U.S. patent application number 11/074497 was filed with the patent office on 2006-06-08 for method for treating or preventing a neural disorder with a neurotrophic growth factor.
Invention is credited to Hugo Alfons Gabriel Geerts, Stefan Leo Jozef Masure, Theo Frans Meert.
Application Number | 20060122135 11/074497 |
Document ID | / |
Family ID | 36575118 |
Filed Date | 2006-06-08 |
United States Patent
Application |
20060122135 |
Kind Code |
A1 |
Gabriel Geerts; Hugo Alfons ;
et al. |
June 8, 2006 |
Method for treating or preventing a neural disorder with a
neurotrophic growth factor
Abstract
There is disclosed an isolated nucleic acid molecule encoding a
human neurotrophic growth factor designated enovin and having the
amino acid sequence illustrated in FIG. 1, 21, 23 or 24 or encoding
a functional equivalent, derivative or bioprecursor of said growth
factor. The growth factor preferably comprises the amino acid
sequence from position 27 to 139 of the sequence illustrated in
FIG. 1, or a functional equivalent, derivative or bioprecursor
thereof. The nucleic acid molecule encoding enovin can be used to
transform a host cell, tissue or organism by including it in an
appropriate vector. The host cell, tissue or organism and the
vector also form part of the invention.
Inventors: |
Gabriel Geerts; Hugo Alfons;
(Berwyn, PA) ; Masure; Stefan Leo Jozef;
(Brasschaat, BE) ; Meert; Theo Frans; (Boom,
BE) |
Correspondence
Address: |
GREENBERG TRAURIG, LLP
MET LIFE BUILDING
200 PARK AVENUE
NEW YORK
NY
10166
US
|
Family ID: |
36575118 |
Appl. No.: |
11/074497 |
Filed: |
March 8, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09357349 |
Jul 14, 1999 |
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11074497 |
Mar 8, 2005 |
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09327668 |
Jun 8, 1999 |
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11074497 |
Mar 8, 2005 |
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09248772 |
Feb 12, 1999 |
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11074497 |
Mar 8, 2005 |
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Current U.S.
Class: |
514/44R ;
435/320.1; 435/325; 435/6.16; 435/69.1; 514/17.7; 514/17.8;
514/17.9; 514/18.2; 514/6.9; 514/8.4; 514/8.9; 530/399;
536/23.5 |
Current CPC
Class: |
C07K 14/475 20130101;
C07K 14/48 20130101; A61K 38/00 20130101; A61P 25/02 20180101; A61P
25/00 20180101 |
Class at
Publication: |
514/044 ;
435/006; 435/069.1; 435/320.1; 435/325; 530/399; 536/023.5;
514/012 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C12Q 1/68 20060101 C12Q001/68; C07H 21/04 20060101
C07H021/04; C12P 21/06 20060101 C12P021/06; C07K 14/475 20060101
C07K014/475; A61K 38/18 20060101 A61K038/18 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 14, 1998 |
GB |
9815283.8 |
Claims
1-57. (canceled)
58. A method for treating or preventing a neural disorder in a
subject the method comprising administering to the subject an
amount of a human neurotrophic growth factor polypeptide comprising
the amino acid sequence of SEQ ID NO:3, or a functional equivalent
derivative thereof, in a sufficient concentration to reduce or
prevent the symptoms of the neural disorder.
59. A method according to claim 58, wherein the neural disorder is
selected from the group consisting of: Parkinson's disease,
Alzheimer's disease, neuronal disorders associated with expanded
polyglutamine sequences such as Huntington's disease, peripheral
neuropathy, neuropathic pain; acute brain injury, nervous system
tumors, multiple sclerosis, amyotrophic lateral sclerosis,
peripheral nerve trauma, injury exposure to neurotoxins, multiple
endocrine neoplasia, familial Hirschsprung disease, prion
associated diseases, Creutzfeld-Jacob disease, cancer or
stroke.
60. A method according to claim 58, wherein the functional
equivalent derivative comprises an amino acid sequence having at
least 90% homology to SEQ ID NO:3.
61. A method according to claim 60, wherein the functional
equivalent derivative comprises SEQ ID NO:9 or SEQ ID NO:10.
Description
[0001] The present invention is concerned with a neurotrophic
factor and, in particular, with cloning and expression of a novel
member of the GDNF family of neurotrophic factors, designated
herein as "enovin" (EVN).
INTRODUCTION
[0002] Neurotrophic factors are involved in neuronal
differentiation, development and maintenance. These proteins can
prevent degeneration and promote survival of different types of
neuronal cells and are thus potential therapeutic agents for
neurodegenerative diseases. Glial cell-line derived neurotrophic
factor (GDNF) was the first member of a growing subfamily of
neurotrophic factors structurally distinct from the neurotrophins.
GDNF is a member of the transforming growth factor .beta.
(TGF-.beta.) superfamily of growth factors, characterized by a
specific pattern of seven highly conserved cysteine residues within
the amino acid sequence (Kingsley, 1994). GDNF was originally
purified using an assay based on its ability to maintain the
survival and function of embryonic ventral midbrain dopaminergic
neurons in vitro (Lin et al., 1993). Other neuronal cell types in
the central (CNS) or peripheral nervous systems (PNS) are also
responsive to the survival effects of GDNF (Henderson et al., 1994,
Buj-Bello et al., 1995, Mount et al., 1995, Oppenheim et al.,
1995). GDNF is produced by cells in an inactive proform, which is
cleaved specifically at a RXXR furin recognition site to produce
active (mature) GDNF (Lin et al., 1993). Exogenous administration
of GDNF has potent neuroprotective effects in animal models of
Parkinson's disease, a common neurodegenerative disorder
characterised by the loss of up to 70% of dopaminergic cells in the
substantia nigra of the brain (Beck et al., 1995, Tomac et al.,
1995, Gash et al., 1996, Choi-Lundberg et al., 1997, Bilang-Bleuel
et al., 1997).
[0003] Recently, additional neurotrophic factors of the GDNF family
have been discovered. Neurturin (NTN) was purified from conditioned
medium from Chinese hamster ovary (CHO) cells using an assay based
on its ability to promote the survival of sympathetic neurons in
culture (Kotzbauer et al., 1996). The mature NTN protein is 57%
similar to mature GDNF. Persephin (PSP) was discovered by cloning
using degenerate primer PCR with genomic DNA as a template. The
mature PSP, like mature GDNF, promotes the survival of ventral
midbrain dopaminergic neurons and of motor neurons in culture
(Milbrandt et al., 1998). The similarity of the mature PSP protein
with mature GDNF and NTN is .apprxeq.50%. Artemin (ARTN) was
discovered by DNA database searching and is a survival factor of
sensory and sympathetic neurons in culture (Baloh et al,
1998b).
[0004] GDNF, NTN, PSP and ARTN require a heterodimeric receptor
complex in order to carry out downstream intracellular signal
transduction. GDNF binds to the GDNF family receptor alpha 1
(GFR.alpha.-1; GFR.alpha. Nomenclature Committee, 1997) subunit, a
glycosyl-phosphatidyl-inositol (glycosyl-PtdIns) anchored membrane
protein (Jing et al., 1996, Treanor et al., 1996, Sanicola et al.,
1997). The GDNF/GFR.alpha.-1 complex subsequently binds to and
activates the cRET proto-oncogene, a membrane bound tyrosine kinase
(Durbec et al., 1996, Trupp et al., 1996), resulting in the
phosphorylation of tyrosine residues in cRET and subsequent
activation of downstream signal transduction pathways (Worby et
al., 1996). Several other members of the GFR.alpha. family of
ligand binding receptors have been characterised (Baloh et al.,
1997, Sanicola et al., 1997, Klein et al., 1997, Buj-Bello et al.,
1997, Suvanto et al., 1997). GFR.alpha.-2 and GFR.alpha.-3 (Jing et
al., 1997, Masure et al., 1998, Woby et al., 1998, Naveilham et
al., 1998, Baloh et al., 1998a) have been identified by a number of
different groups. GFR.alpha.-1 and GFR.alpha.-2 are widely
expressed in almost all tissues and expression may be
developmentally regulated (Sanicola et al., 1997, Widenfalk et al.,
1997).
[0005] GFR.alpha.-3 is not expressed in the developing or adult
central nervous system, but is highly expressed in several
developing and adult sensory and sympathetic ganglia of the
peripheral nervous system (Widenfalk et al., 1998, Naveilhan et
al., 1998, Baloh et al., 1998a). A fourth family member,
GFR.alpha.-4, was cloned from chicken cDNA (Thompson et al., 1998).
GFR.alpha.-1 is the preferred receptor for GDNF, whereas
GFR.alpha.-2 preferentially binds NTN (Jing et al., 1996, Treanor
et al., 1996, Klein et al., 1997). Chicken GFR.alpha.-4 forms a
functional receptor complex for PSP in combination with cRET
(Enokido et al., 1998). Cells expressing both GFR.alpha.-3 and cRET
were shown not to respond to either GDNF, NTN or PSP (Worby et al.,
1998, Baloh et al., 1998a). Recently, ART has been shown to signal
through cRET using GFR.alpha.-3 as the preferred ligand-binding
receptor (Baloh et al., 1998b). Cross-talk between the neurotrophic
factors and GFR.alpha. receptors is possible in vitro, as GDNF can
bind to GFR.alpha.-2 or GFR.alpha.-3 in the presence of cRET
(Sanicola et al., 1997, Trupp et al., 1998) and NTN can bind to
GFR.alpha.-1 with low affinity (Klein et al., 1997). In summary,
GDNF, NTN, PSP and ART are part of a neurotrophic signalling system
whereby different ligand-binding subunits (GFR.alpha.-1 to -4) can
interact with the same Tyrosine kinase subunit (cRET). The
physiological relevance of these in vitro findings was recently
shown in gene knockout studies (reviewed by Rosenthal, 1999), which
clearly show that GDNF interacts with GFR.alpha.-1 in vivo, whereas
NTN is the preferred ligand for GFR.alpha.-2.
[0006] The present inventors have identified, cloned, expressed,
chromosomally localized and characterized Enovin (EVN), the fourth
member of the GDNF family. The knowledge of the mature EVN protein
has been extended with the discovery of different functional and
non-functional mRNA splice variants. Moreover, we present
expression data, binding data of EVN to GFR.alpha.-3 and in vitro
effects of EVN on neurite outgrowth and protection against
taxol-induced neurotoxicity in staurosporine-differentiated SH-SY5Y
human neuroblastoma cell cultures.
SUMMARY OF THE INVENTION
[0007] In the present application, there is provided a nucleic acid
molecule encoding a novel human neurotrophic growth factor,
"enovin", an expression vector comprising said nucleic acid
molecule, a host cell transformed with said vector, a neurotrophic
growth factor encoded by said nucleic acid molecule, isolated
enovin, compounds which act as agonists or antagonists of enovin
and pharmaceutical compositions containing the nucleic acid or the
enovin protein or the agonists or antagonists thereof.
DETAILED DESCRIPTION OF THE INVENTION
[0008] According to a first aspect of the present invention there
is provided a nucleic acid molecule encoding a human neurotrophic
growth factor, designated herein as enovin, having the amino acid
sequence illustrated in FIG. 21, or encoding a functional
equivalent, derivative or bioprecursor of said growth factor.
Preferably, said nucleic acid molecule is DNA and even more
preferably a cDNA molecule.
[0009] Preferably, the nucleic acid according to the invention
comprises the sequence from positions 81 to 419 of the sequence
illustrated in FIG. 1 and more preferably from positions 81 to 422
and even more preferably the complete sequence illustrated in FIG.
1.
[0010] The nucleic acid molecule from position 81 to 419 is
believed to encode the sequence of the mature enovin protein
subsequent to processing of the proform of the protein at the RXXR
processing site present in the stable proform of said enovin
protein.
[0011] There is also provided by the invention an antisense
molecule capable of hybridising to any of the nucleic acid
sequences according to the invention, under high stringency
conditions, which would be well known to those skilled in the
art.
[0012] Stringency of hybridisation as used herein refers to
conditions under which polynucleic acids are stable. The stability
of hybrids is reflected in the melting temperature (Tm) of the
hybrids. Tm can be approximated by the formula: 81.5.degree.
C.-16.6(log 10[Na.sup.+]+0.41(% G&C)-600/l wherein 1 is the
length of the hybrids in nucleotides. Tm decreases approximately by
1-1.5.degree. C. with every 1% decrease in sequence homology.
[0013] Advantageously, the nucleic acid molecule according to the
invention may be used to express the human neurotrophic growth
factor according to the invention, in a host cell or the like using
an appropriate expression vector.
[0014] An expression vector according to the invention includes
vectors capable of expressing DNA operatively linked to regulatory
sequences, such as promoter regions, that are capable of effecting
expression of such DNA fragments.
[0015] Regulatory elements required for expression include promoter
sequences to bind RNA polymerase and transcription initiation
sequences for ribosome binding. For example, a bacterial expression
vector may include a promoter such as the lac promoter and for
transcription initiation the Shine-Dalgarno sequence and the start
codon AUG. Similarly, a eukaryotic expression vector may include a
heterologous or homologous promoter for RNA polymerase II, a
downstream polyadenylation signal, the start codon AUG, and a
termination codon for detachment of the ribosome. Such vectors may
be obtained commercially or assembled from the sequences described
by methods well known in the art.
[0016] Thus, an expression vector refers to a recombinant DNA or
RNA construct, such as a plasmid, a phage, recombinant virus or
other vector that upon introduction into an appropriate host cell
results in expression of the DNA or RNA fragments. Appropriate
expression vectors are well known to those skilled in the art and
include those that are replicable in eukaryotic cells and/or
prokaryotic cells and those that remain episomal or those which
integrate into the host cell genome.
[0017] The antisense molecule capable of hybridising to the nucleic
acid according to the invention may be used as a probe or as a
medicament or in a pharmaceutical composition.
[0018] Nucleic acid molecules according to the invention may be
inserted into the vectors described in an antisense orientation in
order to provide for the production of antisense RNA. Antisense RNA
or other antisense nucleic acids may be produced by synthetic
means.
[0019] A further aspect of the invention comprises the host cell
transformed, transfected or infected with the expression vector
according to the invention, which cell preferably comprises a
eukaryotic cell and more preferably a mammalian cell.
[0020] Incorporation of cloned DNA into a suitable expression
vector for subsequent transformation of said cell and subsequent
selection of the transformed cells is well known to those skilled
in the art as provided in Sambrook et al (1989) Molecular Cloning,
A Laboratory manual, Cold Spring Harbour Laboratory Press.
[0021] A further aspect of the present invention comprises a
nucleic acid molecule having at least 15 nucleotides of the nucleic
acid molecule according to the invention and preferably from 15 to
50 nucleotides.
[0022] These sequences may, advantageously be used as probes or
primers to initiate replication or the like. Such nucleic acid
molecules may be produced according to techniques well known in the
art, such as by recombinant or synthetic means. They may also be
used in diagnostic kits or devices or the like for detecting for
the presence of a nucleic acid according to the invention. These
tests generally comprise contacting the probe with a sample under
hybridising conditions and detecting for the presence of any duplex
formation between the probe and any nucleic acid in the sample.
[0023] According to the present invention these probes may be
anchored to a solid support. Preferably, they are present on an
array so that multiple probes can simultaneously hybridize to a
single biological sample. The probes can be spotted onto the array
or synthesised in situ on the array. (See Lockhart et al., Nature
Biotechnology, vol. 14, December 1996 "Expression monitoring by
hybridisation into high density oligonucleotide arrays". A single
array can contain more than 100, 500 or even 1,000 different probes
in discrete locations.
[0024] Nucleic acid molecules according to the invention may also
be produced using such recombinant or synthetic means, such as, for
example, using PCR cloning mechanisms which generally involve
making a pair of primers, which may be from approximately 10 to 50
nucleotides to a region of the gene which is desired to be cloned,
bringing the primers into contact with mRNA, cDNA, or genomic DNA
from a human cell, performing a polymerase chain reaction under
conditions which bring about amplification of the desired region,
isolating the amplified region or fragment and recovering the
amplified DNA. Generally, such techniques as defined herein are
well known in the art, such as described in Sambrook et al
(Molecular Cloning: a Laboratory Manual, 1989).
[0025] The nucleic acids or oligonucleotides according to the
invention may carry a revealing label. Suitable labels include
radioisotopes such as .sup.32P or .sup.35S, enzyme labels or other
protein labels such as biotin or fluorescent markers. Such lables
may be added to the nucleic acids or oligonucleotides of the
invention and may be detected using known techniques per se.
[0026] Advantageously, human allelic variants or polymorphisms of
the DNA molecule according to the invention may be identified by,
for example, probing cDNA or genomic libraries from a range of
individuals for example from different populations. Furthermore,
nucleic acids and probes according to the invention may be used to
sequence genomic DNA from patients using techniques well known in
the art, such as the Sanger Dideoxy chain termination method, which
may advantageously ascertain any predisposition of a patient to
certain disorders associated with a growth factor according to the
invention.
[0027] Further provided by the present invention is a transgenic
cell, tissue or organism comprising a transgene capable of
expressing the human neurotrophic factor enovin according to the
invention.
[0028] The term "transgene capable of expression" as used herein
means any suitable nucleic acid sequence which leads to expression
of a neurotrophic factor having the same function and/or activity
of a neurotrophic factor according to the invention. The transgene
may include, for example, genomic nucleic acid isolated from human
cells or synthetic nucleic acid including cDNA, integrated into the
chromosome or in an extrachromosomal state.
[0029] Preferably, the transgene comprises a vector according to
the invention, which vector includes a nucleic acid molecule
encoding said neurotrophic factor, or a functional fragment of said
nucleic acid molecule. A "functional fragment" of said nucleic acid
should be taken to mean a fragment of the gene or cDNA encoding
said neurotrophic factor or a functional equivalent thereof, which
fragment is capable of being expressed to produce a functional
neurotrophic growth factor according to the invention. Thus, for
example, fragments of the neurotrophic growth factor according to
the invention which correspond to the specific amino acid residues
interacting with the corresponding receptor also form part of the
present invention and which fragments may serve to function as
agonists activating the corresponding receptor of the growth factor
according to the invention so as to elicit its growth promoting and
survival sustaining effects on cells. This aspect of the invention
also includes differentially spliced isoforms and transcriptional
starts of the nucleic acids according to the invention.
[0030] In accordance with the present invention, a defined nucleic
acid includes not only the identical nucleic acid but also any
minor base variations including in particular, substitutions in
bases which result in a synonymous codon (a different codon
specifying the same amino acid residue) due to the degenerate code
in conservative amino acid substitutions. The term "nucleic acid
molecule" also includes the complementary sequence to any single
stranded sequence given regarding base variations.
[0031] According to a further aspect the invention provides an
isolated human neurotrophic growth factor, encoded by a nucleic
acid molecule as defined herein. Preferably, the growth factor
comprises an amino acid sequence from position 27 to 139 of the
amino acid sequence of FIG. 1 or a functional equivalent,
derivative or bioprecursor thereof.
[0032] A "functional equivalent" as defined herein should be taken
to mean a growth factor that exhibits all of the growth properties
and functionality associated with the growth factor enovin. A
"derivative" of enovin as defined herein is intended to include a
polypeptide in which certain amino acids have been altered or
deleted or replaced with other amino acids and which polypeptide
retains the biological activity of enovin and/or which polypeptide
can react with antibodies raised using enovin according to the
invention as the challenging antigen.
[0033] Encompassed within the scope of the present invention are
hybrid and modified forms of enovin, including fusion proteins and
fragments. The hybrid and modified forms include, for example, when
certain amino acids have been subjected to some modification or
replacement, such as for example, by point mutation yet which
modifications still result in a protein which retains the
biological activity of enovin, according to the invention. Specific
nucleic acid sequences can be altered by those of skill in the art
to produce a growth factor exhibiting the same or substantially
properties to enovin.
[0034] As is well known in the art, many proteins are produced in
vivo with a (pre) signal sequence at the N-terminus of the protein.
Furthermore, such proteins may comprise a further pro sequence that
represents a stable precursor to the mature protein. Such pre and
pro sequences are not generally necessary for biological activity.
The enovin molecule according to the invention includes not only
the full length sequence illustrated in FIG. 21 but from position
27 to 139, which follows the RXXR proteolytic processing site
present in growth factors of this type and which is believed to
represent the mature sequence of enovin.
[0035] A defined protein, polypeptide or amino acid sequence
according to the invention includes not only the identical amino
acid sequence but isomers thereof in addition to minor amino acid
variations from the natural amino acid sequence including
conservative amino acid replacements (a replacement by an amino
acid that is related in its side chains). Also included are amino
acid sequences which vary from the natural amino acid but result in
a polypeptide which is immunologically identical or similar to the
polypeptide encoded by the naturally occurring sequence.
[0036] Proteins or polypeptides according to the invention further
include variants of such sequences, including naturally occurring
allelic variants which are substantially homologous to said
proteins or polypeptides. In this context, substantial homology is
regarded as a sequence which has at least 70%, and preferably 80%,
90% or 95% amino acid homology with the proteins or polypeptides
encoded by the nucleic acid molecules according to the
invention.
[0037] Neurotrophic growth factors expressed by the host cells
according to the invention are also encompassed within the present
invention.
[0038] The present invention is further directed to inhibiting the
neurotrophic growth factor according to the invention in vivo by
the use of antisense technology. Antisense technology can be used
to control gene expression through triple-helix formation or
antisense DNA or RNA, both of which methods are based on binding of
a polynucleotide to DNA or RNA. For example, the part of the DNA
sequence coding for the mature protein of the present invention is
used to design an antisense RNA oligonucleotide of from 10 to 50
base pairs in length. A DNA oligonucleotide is designed to be
complementary to a region of the gene involved in transcription
(triple-helix--see Lee et al. Nucl. Acids. Res., 6:3073 (1979);
Cooney et al., Science, 241:456 (1988); and Dervan et al., Science,
251: 1360 (1991), thereby preventing transcription and the
production of enovin. The antisense RNA oligonucleotide hybridises
to the mRNA in vivo and blocks translation of an mRNA molecule into
enovin.
[0039] Because of the sequence similarity between the growth factor
described herein with previously identified growth factors of the
GDNF family, enovin is also believed to be capable of promoting
cell survival and growth and in treating disorders resulting from
defects in function or expression of said neurotrophic factor.
[0040] The nucleic acid molecules or the neurotrophic factor
according to the invention may, advantageously, therefore be used
to treat or prevent neural disorders in a subject by administering
to said subject an amount of a nucleic acid molecule or growth
factor according to the invention in sufficient concentration to
reduce the symptoms of said disorder. Thus, the nucleic acid
molecules of the invention may be used to promote maintenance and
survival of neuronal cells and for treating neuronal disorders or
neurodegenerative conditions including Parkinson's disease,
Alzheimer's disease, peripheral neuropathy, amyotrophic lateral
sclerosis, peripheral and central nerve trauma or injury and
exposure to neurotoxins.
[0041] The neurotrophic growth factor according to the invention
has, advantageously, been observed to confer a neurotrophic or
neuroprotective effect on neuronal cells or cell populations,
particularly those neuronal cells or cell populations which have
been induced to undergo apoptosis. Accordingly, the nucleic acid or
the enovin growth factor itself according to the invention may
additionally be used in treating neurodegenerative disorders such
as stroke, Huntingdons disease, peripheral neuropathy, acute brain
injury, nervous system tumours, multiple sclerosis, amyotrophic
lateral sclerosis, peripheral nerve trauma, injury exposure to
neurotoxins, multiple endocrine neoplasia, familial Hirschsprung
disease, Prion associated diseases, Creutzfeld-Jacob disease by
administering to a patient in need thereof, an amount of said
nucleic acid or enovin in sufficient concentration to reduce or
prevent the symptoms of the neural disorders described herein.
[0042] Additionally, and which is described in more details in the
example below, enovin has been shown to speed up recovery of
induced sensory deficits, which identifies enovin as a candidate
for treating or alleviating pain syndromes with a peripheral or
central neurogenic component, rheumatic/inflammatory diseases as
well as conductance disturbances, by administration to a patient in
need thereof in sufficient concentration to reduce or prevent the
symptoms of these disorders.
[0043] An alternative method for treating the nerve disorders
described above comprises implanting in a subject cells that
express a human neurotrophic growth factor according to the
invention such as the transgenic cell described herein.
[0044] The nucleic acid molecules and neurotrophic growth factor
according to the invention may also be included in a pharmaceutical
composition together with a pharmaceutically acceptable carrier,
diluent or excipient therefor.
[0045] Antibodies to the neurotrophic factor of the present
invention may, advantageously, be prepared by techniques which are
known in the art. For example, polyclonal antibodies may be
prepared by inoculating a host animal such as a mouse with the
growth factor or an epitope thereof and recovering immune serum.
Monoclonal antibodies may be prepared according to known techniques
such as described by Kohler R. and Milstein C., Nature (1975) 256,
495-497.
[0046] Antibodies according to the invention may, advantageously,
be used in a method of detecting for the presence of a growth
factor according to the invention, which method comprises reacting
the antibody with a sample and identifying any protein bound to
said antibody. A kit is also provided for performing said method
which comprises an antibody according to the invention and means
for reacting the antibody with said sample.
[0047] Also provided by the present invention is a kit or device
for detecting for the presence of a neurotrophic growth factor
according to the invention in a sample, comprising an antibody as
described above and means for reacting said antibody and said
sample.
[0048] Proteins which interact with the neurotrophic factor of the
invention, such as for example it's corresponding cellular receptor
may be identified by investigating protein-protein interactions
using the two-hybrid vector system which is well known to molecular
biologists (Fields & Song, Nature 340:245 1989). This technique
is based on functional reconstitution in vivo of a transcription
factor which activates a reporter gene. More particularly the
technique comprises providing an appropriate host cell with a DNA
construct comprising a reporter gene under the control of a
promoter regulated by a transcription factor having a DNA binding
domain and an activating domain, expressing in the host cell a
first hybrid DNA sequence encoding a first fusion of a fragment or
all of a nucleic acid sequence according to the invention and
either said DNA binding domain or said activating domain of the
transcription factor, expressing in the host at least one second
hybrid DNA sequence, such as a library or the like, encoding
putative binding proteins to be investigated together with the DNA
binding or activating domain of the transcription factor which is
not incorporated in the first fusion; detecting any binding of the
proteins to be investigated with a protein according to the
invention by detecting for the presence of any reporter gene
product in the host cell; optionally isolating second hybrid DNA
sequences encoding the binding protein.
[0049] An example of such a technique utilises the GAL4 protein in
yeast. GAL4 is a transcriptional activator of galactose metabolism
in yeast and has a separate domain for binding to activators
upstream of the galactose metabolising genes as well as a protein
binding domain. Nucleotide vectors may be constructed, one of which
comprises the nucleotide residues encoding the DNA binding domain
of GAL4. These binding domain residues may be fused to a known
protein encoding sequence, such as for example the nucleic acids
according to the invention. The other vector comprises the residues
encoding the protein binding domain of GAL4. These residues are
fused to residues encoding a test protein, preferably from the
signal transduction pathway of the vertebrate in question. Any
interaction between neurotrophic factor encoded by the nucleic acid
according to the invention and the protein to be tested leads to
transcriptional activation of a reporter molecule in a GAL-4
transcription deficient yeast cell into which the vectors have been
transformed. Preferably, a reporter molecule such as
.beta.-galactosidase is activated upon restoration of transcription
of the yeast galactose metabolism genes.
[0050] The receptor for enovin has been identified by the present
inventors as GFR.alpha.3. Assays may therefore be prepared to
identify agonist or antagonistic compounds of enovin. This assay
may also be used in association with other neurotrophic growth
factors and their corresponding receptors. Compounds identified may
be used to treat or prevent disorders such as Parkinson's disease,
Alzheimer's disease, neuronal disorders associated with expanded
polyglutamine sequences, such as, Huntingdon's disease, peripheral
neuropathy, acute brain injury, nervous system tumours, multiple
sclerosis, amyotrophic lateral sclerosis, peripheral nerve trauma
or injury exposure to neurotoxins, multiple endocrine neoplasia and
familial Hirschsprung disease, Prion associated diseases,
Creutzfeld-Jacob disease, stroke, pain syndromes with a
substantially peripheral or central neurogenic component,
rheumatic/inflammatory diseases as well as conductance disturbances
by administering to an individual an amount of said agonist or
antagonist in sufficient concentration to prevent or treat said
neural disorders. Such compounds may also be included in
pharmaceutical compositions together with a pharmaceutically
acceptable carrier, diluent or excipient therefor.
[0051] Agonists or antagonists of a growth factor (such as for
example enovin) may be identified in one embodiment by contacting a
cell tissue or organism expressing an appropriate receptor and cRET
with a candidate compound in the presence of the growth factor and
comparing the levels of RET activation in said cell, tissue or
organism with a control which has not been contacted with said
candidate compound.
[0052] An alternative embodiment of the invention comprises a
method of identifying agonists or antagonists of a neurotrophic
growth factor said method comprising contacting a cell tissue or
organism expressing an appropriate receptor of said growth factor
and cRET with a candidate compound in the presence of said growth
factor, measuring the level of activation of a signalling kinase in
the signal transduction pathway of which said appropriate receptor
is a component following addition of an antibody specific for said
signal kinase conjugated to a reporter molecule compared to a cell
tissue or organism which has not been contacted with said
compound.
[0053] A further aspect of the invention comprises use of a
compound identified as an antagonist according to the invention in
the manufacture of a medicament for treating gastrointestinal
disorders or conditions mediated by increased peristaltic
intestinal movement.
[0054] The compounds identified in the assays of the present
invention may advantageously be used to enhance the
gastrointestinal motility and therefore may be useful in treating
conditions related to a hampered or impaired gastrointestinal
transit.
[0055] Accordingly, such compounds may be useful in treating
warm-blooded animals, including humans, suffering from conditions
related to a hampered or impaired gastric emptying or more
generally suffering from conditions related to a hampered or
impaired gastrointestinal transit. Consequently a method of
treatment is provided for relieving patients from conditions, such
as, for example, gastrooesophageal reflux, dyspepsia,
gastroparesis, post-operative ileus, and intestinal
pseudo-obstruction.
[0056] Dyspepsia is an impairment of the function of digestion,
that can arise as a symptom of a primary gastrointestinal
dysfunction, especially a gastrointestinal dysfunction related to
an increased muscle tone or as a complication due to other
disorders such as appendicitis, galbladder disturbances, or
malnutrition. Dyspeptic symptoms are for example a lack of
appetite, feeling of fullness, early satiety, nausea, vomiting and
bloating.
[0057] Gastroparesis can be brought about by an abnormmaly in the
stomach or as a complication of diseases such as diabetes,
progressive systemic sclerosis, anorexia, nervosa and myotonic
dystrophy.
[0058] Post-operative ileus is an obstruction or a kinetic
impairment in the intestine due to a disruption in muscle tone
following surgery.
[0059] Intestinal pseudo-obstruction is a condition characterized
by constipation, colicky pain, and vomiting, but without evidence
of physical obstruction.
[0060] The compounds of the present invention can thus be used
either to take away the actual cause of the condition or to
alleviate the symptoms of the conditions.
[0061] Additionally some of the compounds being stimulators of
kinetic activity on the colon, may be useful to normalize or to
improve the intestinal transit in subjects suffering from symptoms
related to disturbed motility, e.g. a decreased peristalsis of the
small and large intestine alone or in combination with delayed
gastric emptying.
[0062] In view of the colon kinetic utility of the compounds of the
present invention, there is provided a method of treating
warm-blooded animals, including humans, suffering from motility
disorders of the intestinal system, such as, for example,
constipation, pseudo-obstruction, intestinal atony, post-operative
intestinal atony, irritable bowel sydrome (IBS), and drug-induced
delayed transit.
[0063] Compounds identified as antagonists according to the assays
of the present invention may also be of potential use in the
treatment or prophylaxis of gastrointestinal conditions resulting
from increased peristaltic movements in the intestines such as
diarrhea (including secretory diarrhea, bacterial induced diarrhea,
choleic diarrhea, traveller's diarrhea and psychogenic diarrhea),
Crohn's disease, spastic colon, irritable bowel syndrome (IBS),
diarrhea predominant irritable bowel gastrointestinal
hypersensitivity.
[0064] In view of the utility of the compounds of the invention, it
follows that the present invention also provides a method of
treating warm-blooded animals, including humans suffering from
gastrointestinal conditions such as irritable bowel syndrome (IBS),
especially the diarrhoea aspects of IBS. Consequently a method of
treatment is provided for relieving patients suffering from
conditions such as irritable bowel syndrom (IBS),
diarrheapredonminant irritable bowel syndrome, bowel
hypersensitivity, and the reduction of pain associated with
gastrointestinal hypersensitivity.
[0065] The present compounds may also be of potential use in other
gastrointestinal disorders, such as those associated with upper gut
motility, and as antiemetics for treating emesis, and cytotoxic
drug and radiation induced emesis.
[0066] Inflammatory bowel diseases include, for example, ulcerative
colitis, Crohn's disease and the like.
[0067] A further aspect of the invention also comprises a method of
treating a disorder mediated by expression of enovin according to
the invention by administering to a patient an amount of an
antisense molecule or an antagonist thereof according to the
invention in sufficient concentration to alleviate or reduce the
symptoms of said disorder.
[0068] Disorders mediated by inactivation or inhibiting expression
of enovin may also advantageously be treated by administering to an
individual an amount of a compound identified as an agonist of
enovin in sufficient concentration to reduce or prevent the
symptoms of the disorder.
[0069] In a further aspect, the invention provides a method for
making a pharmaceutical formulation for the treatment of diseases
associated with human neurotrophic growth factor enovin, said
method comprising, selecting a candidate compound identified as an
agonist or antagonist of enovin according to the invention,
manufacturing bulk quantities of said compound and formulating the
compound manufactured in a pharmaceutically acceptable carrier.
[0070] As will be seen in more detail from the examples below,
enovin has been successful in reducing taxol induced sensory
deficits. Enovin may therefore play a possible role in pain
syndromes with a substantially peripheral and central neurogenic
component, rheumatic diseases as well as conductance disturbances
and can play a modulatory role in sensory processes after
transdermal, topical, local central (such as epidural, intrathecal,
ICV, intraplexus, intraneuronal) per oral, rectal and systemic
application. Therefore, in the same manner as described herein for
other conditions mediated by enovin, these conditions may be
alleviated or even prevented by administering either an antisense
molecule, a nucleic acid, enovin protein, pharmaceutical
composition, or a compound identified as an agonist or an
antagonist, as appropriate, according to the invention, in
sufficient concentrations to alleviate or prevent the symptoms of
said disorder(s).
[0071] The therapeutic or pharmaceutical compositions of the
present invention can be administered by any suitable route known
in the art including for example intravenous, subcutaneous,
intramuscular, transdermal, intrathecal or intracerebral or
administration to cells in ex vivo treatment protocols.
Administration can be either rapid as by injection or over a period
of time as by slow infusion or administration of slow release
formulation. For treating tissues in the central nervous system,
administration can be by injection or infusion into the
cerebrospinal fluid (CSF).
[0072] Enovin can also be linked or conjugated with agents that
provide desirable pharmaceutical or pharmacodynamic properties. For
example, it can be coupled to any substance known in the art to
promote penetration or transport across the blood-brain barrier
such as an antibody to the transferrin receptor, and administered
by intravenous injection.
[0073] Enovin, the antisense molecules or indeed the compounds
identified as agonists or antagonists of enovin according to the
invention may be used in the form of a pharmaceutical composition,
which may be prepared according to procedures well known in the
art. Preferred compositions include a pharmaceutically acceptable
vehicle or diluent or excipient, such as for example, a
physiological saline solution. Other pharmaceutically acceptable
carriers including other non-toxic salts, sterile water or the like
may also be used. A suitable buffer may also be present allowing
the compositions to be lyophilized and stored in sterile conditions
prior to reconstitution by the addition of sterile water for
subsequent administration. Incorporation of enovin into a solid or
semi-solid biologically compatible matrix may be carried out which
can be implanted into tissues requiring treatment.
[0074] The carrier can also contain other pharmaceutically
acceptable excipients for modifying other conditions such as pH,
osmolarity, viscosity, sterility, lipophilicity, solubility or the
like. Pharmaceutically acceptable excipients which permit sustained
or delayed release following administration may also be
included.
[0075] The enovin protein or the nucleic acid molecules or
compounds according to the invention may be administered orally. In
this embodiment they may be encapsulated and combined with suitable
carriers in solid dosage forms which would be well known to those
skilled in the art.
[0076] As would be well known to those of skill in the art, the
specific dosage regime may be calculated according to the body
surface area of the patient or the volume of body space to be
occupied, dependent upon the particular route of administration to
be used. The amount of the composition actually administered will,
however, be determined by a medical practitioner, based on the
circumstances pertaining to the disorder to be treated, such as the
severity of the symptoms, the composition to be administered, the
age, weight, and response of the individual patient and the chosen
route of administration.
[0077] The present invention may be more clearly understood by the
following examples which are purely exemplary and by reference to
the accompanying drawings wherein:
[0078] FIG. 1: is partial cDNA sequence of a neurotrophic factor
according to the invention designated as enovin. The consensus
sequence was obtained by PCR amplification with primers PNHsp3 and
PNHapl on different cDNAs and on genomic DNA followed by cloning
and sequence analysis and comparison of the obtained sequences. The
predicted one letter code amino acid sequence is shown above the
DNA sequence. The nucleotide residue number is shown on the right
of the DNA sequence, whereas the amino acid residue number is shown
to the right of the translated protein sequence. The putative RXXR
cleavage site for the prodomain is indicated in bold and
underlined. The putative start of the mature protein is indicated
by an arrow. The seven conserved cysteine residues characteristics
for all members of the TGF-.beta. family are indicated in bold. A
potential N-glycosylation site is double underlined,
[0079] FIG. 2: is alignment of the predicted mature protein
sequences of human GDNF, NTN, PSP and EVN. The sequences were
aligned using the ClustalW alignment program. Amino acid residues
conserved between all three proteins are included in the black
areas. Residues conserved between two or three of the sequences are
shaded in grey. The 7 conserved cysteine residues characteristic
for members of the TGF-.beta. family are indicated by asterisks
above the sequence. Amino acid residues are numbered to the right.
The dashes indicate gaps introduced into the sequence to optimize
the alignment,
[0080] FIG. 3: is partial cDNA sequence of enovin. The consensus
sequence was obtained by PCR amplification (primary PCR with
primers PNHspl and PNHapl and nested PCR with primers PNHsp2 and
PNHap2) on different cDNAs followed by cloning and sequence
analysis and comparison of the obtained sequences. The translated
one letter code amino acid sequence of nucleotides 30 to 284
(reading frame A) is shown above the sequence and numbered to the
right (A1 to A85). This reading frame contains a putative ATG
translation start codon. The translated one letter code amino acid
sequence of nucleotides 334 to 810 (reading frame B) is shown above
the sequence and numbered to the right (B1 to B159). This reading
frame contains the region of homology with GDNF, NTN and PSP. The
nucleotide residue number is shown to the right of the DNA
sequence. The putative RXXR cleavage site for the prodomain is
indicated in bold and underlined. The putative start of the mature
protein is indicated by an arrow. The seven conserved cysteine
residues characteristic for all members of the TGF-.beta. family
are indicated in bold. A potential N-glycosylation site is double
underlined,
[0081] FIG. 4: is an illustration of the chromosomal localisation
of human Enovin. (A) Diagram of FISH mapping results for Enovin.
Each dot represents the double FISH signals detected on human
chromosome 1, region p31.3-p32. (B) Example of FISH mapping of
Enovin. The left panel shows the FISH signals on chromosome 1. The
right panel shows the same mitotic figure stained with
4',6-diamidino-2-phenylindole to identify chromosome 1,
[0082] FIG. 5. is an illustration of expression of Enovin in
different human tissues. (A), (B), (C) Northern blot analysis of
tissue expression of Enovin. The expression of Enovin mRNA in
different human tissues was assessed using a probe corresponding to
part of the coding region of Enovin (including the region coding
for the mature Enovin protein) to analyse blots of human poly(A)
rich RNA. (A) Multiple Tissue Northern (MTN) blot; (B) MTN blot II)
Fetal MTN blot II. Panel (D) shows an autoradiography of the human
RNA master blot probed with the same Enovin cDNA fragment. Panel
(E) shows the location of human tissue mRNA samples on the RNA
master blot from (D),
[0083] FIG. 6: is a graphic illustration of the total survival of
SH-SY5Y cells after 72 hours treatment with 10-6M taxol and the
effect of increasing doses of enovin on this survival, normalised
to the condition of solvent. SH-SY5Y cells are differentiated for 5
days with 25 nM staurosporine before application of taxol. Data are
from two independent experiments in sixtuplate. Mean and st. dev.
is shown,
[0084] FIG. 7: is a graphic representation of the effect of
increasing concentrations of enovin over 48 hours on neurite
outgrowth of staurosporine--differentiated SH-SY5Y cells,
normalised to the condition of solvent. SH-SY5Y cells are
differentiated for 5 days with 25 nM staurosporine before starting
the 48 hour experiment. As a positive control, the differentiating
effect of 25 nM staurosporine is shown. Neurite length is
calculated on at least 5000 cells. Data is provided from the
experiments performed in duplicate. Mean and st. dev. is shown.
[0085] FIGS. 8 to 18: are graphic representations of the effect of
enovin on proliferation of various cell types.
[0086] FIG. 19: is a graphic representation of the effects of
enovin on taxol-induced sensory deficits using the pin prick test.
Given are the average (.+-.1 SEM) cumulative scores over time of
rats treated with either 2 different doses of enovin (23 or 130
.mu.g/ml; n=10 rats/group) or vehicle/saline (n=20 rats) after
taxol. Enovin or saline/vehicle were injected in a volume of 75
.mu.l in the subplantar area of the right hind paw.
[0087] FIG. 20: is a graphic representation of the effects of
enovin on taxol-induced sensory deficits using the pin prick test.
Given are the average (.+-.1 SEM) cumulative scores over time of
rats treated with either 2 different doses of enovin (23 or 130
.mu.g/ml; n=10 rats/group) or vehicle/saline (n=20 rats) before
taxol. Enovin or saline/vehicle were injected in a volume of 75
.mu.l in the subplantar area of the right hind paw.
[0088] FIG. 21: is a DNA sequence of enovin. The consensus sequence
was obtained by amplification with PCR using primers PNHsp5 and
PNHap1 on human frontal cortex cDNA and on human genomic DNA
followed by cloning, sequence analysis and comparison of the
resultant sequences. The predicted amino acid sequence is shown
above the DNA sequence for the only splice variant yielding a
functional Enovin protein after translation. The nucleotide residue
number is shown to the left of the DNA sequence, whereas the amino
acid residue number is shown to the right of the translated protein
sequence. 5' and 3' splice sites detected by comparison of
sequenced cDNA fragments with the genomic sequence are indicated by
vertical lines bending to the left or right, respectively, and are
numbered consecutively. The putative RXXR furin cleavage site for
the prodomain is indicated in bold and underlined. The putative
start of the mature protein is indicated by an arrow. The seven
conserved cysteine residues characteristic for all members of the
TGF-.beta. family are indicated in bold. A potential N-linked
glycosylation site is double underlined. The 5' and 3' splice sites
are numbered and encircled.
[0089] FIG. 22: is an illustration of expression of different
Enovin splice variants in human tissues. (A) schematic diagram of
Enovin splice variants identified by RT-PCR experiments with Enovin
specific primers on RNA derived from different human tissues
followed by cloning and sequence analysis of PCR products. The top
line shows a scale (in bp). The second line represents the Enovin
genomic sequence. The position of the translation start and stop
codon, of the start of the mature Enovin coding sequence and of the
5' and 3' splice sites (see FIG. 21) are indicated. The right part
of the figure shows the PCR products obtained by RT-PCR on ovary
and on frontal cortex RNA together with a 100 bp DNA ladder. The
position of the different mRNA variants is indicated together with
their size (from start to stop codon). The translated proteins are
shown on the left hand side. Boxes delineate regions represented in
the cDNA. Dashed lines represent spliced out genomic DNA. The
shaded region represents the mature Enovin coding sequence. The
dotted line marks the start of the mature Enovin coding sequence.
The two transcripts capable of yielding functional Enovin protein
are indicated by an asterisk at the left hand side. (B) Tissue
distribution of the main splice variants. The photograph shows the
PCR fragments obtained by RT-PCR with Enovin specific primers on
different human cDNAs. The 4 main splice variants (A to D) are
indicated by arrows at the left hand side. Sizes are indicated on
the right hand side based on the 100 bp DNA ladder used as size
reference on the gel.
[0090] FIG. 23: Predicted protein sequence of the long splice
variant of Enovin, obtained by splicing out the two introns from
the DNA sequence of FIG. 21. Splice sites 5'1 and 3'-1 are used to
remove the first intron and splice sites 5'-2 and 3'-3 are used to
remove the second intron. This results in a cDNA sequence having an
open reading frame coding for the 228 amino acid residue protein
shown above.
[0091] FIG. 24: Predicted protein sequence of an alternative
(short) splice variant of Enovin, obtained by splicing out the two
introns from the DNA sequence of FIG. 21. Splice sites 5'-1 and
3'-2 are used to remove the first intron and splice sites 5'-2 and
3'-3 are used to remove the second intron. This results in a cDNA
sequence having an open reading frame coding for the 220 amino acid
residue protein shown above. This protein sequence misses 8 amino
acid residues compared to the sequence of FIG. 23.
[0092] FIG. 25: is a graphic representation of the results obtained
from experiments designed to compare the levels of expression of
enovin in normal diseased tissue. Enovin and GAPDH expression is
represented in brain tissue, in respect of multiple sclerosis and
Alzheimer's disease.
[0093] FIG. 26: is a graphic representation of the results obtained
to detect levels of expression of enovin and GAPDH in Parkinson's
disease and cancer.
[0094] Deposits
[0095] Plasmid EVNmat/pRSETB including the DNA sequence encoding
enovin, was deposited on 6 May 1999 under Accession No. LMBP3931,
at the Belgian Coordinated Collections of Micro-Biologie (BCCM) at
Laboratorium voor Moleculaire-Plasmidencollectie (LMBP) B9000,
Ghent, Belgium, in accordance with the provisions of the Budapest
Treaty of 28 Apr. 1997.
[0096] Materials and Methods
[0097] Materials
[0098] Native Taq polymerase, ampicillin, IPTG
(isopropyl-.beta.-D-thiogalactoside), X-gal
(5-bromo-4-chloro-3-indolyl-.beta.-D-galactopyranoside) and all
restriction enzymes used were from Boehringer Mannheim (Mannhein,
Germany). 10 mM dNTP mix was purchased from Life Technologies
(Gaithersburg, Md., USA). The TOPO-TA cloning kit was purchased
from Invitrogen BV (Leek, The Netherlands). The Qiagen plasmid
mini- or midi-DNA purification kit, the Qiaprep Spin Miniprep kit
and the Qiaquick gel extraction kit were purchased from Qiagen GmbH
(Dusseldorf, Germany). cDNA libraries, Marathon.TM. Ready cDNA
kits, human multiple tissue cDNA (MTC.TM.) panels I and II multiple
tissues northern blots and the Advantage-GC cDNA PCR kit were
obtained from Clontech Laboratories (Palo Alto, Calif., USA). All
PCR reactions were performed in a GeneAmp PCR system 9600 cycler
(Perkin Elmer, Foster City, Calif., USA). LB (Luria-Bertani) medium
consists of 10 g/l of tryptone, 5 g/l of yeast extract and 10 g/l
of NaCl. 2.times.YT/ampicillin plates consist of 16 g/l of
tryptone, 10 g/l of yeast extract, 5 g/l of NaCl, 15 g/l of agar
and 100 mg/1 of ampicillin.
[0099] Database Homology Searching and Sequence Comparison.
[0100] Using the complete human glial cell-line derived
neurotrophic factor (GDNF; accession no. Q99748), neurturin (NTN;
accession no. P39905) and persephin (PSP; accession no. AF040962)
cDNA derived protein sequences as query sequences, a BLAST (Basic
Local Alignment Search Tool; Altschul et al., 1990) search was
performed on the daily update of the EMBL/GenBank human expressed
sequence tag (EST) and genomic databases.
[0101] Additional BLAST searches were performed using the genomic
sequence with accession no. AC005038 and several ESTs present in
the GenBank database and showing homology to this genomic sequence
were detected.
[0102] The percentage identity and percentage similarity between
members of the GDNF family was calculated by pairwise comparison of
the sequences using the BESTFIT program (Genetics Computer Group
sequence analysis software package, version 8.0, University of
Wisconsin, Madison, Wis., USA). Alignments of DNA or protein
sequences were done with the ClustalW alignment program (EMBL,
Heidelberg, Germany).
[0103] Oligonucleotide Synthesis for PCR and DNA Sequencing.
[0104] All oligonucleotide primers were ordered from Eurogentec
(Seraing, Belgium). Insert-specific sequencing primers (15- and
16-mers) and primers for use in PCR reactions were designed
manually. DNA was prepared on Qiagen-tip-20 or -100 anion exchange
or Qiaquick spin columns (Qiagen GmbH, Dusseldorf, Germany) and
recovered from the columns in 30 .mu.l TE-buffer (10 mM Tris.HCl, 1
mM EDTA (sodium salt), pH 8.0).
[0105] Sequencing reactions were done on both strands using the ABI
prism BigDye Terminator Cycle sequencing kit and were run on an
Applied Biosystems 377XL sequencer (Perkin Elmer, ABI Division,
Foster City, Calif., USA). The Sequencher.TM. software was used for
sequence assembly and manual editing (GeneCodes, Ann Arbor, Mich.,
USA).
[0106] Cloning of a Novel GDNF Homologue.
[0107] A DNA region spanning nucleotides 67411 to 68343 of EMBL
accession no. AC005038 of which the translated protein sequence was
homologous to mature NTN and PSP was used to design oligonucleotide
primers for PCR amplification. The different primers used are shown
in Table 1. TABLE-US-00001 TABLE 1 Primers used for the PCR
amplification of fragments of AC005038. Primer name Primer sequence
PNHsp1 5' - CGGTGCACTCAGGTGATTCCTCC - 3' PNHsp2 5' -
GGCAGCAAACCCATTATACTGGAACC - 3' PNHsp3 5' - CGCTGGTGCAGTGGAAGAGCC -
3' PNHsp4 5' - CTGCACCCCCATCTGCTCTTCC - 3' PNHap1 5' -
GCAGGAAGAGCCACCGGTAAGG - 3' PNHap2 5' - CCAGTCTGCAAAGCCCTGGAGC -
3'
[0108] Primers PNHsp3 and PNHapl were used to amplify a fragment of
502 bp on cDNA derived from different human tissues (fetal brain,
whole fetus, prostate or lung Marathon-Ready.TM. cDNA (Clontech
Laboratories), frontal cortex, hippocampus and cerebellum cDNA) and
on human genomic DNA. Based on the genomic sequence from the
EMBL/GenBank database (acc. no. AC005038), the fragment to be
amplified was predicted to have a G+C content of 76%. Therefore,
amplifications were done using the Advantage-GC cDNA PCR kit
(Clontech Laboratories, Palo Alto, Calif., USA) optimized for the
amplification of GC-rich DNA sequences. PCR reactions were
performed in a total volume of 50 .mu.l, containing 1.times.GC cDNA
PCR reaction buffer, 0.2 mM dNTP, 1 M GC-MELT.TM., 200 nM of
primers PNHsp3 and PNHapl, 1 .mu.l of Advantage KlenTaq polymerase
mix and 1 to 5 .mu.l of cDNA or 0.5 .mu.g of genomic DNA. Samples
were heated to 95.degree. C. for 5 min and cycling was done for 45
s at 95.degree. C., 1 min at 58.degree. C. and 40 s at 72.degree.
C. for 35 cycles, with a final step of 7 min at 72.degree. C.
Samples were finally treated with 2.5 U of native Taq DNA
polymerase to add an A-overhang. PCR products were analysed on a 1%
(w/v) agarose gel in 1.times.TAE buffer (40 mM Tris-acetate, 1 mM
EDTA (sodium salt), pH 8.3). PCR fragments of the expected size
(495 bp) were excised from the gel and purified with the Qiaquick
gel extraction kit. The PCR fragments were sequenced to confirm
their identity and cloned into the plasmid vector pCR2.1-TOPO using
the TOPO TA cloning kit according to manufacturer's instructions.
Approximately 20 ng of purified fragment was combined with 1 .mu.l
pCR2.1-TOPO vector in a total volume of 5 .mu.l. Reactions were
incubated at room temperature (20.degree. C.) for 5 min. 2 .mu.l of
the reaction was transformed into TOP1OF' competent cells
(Invitrogen BV) using heat-shock transformation and plated on
2.times.YT/ampicillin plates supplemented with 10 mM IPTG and 2%
(w/v) X-gal for blue-white screening. White colonies after
overnight growth were picked from the plates, grown in 5 ml of LB
medium supplemented with 100 mg/1 ampicillin and plasmid DNA
prepared using the Qiaprep Spin Miniprep kit. The presence of an
insert of the expected size was confirmed by restriction digestion
with EcoRI. The plasmid insert of several positive clones was
sequenced and the obtained sequences compared using the ClustalW
alignment program.
[0109] To obtain additional coding sequence for the novel GDNF
homologue, a fragment with an expected size of 931 bp based on the
EMBL/GenBank sequence (acc. no. AC005038) was amplified by PCR
using primers PNHspl and PNHapl. PCR reactions were performed in a
total volume of 50 .mu.l, containing 1.times.GC cDNA PCR reaction
buffer, 0.2 mM dNTP, 1 M GC-MELT.TM., 200 nM of primers PNHspl and
PNHapl, 1 .mu.l of Advantage KlenTaq polymerase mix and 1 to 5
.mu.l of cDNA from cerebellum, frontal cortex or hippocampus or 0.5
.mu.g of genomic DNA. Samples were heated to 95.degree. C. for 5
min and cycling was done for 45 s at 95.degree. C., 1 min at
58.degree. C. and 1 min 30 s at 72.degree. C. for 35 cycles, with a
final step of 7 min at 72.degree. C. PCR products were analysed on
a 1% (w/v) agarose gel in 1.times.TAE buffer (40 mM Tris-acetate, 1
mM EDTA (sodium salt), pH 8.3). A second round amplification was
performed with nested primers (PNHsp2 and PNHap2). 1 .mu.l of the
first round amplification reaction was used in a total volume of 50
.mu.l, containing 1.times.GC cDNA PCR reaction buffer, 0.2 mM dNTP,
1 M GC-MELT.TM., 200 nM of primers PNHsp2 and PNHap2 and 1 .mu.l of
Advantage KlenTaq polymerase mix. Samples were heated to 95.degree.
C. for 5 min and cycling was done for 45 s at 95.degree. C., 1 min
at 58.degree. C. and 1 min 30 s at 72.degree. C. for 35 cycles,
with a final step of 7 min at 72.degree. C. Samples were analysed
on a 1% (w/v) agarose gel in 1.times.TAE buffer. PCR fragments of
the expected size (870 bp) were excised from the gel and purified
with the Qiaquick gel extraction kit. The PCR fragments were
sequenced to confirm their identity, treated with 2.5 U of Taq
polymerase and cloned into the plasmid vector pCR2.1-TOPO using the
TOPO TA cloning kit according to manufacturer's instructions.
Approximately 20 ng of purified fragment was combined with 1 .mu.l
pCR2.1-TOPO vector in a total volume of 5 .mu.l. Reactions were
incubated at room temperature (20.degree. C.) for 5 min. 2 .mu.l of
the reactions was transformed into TOP1OF' competent cells using
heat-shock transformation and plated on 2.times.YT/ampicillin
plates supplemented with 10 mM IPTG and 2% (w/v) X-gal for
blue-white screening. White colonies after overnight growth were
picked from the plates, grown in 5 ml of LB medium supplemented
with 100 mg/l ampicillin and plasmid DNA prepared using the Qiaprep
Spin Miniprep kit. The presence of an insert of the expected size
was confirmed by restriction digestion with EcoRI. The plasmid
insert of several positive clones was sequenced and the sequences
compared using the ClustalW alignment program.
[0110] Analysis of Enovin Gene Expression by RT-PCR Analysis.
[0111] Oligonucleotide primers PNHsp3 and PNHapl (see table 1) were
used for the specific PCR amplification of a 502 bp fragment from
enovin. PCR amplifications were performed on human multiple tissue
cDNA (MTC.TM.) panels normalised to the mRNA expression levels of
six different housekeeping genes. PCR reactions with enovin
specific primers were performed in a total volume of 50 .mu.l,
containing 5 .mu.l of cDNA, 1.times.GC cDNA PCR reaction buffer,
0.2 mM dNTP, 1 M GC-MELT.TM., 400 nM of primers PNHsp3 and PNHap1
and 1 .mu.l of Advantage KlenTaq polymerase mix. samples were
heated to 95.degree. C. for 30 s and cycling was done for 30 s at
95.degree. C. and 30 s at 68.degree. C. for 35 cycles. Samples were
analysed on a 1.2% (w/v) agarose gel in 1.times.TAE buffer (40 mM
Tris-acetate, 1 mM EDTA (sodium salt), pH 8.3) and images of the
ethidium bromide stained gels were obtained using an Eagle Eye II
Video system (Stratagene, La Jolla, Calif., USA).
[0112] Similarity searching of the daily update of the EMBL/GenBank
database with the human neurturin and persephin protein sequences
yielded a genomic DNA sequence coding for a putative novel protein
similar to the neurotrophic factors GDNF, NTN and PSP which has
been called enovin (EVN). Additional database homology searching
using the genomic DNA sequence surrounding the region coding for
enovin yielded several expressed sequence tag (EST) clones derived
from different human tissues (prostate epithelium [accession no.
AA533512 (ID1322952)], lung carcinoma [accession no. AA931637] and
parathyroid tumor [accession no. AA844072]). These clones contain
DNA sequence outside of the region of homology with GDNF, PSP or
NTN, but confirmed that enovin mRNA is expressed in normal and
tumor tissues.
[0113] Initial PCR amplification using primers (PNHsp3 and PNHapl)
based on the genomic sequence yielded a fragment of .apprxeq.500 bp
from fetus, fetal brain, prostate, frontal cortex, hippocampus,
cerebellum cDNA and from genomic DNA, but not from lung cDNA.
Cloning and sequence analysis of these fragments yielded a DNA
sequence of 474 bp that translated into a predicted protein
sequence of 139 amino acid residues including seven conserved
cysteine residues characteristic of all the members of the
transforming growth factor .beta. (TGF-.beta.) family of proteins
(Kingsley, 1994) (FIG. 1). The sequence also contained a RXXR motif
for cleavage of the prodomain (RAAR, amino acid position 23 to 26)
(Barr, 1991). A similar cleavage site is present in the GDNF, NTN
and PSP protein sequences, at a comparable position in the
prodomain sequence. Assuming cleavage of the enovin prodomain
occurs at this site in vivo, the mature EVN protein sequence
contains 113 amino acid residues (residue 27 to 139 in FIG. 1) and
has a calculated molecular mass of 11965 Da and an isoelectric
point of 11.8. There is one potential N-glycosylation site present
in the mature sequence (NST at amino acid position 121-123).
Moreover, several regions conserved between the mature forms of the
known neurotrophic factors GDNF, NTN and PSP were also present in
enovin (FIG. 2). Table 2 summarizes the results of the comparison
of the mature protein sequences of the GDNF family members by the
BESTFIT program. Percentage identity and percentage similarity are
shown. The GDNF, NTN, PSP and EVN mature sequences used in this
comparison started at the first amino acid residue following the
RXXR cleavage site. TABLE-US-00002 TABLE 2 Pairwise comparison of
mature human GDNF family members using the BESTFIT program.
Comparison % identity % similarity EVN vs GDNF 38.8 47.2 EVN vs NTN
51 56.1 EVN vs PSP 53.3 57.8 GDNF vs NTN 44.8 57.3 GDNF vs PSP 44.3
50 NTN vs PSP 50 54.4
[0114] From these comparisons it is apparent that the mature enovin
protein is more closely related to persephin and to neurturin than
to GDNF.
[0115] Amplification, cloning and sequence analysis of a larger
fragment of the enovin DNA sequence from frontal cortex cDNA using
primers based on the genomic EMBL/GenBank sequence (acc. no.
AC005038) yielded a sequence of 819 bp (FIG. 3). This sequence
contains a putative ATG start codon at nucleotide positions 30-32
and yields an open reading frame (reading frame A in FIG. 3) that
extends up to a stop codon at nucleotide positions 285-287. The
translated protein sequence of this region does not show similarity
to any known protein in the databases. Translation of the cDNA
sequence in the second reading frame (reading frame B in FIG. 3)
yields a predicted protein sequence of 159 amino acid residues.
This sequence contains the RXXR cleavage site (position B43 to B46;
nucleotide position 460-471) and the sequence corresponding to the
mature enovin sequence (position B47 to B159; nucleotide position
472-810). The open reading frame including the RXXR cleavage site
and the mature enovin coding sequence extends from nucleotide
position 334 (preceded-by an in-frame stop codon) to a stop codon
at position 811-813, but does not contain an ATG codon for a
starting methionine residue. In analogy with persephin (Milbrandt
et al., 1998) we hypothesize that an unspliced intron is present in
the majority of the mRNA transcripts from the EVN gene. GDNF and
NTN also have an intron in their respective prodomain coding
regions (Matsushita et al., 1997, Heuckeroth et al., 1997).
[0116] To evaluate the existence of different mRNA transcripts for
Enovin, RT-PCR experiments were performed using primers situated at
the 5' end of the Enovin coding sequence just 5' of a possible
upstream ATG start codon (primer PNHsp5 [5'-GCA AGC TGC CTC AAC AGG
AGG G-3'] and nested primer PNHsp6 [5'-GGT GGG GGA ACA GCT CAA CAA
TGG-3'] and at the 3' end (primer PNHap1 and nested primer PNHap2
[see Table 1]. Experiments were performed on human multiple tissue
cDNA panels (Clontech MTC panels I and II), on a fetal heart cDNA
library (Clontech) and on cDNA derived from human cerebellum,
hippocampus or frontal cortex (Masure et al., 1998). Primary PCR
reactions were performed with primers PNHsp5 and PNHap1 under
GC-rich conditions (Advantage GC-PCR kit, Clontech) for 30 cycles
(95.degree. C.--30 s, 60.degree. C.--30 s, 72.degree. C.--1 min) as
described. Nested PCR reactions were performed on 1 .mu.l of the
primary PCR product using primers PNHsp6 and PNHap2 under the same
conditions for 30 cycles. Resulting PCR products were analysed on a
1.5% agarose gel and ranged in size from .+-.350 bp to .+-.800 bp.
Several bands were purified from the gel and the PCR fragments
sequenced directly. Some purified PCR products were also cloned in
vector pCR2.1-TOPO (TOPO-TA cloning kit, Invitrogen) and then
sequenced. Sequence analysis confirmed the existence of different
mRNA molecules containing Enovin sequence. The obtained fragment
sequences were compared with the genomic Enovin sequence. This
allowed us to identify several possible 5' and 3' splice sites in
the genomic sequence (FIG. 21). All these splice sites corresponded
to the consensus sequences for donor and acceptor splice sites
(Senaphthy, P., Shapiro, M. B. & Harris, N. L. (1990)) splice
junctions, branch point sites, and exons: sequence statistics,
identification, and applications to genome project. Methods
Enzymol. 183, 252-278). The different Enovin splice variants
identified and their presence in different human tissues are
summarized in FIG. 22. Only two of the 5 sequenced transcripts
yield functional Enovin protein upon translation from the ATG start
codon. These two transcripts code for proteins of 228 or 220 amino
acids, respectively with predicted signal peptides of 47 and 39
amino acid residues. The predicted protein sequences of these two
variants are shown in FIG. 23 (long variant) and FIG. 24 (short
variant). The long variant can be deduced from the DNA sequence of
FIG. 21 by splicing out the first intron at locations 5'-1 and 3'-1
and the second intron at 5'-2 and 3'-3. Upon translation of the
open reading frame, the predicted protein sequence of FIG. 23 is
obtained. The shorter variant can be deduced from the DNA sequence
of FIG. 21 by splicing out the first intron at locations 5'-1 and
3'-2 and the second intron at 5'-2 and 3'-3. Upon translation of
the open reading frame, the predicted protein sequence of FIG. 24
is obtained.
[0117] The longest transcript seems to be the most abundant in most
tissues as judged by the band intensity in FIG. 22B. The shorter
transcripts result in frame shifts yielding a translated protein
missing the mature Enovin amino acid sequence homologous with GDNF,
NTN and PSP. The two smallest transcripts even miss part of the
mature coding sequence, including two of the seven highly conserved
cysteine residues. FIG. 22B shows the distribution of the main
splice variants in different human tissues. Functional Enovin mRNA
is expressed in almost all tissues tested, including brain, heart,
kidney, liver, lung, pancreas, skeletal muscle, colon, small
intestine, peripheral blood leukocytes, spleen, thymus, prostate,
testis, ovary, placenta and fetal heart. In some human tissues
(e.g. cerebellum, hippocampus), only non-functional transcripts
could be amplified by PCR. To our knowledge, the occurrence of
non-functional mRNA transcripts to such an extent has not been
described before. The biological significance of this finding
remains to be studied. Although the expression of NTN and PSP in
different tissues has not been fully characterized, their
expression levels seem much lower and the expression more
restricted to certain tissues (Kotzbauer et al., 1996, Milbrandt et
al., 1998).
[0118] Recombinant Expression of Enovin in E. coli Construction of
an Enovin Expression Plasmid
[0119] A 414 bp PCR fragment was amplified from human genomic DNA
with primers PNHsp4 and PNHap2 (Table 1) and cloned in vector
pCR2.1-TOPO using TA-cloning (Invitrogen). The sequence of the
insert was confirmed by sequence analysis. One clone containing an
insert with the Enovin consensus sequence (clone 36) was used for
subsequent construction of an expression plasmid. Two primers were
designed containing appropriate restriction sites at their 5' ends.
Forward primer PNHexp-sp1 (5'-GCG GAT CCG GCT GGG GGC CCG GGC A-3')
contained a BamHI restriction site (underlined) and reverse primer
PNHexp-ap1 (5'-GCC TCG AGT CAG CCC AGG CAG CCG CAG G-3') contained
a XhoI restriction site (also underlined). Using these primers, the
343 bp fragment coding for mature Enovin (position 81 to 422 in
FIG. 1) was amplified from clone 36. The PCR reaction was performed
in a total volume of 50 .mu.l, containing 1.times.GC cDNA PCR
reaction buffer, 0.2 mM dNTP, 1 M GC-MELT.TM. (, 200 nM of primers
PNHexp-sp1 and PNHexp-ap1, 1 .mu.l of Advantage KlenTaq polymerase
mix and 10 ng of plasmid DNA from clone 36. Samples were heated to
94.degree. C. for 5 min and cycling was done for 45 s at 94.degree.
C., 1 min at 58.degree. C. and 30 s at 72.degree. C. for 25 cycles,
with a final step of 7 min at 72.degree. C. The resulting 50 .mu.l
product was purified using the Qiaquick PCR purification kit
(Qiagen) and the DNA eluted in 30 .mu.l. 25 .mu.l of this purified
product was then digested in a 30 .mu.l reaction with 10 U of BamHI
and 10 U of XhoI in 1.times. buffer B (Boehringer Mannheim) for 1 h
at 37.degree. C. After electrophoresis in a 1% (w/v) agarose gel in
1.times.TAE buffer (40 mM Tris-acetate, 1 mM EDTA (sodium salt), pH
8.3), the expected 353 bp band was excised from the gel and
purified using the Qiaquick gel extraction kit. The resulting
fragment was ligated in the vector PRSET B (Invitrogen) linearised
with BamHI and XhoI. The insert of the resulting plasmid construct
(hEVNmat/pRSETB) was confirmed by complete sequence analysis. The
resulting construct codes for a 146 amino acid protein with a
predicted molecular mass of 15704 Da including an NH2-terminal
6.times.His-tag fused in frame to the mature Enovin coding
sequence. The NH2-terminal amino acid sequence of the resulting
protein is thus MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDPAGGPGS (mature
Enovin sequence in bold, 6.times.His tag underlined).
[0120] Expression of Enovin in BL21(DE3) E. coli Cells
[0121] Recombinant production of Enovin protein was performed
essentially as described for Neurturin by Creedon et al. (1997),
with modifications. For the production of recombinant Enovin
protein, the plasmid hEVNmat/pRSETB was transformed in E. coli
strain BL21(DE3) (Novagen) and grown in 2xYT/ampicillin-medium (16
g/l of tryptone, 10 g/l of yeast extract, 5 g/l of NaCl and 100
mg/l of ampicillin) at 30.degree. C. (225 rpm) or 37.degree. C.
(300 rpm) to an OD600 of approximately 0.5 prior to addition of
IPTG to a final concentration of 0.2 mM to induce expression. Cell
pellets were harvested by centrifugation following a 3 h induction
period, washed with phosphate-buffered saline, centrifuged and
stored frozen. For purification and refolding, cell pellets were
resuspended in sonication buffer (20 mM Tris-HCl, pH 8.0, 300 mM
NaCl, 1 mM 2-mercaptoethanol, protease inhibitors (Complete.TM.
protease inhibitor cocktail tablets (Boehringer Mannheim, 1 tablet
per 50 ml buffer) and 1 mg lysozyme per 500 mg cell pellet). Cells
were disrupted by sonication and inclusion bodies harvested by
centrifugation. Inclusion bodies were dissolved and incubated in
buffer A (8 M urea, 20 mM Tris-HCl pH 7.6, 200 mM NaCl, 1 mM
2-mercaptoethanol) for 30 min at 37.degree. C. prior to adding to
Ni-NTA resin (nickel nitrilotriacetic acid, Qiagen). After 40 min
shaking at 37.degree. C., samples were washed once with buffer A
and loaded onto a 5 ml Ni-NTA column. The column was washed
successively with 10 column volumes of buffer A, 10 column volumes
of buffer A at pH 7.2 and 10 column volumes of buffer A at pH
7.2+10 mM imidazole. The Enovin was eluted from the column in 4
column volumes of buffer A at pH 7.2+200 mM imidazole.
[0122] Enovin refolding was performed by stepwise overnight
dialysis at 4.degree. C. in renaturation buffer (0.1M sodium
phosphate, 0.15M NaCl, 3 .mu.M cysteine, 0.02% Tween-20, 10%
glycerol, 0.01M Tris-HCl, pH 8.3) containing decreasing amounts of
urea at each step (6M to 4M to 3M to 2M to 1M to 0.5M to 0M urea).
The purified protein was aliquotted, stored at -20.degree. C. and
further used for functional assays.
[0123] Chromosomal Localization of the Enovin Gene.
[0124] A 3.3 kb fragment of the Enovin gene was amplified from
cerebellum cDNA using primers EVN(7)-sp1 (5'-TTC GCG TGT CTA CAA
ACT CAA CTC CC-3') and PNHap1 (5'-GCA GGA AGA GCC ACC GGT AAG G-3')
designed on the sequence of EMBL accession number AC005038. The PCR
reaction was performed in a total volume of 50 .mu.l, containing
1.times. Expand Long Template PCR reaction buffer (Boehringer
Mannheim), 0.5 mM dNTP, 1 M GC-MELT ((Clontech Laboratories), 400
nM of primers EVN(7)-sp1 and PNHap1 and 1 .mu.l of cerebellum cDNA.
After an initial 2 min at 94.degree. C., 0.75 .mu.l of Expand Long
Template polymerase (Boehringer Mannheim) was added and cycling was
done for 10 s at 94.degree. C., 30 s at 58.degree. C. and 3 min at
68.degree. C. for 10 cycles. Then, 20 additional cycles were
performed increasing the extension time at 68.degree. C. with 20 s
every cycle. A final 7 min at 68.degree. C. were also included. The
resulting 3.3 kb fragment was purified after electrophoresis in a
0.8% agarose/TAE gel and cloned in vector pCR2.1-TOPO using
TA-cloning (Invitrogen). Complete sequence analysis of the 3.3 kb
insert of one clone confirmed that the obtained cDNA sequence
corresponded to the genomic sequence in the EMBL database
(accession number AC005038). No introns were spliced out in the
cDNA obtained from cerebellum cDNA.
[0125] Chromosomal mapping studies were carried out using
fluorescent in situ hybridization (FISH) analysis essentially as
described (Heng et al., 1992, Heng & Tsui, 1993). Human
lymphocytes were cultured at 37.degree. C. for 68-72 h before
treatment with 0.18 mg/ml 5-bromo-2'-deoxyuridine (BrdU) to
synchronize the cell cycles in the cell population. The
synchronized cells were washed and recultured at 37.degree. C. for
6 h. Cells were harvested and slides were prepared using standard
procedures including hypotonic treatment, fixation and air-drying.
The 3.3 kb probe for Enovin was biotinylated and used for FISH
detection. Slides were baked at 55.degree. C. for 1 h, treated with
RNase and denatured in 70% formamide in 2.times.NaCl/Cit
(20.times.NaCl/Cit being 3 M NaCl, 0.3 M disodium citrate, pH 7.0)
for 2 min at 70.degree. C. followed by dehydration with ethanol.
The probe was denatured prior to loading on the denatured
chromosomal slides. After overnight hybridization, slides were
washed and FISH signals and the 4',6-diamidino-2-phenylindole
banding pattern were recorded separately on photographic film, and
the assignment of the FISH mapping data with chromosomal bands was
achieved by superimposition of FISH signals with
4',6-diamidino-2-phenylindole banded chromosomes (Heng & Tsui,
1993). Under the conditions used, the hybridization efficiency was
approximately 72% for this probe (among 100 checked mitotic
figures, 72 of them showed signals on one pair of the chromosomes).
Since the 4',6-diamidino-2-phenylindole banding was used to
identify the specific chromosome, the assignment between the signal
from the probe and the short arm of chromosome 1 was obtained. The
detailed position was further determined based upon the summary
from 10 photographs (FIG. 4A). There was no additional locus picked
by FISH detection under the conditions used, therefore, we conclude
that Enovin is located at human chromosome 1, region p31.3-p32. An
example of the mapping results is presented in FIG. 4B.
[0126] From the gene map data at the National Center for
Biotechnology Information (NCBI,
http://www.ncbi.nlm.nih.gov/genemap), it can be deduced that the
genomic clone containing the Enovin sequence (EMBL accession number
AC005038) is located on chromosome 1, between markers D1S2843 and
D1S417. This corresponds to chromosome 1, region p31.1 to p32.3,
confirming the data obtained by FISH analysis.
[0127] Tissue Distribution of Enovin as Determined by Northern Blot
and Dot Blot Analysis.
[0128] Northern blots containing 2 .mu.g of poly(A)-rich RNA
derived from different human tissues (Clontech Laboratories, Palo
Alto, Calif., USA; MTN.TM. blot, MTN.TM. blot II and Fetal MTN.TM.
blot II) were hybridised according to the manufacturer's
instructions with a (.alpha.-.sup.32P-dCTP random-priming labelled
(HighPrime kit, Boehringer Mannheim) 897 bp Enovin fragment. This
fragment was obtained by PCR amplification with primers PNHsp1 and
PNHap1 on frontal cortex cDNA and subsequent cloning in vector
pCR2.1-TOPO. The fragment contains 897 bp of Enovin sequence up to
the stop codon and includes the complete coding sequence for the
mature Enovin protein.
[0129] Enovin mRNA was detected as a main transcript of
approximately 4.5 kb (FIG. 5A-C). Enovin mRNA was expressed in a
range of tissues, most prominently in heart, skeletal muscle,
pancreas and prostate. Some smaller-sized transcripts are present
in e.g. placenta (4 kb, 2.4 kb and 1.6 kb) and prostate (4 kb and
1.6 kb). In fetal tissue, a prominent 2.4 kb transcript is present
in liver and to a lesser extent lung. Other transcripts are also
present in fetal kidney, liver, lung and brain.
[0130] In addition an RNA master blot (Clontech Laboratories)
containing poly(A) rich RNA from different human tissues and
developmental stages was also hybridized with the 897 bp Enovin
probe. The poly(A) rich RNA samples used for making this blot have
been normalized to the mRNA expression levels of eight different
housekeeping genes by the manufacturer. Enovin mRNA was expressed
ubiquitously, but highest mRNA levels were apparent in prostate,
pituitary gland, trachea, placenta, fetal lung, pancreas and kidney
(FIG. 5D+E).
[0131] Construction of GFR.alpha.-IgG-Fc Fusions Vectors
[0132] cDNA regions of GFR.alpha.-1, GFR.alpha.-2 and GFR.alpha.-3
(coding for amino acids 27 to 427, 20 to 431 and 28 to 371,
respectively) excluding the sequences coding for the signal peptide
and for the COOH-terminal hydrophobic region involved in
GPI-anchoring were cloned in-frame in the expression vector Signal
pIg plus (R&D Systems Europe Ltd). The resulting proteins
expressed from these constructs contain a 17 amino acid
NH.sub.2-terminal CD33 signal peptide, the GFR.alpha. protein
region and a 243 amino acid COOH-terminal human IgG.sub.1-Fc fusion
domain. CHO cells were transfected with GFR.alpha. fusion
constructs and stably transfected cells were selected using 500
.mu.g G418. Permanent clones were selected using anti Fc antibody.
For purification of GFR.alpha. fusion proteins, cells were grown in
serum-free medium and medium was collected after every 3 days.
Medium was centrifuged and applied to a protein A column (Protein A
Sepharose, Pharmacia Biotech). Bound protein was eluted with 0.1 M
Na citrate, pH 3.0 and collected into 1 M Tris buffer, pH 8.4.
Protein concentration was estimated by absorbance at 280 nm using
an extinction coefficient of 1.5. These purified soluble
GFR.alpha.-1 to -3 Fc fusion proteins were used for subsequent
binding studies.
[0133] Surface Plasmon Resonance Analysis
[0134] Surface plasmon resonance (SPR) experiments were performed
at 25.degree. C. using a BIAcore 3000 instrument. Analyses were
performed with enovin and NGF as immobilised ligands. The
carboxylated matrix of a F1 sensor chip was first activated with a
1:1 mixture of 400 mM N-ethyl-N-(dimethylaminopropyl)-carbodiimide
and 100 mM N-hydroxy-succinimide for 10 min. Than, recombinant
enovin and NGF were applied onto the activated surface in 10 mM
acetate buffer, pH 4.5 at a flow rate of 5 .mu.l/min. Unoccupied
reactive groups were inactivated with 1 M ethanolamine
hydrochloride. For binding experiments, soluble GFR.alpha.1-3-Fc
were superfused at concentrations of 10-100 nM in HEPES buffered
saline (150 mM NaCl, 3.5 mM EDTA, 0.05% P-20, 10 mM HEPES, pH 7.4)
at a flow rate of 10 .mu.l/min. The association was monitored for 3
min and dissociation for 1 min, followed by regeneration with 5 mM
NaOH. Dissociation was initiated by superfusion with HEPES buffered
saline. A BIAcore evaluation software, 3.0 was used to calculate
the association rate (k.sub.a), dissociation rate (k.sub.d) and the
equilibrium dissociation constants (K.sub.D, calculated as
k.sub.d/k.sub.a).
[0135] Results
[0136] SPR was used to measure binding of soluble GFR.alpha.1-3 to
immobilised enovin. Specific binding to enovin could be detected
with the soluble GFR.alpha.3 only. GFR.alpha.1 and GFR.alpha.2 did
not bind to the immobilised enovin. The observed binding of
GFR.alpha.3 was specific as there was no binding to NGF. In the
separate control experiment specific binding of TrkA-Fc (NGF
receptor) to the immobilised NGF was detected without binding to
immobilised enovin.
[0137] From the binding curves obtained using three different
concentrations of GFR.alpha., the following constants in Table 3
were derived. These results demonstrate that GFR.alpha.3 binds
specifically to enovin. TABLE-US-00003 TABLE 3 K.sub.a (1/MS)
K.sub.d (1/s) K.sub.D (M) GFR.alpha.3 1.65 10.sup.5 5.08 10.sup.-4
3.1 10.sup.-9
[0138] Since GDNF, NTN and PSP all promote the maintenance and
survival of different types of neuronal cells, it is anticipated
that enovin has similar biological effects on nerve cells and,
possibly, on other cell types too. Therefore, it is envisaged that
the enovin protein may be useful in the treatment of neural
disorders in general, including Parkinson's disease, Alzheimer's
disease, peripheral neuropathy, amyotrophic lateral sclerosis
(ALS), Huntington's disease, acute brain injury, nervous system
tumors, multiple sclerosis, peripheral nerve trauma or injury and
exposure to neurotoxins.
[0139] Enovin could also be useful in various aspects of
neuroprotection. Considering its effect on survival of different
neuronal cell populations and on the observed neurite extensions in
our model of SHSY5Y cells, we propose that this compound could have
neuroprotective and neuroregenerative applications.
[0140] This is based upon the following observations. Taxol induces
neuronal apoptosis in NGF-differentiated PC12 rat pheochromocytoma
cells (Nuydens et al, submitted). Therefore, taxol induced
cytotoxicity has features of neuronal apoptosis, as monitored by
DNA fragmentation, Annexin V labelling and bcl-2 protection. As an
extension, therefore, it can be deduced that taxol induces
apoptosis in differentiated SH-SY5Y cells. Enovin is now shown to
be able to reduce this cell death and therefore may reverse
neuronal apoptosis in general.
[0141] The compound may therefore be helpful in the following
neurodegenerative conditions in which apoptosis has been observed,
stroke (Hakim 1998), Parkinson's disease (Marsden et al 1998),
Alzheimer's disease (Nagy et al 1998), Huntington's disease
(Wellington et al. 1997), Neurotrauma (Smirnova et al. 1998),
Peripheral neuropathies, (Srinivisan et al. 1998).
[0142] As an example for the last clinical indication, we have
shown that this neurotrophic factor actually protects
differentiated SH-SY5Y human neuroblastoma cells against
taxol-induced cell toxicity.
[0143] Methodology of Viability Measurements
[0144] Cell viability was determined by adding 100 .mu.l of a 1
mg/ml 2,3-bis
[2-methoxy-4-nitro-5-sulphophenyl]-2H-tetrazolium-5-carboxanilide
(XTT, Sigma) solution in DMEM (37.degree. C.) supplemented with
0.02 mM phenazine methosulfate (PMS, Sigma) to each well. The
plates were then incubated at 37.degree. C. for 2.5 hours. The
optical densities were read (Molecular devices) at 450 nm, using
650 nm as a reference. The XTT assay is based on the conversion of
the tetrazolium salt XTT into a red colored formazan product. This
reaction is performed by mitochondrial enzymes.
[0145] Methodology of Neuronal Differentiation
[0146] 1. Differentiation in Human Neuroblastoma SHSY5Y Cells
[0147] SHSY5Y cells are differentiated for 5 days with 25 nM
staurosporine. Effect of Enovin is measured 72 hrs after start of
the experiment. (reference Jalava et al. "Protein Kinase inhibitor
staurosporine induces a mature neuronal phenotype in SH-SY5Y human
neuroblastoma cells through an a,b,z PKC independent pathway" Journ
cell Physiol 155, 301-312 (1993)).
[0148] 2. Measurement of Neurite Extension.
[0149] Morphological changes of neurones were automatically
quantified as follows. Briefly, at the appropriate times,
glutaraldehyde was added directly to the medium and left for 30
minutes at room temperature. This ensured that the morphology of
the cells at that time point reflected the real situation. The
cells were observed through transmitted light mode in an Axiovert
microscope (Zeiss Oberkochen, Germany), equipped with a Marzhauser
scanning stage driven by an Indy workstation (Silicon Graphics,
Mountain View, USA). Images were captured using a MX5 video camera
(HCS). About 3000 cells were evaluated in 64 aligned images,
forming a 8.times.8 square matrix of images. The exact alignment of
the images ensured that neurites could be followed from one image
field into the next. Automatic detection of the neurite extensions,
labeled by polyclonal tau antibody was performed using an unbiased
detector of curvilinear structures (Steger 1998). The analysis
software automatically calculated total cell body area, number of
cell bodies and total neurite length.
[0150] To investigate the effect of enovin on different cell types,
two assays were performed, a DNA synthesis assay and a chemotaxis
assay.
[0151] DNA Synthesis Assay
[0152] Cells including human dermal fibroblasts (39SK), human
umbilical vein endothelial cells (HUVEC), human smooth muscle cells
(HSMC), human chondrocytes, and rat osteoblasts were maintained in
DMEM containing 10% FBS (39-SK, HSMC, rat osteoblasts) or defined
media (chondrocytes and HUVEC) at 37.degree. C. with 5% CO.sub.2
and 95% air. For the DNA synthesis assay, cells were seeded in a
96-well tissue culture plate at a density of 5,000 cells/well in
DMEM containing 10% FBS and incubated for 24 h. The culture medium
then was replaced with DMEM containing various concentrations of
Enovin and 0.1% BSA (for 39-SK, osteoblasts, HSMC, chondrocytes) or
DMEM containing various concentrations of Enovin and 0.5% FBS (for
HUVEC) and cells were incubated for 24 h. Subsequently, the culture
medium was replaced with 100 .mu.l of DMEM containing 5% FBS and
0.1 .mu.Ci of [.sup.3H]-thymidine. Following 2 h of pulse
labelling, cells were fixed with methanol/acetic acid (3:1,
vol/vol) for 1 h at room temperature. The fixed cells were washed
twice with 80% methanol. The cells were solubilized in 0.05%
trypsin (100 .mu.l/well) for 30 min and then in 0.5% SDS (100
.mu.l/well) for an additional 30 min. Aliquotes of cell lysates
(180 .mu.l) were combined with 2 ml of scintillation cocktail and
the radioactivity of cell lysates was measured using a liquid
scintillation counter (Wallac 1409).
[0153] Chemotaxis Assay
[0154] Cells were maintained as described in "DNA Synthesis Assay".
The chemotactic activity of Enovin was analyzed using a 12-well
modified Boyden Chamber (McQuillan, D. J., Handley, C. J.,
Campbell, M. A., Bolis, S., Milway, V. E., Herington, A. C.,
(1986), "Stimulation of Proteoglycan biosynthesis by serum and
insulin-like growth factor-I in cultured bovine articular
cartilage", Biochem. J. 240:423-430). Cells were trypsinized using
0.05% trypsin and 0.5 mM EDTA and resuspended in DMEM. To the
bottom wells of a Boyden chamber, aliquots of 150 .mu.l of media
containing various concentrations of Enovin were added. A
polycarbonate membrane (8 .mu.m) coated with 0.1 mg/ml of type I
collagen was placed on the top of the bottom wells, followed by
assembling the top wells. To the top wells, aliquots of 100 .mu.l
of cells (70,000 cells/ml) were added. Following a 6-h incubation
period, the apparatus was disassembled. Cells remaining on the top
of the membrane were removed. The membrane was fixed with 10%
formaldehyde for 15 min, followed by staining with Gill's strength
hemotoxylin. Cells were counted under microscopy (250.times.
magnification), and the average of cell counts from five areas of
each well was used. Each experiment was repeated at least four
times. The results were expressed as the fold of control (DMEM
containing 0.1% BSA).
[0155] As illustrated by the results in FIG. 8 to 18, enovin has no
effect on proliferation in each of the cell types used, or on the
migration of HUVEC cells (FIG. 14) as described above. There was an
effect of enovin on SH-SY-5Y neuroblastoma cells. This demonstrated
enovins selective effect on neuronal cells.
[0156] Both GDNF and NTN have been shown to signal via a signalling
complex composed of a ligand-binding subunit, either GFR.alpha.-1.
or GFR.alpha.-2, and a signalling subunit, the cRET protein
tyrosine kinase. Enovin is expected to exert its biological effects
via a similar signalling complex composed of a GFR.alpha. binding
partner (either GFR.alpha.-1, GFR.alpha.-2, the recently
characterised orphan receptor GFR.alpha.-3 or other as yet
uncharacterized members of the GFR.alpha. family) in combination
with cRET or another signalling partner. Indeed, our binding data
show that enovin can bind specifically to GFR.alpha.-3.
[0157] In humans, germ line mutations in GDNF or cRET can lead to
several disease phenotypes including multiple endocrine neoplasia
and Familial Hirschsprung disease (HSCR) (Romeo et al., 1994, Edery
et al., 1994, Angrist et al., 1996). Both diseases are associated
with gut dismotility, with Hirschsprung disease being the most
common cause of congenital bowel obstruction in infants.
Interestingly, GDNF and cRET knockout mice exhibit remarkably
similar pathologies with renal agenesis and intestinal
aganglionosis (Sanchez et al., 1996; Moore et al., 1996; Pichel et
al., 1996). Enovin could be involved in similar disorders of the
gut or the kidneys or, since it is ubiquitously expressed, could be
important in the development of other peripheral organs in the
body.
[0158] The interaction of ligands with their receptors is generally
achieved by the interaction of specific bonds from particular
residues in both proteins. Fragments of a protein can serve as
agonists activating the receptor to elicit its growth promoting and
survival sustaining effects on cells. Parts of enovin or synthetic
peptides based on the enovin protein sequence can therefore be
useful as agonists or antagonists to regulate its receptor
GFR.alpha.3. Using peptide synthesis or recombinant techniques,
hybrid growth factors composed of parts of GDNF, NTN or PSP or any
other neurotrophic or growth factor with parts of enovin can be
produced to yield a novel synthetic growth factor with new
properties.
[0159] Two pilot trials were conducted to test whether enovin is
able to change the taxol-induced sensory deficits in rats after
subplantar injections in rats. In a first experiment, it was tested
whether a single treatment with enovin could reverse the
taxol-induced sensory deficit, whereas in a second trial it was
tested whether enovin could prevent the development of the
taxol-induced deficits.
[0160] Reversal Over Time of Taxol-Induced Sensory Dysfunction.
[0161] Procedure
[0162] Male Sprague-Dawley rats, weighing 300-340 gram, were used.
The animals were housed individually with food and water ad lib.
Before the start of the experiment, the animals were placed in
standard observation cages and after a habituation period of 15
min, the pin prick reflex was evaluated. To do so, the plantar
surface of the right paw of the animal was stimulated with a needle
and the reactivity to this pin-prick was scored as either present
(score=1) or absent (score=0). Within one session, the procedure
was repeated three times with a time interval of 1 min between 2
consecutive stimulus presentations; as such the pin prick test
consisted of 3 measures of reactivity to a pin prick. Only rats
having normal reactions on the 3 pin pricks were included in the
experiment.
[0163] On the 3 consecutive days in the morning, the animals
received daily a subplantar injection of 50 .mu.l of taxol (3 mg/ml
paclitaxel dissolved in cremophor and dehydrated alcohol plus
water) in the right hind paw. During the next morning, the pin
prick reflex was re-evaluated and animals not showing any
reactivity to the 3 stimulus presentations were selected. These
animals were randomly divided in subgroups (n=10/group) receiving a
subplantar injection in the right hind paw of 75 .mu.l of either
vehicle, saline or 23 or 130 .mu.g/ml enovin. Because no
differences were observed between the results of the vehicle and
saline treated animals, both groups were joined (control group). At
days 1, 4, 5 and 7 after the last treatment, the pin prick test was
performed both in the morning (between 8 and 9 a m) and the evening
(between 3.30 and 4.30 p m). On day 8, a last pin prick test was
taken during the morning. For each animal, the cumulative score of
reactivity to the pin prick was measured over time. Because in
total 9 pin prick tests (each consisting of 3 pin prick
presentations) were performed after the last drug treatment, the
maximal score to be reached over the total time period of the
experiment is 27.
[0164] Results
[0165] Repeated subplantar injections of taxol over 3 consecutive
days results in an acute inflammatory reaction with a lack of
responding to a pin prick stimulation in the majority of animals. A
subplantar injection of saline or vehicle did not affect the
taxol-induced deficit. At the first measurement, only 4 out of 20
controls showed at least 1 reaction to the three pin pricks and the
mean (.+-.SEM) pin prick score of the controls at the first
measurement was 0.25 (.+-.0.12); this in contrast to the starting
of the experiment where the mean score was 3.0 (.+-.0.0) because
all animals responded to the pin prick. Even after 8 days of
measurement, the reactivity in the controls was still impaired with
11 out of 20 rats responding at least once and with a mean pin
prick score of 0.75 (.+-.0.18). Within this control group, none of
the rats displayed a normal reactivity to all 3 stimuli. The
cumulative pin prick score of the controls over time is presented
in FIG. 19. Because the animals were tested 9 times over an 8 days
period, the maximal score to be reached with 3 pin pricks at each
test is 27. As seen on the graph, a subplantar injection of saline
or vehicle was unable to reverse the taxol-induced deficit over the
time period tested. The mean total cumulative score of the controls
at the end of the experiment was 5.10 (.+-.0.87); being 18.9% of
the maximal score to be reached.
[0166] A single subplantar injection of 75 .mu.l of 23 .mu.g/ml
enovin, resulted after the first measurement in 4 out of 10 rats
responding at least once, with a mean pin prick score of 0.70
(.+-.0.33). At day 8, all 10 animals responded at least once to the
pin prick, and a normal reactivity was present in 5 out of 10 rats.
The average pin prick score of this group at day 8 was 2.20
(.+-.0.29). As compared to the controls, the average cumulative
score at the end of the 8 days of measurement was significantly
increased (Mann-Whitney U-test, two-tailed, p<0.01), reaching a
mean pin prick score of 14.50 (.+-.1.96) (FIG. 19). This is 53.7%
of the maximal score.
[0167] Also with an subplantar injection of 130 .mu.g/ml enovin
there was improved efficacy against the controls. At the first
measurement after 130 .mu.g/ml enovin, 6 out of 10 rats reponded at
least once with a mean pin prick score of 1.10 (.+-.0.35). At day
8, all 10 animals responded to at least one pin prick with a mean
score of 2.60 (.+-.0.22). A normal reactivity to the 3 pin pricks
was present in 8 out of 10 rats. The average cumulative total pin
prick score at the end of the experiment in this group was 17.20
(.+-.1.94). This is 63.7% of the total possible score and
significantly improved as compared to the control group
(p<0.01).
[0168] Prevention Over Time of Taxol-Induced Sensory
Dysfunction.
[0169] Procedure
[0170] Male Sprague-Dawley rats, weighing 300-340 gram, were used.
The animals were housed individually with food and water ad lib.
Before the start of the experiment, the animals were placed in
standard observation cages and after a habituation period of 15
min, the pin prick reflex was evaluated. To do so, the plantar
surface of the right paw of the animal was stimulated with a needle
and the reactivity to this pin-prick was scored as either present
(score=1) or absent (score=0). Within one session, the procedure
was repeated three times with a time interval of 1 min between 2
consecutive stimulus presentations; as such the pin prick test
consisted of 3 measures of reactivity to a pin prick. Only rats
having normal reactions on the 3 pin pricks were included in the
experiment (pin prick score=3). After this control measurement, the
animals were randomly divided in subgroups (n=10/group) receiving
an subplantar injection in the right hind paw of 75 .mu.l of either
vehicle, saline or 23 or 130 .mu.g/ml enovin. Because no
differences were observed between the results of the vehicle and
saline treated animals, both groups were joined (control group).
During the 3 consecutive days, the animals received daily a
subplantar injection of 50 .mu.l of taxol (3 mg/ml paclitaxel
dissolved in cremophor and dehydrated alcohol plus water) in the
right hind paw. At days 1, 4, 5 and 7 after taxol, the pin prick
test was performed both in the morning (between 8 and 9 a m) and
the evening (between 3.30 and 4.30 p m). On day 8, a last pin prick
test was done during the morning. For each animal, the cumulative
score of reactivity to the pin prick was measured over time.
Because in total 9 pin prick tests (each consisting of 3 pin prick
presentations) were performed after the taxol treatment, the
maximal cumulative score to be reached over the total time period
of the experiment is 27.
[0171] Results
[0172] A subplantar injection of saline or vehicle before taxol did
not prevent the taxol-induced deficit in the pin prick test. At the
first testing after taxol, 8 out of 20 rats responded at least once
to the pin prick, with a mean pin prick score of 0.60 (.+-.0.18).
At day 8, the taxol-induced deficit was still present, with only 8
out of 20 animals responding and having a mean score of 0.8
(.+-.0.25). Within two animals, a normalised pin prick reflex was
present. Over time, the cumulative pin prick score was also
reduced, resulting in a mean value of 6.55 (.+-.1.08), which is
24.3% of the maximal score (FIG. 20).
[0173] Pretreatment with 23 .mu.g/ml enovin reduced the
taxol-induced deficits on the pin prick. At day 1, 8 out of 10
animals responded at least once, and the average pin prick score
was 1.70 (.+-.0.40). At day 8, all animals were responding with a
mean score of 2.50 (.+-.0.27). Here 7 animals revealed a normal
reactivity on all pin prick exposures. With regard to the
cumulative responding over time (FIG. 20), the mean total score was
significantly improved (p<0.01) over the control level to 18.40
(.+-.1.73); this is 68.1% of the maximal value.
[0174] Comparable results were obtained after a pretreatment with
130 .mu.g/ml enovin. Here, 6 out of 10 animals responded during the
first testing with a mean pin prick score of 1.70 (.+-.0.31). At
day 8, all animals were reacting at least once to a pin prick
stimulation with a mean score of 2.40 (.+-.0.22) and all 3
reactions were normal in half of the animals. With regard to the
cumulative score, the mean score obtained at day 8 is 17.70
(.+-.1.92), representing 65.5% of the total score.
[0175] The present series of experiments indicate that a single
subplantar injection of enovin is able to reduce the taxol-induced
sensory deficits as measured by a pin prick test. Activity is seen
when the drug was applied both before and after taxol.
[0176] Enovin is a possible candidate for pain syndromes with
mainly a peripheral and central neurogenic component,
rheumatic/inflammatory diseases as well as conductance
disturbances, and can play a modulatory role in sensory processes
after transdermal, topical, local, central (such as epidural,
intrathecal, and the like) and systemic application. Further it is
worthwhile to use enovin as a diagnostic tool to screen for
physiophatological changes in the area's mentioned above.
[0177] Comparison of Enovin mRNA Expression in Normal Versus
Diseased Tissues
[0178] The expression of Enovin mRNA was quantitatively analysed
using the ABI Prism 7700 Sequence Detection System (TaqMan; Perkin
Elmer) using proprietary technology developed and carried out at
Pharmagene Laboratories Ltd. Royston, United Kingdom. The system
uses a fluorogenic probe to generate sequence specific fluorescent
signals during PCR. The probe is an oligonucleotide with
fluorescent reporter and quencher dyes attached, it is positioned
between the forward and reverse PCR primers. While intact, the
intensity of reporter fluorescence is suppressed by the quencher.
Should the probe form part of a replication complex, the
fluorescent reporter is cleaved from the quencher by a 5' to 3'
exonuclease activity inherent in Taq polymerase. The increase in
fluorescent reporter signal within a reaction is a direct measure
of the accumulation of PCR product. The starting copy number of an
mRNA target sequence (Cn) is established by determining the
fractional PCR cycle number (Ct) at which a PCR product is first
detected--the point at which the fluorescence signal passes above a
threshold baseline. Quantification of the amount of target mRNA in
each sample is established through comparison of experimental Ct
values with a standard curve.
[0179] RNA Preparation and Quality Control
[0180] Total RNA was isolated from whole and sub-dissected tissue,
using Tri-Zol reagent (Life Technologies, Gaithersburg, Md., USA)
according to the suppliers' protocol. Quality control procedures
for all RNA samples included an assessment of integrity (intact 18S
and 28S ribosomal RNA) and determination of the presence of high
abundance (actin) and low abundance (transferrin receptor)
transcripts.
[0181] Primer/Probe Design
[0182] A pair of primers and a TaqMan probe were designed to
amplify a specific sequence from Enovin TABLE-US-00004 Primer 1: 5'
ACGGTTCTCCAGGTGCTGT 3' Primer 3: 5' TGCTGCCGACCCACG 3' Probe 5: 5'
CTACGAAGCGGTCTCCTTCATGGACG 3'
[0183] In addition a pair of primers and a TaqMan probe were
designed which span an intron and amplify a portion of the human
GAPDH gene TABLE-US-00005 Primer 2: 5' CAGAGTTAAAAGCAGCCCTGGT 3'
Primer 4: 5' GAAGGTGAAGGTCGGAGTCAAC 3' Probe 6: 5'
TTTGGTCCGTATTGGGCGCCT 3'
[0184] Probe 5 is labelled with the fluor FAM while probe 6 is
labelled with the fluor VIC.
[0185] DNase Treatment of Total RNA
[0186] For each tissue tested 2.2 .mu.g of total RNA was digested
with 2 units of RNase free DNase (Gibco BRL) for 15 minutes at room
temperature in a 20 .mu.l volume of 1.times. DNase buffer (Gibco
BRL). The reaction was stopped by addition of 2 .mu.l of 25 mM EDTA
solution. The samples were then incubated at 65.degree. C. for 10
minutes to inactivate the enzyme.
[0187] First Strand cDNA Synthesis
[0188] For each tissue tested 100 ng of total RNA was used as
template for first strand cDNA synthesis. The RNA in a volume of 4
ml and in the presence of 50 nM primers 1 and 2, 1.times.PCR buffer
II (Perkin Elmer) and 5 mM MgCl.sub.2 was heated to 72.degree. C.
for 5 minutes and cooled slowly to 55.degree. C. After addition of
all other reagents, the 6 ml reaction was incubated at 48.degree.
C. for 30 minutes followed by an enzyme inactivation step of
90.degree. C. for 5 minutes. The final reaction conditions were as
follows: 1.times.PCR buffer II, 5 mM MgCl.sub.2, 1 mM dATP, dTTP,
dGTP, dCTP, 12.5 units MuLV reverse transcriptase (Gibco BRL).
[0189] PCR Amplification of First Strand cDNA Products
[0190] The cDNA derived from 100 ng total RNA for each sample was
subjected to PCR amplification in a single reaction to identify
both target and GAPDH transcripts. The final primer/probe
concentrations for target were 300 nM primer 1, 300 nM primer 3 and
200 nM probe 5, those for GAPDH were 20 nM primer 2, 20 nM primer 4
and 100 nM probe 6. The final concentration of other reagents in
the reaction were 4.5% glycerol, 1.times. TaqMan buffer A (Perkin
Elmer), 6.25 mM MgCl.sub.2, 430M dATP, dUTP, dGTP, dCTP, 2.5 units
AmpliTaq Gold. The PCR amplification was carried out in the ABI
7700 sequence detection system, an initial enzyme activation step
of 94.degree. C. for 12 min was followed by 45 cycles of 94.degree.
C. 15 secs, 60.degree. C. 1 min (minimum ramp time).
[0191] Diseases and Tissues Tested
[0192] Enovin mRNA expression was compared in tissues derived from
disease patients and normal control individuals (FIGS. 25 and 26).
The table below shows the diseases and corresponding tissues that
have been investigated. For each condition, three diseased and
three control samples were analysed. TABLE-US-00006 Patholog Tissue
1 Tissue 2 Tissue 3 Alzheimer's temporal hippocampus occipital
disease cortex cortex Multiple spinal cord periventicular
cerebellum sclerosis white matter Parkinson's substantia putamen
cerebellum disease nigra Cancer Colon breast ductal lung
adenocarcinoma adenocarcinoma squamous cell carcinoma
[0193] Statistical Analysis
[0194] For each group of 3 tissues, the mean and standard deviation
were calculated on the Ct values (which are normally distributed)
and were then converted into Cn values according to the formula
Cn=10.sup.((Ct-40.007)/-3.623). Analysis of variance (ANOVA) was
performed on the Ct values also to compare the mean Enovin mRNA
expression levels in normal versus diseased tissues.
[0195] FIGS. 25 and 26 show the mean Enovin mRNA copy numbers
(.+-.SD; n=3) in diseased versus control tissues. Statistical
analysis showed a significant increase in the Enovin expression
level in the periventricular white matter of patients with multiple
sclerosis (p=0.013). The internal GAPDH control showed no
significant difference (p=0.79). Although the Enovin expression
level in the periventricular white matter is quite low in normal
tissue (270 copies per 100 ng total RNA on average versus 200000
copies of GAPDH), the level is three times higher (825) in patients
with multiple sclerosis.
[0196] Only one other diseased tissue showed a significant
difference versus normal control: in breast ductal adenocarcinoma,
the Enovin mRNA expression level is 6 times higher (6000 versus
1000; p=0.007), but the GAPDH control value is also significantly
increased (165000 versus 44000; p=0.03), probably representing a
general increase in mRNA levels.
[0197] In conclusion, we have found Enovin mRNA levels to be
upregulated in the periventricular white matter of patients with
multiple sclerosis.
[0198] Use of Phospho-Specific Antibody Cell-Based ELISA for
Screening of Enovin Mimetic on GFR.alpha.3/cRET Receptor
Complex.
[0199] Method can Also be Used for Identification of Agonist or
Antagonist of Other Neurotrophin Receptors, such as GFR.alpha.1,
GFR.alpha.2, GFR.alpha.4, TrkA, TrkB and TrkC.
[0200] Assay
[0201] Using this assay we can identify agonist or antagonist
compounds of neurotrophic growth factors by measuring the
activation of key signalling kinases activated in the neurotrophic
pathway or by measuring the activation of cRET receptor kinase. The
activation is measured by detecting the amount of phosphorylated
kinase or receptor kinase using phospho-specific antibodies. We
will use NIH 3T3 cells expressing transiently or permanently TrkA,
TrkB, TrkC, GFRa1/cRET, GFRa2/cRET, GFRa3/cRET or GFRa4/cRET.
[0202] The activation of p42/p44 MAP kinase, PKB kinase, c-jun,
CREB, JNK/SAPK kinase and other kinases is detected using
commercialy available phospho-specific antibodies. In addition,
cRET activation can be deleted using phospho-specific cRET
antibody.
[0203] The protocol used was as follows:
[0204] Plate NIH 3T3 cells in 96-wells in 10% calf serum, cells
have to be 80% confluent before stimulation.
[0205] Next day, replace medium with serum-free medium and starve
cells for 18-24 h.
[0206] After starvation stimulate cells with compounds and
neurotrophic factors as positive control (10 ng/ml for neurotrophic
factors)
[0207] Fix cells with 4% formaldehyde in PBS at 4.degree. C. for 20
min.
[0208] Wash cells 3.times. with 200 .mu.l PBS/0.1% Triton for 5
min.
[0209] Quench the cells with 100 .mu.l 0.6% H.sub.2O.sub.2 in
PBS/0.1% Triton for 20 min.
[0210] Wash cells 3.times. with 200 .mu.l PBS/0.1% Triton for 5
min.
[0211] Block the cells with 100 .mu.l 10% foetal calf serum in
PBS/0.1% Triton for 60 min.
[0212] Incubate the cells with phosphospecific antibody in 50 .mu.l
5% BSA//PBS/0.1% Triton, over night at 4.degree. C. Antibody
dilution should be experimentally determined, suggested range
1:100-1:250.
[0213] Wash cells 3.times. with 200 .mu.l PBS/0.1% Triton for 5
min.
[0214] Incubate with secondary antibody HRP linked, dilution 1:100
in 50 ul 5% BSA/PBS/0.1% Triton, for 1 h at room temperature.
[0215] Wash cells 3.times. with 200 .mu.l PBS/0.1% Triton for 5
min.
[0216] Dissolve 1 tablet of OPD (Sigma) in 25 ml buffer (3.65 g
citric acid-H.sub.2O and 5.9 g Na.sub.2HPO.sub.4-.2H.sub.2O in 0.5
l H.sub.2O, pH 5.6) and add 12.5 .mu.l H.sub.2O.sub.2. Add 50 .mu.l
to each well and incubate for 15 min on shaker (200 rpm), covered
with aluminium foil.
[0217] Stop the reaction with 25 .mu.l H.sub.2SO.sub.4.
[0218] Measure OD.sub.490-650 on the ELISA reader.
[0219] Mesencephalic Dopaminergic Neuronal Culture
[0220] Neuronal Culture
[0221] Neuronal cultures were prepared from the ventral
mesencephalon of foetal rat by enzymatic and mechanical dispersion.
The tissue was collected, washed in ice-cold Ca.sup.2+- and
Mg.sup.2+-free phosphate buffered saline containing 0.6% glucose
(PBSG) and incubated for 30 min with PBSG containing 0.1% trypsin
at 37.degree. C. The cell suspension was plated at a density of 2.5
10.sup.5 cells/cm.sup.2 onto 96 well NUNC tissue culture plates. In
advance, culture plates were coated with poly-L-ornithine and CDM
containing 10% foetal calf serum. The cultures were maintained in
chemically defined medium (CDM), composed of a 1:1 mixture of
Dulbecco's Modified Eagles medium and F12 Nutrient supplemented
with glucose (0.6%), glutamine (2 mM), sodium bicarbonate (3 mM),
HEPES (5 mM), insulin (25 .mu.g/ml), human transferrin (100
.mu.g/ml), putrescine (60 .mu.g/ml), sodium selenite (30 nM),
streptomycin (100 .mu.g/ml) and penicillin (100 IU/ml).
[0222] Treatment with Neurotrophic Factors
[0223] Neurotrophins were dissolved in 0.5% bovine serum albumin as
a stock. Neurotrophins were added 3 h after initial plating and
after 5 days in culture. The same amount of 0.5% bovine serum
albumin was added to the control wells.
[0224] High-Affinity Dopamine Uptake
[0225] Dopamine uptake was measured after 10 days. For the uptake,
cells were washed twice with pre-warmed PBS supplemented with
glucose (5 mM), ascorbic acid (100 mM) and pargyline (100 mM) and
pre-incubated for 10 min with the same solution. The pre-incubation
solution was replaced with the same solution containing 50 nM
[.sup.3H]DA and incubation continued for 15 min at 37.degree. C.
Uptake was stopped by 3 rapid washes with ice-cold PBS. The
accumulated [.sup.3H]dopamine was released by incubating with
acidified ethanol for 30 min at room temperature. Radioactivity was
determined after addition of 4 ml of scintillation liquid (Packard
ultima gold MV) using Packard scintillation counter. Non-specific
uptake was determined by adding 20 .mu.M cocaine. TABLE-US-00007
TABLE 4 Effect of enovin on [.sup.3H] dopamine uptake Percent
control Treatment [.sup.3H] dopamine uptake n control 100 5 enovin
300 ng/ml 111 4 enovin 1000 ng/ml 127 5 enovin 2000 ng/ml 152 5
enovin 4000 ng/ml 161 1 enovin 10000 ng/ml 165 2
Cells were grown for 10 days in the presence or absence of enovin.
Untreated controls were set as 100%. Results are obtained in 1-5
independent experiments.
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LIST OF ABBREVIATIONS
[0279] [0280] BLAST basic local alignment search tool [0281] bp
base pairs [0282] cDNA complementary DNA [0283] CNS central nervous
system [0284] EST expressed sequence tag [0285] EVN enovin [0286]
GDNF glial cell-line derived neurotrophic factor [0287] GFR.alpha.
GDNF family receptor .alpha. [0288] GPI glycosyl phosphatidyl
inositol [0289] MTC multiple tissue cDNA [0290] NTN neurturin
[0291] PCR polymerase chain reaction [0292] PNS peripheral nervous
system [0293] PSP persephin [0294] RT-PCR reverse transcription PCR
[0295] TGF-.beta. transforming growth factor .beta. [0296] FISH
fluorescent in situ hybridisation [0297] MTN multiple tissue
northern [0298] NGF nerve growth factor [0299] SPR surface plasmon
resonance
Sequence CWU 1
1
49 1 339 DNA Homo sapiens 1 gctgggggcc cgggcagccg cgctcgggca
gcgggggcgc ggggctgccg cctgcgctcg 60 cagctggtgc cggtgcgcgc
gctcggcctg ggccaccgct ccgacgagct ggtgcgtttc 120 cgcttctgca
gcggctcctg ccgccgcgcg cgctctccac acgacctcag cctggccagc 180
ctactgggcg ccggggccct gcgaccgccc ccgggctccc ggcccgtcag ccagccctgc
240 tgccgaccca cgcgctacga agcggtctcc ttcatggacg tcaacagcac
ctggagaacc 300 gtggaccgcc tctccgccac cgcctgcggc tgcctgggc 339 2 474
DNA Homo sapiens 2 cgccgccgca gccttctcgg cccgcgcccc cgccgcctgc
acccccatct gctcttcccc 60 gcgggggccg cgcggcgcgg gctgggggcc
cgggcagccg cgctcgggca gcgggggcgc 120 ggggctgccg cctgcgctcg
cagctggtgc cggtgcgcgc gctcggcctg ggccaccgct 180 ccgacgagct
ggtgcgtttc cgcttctgca gcggctcctg ccgccgcgcg cgctctccac 240
acgacctcag cctggccagc ctactgggcg ccggggccct gcgaccgccc ccgggctccc
300 ggcccgtcag ccagccctgc tgccgaccca cgcgctacga agcggtctcc
ttcatggacg 360 tcaacagcac ctggagaacc gtggaccgcc tctccgccac
cgcctgcggc tgcctgggct 420 gagggctcgc tccagggctt tgcagactgg
acccttaccg gtggctcttc ctgc 474 3 113 PRT Homo sapiens 3 Ala Gly Gly
Pro Gly Ser Arg Ala Arg Ala Ala Gly Ala Arg Gly Cys 1 5 10 15 Arg
Leu Arg Ser Gln Leu Val Pro Val Arg Ala Leu Gly Leu Gly His 20 25
30 Arg Ser Asp Glu Leu Val Arg Phe Arg Phe Cys Ser Gly Ser Cys Arg
35 40 45 Arg Ala Arg Ser Pro His Asp Leu Ser Leu Ala Ser Leu Leu
Gly Ala 50 55 60 Gly Ala Leu Arg Pro Pro Pro Gly Ser Arg Pro Val
Ser Gln Pro Cys 65 70 75 80 Cys Arg Pro Thr Arg Tyr Glu Ala Val Ser
Phe Met Asp Val Asn Ser 85 90 95 Thr Trp Arg Thr Val Asp Arg Leu
Ser Ala Thr Ala Cys Gly Cys Leu 100 105 110 Gly 4 139 PRT Homo
sapiens 4 Pro Pro Gln Pro Ser Arg Pro Ala Pro Pro Pro Pro Ala Pro
Pro Ser 1 5 10 15 Ala Leu Pro Arg Gly Gly Arg Ala Ala Arg Ala Gly
Gly Pro Gly Ser 20 25 30 Arg Ala Arg Ala Ala Gly Ala Arg Gly Cys
Arg Leu Arg Ser Gln Leu 35 40 45 Val Pro Val Arg Ala Leu Gly Leu
Gly His Arg Ser Asp Glu Leu Val 50 55 60 Arg Phe Arg Phe Cys Ser
Gly Ser Cys Arg Arg Ala Arg Ser Pro His 65 70 75 80 Asp Leu Ser Leu
Ala Ser Leu Leu Gly Ala Gly Ala Leu Arg Pro Pro 85 90 95 Pro Gly
Ser Arg Pro Val Ser Gln Pro Cys Cys Arg Pro Thr Arg Tyr 100 105 110
Glu Ala Val Ser Phe Met Asp Val Asn Ser Thr Trp Arg Thr Val Asp 115
120 125 Arg Leu Ser Ala Thr Ala Cys Gly Cys Leu Gly 130 135 5 819
DNA Homo sapiens 5 gagtttcccc tccacacagc taggagccca tgcccggcct
gatctcagcc cgaggacagc 60 ccctccttga ggtccttcct ccccaagccc
acctgggtgc cctctttctc cctgaggctc 120 cacttggtct ctccgcgcag
cctgccctgt ggcccaccct ggccgctctg gctctgctga 180 gcagcgtcgc
agaggcctcc ctgggctccg cgccccgcag ccctgccccc cgcgaaggcc 240
ccccgcctgt cctggcgtcc cccgccggcc acctgccggg taggtgagag ggcgaggggg
300 cggggcgggg ctggcccggg acaccgcgcg tgactgggtc tcattccagg
gggacgcacg 360 gcccgctggt gcagtggaag agcccggcgg ccgccgccgc
agccttctcg gcccgcgccc 420 ccgccgcctg cacccccatc tgctcttccc
cgcgggggcc gcgcggcgcg ggctgggggc 480 ccgggcagcc gcgctcgggc
agcgggggcg cggggctgcc gcctgcgctc gcagctggtg 540 ccggtgcgcg
cgctcggcct gggccaccgc tccgacgagc tggtgcgttt ccgcttctgc 600
agcggctcct gccgccgcgc gcgctctcca cacgacctca gcctggccag cctactgggc
660 gccggggccc tgcgaccgcc cccgggctcc cggcccgtca gccagccctg
ctgccgaccc 720 acgcgctacg aagcggtctc cttcatggac gtcaacagca
cctggagaac cgtggaccgc 780 ctctccgcca ccgcctgcgg ctgcctgggc
tgagggctc 819 6 85 PRT Homo sapiens 6 Met Pro Gly Leu Ile Ser Ala
Arg Gly Gln Pro Leu Leu Glu Val Leu 1 5 10 15 Pro Pro Gln Ala His
Leu Gly Ala Leu Phe Leu Pro Glu Ala Pro Leu 20 25 30 Gly Leu Ser
Ala Gln Pro Ala Leu Trp Pro Thr Leu Ala Ala Leu Ala 35 40 45 Leu
Leu Ser Ser Val Ala Glu Ala Ser Leu Gly Ser Ala Pro Arg Ser 50 55
60 Pro Ala Pro Arg Glu Gly Pro Pro Pro Val Leu Ala Ser Pro Ala Gly
65 70 75 80 His Leu Pro Gly Arg 85 7 159 PRT Homo sapiens 7 Leu Gly
Leu Ile Pro Gly Gly Arg Thr Ala Arg Trp Cys Ser Gly Arg 1 5 10 15
Ala Arg Arg Pro Pro Pro Gln Pro Ser Arg Pro Ala Pro Pro Pro Pro 20
25 30 Ala Pro Pro Ser Ala Leu Pro Arg Gly Gly Arg Ala Ala Arg Ala
Gly 35 40 45 Gly Pro Gly Ser Arg Ala Arg Ala Ala Gly Ala Arg Gly
Cys Arg Leu 50 55 60 Arg Ser Gln Leu Val Pro Val Arg Ala Leu Gly
Leu Gly His Arg Ser 65 70 75 80 Asp Glu Leu Val Arg Phe Arg Phe Cys
Ser Gly Ser Cys Arg Arg Ala 85 90 95 Arg Ser Pro His Asp Leu Ser
Leu Ala Ser Leu Leu Gly Ala Gly Ala 100 105 110 Leu Arg Pro Pro Pro
Gly Ser Arg Pro Val Ser Gln Pro Cys Cys Arg 115 120 125 Pro Thr Arg
Tyr Glu Ala Val Ser Phe Met Asp Val Asn Ser Thr Trp 130 135 140 Arg
Thr Val Asp Arg Leu Ser Ala Thr Ala Cys Gly Cys Leu Gly 145 150 155
8 1188 DNA Homo sapiens 8 ctgatgggcg ctcctggtgt tgatagagat
ggaacttgga cttggaggcc tctccacgct 60 gtcccactgc ccctggccta
ggcggcaggt gagtggttct cccagtgact cctacctggt 120 actgaggaaa
ggcggcttga ctggtgaggg agagcagggc ttggcttggg cagcggttag 180
gtgtgggagg gaaaatggtc agggagggac caggtgaatg ggaggaggag cgggacttct
240 ctgaatggtc ggtgcactca ggtgattcct cccctgggct cccagaggca
gcaaacccat 300 tatactggaa cctaggccct tcctgagttt cccctccaca
cagctaggag cccatgcccg 360 gcctgatctc agcccgagga cagcccctcc
ttgaggtcct tcctccccaa gcccacctgg 420 gtgccctctt tctccctgag
gctccacttg gtctctccgc gcagcctgcc ctgtggccca 480 ccctggccgc
tctggctctg ctgagcagcg tcgcagaggc ctccctgggc tccgcgcccc 540
gcagccctgc cccccgcgaa ggccccccgc ctgtcctggc gtcccccgcc ggccacctgc
600 cgggtaggtg agagggcgag ggggcggggc ggggctggcc cgggacaccg
cgcgtgactg 660 ggtctcattc cagggggacg cacggcccgc tggtgcagtg
gaagagcccg gcggccgccg 720 ccgcagcctt ctcggcccgc gcccccgccg
cctgcacccc catctgctct tccccgcggg 780 ggccgcgcgg cgcgggctgg
gggcccgggc agccgcgctc gggcagcggg ggcgcggggc 840 tgccgcctgc
gctcgcagct ggtgccggtg cgcgcgctcg gcctgggcca ccgctccgac 900
gagctggtgc gtttccgctt ctgcagcggc tcctgccgcc gcgcgcgctc tccacacgac
960 ctcagcctgg ccagcctact gggcgccggg gccctgcgac cgcccccggg
ctcccggccc 1020 gtcagccagc cctgctgccg acccacgcgc tacgaagcgg
tctccttcat ggacgtcaac 1080 agcacctgga gaaccgtgga ccgcctctcc
gccaccgcct gcggctgcct gggctgaggg 1140 ctcgctccag ggctttgcag
actggaccct taccggtggc tcttcctg 1188 9 228 PRT Homo sapiens 9 Met
Glu Leu Gly Leu Gly Gly Leu Ser Thr Leu Ser His Cys Pro Trp 1 5 10
15 Pro Arg Arg Gln Ala Pro Leu Gly Leu Ser Ala Gln Pro Ala Leu Trp
20 25 30 Pro Thr Leu Ala Ala Leu Ala Leu Leu Ser Ser Val Ala Glu
Ala Ser 35 40 45 Leu Gly Ser Ala Pro Arg Ser Pro Ala Pro Arg Glu
Gly Pro Pro Pro 50 55 60 Val Leu Ala Ser Pro Ala Gly His Leu Pro
Gly Gly Arg Thr Ala Arg 65 70 75 80 Trp Cys Ser Gly Arg Ala Arg Arg
Pro Pro Pro Gln Pro Ser Arg Pro 85 90 95 Ala Pro Pro Pro Pro Ala
Pro Pro Ser Ala Leu Pro Arg Gly Gly Arg 100 105 110 Ala Ala Arg Ala
Gly Gly Pro Gly Ser Arg Ala Arg Ala Ala Gly Ala 115 120 125 Arg Gly
Cys Arg Leu Arg Ser Gln Leu Val Pro Val Arg Ala Leu Gly 130 135 140
Leu Gly His Arg Ser Asp Glu Leu Val Arg Phe Arg Phe Cys Ser Gly 145
150 155 160 Ser Cys Arg Arg Ala Arg Ser Pro His Asp Leu Ser Leu Ala
Ser Leu 165 170 175 Leu Gly Ala Gly Ala Leu Arg Pro Pro Pro Gly Ser
Arg Pro Val Ser 180 185 190 Gln Pro Cys Cys Arg Pro Thr Arg Tyr Glu
Ala Val Ser Phe Met Asp 195 200 205 Val Asn Ser Thr Trp Arg Thr Val
Asp Arg Leu Ser Ala Thr Ala Cys 210 215 220 Gly Cys Leu Gly 225 10
220 PRT Homo sapiens 10 Met Glu Leu Gly Leu Gly Gly Leu Ser Thr Leu
Ser His Cys Pro Trp 1 5 10 15 Pro Arg Arg Gln Pro Ala Leu Trp Pro
Thr Leu Ala Ala Leu Ala Leu 20 25 30 Leu Ser Ser Val Ala Glu Ala
Ser Leu Gly Ser Ala Pro Arg Ser Pro 35 40 45 Ala Pro Arg Glu Gly
Pro Pro Pro Val Leu Ala Ser Pro Ala Gly His 50 55 60 Leu Pro Gly
Gly Arg Thr Ala Arg Trp Cys Ser Gly Arg Ala Arg Arg 65 70 75 80 Pro
Pro Pro Gln Pro Ser Arg Pro Ala Pro Pro Pro Pro Ala Pro Pro 85 90
95 Ser Ala Leu Pro Arg Gly Gly Arg Ala Ala Arg Ala Gly Gly Pro Gly
100 105 110 Ser Arg Ala Arg Ala Ala Gly Ala Arg Gly Cys Arg Leu Arg
Ser Gln 115 120 125 Leu Val Pro Val Arg Ala Leu Gly Leu Gly His Arg
Ser Asp Glu Leu 130 135 140 Val Arg Phe Arg Phe Cys Ser Gly Ser Cys
Arg Arg Ala Arg Ser Pro 145 150 155 160 His Asp Leu Ser Leu Ala Ser
Leu Leu Gly Ala Gly Ala Leu Arg Pro 165 170 175 Pro Pro Gly Ser Arg
Pro Val Ser Gln Pro Cys Cys Arg Pro Thr Arg 180 185 190 Tyr Glu Ala
Val Ser Phe Met Asp Val Asn Ser Thr Trp Arg Thr Val 195 200 205 Asp
Arg Leu Ser Ala Thr Ala Cys Gly Cys Leu Gly 210 215 220 11 766 DNA
Homo sapiens 11 ctgatgggcg ctcctggtgt tgatagagat ggaacttgga
cttggaggcc tctccacgct 60 gtcccactgc ccctggccta ggcggcaggc
tccacttggt ctctccgcgc agcctgccct 120 gtggcccacc ctggccgctc
tggctctgct gagcagcgtc gcagaggcct ccctgggctc 180 cgcgccccgc
agccctgccc cccgcgaagg ccccccgcct gtcctggcgt cccccgccgg 240
ccacctgccg gggggacgca cggcccgctg gtgcagtgga agagcccggc ggccgccgcc
300 gcagccttct cggcccgcgc ccccgccgcc tgcaccccca tctgctcttc
cccgcggggg 360 ccgcgcggcg cgggctgggg gcccgggcag ccgcgctcgg
gcagcggggg cgcggggctg 420 ccgcctgcgc tcgcagctgg tgccggtgcg
cgcgctcggc ctgggccacc gctccgacga 480 gctggtgcgt ttccgcttct
gcagcggctc ctgccgccgc gcgcgctctc cacacgacct 540 cagcctggcc
agcctactgg gcgccggggc cctgcgaccg cccccgggct cccggcccgt 600
cagccagccc tgctgccgac ccacgcgcta cgaagcggtc tccttcatgg acgtcaacag
660 cacctggaga accgtggacc gcctctccgc caccgcctgc ggctgcctgg
gctgagggct 720 cgctccaggg ctttgcagac tggaccctta ccggtggctc ttcctg
766 12 742 DNA Homo sapiens 12 ctgatgggcg ctcctggtgt tgatagagat
ggaacttgga cttggaggcc tctccacgct 60 gtcccactgc ccctggccta
ggcggcagcc tgccctgtgg cccaccctgg ccgctctggc 120 tctgctgagc
agcgtcgcag aggcctccct gggctccgcg ccccgcagcc ctgccccccg 180
cgaaggcccc ccgcctgtcc tggcgtcccc cgccggccac ctgccggggg gacgcacggc
240 ccgctggtgc agtggaagag cccggcggcc gccgccgcag ccttctcggc
ccgcgccccc 300 gccgcctgca cccccatctg ctcttccccg cgggggccgc
gcggcgcggg ctgggggccc 360 gggcagccgc gctcgggcag cgggggcgcg
gggctgccgc ctgcgctcgc agctggtgcc 420 ggtgcgcgcg ctcggcctgg
gccaccgctc cgacgagctg gtgcgtttcc gcttctgcag 480 cggctcctgc
cgccgcgcgc gctctccaca cgacctcagc ctggccagcc tactgggcgc 540
cggggccctg cgaccgcccc cgggctcccg gcccgtcagc cagccctgct gccgacccac
600 gcgctacgaa gcggtctcct tcatggacgt caacagcacc tggagaaccg
tggaccgcct 660 ctccgccacc gcctgcggct gcctgggctg agggctcgct
ccagggcttt gcagactgga 720 cccttaccgg tggctcttcc tg 742 13 603 DNA
Homo sapiens 13 ctgatgggcg ctcctggtgt tgatagagat ggaacttgga
cttggaggcc tctccacgct 60 gtcccactgc ccctggccta ggcggcaggg
ggacgcacgg cccgctggtg cagtggaaga 120 gcccggcggc cgccgccgca
gccttctcgg cccgcgcccc cgccgcctgc acccccatct 180 gctcttcccc
gcgggggccg cgcggcgcgg gctgggggcc cgggcagccg cgctcgggca 240
gcgggggcgc ggggctgccg cctgcgctcg cagctggtgc cggtgcgcgc gctcggcctg
300 ggccaccgct ccgacgagct ggtgcgtttc cgcttctgca gcggctcctg
ccgccgcgcg 360 cgctctccac acgacctcag cctggccagc ctactgggcg
ccggggccct gcgaccgccc 420 ccgggctccc ggcccgtcag ccagccctgc
tgccgaccca cgcgctacga agcggtctcc 480 ttcatggacg tcaacagcac
ctggagaacc gtggaccgcc tctccgccac cgcctgcggc 540 tgcctgggct
gagggctcgc tccagggctt tgcagactgg acccttaccg gtggctcttc 600 ctg 603
14 489 DNA Homo sapiens 14 ctgatgggcg ctcctggtgt tgatagagat
ggaacttgga cttggaggcc tctccacgct 60 gtcccactgc ccctggccta
ggcggcagcc tgccctgtgg cccaccctgg ccgctctggc 120 tctgctgagc
agcgtcgcag aggcctccct gggctccgcg ccccgcagcc ctgccccccg 180
cgaaggcccc ccgcctgtcc tggcgtcccc cgccggccac ctgccggcgg ctcctgccgc
240 cgcgcgcgct ctccacacga cctcagcctg gccagcctac tgggcgccgg
ggccctgcga 300 ccgcccccgg gctcccggcc cgtcagccag ccctgctgcc
gacccacgcg ctacgaagcg 360 gtctccttca tggacgtcaa cagcacctgg
agaaccgtgg accgcctctc cgccaccgcc 420 tgcggctgcc tgggctgagg
gctcgctcca gggctttgca gactggaccc ttaccggtgg 480 ctcttcctg 489 15
350 DNA Homo sapiens 15 ctgatgggcg ctcctggtgt tgatagagat ggaacttgga
cttggaggcc tctccacgct 60 gtcccactgc ccctggccta ggcggcagcg
gctcctgccg ccgcgcgcgc tctccacacg 120 acctcagcct ggccagccta
ctgggcgccg gggccctgcg accgcccccg ggctcccggc 180 ccgtcagcca
gccctgctgc cgacccacgc gctacgaagc ggtctccttc atggacgtca 240
acagcacctg gagaaccgtg gaccgcctct ccgccaccgc ctgcggctgc ctgggctgag
300 ggctcgctcc agggctttgc agactggacc cttaccggtg gctcttcctg 350 16
74 PRT Homo sapiens 16 Ser Pro Asp Lys Gln Met Ala Val Leu Pro Arg
Arg Glu Arg Asn Arg 1 5 10 15 Gln Ala Ala Ala Ala Asn Pro Glu Asn
Ser Arg Gly Lys Gly Arg Arg 20 25 30 Gly Gln Arg Gly Lys Asn Arg
Gly Cys Val Leu Thr Ala Ile His Leu 35 40 45 Asn Val Thr Asp Leu
Gly Leu Gly Tyr Glu Thr Lys Glu Glu Leu Ile 50 55 60 Phe Arg Tyr
Cys Ser Gly Ser Cys Asp Ala 65 70 17 20 PRT Homo sapiens 17 Ala Glu
Thr Thr Tyr Asp Lys Ile Leu Lys Asn Leu Ser Arg Asn Arg 1 5 10 15
Arg Leu Val Ser 20 18 40 PRT Homo sapiens 18 Asp Lys Val Gly Gln
Ala Cys Cys Arg Pro Ile Ala Phe Asp Asp Asp 1 5 10 15 Leu Ser Phe
Leu Asp Asp Asn Leu Val Tyr His Ile Leu Arg Lys His 20 25 30 Ser
Ala Lys Arg Cys Gly Cys Ile 35 40 19 41 PRT Homo sapiens 19 Ala Arg
Leu Gly Ala Arg Pro Cys Gly Leu Arg Glu Leu Glu Val Arg 1 5 10 15
Val Ser Glu Leu Gly Leu Gly Tyr Ala Ser Asp Glu Thr Val Leu Phe 20
25 30 Arg Tyr Cys Ala Gly Ala Cys Glu Ala 35 40 20 20 PRT Homo
sapiens 20 Ala Ala Arg Val Tyr Asp Leu Gly Leu Arg Arg Leu Arg Gln
Arg Arg 1 5 10 15 Arg Leu Arg Arg 20 21 41 PRT Homo sapiens 21 Glu
Arg Val Arg Ala Gln Pro Cys Cys Arg Pro Thr Ala Tyr Glu Asp 1 5 10
15 Glu Val Ser Phe Leu Asp Ala His Ser Arg Tyr His Thr Val His Glu
20 25 30 Leu Ser Ala Arg Glu Cys Ala Cys Val 35 40 22 56 PRT Homo
sapiens 22 Ala Leu Ser Gly Pro Cys Gln Leu Trp Ser Leu Thr Leu Ser
Val Ala 1 5 10 15 Glu Leu Gly Leu Gly Tyr Ala Ser Glu Glu Lys Val
Ile Phe Arg Tyr 20 25 30 Cys Ala Gly Ser Cys Pro Arg Gly Ala Arg
Thr Gln His Gly Leu Ala 35 40 45 Leu Ala Arg Leu Gln Gly Gln Gly 50
55 23 14 PRT Homo sapiens 23 Arg Ala His Gly Gly Pro Cys Cys Arg
Pro Thr Arg Tyr Thr 1 5 10 24 26 PRT Homo sapiens 24 Asp Val Ala
Phe Leu Asp Asp Arg His Arg Trp Gln Arg Leu Pro Gln 1 5 10 15 Leu
Ser Ala Ala Ala Cys Gly Cys Gly Gly 20 25 25 49 PRT Homo sapiens 25
Ala Gly Gly Pro Gly Ser Arg Ala Arg Ala Ala Gly Ala Arg Gly Cys 1 5
10 15 Arg Leu Arg Ser Gln Leu Val Pro Val Arg Ala Leu Gly Leu Gly
His 20 25 30 Arg Ser Asp Glu Leu Val Arg Phe Arg Phe Cys Ser Gly
Ser Cys Arg 35 40 45 Arg 26 38 PRT Homo sapeins 26 Ala Arg Ser Pro
His Asp Leu Ser Leu Ala Ser Leu Leu Gly Ala Gly 1 5 10 15 Ala Leu
Arg Pro Pro Pro Gly Ser Arg Pro Val Ser Gln Pro Cys Cys 20 25 30
Arg Pro Thr Arg Tyr Glu 35 27 26 PRT Homo sapiens 27 Ala Val Ser
Phe Met Asp Val Asn Ser Thr Trp Arg Thr Val Asp Arg 1 5 10 15
Leu
Ser Ala Thr Ala Cys Gly Cys Leu Gly 20 25 28 20 PRT Homo sapiens 28
Met Glu Leu Gly Leu Gly Gly Leu Ser Thr Leu Ser His Cys Pro Trp 1 5
10 15 Pro Arg Arg Gln 20 29 54 PRT Homo sapiens 29 Ala Pro Leu Gly
Leu Ser Ala Gln Pro Ala Leu Trp Pro Thr Leu Ala 1 5 10 15 Ala Leu
Ala Leu Leu Ser Ser Val Ala Glu Ala Ser Leu Gly Ser Ala 20 25 30
Pro Arg Ser Pro Ala Pro Arg Glu Gly Pro Pro Pro Val Leu Ala Ser 35
40 45 Pro Ala Gly His Leu Pro 50 30 154 PRT Homo sapiens 30 Gly Gly
Arg Thr Ala Arg Trp Cys Ser Gly Arg Ala Arg Arg Pro Pro 1 5 10 15
Pro Gln Pro Ser Arg Pro Ala Pro Pro Pro Pro Ala Pro Pro Ser Ala 20
25 30 Leu Pro Arg Gly Gly Arg Ala Ala Arg Ala Gly Gly Pro Gly Ser
Arg 35 40 45 Ala Arg Ala Ala Gly Ala Arg Gly Cys Arg Leu Arg Ser
Gln Leu Val 50 55 60 Pro Val Arg Ala Leu Gly Leu Gly His Arg Ser
Asp Glu Leu Val Arg 65 70 75 80 Phe Arg Phe Cys Ser Gly Ser Cys Arg
Arg Ala Arg Ser Pro His Asp 85 90 95 Leu Ser Leu Ala Ser Leu Leu
Gly Ala Gly Ala Leu Arg Pro Pro Pro 100 105 110 Gly Ser Arg Pro Val
Ser Gln Pro Cys Cys Arg Pro Thr Arg Tyr Glu 115 120 125 Ala Val Ser
Phe Met Asp Val Asn Ser Thr Trp Arg Thr Val Asp Arg 130 135 140 Leu
Ser Ala Thr Ala Cys Gly Cys Leu Gly 145 150 31 23 DNA Artificial
Primer PNHsp1 31 cggtgcactc aggtgattcc tcc 23 32 26 DNA Artificial
Primer PNHsp2 32 ggcagcaaac ccattatact ggaacc 26 33 21 DNA
Artificial Primer PNHsp3 33 cgctggtgca gtggaagagc c 21 34 22 DNA
Artificial Primer PHNsp4 34 ctgcaccccc atctgctctt cc 22 35 22 DNA
Artificial Primer PNHap1 35 gcaggaagag ccaccggtaa gg 22 36 22 DNA
Artificial Primer PNHap2 36 ccagtctgca aagccctgga gc 22 37 22 DNA
Artificial Primer PNHsp5 37 gcaagctgcc tcaacaggag gg 22 38 24 DNA
Artificial Nested Primer PNHsp6 38 ggtgggggaa cagctcaaca atgg 24 39
25 DNA Artificial Forward Primer PNHexp-sp1 39 gcggatccgg
ctgggggccc gggca 25 40 28 DNA Artificial Reverse Primer PNHexp-ap1
40 gcctcgagtc agcccaggca gccgcagg 28 41 39 PRT Artificial Plasmid
NH2-terminal 41 Met Arg Gly Ser His His His His His His Gly Met Ala
Ser Met Thr 1 5 10 15 Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp
Asp Asp Asp Lys Asp 20 25 30 Pro Ala Gly Gly Pro Gly Ser 35 42 26
DNA Artificial Primer EVN(7)-sp1 42 ttcgcgtgtc tacaaactca actccc 26
43 22 DNA Artificial Primer PNHap1 43 gcaggaagag ccaccggtaa gg 22
44 19 DNA Artificial Primer 1 44 acggttctcc aggtgctgt 19 45 15 DNA
Artificial Primer 3 45 tgctgccgac ccacg 15 46 26 DNA Artificial
TaqMan Probe 5 46 ctacgaagcg gtctccttca tggacg 26 47 22 DNA
Artificial Primer 2 47 cagagttaaa agcagccctg gt 22 48 22 DNA
Artificial Primer 4 48 gaaggtgaag gtcggagtca ac 22 49 21 DNA
Artificial TaqMan Probe 6 49 tttggtccgt attgggcgcc t 21
* * * * *
References