine hydroxylase immunoreactive neurons than
HiB5-C co-cultures (P<0.05) (FIG. 5C). This indicates, that
neublastin secreted from the HiB5pUbi1zNBN22 clone has a potent
survival effect on embryonic porcine dopaminergic neurons.
Example 8
Survival of Dorsal Root Ganglion Cells in Serum-Free Medium
[0310] This example shows the neurotrophic activity of a neublastin
polypeptide in comparison with known neurotrophic factors.
[0311] Pregnant female mice were killed by cervical dislocation.
The embryos were processed for culture as follows.
[0312] Electrolytically sharpened tungsten needles were used to
dissect dorsal root ganglia from indicated stages of C57/B16 mice
(Mollegaard Breeding, Denmark). Embryonic ganglia were incubated
for 5 minutes at 37.degree. C. with 0.05% trypsin (Gibco/BRL) in
calcium and magnesium-free Hanks balanced salt solution. Postnatal
ganglia were treated with collagenase/dispase 1 mg/ml for 30 to 45
minutes and then trypsin/DNAse 0.25% for 15 minutes. After removal
of the trypsin solution, the ganglia were washed once with 10 ml of
DMEM containing 10% heat inactivated horse serum, and were gently
triturated with a fire-polished Pasteur pipette to give a single
cell suspension.
[0313] The cells were plated on 24 well plates (Nunc), that were
precoated with polyornithine (0.5 mg/ml, overnight) and laminin (20
mg/ml for 4 h; Gibco/BRL). The neurons were incubated at 37.degree.
C. in a humidified 5% CO, incubator in a defined medium consisting
of Hams F14 supplemented with 2 mM glutamine, 0.35% bovine serum
albumin, 60 ng/ml progesterone, 16 mg/ml putrescine, 400 ng/ml
L-thyroxine, 38 ng/ml sodium selenite, 340 ng/ml triiodo-thyronine,
60 mg/ml penicillin and 100 mg/ml streptomycin.
[0314] After 48 hours of incubation, neurons were clearly
recognised by their bipolar morphology under phase-contrast optics.
The percentage neuronal survival in the absence or presence of
trophic factors (added to the culture medium prior to plating the
neurons at 10 ng/ml), or of conditioned medium from the neublastin
producing HiB5pUbi1zNBN22 cells, was assessed by counting the
neurons in the wells at 48 hours.
[0315] The results of these experiments are presented in FIG. 9, in
which figure:
[0316] 0 represents the control experiment (in absence of
factors);
[0317] 1 represents experiments in the presence of GDNF;
[0318] 2 represents experiments in the presence of Neuturin;
[0319] 3 represents experiments in the presence of Neublastin of
the invention;
[0320] E12 represents data from experiments carried out on DRG
cells isolated from embryonic day 12;
[0321] E16 represents data from experiments carried out on DRG
cells isolated from embryonic day 16;
[0322] P0 represents data from experiments carried out on DRG cells
isolated from the day of birth;
[0323] P7 represents data from experiments carried out on DRG cells
isolated from day 7 after birth; and
[0324] P15 represents data from experiments carried out on DRG
cells isolated from day 15 after birth.
[0325] These results clearly show that the neurotrophic factor of
the invention show activities comparable to, or even better than
those of the well established neurotrophic factors.
Example 9
In Vivo Effects of Neublastin on Nigral Dopamine Neurons
[0326] In order to test the ability of neublastin (neublastin) to
protect adult nigral dopamine (DA) neurons from 6-hydroxydopamine
induced degeneration, we employed a rat model of Parkinson's
disease ([Sauer and Oertel, Neuroscience 1994 59, 401-415) and
lentiviral gene transfer of neublastin.
[0327] Lentivirus Production:
[0328] To generate a lentiviral transfer vector encoding
neublastin, pHR'-neublastin, a 1331 bp BamH1 fragment from
neublastin cDNA was subcloned in the BamH1/Bgl II site of pSL301
(Invitrogen). From this construct a 1519 bp BamH1/Xho1 fragment was
cut out and ligated in the BamH1/Xho1 site of pHR' carrying a
woodchuck hepatitis virus post-translational fragment [Zufferey R,
Donello J E, Trono D, Hope T J: Woodchuck hepatitis virus
posttranscriptional regulatory element enhances expression of
transgenes delivered by retroviral vectors"; J. Virol. 1999 73 (4)
2886-2892]. To generate pHR-GDNF a 701 bp BamH1/Xho1 fragment from
pUbilz-GDNF was ligated in the BamH1/Xho1 site of pHR'.
[0329] Production of the lentiviral vector have been described by
e.g. Zufferey et al. [Zufferey R, Nagy D, Mandel R J, Naldini L.
Trono D: "Multiply attenuated lentiviral vector achieves efficient
gene delivery in vivo; Nat. Biotechnol. 1997 15 (9) 871-875].
Briefly, the transfer constructs and the helper plasmids pR8.91 and
pMDG were co-transfected into 293T cells. Virions released into the
media were collected at 48 and 72 hrs post-transfection. To
concentrate the virus, the media was centrifuged 1.5 hrs at 141 000
g, and the pellet dissolved in DMEM. The titer of a control
carrying the gene for Green Fluorescent Protein ("GFP") was
determined to be 10.sup.8 transforming units (TU)/ml by GFP
fluorescence in 293T cells. A RNA slot blot technique [von
Schwedler U, Song J, Aiken C, Trono D: "Vif is crucial for human
immunodeficiency virus type 1 proviral DNA synthesis in infected
cells"; J. Virol. 1993 67 (8) 4945-4955] was used to determine
viral particle titer. In the GDNF supernatant and neublastin
supernatant there was 10 times less particles as compared to the
GFP supernatant.
[0330] Surgical Procedures:
[0331] All work involving animals was conducted according to the
rules set by the Ethical Committee for Use of Laboratory Animals at
Lund University.
[0332] A total of 21 young adult female Sprague-Dawley rats
(B&K Universal, Stockholm, Sweden) were used and housed under
12 hours light:dark cycle with free access to rat chow and water.
Retrograde labelling and 6-OHDA lesions were performed 3 weeks
prior to lesion according to Sauer and Oertel [Sauer and Oertel,
Neuroscience 1994 59:401-415]. Briefly, under Equithesin
anaesthesia (0.3 ml/100 g) the rats were injected bilaterally with
0.2 .mu.l of a 2% solution (dissolved in 0.9% NaCl) of the
retrograde tracer Fluoro-Gold (FG; Fluorochrome, Inc., Englewood,
Colo.). Injections were made using a 2 .mu.l Hamilton syringe at
co-ordinates: AP=+0.5 mm; ML=.+-.3.4 mm relative to bregma; DV=-5.0
mm relative to the dura and incisor bar set to 0.0 mm. In addition,
0.05 .mu.l/min was injected with another 5 min left before the
needle was retracted.
[0333] Fourteen days after the FG injections animals received a
total of 5 deposits (1 .mu.l/deposit) of a lentiviral vector
carrying the gene for green fluorescent protein (GFP), neublastin
or GDNF. Four of the deposits were into the striatum along two
needle tracts at the following co-ordinates: AP=+1.0 mm, ML=-2.6
mm, DV.sub.1=-5.0 mm DV.sub.2=-4.5 mm and AP=0.0 mm, ML=-3.7 mm,
DV.sub.1=-5.0 mm DV.sub.2=-4.5 mm. The supranigral deposit was made
at AP=-5.2 mm, ML=-2.0 mm, DV.sub.1=-6.3 mm. Tooth bar was set at
-2.3 mm.
[0334] Twenty-one days after retrograde labelling, and 7 days after
lentiviral injections the animals were re-anaesthetised and with a
10 .mu.l Hamilton syringe a single deposit of 20 .mu.g 6-OHDA
(Sigma; calculated as free base and dissolved in 3 .mu.l ice cold
saline supplemented with 0.02% ascorbic acid) was injected into the
right striatum in the same location as the FG deposits. The
injection rate was 1 .mu.l/min, leaving another 3 min before
retracting the needle.
[0335] Tissue Processing:
[0336] At 21 days after the 6-OHDA injection the animals were
deeply anaesthetised with chloral hydrate and transcardially
perfused with saline (pH 7.4; room temperature) for one min
followed by 200 ml ice cold formaldehyde solution (4%
paraformaldehyde in 0.1M phosphate buffer, pH 7.4). The brains were
dissected and postfixed in the same fixative for 3-4 hours and then
transferred into 25% sucrose/0.1M phosphate buffer for 48 hours.
Five series of 40 .mu.m sections through the striatum and
substantia nigra (SN) were cut on a freezing microtome.
[0337] Quantitative Assessment of Dopaminergic Neurons in the
SN:
[0338] The number of FG-labelled in the SN pars compacta was
assessed by a blinded observer as described previously [Sauer and
Oertel, Neuroscience 1994 59, 401-415]. In brief, three consecutive
sections centred around the level of the medial terminal nucleus of
the accessory optic tract (MTN; -5.3 in the atlas of Paxinos and
Watson (1997)) were used and all labelled/stained neurons laterally
to the MTN was counted at 40.times. magnification (n=6-7/group).
FG-labelled neurons were included if they were brightly fluorescent
under epi-illumination at 330 nm, displayed a neuronal profile and
extend at least one neuritic process.
[0339] On the lesion side in animals receiving injections of
lentivirus carrying GFP the number of FG-positive nigral neurons
were reduced to 18% of that on the intact side. In contrast,
animals injected with lenti-neublastin showed a near complete
protection of the number of FG-positive nigral neurons (89%). This
was as efficient as lenti-GDNF treated animals where 87% of the
retrogradely labelled neurons remained on the lesioned side. This
shows that neublastin is a potent survival factor for lesioned
adult nigral dopamine neurons and that it is as potent as GDNF.
[0340] FIG. 6 is an illustration of the in vivo effect of
lentiviral-produced neublastin on nigral dopamine neurons. Neurons
of the SN pars compacta, in female Sprague Dawley rats, were
retrogradely-labelled with Fluorogold (FG), 3 weeks prior to a
single injection of 6-hydroxydopamine (6-OHDA) in the right
striatum. One week before the 6-OHDA injection, the animals
received injections with lentiviral vectors expressing neublastin
[neublastin], GDNF [GDNF] or the Green Fluorescent Protein [GFP] as
indicated in the figure. Twenty one days after the 6-OHDA
injections, the number of FG-labelled neurons in both sides of the
striata were determined. The figure shows the percentage [% FG
lesion/intact] of FG-labelled neurons in the lesioned (right) side
verses the intact (left) side of the striata of the three groups of
animals.
Example 10
Production of Antibodies
[0341] To prepare antibodies against neublastin, two rabbits were
immunised with either peptide 1: CRPTRYEAVSFMDVNST (amino acids
108-124 of SEQ ID NO: 9); or peptide 2: ALRPPPGSRPVSQPC (amino
acids 93-107 of SEQ ID NO: 9) conjugated to carrier protein at 3
week intervals. Two rabbits for each peptide were immunized at week
0, 3, 6 and 10, and bleeds were collected at week 7 and 11. The
second bleed was affinity purified via a peptide affinity column.
The antibodies were named Ab-1 and Ab-2, according to the
peptide.
Western Blot:
[0342] 2.times.10.sup.6 HiB5 cells, stably transfected with the
cDNA for neublastin (Hib5pUbi1zNBN22), or untransfected HiB5 cells,
were incubated overnight in serum free medium with N.sub.2
supplement (GIBCO). The medium was concentrated on small
concentrators with cut-off membranes of 5 kDa (Millipore, Bedford,
Mass.). Concentrated samples were added 5.times. Laemmli sample
buffer and were heated to 95.degree. C. for 5 minutes. Samples were
separated by SDS polyacrylamide gel electrophoresis on 15%
acrylamide gels and transferred to PVDF-membranes. Residual
protein-binding sites were blocked with 5% non-fat dry milk in PBS
with 0.1% Tween-20. Membranes were incubated overnight with
neublastin antibody (1:1000), followed by incubation with a
secondary anti-rabbit or anti-mouse IgG antibody conjugated to
horseradish peroxidase (1:2000).
[0343] Immunostaining was visualized using enhanced
chemoluminiscence Plus (ECL+) according to the manufacturer's
instructions (Amersham). The results of these experiments are shown
in FIG. 3 and Example 5.
[0344] Using standard techniques, we also raised rabbit polyclonal
antibodies against the following peptides:
TABLE-US-00011 (amino acids 30-43 of SEQ ID NO: 9) Peptide R27:
GPGSRARAAGARGC; (amino acids 57-70 of SEQ ID NO: 9) Peptide R28:
LGHRSDELVRFRFC; (amino acids 74-85 of SEQ ID NO: 9) Peptide R29:
CRRARSPHDLSL; (amino acids 94-107 of SEQ ID NO: 9) Peptide R30:
LRPPPGSRPVSQPC; and (amino acids 123-136 of SEQ ID NO: 9) Peptide
R31: STWRTVDRLSATAC.
[0345] Only peptides R30 and R31, relatively close to the
C-terminus, recognized the denatured protein under reducing
conditions on a Western blot.
TABLE-US-00012 Description of Sequences Contained in the Sequence
Listing SEQ ID NO.: 1 Humar neublastin nucleic acid. 865 bp SEQ ID
NO.: 2 Human neublastin polypeptide from sequence 1. 200 aa SEQ ID
NO.: 3 Coding region (CDS) of a human pre-pro-polypeptide. 861 bp
SEQ ID NO.: 4 Human neublastin polypeptide from sequence 3. 238 aa
SEQ ID NO.: 5 Variant of human neublastin in sequence 4 (Xaa is Asn
or Thr; 140 aa Yaa is Ala or Pro). SEQ ID NO.: 6 Variant of human
neublastin in sequence 4 (Xaa is Asn or Thr; 116 aa Yaa is Ala or
Pro). SEQ ID NO.: 7 Variant of human neublastin in sequence 4 (Xaa
is Asn or Thr; 113 aa Yaa is Ala or Pro). SEQ ID NO.: 8 cDNA from
positive colony PCR of human fetal brain cDNA. 861 bp SEQ ID NO.: 9
human fetal brain pre-pro-neublastin polypeptide including "stop"
221 aa (corresponds to seq. 8) SEQ ID NO.: 10 Variant of
pre-pro-neublastin (seq. 9) NBN140, 14.7 kD. 140 aa SEQ ID NO.: 11
Variant of pre-pro-neublastin (seq. 9) NBN116, 12.4 kD. 116 aa SEQ
ID NO.: 12 Variant of pre-pro-neublastin (seq. 9) NBN113, 12.1 kD.
113 aa SEQ ID NO.: 13 PCR product from screen of human fetal brain
cDNA master plate 102 bp using SEQ. ID. NOS. 17 and 18 as primers.
SEQ ID NO.: 14 PCR product from screen of mouse fetal cDNA master
plate 220 bp using SEQ. ID. NOS. 21 and 22 as primers. SEQ ID NO.:
15 Full length mouse neublastin cDNA. 2136 bp SEQ ID NO.: 16 Mouse
pre-pro-neublastin polypeptide. 224 aa SEQ ID NO.: 17
"NBNint.sence" Top Primer for NBN from human fetal brain cDNA 18 nt
complementary to bases 551-568 of SEQ. ID. NO. 1 SEQ ID NO.: 18
"NBNint.antisence" Bottom Primer for NBN from human fetal brain
cDNA 20 nt reverse complement to bases 633-652 of SEQ. ID. NO. 1
SEQ ID NO.: 19 "NBNext.sence" Top Primer for whole human brain mRNA
RT-PCR 17 nt complementary to bases 58-74 of SEQ. ID. NO. 8. SEQ ID
NO.: 20 "NBNext.antisence" Bottom Primer for whole human brain mRNA
RT-PCR 16 nt reverse complement to bases 850-865 of SEQ. ID. NO. 8.
SEQ ID NO.: 21 "NBNint.sence" NBN C2 Primer for screening mouse
fetal cDNA master plate 18 nt complementary to bases 1398-1415 of
SEQ. ID. NO. 15. SEQ ID NO.: 22 "NBNint.antisence" NBN C2as Primer
for screening mouse fetal cDNA master 20 nt plate. Reverse
complement to bases 1598-1617 of SEQ. ID. NO. 15. SEQ ID NO.: 23
Primer Pair 1 Sense PCR Primer for human genomic DNA amplification
29 nt complementary to bases 60-88 of SEQ. ID. NO. 3. SEQ ID NO.:
24 Primer Pair 1 Antisense PCR Primer for human genomic DNA
amplification 27 nt Reverse complement to bases 835-861 of SEQ. ID.
NO. 3. SEQ ID NO.: 25 Primer Pair 2 Sense PCR Primer for human
genomic DNA amplification 35 nt complementary to bases 1-35 of SEQ.
ID. NO. 3. SEQ ID NO.: 26 Primer Pair 2 Antisense PCR Primer for
human genomic DNA amplification 34 nt reverse complement to bases
786-819 of SEQ. ID. NO. 3. SEQ ID NO.: 27 Antisense alkaline
phosphatase conjugated hybridization probe, 30 nt complimentary to
bases 1140-1169 of mouse neuroblastin cDNA. SEQ ID NO.: 28
"NBNext.sence" Top Primer for whole human brain mRNA RT-PCR 16 nt
complementary to bases 1-16 of SEQ. ID. NO. 1 SEQ ID NO.: 29
Syngene from FIG. 14 of neublastin. 351 nt SEQ ID NO.: 30 Syngene
from FIG. 15 of Hisneublastin. 414 nt SEQ ID NO.: 31 Primer for
isolating neublastin. 39 nt SEQ ID NO.: 32 Primer for isolating
neublastin. 39 nt SEQ ID NO.: 33 "NBNint.antisence" NBN primer;
reverse complement to bases 715-730 16 nt of SEQ. ID. NO. 8.
Sequence CWU 1
1
431865DNAHomo
sapiensCDS(120)..(719)5'UTR(1)..(119)3'UTR(721)..(865)sig_peptide(120)..(-
179)mat_peptide(405)..(719)misc_structure(661)..(663)CARBOHYD
Glycosylated Asparagine at Asn87 1ctaggagccc atgcccggcc tgatctcagc
ccgaggacag cccctccttg aggtccttcc 60tccccaagcc cacctgggtg ccctctttct
ccctgaggct ccacttggtc tctccgcgc 119atg cct gcc ctg tgg ccc acc ctg
gcc gct ctg gct ctg ctg agc agc 167Met Pro Ala Leu Trp Pro Thr Leu
Ala Ala Leu Ala Leu Leu Ser Ser-95 -90 -85 -80gtc gca gag gcc tcc
ctg ggc tcc gcg ccc cgc agc cct gcc ccc cgc 215Val Ala Glu Ala Ser
Leu Gly Ser Ala Pro Arg Ser Pro Ala Pro Arg -75 -70 -65gaa ggc ccc
ccg cct gtc ctg gcg tcc ccc gcc ggc cac ctg ccg ggg 263Glu Gly Pro
Pro Pro Val Leu Ala Ser Pro Ala Gly His Leu Pro Gly -60 -55 -50gga
cgc acg gcc cgc tgg tgc agt gga aga gcc cgg cgg ccg cgc cgc 311Gly
Arg Thr Ala Arg Trp Cys Ser Gly Arg Ala Arg Arg Pro Arg Arg -45 -40
-35aga cac ttc tcg gcc cgc gcc ccc gcc gcc tgc acc ccc atc tgc tct
359Arg His Phe Ser Ala Arg Ala Pro Ala Ala Cys Thr Pro Ile Cys Ser
-30 -25 -20tcc ccg cgg gtc cgc gcg gcg cgg ctg ggg ggc cgg gca gcg
cgc tcg 407Ser Pro Arg Val Arg Ala Ala Arg Leu Gly Gly Arg Ala Ala
Arg Ser-15 -10 -5 -1 1ggc agc ggg ggc gcg ggg tgc cgc ctg cgc tcg
cag ctg gtg ccg gtg 455Gly Ser Gly Gly Ala Gly Cys Arg Leu Arg Ser
Gln Leu Val Pro Val 5 10 15cgc gcg ctc ggc ctg ggc cac cgc tcc gac
gag ctg gtg cgt ttc cgc 503Arg Ala Leu Gly Leu Gly His Arg Ser Asp
Glu Leu Val Arg Phe Arg 20 25 30ttc tgc acc ggc tcc tgc ccg cgc gcg
cgc tct cca cac gac ctc agc 551Phe Cys Thr Gly Ser Cys Pro Arg Ala
Arg Ser Pro His Asp Leu Ser 35 40 45ctg gcc agc cta ctg ggc gcc ggg
gcc ctg cga ccg ccc ccg ggc tcc 599Leu Ala Ser Leu Leu Gly Ala Gly
Ala Leu Arg Pro Pro Pro Gly Ser50 55 60 65cgg ccc gtc agc cag ccc
tgc tgc cga ccc acg cgc tac gaa gcg gtc 647Arg Pro Val Ser Gln Pro
Cys Cys Arg Pro Thr Arg Tyr Glu Ala Val 70 75 80tcc ttc atg gac gtc
aac agc acc tgg aga acc gtg gac cgc ctc tcc 695Ser Phe Met Asp Val
Asn Ser Thr Trp Arg Thr Val Asp Arg Leu Ser 85 90 95gcc acc gcc tgc
ggc tgc ctg ggc tgagggctcg ctccagggct ttgcagactg 749Ala Thr Ala Cys
Gly Cys Leu Gly 100 105gacccttacc ggtggctctt cctgcctggg accctcccgc
agagtcccac tagccagcgg 809cctcagccag ggacgaaggc ctcaaagctg
agaggcccct gccggtgggt gatgga 8652200PRTHomo sapiens 2Met Pro Ala
Leu Trp Pro Thr Leu Ala Ala Leu Ala Leu Leu Ser Ser-95 -90 -85
-80Val Ala Glu Ala Ser Leu Gly Ser Ala Pro Arg Ser Pro Ala Pro Arg
-75 -70 -65Glu Gly Pro Pro Pro Val Leu Ala Ser Pro Ala Gly His Leu
Pro Gly -60 -55 -50Gly Arg Thr Ala Arg Trp Cys Ser Gly Arg Ala Arg
Arg Pro Arg Arg -45 -40 -35Arg His Phe Ser Ala Arg Ala Pro Ala Ala
Cys Thr Pro Ile Cys Ser -30 -25 -20Ser Pro Arg Val Arg Ala Ala Arg
Leu Gly Gly Arg Ala Ala Arg Ser-15 -10 -5 -1 1Gly Ser Gly Gly Ala
Gly Cys Arg Leu Arg Ser Gln Leu Val Pro Val 5 10 15Arg Ala Leu Gly
Leu Gly His Arg Ser Asp Glu Leu Val Arg Phe Arg 20 25 30Phe Cys Thr
Gly Ser Cys Pro Arg Ala Arg Ser Pro His Asp Leu Ser 35 40 45Leu Ala
Ser Leu Leu Gly Ala Gly Ala Leu Arg Pro Pro Pro Gly Ser50 55 60
65Arg Pro Val Ser Gln Pro Cys Cys Arg Pro Thr Arg Tyr Glu Ala Val
70 75 80Ser Phe Met Asp Val Asn Ser Thr Trp Arg Thr Val Asp Arg Leu
Ser 85 90 95Ala Thr Ala Cys Gly Cys Leu Gly 100 1053861DNAHomo
sapiensCDS(7)..(717)5'UTR(1)..(6)3'UTR(718)..(861)sig_peptide(7)..(174)ma-
t_peptide(298)..(717)mat_peptide(370)..(717)mat_peptide(379)..(717)misc_st-
ructure(661)..(663)CARBOHYD glycosylated Asparagine as Asn122
3gagccc atg ccc ggc ctg atc tca gcc cga gga cag ccc ctc ctt gag 48
Met Pro Gly Leu Ile Ser Ala Arg Gly Gln Pro Leu Leu Glu -95 -90
-85gtc ctt cct ccc caa gcc cac ctg ggt gcc ctc ttt ctc cct gag gct
96Val Leu Pro Pro Gln Ala His Leu Gly Ala Leu Phe Leu Pro Glu Ala
-80 -75 -70cca ctt ggt ctc tcc gcg cag cct gcc ctg tgg ccc acc ctg
gcc gct 144Pro Leu Gly Leu Ser Ala Gln Pro Ala Leu Trp Pro Thr Leu
Ala Ala -65 -60 -55ctg gct ctg ctg agc agc gtc gca gag gcc tcc ctg
ggc tcc gcg ccc 192Leu Ala Leu Leu Ser Ser Val Ala Glu Ala Ser Leu
Gly Ser Ala Pro -50 -45 -40cgc agc cct gcc ccc cgc gaa ggc ccc ccg
cct gtc ctg gcg tcc ccc 240Arg Ser Pro Ala Pro Arg Glu Gly Pro Pro
Pro Val Leu Ala Ser Pro-35 -30 -25 -20gcc ggc cac ctg ccg ggg gga
cgc acg gcc cgc tgg tgc agt gga aga 288Ala Gly His Leu Pro Gly Gly
Arg Thr Ala Arg Trp Cys Ser Gly Arg -15 -10 -5gcc cgg cgg ccg ccg
ccg cag cct tct cgg ccc gcg ccc ccg ccg cct 336Ala Arg Arg Pro Pro
Pro Gln Pro Ser Arg Pro Ala Pro Pro Pro Pro -1 1 5 10gca ccc cca
tct gct ctt ccc cgc ggg ggc cgc gcg gcg cgg gct ggg 384Ala Pro Pro
Ser Ala Leu Pro Arg Gly Gly Arg Ala Ala Arg Ala Gly 15 20 25ggc ccg
ggc aac cgc gct cgg gca gcg ggg gcg cgg ggc tgc cgc ctg 432Gly Pro
Gly Asn Arg Ala Arg Ala Ala Gly Ala Arg Gly Cys Arg Leu30 35 40
45cgc tcg cag ctg gtg ccg gtg cgc gcg ctc ggc ctg ggc cac cgc tcc
480Arg Ser Gln Leu Val Pro Val Arg Ala Leu Gly Leu Gly His Arg Ser
50 55 60gac gag ctg gtg cgt ttc cgc ttc tgc agc ggc tcc tgc cgc cgc
gcg 528Asp Glu Leu Val Arg Phe Arg Phe Cys Ser Gly Ser Cys Arg Arg
Ala 65 70 75cgc tct cca cac gac ctc agc ctg gcc agc cta ctg ggc gcc
ggg gcc 576Arg Ser Pro His Asp Leu Ser Leu Ala Ser Leu Leu Gly Ala
Gly Ala 80 85 90ctg cga ccg ccc ccg ggc tcc cgg ccc gtc agc cag ccc
tgc tgc cga 624Leu Arg Pro Pro Pro Gly Ser Arg Pro Val Ser Gln Pro
Cys Cys Arg 95 100 105ccc acg cgc tac gaa gcg gtc tcc ttc atg gac
gtc aac agc acc tgg 672Pro Thr Arg Tyr Glu Ala Val Ser Phe Met Asp
Val Asn Ser Thr Trp110 115 120 125aga acc gtg gac cgc ctc tcc gcc
aac ccc tgc ggc tgc ctg ggc 717Arg Thr Val Asp Arg Leu Ser Ala Asn
Pro Cys Gly Cys Leu Gly 130 135 140tgagggctcg ctccagggct ttgcagactg
gacccttacc ggtggctctt cctgcctggg 777accctcccgc agagtcccac
tagccagcgg cctcagccag ggacgaaggc ctcaaagctg 837agaggcccct
gccggtgggt gatg 8614237PRTHomo sapiens 4Met Pro Gly Leu Ile Ser Ala
Arg Gly Gln Pro Leu Leu Glu Val Leu -95 -90 -85Pro Pro Gln Ala His
Leu Gly Ala Leu Phe Leu Pro Glu Ala Pro Leu -80 -75 -70Gly Leu Ser
Ala Gln Pro Ala Leu Trp Pro Thr Leu Ala Ala Leu Ala-65 -60 -55
-50Leu Leu Ser Ser Val Ala Glu Ala Ser Leu Gly Ser Ala Pro Arg Ser
-45 -40 -35Pro Ala Pro Arg Glu Gly Pro Pro Pro Val Leu Ala Ser Pro
Ala Gly -30 -25 -20His Leu Pro Gly Gly Arg Thr Ala Arg Trp Cys Ser
Gly Arg Ala Arg -15 -10 -5Arg Pro Pro Pro Gln Pro Ser Arg Pro Ala
Pro Pro Pro Pro Ala Pro-1 1 5 10 15Pro Ser Ala Leu Pro Arg Gly Gly
Arg Ala Ala Arg Ala Gly Gly Pro 20 25 30Gly Asn Arg Ala Arg Ala Ala
Gly Ala Arg Gly Cys Arg Leu Arg Ser 35 40 45Gln Leu Val Pro Val Arg
Ala Leu Gly Leu Gly His Arg Ser Asp Glu 50 55 60Leu Val Arg Phe Arg
Phe Cys Ser Gly Ser Cys Arg Arg Ala Arg Ser 65 70 75Pro His Asp Leu
Ser Leu Ala Ser Leu Leu Gly Ala Gly Ala Leu Arg80 85 90 95Pro Pro
Pro Gly Ser Arg Pro Val Ser Gln Pro Cys Cys Arg Pro Thr 100 105
110Arg Tyr Glu Ala Val Ser Phe Met Asp Val Asn Ser Thr Trp Arg Thr
115 120 125Val Asp Arg Leu Ser Ala Asn Pro Cys Gly Cys Leu Gly 130
135 1405140PRTHomo sapiensVARIANT(134)Wherein Xaa at position 134
designates Asn or Thr 5Pro Pro Pro Gln Pro Ser Arg Pro Ala Pro Pro
Pro Pro Ala Pro Pro1 5 10 15Ser Ala Leu Pro Arg Gly Gly Arg Ala Ala
Arg Ala Gly Gly Pro Gly 20 25 30Asn Arg Ala Arg Ala Ala Gly Ala Arg
Gly Cys Arg Leu Arg Ser Gln 35 40 45Leu Val Pro Val Arg Ala Leu Gly
Leu Gly His Arg Ser Asp Glu Leu 50 55 60Val Arg Phe Arg Phe Cys Ser
Gly Ser Cys Arg Arg Ala Arg Ser Pro65 70 75 80His Asp Leu Ser Leu
Ala Ser Leu Leu Gly Ala Gly Ala Leu Arg Pro 85 90 95Pro Pro Gly Ser
Arg Pro Val Ser Gln Pro Cys Cys Arg Pro Thr Arg 100 105 110Tyr Glu
Ala Val Ser Phe Met Asp Val Asn Ser Thr Trp Arg Thr Val 115 120
125Asp Arg Leu Ser Ala Xaa Xaa Cys Gly Cys Leu Gly 130 135
1406116PRTHomo sapiensVARIANT(110)Wherein Xaa at position 110
designates Asn or Thr 6Ala Ala Arg Ala Gly Gly Pro Gly Asn Arg Ala
Arg Ala Ala Gly Ala1 5 10 15Arg Gly Cys Arg Leu Arg Ser Gln Leu Val
Pro Val Arg Ala Leu Gly 20 25 30Leu Gly His Arg Ser Asp Glu Leu Val
Arg Phe Arg Phe Cys Ser Gly 35 40 45Ser Cys Arg Arg Ala Arg Ser Pro
His Asp Leu Ser Leu Ala Ser Leu 50 55 60Leu Gly Ala Gly Ala Leu Arg
Pro Pro Pro Gly Ser Arg Pro Val Ser65 70 75 80Gln Pro Cys Cys Arg
Pro Thr Arg Tyr Glu Ala Val Ser Phe Met Asp 85 90 95Val Asn Ser Thr
Trp Arg Thr Val Asp Arg Leu Ser Ala Xaa Xaa Cys 100 105 110Gly Cys
Leu Gly 1157113PRTHomo sapiensVARIANT(107)Wherein Xaa at position
107 designates Asn or Thr 7Ala Gly Gly Pro Gly Asn Arg Ala Arg Ala
Ala Gly Ala Arg Gly Cys1 5 10 15Arg Leu Arg Ser Gln Leu Val Pro Val
Arg Ala Leu Gly Leu Gly His 20 25 30Arg Ser Asp Glu Leu Val Arg Phe
Arg Phe Cys Ser Gly Ser Cys Arg 35 40 45Arg Ala Arg Ser Pro His Asp
Leu Ser Leu Ala Ser Leu Leu Gly Ala 50 55 60Gly Ala Leu Arg Pro Pro
Pro Gly Ser Arg Pro Val Ser Gln Pro Cys65 70 75 80Cys Arg Pro Thr
Arg Tyr Glu Ala Val Ser Phe Met Asp Val Asn Ser 85 90 95Thr Trp Arg
Thr Val Asp Arg Leu Ser Ala Xaa Xaa Cys Gly Cys Leu 100 105 110Gly
8861DNAHomo
sapiensCDS(58)..(717)5'UTR(1)..(57)3'UTR(718)..(861)sig_peptide(58)..(174-
)mat_peptide(298)..(717)mat_peptide(370)..(717)mat_peptide(379)..(717)misc-
_structure(661)..(663)CARBOHYD glycosylated asparagine at Asn122
8aggagggtgg gggaacagct caacaatggc tgatgggcgc tcctggtgtt gatagag
57atg gaa ctt gga ctt gga ggc ctc tcc acg ctg tcc cac tgc ccc tgg
105Met Glu Leu Gly Leu Gly Gly Leu Ser Thr Leu Ser His Cys Pro
Trp-80 -75 -70 -65cct agg cgg cag cct gcc ctg tgg ccc acc ctg gcc
gct ctg gct ctg 153Pro Arg Arg Gln Pro Ala Leu Trp Pro Thr Leu Ala
Ala Leu Ala Leu -60 -55 -50ctg agc agc gtc gca gag gcc tcc ctg ggc
tcc gcg ccc cgc agc cct 201Leu Ser Ser Val Ala Glu Ala Ser Leu Gly
Ser Ala Pro Arg Ser Pro -45 -40 -35gcc ccc cgc gaa ggc ccc ccg cct
gtc ctg gcg tcc ccc gcc ggc cac 249Ala Pro Arg Glu Gly Pro Pro Pro
Val Leu Ala Ser Pro Ala Gly His -30 -25 -20ctg ccg ggg gga cgc acg
gcc cgc tgg tgc agt gga aga gcc cgg cgg 297Leu Pro Gly Gly Arg Thr
Ala Arg Trp Cys Ser Gly Arg Ala Arg Arg -15 -10 -5 -1ccg ccg ccg
cag cct tct cgg ccc gcg ccc ccg ccg cct gca ccc cca 345Pro Pro Pro
Gln Pro Ser Arg Pro Ala Pro Pro Pro Pro Ala Pro Pro1 5 10 15tct gct
ctt ccc cgc ggg ggc cgc gcg gcg cgg gct ggg ggc ccg ggc 393Ser Ala
Leu Pro Arg Gly Gly Arg Ala Ala Arg Ala Gly Gly Pro Gly 20 25 30agc
cgc gct cgg gca gcg ggg gcg cgg ggc tgc cgc ctg cgc tcg cag 441Ser
Arg Ala Arg Ala Ala Gly Ala Arg Gly Cys Arg Leu Arg Ser Gln 35 40
45ctg gtg ccg gtg cgc gcg ctc ggc ctg ggc cac cgc tcc gac gag ctg
489Leu Val Pro Val Arg Ala Leu Gly Leu Gly His Arg Ser Asp Glu Leu
50 55 60gtg cgt ttc cgc ttc tgc agc ggc tcc tgc cgc cgc gcg cgc tct
cca 537Val Arg Phe Arg Phe Cys Ser Gly Ser Cys Arg Arg Ala Arg Ser
Pro65 70 75 80cac gac ctc agc ctg gcc agc cta ctg ggc gcc ggg gcc
ctg cga ccg 585His Asp Leu Ser Leu Ala Ser Leu Leu Gly Ala Gly Ala
Leu Arg Pro 85 90 95ccc ccg ggc tcc cgg ccc gtc agc cag ccc tgc tgc
cga ccc acg cgc 633Pro Pro Gly Ser Arg Pro Val Ser Gln Pro Cys Cys
Arg Pro Thr Arg 100 105 110tac gaa gcg gtc tcc ttc atg gac gtc aac
agc acc tgg aga acc gtg 681Tyr Glu Ala Val Ser Phe Met Asp Val Asn
Ser Thr Trp Arg Thr Val 115 120 125gac cgc ctc tcc gcc acc gcc tgc
ggc tgc ctg ggc tgagggctcg 727Asp Arg Leu Ser Ala Thr Ala Cys Gly
Cys Leu Gly 130 135 140ctccagggct ttgcagactg gacccttacc ggtggctctt
cctgcctggg accctcccgc 787agagtcccac tagccagcgg cctcagccag
ggacgaaggc ctcaaagctg agaggcccct 847accggtgggt gatg 8619220PRTHomo
sapiens 9Met Glu Leu Gly Leu Gly Gly Leu Ser Thr Leu Ser His Cys
Pro Trp-80 -75 -70 -65Pro Arg Arg Gln Pro Ala Leu Trp Pro Thr Leu
Ala Ala Leu Ala Leu -60 -55 -50Leu Ser Ser Val Ala Glu Ala Ser Leu
Gly Ser Ala Pro Arg Ser Pro -45 -40 -35Ala Pro Arg Glu Gly Pro Pro
Pro Val Leu Ala Ser Pro Ala Gly His -30 -25 -20Leu Pro Gly Gly Arg
Thr Ala Arg Trp Cys Ser Gly Arg Ala Arg Arg -15 -10 -5 -1Pro Pro
Pro Gln Pro Ser Arg Pro Ala Pro Pro Pro Pro Ala Pro Pro 1 5 10
15Ser Ala Leu Pro Arg Gly Gly Arg Ala Ala Arg Ala Gly Gly Pro Gly
20 25 30Ser Arg Ala Arg Ala Ala Gly Ala Arg Gly Cys Arg Leu Arg Ser
Gln 35 40 45Leu Val Pro Val Arg Ala Leu Gly Leu Gly His Arg Ser Asp
Glu Leu 50 55 60Val Arg Phe Arg Phe Cys Ser Gly Ser Cys Arg Arg Ala
Arg Ser Pro65 70 75 80His Asp Leu Ser Leu Ala Ser Leu Leu Gly Ala
Gly Ala Leu Arg Pro 85 90 95Pro Pro Gly Ser Arg Pro Val Ser Gln Pro
Cys Cys Arg Pro Thr Arg 100 105 110Tyr Glu Ala Val Ser Phe Met Asp
Val Asn Ser Thr Trp Arg Thr Val 115 120 125Asp Arg Leu Ser Ala Thr
Ala Cys Gly Cys Leu Gly 130 135 14010140PRTHomo
sapiensCARBOHYD(122)glycosylated asparagine 10Pro Pro Pro Gln Pro
Ser Arg Pro Ala Pro Pro Pro Pro Ala Pro Pro1 5 10 15Ser Ala Leu Pro
Arg Gly Gly Arg Ala Ala Arg Ala Gly Gly Pro Gly 20 25 30Ser Arg Ala
Arg Ala Ala Gly Ala Arg Gly Cys Arg Leu Arg Ser Gln 35 40 45Leu Val
Pro Val Arg Ala Leu Gly Leu Gly His Arg Ser Asp Glu Leu 50 55 60Val
Arg Phe Arg Phe Cys Ser Gly Ser Cys Arg Arg Ala Arg Ser Pro65 70 75
80His Asp Leu Ser Leu Ala Ser Leu Leu Gly Ala Gly Ala Leu Arg Pro
85 90 95Pro Pro Gly Ser Arg Pro Val Ser Gln Pro Cys Cys Arg Pro Thr
Arg 100
105 110Tyr Glu Ala Val Ser Phe Met Asp Val Asn Ser Thr Trp Arg Thr
Val 115 120 125Asp Arg Leu Ser Ala Thr Ala Cys Gly Cys Leu Gly 130
135 14011116PRTHomo sapiensCARBOHYD(98)glycosylated asparagine
11Ala Ala Arg Ala Gly Gly Pro Gly Ser Arg Ala Arg Ala Ala Gly Ala1
5 10 15Arg Gly Cys Arg Leu Arg Ser Gln Leu Val Pro Val Arg Ala Leu
Gly 20 25 30Leu Gly His Arg Ser Asp Glu Leu Val Arg Phe Arg Phe Cys
Ser Gly 35 40 45Ser Cys Arg Arg Ala Arg Ser Pro His Asp Leu Ser Leu
Ala Ser Leu 50 55 60Leu Gly Ala Gly Ala Leu Arg Pro Pro Pro Gly Ser
Arg Pro Val Ser65 70 75 80Gln Pro Cys Cys Arg Pro Thr Arg Tyr Glu
Ala Val Ser Phe Met Asp 85 90 95Val Asn Ser Thr Trp Arg Thr Val Asp
Arg Leu Ser Ala Thr Ala Cys 100 105 110Gly Cys Leu Gly
11512113PRTHomo sapiensCARBOHYD(95)glycosylated asparagine 12Ala
Gly Gly Pro Gly Ser Arg Ala Arg Ala Ala Gly Ala Arg Gly Cys1 5 10
15Arg Leu Arg Ser Gln Leu Val Pro Val Arg Ala Leu Gly Leu Gly His
20 25 30Arg Ser Asp Glu Leu Val Arg Phe Arg Phe Cys Ser Gly Ser Cys
Arg 35 40 45Arg Ala Arg Ser Pro His Asp Leu Ser Leu Ala Ser Leu Leu
Gly Ala 50 55 60Gly Ala Leu Arg Pro Pro Pro Gly Ser Arg Pro Val Ser
Gln Pro Cys65 70 75 80Cys Arg Pro Thr Arg Tyr Glu Ala Val Ser Phe
Met Asp Val Asn Ser 85 90 95Thr Trp Arg Thr Val Asp Arg Leu Ser Ala
Thr Ala Cys Gly Cys Leu 100 105 110Gly 13102DNAHomo sapiens
13cctggccagc ctactgggcg ccggggccct gcgaccgccc ccgggctccc ggcccgtcag
60ccagccctgc tgccgaccca cgcgctacga agcggtctcc tt 10214220DNAMurinae
gen. sp. 14ggccaccgct ccgacgagct gatacgtttc cgcttctgca gcggctcgtg
ccgccgagca 60cgctcccagc acgatctcag tctggccagc ctactgggcg ctggggccct
acggtcgcct 120cccgggtccc ggccgatcag ccagccctgc tgccggccca
ctcgctatga ggccgtctcc 180ttcatggacg tgaacagcac ctggagaacc
gtggaccgcc 220152136DNAMurinae gen. sp.CDS(975)..(1646)
15gcggccgcga attcggcacg agggcgtctc gctgcagccc gcgatctcta ctctgcctcc
60tggggtcttc tccaaatgtc tagcccccac ctagagggac ctagcctagc cagcggggac
120cggatccgga gggtggagcg gccaggtgag ccctgaaagg tggggcgggg
cgggggcgct 180ctgggcccca ccccgggatc tggtgacgcc ggggctggaa
tttgacaccg gacggcggcg 240ggcaggaggc tgctgaggga tggagttggg
ctcggccccc agatgcggcc cgcgggctct 300gccagcaaca agtccctcgg
gccccagccc tcgctgcgac tggggcttgg agccctgcac 360ccaagggcac
agaccggctg ccaaggcccc acttttaact aaaagaggcg ctgccaggtg
420cacaactctg ggcatgatcc acttgagctt cgggggaaag cccagcactg
gtcccaggag 480aggcgcctag aaggacacgg accaggaccc ctttggtatg
gagtgaacgc tgagcatgga 540gtggaaggaa ctcaagttac tactttctcc
aaccaccctg gtaccttcag ccctgaagta 600cagagcagaa gggtcttaga
agacaggacc acagctgtgt gagtctcccc cctgaggcct 660tagacgatct
ctgagctcag ctgagctttg tttgcccatc tggagaagtg agccattgat
720tgaccttgtg gcatcgcgaa ggaacaggtc ctgccaagca cctaacacag
agagcaaggt 780tctccatcgc agctaccgct gctgagttga ctctagctac
tccaacctcc tgggtcgctt 840cgagagactg gagtggaagg aggaataccc
caaaggataa ctaactcatc tttcagtttg 900caagctgccg caggaagagg
gtggggaaac gggtccacga aggcttctga tgggagcttc 960tggagccgaa agct atg
gaa ctg gga ctt gca gag cct act gca ttg tcc 1010 Met Glu Leu Gly
Leu Ala Glu Pro Thr Ala Leu Ser 1 5 10cac tgc ctc cgg cct agg tgg
cag tca gcc tgg tgg cca acc cta gct 1058His Cys Leu Arg Pro Arg Trp
Gln Ser Ala Trp Trp Pro Thr Leu Ala 15 20 25gtt cta gcc ctg ctg agc
tgc gtc aca gaa gct tcc ctg gac cca atg 1106Val Leu Ala Leu Leu Ser
Cys Val Thr Glu Ala Ser Leu Asp Pro Met 30 35 40tcc cgc agc ccc gcc
gct cgc gac ggt ccc tca ccg gtc ttg gcg ccc 1154Ser Arg Ser Pro Ala
Ala Arg Asp Gly Pro Ser Pro Val Leu Ala Pro45 50 55 60ccc acg gac
cac ctg cct ggg gga cac act gcg cat ttg tgc agc gaa 1202Pro Thr Asp
His Leu Pro Gly Gly His Thr Ala His Leu Cys Ser Glu 65 70 75aga acc
ctg cga ccc ccg cct cag tct cct cag ccc gca ccc ccg ccg 1250Arg Thr
Leu Arg Pro Pro Pro Gln Ser Pro Gln Pro Ala Pro Pro Pro 80 85 90cct
ggt ccc gcg ctc cag tct cct ccc gct gcg ctc cgc ggg gca cgc 1298Pro
Gly Pro Ala Leu Gln Ser Pro Pro Ala Ala Leu Arg Gly Ala Arg 95 100
105gcg gcg cgt gca gga acc cgg agc agc cgc gca cgg acc aca gat gcg
1346Ala Ala Arg Ala Gly Thr Arg Ser Ser Arg Ala Arg Thr Thr Asp Ala
110 115 120cgc ggc tgc cgc ctg cgc tcg cag ctg gtg ccg gtg agc gcg
ctc ggc 1394Arg Gly Cys Arg Leu Arg Ser Gln Leu Val Pro Val Ser Ala
Leu Gly125 130 135 140cta ggc cac agc tcc gac gag ctg ata cgt ttc
cgc ttc tgc agc ggc 1442Leu Gly His Ser Ser Asp Glu Leu Ile Arg Phe
Arg Phe Cys Ser Gly 145 150 155tcg tgc cgc cga gca cgc tcc cag cac
gat ctc agt ctg gcc agc cta 1490Ser Cys Arg Arg Ala Arg Ser Gln His
Asp Leu Ser Leu Ala Ser Leu 160 165 170ctg ggc gct ggg gcc cta cgg
tcg cct ccc ggg tcc cgg ccg atc agc 1538Leu Gly Ala Gly Ala Leu Arg
Ser Pro Pro Gly Ser Arg Pro Ile Ser 175 180 185cag ccc tgc tgc cgg
ccc act cgc tat gag gcc gtc tcc ttc atg gac 1586Gln Pro Cys Cys Arg
Pro Thr Arg Tyr Glu Ala Val Ser Phe Met Asp 190 195 200gtg aac agc
acc tgg agg acc gtg gac cac ctc tcc gcc act gcc tgc 1634Val Asn Ser
Thr Trp Arg Thr Val Asp His Leu Ser Ala Thr Ala Cys205 210 215
220ggc tgt ctg ggc tgaggatgat ctatctccaa gcctttgcac actagaccca
1686Gly Cys Leu Glytgtgttgccc tacctggaac agctccaccg ggcctcacta
accaggagcc tcaactcagc 1746aggatatgga ggctgcagag ctcaggcccc
aggccggtga gtgacagacg tcgtcggcat 1806gacagacaga gtgaaagatg
tcggaaccac tgaccaacag tcccaagttg ttcatggatc 1866ccagctctac
agacaggaga aacctcagct aaagagaact cctctgggag aatccagaaa
1926tggccctctg tcctggggaa tgaattttga agagatatat atacatatat
acattgtagt 1986cgcgttgctg gaccagcctg tgctgaaacc agtcccgtgt
tcacttgtgg aagccgaagc 2046cctatttatt atttctaaat tatttattta
ctttgaaaaa aaacggccaa gtcggcctcc 2106ctttagtgag ggttaatttg
tgatcccggg 213616224PRTMurinae gen. sp. 16Met Glu Leu Gly Leu Ala
Glu Pro Thr Ala Leu Ser His Cys Leu Arg1 5 10 15Pro Arg Trp Gln Ser
Ala Trp Trp Pro Thr Leu Ala Val Leu Ala Leu 20 25 30Leu Ser Cys Val
Thr Glu Ala Ser Leu Asp Pro Met Ser Arg Ser Pro 35 40 45Ala Ala Arg
Asp Gly Pro Ser Pro Val Leu Ala Pro Pro Thr Asp His 50 55 60Leu Pro
Gly Gly His Thr Ala His Leu Cys Ser Glu Arg Thr Leu Arg65 70 75
80Pro Pro Pro Gln Ser Pro Gln Pro Ala Pro Pro Pro Pro Gly Pro Ala
85 90 95Leu Gln Ser Pro Pro Ala Ala Leu Arg Gly Ala Arg Ala Ala Arg
Ala 100 105 110Gly Thr Arg Ser Ser Arg Ala Arg Thr Thr Asp Ala Arg
Gly Cys Arg 115 120 125Leu Arg Ser Gln Leu Val Pro Val Ser Ala Leu
Gly Leu Gly His Ser 130 135 140Ser Asp Glu Leu Ile Arg Phe Arg Phe
Cys Ser Gly Ser Cys Arg Arg145 150 155 160Ala Arg Ser Gln His Asp
Leu Ser Leu Ala Ser Leu Leu Gly Ala Gly 165 170 175Ala Leu Arg Ser
Pro Pro Gly Ser Arg Pro Ile Ser Gln Pro Cys Cys 180 185 190Arg Pro
Thr Arg Tyr Glu Ala Val Ser Phe Met Asp Val Asn Ser Thr 195 200
205Trp Arg Thr Val Asp His Leu Ser Ala Thr Ala Cys Gly Cys Leu Gly
210 215 2201718DNAArtificial SequenceDescription of Artificial
Sequence PCR Primer 17cctggccagc ctactggg 181820DNAArtificial
SequenceDescription of Artificial Sequence PCR Primer 18aaggagaccg
cttcgtagcg 201917DNAArtificial SequenceDescription of Artificial
Sequence PCR Primer 19atggaacttg gacttgg 172016DNAArtificial
SequenceDescription of Artificial Sequence PCR Primer 20tccatcaccc
accggc 162118DNAArtificial SequenceDescription of Artificial
Sequence PCR Primer 21ggccaccgct ccgacgag 182220DNAArtificial
SequenceDescription of Artificial Sequence PCR Primer 22ggcggtccac
ggttctccag 202329DNAArtificial SequenceDescription of Artificial
Sequence PCR Primer 23ccaagcccac ctgggtgccc tctttctcc
292427DNAArtificial SequenceDescription of Artificial Sequence PCR
Primer 24catcacccac cggcaggggc ctctcag 272535DNAArtificial
SequenceDescription of Artificial Sequence PCR Primer 25gagcccatgc
ccggcctgat ctcagcccga ggaca 352634DNAArtificial SequenceDescription
of Artificial Sequence PCR Primer 26ccctggctga ggccgctggc
tagtgggact ctgc 342731DNAArtificial SequenceDescription of
Artificial Sequence Hybridization Probe 27ncaggtggtc cgtggggggc
gccaagaccg g 312816DNAArtificial SequenceDescription of Artificial
Sequence PCR primer 28ctaggagccc atgccc 1629351DNAHomo sapiens
29atggctggag gaccgggatc tcgtgctcgt gcagcaggag cacgtggctg tcgtctgcgt
60tctcaactag tgccggtgcg tgcactcgga ctgggacacc gttccgacga actagtacgt
120tttcgttttt gttcaggatc ttgtcgtcgt gcacgttctc cgcatgatct
atctctagca 180tctctactag gagccggagc actaagaccg ccgccgggat
ctagacctgt atctcaacct 240tgttgtagac ctactagata cgaagcagta
tctttcatgg acgtaaactc tacatggaga 300accgtagata gactatctgc
aaccgcatgt ggctgtctag gatgataata g 35130414DNAHomo sapiens
30atgggccatc atcatcatca tcatcatcat catcactcga gcggccatat cgacgacgac
60gacaaggctg gaggaccggg atctcgtgct cgtgcagcag gagcacgtgg ctgtcgtctg
120cgttctcaac tagtgccggt gcgtgcactc ggactgggac accgttccga
cgaactagta 180cgttttcgtt tttgttcagg atcttgtcgt cgtgcacgtt
ctccgcatga tctatctcta 240gcatctctac taggagccgg agcactaaga
ccgccgccgg gatctagacc tgtatctcaa 300ccttgttgta gacctactag
atacgaagca gtatctttca tggacgtaaa ctctacatgg 360agaaccgtag
atagactatc tgcaaccgca tgtggctgtc taggatgata atag
4143139DNAArtificial SequenceDescription of Artificial Sequence PCR
primer 31aaggaaaaaa gcggccgcca tggaacttgg acttggagg
393239DNAArtificial SequenceDescription of Artificial Sequence PCR
primer 32ttttttcctt ggcggccgct cagcccaggc agccgcagg
393316DNAArtificial SequenceDescription of Artificial Sequence
primer 33gagcgagccc tcagcc 1634197PRTHomo sapiens 34Met Gln Arg Trp
Lys Ala Ala Ala Leu Ala Ser Val Leu Cys Ser Ser1 5 10 15Val Leu Ser
Ile Trp Met Cys Arg Glu Gly Leu Leu Leu Ser His Arg 20 25 30Leu Gly
Pro Ala Leu Val Pro Leu His Arg Leu Pro Arg Thr Leu Asp 35 40 45Ala
Arg Ile Ala Arg Leu Ala Gln Tyr Arg Ala Leu Leu Gln Gly Ala 50 55
60Pro Asp Ala Met Glu Leu Arg Glu Leu Thr Pro Trp Ala Gly Arg Pro65
70 75 80Pro Gly Pro Arg Arg Arg Ala Gly Pro Arg Arg Arg Arg Ala Arg
Ala 85 90 95Arg Leu Gly Ala Arg Pro Cys Gly Leu Arg Glu Leu Glu Val
Arg Val 100 105 110Ser Glu Leu Gly Leu Gly Tyr Ala Ser Asp Glu Thr
Val Leu Phe Arg 115 120 125Tyr Cys Ala Gly Ala Cys Glu Ala Ala Ala
Arg Val Tyr Asp Leu Gly 130 135 140Leu Arg Arg Leu Arg Gln Arg Arg
Arg Leu Arg Arg Glu Arg Val Arg145 150 155 160Ala Gln Pro Cys Cys
Arg Pro Thr Ala Tyr Glu Asp Glu Val Ser Phe 165 170 175Leu Asp Ala
His Ser Arg Tyr His Thr Val His Glu Leu Ser Ala Arg 180 185 190Glu
Cys Ala Cys Val 19535220PRTHomo sapiens 35Met Glu Leu Gly Leu Gly
Gly Leu Ser Thr Leu Ser His Cys Pro Trp1 5 10 15Pro Arg Arg Gln Pro
Ala Leu Trp Pro Thr Leu Ala Ala Leu Ala Leu 20 25 30Leu Ser Ser Val
Ala Glu Ala Ser Leu Gly Ser Ala Pro Arg Ser Pro 35 40 45Ala Pro Arg
Glu Gly Pro Pro Pro Val Leu Ala Ser Pro Ala Gly His 50 55 60Leu Pro
Gly Gly Arg Thr Ala Arg Trp Cys Ser Gly Arg Ala Arg Arg65 70 75
80Pro Pro Pro Gln Pro Ser Arg Pro Ala Pro Pro Pro Pro Ala Pro Pro
85 90 95Ser Ala Leu Pro Arg Gly Gly Arg Ala Ala Arg Ala Gly Gly Pro
Gly 100 105 110Ser Arg Ala Arg Ala Ala Gly Ala Arg Gly Cys Arg Leu
Arg Ser Gln 115 120 125Leu Val Pro Val Arg Ala Leu Gly Leu Gly His
Arg Ser Asp Glu Leu 130 135 140Val Arg Phe Arg Phe Cys Ser Gly Ser
Cys Arg Arg Ala Arg Ser Pro145 150 155 160His Asp Leu Ser Leu Ala
Ser Leu Leu Gly Ala Gly Ala Leu Arg Pro 165 170 175Pro Pro Gly Ser
Arg Pro Val Ser Gln Pro Cys Cys Arg Pro Thr Arg 180 185 190Tyr Glu
Ala Val Ser Phe Met Asp Val Asn Ser Thr Trp Arg Thr Val 195 200
205Asp Arg Leu Ser Ala Thr Ala Cys Gly Cys Leu Gly 210 215
22036156PRTHomo sapiens 36Met Ala Val Gly Lys Phe Leu Leu Gly Ser
Leu Leu Leu Leu Ser Leu1 5 10 15Gln Leu Gly Gln Gly Trp Gly Pro Asp
Ala Arg Gly Val Pro Val Ala 20 25 30Asp Gly Glu Phe Ser Ser Glu Gln
Val Ala Lys Ala Gly Gly Thr Trp 35 40 45Leu Gly Thr His Arg Pro Leu
Ala Arg Leu Arg Arg Ala Leu Ser Gly 50 55 60Pro Cys Gln Leu Trp Ser
Leu Thr Leu Ser Val Ala Glu Leu Gly Leu65 70 75 80Gly Tyr Ala Ser
Glu Glu Lys Val Ile Phe Arg Tyr Cys Ala Gly Ser 85 90 95Cys Pro Arg
Gly Ala Arg Thr Gln His Gly Leu Ala Leu Ala Arg Leu 100 105 110Gln
Gly Gln Gly Arg Ala His Gly Gly Pro Cys Cys Arg Pro Thr Arg 115 120
125Tyr Thr Asp Val Ala Phe Leu Asp Asp Arg His Arg Trp Gln Arg Leu
130 135 140Pro Gln Leu Ser Ala Ala Ala Cys Gly Cys Gly Gly145 150
15537211PRTHomo sapiens 37Met Lys Leu Trp Asp Val Val Ala Val Cys
Leu Val Leu Leu His Thr1 5 10 15Ala Ser Ala Phe Pro Leu Pro Ala Gly
Lys Arg Pro Pro Glu Ala Pro 20 25 30Ala Glu Asp Arg Ser Leu Gly Arg
Arg Arg Ala Pro Phe Ala Leu Ser 35 40 45Ser Asp Ser Asn Met Pro Glu
Asp Tyr Pro Asp Gln Phe Asp Asp Val 50 55 60Met Asp Phe Ile Gln Ala
Thr Ile Lys Arg Leu Lys Arg Ser Pro Asp65 70 75 80Lys Gln Met Ala
Val Leu Pro Arg Arg Glu Arg Asn Arg Gln Ala Ala 85 90 95Ala Ala Asn
Pro Glu Asn Ser Arg Gly Lys Gly Arg Arg Gly Gln Arg 100 105 110Gly
Lys Asn Arg Gly Cys Val Leu Thr Ala Ile His Leu Asn Val Thr 115 120
125Asp Leu Gly Leu Gly Tyr Glu Thr Lys Glu Glu Leu Ile Phe Arg Tyr
130 135 140Cys Ser Gly Ser Cys Asp Ala Ala Glu Thr Thr Tyr Asp Lys
Ile Leu145 150 155 160Lys Asn Leu Ser Arg Asn Arg Arg Leu Val Ser
Asp Lys Val Gly Gln 165 170 175Ala Cys Cys Arg Pro Ile Ala Phe Asp
Asp Asp Leu Ser Phe Leu Asp 180 185 190Asp Asn Leu Val Tyr His Ile
Leu Arg Lys His Ser Ala Lys Arg Cys 195 200 205Gly Cys Ile
2103818DNAArtificial SequenceDescription of Artificial Sequence
primer 38gctggcccgg ctgcaggg 183918DNAArtificial
SequenceDescription of Artificial Sequence primer 39gctgcgacga
ctgcgcca 184018DNAArtificial SequenceDescription of Artificial
Sequence primer 40attgaaaaac ttatccag
184120DNAArtificial SequenceDescription of Artificial Sequence
primer 41taggccacgt cggtgtagcg 204223DNAArtificial
SequenceDescription of Artificial Sequence primer 42aaggacacct
cgtcctcgta ggc 234323DNAArtificial SequenceDescription of
Artificial Sequence primer 43aacgacaggt catcatcaaa ggc 23
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