U.S. patent application number 11/835262 was filed with the patent office on 2008-01-31 for human ependymin.
This patent application is currently assigned to Human Genome Sciences, Inc.. Invention is credited to Reinhard Ebner, Steven M. Ruben.
Application Number | 20080026989 11/835262 |
Document ID | / |
Family ID | 26752103 |
Filed Date | 2008-01-31 |
United States Patent
Application |
20080026989 |
Kind Code |
A1 |
Ebner; Reinhard ; et
al. |
January 31, 2008 |
Human Ependymin
Abstract
The present invention relates to a novel human Ependymin protein
which is a member of the ependymin family. In particular, isolated
nucleic acid molecules are provided encoding the human Ependymin
protein. Ependymin polypeptides are also provided as are vectors,
host cells and recombinant methods for producing the same. The
invention further relates to screening methods for identifying
agonists and antagonists of Ependymin activity. Also provided are
diagnostic methods for detecting nervous system-related disorders
and therapeutic methods for treating nervous system-related
disorders.
Inventors: |
Ebner; Reinhard;
(Gaithersburg, MD) ; Ruben; Steven M.;
(Brookeville, MD) |
Correspondence
Address: |
HUMAN GENOME SCIENCES INC.;INTELLECTUAL PROPERTY DEPT.
14200 SHADY GROVE ROAD
ROCKVILLE
MD
20850
US
|
Assignee: |
Human Genome Sciences, Inc.
Rockville
MD
|
Family ID: |
26752103 |
Appl. No.: |
11/835262 |
Filed: |
August 7, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10733646 |
Dec 12, 2003 |
7252956 |
|
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11835262 |
Aug 7, 2007 |
|
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10187904 |
Jul 3, 2002 |
6683161 |
|
|
10733646 |
Dec 12, 2003 |
|
|
|
09229583 |
Jan 13, 1999 |
6489138 |
|
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10187904 |
Jul 3, 2002 |
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60071330 |
Jan 14, 1998 |
|
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60075278 |
Feb 19, 1998 |
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Current U.S.
Class: |
424/133.1 ;
435/320.1; 435/325; 435/455; 435/6.14; 435/69.1; 435/7.21;
514/19.4; 530/387.9; 530/395; 536/23.5 |
Current CPC
Class: |
A61P 43/00 20180101;
G01N 2800/28 20130101; G01N 2500/00 20130101; C07K 14/475 20130101;
G01N 33/6893 20130101; A61K 38/00 20130101; Y10S 530/857
20130101 |
Class at
Publication: |
514/008 ;
435/320.1; 435/325; 435/455; 435/006; 435/069.1; 435/007.21;
530/387.9; 530/395; 536/023.5 |
International
Class: |
A61K 38/00 20060101
A61K038/00; A61P 43/00 20060101 A61P043/00; C07K 14/00 20060101
C07K014/00; C12N 15/00 20060101 C12N015/00; C12N 15/11 20060101
C12N015/11; C12N 15/87 20060101 C12N015/87; C12N 5/06 20060101
C12N005/06; C12P 21/04 20060101 C12P021/04; C12Q 1/68 20060101
C12Q001/68; G01N 33/53 20060101 G01N033/53 |
Claims
1. An isolated nucleic acid molecule nucleic acid molecule
comprising a polynucleotide having a nucleotide sequence at least
95% identical to a sequence selected from the group consisting of:
(a) a nucleotide sequence encoding the Ependymin polypeptide having
the complete amino acid sequence in SEQ ID NO:2 (i.e., positions
-37 to 187 of SEQ ID NO:2); (b) a nucleotide sequence encoding the
Ependymin polypeptide having the complete amino acid sequence in
SEQ ID NO:2 excepting the N-terminal methionine (i.e., positions
-36 to 187 of SEQ ID NO:2); (c) a nucleotide sequence encoding the
predicted mature Ependymin polypeptide having the amino acid
sequence at positions 1 to 187 in SEQ ID NO:2; (d) a nucleotide
sequence encoding the Ependymin polypeptide having the complete
amino acid sequence encoded by the cDNA clone contained in ATCC.TM.
Deposit No. 209464; (e) a nucleotide sequence encoding the
Ependymin polypeptide having the complete amino acid sequence
excepting the N-terminal methionine encoded by the cDNA clone
contained in ATCC.TM. Deposit No. 209464; (f) a nucleotide sequence
encoding the mature Ependymin polypeptide having the amino acid
sequence encoded by the cDNA clone contained in ATCC.TM. Deposit
No. 209464; and (g) a nucleotide sequence complementary to any of
the nucleotide sequences in (a), (b), (c), (d), (e) or (f),
above.
2. The nucleic acid molecule of claim 1 wherein said polynucleotide
has the complete nucleotide sequence in FIGS. 1A, 1B, and 1C (SEQ
ID NO:1).
3. The nucleic acid molecule of claim 1 wherein said polynucleotide
has the nucleotide sequence in FIGS. 1A, 1B, and 1C (SEQ ID NO:1)
encoding the mature Ependymin polypeptide having the amino acid
sequence from about 1 to about 187 in SEQ ID NO:2.
4. The nucleic acid molecule of claim 1 wherein said polynucleotide
has the complete nucleotide sequence of the cDNA clone contained in
ATCC.TM. Deposit No. 209464.
5. The nucleic acid molecule of claim 1 wherein said polynucleotide
has the nucleotide sequence encoding the Ependymin polypeptide
having the complete amino acid sequence excepting the N-terminal
methionine encoded by the cDNA clone contained in ATCC.TM. Deposit
No. 209464.
6. The nucleic acid molecule of claim 1 wherein said polynucleotide
has the nucleotide sequence encoding the mature Ependymin
polypeptide having the amino acid sequence encoded by the cDNA
clone contained in ATCC.TM. Deposit No. 209464.
7. An isolated nucleic acid molecule comprising a polynucleotide
which hybridizes under stringent hybridization conditions to a
polynucleotide having a nucleotide sequence identical to a
nucleotide sequence in (a), (b), (c), (d), (e), (f) or (g) of claim
1 wherein said polynucleotide which hybridizes does not hybridize
under stringent hybridization conditions to a polynucleotide having
a nucleotide sequence consisting of only A residues or of only T
residues.
8. An isolated nucleic acid molecule comprising a polynucleotide
which encodes the amino acid sequence of an epitope-bearing portion
of a Ependymin polypeptide having an amino acid sequence in (a),
(b), (c), (d), (e) or (f) of claim 1.
9. The isolated nucleic acid molecule of claim 10, which encodes an
epitope-bearing portion of a Ependymin polypeptide wherein the
amino acid sequence of said portion is selected from the group of
sequences in SEQ ID NO:2 consisting of: about Ala-1 to about Gln-9;
about Pro-8 to about Val-16; about Gln-19 to about Arg-27; about
Ile-69 to about Ser-77; about Asp-86 to about Glu-107; about
Glu-113 to about Tyr-123; about Thr-131 to about Gln-139; about
Leu-159 to about Phe-167; and about Leu-178 to about Ser-186.
10. A method for making a recombinant vector comprising inserting
an isolated nucleic acid molecule of claim 1 into a vector.
11. A recombinant vector produced by the method of claim 10.
12. A method of making a recombinant host cell comprising
introducing the recombinant vector of claim 11 into a host
cell.
13. A recombinant host cell produced by the method of claim 12.
14. A recombinant method for producing an Ependymin polypeptide,
comprising culturing the recombinant host cell of claim 13 under
conditions such that said polypeptide is expressed and recovering
said polypeptide.
15. An isolated Ependymin polypeptide comprising an amino acid
sequence at least 95% identical to a sequence selected from the
group consisting of: (a) the amino acid sequence of the full-length
Ependymin polypeptide having the complete amino acid sequence shown
in SEQ ID NO:2 (i.e., positions -37 to 187 of SEQ ID NO:2); (b) the
amino acid sequence of the full-length Ependymin polypeptide having
the complete amino acid sequence shown in SEQ ID NO:2 excepting the
N-terminal methionine (i.e., positions -36 to 187 of SEQ ID NO:2);
(c) the amino acid sequence of the predicted mature Ependymin
polypeptide having the amino acid sequence at positions 1 to 187 in
SEQ ID NO:2; (d) the complete amino acid sequence encoded by the
cDNA clone contained in the ATCC.TM. Deposit No. 209464; (e) the
complete amino acid sequence excepting the N-terminal methionine
encoded by the cDNA clone contained in the ATCC.TM. Deposit No.
209464; and (f) the complete amino acid sequence of the predicted
mature Ependymin polypeptide encoded by the cDNA clone contained in
the ATCC.TM. Deposit No. 209464.
16. An isolated antibody that binds specifically to a Ependymin
polypeptide of claim 15.
17. An isolated nucleic acid molecule comprising a polynucleotide
having a sequence at least 95% identical to a sequence selected
from the group consisting of: (a) the nucleotide sequence of SEQ ID
NO:15; (b) the nucleotide sequence of SEQ ID NO:16; (c) the
nucleotide sequence of SEQ ID NO:17; (d) the nucleotide sequence of
SEQ ID NO:18; (e) the nucleotide sequence of SEQ ID NO:19; (f) the
nucleotide sequence of SEQ ID NO:20; (g) the nucleotide sequence of
a portion of the sequence shown in FIGS. 1A, 1B, and 1C (SEQ ID
NO:1) wherein said portion comprises at least 50 contiguous
nucleotides from nucleotide 1 to nucleotide 2505; and (h) a
nucleotide sequence complementary to any of the nucleotide
sequences in (a), (b), (c), (d), (e), (f) or (g), above.
18. A method for the treatment of a patient having need of human
ependymin polypeptide comprising: (a) isolating the polypeptide of
claim 1, and (b) administering to the patient a therapeutically
effective amount of said polypeptide.
19. A method for identifying compounds which bind to and inhibit
activation of the polypeptide of claim 1 comprising: (a) contacting
a cell expressing on the surface thereof a receptor for the
polypeptide, said receptor being associated with a second component
capable of providing a detectable signal in response to the binding
of a compound to said receptor, with an analytically detectable
human cytokine polypeptide and a compound under conditions to
permit binding to the receptor; and (b) determining whether the
compound binds to and inhibits the receptor by detecting the
absence of a signal generated from the interaction of the human
ependymin polypeptide with the receptor.
20. A process for diagnosing a disease or a susceptibility to a
disease related to an under-expression of the polypeptide of claim
1 comprising: (a) isolating a sample of the nucleic acid sequence
encoding said polypeptide, and (b) determining a mutation in a
nucleic acid sequence encoding said polypeptide.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 10/733,646, filed Dec. 12, 2003 (now U.S. Pat. No. 7,252,956,
issued Aug. 7, 2007), which is a divisional of U.S. application
Ser. No. 10/187,904, filed Jul. 3, 2002 (now U.S. Pat. No.
6,683,161, issued Jan. 27, 2004), which is a divisional of U.S.
application Ser. No. 09/229,583, filed Jan. 13, 1999 (now U.S. Pat.
No. 6,489,138, issued Dec. 3, 2002), which claims benefit under 35
U.S.C. .sctn. 119(e) of U.S. Provisional Application Nos.
60/071,330, filed on Jan. 14, 1998, and 60/075,278, filed on Feb.
19, 1998, each of which is hereby incorporated by reference in its
entirety.
REFERENCE TO SEQUENCE LISTING AS TEXT FILE
[0002] This application refers to a "Sequence Listing" listed
below, which is provided as a text file. The text file contains a
document entitled "PF403D3-SequenceListing.txt" (27,516 bytes,
created Aug. 7, 2007), which is incorporated by reference in its
entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to a novel human gene encoding
a polypeptide which is a member of the Ependymin family. More
specifically, isolated nucleic acid molecules are provided encoding
a human polypeptide named Ependymin. Ependymin polypeptides are
also provided, as are vectors, host cells and recombinant methods
for producing the same. Also provided are diagnostic methods for
detecting disorders related to the nervous system, and therapeutic
methods for treating such disorders.
[0004] The invention further relates to screening methods for
identifying agonists and antagonists of Ependymin activity.
BACKGROUND OF THE INVENTION
[0005] Within the last several years, a number of ependymins have
been molecularly cloned from a variety of teleost fish including
Oncorhynchus mykiss (rainbow trout; Muller-Schmid, A., et al., Gene
118:189-196 (1992)), Salmo salar (Atlantic salmon; Muller-Schmid,
A., et al., Gene 118:189-196 (1992)), Esox lucius (pike;
Muller-Schmid, A., et al., J. Molec. Evol. 36:578-585 (1993)),
Carassius auratus (goldfish; Konigstorfer, A., et al., J.
Neurochem. 52:310-312 (1989); Konigstorfer, A., et al., J. Biol.
Chem. 264:13689-13692 (1989)), Brachydanio rerio (zebrafish;
Sterrer, S., et al., Neurosci. 37:277-284 (1990)), and Clupea
harengus (herring, Muller-Schmid, et al., J. Molec. Evol.
36:578-585 (1993)). The ependymins produced by these organisms are
synthesized as precursors which contain N-terminal, hydrophobic
signal sequences. Each of these molecules contains multiple
N-linked glycosylation sites, only some of which are conserved
between species (Schmidt, R. and Shashoua, V. E. J. Neurochem.
36:1368-1377 (1981); Schmidt, R. and Shashoua, V. E. J. Neurochem.
40:652-660 (1983); Ganb, B. and Hoffman, W. Eur. J. Biochem.
217:275-280 (1993)). The precursor ependymins range in apparent
molecular mass from 23.7 to 24.5 kDa, while the secreted mature
forms of these molecules are typically 21.6-22.3 kDa in size.
[0006] The piscine ependymins characterized thus far may be
categorized according to the number of cysteine residues present in
the mature polypeptide. Mature salmoniform (O. mykiss, S. salar,
and E. lucius) ependymin polypeptides contain only four cysteine
residues, whereas mature cypriniform (C. auratus and B. rerio) and
clupeiform (C. harengus) ependymin polypeptides contain five and
six cysteine residues, respectively (Hoffmann, W. Int. J. Biochem.
26:607-619 (1994)). Correspondingly, disulfide-linked dimerization
of the salmoniform ependymin polypeptides is not observed after
non-reducing SDS-PAGE. However, cypriniform and clupeiform
ependymins are observed as disulfide-linked dimers under
non-reducing conditions. It is speculated that the dimerization
occurs via the cysteine residue conserved only between the
salmoniform ependymins (this cysteine residue aligns with the
lysine residue at location 133 of human ependymin of the present
invention as shown in SEQ ID NO:2).
[0007] Several lines of evidence have provided the basis for an
understanding of the functional role(s) of the ependymins.
Ca.sup.2+-binding has been demonstrated for at least goldfish and
rainbow trout ependymins (Schmidt, R. and Makiola, E. Neuro. Chem.
(Life Sci. Adv.) 10:161-171 (1991); Ganb, B. and Hoffman, supra).
Further, ependymins are the primary cerebrospinal fluid component
in a number of teleost fish (Schmidt, R. and Lapp, H. Neurochem.
Int. 10:383-390 1987). Finally, roughly two-thirds of Ca2+ in the
CSF of rainbow trout is protein-bound (Ganb, B. and Hoffman,
supra). As a result, it is thought that ependymins may function in
Ca.sup.2+ homeostasis of the teleost piscine brain (Hoffman, W.,
supra).
[0008] In situ hybridization analyses have shown that ependymins
are apparently synthesized exclusively in miningeal fibroblasts of
the mininx (also termed the endomeninx or leptomeninx) of teleost
fish (Konigstorfer, A., et al., Cell Tissue Res. 261:59-64 (1990)).
Ependymins have also been found to associate with collagen fibrils
of the extracellular matrix (ECM; Schwarz, H., et al. Cell Tissue
Res. 273:417-425 (1993)), and, further, have the capacity to serve
as a substrate for outgrowing retinal axons (Schmidt, J. T., et
al., J. Neurobiol. 22:40-54 (1991)).
[0009] An additional role for ependymins has been identified in the
field of learning and memory. Using an experimental approach in
which goldfish learn to swim to a specific compartment of its
environment to avoid an electric shock, investigators have
determined that the amount of unbound or unincorporated
extracellular ependymins decreases after learning (Piront, M.-L.,
and Schmidt, R. Brain Res. 442:53-62 (1988); Schmidt, R. J.
Neurochem. 48:1870-1878 (1987)). Further, blockage of functional
ependymin molecules, either with antibodies or antisense
polynucleotides, resulted in the reversible inability of the
experimental animal to remember the task which it had learned.
Removal of the inhibitory substance then resulted in a reappearance
of the learned ability (Schmidt, R. J. supra; Shashoua, V. E. and
Moore, M. E. Brain Res. 148:441-449 (1978)).
[0010] Thus, there is a need for polypeptides that function as
neurotrophic factors in the regeneration of the optic and other
nerves and in long-term memory consolidation, since disturbances of
such regulation may be involved in disorders relating to the
complex molecular and cellular process regulating neuronal and
nervous system function. Such disorders may include Parkinson's
disease, Alzheimer's disease, amyotrophic lateral sclerosis, pain,
stroke, depression, anxiety, epilepsy, and other neurological and
psychiatric disorders. Therefore, there is a need for
identification and characterization of such human polypeptides
which can play a role in detecting, preventing, ameliorating or
correcting such disorders.
SUMMARY OF THE INVENTION
[0011] The present invention provides isolated nucleic acid
molecules comprising a polynucleotide encoding at least a portion
of the Ependymin polypeptide having the complete amino acid
sequence shown in SEQ ID NO:2 or the complete amino acid sequence
encoded by the cDNA clone deposited as plasmid DNA as ATCC.TM.
Deposit Number 209464 on Nov. 14, 1997. The nucleotide sequence
determined by sequencing the deposited Ependymin clone, which is
shown in FIGS. 1A, 1B, and 1C (SEQ ID NO:1), contains an open
reading frame encoding a complete polypeptide of 224 amino acid
residues, including an initiation codon encoding an N-terminal
methionine at nucleotide positions 296-298, and a predicted
molecular weight of about 25.4 kDa. Nucleic acid molecules of the
invention include those encoding the complete amino acid sequence
excepting the N-terminal methionine shown in SEQ ID NO:2, or the
complete amino acid sequence excepting the N-terminal methionine
encoded by the cDNA clone in ATCC.TM. Deposit Number 209464, which
molecules also can encode additional amino acids fused to the
N-terminus of the Ependymin amino acid sequence.
[0012] The encoded polypeptide has a predicted leader sequence of
37 amino acids underlined in FIGS. 1A, 1B, and 1C; and the amino
acid sequence of the predicted mature Ependymin protein is also
shown in FIGS. 1A, 1B, and 1C, as amino acid residues 38-224 and as
residues 1-187 in SEQ ID NO:2.
[0013] Thus, one aspect of the invention provides an isolated
nucleic acid molecule comprising a polynucleotide having a
nucleotide sequence selected from the group consisting of: (a) a
nucleotide sequence encoding the Ependymin polypeptide having the
complete amino acid sequence in SEQ ID NO:2 (i.e., positions -37 to
187 of SEQ ID NO:2); (b) a nucleotide sequence encoding the
Ependymin polypeptide having the complete amino acid sequence in
SEQ ID NO:2 excepting the N-terminal methionine (i.e., positions
-36 to 187 of SEQ ID NO:2); (c) a nucleotide sequence encoding the
predicted mature Ependymin polypeptide having the amino acid
sequence at positions 1 to 187 in SEQ ID NO:2; (d) a nucleotide
sequence encoding the Ependymin polypeptide having the complete
amino acid sequence encoded by the cDNA clone contained in ATCC.TM.
Deposit No. 209464; (e) a nucleotide sequence encoding the
Ependymin polypeptide having the complete amino acid sequence
excepting the N-terminal methionine encoded by the cDNA clone
contained in ATCC.TM. Deposit No. 209464; (f) a nucleotide sequence
encoding the mature Ependymin polypeptide having the amino acid
sequence encoded by the cDNA clone contained in ATCC.TM. Deposit
No. 209464; and (g) a nucleotide sequence complementary to any of
the nucleotide sequences in (a), (b), (c), (d), (e) or (f),
above.
[0014] Further embodiments of the invention include isolated
nucleic acid molecules that comprise a polynucleotide having a
nucleotide sequence at least 90% identical, and more preferably at
least 95%, 96%, 97%, 98% or 99% identical, to any of the nucleotide
sequences in (a), (b), (c), (d), (e), (f) or (g), above, or a
polynucleotide which hybridizes under stringent hybridization
conditions to a polynucleotide in (a), (b), (c), (d), (e), (f) or
(g), above. This polynucleotide which hybridizes does not hybridize
under stringent hybridization conditions to a polynucleotide having
a nucleotide sequence consisting of only A residues or of only T
residues.
[0015] An additional nucleic acid embodiment of the invention
relates to an isolated nucleic acid molecule comprising a
polynucleotide which encodes the amino acid sequence of an
epitope-bearing portion of a Ependymin polypeptide having an amino
acid sequence in (a), (b), (c), (d), (e) or (f), above. A further
nucleic acid embodiment of the invention relates to an isolated
nucleic acid molecule comprising a polynucleotide which encodes the
amino acid sequence of a Ependymin polypeptide having an amino acid
sequence which contains at least one conservative amino acid
substitution, but not more than 50 conservative amino acid
substitutions, even more preferably, not more than 40 conservative
amino acid substitutions, still more preferably, not more than 30
conservative amino acid substitutions, and still even more
preferably, not more than 20 conservative amino acid substitutions.
Of course, in order of ever-increasing preference, it is highly
preferable for a polynucleotide which encodes the amino acid
sequence of a Ependymin polypeptide to have an amino acid sequence
which contains not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1
conservative amino acid substitutions.
[0016] The present invention also relates to recombinant vectors,
which include the isolated nucleic acid molecules of the present
invention, and to host cells containing the recombinant vectors, as
well as to methods of making such vectors and host cells and for
using them for production of Ependymin polypeptides or peptides by
recombinant techniques.
[0017] The invention further provides an isolated Ependymin
polypeptide comprising an amino acid sequence selected from the
group consisting of: (a) the amino acid sequence of the full-length
Ependymin polypeptide having the complete amino acid sequence shown
in SEQ ID NO:2 (i.e., positions -37 to 187 of SEQ ID NO:2); (b) the
amino acid sequence of the full-length Ependymin polypeptide having
the complete amino acid sequence shown in SEQ ID NO:2 excepting the
N-terminal methionine (i.e., positions -36 to 187 of SEQ ID NO:2);
(c) the amino acid sequence of the predicted mature Ependymin
polypeptide having the amino acid sequence at positions 1 to 187 in
SEQ ID NO:2; (d) the complete amino acid sequence encoded by the
cDNA clone contained in the ATCC.TM. Deposit No. 209464; (e) the
complete amino acid sequence excepting the N-terminal methionine
encoded by the cDNA clone contained in the ATCC.TM. Deposit No.
209464; and (f) the complete amino acid sequence of the predicted
mature Ependymin polypeptide encoded by the cDNA clone contained in
the ATCC.TM. Deposit No. 209464. The polypeptides of the present
invention also include polypeptides having an amino acid sequence
at least 80% identical, more preferably at least 90% identical, and
still more preferably 95%, 96%, 97%, 98% or 99% identical to those
described in (a), (b), (c), (d), (e) or (f), above, as well as
polypeptides having an amino acid sequence with at least 90%
similarity, and more preferably at least 95% similarity, to those
above.
[0018] An additional embodiment of this aspect of the invention
relates to a peptide or polypeptide which comprises the amino acid
sequence of an epitope-bearing portion of a Ependymin polypeptide
having an amino acid sequence described in (a), (b), (c), (d), (e)
or (f), above. Peptides or polypeptides having the amino acid
sequence of an epitope-bearing portion of a Ependymin polypeptide
of the invention include portions of such polypeptides with at
least six or seven, preferably at least nine, and more preferably
at least about 30 amino acids to about 50 amino acids, although
epitope-bearing polypeptides of any length up to and including the
entire amino acid sequence of a polypeptide of the invention
described above also are included in the invention.
[0019] A further embodiment of the invention relates to a peptide
or polypeptide which comprises the amino acid sequence of a
Ependymin polypeptide having an amino acid sequence which contains
at least one conservative amino acid substitution, but not more
than 50 conservative amino acid substitutions, even more
preferably, not more than 40 conservative amino acid substitutions,
still more preferably, not more than 30 conservative amino acid
substitutions, and still even more preferably, not more than 20
conservative amino acid substitutions. Of course, in order of
ever-increasing preference, it is highly preferable for a peptide
or polypeptide to have an amino acid sequence which comprises the
amino acid sequence of a Ependymin polypeptide, which contains at
least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1
conservative amino acid substitutions.
[0020] In another embodiment, the invention provides an isolated
antibody that binds specifically to a Ependymin polypeptide having
an amino acid sequence described in (a), (b), (c), (d), (e) or (f)
above. The invention further provides methods for isolating
antibodies that bind specifically to a Ependymin polypeptide having
an amino acid sequence as described herein. Such antibodies are
useful diagnostically or therapeutically as described below.
[0021] The invention also provides for pharmaceutical compositions
comprising Ependymin polypeptides, particularly human Ependymin
polypeptides, which may be employed, for instance, to treat
Parkinson's disease, Alzheimer's disease, amyotrophic lateral
sclerosis, pain, stroke, depression, anxiety, epilepsy, and other
neurological and psychiatric disorders. Methods of treating
individuals in need of Ependymin polypeptides are also
provided.
[0022] The invention further provides compositions comprising a
Ependymin polynucleotide or an Ependymin polypeptide for
administration to cells in vitro, to cells ex vivo and to cells in
vivo, or to a multicellular organism. In certain particularly
preferred embodiments of this aspect of the invention, the
compositions comprise a Ependymin polynucleotide for expression of
a Ependymin polypeptide in a host organism for treatment of
disease. Particularly preferred in this regard is expression in a
human patient for treatment of a dysfunction associated with
aberrant endogenous activity of a Ependymin
[0023] In another aspect, a screening assay for agonists and
antagonists is provided which involves determining the effect a
candidate compound has on Ependymin binding to a receptor. In
particular, the method involves contacting the receptor with a
Ependymin polypeptide and a candidate compound and determining
whether Ependymin polypeptide binding to the receptor is increased
or decreased due to the presence of the candidate compound. In this
assay, an increase in binding of Ependymin over the standard
binding indicates that the candidate compound is an agonist of
Ependymin binding activity and a decrease in Ependymin binding
compared to the standard indicates that the compound is an
antagonist of Ependymin binding activity.
[0024] In yet another aspect, the Ependymin may bind to a cell
surface protein which also function as a viral receptor or
coreceptor. Thus, Ependymin, or agonists or antagonists thereof,
may be used to regulate viral infectivity at the level of viral
binding or interaction with the Ependymin receptor or coreceptor or
during the process of viral internalization or entry into the
cell.
[0025] It has been discovered that Ependymin is expressed not only
in primary dendritic cells, but also in the KMH2 cell line,
placenta, fetal and adult liver, spinal cord, osteoclastoma,
cerebellum, synovial fibroblasts, 12 week old early stage human
embryo, adrenal gland tumor, whole brain, Hodgkin's Lymphoma
tissue, macrophages, HEL cell line, and chondrosarcoma. Therefore,
nucleic acids of the invention are useful as hybridization probes
for differential identification of the tissue(s) or cell type(s)
present in a biological sample. Similarly, polypeptides and
antibodies directed to those polypeptides are useful to provide
immunological probes for differential identification of the
tissue(s) or cell type(s). In addition, for a number of disorders
of the above tissues or cells, particularly of the nervous system,
significantly higher or lower levels of Ependymin gene expression
may be detected in certain tissues (e.g., cancerous and wounded
tissues) or bodily fluids (e.g., serum, plasma, urine, synovial
fluid or spinal fluid) taken from an individual having such a
disorder, relative to a "standard" Ependymin gene expression level,
i.e., the Ependymin expression level in healthy tissue from an
individual not having the nervous system disorder. Thus, the
invention provides a diagnostic method useful during diagnosis of
such a disorder, which involves: (a) assaying Ependymin gene
expression level in cells or body fluid of an individual; (b)
comparing the Ependymin gene expression level with a standard
Ependymin gene expression level, whereby an increase or decrease in
the assayed Ependymin gene expression level compared to the
standard expression level is indicative of disorder in the nervous
system.
[0026] An additional aspect of the invention is related to a method
for treating an individual in need of an increased level of
Ependymin activity in the body comprising administering to such an
individual a composition comprising a therapeutically effective
amount of an isolated Ependymin polypeptide of the invention or an
agonist thereof.
[0027] A still further aspect of the invention is related to a
method for treating an individual in need of a decreased level of
Ependymin activity in the body comprising, administering to such an
individual a composition comprising a therapeutically effective
amount of an Ependymin antagonist. Preferred antagonists for use in
the present invention are Ependymin-specific antibodies.
BRIEF DESCRIPTION OF THE FIGURES
[0028] FIGS. 1A, 1B, and 1C show the nucleotide sequence (SEQ ID
NO:1) and deduced amino acid sequence (SEQ ID NO:2) of Ependymin.
The predicted leader sequence of about 37 amino acids is
underlined. Note that the methionine residue at the beginning of
the leader sequence in FIGS. 1A, 1B, and 1C is shown in position
number (positive) 1, whereas the leader positions in the
corresponding sequence of SEQ ID NO:2 are designated with negative
position numbers. Thus, the leader sequence positions 1 to 37 in
FIGS. 1A, 1B, and 1C correspond to positions -37 to -1 in SEQ ID
NO:2.
[0029] Two potential asparagine-linked glycosylation sites are
marked in the amino acid sequence of Ependymin. The sites are
asparagine-130 and asparagine-182 in FIGS. 1A, 1B, and 1C
(asparagine-93 and asparagine-145 in SEQ ID NO:2), and are labeled
with the bold pound symbol (#) above the nucleotide sequence
coupled with a bolded one letter abbreviation for the asparagine
(N) in the amino acid sequence in FIGS. 1A, 1B, and 1C; that is,
the actual asparagine residues which are potentially glycosylated
is bolded in FIGS. 1A, 1B, and 1C.
[0030] A polyadenylation signal sequence is present in the 3'
untranslated region of the nucleotide sequence of Ependymin of the
present invention. The polyadenylation signal sequence is
delineated by a double underline in the Ependymin sequence shown in
FIGS. 1A, 1B, and 1C.
[0031] Regions of high identity between Human Ependymin of the
present invention and several previously identified piscine
ependymins (an alignment of these sequences is presented in FIG. 2)
are delineated in FIGS. 1A, 1B, and 1C with a double underline.
These regions are not limiting and are labeled as Conserved Domain
(CD)-II, CD-III, CD-IV, CD-V, CD-VI, CD-VII, CD-IX, and CD-X in
FIGS. 1A, 1B, and 1C.
[0032] FIG. 2 shows the regions of identity between the amino acid
sequences of the Ependymin protein and translation product of the
Zebrafish mRNA for Ependymin (SEQ ID NO:6), determined by the
computer program Bestfit (Wisconsin Sequence Analysis Package,
Version 8 for Unix, Genetics Computer Group, University Research
Park, 575 Science Drive, Madison, Wis. 53711) using the default
parameters.
[0033] FIGS. 3A and 3B are a multiple sequence alignment
illustrating a comparison of the amino acid sequences of the novel
human ependymin of the present invention (SEQ ID NO:2) and five
previously described ependymin-like molecules. Conserved residues
are highlighted in black. The ependymin homologues shown in the
figure are goldfish ependymin II (SEQ ID NO:3; GenBank Accession
No. J04986), rainbow trout ependymin II (SEQ ID NO:4; GenBank
Accession No. M93698), common carp ependymin (SEQ ID NO:5; GenBank
Accession No. U00432), zebrafish ependymin (SEQ ID NO:6; GenBank
Accession No. X52502), and Atlantic herring ependymin (SEQ ID NO:7;
GenBank Accession No. L09065).
[0034] FIG. 4 shows an analysis of the Ependymin amino acid
sequence. Alpha, beta, turn and coil regions; hydrophilicity and
hydrophobicity; amphipathic regions; flexible regions; antigenic
index and surface probability are shown. In the "Antigenic Index or
Jameson-Wolf" graph, the positive peaks indicate locations of the
highly antigenic regions of the Ependymin protein, i.e., regions
from which epitope-bearing peptides of the invention can be
obtained.
[0035] The data presented in FIG. 4 are also represented in tabular
form in Table I. The columns are labeled with the headings "Res",
"Position", and Roman Numerals I-XIV. The column headings refer to
the following features of the amino acid sequence presented in FIG.
4 and Table I: "Res": amino acid residue of FIGS. 1A, 1B, and 1C
(which is the identical sequence shown in SEQ ID NO:2, with the
exception that the residues are numbered 1-224 in FIGS. 1A, 1B, and
1C and -37 through 187 in SEQ ID NO:2); "Position": position of the
corresponding residue within FIGS. 1A, 1B, and 1C (which is the
identical sequence shown in SEQ ID NO:2, with the exception that
the residues are numbered 1-224 in FIGS. 1A, 1B, and 1C and -37
through 187 in SEQ ID NO:2); I: Alpha, Regions--Garnier-Robson; II:
Alpha, Regions--Chou-Fasman; III: Beta, Regions--Garnier-Robson;
IV: Beta, Regions--Chou-Fasman; V: Turn, Regions--Garnier-Robson;
VI: Turn, Regions--Chou-Fasman; VII: Coil, Regions--Garnier-Robson;
VIII: Hydrophilicity Plot--Kyte-Doolittle; IX: Hydrophobicity
Plot--Hopp-Woods; X: Alpha, Amphipathic Regions--Eisenberg; XI:
Beta, Amphipathic Regions--Eisenberg; XII: Flexible
Regions--Karplus-Schulz; XIII: Antigenic Index--Jameson-Wolf; and
XIV: Surface Probability Plot--Emini.
DETAILED DESCRIPTION
[0036] The present invention provides isolated nucleic acid
molecules comprising a polynucleotide encoding a Ependymin
polypeptide having the amino acid sequence shown in SEQ ID NO:2,
which was determined by sequencing a cloned cDNA. The nucleotide
sequence shown in FIGS. 1A, 1B, and 1C (SEQ ID NO:1) was obtained
by sequencing the HDPIE88 clone, which was deposited on Nov. 14,
1997 at the American Type Culture Collection, 10801 University
Boulevard, Manassas, Va. 20110-2209, and given accession number
ATCC.TM. 209464. The deposited clone is contained in the
pBluescript.TM. SK(-) plasmid (STRATAGENE.TM., La Jolla,
Calif.).
[0037] The Ependymin protein of the present invention shares
sequence homology with the translation products of ependymins from
a number of teleost fish including the Zebrafish mRNA for ependymin
(FIG. 2; SEQ ID NO:6). Zebrafish ependymin, and other ependymins,
are thought to be involved in the regulation of extracellular
Ca.sup.2+-binding. In fact, as the predominant cerebrospinal fluid
constituents in many teleost fish, ependymins are believed to be
antiadhesive extracellular matrix glycoproteins which function in
the processes of cell contact and regeneration.
Nucleic Acid Molecules
[0038] Unless otherwise indicated, all nucleotide sequences
determined by sequencing a DNA molecule herein were determined
using an automated DNA sequencer (such as the Model 373 from
Applied Biosystems, Inc., Foster City, Calif.), and all amino acid
sequences of polypeptides encoded by DNA molecules determined
herein were predicted by translation of a DNA sequence determined
as above. Therefore, as is known in the art for any DNA sequence
determined by this automated approach, any nucleotide sequence
determined herein may contain some errors. Nucleotide sequences
determined by automation are typically at least about 90%
identical, more typically at least about 95% to at least about
99.9% identical to the actual nucleotide sequence of the sequenced
DNA molecule. The actual sequence can be more precisely determined
by other approaches including manual DNA sequencing methods well
known in the art. As is also known in the art, a single insertion
or deletion in a determined nucleotide sequence compared to the
actual sequence will cause a frame shift in translation of the
nucleotide sequence such that the predicted amino acid sequence
encoded by a determined nucleotide sequence will be completely
different from the amino acid sequence actually encoded by the
sequenced DNA molecule, beginning at the point of such an insertion
or deletion.
[0039] By "nucleotide sequence" of a nucleic acid molecule or
polynucleotide is intended, for a DNA molecule or polynucleotide, a
sequence of deoxyribonucleotides, and for an RNA molecule or
polynucleotide, the corresponding sequence of ribonucleotides (A,
G, C and U), where each thymidine deoxyribonucleotide (T) in the
specified deoxyribonucleotide sequence is replaced by the
ribonucleotide uridine (U).
[0040] Using the information provided herein, such as the
nucleotide sequence in FIGS. 1A, 1B, and 1C (SEQ ID NO: 1), a
nucleic acid molecule of the present invention encoding a Ependymin
polypeptide may be obtained using standard cloning and screening
procedures, such as those for cloning cDNAs using mRNA as starting
material. Illustrative of the invention, the nucleic acid molecule
described in FIGS. 1A, 1B, and 1C (SEQ ID NO:1) was discovered in a
cDNA library derived from primary dendritic cells.
[0041] Additional clones of the same gene were also identified in
cDNA libraries from the following tissues: the KMH2 cell line,
placenta, fetal and adult liver, spinal cord, osteoclastoma,
cerebellum, synovial fibroblasts, 12 week old early stage human
embryo, adrenal gland tumor, whole brain, Hodgkin's Lymphoma
tissue, macrophages, HEL cell line, and chondrosarcoma.
[0042] The determined nucleotide sequence of the Ependymin cDNA of
FIGS. 1A, 1B, and 1C (SEQ ID NO:1) contains an open reading frame
encoding a protein of 224 amino acid residues, with an initiation
codon at nucleotide positions 296-298 of the nucleotide sequence in
FIGS. 1A, 1B, and 1C (SEQ ID NO:1), and a deduced molecular weight
of about 25.4 kDa. The amino acid sequence of the Ependymin protein
shown in SEQ ID NO:2 is about 22.5% identical to Zebrafish mRNA for
ependymin (FIG. 2; Sterrer, S., et al., Neurosci. 37:277-284
(1990); GenBank Accession No. X52502).
[0043] As one of ordinary skill would appreciate, due to the
possibilities of sequencing errors discussed above, the actual
complete Ependymin polypeptide encoded by the deposited cDNA, which
comprises about 224 amino acids, may be somewhat longer or shorter.
It will further be appreciated that, depending on the analytical
criteria used for identifying various functional domains, the exact
"address" of the signal peptide and mature regions of the Ependymin
polypeptide may differ slightly from the predicted positions above.
For example, the exact location of the Ependymin signal peptide in
SEQ ID NO:2 may vary slightly (e.g., the address may "shift" by
about 1 to about 15 residues, more likely about 1 to about 10
residues, and even more likely about 1 to about 5 residues)
depending on the criteria used to define the domain. In this case,
the end of the signal peptide and the beginning of the mature
Ependymin molecule were predicted using the Human Genome Sciences,
Inc. (HGSI) SignalP computer algorithm (Pedersen, A. G. and
Nielsen, H. ISMB 5:226-233 (1997)). One of skill in the art will
realize that another widely accepted computer algorithm used to
predict potential sites of polypeptide cleavage, PSORT, will
predict the cleavage of an N-terminal signal peptide from the
Ependymin polypeptide at a point slightly different from that
predicted by the HGSI SignalP algorithm. In any event, as discussed
further below, the invention further provides polypeptides having
various residues deleted from the N-terminus of the complete
polypeptide, including polypeptides lacking one or more amino acids
from the N-terminus of the mature polypeptide described herein.
Leader and Mature Sequences
[0044] The amino acid sequence of the complete Ependymin protein
includes a leader sequence and a mature protein, as shown in SEQ ID
NO:2. More in particular, the present invention provides nucleic
acid molecules encoding a mature form of the Ependymin protein.
Thus, according to the signal hypothesis, once export of the
growing protein chain across the rough endoplasmic reticulum has
been initiated, proteins secreted by mammalian cells have a signal
or secretory leader sequence which is cleaved from the complete
polypeptide to produce a secreted "mature" form of the protein.
Most mammalian cells and even insect cells cleave secreted proteins
with the same specificity. However, in some cases, cleavage of a
secreted protein is not entirely uniform, which results in two or
more mature species of the protein. Further, it has long been known
that the cleavage specificity of a secreted protein is ultimately
determined by the primary structure of the complete protein, that
is, it is inherent in the amino acid sequence of the polypeptide.
Therefore, the present invention provides a nucleotide sequence
encoding the mature Ependymin polypeptide having the amino acid
sequence encoded by the cDNA clone contained in ATCC.TM. Deposit
No. 209464. By the "mature Ependymin polypeptide having the amino
acid sequence encoded by the cDNA clone in ATCC.TM. Deposit No.
209464" is meant the mature form(s) of the Ependymin protein
produced by expression in a mammalian cell (e.g., COS cells, as
described below) of the complete open reading frame encoded by the
human DNA sequence of the deposited clone.
[0045] In addition, methods for predicting whether a protein has a
secretory leader as well as the cleavage point for that leader
sequence are available. For instance, the method of McGeoch (Virus
Res. 3:271-286 (1985)) uses the information from a short N-terminal
charged region and a subsequent uncharged region of the complete
(uncleaved) protein. The method of von Heinje (Nucleic Acids Res.
14:4683-4690 (1986)) uses the information from the residues
surrounding the cleavage site, typically residues -13 to +2 where
+1 indicates the amino terminus of the mature protein. The accuracy
of predicting the cleavage points of known mammalian secretory
proteins for each of these methods is in the range of 75-80% (von
Heinje, supra). However, the two methods do not always produce the
same predicted cleavage point(s) for a given protein.
[0046] In the present case, the deduced amino acid sequence of the
complete Ependymin polypeptide was analyzed by the HGSI SignalP
computer algorithm (Pedersen, A. G. and Nielsen, H. ISMB 5:226-233
(1997)). Using this computer analysis tool, a likely site of signal
peptide cleavage was predicted between amino acid residues 37 and
38 of the Ependymin sequence (SEQ ID NO:2). However, the deduced
amino acid sequence of the complete Ependymin polypeptide was also
analyzed by a computer program designated "PSORT", available from
Dr. Kenta Nakai of the Institute for Chemical Research, Kyoto
University (Nakai, K. and Kanehisa, M. Genomics 14:897-911 (1992)),
which is an expert system for predicting the cellular location of a
protein based on the amino acid sequence. As part of this
computational prediction of localization, the methods of McGeoch
and von Heinje are incorporated. Thus, the computation analysis
above predicted a single cleavage site within the complete amino
acid sequence shown in SEQ ID NO:2.
[0047] As one of ordinary skill would appreciate from the above
discussions, due to the possibilities of sequencing errors as well
as the variability of cleavage sites in different known proteins,
the mature Ependymin polypeptide encoded by the deposited cDNA is
expected to consist of about 187 amino acids (presumably residues 1
to 187 of SEQ ID NO:2) based on analysis of the Ependymin amino
acid sequence using the SignalP computer algorithm, but may consist
of any number of amino acids in the range of about 187 to 200 amino
acids (the mature polypeptide is predicted to be 200 amino acids
using the PSORT computer algorithm). Further, the actual leader
sequence(s) of this protein is expected to be 37 amino acids
(presumably residues -37 through -1 of SEQ ID NO:2) based on
analysis of the Ependymin amino acid sequence using the SignalP
computer algorithm, but may consist of any number of amino acids in
the range of 24-37 amino acids (the signal peptide is predicted to
be 24 amino acids using the PSORT computer algorithm).
[0048] As indicated, nucleic acid molecules of the present
invention may be in the form of RNA, such as mRNA, or in the form
of DNA, including, for instance, cDNA and genomic DNA obtained by
cloning or produced synthetically. The DNA may be double-stranded
or single-stranded. Single-stranded DNA or RNA may be the coding
strand, also known as the sense strand, or it may be the non-coding
strand, also referred to as the anti-sense strand.
[0049] By "isolated" nucleic acid molecule(s) is intended a nucleic
acid molecule, DNA or RNA, which has been removed from its native
environment For example, recombinant DNA molecules contained in a
vector are considered isolated for the purposes of the present
invention. Further examples of isolated DNA molecules include
recombinant DNA molecules maintained in heterologous host cells or
purified (partially or substantially) DNA molecules in solution.
Isolated RNA molecules include in vivo or in vitro RNA transcripts
of the DNA molecules of the present invention. Isolated nucleic
acid molecules according to the present invention further include
such molecules produced synthetically.
[0050] Isolated nucleic acid molecules of the present invention
include DNA molecules comprising an open reading frame (ORF) with
an initiation codon at positions 288-290 of the nucleotide sequence
shown in FIGS. 1A, 1B, and 1C (SEQ ID NO:1).
[0051] Also included are DNA molecules comprising the coding
sequence for the predicted mature Ependymin protein shown at
positions 1-187 of SEQ ID NO:2.
[0052] In addition, isolated nucleic acid molecules of the
invention include DNA molecules which comprise a sequence
substantially different from those described above but which, due
to the degeneracy of the genetic code, still encode the Ependymin
protein. Of course, the genetic code and species-specific codon
preferences are well known in the art. Thus, it would be routine
for one skilled in the art to generate the degenerate variants
described above, for instance, to optimize codon expression for a
particular host (e.g., change codons in the human mRNA to those
preferred by a bacterial host such as E. coli).
[0053] In another aspect, the invention provides isolated nucleic
acid molecules encoding the Ependymin polypeptide having an amino
acid sequence encoded by the cDNA clone contained in the plasmid
deposited as ATCC.TM. Deposit No. 209464 on Nov. 14, 1997.
[0054] Preferably, this nucleic acid molecule will encode the
mature polypeptide encoded by the above-described deposited cDNA
clone.
[0055] The invention further provides an isolated nucleic acid
molecule having the nucleotide sequence shown in FIGS. 1A, 1B, and
1C (SEQ ID NO:1) or the nucleotide sequence of the Ependymin cDNA
contained in the above-described deposited clone, or a nucleic acid
molecule having a sequence complementary to one of the above
sequences. Such isolated molecules, particularly DNA molecules, are
useful as probes for gene mapping, by in situ hybridization with
chromosomes, and for detecting expression of the Ependymin gene in
human tissue, for instance, by Northern blot analysis.
[0056] The present invention is further directed to nucleic acid
molecules encoding portions of the nucleotide sequences described
herein as well as to fragments of the isolated nucleic acid
molecules described herein. In particular, the invention provides a
polynucleotide having a nucleotide sequence representing the
portion of SEQ ID NO:1 which consists of positions 1-967 of SEQ ID
NO:1.
[0057] In addition, the invention provides nucleic acid molecules
having nucleotide sequences related to extensive portions of SEQ ID
NO:1 which have been determined from the following related cDNA
clones: HATBS80R (SEQ ID NO:15); HSRAN11R (SEQ ID NO:16); HCECE56R
(SEQ ID NO:17); HSNBF20R (SEQ ID NO:18); HPMDJ94R (SEQ ID NO:19);
and HE2FK31R (SEQ ID NO:20).
[0058] Further, the invention includes a polynucleotide comprising
any portion of at least about 30 nucleotides, preferably at least
about 50 nucleotides, of SEQ ID NO:1 from residue 1 to 2505.
[0059] More generally, by a fragment of an isolated nucleic acid
molecule having the nucleotide sequence of the deposited cDNA or
the nucleotide sequence shown in FIGS. 1A, 1B, and 1C (SEQ ID NO:1)
is intended fragments at least about 15 nt, and more preferably at
least about 20 nt, still more preferably at least about 30 nt, and
even more preferably, at least about 40 nt in length which are
useful as diagnostic probes and primers as discussed herein. Of
course, larger fragments 50-300 nt in length are also useful
according to the present invention as are fragments corresponding
to most, if not all, of the nucleotide sequence of the deposited
cDNA or as shown in FIGS. 1A, 1B, and 1C (SEQ ID NO:1). By a
fragment at least 20 nt in length, for example, is intended
fragments which include 20 or more contiguous bases from the
nucleotide sequence of the deposited cDNA or the nucleotide
sequence as shown in FIGS. 1A, 1B, and 1C (SEQ ID NO:1). Preferred
nucleic acid fragments of the present invention include nucleic
acid molecules encoding epitope-bearing portions of the Ependymin
polypeptide as identified in FIGS. 3A and 3B and described in more
detail below.
[0060] Preferred nucleic acid fragments of the present invention
also include nucleic acid molecules encoding one or more of the
following domains of Human Ependymin: amino acid residues -20
through -10 of SEQ ID NO:2; amino acid residues 1 through 10 of SEQ
ID NO:2; amino acid residues 31 through 40 of SEQ ID NO:2; amino
acid residues 64 through 70 of SEQ ID NO:2; amino acid residues 76
through 82 of SEQ ID NO:2; amino acid residues 88 through 105 of
SEQ ID NO:2; amino acid residues 106 through 117 of SEQ ID NO:2;
amino acid residues 129 through 143 of SEQ ID NO:2; amino acid
residues 150 through 156 of SEQ ID NO:2; and amino acid residues
162 through 184 of SEQ ID NO:2. These domains are represented as
conserved domains CD-I through CD-X in FIGS. 1A, 1B, and 1C.
[0061] In specific embodiments, the polynucleotide fragments of the
invention encode antigenic regions. Non-limiting examples of
antigenic polypeptides or peptides that can be used to generate
Ependymin-specific antibodies include: a polypeptide comprising
amino acid residues from about Ala-1 to about Gln-9 in SEQ ID NO:2;
a polypeptide comprising amino acid residues from about Pro-8 to
about Val-16 in SEQ ID NO:2; a polypeptide comprising amino acid
residues from about Gln-19 to about Arg-27 in SEQ ID NO:2; a
polypeptide comprising amino acid residues from about Ile-69 to
about Ser-77 in SEQ ID NO:2; a polypeptide comprising amino acid
residues from about Asp-86 to about Glu-107 in SEQ ID NO:2; a
polypeptide comprising amino acid residues from about Glu-113 to
about Tyr-123 in SEQ ID NO:2; a polypeptide comprising amino acid
residues from about Thr-131 to about Gln-139 in SEQ ID NO:2; a
polypeptide comprising amino acid residues from about Leu-159 to
about Phe-167 in SEQ ID NO:2; and a polypeptide comprising amino
acid residues from about Leu-178 to about Ser-186 in SEQ ID
NO:2.
[0062] In additional embodiments, the polynucleotides of the
invention encode functional attributes of Human Ependymin.
Preferred embodiments of the invention in this regard include
fragments that comprise alpha-helix and alpha-helix forming regions
("alpha-regions"), beta-sheet and beta-sheet forming regions
("beta-regions"), turn and turn-forming regions ("turn-regions"),
coil and coil-forming regions ("coil-regions"), hydrophilic
regions, hydrophobic regions, alpha amphipathic regions, beta
amphipathic regions, flexible regions, surface-forming regions and
high antigenic index regions of Human Ependymin.
[0063] The data representing the structural or functional
attributes of Human Ependymin set forth in FIG. 4 and/or Table I,
as described above, was generated using the various modules and
algorithms of the DNA*STAR set on default parameters. In a
preferred embodiment, the data presented in columns VIII, IX, XIII,
and XIV of Table I can be used to determine regions of Human
Ependymin which exhibit a high degree of potential for
antigenicity. Regions of high antigenicity are determined from the
data presented in columns VIII, IX, XIII, and/or IV by choosing
values which represent regions of the polypeptide which are likely
to be exposed on the surface of the polypeptide in an environment
in which antigen recognition may occur in the process of initiation
of an immune response.
[0064] Certain preferred regions in these regards are set out in
FIG. 4, but may, as shown in Table I, be represented or identified
by using tabular representations of the data presented in FIG. 4.
The DNA*STAR computer algorithm used to generate FIG. 4 (set on the
original default parameters) was used to present the data in FIG. 4
in a tabular format (See Table I). The tabular format of the data
in FIG. 4 may be used to easily determine specific boundaries of a
preferred region.
[0065] The above-mentioned preferred regions set out in FIG. 4 and
in Table I include, but are not limited to, regions of the
aforementioned types identified by analysis of the amino acid
sequence set out in FIGS. 1A, 1B, and 1C. As set out in FIG. 4 and
in Table I, such preferred regions include Garnier-Robson
alpha-regions, beta-regions, turn-regions, and coil-regions,
Chou-Fasman alpha-regions, beta-regions, and coil-regions,
Kyte-Doolittle hydrophilic regions and hydrophobic regions,
Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz
flexible regions, Emini surface-forming regions and Jameson-Wolf
regions of high antigenic index. TABLE-US-00001 TABLE I Res
Position I II III IV V VI VII VIII IX X XI XII XIII XIV Met 1 . . B
. . T 0.31 -0.24 * * . 1.07 1.11 Pro 2 . . . . . T C 0.49 -0.17 . *
. 1.34 0.88 Gly 3 . . . . T T . 0.07 -0.17 * * . 1.91 1.06 Arg 4 .
. B . . T . 0.57 0.09 * * . 0.98 0.89 Ala 5 . . B . . . . 0.64
-0.53 * * F 2.20 1.12 Pro 6 . . B B . . . 0.39 -0.47 * * F 1.48
1.63 Leu 7 . . B B . . . 0.39 -0.26 * * . 0.96 0.62 Arg 8 . . B B .
. . 0.39 0.17 * * F 0.29 0.95 Thr 9 . . B B . . . -0.31 0.10 * * F
0.07 0.61 Val 10 . . B . . T . -0.53 0.17 * * F 0.25 0.74 Pro 11 .
. B . . T . -0.67 0.17 * . F 0.25 0.31 Gly 12 . . B . . T . -0.44
0.60 * . F -0.05 0.21 Ala 13 . . B . . T . -0.84 0.61 * . . -0.20
0.29 Leu 14 . A B B . . . -1.34 0.89 . . . -0.60 0.20 Gly 15 . A B
B . . . -1.30 1.14 . . . -0.60 0.17 Ala 16 . A B B . . . -1.43 1.40
. . . -0.60 0.14 Trp 17 . A B B . . . -1.43 1.33 . . . -0.60 0.16
Leu 18 . A B B . . . -1.66 1.07 . . . -0.60 0.16 Leu 19 . A B B . .
. -1.13 1.33 . . . -0.60 0.13 Gly 20 . A . B T . . -1.38 1.74 . . .
-0.20 0.13 Gly 21 . . . B . . C -1.08 1.33 . . . -0.40 0.16 Leu 22
. . . B T . . -1.10 1.56 . . . -0.20 0.21 Trp 23 . . . B T . .
-1.10 1.36 . . . -0.20 0.30 Ala 24 . . B B . . . -0.96 1.61 . . .
-0.60 0.25 Trp 25 . . B B . . . -0.96 1.76 . . . -0.60 0.16 Thr 26
. . B B . . . -1.42 1.50 . . . -0.60 0.15 Leu 27 . . B B . . .
-1.28 1.27 . . . -0.60 0.13 Cys 28 . . . B T . . -1.29 1.34 . . .
-0.20 0.06 Gly 29 . . . B T . . -1.51 0.81 . . . -0.20 0.06 Leu 30
. . . B T . . -1.57 1.01 . . . -0.20 0.06 Cys 31 . . B . . T .
-1.84 0.76 . . . -0.20 0.11 Ser 32 . . B . . T . -1.89 0.69 . . .
-0.20 0.11 Leu 33 . . B . . T . -1.57 0.90 . . . -0.20 0.10 Gly 34
. . B . . T . -1.81 0.64 . . . -0.20 0.19 Ala 35 . . B B . . .
-1.21 0.57 . . . -0.60 0.14 Val 36 . . B B . . . -0.43 0.61 . . .
-0.60 0.26 Gly 37 . . B B . . . -0.34 -0.07 . . . 0.58 0.52 Ala 38
. . B . . . . -0.20 -0.07 . . F 1.21 0.80 Pro 39 . . B . . . . 0.14
-0.00 * . F 1.49 0.58 Arg 40 . . . . . T C 0.14 -0.24 * . F 2.32
1.01 Pro 41 . . . . T T . 0.79 -0.17 . . F 2.80 1.01 Cys 42 . . . .
T T . 1.13 -0.24 * . F 2.52 1.01 Gln 43 . . B . . T . 1.72 -0.27 *
. F 1.69 0.89 Ala 44 . . B . . . . 1.64 0.13 * * F 0.61 1.00 Pro 45
. . . . . . C 1.53 0.61 * * F 0.58 1.96 Gln 46 . . . . T . . 1.40
0.04 * * F 1.00 1.96 Gln 47 . . . . T . . 2.18 0.07 . * F 1.20 1.92
Trp 48 . . . . T . . 2.18 -0.43 . * F 2.00 2.43 Glu 49 . . . . . .
C 1.91 -0.46 . * F 2.00 2.43 Gly 50 . . . B T . . 1.52 -0.21 . * F
1.80 1.04 Arg 51 . . . B T . . 1.28 -0.00 . * F 1.45 0.98 Gln 52 .
. B B . . . 1.28 -0.16 . * . 0.70 0.89 Val 53 . . B B . . . 1.57
0.24 . * . 0.05 1.55 Met 54 . . B B . . . 1.27 0.21 . . . -0.15
1.37 Tyr 55 . . B B . . . 1.31 0.60 * . . -0.45 1.06 Gln 56 . . B B
. . . 0.86 0.59 * . F 0.04 1.91 Gln 57 . . B B . . . 0.97 0.37 . .
F 0.68 1.91 Ser 58 . . . . . T C 1.82 -0.24 . . F 2.22 2.39 Ser 59
. . . . . T C 2.12 -0.60 * . F 2.86 2.22 Gly 60 . . . . T T . 2.48
-0.61 * . F 3.40 1.72 Arg 61 . . . . T T . 1.89 -1.01 * . F 3.06
2.51 Asn 62 . . . . . T C 1.08 -0.90 * . F 2.52 1.89 Ser 63 . . B .
. T . 0.57 -0.60 * . F 1.98 1.58 Arg 64 . . B . . T . 0.57 -0.34 *
. F 1.19 0.66 Ala 65 . . B . . T . 0.67 0.04 * * . 0.10 0.55 Leu 66
. . B . . . . 0.56 0.40 * * . -0.10 0.65 Leu 67 . . B . . . . 0.21
0.01 . * . -0.10 0.55 Ser 68 . . B . . T . -0.30 0.44 . * . -0.20
0.54 Tyr 69 . . B . . T . -0.41 0.63 . * . -0.20 0.54 Asp 70 . . .
. T T . 0.18 0.34 . . F 0.80 1.06 Gly 71 . . . . T T . 1.10 0.06 .
* F 0.80 1.36 Leu 72 . . . . . . C 1.06 -0.33 * * F 1.00 1.70 Asn
73 . . B B . . . 1.47 -0.44 * * F 0.45 0.76 Gln 74 . . B B . . .
0.86 -0.44 * * F 0.60 1.50 Arg 75 . . B B . . . 0.04 -0.23 * * F
0.60 1.35 Val 76 . A B B . . . 0.39 -0.23 * * . 0.30 0.69 Arg 77 .
A B B . . . 1.20 -0.63 * * . 0.60 0.67 Val 78 . A B B . . . 1.31
-1.03 . * . 0.60 0.59 Leu 79 . A B B . . . 1.36 -1.03 . * . 0.75
1.56 Asp 80 . A B B . . . 0.66 -1.67 . * F 0.90 1.59 Glu 81 . A B .
T . . 0.70 -1.17 * . F 1.30 2.16 Arg 82 . A . . T . . -0.30 -1.13 *
. F 1.30 2.16 Lys 83 . A . . T . . 0.34 -1.13 . . F 1.15 0.91 Ala
84 . A . . T . . 0.49 -0.70 . * . 1.00 0.81 Leu 85 . A B . . . .
0.53 -0.13 * . . 0.30 0.22 Ile 86 . . B . . T . 0.64 -0.13 * . .
0.70 0.22 Pro 87 . . B . . T . -0.28 -0.13 * . . 0.70 0.43 Cys 88 .
. B . . T . -1.02 0.06 * . . 0.10 0.43 Lys 89 . . B . . T . -0.43
0.16 * . . 0.10 0.53 Arg 90 . . B . . . . 0.13 -0.53 * . . 0.80
0.60 Leu 91 . . B B . . . 0.13 -0.20 * . . 0.45 1.74 Phe 92 . . B B
. . . -0.47 -0.09 * . . 0.30 0.61 Glu 93 . . B B . . . -0.61 0.60 *
. . -0.60 0.26 Tyr 94 . . B B . . . -0.90 1.29 * . . -0.60 0.26 Ile
95 . . B B . . . -0.97 1.36 * . . -0.60 0.46 Leu 96 . . B B . . .
-0.16 0.57 . . . -0.60 0.54 Leu 97 . . B B . . . 0.20 0.57 . . .
-0.60 0.57 Tyr 98 . . . . T T . -0.66 0.24 . . . 0.50 0.81 Lys 99 .
. . . T T . -1.01 0.20 . . . 0.50 0.73 Asp 100 . . . . T T . -0.82
0.13 . . . 0.50 0.87 Gly 101 . . B . . T . -0.01 0.23 . * . 0.10
0.48 Val 102 . . B B . . . -0.09 -0.13 . * . 0.30 0.42 Met 103 . .
B B . . . 0.16 0.56 . * . -0.60 0.18 Phe 104 . . B B . . . 0.11
0.56 . * . -0.60 0.30 Gln 105 . . B B . . . -0.48 0.53 * . . -0.30
0.69 Ile 106 . . B B . . . -0.44 0.39 * * . 0.30 0.70 Asp 107 . . B
B . . . 0.46 0.26 * * F 0.90 1.17 Gln 108 . . . . T . . 1.06 -0.53
* * F 2.70 1.36 Ala 109 . . . . T . . 1.09 -0.53 * * F 3.00 3.35
Thr 110 . . . . T . . 0.79 -0.64 * . F 2.70 1.07 Lys 111 . . . . T
. . 1.72 -0.26 * . F 1.95 0.83 Gln 112 . . . . T . . 1.12 -0.66 * .
F 2.10 1.65 Cys 113 . . . . T . . 0.81 -0.54 * * F 1.80 1.13 Ser
114 . . B . . . . 0.59 -0.54 * * F 0.95 0.81 Lys 115 . . B . . . .
0.59 0.14 . . F 0.05 0.39 Met 116 . . B . . . . 0.54 0.23 . . .
0.05 1.04 Thr 117 . . B . . . . 0.33 0.06 . . . 0.05 1.35 Leu 118 .
. B . . . . 0.71 0.10 . * F 0.20 1.04 Thr 119 . . B . . . . 1.01
1.01 . * F -0.10 1.11 Gln 120 . . B . . . . 0.76 0.40 * * F 0.20
1.28 Pro 121 . . . . T . . 0.54 0.34 * . F 0.60 2.41 Trp 122 . . .
. T . . 0.86 0.34 * . F 0.60 1.37 Asp 123 . . . . . T C 0.78 -0.14
* . F 1.20 1.33 Pro 124 . . B . . T . 0.88 0.14 . . F 0.25 0.60 Leu
125 . . . . T T . 0.88 0.14 . . F 0.93 0.88 Asp 126 . . B . . T .
1.09 -0.37 . . F 1.41 0.92 Ile 127 . . . . . . C 1.08 0.03 . . F
1.09 0.95 Pro 128 . . . . . T C 0.77 -0.01 . * F 2.32 1.55 Gln 129
. . . . T T . 0.28 -0.21 . * F 2.80 1.34 Asn 130 . . . . . T C 1.09
0.57 . * F 1.42 1.65 Ser 131 . . . . . T C 1.09 -0.11 . * F 2.04
1.85 Thr 132 . . B . . . . 1.98 -0.54 . . F 1.66 1.79 Phe 133 . . B
. . . . 1.94 -0.54 . . F 1.60 1.92 Glu 134 . . B . . . . 1.64 -0.19
. * F 1.24 2.25 Asp 135 . . B . . T . 0.76 -0.19 . * F 1.66 2.09
Gln 136 . . B . . T . 0.71 0.01 . * . 1.13 1.69 Tyr 137 . . . . T T
. 0.68 -0.34 . * . 2.20 0.97 Ser 138 . . . . T T . 1.17 0.09 . * .
1.38 0.57 Ile 139 . . . . T . . 1.17 0.51 . . F 0.97 0.51 Gly 140 .
. . . . . C 1.17 0.51 . . F 0.71 0.56 Gly 141 . . . . . . C 1.17
-0.24 . * F 1.55 0.73 Pro 142 . . . . . . C 0.52 -0.23 . . F 1.64
1.80 Gln 143 . . . B . . C 0.51 -0.23 . * F 1.60 1.28 Glu 144 . . B
B . . . 0.54 -0.17 . * F 1.24 1.86 Gln 145 . . B B . . . 0.89 0.04
. * F 0.33 0.89 Ile 146 . . B B . . . 1.23 0.01 . * F 0.17 0.89 Thr
147 . . B B . . . 1.16 -0.39 . * . 0.46 0.89 Val 148 . . B B . . .
0.86 0.53 . * . -0.60 0.54 Gln 149 . . B B . . . 0.86 0.51 . * .
-0.11 1.04 Glu 150 . . B B . . . 0.97 -0.17 . * F 1.28 1.20 Trp 151
. . . . T T . 1.90 -0.66 . . F 2.72 3.17 Ser 152 . . . . . T C 1.91
-1.30 . . F 2.86 3.66 Asp 153 . . . . T T . 2.18 -1.31 * * F 3.40
2.83 Arg 154 . . . . T T . 2.29 -0.81 * * F 3.06 2.72 Lys 155 . . .
. T . . 1.99 -1.73 * * F 2.72 3.98 Ser 156 . . . . . . C 2.03 -1.73
* * F 2.38 3.19 Ala 157 . . . . . T C 2.33 -0.97 * . F 2.44 2.55
Arg 158 . . . . . T C 2.02 -0.97 * * F 2.30 2.21 Ser 159 . . B . .
T . 1.62 -0.49 * * F 2.00 2.38 Tyr 160 . . B . . T . 0.69 0.04 * .
F 1.20 2.48 Glu 161 . . B B . . . 0.64 0.23 * * F 0.45 0.89 Thr 162
. . B B . . . 0.34 0.66 * . . -0.20 0.65 Trp 163 . . B B . . .
-0.01 0.96 * . . -0.40 0.29 Ile 164 . . B B . . . -0.02 0.96 . . .
-0.60 0.27 Gly 165 . . B B . . . -0.63 1.44 . . . -0.60 0.27 Ile
166 . . B B . . . -0.59 1.60 . . . -0.60 0.19 Tyr 167 . . B B . . .
-0.28 0.69 . . . -0.60 0.53 Thr 168 . . B B . . . -0.66 -0.00 . . .
0.30 0.90 Val 169 . . . B T . . -0.01 0.14 . . . 0.24 0.69 Lys 170
. . . . T T . 0.12 0.21 * . F 0.93 0.69 Asp 171 . . . . T T . 0.16
-0.11 . * . 1.52 0.74 Cys 172 . . B . . T . 0.40 0.04 . * . 0.66
0.74 Tyr 173 . . B . . T . 0.71 -0.20 * . . 1.40 0.64 Pro 174 . . B
. . . . 1.26 -0.20 * * . 1.06 0.66 Val 175 . . B B . . . 0.51 0.29
* . . 0.27 1.78 Gln 176 . . B B . . . 0.20 0.50 . * F -0.17 0.99
Glu 177 . . B B . . . -0.02 0.23 . * F -0.01 0.92 Thr 178 . . B B .
. . 0.22 0.49 . * F -0.45 0.87 Phe 179 . . B B . . . 0.19 0.24 . *
. -0.30 0.81 Thr 180 . . B B . . . 0.74 0.60 . * . -0.60 0.73 Ile
181 . . B B . . . -0.11 0.99 . * . -0.60 0.68 Asn 182 . . B B . . .
-1.00 1.14 . * . -0.60 0.58 Tyr 183 . . B B . . . -1.50 1.04 . * .
-0.60 0.28 Ser 184 . . B B . . . -1.10 1.24 . * . -0.60 0.33 Val
185 . . B B . . . -1.10 0.94 * * . -0.60 0.28 Ile 186 . . B B . . .
-0.10 1.03 * * . -0.60 0.25 Leu 187 . . B B . . . -0.80 0.27 * * .
-0.30 0.37 Ser 188 . . B B . . . -1.26 0.67 * * . -0.60 0.43 Thr
189 . . B B . . . -0.96 0.81 * * F -0.45 0.54 Arg 190 . . B B . . .
-0.99 0.13 . * . -0.15 1.09 Phe 191 . . B B . . . -0.10 0.13 . * .
-0.30 0.57 Phe 192 . . B B . . . -0.10 0.14 . * . -0.30 0.68 Asp
193 . . B B . . . -0.14 0.34 * * . -0.30 0.29 Ile 194 . . B B . . .
-0.72 0.77 . * . -0.60 0.33 Gln 195 . . B B . . . -0.79 0.67 * * .
-0.60 0.27 Leu 196 . . B B . . . -0.09 -0.11 * * . 0.51 0.32 Gly
197 . . . . T . . 0.40 -0.11 . * . 1.32 0.76 Ile 198 . . . . T . .
0.10 -0.37 . * . 1.53 0.68 Lys 199 . . . . . . C 0.13 -0.39 * * F
1.84 1.10 Asp 200 . . . . . T C -0.57 -0.43 . * F 2.10 0.83 Pro 201
. . B . . T . -0.07 -0.07 . . F 1.84 1.02 Ser 202 . . B . . T .
0.07 -0.27 . . F 1.48 0.74 Val 203 . . B . . T . 0.74 0.16 . . .
0.52 0.68 Phe 204 . . B . . . . 0.40 0.59 . . . -0.19 0.68 Thr 205
. . . . . . C 0.09 0.54 . . F -0.05 0.68 Pro 206 . . . . . T C
-0.37 0.64 . . F 0.30 1.32 Pro 207 . . . . T T . -0.07 0.57 . . F
0.35 0.82 Ser 208 . . . . T T . 0.19 0.19 . . F 0.65 0.98 Thr 209 .
. . . T T . 0.30 0.31 . . F 0.65 0.63 Cys 210 . A B . . . . 0.61
0.39 . . . -0.30 0.41 Gln 211 . A B . . . . 0.01 0.36 . . . -0.30
0.53 Met 212 . A B . . . . 0.22 0.66 . . . -0.60 0.30 Ala 213 . A B
. . . . 0.57 0.17 . . . -0.30 0.98 Gln 214 A A . . . . . 0.28 -0.40
. . . 0.45 1.13 Leu 215 A A . . . . . 0.64 -0.19 . . . 0.76 1.13
Glu 216 . A B . . . . 0.64 -0.41 * . . 1.07 1.50 Lys 217 . A B . .
. . 1.24 -0.91 * . F 1.83 1.50 Met 218 . A . . T . . 1.17 -1.31 * .
F 2.54 3.05 Ser 219 . . . . T T . 0.87 -1.43 * . F 3.10 0.94 Glu
220 . . . . T T . 1.39 -1.04 * . F 2.79 0.63 Asp 221 . . . . T T .
1.00 -0.13 . * F 2.18 0.67 Cys 222 . . . . T T . 0.57 -0.31 . * .
1.72 0.64 Ser 223 . . . . T . . 0.78 -0.27 . . . 1.21 0.47 Trp 224
. . . . T . . 0.69 0.16 . . . 0.30 0.36
[0066] Among highly preferred fragments in this regard are those
that comprise regions of Human Ependymin that combine several
structural features, such as several of the features set out
above.
[0067] In another aspect, the invention provides an isolated
nucleic acid molecule comprising a polynucleotide which hybridizes
under stringent hybridization conditions to a portion of the
polynucleotide in a nucleic acid molecule of the invention
described above, for instance, the cDNA clone contained in ATCC.TM.
Deposit No. 209464. By "stringent hybridization conditions" is
intended overnight incubation at 42.degree. C. in a solution
comprising: 50% formamide, 5.times.SSC (750 mM NaCl, 75 mM
trisodium citrate), 50 mM sodium phosphate (pH 7.6),
5.times.Denhardt's solution, 10% dextran sulfate, and 20 .mu.g/ml
denatured, sheared salmon sperm DNA, followed by washing the
filters in 0.1.times.SSC at about 65.degree. C.
[0068] By a polynucleotide which hybridizes to a "portion" of a
polynucleotide is intended a polynucleotide (either DNA or RNA)
hybridizing to at least about 15 nucleotides (nt), and more
preferably at least about 20 nt, still more preferably at least
about 30 nt, and even more preferably about 30-70 (e.g., 50) nt of
the reference polynucleotide. These are useful as diagnostic probes
and primers as discussed above and in more detail below.
[0069] By a portion of a polynucleotide of "at least 20 nt in
length," for example, is intended 20 or more contiguous nucleotides
from the nucleotide sequence of the reference polynucleotide (e.g.,
the deposited cDNA or the nucleotide sequence as shown in FIGS. 1A,
1B, and 1C (SEQ ID NO:1)). Of course, a polynucleotide which
hybridizes only to a poly A sequence (such as the 3' terminal
poly(A) tract of the Ependymin cDNA shown in FIGS. 1A, 1B, and 1C
(SEQ ID NO:1)), or to a complementary stretch of T (or U) residues,
would not be included in a polynucleotide of the invention used to
hybridize to a portion of a nucleic acid of the invention, since
such a polynucleotide would hybridize to any nucleic acid molecule
containing a poly (A) stretch or the complement thereof (e.g.,
practically any double-stranded cDNA clone).
[0070] As indicated, nucleic acid molecules of the present
invention which encode a Ependymin polypeptide may include, but are
not limited to those encoding the amino acid sequence of the mature
polypeptide, by itself; and the coding sequence for the mature
polypeptide and additional sequences, such as those encoding the
about 37 amino acid leader or secretory sequence, such as a pre-,
or pro- or prepro-protein sequence; the coding sequence of the
mature polypeptide, with or without the aforementioned additional
coding sequences.
[0071] Also encoded by nucleic acids of the invention are the above
protein sequences together with additional, non-coding sequences,
including for example, but not limited to introns and non-coding 5'
and 3' sequences, such as the transcribed, non-translated sequences
that play a role in transcription, mRNA processing, including
splicing and polyadenylation signals, for example--ribosome binding
and stability of mRNA; an additional coding sequence which codes
for additional amino acids, such as those which provide additional
functionalities.
[0072] Thus, the sequence encoding the polypeptide may be fused to
a marker sequence, such as a sequence encoding a peptide which
facilitates purification of the fused polypeptide. In certain
preferred embodiments of this aspect of the invention, the marker
amino acid sequence is a hexa-histidine peptide, such as the tag
provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue,
Chatsworth, Calif., 91311), among others, many of which are
commercially available. As described by Gentz and colleagues (Proc.
Natl. Acad. Sci. USA 86:821-824 (1989)), for instance,
hexa-histidine provides for convenient purification of the fusion
protein. The "HA" tag is another peptide useful for purification
which corresponds to an epitope derived from the influenza
hemagglutinin protein, which has been described by Wilson and
coworkers (Cell 37:767 (1984)). As discussed below, other such
fusion proteins include the Ependymin fused to Fc at the N- or
C-terminus.
Variant and Mutant Polynucleotides
[0073] The present invention further relates to variants of the
nucleic acid molecules of the present invention, which encode
portions, analogs or derivatives of the Ependymin protein. Variants
may occur naturally, such as a natural allelic variant. By an
"allelic variant" is intended one of several alternate forms of a
gene occupying a given locus on a chromosome of an organism (Genes
II, Lewin, B., ed., John Wiley & Sons, New York (1985)).
Non-naturally occurring variants may be produced using art-known
mutagenesis techniques.
[0074] Such variants include those produced by nucleotide
substitutions, deletions or additions. The substitutions, deletions
or additions may involve one or more nucleotides. The variants may
be altered in coding regions, non-coding regions, or both.
Alterations in the coding regions may produce conservative or
non-conservative amino acid substitutions, deletions or additions.
Especially preferred among these are silent substitutions,
additions and deletions, which do not alter the properties and
activities of the Ependymin protein or portions thereof. Also
especially preferred in this regard are conservative
substitutions.
[0075] Most highly preferred are nucleic acid molecules encoding
the mature protein having the amino acid sequence shown in SEQ ID
NO:2 or the mature Ependymin amino acid sequence encoded by the
deposited cDNA clone.
[0076] Thus, one aspect of the invention provides an isolated
nucleic acid molecule comprising a polynucleotide having a
nucleotide sequence selected from the group consisting of: (a) a
nucleotide sequence encoding the Ependymin polypeptide having the
complete amino acid sequence in SEQ ID NO:2 (i.e., positions -37 to
187 of SEQ ID NO:2); (b) a nucleotide sequence encoding the
Ependymin polypeptide having the complete amino acid sequence in
SEQ ID NO:2 excepting the N-terminal methionine (i.e., positions
-36 to 187 of SEQ ID NO:2); (c) a nucleotide sequence encoding the
predicted mature Ependymin polypeptide having the amino acid
sequence at positions 1 to 187 in SEQ ID NO:2; (d) a nucleotide
sequence encoding the Ependymin polypeptide having the complete
amino acid sequence encoded by the cDNA clone contained in ATCC.TM.
Deposit No. 209464; (e) a nucleotide sequence encoding the
Ependymin polypeptide having the complete amino acid sequence
excepting the N-terminal methionine encoded by the cDNA clone
contained in ATCC.TM. Deposit No. 209464; (f) a nucleotide sequence
encoding the mature Ependymin polypeptide having the amino acid
sequence encoded by the cDNA clone contained in ATCC.TM. Deposit
No. 209464; and (g) a nucleotide sequence complementary to any of
the nucleotide sequences in (a), (b), (c), (d), (e) or (f),
above.
[0077] Further embodiments of the invention include isolated
nucleic acid molecules that comprise a polynucleotide having a
nucleotide sequence at least 90% identical, and more preferably at
least 95%, 96%, 97%, 98% or 99% identical, to any of the nucleotide
sequences in (a), (b), (c), (d), (e), (f) or (g), above, or a
polynucleotide which hybridizes under stringent hybridization
conditions to a polynucleotide in (a), (b), (c), (d), (e), (f) or
(g), above. This polynucleotide which hybridizes does not hybridize
under stringent hybridization conditions to a polynucleotide having
a nucleotide sequence consisting of only A residues or of only T
residues. An additional nucleic acid embodiment of the invention
relates to an isolated nucleic acid molecule comprising a
polynucleotide which encodes the amino acid sequence of an
epitope-bearing portion of a Ependymin polypeptide having an amino
acid sequence in (a), (b), (c), (d), (e) or (f), above. A further
nucleic acid embodiment of the invention relates to an isolated
nucleic acid molecule comprising a polynucleotide which encodes the
amino acid sequence of a Ependymin polypeptide having an amino acid
sequence which contains at least one conservative amino acid
substitution, but not more than 50 conservative amino acid
substitutions, even more preferably, not more than 40 conservative
amino acid substitutions, still more preferably, not more than 30
conservative amino acid substitutions, and still even more
preferably, not more than 20 conservative amino acid substitutions.
Of course, in order of ever-increasing preference, it is highly
preferable for a polynucleotide which encodes the amino acid
sequence of a Ependymin polypeptide to have an amino acid sequence
which contains not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1
conservative amino acid substitutions.
[0078] The present invention also relates to recombinant vectors,
which include the isolated nucleic acid molecules of the present
invention, and to host cells containing the recombinant vectors, as
well as to methods of making such vectors and host cells and for
using them for production of Ependymin polypeptides or peptides by
recombinant techniques.
[0079] By a polynucleotide having a nucleotide sequence at least,
for example, 95% "identical" to a reference nucleotide sequence
encoding a Ependymin polypeptide is intended that the nucleotide
sequence of the polynucleotide is identical to the reference
sequence except that the polynucleotide sequence may include up to
five point mutations per each 100 nucleotides of the reference
nucleotide sequence encoding the Ependymin polypeptide. In other
words, to obtain a polynucleotide having a nucleotide sequence at
least 95% identical to a reference nucleotide sequence, up to 5% of
the nucleotides in the reference sequence may be deleted or
substituted with another nucleotide, or a number of nucleotides up
to 5% of the total nucleotides in the reference sequence may be
inserted into the reference sequence. These mutations of the
reference sequence may occur at the 5' or 3' terminal positions of
the reference nucleotide sequence or anywhere between those
terminal positions, interspersed either individually among
nucleotides in the reference sequence or in one or more contiguous
groups within the reference sequence.
[0080] As a practical matter, whether any particular nucleic acid
molecule is at least 90%, 95%, 96%, 97%, 98% or 99% identical to,
for instance, the nucleotide sequence shown in FIGS. 1A, 1B, and
1C, or to the nucleotides sequence of the deposited cDNA clone
HDPIE88 can be determined conventionally using known computer
programs such as the Bestfit program (Wisconsin Sequence Analysis
Package, Version 8 for Unix, Genetics Computer Group, University
Research Park, 575 Science Drive, Madison, Wis. 53711). Bestfit
uses the local homology algorithm of Smith and Waterman to find the
best segment of homology between two sequences (Advances in Applied
Mathematics 2:482-489 (1981)). When using Bestfit or any other
sequence alignment program to determine whether a particular
sequence is, for instance, 95% identical to a reference sequence
according to the present invention, the parameters are set, of
course, such that the percentage of identity is calculated over the
full length of the reference nucleotide sequence and that gaps in
homology of up to 5% of the total number of nucleotides in the
reference sequence are allowed. A preferred method for determining
the best overall match between a query sequence (a sequence of the
present invention) and a subject sequence, also referred to as a
global sequence alignment, can be determined using the FASTDB
computer program based on the algorithm of Brutlag and colleagues
(Comp. App. Biosci. 6:237-245 (1990)). In a sequence alignment the
query and subject sequences are both DNA sequences. An RNA sequence
can be compared by converting U's to T's. The result of said global
sequence alignment is in percent identity. Preferred parameters
used in a FASTDB alignment of DNA sequences to calculate percent
identity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1,
Joining Penalty=30, Randomization Group Length=0, Cutoff Score=1,
Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the length
of the subject nucleotide sequence, whichever is shorter.
[0081] If the subject sequence is shorter than the query sequence
because of 5' or 3' deletions, not because of internal deletions, a
manual correction must be made to the results. This is because the
FASTDB program does not account for 5' and 3' truncations of the
subject sequence when calculating percent identity. For subject
sequences truncated at the 5' or 3' ends, relative to the query
sequence, the percent identity is corrected by calculating the
number of bases of the query sequence that are 5' and 3' of the
subject sequence, which are not matched/aligned, as a percent of
the total bases of the query sequence. Whether a nucleotide is
matched/aligned is determined by results of the FASTDB sequence
alignment. This percentage is then subtracted from the percent
identity, calculated by the above FASTDB program using the
specified parameters, to arrive at a final percent identity score.
This corrected score is what is used for the purposes of the
present invention. Only bases outside the 5' and 3' bases of the
subject sequence, as displayed by the FASTDB alignment, which are
not matched/aligned with the query sequence, are calculated for the
purposes of manually adjusting the percent identity score.
[0082] For example, a 90 base subject sequence is aligned to a 100
base query sequence to determine percent identity. The deletions
occur at the 5' end of the subject sequence and therefore, the
FASTDB alignment does not show a matched/alignment of the first 10
bases at 5' end. The 10 unpaired bases represent 10% of the
sequence (number of bases at the 5' and 3' ends not matched/total
number of bases in the query sequence) so 10% is subtracted from
the percent identity score calculated by the FASTDB program. If the
remaining 90 bases were perfectly matched the final percent
identity would be 90%. In another example, a 90 base subject
sequence is compared with a 100 base query sequence. This time the
deletions are internal deletions so that there are no bases on the
5' or 3' of the subject sequence which are not matched/aligned with
the query. In this case the percent identity calculated by FASTDB
is not manually corrected. Once again, only bases 5' and 3' of the
subject sequence which are not matched/aligned with the query
sequence are manually corrected for. No other manual corrections
are to made for the purposes of the present invention.
[0083] The present application is directed to nucleic acid
molecules at least 90%, 95%, 96%, 97%, 98% or 99% identical to the
nucleic acid sequence shown in FIGS. 1A, 1B, and 1C (SEQ ID NO:1)
or to the nucleic acid sequence of the deposited cDNA, irrespective
of whether they encode a polypeptide having Ependymin activity.
This is because even where a particular nucleic acid molecule does
not encode a polypeptide having Ependymin activity, one of skill in
the art would still know how to use the nucleic acid molecule, for
instance, as a hybridization probe or a polymerase chain reaction
(PCR) primer. Uses of the nucleic acid molecules of the present
invention that do not encode a polypeptide having Ependymin
activity include, inter alia, (1) isolating the Ependymin gene or
allelic variants thereof in a cDNA library; (2) in situ
hybridization (e.g., "FISH") to metaphase chromosomal spreads to
provide precise chromosomal location of the Ependymin gene, as
described by Verma and colleagues (Human Chromosomes: A Manual of
Basic Techniques, Pergamon Press, New York (1988)); and Northern
Blot analysis for detecting Ependymin mRNA expression in specific
tissues.
[0084] Preferred, however, are nucleic acid molecules having
sequences at least 90%, 95%, 96%, 97%, 98% or 99% identical to the
nucleic acid sequence shown in FIGS. 1A, 1B, and 1C (SEQ ID NO:1)
or to the nucleic acid sequence of the deposited cDNA which do, in
fact, encode a polypeptide having Ependymin protein activity. By "a
polypeptide having Ependymin activity" is intended polypeptides
exhibiting activity similar, but not necessarily identical, to an
activity of the mature Ependymin protein of the invention, as
measured in a particular biological assay. For example, the
Ependymin protein of the present invention binds Ca.sup.2+ to a
high degree. Maruyama and colleagues (J. Biochem. 95:511-519
(1984)), Ganss and Hoffman (Eur. J. Biochem. 217:275-280 (1993)),
and Schmidt and Makiola (Neuro. Chem. (Life Sci. Adv.) 10: 161-171
(1991)) each demonstrate a convenient laboratory method to
determine and roughly quantitate the amount of radiolabeled
Ca.sup.2+ which a given polypeptide, such as Ependymin of the
present invention, can bind. Briefly, 5-10 .mu.g protein is used
for each analysis. The protein samples are separated by
conventional SDS/PAGE using a 13% polyacrylamide gel according to
methods which are well-known to one of ordinary skill in the art.
Following SDS/PAGE, the proteins are transferred to a
nitrocellulose filter also according to methods which are
well-known to one of ordinary skill in the art (for example, such a
method is provided by Towbin, H., et al., Proc. Natl. Acad. Sci.
USA 76:4350-4354 (1979)). After the protein is transferred to the
nitrocellulose filter, the blot is hybridized with 200 ml
.sup.45CaCl.sub.2 (300 .mu.Ci) in the presence of 5 mM MgCl.sub.2.
To reduce non-specific binding of Ca.sup.2+, the transfer buffer is
washed out by three changes (20 minutes each) of 10 mM
imidazole/HCl buffer, pH 6.8, containing 5 mM MgCl.sub.2 and 60 mM
KCl. In such analyses, it is convenient to use a well-known, strong
Ca.sup.2+-binding protein, such as calmodulin (Pharmacia) and a
well-known, protein which does not bind Ca.sup.2+, such as bovine
serum albumin (Sigma) as controls. To quantitate the amount of
Ca.sup.2+ bound to a specific protein, the region of the
nitrocellulose filter which to which the protein is bound is cut
out and placed in a standard scintillation vial. The vial is then
filled with 4 ml scintillation cocktail (for example, Quickszint
361; Zinsser, Maidenhead, U. K.) and counted in a liquid
scintillation counter (for example, the Wallac 1410, Pharmacia).
Using such an analysis, one of ordinary skill in the art may easily
ascertain useful qualitative and quantitative information regarding
proteins such as Ependymin, or muteins thereof, of the present
invention such as the amount of Ca.sup.2+ that the protein will
bind and the apparent molecular mass of the protein.
Ca.sup.2+-binding activity is a useful indicator of the potential
for more complex biological activities.
[0085] Ependymin protein binds Ca.sup.2+ in a dose-dependent manner
in the above-described assay. Thus, "a polypeptide having Ependymin
protein activity" includes polypeptides that also exhibit any of
the same Ca.sup.2+-binding activities in the above-described assays
in a dose-dependent manner. Although the degree of dose-dependent
activity need not be identical to that of the Ependymin protein,
preferably, "a polypeptide having Ependymin protein activity" will
exhibit substantially similar dose-dependence in a given activity
as compared to the Ependymin protein (i.e., the candidate
polypeptide will exhibit greater activity or not more than about
25-fold less and, preferably, not more than about tenfold less
activity relative to the reference Ependymin protein).
[0086] Of course, due to the degeneracy of the genetic code, one of
ordinary skill in the art will immediately recognize that a large
number of the nucleic acid molecules having a sequence at least
90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid
sequence of the deposited cDNA or the nucleic acid sequence shown
in FIGS. 1A, 1B, and 1C (SEQ ID NO:1) will encode a polypeptide
"having Ependymin protein activity." In fact, since degenerate
variants of these nucleotide sequences all encode the same
polypeptide, this will be clear to the skilled artisan even without
performing the above described comparison assay. It will be further
recognized in the art that, for such nucleic acid molecules that
are not degenerate variants, a reasonable number will also encode a
polypeptide having Ependymin protein activity. This is because the
skilled artisan is fully aware of amino acid substitutions that are
either less likely or not likely to significantly effect protein
function (e.g., replacing one aliphatic amino acid with a second
aliphatic amino acid), as further described below.
Vectors and Host Cells
[0087] The present invention also relates to vectors which include
the isolated DNA molecules of the present invention, host cells
which are genetically engineered with the recombinant vectors, and
the production of Ependymin polypeptides or fragments thereof by
recombinant techniques. The vector may be, for example, a phage,
plasmid, viral or retroviral vector. Retroviral vectors may be
replication competent or replication defective. In the latter case,
viral propagation generally will occur only in complementing host
cells.
[0088] The polynucleotides may be joined to a vector containing a
selectable marker for propagation in a host. Generally, a plasmid
vector is introduced in a precipitate, such as a calcium phosphate
precipitate, or in a complex with a charged lipid. If the vector is
a virus, it may be packaged in vitro using an appropriate packaging
cell line and then transduced into host cells.
[0089] The DNA insert should be operatively linked to an
appropriate promoter, such as the phage lambda PL promoter, the E.
coli lac, trp, phoA and tac promoters, the SV40 early and late
promoters and promoters of retroviral LTRs, to name a few. Other
suitable promoters will be known to the skilled artisan. The
expression constructs will further contain sites for transcription
initiation, termination and, in the transcribed region, a ribosome
binding site for translation. The coding portion of the transcripts
expressed by the constructs will preferably include a translation
initiating codon at the beginning and a termination codon (UAA, UGA
or UAG) appropriately positioned at the end of the polypeptide to
be translated.
[0090] As indicated, the expression vectors will preferably include
at least one selectable marker. Such markers include dihydrofolate
reductase, G418 or neomycin resistance for eukaryotic cell culture
and tetracycline, kanamycin or ampicillin resistance genes for
culturing in E. coli and other bacteria. Representative examples of
appropriate hosts include, but are not limited to, bacterial cells,
such as E. coli, Streptomyces and Salmonella typhimurium cells;
fungal cells, such as yeast cells; insect cells such as Drosophila
S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, 293 and
Bowes melanoma cells; and plant cells. Appropriate culture mediums
and conditions for the above-described host cells are known in the
art.
[0091] Vectors preferred for use in bacteria include pHE4-5
(ATCC.TM. Accession No. 209311; and variations thereof), pQE70,
pQE60 and pQE-9 (QIAGEN, Inc., supra); pBS vectors, Phagescript
vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A
(STRATAGENE.TM.); and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5
(Pharmacia). Among preferred eukaryotic vectors are pWLNEO,
pSV2CAT, pOG44, pXT1, and pSG (STRATAGENE.TM.); and pSVK3, pBPV,
pMSG and pSVL (Pharmacia). Other suitable vectors will be readily
apparent to the skilled artisan.
[0092] Introduction of the construct into the host cell can be
effected by calcium phosphate transfection, DEAE-dextran mediated
transfection, cationic lipid-mediated transfection,
electroporation, transduction, infection or other methods. Such
methods are described in many standard laboratory manuals (for
example, Davis, et al., Basic Methods In Molecular Biology
(1986)).
[0093] The polypeptide may be expressed in a modified form, such as
a fusion protein, and may include not only secretion signals, but
also additional heterologous functional regions. For instance, a
region of additional amino acids, particularly charged amino acids,
may be added to the N-terminus of the polypeptide to improve
stability and persistence in the host cell, during purification, or
during subsequent handling and storage. Also, peptide moieties may
be added to the polypeptide to facilitate purification. Such
regions may be removed prior to final preparation of the
polypeptide. The addition of peptide moieties to polypeptides to
engender secretion or excretion, to improve stability and to
facilitate purification, among others, are familiar and routine
techniques in the art. A preferred fusion protein comprises a
heterologous region from immunoglobulin that is useful to stabilize
and purify proteins. For example, EP-A-O 464 533 (Canadian
counterpart 2045869) discloses fusion proteins comprising various
portions of constant region of immunoglobulin molecules together
with another human protein or part thereof. In many cases, the Fc
part in a fusion protein is thoroughly advantageous for use in
therapy and diagnosis and thus results, for example, in improved
pharmacokinetic properties (EP-A 0232 262). On the other hand, for
some uses it would be desirable to be able to delete the Fc part
after the fusion protein has been expressed, detected and purified
in the advantageous manner described. This is the case when Fc
portion proves to be a hindrance to use in therapy and diagnosis,
for example when the fusion protein is to be used as antigen for
immunizations. In drug discovery, for example, human proteins, such
as hIL-5, have been fused with Fc portions for the purpose of
high-throughput screening assays to identify antagonists of hIL-5
(Bennett, D., et al., J. Molecular Recognition 8:52-58 (1995);
Johanson, K., et al., J. Biol. Chem. 270:9459-9471 (1995)).
[0094] The Ependymin protein can be recovered and purified from
recombinant cell cultures by well-known methods including ammonium
sulfate or ethanol precipitation, acid extraction, anion or cation
exchange chromatography, phosphocellulose chromatography,
hydrophobic interaction chromatography, affinity chromatography,
hydroxylapatite chromatography and lectin chromatography. Most
preferably, high performance liquid chromatography ("HPLC") is
employed for purification. Polypeptides of the present invention
include: products purified from natural sources, including bodily
fluids, tissues and cells, whether directly isolated or cultured;
products of chemical synthetic procedures; and products produced by
recombinant techniques from a prokaryotic or eukaryotic host,
including, for example, bacterial, yeast, higher plant, insect and
mammalian cells. Depending upon the host employed in a recombinant
production procedure, the polypeptides of the present invention may
be glycosylated or may be non-glycosylated. In addition,
polypeptides of the invention may also include an initial modified
methionine residue, in some cases as a result of host-mediated
processes. Thus, it is well known in the art that the N-terminal
methionine encoded by the translation initiation codon generally is
removed with high efficiency from any protein after translation in
all eukaryotic cells. While the N-terminal methionine on most
proteins also is efficiently removed in most prokaryotes, for some
proteins this prokaryotic removal process is inefficient, depending
on the nature of the amino acid to which the N-terminal methionine
is covalently linked.
Polypeptides and Fragments
[0095] The invention further provides an isolated Ependymin
polypeptide having the amino acid sequence encoded by the deposited
cDNA, or the amino acid sequence in SEQ ID NO:2, or a peptide or
polypeptide comprising a portion of the above polypeptides.
Variant and Mutant Polypeptides
[0096] To improve or alter the characteristics of Ependymin
polypeptides, protein engineering may be employed. Recombinant DNA
technology known to those skilled in the art can be used to create
novel mutant proteins or muteins including single or multiple amino
acid substitutions, deletions, additions or fusion proteins. Such
modified polypeptides can show, e.g., enhanced activity or
increased stability. In addition, they may be purified in higher
yields and show better solubility than the corresponding natural
polypeptide, at least under certain purification and storage
conditions.
N-Term in al and C-Terminal Deletion Mutants
[0097] For instance, for many proteins, including the extracellular
domain of a membrane associated protein or the mature form(s) of a
secreted protein, it is known in the art that one or more amino
acids may be deleted from the N-terminus or C-terminus without
substantial loss of biological function. For instance, Ron and
colleagues (J. Biol. Chem., 268:2984-2988 (1993)) reported modified
KGF proteins that had heparin binding activity even if 3, 8, or 27
N-terminal amino acid residues were missing. In the present case,
since the protein of the invention is a member of the ependymin
polypeptide family, deletions of N-terminal amino acids up to the
cysteine at position 5 of SEQ ID NO:2 may retain some biological
activity such as Ca.sup.2+-binding or the ability to modulate
regeneration. Polypeptides having further N-terminal deletions
including the Cys-5 residue in SEQ ID NO:2 would not be expected to
retain such biological activities because this residue is conserved
in each of the ependymin-related polypeptides shown in FIGS. 3A and
3B and it is likely that a cysteine in such a position may be
required for forming a disulfide bridge to provide structural
stability which is needed for receptor binding and signal
transduction.
[0098] However, even if deletion of one or more amino acids from
the N-terminus of a protein results in modification or loss of one
or more biological functions of the protein, other biological
activities may still be retained. Thus, the ability of the
shortened protein to induce and/or bind to antibodies which
recognize the complete or mature of the protein generally will be
retained when less than the majority of the residues of the
complete or mature protein are removed from the N-terminus. Whether
a particular polypeptide lacking N-terminal residues of a complete
protein retains such immunologic activities can readily be
determined by routine methods described herein and otherwise known
in the art.
[0099] Accordingly, the present invention further provides
polypeptides having one or more residues deleted from the amino
terminus of the amino acid sequence of the Ependymin shown in SEQ
ID NO:2, up to the cysteine residue at position number 5, and
polynucleotides encoding such polypeptides. In particular, the
present invention provides polypeptides comprising the amino acid
sequence of residues n.sup.1-187 of SEQ ID NO:2, where n.sup.1 is
an integer in the range of -37 to 4, and 5 is the position of the
first residue from the N-terminus of the complete Ependymin
polypeptide (shown in SEQ ID NO:2) believed to be required for the
Ca.sup.2+-binding or the regeneration modulatory activities of the
Ependymin protein.
[0100] More in particular, the invention provides polynucleotides
encoding polypeptides having the amino acid sequence of residues of
-37-187, -36-187, -35-187, -34-187, -33-187, -32-187, -31-187,
-30-187, -29-187, -28-187, -27-187, -26-187, -25-187, -24-187,
-23-187, -22-187, -21-187, -20-187, -19-187, -18-187, -17-187,
-16-187, -15-187, -14-187, -13-187, -12-187, -11-187, -10-187,
-9-187, -8-187, -7-187, -6-187, -5-187, -4-187, -3-187, -2-187,
-1-187, 1-187, 2-187, 3-187, 4-187, and 5-187 of SEQ ID NO:2.
Polynucleotides encoding these polypeptides also are provided.
[0101] Similarly, many examples of biologically functional
C-terminal deletion muteins are known. For instance, Interferon
gamma shows up to ten times higher activities by deleting 8-10
amino acid residues from the carboxy terminus of the protein
(Dobeli, et al., J. Biotechnology 7:199-216 (1988)). In the present
case, since the protein of the invention is a member of the
ependymin polypeptide family, deletions of C-terminal amino acids
up to the cysteine at position 173 of SEQ ID NO:2 may retain some
biological activity such as Ca.sup.2+-binding or the ability to
modulate regeneration. Polypeptides having further C-terminal
deletions including the cysteine residue at position 173 of SEQ ID
NO:2 would not be expected to retain such biological activities
because this residue is conserved in each of the ependymin-related
polypeptides shown in FIGS. 3A and 3B and it is likely that a
cysteine in such a position may be required for forming a disulfide
bridge to provide structural stability which is needed for receptor
binding and signal transduction.
[0102] However, even if deletion of one or more amino acids from
the C-terminus of a protein results in modification of loss of one
or more biological functions of the protein, other biological
activities may still be retained. Thus, the ability of the
shortened protein to induce and/or bind to antibodies which
recognize the complete or mature of the protein generally will be
retained when less than the majority of the residues of the
complete or mature protein are removed from the C-terminus. Whether
a particular polypeptide lacking C-terminal residues of a complete
protein retains such immunologic activities can readily be
determined by routine methods described herein and otherwise known
in the art.
[0103] Accordingly, the present invention further provides
polypeptides having one or more residues from the carboxy terminus
of the amino acid sequence of the Ependymin shown in SEQ ID NO:2,
up to the cysteine residue at position 173 of SEQ ID NO:2, and
polynucleotides encoding such polypeptides. In particular, the
present invention provides polypeptides having the amino acid
sequence of residues -37-m.sup.1 of the amino acid sequence in SEQ
ID NO:2, where m.sup.1 is any integer in the range of 173 to 187,
and residue 173 is the position of the first residue from the
C-terminus of the complete Ependymin polypeptide (shown in SEQ ID
NO:2) believed to be required for the Ca.sup.2+-binding or the
regeneration modulatory abilities of the Ependymin protein.
[0104] More in particular, the invention provides polynucleotides
encoding polypeptides having the amino acid sequence of residues
-37-173, -37-174, -37-175, -37-176, -37-177, -37-178, -37-179,
-37-180, -37-181, -37-182, -37-183, -37-184, -37-185, -37-186, and
-37-187 of SEQ ID NO:2. Polynucleotides encoding these polypeptides
also are provided.
[0105] The invention also provides polypeptides having one or more
amino acids deleted from both the amino and the carboxyl termini,
which may be described generally as having residues n.sup.1-m.sup.1
of SEQ ID NO:2, where n.sup.1 and m.sup.1 are integers as described
above.
[0106] Also included are a nucleotide sequence encoding a
polypeptide consisting of a portion of the complete Ependymin amino
acid sequence encoded by the cDNA clone contained in ATCC.TM.
Deposit No. 209464, where this portion excludes from 1 to about 42
amino acids from the amino terminus of the complete amino acid
sequence encoded by the cDNA clone contained in ATCC.TM. Deposit
No. 209464, or from 1 to about 15 amino acids from the carboxy
terminus, or any combination of the above amino terminal and
carboxy terminal deletions, of the complete amino acid sequence
encoded by the cDNA clone contained in ATCC.TM. Deposit No. 209464.
Polynucleotides encoding all of the above deletion mutant
polypeptide forms also are provided.
[0107] As mentioned above, even if deletion of one or more amino
acids from the N-terminus of a protein results in modification of
loss of one or more biological functions of the protein, other
biological activities may still be retained. Thus, the ability of
the shortened Human Ependymin mutein to induce and/or bind to
antibodies which recognize the complete or mature of the protein
generally will be retained when less than the majority of the
residues of the complete or mature protein are removed from the
N-terminus. Whether a particular polypeptide lacking N-terminal
residues of a complete protein retains such immunologic activities
can readily be determined by routine methods described herein and
otherwise known in the art. It is not unlikely that a Human
Ependymin mutein with a large number of deleted N-terminal amino
acid residues may retain some biological or immungenic activities.
In fact, peptides composed of as few as six Human Ependymin amino
acid residues may often evoke an immune response.
[0108] Accordingly, the present invention further provides
polypeptides having one or more residues deleted from the amino
terminus of the Human Ependymin amino acid sequence shown in SEQ ID
NO:2, up to the serine acid residue at position number 219 and
polynucleotides encoding such polypeptides. In particular, the
present invention provides polypeptides comprising the amino acid
sequence of residues n.sup.2-224 of FIGS. 1A, 1B, and 1C (SEQ ID
NO:2), where n.sup.2 is an integer in the range of 2 to 219, and
220 is the position of the first residue from the N-terminus of the
complete Human Ependymin polypeptide believed to be required for at
least immunogenic activity of the Human Ependymin protein.
[0109] More in particular, the invention provides polynucleotides
encoding polypeptides comprising, or alternatively consisting of,
the amino acid sequence of residues of P-2 to W-224; G-3 to W-224;
R-4 to W-224; A-5 to W-224; P-6 to W-224; L-7 to W-224; R-8 to
W-224; T-9 to W-224; V-10 to W-224; P-1 to W-224; G-12 to W-224;
A-13 to W-224; L-14 to W-224; G-15 to W-224; A-16 to W-224; W-17 to
W-224; L-18 to W-224; L-19 to W-224; G-20 to W-224; G-21 to W-224;
L-22 to W-224; W-23 to W-224; A-24 to W-224; W-25 to W-224; T-26 to
W-224; L-27 to W-224; C-28 to W-224; G-29 to W-224; L-30 to W-224;
C-31 to W-224; S-32 to W-224; L-33 to W-224; G-34 to W-224; A-35 to
W-224; V-36 to W-224; G-37 to W-224; A-38 to W-224; P-39 to W-224;
R-40 to W-224; P-41 to W-224; C-42 to W-224; Q-43 to W-224; A-44 to
W-224; P-45 to W-224; Q-46 to W-224; Q-47 to W-224; W-48 to W-224;
E-49 to W-224; G-50 to W-224; R-51 to W-224; Q-52 to W-224; V-53 to
W-224; M-54 to W-224; Y-55 to W-224; Q-56 to W-224; Q-57 to W-224;
S-58 to W-224; S-59 to W-224; G-60 to W-224; R-61 to W-224; N-62 to
W-224; S-63 to W-224; R-64 to W-224; A-65 to W-224; L-66 to W-224;
L-67 to W-224; S-68 to W-224; Y-69 to W-224; D-70 to W-224; G-71 to
W-224; L-72 to W-224; N-73 to W-224; Q-74 to W-224; R-75 to W-224;
V-76 to W-224; R-77 to W-224; V-78 to W-224; L-79 to W-224; D-80 to
W-224; E-81 to W-224; R-82 to W-224; K-83 to W-224; A-84 to W-224;
L-85 to W-224; 1-86 to W-224; P-87 to W-224; C-88 to W-224; K-89 to
W-224; R-90 to W-224; L-91 to W-224; F-92 to W-224; E-93 to W-224;
Y-94 to W-224; 1-95 to W-224; L-96 to W-224; L-97 to W-224; Y-98 to
W-224; K-99 to W-224; D-100 to W-224; G-101 to W-224; V-102 to
W-224; M-103 to W-224; F-104 to W-224; Q-105 to W-224; 1-106 to
W-224; D-107 to W-224; Q-108 to W-224; A-109 to W-224; T-110 to
W-224; K-111 to W-224; Q-112 to W-224; C-113 to W-224; S-114 to
W-224; K-115 to W-224; M-116 to W-224; T-117 to W-224; L-118 to
W-224; T-119 to W-224; Q-120 to W-224; P-121 to W-224; W-122 to
W-224; D-123 to W-224; P-124 to W-224; L-125 to W-224; D-126 to
W-224; I-127 to W-224; P-128 to W-224; Q-129 to W-224; N-130 to
W-224; S-131 to W-224; T-132 to W-224; F-133 to W-224; E-134 to
W-224; D-135 to W-224; Q-136 to W-224; Y-137 to W-224; S-138 to
W-224; I-139 to W-224; G-140 to W-224; G-141 to W-224; P-142 to
W-224; Q-143 to W-224; E-144 to W-224; Q-145 to W-224; 1-146 to
W-224; T-147 to W-224; V-148 to W-224; Q-149 to W-224; E-150 to
W-224; W-151 to W-224; S-152 to W-224; D-153 to W-224; R-154 to
W-224; K-155 to W-224; S-156 to W-224; A-157 to W-224; R-158 to
W-224; S-159 to W-224; Y-160 to W-224; E-161 to W-224; T-162 to
W-224; W-163 to W-224; I-164 to W-224; G-165 to W-224; I-166 to
W-224; Y-167 to W-224; T-168 to W-224; V-169 to W-224; K-170 to
W-224; D-171 to W-224; C-172 to W-224; Y-173 to W-224; P-174 to
W-224; V-175 to W-224; Q-176 to W-224; E-177 to W-224; T-178 to
W-224; F-179 to W-224; T-180 to W-224; I-181 to W-224; N-182 to
W-224; Y-183 to W-224; S-184 to W-224; V-185 to W-224; I-186 to
W-224; L-187 to W-224; S-188 to W-224; T-189 to W-224; R-190 to
W-224; F-191 to W-224; F-192 to W-224; D-193 to W-224; 1-194 to
W-224; Q-195 to W-224; L-196 to W-224; G-197 to W-224; 1-198 to
W-224; K-199 to W-224; D-200 to W-224; P-201 to W-224; S-202 to
W-224; V-203 to W-224; F-204 to W-224; T-205 to W-224; P-206 to
W-224; P-207 to W-224; S-208 to W-224; T-209 to W-224; C-210 to
W-224; Q-211 to W-224; M-212 to W-224; A-213 to W-224; Q-214 to
W-224; L-215 to W-224; E-216 to W-224; K-217 to W-224; M-218 to
W-224; and S-219 to W-224 of the Human Ependymin amino acid
sequence shown in FIGS. 1A, 1B, and 1C (which is identical to the
sequence shown as SEQ ID NO:2, with the exception that the amino
acid residues in FIGS. 1A, 1B, and 1C are numbered consecutively
from 1 through 224 from the N-terminus to the C-terminus, while the
amino acid residues in SEQ ID NO:2 are numbered consecutively from
-37 through 187 to reflect the position of the predicted signal
peptide). Polynucleotides encoding these polypeptides are also
encompassed by the invention.
[0110] Also as mentioned above, even if deletion of one or more
amino acids from the C-terminus of a protein results in
modification of loss of one or more biological functions of the
protein, other biological activities may still be retained. Thus,
the ability of the shortened Human Ependymin mutein to induce
and/or bind to antibodies which recognize the complete or mature of
the protein generally will be retained when less than the majority
of the residues of the complete or mature protein are removed from
the C-terminus. Whether a particular polypeptide lacking C-terminal
residues of a complete protein retains such immunologic activities
can readily be determined by routine methods described herein and
otherwise known in the art. It is not unlikely that a Human
Ependymin mutein with a large number of deleted C-terminal amino
acid residues may retain some biological or immungenic activities.
In fact, peptides composed of as few as six Human Ependymin amino
acid residues may often evoke an immune response.
[0111] Accordingly, the present invention further provides
polypeptides having one or more residues deleted from the carboxy
terminus of the amino acid sequence of the Human Ependymin shown in
SEQ ID NO:2, up to the proline residue at position number 6, and
polynucleotides encoding such polypeptides. In particular, the
present invention provides polypeptides comprising the amino acid
sequence of residues 1-m.sup.2 of SEQ ID NO:2, where m.sup.2 is an
integer in the range of 6 to 224, and 6 is the position of the
first residue from the C-terminus of the complete Human Ependymin
polypeptide believed to be required for at least immunogenic
activity of the Human Ependymin protein.
[0112] More in particular, the invention provides polynucleotides
encoding polypeptides comprising, or alternatively consisting of,
the amino acid sequence of residues M-1 to S-223; M-1 to C-222; M-1
to D-221; M-1 to E-220; M-1 to S-219; M-1 to M-218; M-1 to K-217;
M-1 to E-216; M-1 to L-215; M-1 to Q-214; M-1 to A-213; M-1 to
M-212; M-1 to Q-211; M-1 to C-210; M-1 to T-209; M-1 to S-208; M-1
to P-207; M-1 to P-206; M-1 to T-205; M-1 to F-204; M-1 to V-203;
M-1 to S-202; M-1 to P-201; M-1 to D-200; M-1 to K-199; M-1 to
1-198; M-1 to G-197; M-1 to L-196; M-1 to Q-195; M-1 to 1-194; M-1
to D-193; M-1 to F-192; M-1 to F-191; M-1 to R-190; M-1 to T-189;
M-1 to S-188; M-1 to L-187; M-1 to 1-186; M-1 to V-185; M-1 to
S-184; M-1 to Y-183; M-1 to N-182; M-1 to 1-181; M-1 to T-180; M-1
to F-179; M-1 to T-178; M-1 to E-177; M-1 to Q-176; M-1 to V-175;
M-1 to P-174; M-1 to Y-173; M-1 to C-172; M-1 to D-171; M-1 to
K-170; M-1 to V-169; M-1 to T-168; M-1 to Y-167; M-1 to 1-166; M-1
to G-165; M-1 to 1-164; M-1 to W-163; M-1 to T-162; M-1 to E-161;
M-1 to Y-160; M-1 to S-159; M-1 to R-158; M-1 to A-157; M-1 to
S-156; M-1 to K-155; M-1 to R-154; M-1 to D-153; M-1 to S-152; M-1
to W-151; M-1 to E-150; M-1 to Q-149; M-1 to V-148; M-1 to T-147;
M-1 to I-146; M-1 to Q-145; M-1 to E-144; M-1 to Q-143; M-1 to
P-142; M-1 to G-141; M-1 to G-140; M-1 to 1-139; M-1 to S-138; M-1
to Y-137; M-1 to Q-136; M-1 to D-135; M-1 to E-134; M-1 to F-133;
M-1 to T-132; M-1 to S-131; M-1 to N-130; M-1 to Q-129; M-1 to
P-128; M-1 to 1-127; M-1 to D-126; M-1 to L-125; M-1 to P-124; M-1
to D-123; M-1 to W-122; M-1 to P-121; M-1 to Q-120; M-1 to T-119;
M-1 to L-118; M-1 to T-117; M-1 to M-116; M-1 to K-115; M-1 to
S-114; M-1 to C-113; M-1 to Q-112; M-1 to K-111; M-1 to T-110; M-1
to A-109; M-1 to Q-108; M-1 to D-107; M-1 to 1-106; M-1 to Q-105;
M-1 to F-104; M-1 to M-103; M-1 to V-102; M-1 to G-101; M-1 to
D-100; M-1 to K-99; M-1 to Y-98; M-1 to L-97; M-1 to L-96; M-1 to
1-95; M-1 to Y-94; M-1 to E-93; M-1 to F-92; M-1 to L-91; M-1 to
R-90; M-1 to K-89; M-1 to C-88; M-1 to P-87; M-1 to 1-86; M-1 to
L-85; M-1 to A-84; M-1 to K-83; M-1 to R-82; M-1 to E-81; M-1 to
D-80; M-1 to L-79; M-1 to V-78; M-1 to R-77; M-1 to V-76; M-1 to
R-75; M-1 to Q-74; M-1 to N-73; M-1 to L-72; M-1 to G-71; M-1 to
D-70; M-1 to Y-69; M-1 to S-68; M-1 to L-67; M-1 to L-66; M-1 to
A-65; M-1 to R-64; M-1 to S-63; M-1 to N-62; M-1 to R-61; M-1 to
G-60; M-1 to S-59; M-1 to S-58; M-1 to Q-57; M-1 to Q-56; M-1 to
Y-55; M-1 to M-54; M-1 to V-53; M-1 to Q-52; M-1 to R-51; M-1 to
G-50; M-1 to E-49; M-1 to W-48; M-1 to Q-47; M-1 to Q-46; M-1 to
P-45; M-1 to A-44; M-1 to Q-43; M-1 to C-42; M-1 to P-41; M-1 to
R-40; M-1 to P-39; M-1 to A-38; M-1 to G-37; M-1 to V-36; M-1 to
A-35; M-1 to G-34; M-1 to L-33; M-1 to S-32; M-1 to C-31; M-1 to
L-30; M-1 to G-29; M-1 to C-28; M-1 to L-27; M-1 to T-26; M-1 to
W-25; M-1 to A-24; M-1 to W-23; M-1 to L-22; M-1 to G-21; M-1 to
G-20; M-1 to L-19; M-1 to L-18; M-1 to W-17; M-1 to A-16; M-1 to
G-15; M-1 to L-14; M-1 to A-13; M-1 to G-12; M-1 to P-11; M-1 to
V-10; M-1 to T-9; M-1 to R-8; M-1 to L-7; M-1 to P-6 of the
sequence of the Human Ependymin sequence shown in FIGS. 1A, 1B, and
1C (which is identical to the sequence shown as SEQ ID NO:2, with
the exception that the amino acid residues in FIGS. 1A, 1B, and 1C
are numbered consecutively from 1 through 224 from the N-terminus
to the C-terminus, while the amino acid residues in SEQ ID NO:2 are
numbered consecutively from -37 through 187 to reflect the position
of the predicted signal peptide). Polynucleotides encoding these
polypeptides also are provided.
[0113] The invention also provides polypeptides having one or more
amino acids deleted from both the amino and the carboxyl termini of
a Human Ependymin polypeptide, which may be described generally as
having residues n.sup.2-m.sup.2 of FIGS. 1A, 1B, and 1C (SEQ ID
NO:2), where n.sup.2 and m.sup.2 are integers as described
above.
Other Mutants
[0114] In addition to terminal deletion forms of the protein
discussed above, it also will be recognized by one of ordinary
skill in the art that some amino acid sequences of the Ependymin
polypeptide can be varied without significant effect of the
structure or function of the protein. If such differences in
sequence are contemplated, it should be remembered that there will
be critical areas on the protein which determine activity.
[0115] Thus, the invention further includes variations of the
Ependymin polypeptide which show substantial Ependymin polypeptide
activity or which include regions of Ependymin protein such as the
protein portions discussed below. Such mutants include deletions,
insertions, inversions, repeats, and type substitutions selected
according to general rules known in the art so as have little
effect on activity. For example, guidance concerning how to make
phenotypically silent amino acid substitutions is provided wherein
the authors indicate that there are two main approaches for
studying the tolerance of an amino acid sequence to change (Bowie,
J. U., et al., Science 247:1306-1310 (1990)). The first method
relies on the process of evolution, in which mutations are either
accepted or rejected by natural selection. The second approach uses
genetic engineering to introduce amino acid changes at specific
positions of a cloned gene and selections or screens to identify
sequences that maintain functionality.
[0116] As the authors state, these studies have revealed that
proteins are surprisingly tolerant of amino acid substitutions. The
authors further indicate which amino acid changes are likely to be
permissive at a certain position of the protein. For example, most
buried amino acid residues require nonpolar side chains, whereas
few features of surface side chains are generally conserved. Other
such phenotypically silent substitutions are described by Bowie and
coworkers (supra) and the references cited therein. Typically seen
as conservative substitutions are the replacements, one for
another, among the aliphatic amino acids Ala, Val, Leu and Ile;
interchange of the hydroxyl residues Ser and Thr, exchange of the
acidic residues Asp and Glu, substitution between the amide
residues Asn and Gln, exchange of the basic residues Lys and Arg
and replacements among the aromatic residues Phe, Tyr.
[0117] Thus, the fragment, derivative or analog of the polypeptide
of SEQ ID NO:2, or that encoded by the deposited cDNA, may be (i)
one in which one or more of the amino acid residues are substituted
with a conserved or non-conserved amino acid residue (preferably a
conserved amino acid residue) and such substituted amino acid
residue may or may not be one encoded by the genetic code, or (ii)
one in which one or more of the amino acid residues includes a
substituent group, or (iii) one in which the mature polypeptide is
fused with another compound, such as a compound to increase the
half-life of the polypeptide (for example, polyethylene glycol), or
(iv) one in which the additional amino acids are fused to the above
form of the polypeptide, such as an IgG Fc fusion region peptide or
leader or secretory sequence or a sequence which is employed for
purification of the above form of the polypeptide or a proprotein
sequence. Such fragments, derivatives and analogs are deemed to be
within the scope of those skilled in the art from the teachings
herein.
[0118] Thus, the Ependymin of the present invention may include one
or more amino acid substitutions, deletions or additions, either
from natural mutations or human manipulation. As indicated, changes
are preferably of a minor nature, such as conservative amino acid
substitutions that do not significantly affect the folding or
activity of the protein (see Table II). TABLE-US-00002 TABLE II
Conservative Amino Acid Substitutions. Aromatic Phenylalanine
Tryptophan Tyrosine Hydrophobic Leucine Isoleucine Valine Polar
Glutamine Asparagine Basic Arginine Lysine Histidine Acidic
Aspartic Acid Glutamic Acid Small Alanine Serine Threonine
Methionine Glycine
[0119] Amino acids in the Ependymin protein of the present
invention that are essential for function can be identified by
methods known in the art, such as site-directed mutagenesis or
alanine-scanning mutagenesis (Cunningham and Wells, Science
244:1081-1085 (1989)). The latter procedure introduces single
alanine mutations at every residue in the molecule. The resulting
mutant molecules are then tested for biological activity such as
receptor binding or in vitro proliferative activity.
Polypeptides of the invention also can be purified from natural or
recombinant sources using anti-Ependymin antibodies of the
invention in methods which are well known in the art of protein
purification.
[0120] The invention further provides an isolated Ependymin
polypeptide comprising an amino acid sequence selected from the
group consisting of: (a) the amino acid sequence of the full-length
Ependymin polypeptide having the complete amino acid sequence shown
in SEQ ID NO:2 (i.e., positions -37 to 187 of SEQ ID NO:2); (b) the
amino acid sequence of the full-length Ependymin polypeptide having
the complete amino acid sequence shown in SEQ ID NO:2 excepting the
N-terminal methionine (i.e., positions -36 to 187 of SEQ ID NO:2);
(c) the amino acid sequence of the predicted mature Ependymin
polypeptide having the amino acid sequence at positions 1 to 187 in
SEQ ID NO:2; (d) the complete amino acid sequence encoded by the
cDNA clone contained in the ATCC.TM. Deposit No. 209464; (e) the
complete amino acid sequence excepting the N-terminal methionine
encoded by the cDNA clone contained in the ATCC.TM. Deposit No.
209464; and (f) the complete amino acid sequence of the predicted
mature Ependymin polypeptide encoded by the cDNA clone contained in
the ATCC.TM. Deposit No. 209464. The polypeptides of the present
invention also include polypeptides having an amino acid sequence
at least 80% identical, more preferably at least 90% identical, and
still more preferably 95%, 96%, 97%, 98% or 99% identical to those
described in (a), (b), (c), (d), (e) or (f), above, as well as
polypeptides having an amino acid sequence with at least 90%
similarity, and more preferably at least 95% similarity, to those
above.
[0121] Further polypeptides of the present invention include
polypeptides which have at least 90% similarity, more preferably at
least 95% similarity, and still more preferably at least 96%, 97%,
98% or 99% similarity to those described above. The polypeptides of
the invention also comprise those which are at least 80% identical,
more preferably at least 90% or 95% identical, still more
preferably at least 96%, 97%, 98% or 99% identical to the
polypeptide encoded by the deposited cDNA or to the polypeptide of
SEQ ID NO:2, and also include portions of such polypeptides with at
least 30 amino acids and more preferably at least 50 amino
acids.
[0122] A further embodiment of the invention relates to a peptide
or polypeptide which comprises the amino acid sequence of a
Ependymin polypeptide having an amino acid sequence which contains
at least one conservative amino acid substitution, but not more
than 50 conservative amino acid substitutions, even more
preferably, not more than 40 conservative amino acid substitutions,
still more preferably, not more than 30 conservative amino acid
substitutions, and still even more preferably, not more than 20
conservative amino acid substitutions. Of course, in order of
ever-increasing preference, it is highly preferable for a peptide
or polypeptide to have an amino acid sequence which comprises the
amino acid sequence of a Ependymin polypeptide, which contains at
least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1
conservative amino acid substitutions.
[0123] By "% similarity" for two polypeptides is intended a
similarity score produced by comparing the amino acid sequences of
the two polypeptides using the Bestfit program (Wisconsin Sequence
Analysis Package, Version 8 for Unix, Genetics Computer Group,
University Research Park, 575 Science Drive, Madison, Wis. 53711)
and the default settings for determining similarity. Bestfit uses
the local homology algorithm of Smith and Waterman (Advances in
Applied Mathematics 2:482-489, 1981) to find the best segment of
similarity between two sequences.
[0124] Of special interest are substitutions of charged amino acids
with other charged or neutral amino acids which may produce
proteins with highly desirable improved characteristics, such as
less aggregation. Aggregation may not only reduce activity but also
be problematic when preparing pharmaceutical formulations, because
aggregates can be immunogenic (Pinckard, et al., Clin. Exp.
Immunol. 2:331-340 (1967); Robbins, et al., Diabetes 36:838-845
(1987); Cleland, et al., Crit. Rev. Therapeutic Drug Carrier
Systems 10:307-377 (1993)).
[0125] Replacement of amino acids can also change the selectivity
of the binding of a ligand to cell surface receptors (for example,
Ostade, et al., Nature 361:266-268 (1993)) describes certain
mutations resulting in selective binding of TNF-.alpha. to only one
of the two known types of TNF receptors. Sites that are critical
for ligand-receptor binding can also be determined by structural
analysis such as crystallization, nuclear magnetic resonance or
photoaffinity labeling (Smith, et al., J. Mol. Biol. 224:899-904
(1992); de Vos, et al. Science 255:306-312 (1992)).
[0126] As described above, ependymin function relies, in part, on
asparagine-linked glycosylation. Although the relative locations of
the two potential N-linked glycosylation sites of Ependymin of the
present invention are not conserved with respect to the other known
ependymins, it is likely that disruption of the either or both of
the Ependymin glycosylation sites will, at least, modulate
Ependymin activity. As a result, mutation of the asparagine amino
acid residues at positions 93 and 145 of SEQ ID NO:2 or the
threonine or serine amino acid residues at positions 95 and 147,
respectively, will, at least modulate the biological activity of
Ependymin of the present invention.
[0127] In addition, as depicted in FIGS. 2 and 3, five cysteine
amino acid residues are conserved between Ependymin of the present
invention and five piscine ependymin homologs. Since cysteine amino
acid residues often function in a structural capacity with respect
to the tertiary structure of a polypeptide (or the quaternary
structure of the same polypeptide in cases where polymerization is
expected), it is expected that mutation of any or all of the four
conserved cysteine amino acid residues will result in, at least,
modulation of the biological activity of Ependymin of the present
invention. As shown in FIGS. 3A and 3B, the conserved cysteine
residues are located at positions 5, 51, 76, 135, and 173 of SEQ ID
NO:2.
[0128] Similarly, several additional amino acids are highly
conserved between Ependymin of the present invention and the five
piscine ependymins presented in FIGS. 3A and 3B. These are
Proline-8, Tyrosine-32, Aspartic Acid-33, Glycine-64,
Isoleucine-69, Aspartic Acid-70, Lysine-78, Leucine-81, Proline-91,
Glycine-103, Tryptophan-114, Threonine-131, Phenylalanine-167,
Proline-170, and Glutamic Acid-179. It is expected that mutation of
one, several, or all of these amino acid residues will modulate the
biological activity of Ependymin of the present invention.
[0129] Mutations which may also modulate, and are less likely to
completely eliminate, the biological activity of Ependymin of the
present invention may also be made by changing one or more
non-conserved amino acid residues throughout the Ependymin
polypeptide. Also forming part of the present invention are
isolated polynucleotides comprising nucleic acid sequences which
encode the each of the above Ependymin mutants.
[0130] The polypeptides of the present invention are preferably
provided in an isolated form, and preferably are substantially
purified. A recombinantly produced version of the Ependymin
polypeptide can be substantially purified by the one-step method
described by Smith and Johnson (Gene 67:31-40 (1988)).
[0131] By a polypeptide having an amino acid sequence at least, for
example, 95% "identical" to a reference amino acid sequence of a
Ependymin polypeptide is intended that the amino acid sequence of
the polypeptide is identical to the reference sequence except that
the polypeptide sequence may include up to five amino acid
alterations per each 100 amino acids of the reference amino acid of
the Ependymin polypeptide. In other words, to obtain a polypeptide
having an amino acid sequence at least 95% identical to a reference
amino acid sequence, up to 5% of the amino acid residues in the
reference sequence may be deleted or substituted with another amino
acid, or a number of amino acids up to 5% of the total amino acid
residues in the reference sequence may be inserted into the
reference sequence. These alterations of the reference sequence may
occur at the amino or carboxy terminal positions of the reference
amino acid sequence or anywhere between those terminal positions,
interspersed either individually among residues in the reference
sequence or in one or more contiguous groups within the reference
sequence.
[0132] As a practical matter, whether any particular polypeptide is
at least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance,
the amino acid sequence shown in FIGS. 1A, 1B, and 1C (SEQ ID
NO:2), the amino acid sequence encoded by deposited cDNA clone
HDPIE88, or fragments thereof, can be determined conventionally
using known computer programs such the Bestfit program (Wisconsin
Sequence Analysis Package, Version 8 for Unix, Genetics Computer
Group, University Research Park, 575 Science Drive, Madison, Wis.
53711). When using Bestfit or any other sequence alignment program
to determine whether a particular sequence is, for instance, 95%
identical to a reference sequence according to the present
invention, the parameters are set, of course, such that the
percentage of identity is calculated over the full length of the
reference amino acid sequence and that gaps in homology of up to 5%
of the total number of amino acid residues in the reference
sequence are allowed.
[0133] In a specific embodiment, the identity between a reference
(query) sequence (a sequence of the present invention) and a
subject sequence, also referred to as a global sequence alignment,
is determined using the FASTDB computer program based on the
algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)).
Preferred parameters used in a FASTDB amino acid alignment are:
Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty 20,
Randomization Group Length=0, Cutoff Score=1, Window Size=sequence
length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or
the length of the subject amino acid sequence, whichever is
shorter. According to this embodiment, if the subject sequence is
shorter than the query sequence due to N- or C-terminal deletions,
not because of internal deletions, a manual correction is made to
the results to take into consideration the fact that the FASTDB
program does not account for N- and C-terminal truncations of the
subject sequence when calculating global percent identity. For
subject sequences truncated at the N- and C-termini, relative to
the query sequence, the percent identity is corrected by
calculating the number of residues of the query sequence that are
N- and C-terminal of the subject sequence, which are not
matched/aligned with a corresponding subject residue, as a percent
of the total bases of the query sequence. A determination of
whether a residue is matched/aligned is determined by results of
the FASTDB sequence alignment. This percentage is then subtracted
from the percent identity, calculated by the above FASTDB program
using the specified parameters, to arrive at a final percent
identity score. This final percent identity score is what is used
for the purposes of this embodiment. Only residues to the N- and
C-termini of the subject sequence, which are not matched/aligned
with the query sequence, are considered for the purposes of
manually adjusting the percent identity score. That is, only query
residue positions outside the farthest N- and C-terminal residues
of the subject sequence. For example, a 90 amino acid residue
subject sequence is aligned with a 100 residue query sequence to
determine percent identity. The deletion occurs at the N-terminus
of the subject sequence and therefore, the FASTDB alignment does
not show a matching/alignment of the first 10 residues at the
N-terminus. The 10 unpaired residues represent 10% of the sequence
(number of residues at the N- and C-termini not matched/total
number of residues in the query sequence) so 10% is subtracted from
the percent identity score calculated by the FASTDB program. If the
remaining 90 residues were perfectly matched the final percent
identity would be 90%. In another example, a 90 residue subject
sequence is compared with a 100 residue query sequence. This time
the deletions are internal deletions so there are no residues at
the N- or C-termini of the subject sequence which are not
matched/aligned with the query. In this case the percent identity
calculated by FASTDB is not manually corrected. Once again, only
residue positions outside the N- and C-terminal ends of the subject
sequence, as displayed in the FASTDB alignment, which are not
matched/aligned with the query sequence are manually corrected for.
No other manual corrections are made for the purposes of this
embodiment.
[0134] The polypeptide of the present invention could be used as a
molecular weight marker on SDS-PAGE gels or on molecular sieve gel
filtration columns using methods well known to those of skill in
the art.
[0135] As described in detail below, the polypeptides of the
present invention can also be used to raise polyclonal and
monoclonal antibodies, which are useful in assays for detecting
Ependymin protein expression as described below or as agonists and
antagonists capable of enhancing or inhibiting Ependymin protein
function. Further, such polypeptides can be used in the yeast
two-hybrid system to "capture" Ependymin protein binding proteins
which are also candidate agonists and antagonists according to the
present invention. The yeast two hybrid system is described by
Fields and Song (Nature 340:245-246 (1989)).
Epitope-Bearing Portions
[0136] In another aspect, the invention provides a peptide or
polypeptide comprising an epitope-bearing portion of a polypeptide
of the invention. The epitope of this polypeptide portion is an
immunogenic or antigenic epitope of a polypeptide of the invention.
An "immunogenic epitope" is defined as a part of a protein that
elicits an antibody response when the whole protein is the
immunogen. On the other hand, a region of a protein molecule to
which an antibody can bind is defined as an "antigenic epitope."
The number of immunogenic epitopes of a protein generally is less
than the number of antigenic epitopes (see, for instance, Geysen,
et al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983)).
[0137] As to the selection of peptides or polypeptides bearing an
antigenic epitope (i.e., that contain a region of a protein
molecule to which an antibody can bind), it is well known in that
art that relatively short synthetic peptides that mimic part of a
protein sequence are routinely capable of eliciting an antiserum
that reacts with the partially mimicked protein (see, for instance,
Sutcliffe, J. G., et al., Science 219:660-666 (1983)). Peptides
capable of eliciting protein-reactive sera are frequently
represented in the primary sequence of a protein, can be
characterized by a set of simple chemical rules, and are confined
neither to immunodominant regions of intact proteins (i.e.,
immunogenic epitopes) nor to the amino or carboxyl terminals.
Antigenic epitope-bearing peptides and polypeptides of the
invention are therefore useful to raise antibodies, including
monoclonal antibodies, that bind specifically to a polypeptide of
the invention (see, for instance, Wilson, et al., Cell 37:767-778
(1984)).
[0138] Antigenic epitope-bearing peptides and polypeptides of the
invention preferably contain a sequence of at least seven, more
preferably at least nine and most preferably between about 15 to
about 30 amino acids contained within the amino acid sequence of a
polypeptide of the invention. Non-limiting examples of antigenic
polypeptides or peptides that can be used to generate
Ependymin-specific antibodies include: a polypeptide comprising
amino acid residues from about Ala-1 to about Gln-9 in SEQ ID NO:2;
a polypeptide comprising amino acid residues from about Pro-8 to
about Val-16 in SEQ ID NO:2; a polypeptide comprising amino acid
residues from about Gln-19 to about Arg-27 in SEQ ID NO:2; a
polypeptide comprising amino acid residues from about Ile-69 to
about Ser-77 in SEQ ID NO:2; a polypeptide comprising amino acid
residues from about Asp-86 to about Glu-107 in SEQ ID NO:2; a
polypeptide comprising amino acid residues from about Glu-113 to
about Tyr-123 in SEQ ID NO:2; a polypeptide comprising amino acid
residues from about Thr-131 to about Gln-139 in SEQ ID NO:2; a
polypeptide comprising amino acid residues from about Leu-159 to
about Phe-167 in SEQ ID NO:2; and a polypeptide comprising amino
acid residues from about Leu-178 to about Ser-186 in SEQ ID NO:2.
These polypeptide fragments have been determined to bear antigenic
epitopes of the Ependymin protein by the analysis of the
Jameson-Wolf antigenic index, as shown in FIG. 4 and Table I,
above.
[0139] The epitope-bearing peptides and polypeptides of the
invention may be produced by any conventional means (see, for
example, Houghten, R. A., et al., Proc. Natl. Acad. Sci. USA
82:5131-5135 (1985); and U.S. Pat. No. 4,631,211 to Houghten, et
al. (1986)).
[0140] Epitope-bearing peptides and polypeptides of the invention
are used to induce antibodies according to methods well known in
the art (see, for instance, Sutcliffe, et al., supra; Wilson, et
al., supra; Chow, M., et al., Proc. Natl. Acad. Sci. USA
82:910-914; and Bittle, F. J., et al., J. Gen. Virol. 66:2347-2354
(1985)). Immunogenic epitope-bearing peptides of the invention,
i.e., those parts of a protein that elicit an antibody response
when the whole protein is the immunogen, are identified according
to methods known in the art (see, for instance, Geysen, et al.,
supra). Further still, U.S. Pat. No. 5,194,392, issued to Geysen,
describes a general method of detecting or determining the sequence
of monomers (amino acids or other compounds) which is a topological
equivalent of the epitope (i.e., a "mimotope") which is
complementary to a particular paratope (antigen binding site) of an
antibody of interest. More generally, U.S. Pat. No. 4,433,092,
issued to Geysen, describes a method of detecting or determining a
sequence of monomers which is a topographical equivalent of a
ligand which is complementary to the ligand binding site of a
particular receptor of interest. Similarly, U.S. Pat. No.
5,480,971, issued to Houghten and colleagues, on Peralkylated
Oligopeptide Mixtures discloses linear C1-C7-alkyl peralkylated
oligopeptides and sets and libraries of such peptides, as well as
methods for using such oligopeptide sets and libraries for
determining the sequence of a peralkylated oligopeptide that
preferentially binds to an acceptor molecule of interest. Thus,
non-peptide analogs of the epitope-bearing peptides of the
invention also can be made routinely by these methods.
Fusion Proteins
[0141] As one of skill in the art will appreciate, Ependymin
polypeptides of the present invention and the epitope-bearing
fragments thereof described above can be combined with parts of the
constant domain of immunoglobulins (IgG), resulting in chimeric
polypeptides. These fusion proteins facilitate purification and
show an increased half-life in vivo. This has been shown, e.g., for
chimeric proteins consisting of the first two domains of the human
CD4-polypeptide and various domains of the constant regions of the
heavy or light chains of mammalian immunoglobulins (EP A 394,827;
Traunecker, et al., Nature 331:84-86 (1988)). Fusion proteins that
have a disulfide-linked dimeric structure due to the IgG part can
also be more efficient in binding and neutralizing other molecules
than the monomeric Ependymin protein or protein fragment alone
(Fountoulakis, et al., J. Biochem. 270:3958-3964 (1995)).
Antibodies
[0142] Ependymin protein-specific antibodies for use in the present
invention can be raised against the intact Ependymin protein or an
antigenic polypeptide fragment thereof, which may be presented
together with a carrier protein, such as an albumin, to an animal
system (such as rabbit or mouse) or, if it is long enough (at least
about 25 amino acids), without a carrier.
[0143] As used herein, the term "antibody" (Ab) or "monoclonal
antibody" (Mab) is meant to include intact molecules as well as
antibody fragments (such as, for example, Fab and F(ab')2
fragments) which are capable of specifically binding to Ependymin
protein. Fab and F(ab')2 fragments lack the Fc fragment of intact
antibody, clear more rapidly from the circulation, and may have
less non-specific tissue binding of an intact antibody (Wahl, et
al., J. Nucl. Med. 24:316-325 (1983)). Thus, these fragments are
preferred.
[0144] The antibodies of the present invention may be prepared by
any of a variety of methods. For example, cells expressing the
Ependymin protein or an antigenic fragment thereof can be
administered to an animal in order to induce the production of sera
containing polyclonal antibodies. In a preferred method, a
preparation of Ependymin protein is prepared and purified to render
it substantially free of natural contaminants. Such a preparation
is then introduced into an animal in order to produce polyclonal
antisera of greater specific activity.
[0145] In the most preferred method, the antibodies of the present
invention are monoclonal antibodies (or Ependymin protein binding
fragments thereof). Such monoclonal antibodies can be prepared
using hybridoma technology (Kohler, et al., Nature 256:495 (1975);
Kohler, et al., Eur. J. Immunol. 6:511 (1976); Kohler, et al., Eur.
J. Immunol. 6:292 (1976); Hammerling, et al., in: Monoclonal
Antibodies and T-Cell Hybridomas, Elsevier, N.Y., (1981) pp.
563-681)). In general, such procedures involve immunizing an animal
(preferably a mouse) with a Ependymin protein antigen or, more
preferably, with a Ependymin protein-expressing cell. Suitable
cells can be recognized by their capacity to bind anti-Ependymin
protein antibody. Such cells may be cultured in any suitable tissue
culture medium; however, it is preferable to culture cells in
Earle's modified Eagle's medium supplemented with 10% fetal bovine
serum (inactivated at about 56.degree. C.), and supplemented with
about 10 .mu.l of nonessential amino acids, about 1,000 U/ml of
penicillin, and about 100 .mu.g/ml of streptomycin. The splenocytes
of such mice are extracted and fused with a suitable myeloma cell
line. Any suitable myeloma cell line may be employed in accordance
with the present invention; however, it is preferable to employ the
parent myeloma cell line (SP2O), available from the American Type
Culture Collection, Manassas, Va. After fusion, the resulting
hybridoma cells are selectively maintained in HAT medium, and then
cloned by limiting dilution as described by Wands and colleagues
(Gastroenterology 80:225-232 (1981)). The hybridoma cells obtained
through such a selection are then assayed to identify clones which
secrete antibodies capable of binding the Ependymin protein
antigen.
[0146] Alternatively, additional antibodies capable of binding to
the Ependymin protein antigen may be produced in a two-step
procedure through the use of anti-idiotypic antibodies. Such a
method makes use of the fact that antibodies are themselves
antigens, and that, therefore, it is possible to obtain an antibody
which binds to a second antibody. In accordance with this method,
Ependymin protein-specific antibodies are used to immunize an
animal, preferably a mouse. The splenocytes of such an animal are
then used to produce hybridoma cells, and the hybridoma cells are
screened to identify clones which produce an antibody whose ability
to bind to the Ependymin protein-specific antibody can be blocked
by the Ependymin protein antigen. Such antibodies comprise
anti-idiotypic antibodies to the Ependymin protein-specific
antibody and can be used to immunize an animal to induce formation
of further Ependymin protein-specific antibodies.
[0147] It will be appreciated that Fab and F(ab')2 and other
fragments of the antibodies of the present invention may be used
according to the methods disclosed herein. Such fragments are
typically produced by proteolytic cleavage, using enzymes such as
papain (to produce Fab fragments) or pepsin (to produce F(ab')2
fragments). Alternatively, Ependymin protein-binding fragments can
be produced through the application of recombinant DNA technology
or through synthetic chemistry.
[0148] For in vivo use of anti-Ependymin in humans, it may be
preferable to use "humanized" chimeric monoclonal antibodies. Such
antibodies can be produced using genetic constructs derived from
hybridoma cells producing the monoclonal antibodies described
above. Methods for producing chimeric antibodies are known in the
art (Morrison, Science 229:1202 (1985); Oi, et al., BioTechniques
4:214 (1986); Cabilly, et al., U.S. Pat. No. 4,816,567; Taniguchi,
et al., EP 171496; Morrison, et al., EP 173494; Neuberger, et al.,
WO 8601533; Robinson, et al., WO 8702671; Boulianne, et al., Nature
312:643 (1984); Neuberger, et al., Nature 314:268 (1985).
Nervous System-Related Disorders
Diagnosis
[0149] The present inventors have discovered that Ependymin is
expressed in primary dendritic cells, the KMH2 cell line, placenta,
fetal and adult liver, spinal cord, osteoclastoma, cerebellum,
synovial fibroblasts, 12 week old early stage human embryo, adrenal
gland tumor, whole brain, Hodgkin's Lymphoma tissue, macrophages,
HEL cell line, and chondrosarcoma. For a number of nervous
system-related disorders, substantially altered (increased or
decreased) levels of Ependymin gene expression can be detected in
nervous system tissue or other cells or bodily fluids (e.g., sera,
plasma, urine, synovial fluid or spinal fluid) taken from an
individual having such a disorder, relative to a "standard"
Ependymin gene expression level, that is, the Ependymin expression
level in nervous system tissues or bodily fluids from an individual
not having the nervous system disorder. Thus, the invention
provides a diagnostic method useful during diagnosis of a nervous
system disorder, which involves measuring the expression level of
the gene encoding the Ependymin protein in nervous system tissue or
other cells or body fluid from an individual and comparing the
measured gene expression level with a standard Ependymin gene
expression level, whereby an increase or decrease in the gene
expression level compared to the standard is indicative of an
nervous system disorder.
[0150] In particular, it is believed that certain tissues in
mammals with cancer of the nervous express significantly reduced
levels of the Ependymin protein and mRNA encoding the Ependymin
protein when compared to a corresponding "standard" level. Further,
it is believed that enhanced levels of the Ependymin protein can be
detected in certain body fluids (e.g., sera, plasma, urine, and
spinal fluid) from mammals with such a cancer when compared to sera
from mammals of the same species not having the cancer.
[0151] Thus, the invention provides a diagnostic method useful
during diagnosis of a nervous system disorder, including cancers of
this system, which involves measuring the expression level of the
gene encoding the Ependymin protein in nervous system tissue or
other cells or body fluid from an individual and comparing the
measured gene expression level with a standard Ependymin gene
expression level, whereby an increase or decrease in the gene
expression level compared to the standard is indicative of a
nervous system disorder.
[0152] Where a diagnosis of a disorder in the nervous system,
including diagnosis of a tumor, has already been made according to
conventional methods, the present invention is useful as a
prognostic indicator, whereby patients exhibiting depressed
Ependymin gene expression will experience a worse clinical outcome
relative to patients expressing the gene at a level nearer the
standard level.
[0153] By "assaying the expression level of the gene encoding the
Ependymin protein" is intended qualitatively or quantitatively
measuring or estimating the level of the Ependymin protein or the
level of the mRNA encoding the Ependymin protein in a first
biological sample either directly (e.g., by determining or
estimating absolute protein level or mRNA level) or relatively
(e.g., by comparing to the Ependymin protein level or mRNA level in
a second biological sample). Preferably, the Ependymin protein
level or mRNA level in the first biological sample is measured or
estimated and compared to a standard Ependymin protein level or
mRNA level, the standard being taken from a second biological
sample obtained from an individual not having the disorder or being
determined by averaging levels from a population of individuals not
having a disorder of the nervous system. As will be appreciated in
the art, once a standard Ependymin protein level or mRNA level is
known, it can be used repeatedly as a standard for comparison.
[0154] By "biological sample" is intended any biological sample
obtained from an individual, body fluid, cell line, tissue culture,
or other source which contains Ependymin protein or mRNA. As
indicated, biological samples include body fluids (such as sera,
plasma, urine, synovial fluid and spinal fluid) which contain free
Ependymin protein, nervous system tissue, and other tissue sources
found to express complete or mature Ependymin or a Ependymin
receptor. Methods for obtaining tissue biopsies and body fluids
from mammals are well known in the art. Where the biological sample
is to include mRNA, a tissue biopsy is the preferred source.
[0155] The present invention is useful for diagnosis or treatment
of various nervous system-related disorders in mammals, preferably
humans. Such disorders include any disregulations of nervous cell
function including, but not limited to, Parkinson's disease,
Alzheimer's disease, amyotrophic lateral sclerosis, pain, stroke,
depression, anxiety, epilepsy, other neurological and psychiatric
disorders, and the like.
[0156] Total cellular RNA can be isolated from a biological sample
using any suitable technique such as the single-step
guanidinium-thiocyanate-phenol-chloroform method described by
Chomczynski and Sacchi (Anal. Biochem. 162:156-159 (1987)). Levels
of mRNA encoding the Ependymin protein are then assayed using any
appropriate method. These include Northern blot analysis, S1
nuclease mapping, the polymerase chain reaction (PCR), reverse
transcription in combination with the polymerase chain reaction
(RT-PCR), and reverse transcription in combination with the ligase
chain reaction (RT-LCR).
[0157] Assaying Ependymin protein levels in a biological sample can
occur using antibody-based techniques. For example, Ependymin
protein expression in tissues can be studied with classical
immunohistological methods (Jalkanen, M., et al., J. Cell Biol.
101:976-985 (1985); Jalkanen, M., et al., J. Cell Biol.
105:3087-3096 (1987)). Other antibody-based methods useful for
detecting Ependymin protein gene expression include immunoassays,
such as the enzyme linked immunosorbent assay (ELISA) and the
radioimmunoassay (RIA). Suitable antibody assay labels are known in
the art and include enzyme labels, such as, glucose oxidase, and
radioisotopes, such as iodine (.sup.125I, .sup.121I), carbon
(.sup.14C), sulfur (.sup.35S), tritium (.sup.3H), indium
(.sup.112In), and technetium (.sup.99mTc), and fluorescent labels,
such as fluorescein and rhodamine, and biotin.
[0158] In addition to assaying Ependymin protein levels in a
biological sample obtained from an individual, Ependymin protein
can also be detected in vivo by imaging. Antibody labels or markers
for in vivo imaging of Ependymin protein include those detectable
by X-radiography, NMR or ESR. For X-radiography, suitable labels
include radioisotopes such as barium or cesium, which emit
detectable radiation but are not overtly harmful to the subject.
Suitable markers for NMR and ESR include those with a detectable
characteristic spin, such as deuterium, which may be incorporated
into the antibody by labeling of nutrients for the relevant
hybridoma.
[0159] A Ependymin protein-specific antibody or antibody fragment
which has been labeled with an appropriate detectable imaging
moiety, such as a radioisotope (for example, .sup.131I, .sup.112In,
.sup.99mTc), a radio-opaque substance, or a material detectable by
nuclear magnetic resonance, is introduced (for example,
parenterally, subcutaneously or intraperitoneally) into the mammal
to be examined for immune system disorder. It will be understood in
the art that the size of the subject and the imaging system used
will determine the quantity of imaging moiety needed to produce
diagnostic images. In the case of a radioisotope moiety, for a
human subject, the quantity of radioactivity injected will normally
range from about 5 to 20 millicuries of .sup.99mTc. The labeled
antibody or antibody fragment will then preferentially accumulate
at the location of cells which contain Ependymin protein. In vivo
tumor imaging is described by Burchiel and coworkers (Chapter 13 in
Tumor Imaging: The Radiochemical Detection of Cancer, Burchiel, S.
W. and Rhodes, B. A., eds., Masson Publishing Inc. (1982)).
Treatment
[0160] As noted above, Ependymin polynucleotides and polypeptides
are useful for diagnosis of conditions involving abnormally high or
low expression of Ependymin activities. Given the cells and tissues
where Ependymin is expressed as well as the activities modulated by
Ependymin, it is readily apparent that a substantially altered
(increased or decreased) level of expression of Ependymin in an
individual compared to the standard or "normal" level produces
pathological conditions related to the bodily system(s) in which
Ependymin is expressed and/or is active.
[0161] It is well-known in the art that, in addition to a specific
cellular function, cellular receptor molecules may also often be
exploited by a virus as a means of initiating entry into a
potential host cell. For example, it was recently discovered by Wu
and colleagues (J. Exp. Med. 185:1681-1691 (1997)) that the
cellular chemokine receptor CCR5 functions not only as a cellular
chemokine receptor, but also as a receptor for macrophage-tropic
human immunodeficiency virus (HIV)-1. In addition, RANTES, MIP-1a,
and MIP-1b, which are agonists for the cellular chemokine receptor
CCR5, inhibit entry of various strains of HIV-1 into susceptible
cell lines (Cocchi, F., et al., Science 270:1811-1815 (1995)).
Thus, the invention also provides a method of treating an
individual exposed to, or infected with, a virus through the
prophylactic or therapeutic administration of Ependymin, or an
agonist or antagonist thereof, to block or disrupt the interaction
of a viral particle with the Ependymin receptor and, as a result,
block the initiation or continuation of viral infectivity.
[0162] The Ependymin of the present invention binds to the
Ependymin receptor and, as such, is likely to block neurotropic
viral infections. Further, Ependymin expression is also observed in
many bone and cartilage tissues, and, as such, Ependymin is also
likely to block initiation of infectious cycle of many viruses
which infect bone or cartilage. More specifically, a non-limiting
list of viruses whose infectious life cycles may be altered by
Ependymin includes retroviruses such as HIV-1, HIV-2, human T-cell
lymphotropic virus (HTLV)-I, and HTLV-II, as well as other DNA and
RNA viruses including herpes simplex virus (HSV)-1, HSV-2, HSV-6,
cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes samirii,
adenoviruses, rhinoviruses, influenza viruses, reoviruses, and the
like.
[0163] The ability of Ependymin of the present invention, or
agonists or antagonists thereof, to prophylactically or
therapeutically block viral infection may be easily tested by the
skilled artisan. For example, Simmons and coworkers (Science
276:276-279 (1997)) and Arenzana-Seisdedos and colleagues (Nature
383:400 (1996)) each outline a method of observing suppression of
HIV-1 infection by an antagonist of the CCR5 chemokine receptor and
of the CC chemokine RANTES, respectively, in cultured peripheral
blood mononuclear cells. Cells are cultured and infected with a
virus, HIV-1 in both cases noted above. An agonist or antagonist of
the CC chemokine or its receptor is then immediately added to the
culture medium. Evidence of the ability of the agonist or
antagonist of the chemokine or cellular receptor is determined by
evaluating the relative success of viral infection at 3, 6, and 9
days postinfection.
[0164] Administration of a pharmaceutical composition comprising an
amount of an isolated Ependymin, or an agonist or antagonist
thereof, of the invention to an individual either infected with a
virus or at risk for infection with a virus is performed as
described below.
[0165] It will also be appreciated by one of ordinary skill that,
since the Ependymin protein of the invention is a member of the
ependymin polypeptide family, the mature secreted form of the
protein may be released in soluble form from the cells which
express the Ependymin by proteolytic cleavage. Therefore, when
Ependymin mature form is added from an exogenous source to cells,
tissues or the body of an individual, the protein will exert its
physiological activities on its target cells of that
individual.
[0166] Therefore, it will be appreciated that conditions caused by
a decrease in the standard or normal level of Ependymin activity in
an individual, particularly disorders of the nervous system, can be
treated by administration of Ependymin polypeptide (in the form of
the mature protein). Thus, the invention also provides a method of
treatment of an individual in need of an increased level of
Ependymin activity comprising administering to such an individual a
pharmaceutical composition comprising an amount of an isolated
Ependymin polypeptide of the invention, particularly a mature form
of the Ependymin protein of the invention, effective to increase
the Ependymin activity level in such an individual.
[0167] Ependymin is found bound to collagen fibrils in the
extracellular space of the mammalian leptomeninges. Ependymin, or
agonists or antagonists thereof, may thus be employed in the
treatment of ependymitis or other inflammation of the cellular
membrane lining the central canal of the spinal cord and the brain
ventricles. Ependymin is also found in collagen fibrils covering
the endothelial cells of numerous blood vessels. Similarly,
Ependymin, or agonists or antagonists thereof, may be used to
regulate angiogenesis, and other processes or disorders which
involve the formation, maintenance or disorganization of blood
vessels, and to treat a variety of cancers which involve the
formation of new blood vessels. A list of such cancers may include,
but is not limited by, breast cancer, colon cancer, cardiac tumors,
pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung
cancer, intestinal cancer, testicular cancer, stomach cancer,
neuroblastoma, myxoma, myoma, lymphoma, endothelioma,
osteoblastoma, osteoclastoma, adenoma, and the like.
[0168] Ependymins and other antiadhesive extracellular matrix
proteins are involved in the mechanism whereby meningeal cells
influence endfoot formation of Bergmann glial cells, which organize
the superficial glia limitans surrounding the CNS. This function is
essential in establishing accurate early development of the CNS
and, as such, Ependymin of the present invention, or agonists or
antagonists thereof, may be used to treat developmental disorders
of the CNS and the brain. Ependymin may also be used to treat
additional neurodegenerative disorders (such as Alzheimer's
disease, Parkinson's disease, Amyotrophic lateral sclerosis,
Retinitis pigmentosa, Cerebellar degeneration, and the like.
Further, Ependymin of the present invention, or agonists or
antagonists thereof, may be used to modulate long-term memory
consolidation.
[0169] Ependymin of the present invention, or agonists or
antagonists thereof, may be used to enhance the regeneration of the
optic or other nerves. Several scientific studies have revealed
that goldfish ependymin expression increases during optic nerve
regeneration and that anti-ependymin antibodies can prevent the
sharpening of the regenerating retinotectal projection (Schmidt, R.
and Shashoua, V. E. J. Neurochem. 36:1368-1377 (1988); Thomodsson,
F. R., et al., Exp. Neurol. 117:260-268 (1992)).
[0170] Ependymin of the present invention, or agonists or
antagonists thereof, may be used to treat disorders resulting from
axon ingrowth, for example, along blood vessels or into the meninx,
or prophylactically, to prevent undesired or incorrect neuronal
growth.
[0171] Ependymin of the present invention, or agonists or
antagonists thereof, may be used to treat disorders of the
blood-brain barrier since ependymin participates in the endothelial
cell barrier by modulating cell-matrix interactions.
Formulations
[0172] The Ependymin polypeptide composition will be formulated and
dosed in a fashion consistent with good medical practice, taking
into account the clinical condition of the individual patient
(especially the side effects of treatment with Ependymin
polypeptide alone), the site of delivery of the Ependymin
polypeptide composition, the method of administration, the
scheduling of administration, and other factors known to
practitioners. The "effective amount" of Ependymin polypeptide for
purposes herein is thus determined by such considerations.
[0173] As a general proposition, the total pharmaceutically
effective amount of Ependymin polypeptide administered parenterally
per dose will be in the range of about 1 .mu.g/kg/day to 10
mg/kg/day of patient body weight, although, as noted above, this
will be subject to therapeutic discretion. More preferably, this
dose is at least 0.01 mg/kg/day, and most preferably for humans
between about 0.01 and 1 mg/kg/day for the hormone. If given
continuously, the Ependymin polypeptide is typically administered
at a dose rate of about 1 .mu.g/kg/hour to about 50 .mu.g/kg/hour,
either by 1-4 injections per day or by continuous subcutaneous
infusions, for example, using a mini-pump. An intravenous bag
solution may also be employed. The length of treatment needed to
observe changes and the interval following treatment for responses
to occur appears to vary depending on the desired effect.
[0174] Pharmaceutical compositions containing the Ependymin of the
invention may be administered orally, rectally, parenterally,
intracistemally, intravaginally, intraperitoneally, topically (as
by powders, ointments, drops or transdermal patch), bucally, or as
an oral or nasal spray. By "pharmaceutically acceptable carrier" is
meant a non-toxic solid, semisolid or liquid filler, diluent,
encapsulating material or formulation auxiliary of any type. The
term "parenteral" as used herein refers to modes of administration
which include intravenous, intramuscular, intraperitoneal,
intrasternal, subcutaneous and intraarticular injection and
infusion.
[0175] The Ependymin polypeptide is also suitably administered by
sustained-release systems. Suitable examples of sustained-release
compositions include semi-permeable polymer matrices in the form of
shaped articles, e.g., films, or microcapsules. Sustained-release
matrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481),
copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman,
U., et al, Biopolymers 22:547-556 (1983)), poly (2-hydroxyethyl
methacrylate; Langer, R., et al., J. Biomed. Mater. Res. 15:167-277
(1981), and Langer, R., Chem. Tech. 12:98-105 (1982)), ethylene
vinyl acetate (Langer, R., et al., Id.) or
poly-D-(-)-3-hydroxybutyric acid (EP 133,988). Sustained-release
Ependymin polypeptide compositions also include liposomally
entrapped Ependymin polypeptide. Liposomes containing Ependymin
polypeptide are prepared by methods known in the art (DE 3,218,121;
Epstein, et al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985);
Hwang, et al., Proc. Natl. Acad. Sci. (USA) 77:4030-4034 (1980); EP
52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat.
Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP
102,324). Ordinarily, the liposomes are of the small (about 200-800
Angstroms) unilamellar type in which the lipid content is greater
than about 30 mol. percent cholesterol, the selected proportion
being adjusted for the optimal Ependymin polypeptide therapy.
[0176] For parenteral administration, in one embodiment, the
Ependymin polypeptide is formulated generally by mixing it at the
desired degree of purity, in a unit dosage injectable form
(solution, suspension, or emulsion), with a pharmaceutically
acceptable carrier, i.e., one that is non-toxic to recipients at
the dosages and concentrations employed and is compatible with
other ingredients of the formulation. For example, the formulation
preferably does not include oxidizing agents and other compounds
that are known to be deleterious to polypeptides.
[0177] Generally, the formulations are prepared by contacting the
Ependymin polypeptide uniformly and intimately with liquid carriers
or finely divided solid carriers or both. Then, if necessary, the
product is shaped into the desired formulation. Preferably the
carrier is a parenteral carrier, more preferably a solution that is
isotonic with the blood of the recipient. Examples of such carrier
vehicles include water, saline, Ringer's solution, and dextrose
solution. Non-aqueous vehicles such as fixed oils and ethyl oleate
are also useful herein, as well as liposomes.
[0178] The carrier suitably contains minor amounts of additives
such as substances that enhance isotonicity and chemical stability.
Such materials are non-toxic to recipients at the dosages and
concentrations employed, and include buffers such as phosphate,
citrate, succinate, acetic acid, and other organic acids or their
salts; antioxidants such as ascorbic acid; low molecular weight
(less than about ten residues) polypeptides, e.g., polyarginine or
tripeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids, such as glycine, glutamic acid, aspartic acid, or
arginine; monosaccharides, disaccharides, and other carbohydrates
including cellulose or its derivatives, glucose, mannose, or
dextrins; chelating agents such as EDTA; sugar alcohols such as
mannitol or sorbitol; counterions such as sodium; and/or nonionic
surfactants such as polysorbates, poloxamers, or PEG.
[0179] The Ependymin polypeptide is typically formulated in such
vehicles at a concentration of about 0.1 mg/ml to 100 mg/ml,
preferably 1-10 mg/ml, at a pH of about 3 to 8. It will be
understood that the use of certain of the foregoing excipients,
carriers, or stabilizers will result in the formation of Ependymin
polypeptide salts.
[0180] Ependymin polypeptide to be used for therapeutic
administration must be sterile. Sterility is readily accomplished
by filtration through sterile filtration membranes (e.g., 0.2
micron membranes). Therapeutic Ependymin polypeptide compositions
generally are placed into a container having a sterile access port,
for example, an intravenous solution bag or vial having a stopper
pierceable by a hypodermic injection needle.
[0181] Ependymin polypeptide ordinarily will be stored in unit or
multi-dose containers, for example, sealed ampoules or vials, as an
aqueous solution or as a lyophilized formulation for
reconstitution. As an example of a lyophilized formulation, 10-ml
vials are filled with 5 ml of sterile-filtered 1% (w/v) aqueous
Ependymin polypeptide solution, and the resulting mixture is
lyophilized. The infusion solution is prepared by reconstituting
the lyophilized Ependymin polypeptide using bacteriostatic
water-for-injection (WFI).
[0182] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions of the invention.
Associated with such container(s) can be a notice in the form
prescribed by a governmental agency regulating the manufacture, use
or sale of pharmaceuticals or biological products, which notice
reflects approval by the agency of manufacture, use or sale for
human administration. In addition, the polypeptides of the present
invention may be employed in conjunction with other therapeutic
compounds.
Agonists and Antagonists--Assays and Molecules
[0183] The invention also provides a method of screening compounds
to identify those which enhance or block the action of Ependymin on
cells, such as its interaction with Ependymin-binding molecules
such as receptor molecules. An agonist is a compound which
increases the natural biological functions of Ependymin or which
functions in a manner similar to Ependymin, while antagonists
decrease or eliminate such functions.
[0184] In another aspect of this embodiment the invention provides
a method for identifying a receptor protein or other ligand-binding
protein which binds specifically to a Ependymin polypeptide. For
example, a cellular compartment, such as a membrane or a
preparation thereof, may be prepared from a cell that expresses a
molecule that binds Ependymin. The preparation is incubated with
labeled Ependymin and complexes of Ependymin bound to the receptor
or other binding protein are isolated and characterized according
to routine methods known in the art. Alternatively, the Ependymin
polypeptide may be bound to a solid support so that binding
molecules solubilized from cells are bound to the column and then
eluted and characterized according to routine methods.
[0185] In the assay of the invention for agonists or antagonists, a
cellular compartment, such as a membrane or a preparation thereof,
may be prepared from a cell that expresses a molecule that binds
Ependymin, such as a molecule of a signaling or regulatory pathway
modulated by Ependymin. The preparation is incubated with labeled
Ependymin in the absence or the presence of a candidate molecule
which may be a Ependymin agonist or antagonist. The ability of the
candidate molecule to bind the binding molecule is reflected in
decreased binding of the labeled ligand. Molecules which bind
gratuitously, i.e., without inducing the effects of Ependymin on
binding the Ependymin binding molecule, are most likely to be good
antagonists. Molecules that bind well and elicit effects that are
the same as or closely related to Ependymin are agonists.
[0186] Ependymin-like effects of potential agonists and antagonists
may by measured, for instance, by determining activity of a second
messenger system following interaction of the candidate molecule
with a cell or appropriate cell preparation, and comparing the
effect with that of Ependymin or molecules that elicit the same
effects as Ependymin. Second messenger systems that may be useful
in this regard include but are not limited to AMP guanylate
cyclase, ion channel or phosphoinositide hydrolysis second
messenger systems.
[0187] Another example of an assay for Ependymin antagonists is a
competitive assay that combines Ependymin and a potential
antagonist with membrane-bound Ependymin receptor molecules or
recombinant Ependymin receptor molecules under appropriate
conditions for a competitive inhibition assay. Ependymin can be
labeled, such as by radioactivity, such that the number of
Ependymin molecules bound to a receptor molecule can be determined
accurately to assess the effectiveness of the potential
antagonist.
[0188] Potential antagonists include small organic molecules,
peptides, polypeptides and antibodies that bind to a polypeptide of
the invention and thereby inhibit or extinguish its activity.
Potential antagonists also may be small organic molecules, a
peptide, a polypeptide such as a closely related protein or
antibody that binds the same sites on a binding molecule, such as a
receptor molecule, without inducing Ependymin-induced activities,
thereby preventing the action of Ependymin by excluding Ependymin
from binding.
[0189] Other potential antagonists include antisense molecules.
Antisense technology can be used to control gene expression through
antisense DNA or RNA or through triple-helix formation. Antisense
techniques are discussed in a number of studies (for example,
Okano, J. Neurochem. 56:560 (1991); "Oligodeoxynucleotides as
Antisense Inhibitors of Gene Expression." CRC Press, Boca Raton,
Fla. (1988)). Triple helix formation is discussed in a number of
studies, as well (for instance, Lee, et al., Nucleic Acids Research
6:3073 (1979); Cooney, et al., Science 241:456 (1988); Dervan, et
al., Science 251:1360 (1991)). The methods are based on binding of
a polynucleotide to a complementary DNA or RNA. For example, the 5'
coding portion of a polynucleotide that encodes the mature
polypeptide of the present invention may be used to design an
antisense RNA oligonucleotide of from about 10 to 40 base pairs in
length. A DNA oligonucleotide is designed to be complementary to a
region of the gene involved in transcription thereby preventing
transcription and the production of Ependymin. The antisense RNA
oligonucleotide hybridizes to the mRNA in vivo and blocks
translation of the mRNA molecule into Ependymin polypeptide. The
oligonucleotides described above can also be delivered to cells
such that the antisense RNA or DNA may be expressed in vivo to
inhibit production of Ependymin protein.
[0190] The agonists and antagonists may be employed in a
composition with a pharmaceutically acceptable carrier, e.g., as
described above.
[0191] The antagonists may be employed for instance to inhibit the
formation of Ependymin-collagen fibrils which typically cover the
endothelial cells of numerous blood vessels. As a result,
anti-Ependymin antibodies may be used to regulate angiogenesis, and
other processes or disorders which involve the formation,
maintenance or disorganization of blood vessels, and to treat a
variety of cancers which involve the formation of new blood
vessels. Antibodies against Ependymin may also be employed to bind
to and inhibit Ependymin activity to treat Parkinson's disease,
Alzheimer's disease, amyotrophic lateral sclerosis, pain, stroke,
depression, anxiety, epilepsy, and other neurological and
psychiatric disorders. Any of the above antagonists may be employed
in a composition with a pharmaceutically acceptable carrier, e.g.,
as hereinafter described.
Gene Mapping
[0192] The nucleic acid molecules of the present invention are also
valuable for chromosome identification. The sequence is
specifically targeted to and can hybridize with a particular
location on an individual human chromosome. Moreover, there is a
current need for identifying particular sites on the chromosome.
Few chromosome marking reagents based on actual sequence data
(repeat polymorphisms) are presently available for marking
chromosomal location. The mapping of DNAs to chromosomes according
to the present invention is an important first step in correlating
those sequences with genes associated with disease.
[0193] In certain preferred embodiments in this regard, the cDNA
herein disclosed is used to clone genomic DNA of a Ependymin
protein gene. This can be accomplished using a variety of well
known techniques and libraries, which generally are available
commercially. The genomic DNA then is used for in situ chromosome
mapping using well known techniques for this purpose.
[0194] In addition, in some cases, sequences can be mapped to
chromosomes by preparing PCR primers (preferably 15-25 bp) from the
cDNA. Computer analysis of the 3' untranslated region of the gene
is used to rapidly select primers that do not span more than one
exon in the genomic DNA, thus complicating the amplification
process. These primers are then used for PCR screening of somatic
cell hybrids containing individual human chromosomes. Fluorescence
in situ hybridization ("FISH") of a cDNA clone to a metaphase
chromosomal spread can be used to provide a precise chromosomal
location in one step. This technique can be used with probes from
the cDNA as short as 50 or 60 bp (for a review of this technique,
see Verma, et al., Human Chromosomes: A Manual Of Basic Techniques,
Pergamon Press, New York (1988)).
[0195] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data. Such data are found, for
example, on the World Wide Web (McKusick, V. Mendelian Inheritance
In Man, available on-line through Johns Hopkins University, Welch
Medical Library). The relationship between genes and diseases that
have been mapped to the same chromosomal region are then identified
through linkage analysis (coinheritance of physically adjacent
genes).
[0196] Next, it is necessary to determine the differences in the
cDNA or genomic sequence between affected and unaffected
individuals. If a mutation is observed in some or all of the
affected individuals but not in any normal individuals, then the
mutation is likely to be the causative agent of the disease.
[0197] Having generally described the invention, the same will be
more readily understood by reference to the following examples,
which are provided by way of illustration and are not intended as
limiting.
EXAMPLES
Example 1(a)
Expression and Purification of "His-tagged" Ependymin in E.
coli
[0198] The novel pHE4 series of bacterial expression vectors, in
particular, the pHE4-5 vector may be used for bacterial expression
in this example. pHE4-5/MPIFD23 vector plasmid DNA contains an
insert which encodes another ORF. The construct was deposited with
the American Type Culture Collection, 10801 University Boulevard,
Manassas, Va. 20110-2209, on Sep. 30, 1997 and given Accession No.
209311. Using the Nde I and Asp 718 restriction sites flanking the
irrelevant MPIF ORF insert, the pHE4-5 is linearized and the MPIF
ORF is removed.
[0199] The pHE4-5 bacterial expression vector includes a neomycin
phosphotransferase gene for selection, an E. coli origin of
replication, a T5 phage promoter sequence, two lac operator
sequences, a Shine-Dalgarno sequence, and the lactose operon
repressor gene (lacIq). These elements are arranged such that an
inserted DNA fragment encoding a polypeptide expresses that
polypeptide with the six His residues (i.e., a "6.times.His tag")
covalently linked to the amino terminus of that polypeptide. The
promoter and operator sequences of the pHE4-5 vector were made
synthetically. Synthetic production of nucleic acid sequences is
well known in the art (CLONETECH 95/96 Catalog, pages 215-216,
CLONETECH, 1020 East Meadow Circle, Palo Alto, Calif. 94303).
[0200] The DNA sequence encoding the desired portion of the
Ependymin protein comprising the mature form of the Ependymin amino
acid sequence is amplified from the deposited cDNA clone using PCR
oligonucleotide primers which anneal to the amino terminal
sequences of the desired portion of the Ependymin protein and to
sequences in the deposited construct 3' to the cDNA coding
sequence. Additional nucleotides containing restriction sites to
facilitate cloning in the pHE4-5 vector are added to the 5' and 3'
primer sequences, respectively.
[0201] For cloning the mature form of the Ependymin protein, the 5'
primer has the sequence 5' GCG CAT ATG GCC CCG CGC CCG TGC 3' (SEQ
ID NO:8) containing the underlined Nde I restriction site followed
by 15 nucleotides of the amino terminal coding sequence of the
mature Ependymin sequence in SEQ ID NO:2. One of ordinary skill in
the art would appreciate, of course, that the point in the protein
coding sequence where the 5' primer begins may be varied to amplify
a DNA segment encoding any desired portion of the complete
Ependymin protein shorter or longer than the mature form of the
protein. The 3' primer has the sequence 5' GCG GGT ACC TCA CCA GGA
GCA GTC TTC GC 3' (SEQ ID NO:9) containing the underlined Asp 718
restriction site followed by 20 nucleotides complementary to the 3'
end of the coding sequence of the Ependymin DNA sequence in FIGS.
1A, 1B, and 1C.
[0202] The amplified Ependymin DNA fragment and the vector pHE4-5
are digested with Nde I and Asp 718 and the digested DNAs are then
ligated together. Insertion of the Ependymin DNA into the
restricted pHE4-5 vector places the Ependymin protein coding region
downstream from the IPTG-inducible promoter and in-frame with an
initiating AUG and the six histidine codons.
[0203] The ligation mixture is transformed into competent E. coli
cells using standard procedures such as those described by Sambrook
and colleagues (Molecular Cloning: a Laboratory Manual, 2nd Ed.;
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1989)). E. coli strain M15/rep4, containing multiple copies of the
plasmid pREP4, which expresses the lac repressor and confers
kanamycin resistance ("Kanr"), is used in carrying out the
illustrative example described herein. This strain, which is only
one of many that are suitable for expressing Ependymin protein, is
available commercially (QIAGEN, Inc., supra). Transformants are
identified by their ability to grow on LB plates in the presence of
ampicillin and kanamycin. Plasmid DNA is isolated from resistant
colonies and the identity of the cloned DNA confirmed by
restriction analysis, PCR and DNA sequencing.
[0204] Clones containing the desired constructs are grown overnight
("O/N") in liquid culture in LB media supplemented with both
ampicillin (100 .mu.g/ml) and kanamycin (25 .mu.g/ml). The O/N
culture is used to inoculate a large culture, at a dilution of
approximately 1:25 to 1:250. The cells are grown to an optical
density at 600 nm ("OD600") of between 0.4 and 0.6.
Isopropyl-.beta.-D-thiogalactopyranoside ("IPTG") is then added to
a final concentration of 1 mM to induce transcription from the lac
repressor sensitive promoter, by inactivating the lacI repressor.
Cells subsequently are incubated further for 3 to 4 hours. Cells
then are harvested by centrifugation.
[0205] The cells are then stirred for 3-4 hours at 4.degree. C. in
6M guanidine-HCl, pH 8. The cell debris is removed by
centrifugation, and the supernatant containing the Ependymin is
loaded onto a nickel-nitrilo-tri-acetic acid ("Ni-NTA") affinity
resin column (QIAGEN, Inc., supra). Proteins with a 6.times.His tag
bind to the Ni-NTA resin with high affinity and can be purified in
a simple one-step procedure (for details see: The QIAexpressionist,
1995, QIAGEN, Inc., supra). Briefly the supernatant is loaded onto
the column in 6 M guanidine-HCl, pH 8, the column is first washed
with 10 volumes of 6 M guanidine-HCl, pH 8, then washed with 10
volumes of 6 M guanidine-HCl pH 6, and finally the Ependymin is
eluted with 6 M guanidine-HCl, pH 5.
[0206] The purified protein is then renatured by dialyzing it
against phosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6
buffer plus 200 mM NaCl. Alternatively, the protein can be
successfully refolded while immobilized on the Ni-NTA column. The
recommended conditions are as follows: renature using a linear
6M-1M urea gradient in 500 mM NaCl, 20% glycerol, 20 mM Tris/HCl pH
7.4, containing protease inhibitors. The renaturation should be
performed over a period of 1.5 hours or more. After renaturation
the proteins can be eluted by the addition of 250 mM imidazole.
Imidazole is removed by a final dialyzing step against PBS or 50 mM
sodium acetate pH 6 buffer plus 200 mM NaCl. The purified protein
is stored at 4.degree. C. or frozen at -80.degree. C.
[0207] The following alternative method may be used to purify
Ependymin expressed in E coli when it is present in the form of
inclusion bodies. Unless otherwise specified, all of the following
steps are conducted at 4-10.degree. C.
[0208] Upon completion of the production phase of the E. coli
fermentation, the cell culture is cooled to 4-10.degree. C. and the
cells are harvested by continuous centrifugation at 15,000 rpm
(Heraeus Sepatech). On the basis of the expected yield of protein
per unit weight of cell paste and the amount of purified protein
required, an appropriate amount of cell paste, by weight, is
suspended in a buffer solution containing 100 mM Tris, 50 mM EDTA,
pH 7.4. The cells are dispersed to a homogeneous suspension using a
high shear mixer.
[0209] The cells ware then lysed by passing the solution through a
microfluidizer (Microfluidics, Corp. or APV Gaulin, Inc.) twice at
4000-6000 psi. The homogenate is then mixed with NaCl solution to a
final concentration of 0.5 M NaCl, followed by centrifugation at
7000.times.g for 15 min. The resultant pellet is washed again using
0.5M NaCl, 100 mM Tris, 50 mM EDTA, pH 7.4.
[0210] The resulting washed inclusion bodies are solubilized with
1.5 M guanidine hydrochloride (GuHCl) for 2-4 hours. After
7000.times.g centrifugation for 15 min., the pellet is discarded
and the Ependymin polypeptide-containing supernatant is incubated
at 4.degree. C. overnight to allow further GuHCl extraction.
[0211] Following high speed centrifugation (30,000.times.g) to
remove insoluble particles, the GuHCl solubilized protein is
refolded by quickly mixing the GuHCl extract with 20 volumes of
buffer containing 50 mM sodium, pH 4.5, 150 mM NaCl, 2 mM EDTA by
vigorous stirring. The refolded diluted protein solution is kept at
4.degree. C. without mixing for 12 hours prior to further
purification steps.
[0212] To clarify the refolded Ependymin polypeptide solution, a
previously prepared tangential filtration unit equipped with 0.16
.mu.m membrane filter with appropriate surface area (e.g.,
Filtron), equilibrated with 40 mM sodium acetate, pH 6.0 is
employed. The filtered sample is loaded onto a cation exchange
resin (e.g., Poros HS-50, Perseptive Biosystems). The column is
washed with 40 mM sodium acetate, pH 6.0 and eluted with 250 mM,
500 mM, 1000 mM, and 1500 mM NaCl in the same buffer, in a stepwise
manner. The absorbance at 280 mm of the effluent is continuously
monitored. Fractions are collected and further analyzed by
SDS-PAGE.
[0213] Fractions containing the Ependymin polypeptide are then
pooled and mixed with 4 volumes of water. The diluted sample is
then loaded onto a previously prepared set of tandem columns of
strong anion (Poros HQ-50, Perseptive Biosystems) and weak anion
(Poros CM-20, Perseptive Biosystems) exchange resins. The columns
are equilibrated with 40 mM sodium acetate, pH 6.0. Both columns
are washed with 40 mM sodium acetate, pH 6.0, 200 mM NaCl. The
CM-20 column is then eluted using a 10 column volume linear
gradient ranging from 0.2 M NaCl, 50 mM sodium acetate, pH 6.0 to
1.0 M NaCl, 50 mM sodium acetate, pH 6.5. Fractions are collected
under constant A.sub.280 monitoring of the effluent. Fractions
containing the Ependymin polypeptide (determined, for instance, by
16% SDS-PAGE) are then pooled.
[0214] The resultant Ependymin polypeptide exhibits greater than
95% purity after the above refolding and purification steps. No
major contaminant bands are observed from Commassie blue stained
16% SDS-PAGE gel when 5 .mu.g of purified protein is loaded. The
purified protein is also tested for endotoxin/LPS contamination,
and typically the LPS content is less than 0.1 ng/ml according to
LAL assays.
Example 2
Cloning and Expression of Ependymin Protein in a Baculovirus
Expression System
[0215] In this illustrative example, the plasmid shuttle vector pA2
is used to insert the cloned DNA encoding complete protein,
including its naturally associated secretory signal (leader)
sequence, into a baculovirus to express the mature Ependymin
protein, using standard methods as described by Summers and
colleagues (A Manual of Methods for Baculovirus Vectors and Insect
Cell Culture Procedures, Texas Agricultural Experimental Station
Bulletin No. 1555 (1987)). This expression vector contains the
strong polyhedrin promoter of the Autographa californica nuclear
polyhedrosis virus (AcMNPV) followed by convenient restriction
sites such as Bam HI, Xba I and Asp 718. The polyadenylation site
of the simian virus 40 ("SV40") is used for efficient
polyadenylation. For easy selection of recombinant virus, the
plasmid contains the beta-galactosidase gene from E. coli under
control of a weak Drosophila promoter in the same orientation,
followed by the polyadenylation signal of the polyhedrin gene. The
inserted genes are flanked on both sides by viral sequences for
cell-mediated homologous recombination with wild-type viral DNA to
generate a viable virus that express the cloned polynucleotide.
[0216] Many other baculovirus vectors could be used in place of the
vector above, such as pAc373, pVL941 and pAcIM1, as one skilled in
the art would readily appreciate, as long as the construct provides
appropriately located signals for transcription, translation,
secretion and the like, including a signal peptide and an in-frame
AUG as required. Such vectors are described, for instance, by
Luckow and coworkers (Virology 170:31-39 (1989)).
[0217] The cDNA sequence encoding the full length Ependymin protein
in the deposited clone, including the AUG initiation codon and the
naturally associated leader sequence shown in SEQ ID NO:2, is
amplified using PCR oligonucleotide primers corresponding to the 5'
and 3' sequences of the gene. The 5' primer has the sequence 5' GAT
CGC TCT AGA TCC GCC ACC ATG CCA GGA CGC GCT CCC CTC CGC ACC GTC 3'
(SEQ ID NO:10) containing the underlined Xba I restriction enzyme
site, an efficient signal for initiation of translation in
eukaryotic cells (Kozak, M., J. Mol. Biol. 196:947-950 (1987)),
followed by 27 of nucleotides of the sequence of the complete
Ependymin protein shown in FIGS. 1A, 1B, and 1C, beginning with the
AUG initiation codon. The 3' primer has the sequence 5' GAT CGC GGT
ACC TTA TCA CCA GGA GCA GTC TTC GCT CAT CTT CTC CAG 3' (SEQ ID
NO:11) containing the underlined Asp 718 restriction site followed
by 30 nucleotides complementary to the 3' noncoding sequence in
FIGS. 1A, 1B, and 1C.
[0218] The amplified fragment is isolated from a 1% agarose gel
using a commercially available kit ("GENECLEAN.TM.," BIO 101 Inc.,
La Jolla, Calif.). The fragment then is digested with Xba I and Asp
718 and again is purified on a 1% agarose gel. This fragment is
designated herein F1.
[0219] The plasmid is digested with the restriction enzymes Xba I
and Asp 718 and optionally, can be dephosphorylated using calf
intestinal phosphatase, using routine procedures known in the art.
The DNA is then isolated from a 1% agarose gel using a commercially
available kit ("GENECLEAN.TM." BIO 101 Inc., La Jolla, Calif.).
This vector DNA is designated herein "V1".
[0220] Fragment F1 and the dephosphorylated plasmid V1 are ligated
together with T4 DNA ligase. E. coli HB101 or other suitable E.
coli hosts such as XL-1 Blue (STRATAGENE.TM. Cloning Systems, La
Jolla, Calif.) cells are transformed with the ligation mixture and
spread on culture plates. Bacteria are identified that contain the
plasmid with the human Ependymin gene by digesting DNA from
individual colonies using Xba I and Asp 718 and then analyzing the
digestion product by gel electrophoresis. The sequence of the
cloned fragment is confirmed by DNA sequencing. This plasmid is
designated herein pA2Ependymin.
[0221] Five .mu.g of the plasmid pA2Ependymin is co-transfected
with 1.0 .mu.g of a commercially available linearized baculovirus
DNA ("BaculoGod.TM. baculovirus DNA", Pharmingen, San Diego,
Calif.), using the lipofection method described by Felgner and
colleagues (Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987)). One
.mu.g of BaculoGold.TM. virus DNA and 5 .mu.g of the plasmid
pA2Ependymin are mixed in a sterile well of a microtiter plate
containing 50 .mu.l of serum-free Grace's medium (Life Technologies
Inc., Frederick, Md.). Afterwards, 10 .mu.l LIPOFECTIN.TM. plus 90
.mu.l Grace's medium are added, mixed and incubated for 15 minutes
at room temperature. Then the transfection mixture is added
drop-wise to Sf9 insect cells (ATCC.TM. CRL 1711) seeded in a 35 mm
tissue culture plate with 1 ml Grace's medium without serum. The
plate is then incubated for 5 hours at 27.degree. C. The
transfection solution is then removed from the plate and 1 ml of
Grace's insect medium supplemented with 10% fetal calf serum is
added. Cultivation is then continued at 27.degree. C. for four
days.
[0222] After four days the supernatant is collected and a plaque
assay is performed, as described by Summers and Smith (supra). An
agarose gel with "Blue Gal" (Life Technologies Inc., Frederick,
Md.) is used to allow easy identification and isolation of
gal-expressing clones, which produce blue-stained plaques. (A
detailed description of a "plaque assay" of this type can also be
found in the user's guide for insect cell culture and
baculovirology distributed by Life Technologies Inc., Frederick,
Md., page 9-10). After appropriate incubation, blue stained plaques
are picked with the tip of a micropipettor (e.g., Eppendorf). The
agar containing the recombinant viruses is then resuspended in a
microcentrifuge tube containing 200 .mu.l of Grace's medium and the
suspension containing the recombinant baculovirus is used to infect
Sf9 cells seeded in 35 mm dishes. Four days later the supernatants
of these culture dishes are harvested and then they are stored at
4.degree. C. The recombinant virus is called V-Ependymin.
[0223] To verify the expression of the Ependymin gene Sf9 cells are
grown in Grace's medium supplemented with 10% heat-inactivated FBS.
The cells are infected with the recombinant baculovirus V-Ependymin
at a multiplicity of infection ("MOI") of about 2. If radiolabeled
proteins are desired, 6 hours later the medium is removed and is
replaced with SF900 II medium minus methionine and cysteine
(available from Life Technologies Inc., Frederick, Md.). After 42
hours, 5 .mu.Ci of .sup.35S-methionine and 5 .mu.Ci
.sup.35S-cysteine (available from Amersham) are added. The cells
are further incubated for 16 hours and then are harvested by
centrifugation. The proteins in the supernatant as well as the
intracellular proteins are analyzed by SDS-PAGE followed by
autoradiography (if radiolabeled).
[0224] Microsequencing of the amino acid sequence of the amino
terminus of purified protein may be used to determine the amino
terminal sequence of the mature form of the Ependymin protein and
thus the cleavage point and length of the naturally associated
secretory signal peptide.
Example 3
Cloning and Expression of Ependymin in Mammalian Cells
[0225] A typical mammalian expression vector contains the promoter
element, which mediates the initiation of transcription of mRNA,
the protein coding sequence, and signals required for the
termination of transcription and polyadenylation of the transcript.
Additional elements include enhancers, Kozak sequences and
intervening sequences flanked by donor and acceptor sites for RNA
splicing. Highly efficient transcription can be achieved with the
early and late promoters from SV40, the long terminal repeats
(LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI and the early
promoter of the cytomegalovirus (CMV). However, cellular elements
can also be used (e.g., the human actin promoter). Suitable
expression vectors for use in practicing the present invention
include, for example, vectors such as pSVL and pMSG (Pharmacia,
Uppsala, Sweden), pRSVcat (ATCC.TM. 37152), pSV2dhfr (ATCC.TM.
37146) and pBC12MI (ATCC.TM. 67109). Mammalian host cells that
could be used include, human Hela, 293, H9 and Jurkat cells, mouse
NIH3T3 and C127 cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells,
mouse L cells and Chinese hamster ovary (CHO) cells.
[0226] Alternatively, the gene can be expressed in stable cell
lines that contain the gene integrated into a chromosome. The
co-transfection with a selectable marker such as dhfr, gpt,
neomycin, hygromycin allows the identification and isolation of the
transfected cells.
[0227] The transfected gene can also be amplified to express large
amounts of the encoded protein. The DHFR (dihydrofolate reductase)
marker is useful to develop cell lines that carry several hundred
or even several thousand copies of the gene of interest. Another
useful selection marker is the enzyme glutamine synthase (GS;
Murphy, et al., Biochem J. 227:277-279 (1991); Bebbington, et al.,
Bio/Technology 10:169-175 (1992)). Using these markers, the
mammalian cells are grown in selective medium and the cells with
the highest resistance are selected. These cell lines contain the
amplified gene(s) integrated into a chromosome. Chinese hamster
ovary (CHO) and NSO cells are often used for the production of
proteins.
[0228] The expression vectors pC1 and pC4 contain the strong
promoter (LTR) of the Rous Sarcoma Virus (Cullen, et al., Mol. Cel.
Biol. 5:438-447 (1985)) plus a fragment of the CMV-enhancer
(Boshart, et al., Cell 41:521-530 (1985)). Multiple cloning sites,
e.g., with the restriction enzyme cleavage sites Bam HI, Xba I and
Asp 718, facilitate the cloning of the gene of interest. The
vectors contain in addition the 3' intron, the polyadenylation and
termination signal of the rat preproinsulin gene.
Example 3(a)
Cloning and Expression in COS Cells
[0229] The expression plasmid, pEpendyminHA, is made by cloning a
portion of the cDNA encoding the complete Ependymin protein into
the expression vector pcDNAI/Amp or pcDNAIII (which can be obtained
from Invitrogen, Inc.).
[0230] The expression vector pcDNAI/amp contains: (1) an E. coli
origin of replication effective for propagation in E. coli and
other prokaryotic cells; (2) an ampicillin resistance gene for
selection of plasmid-containing prokaryotic cells; (3) an SV40
origin of replication for propagation in eukaryotic cells; (4) a
CMV promoter, a polylinker, an SV40 intron; (5) several codons
encoding a hemagglutinin fragment (i.e., an "HA" tag to facilitate
purification) followed by a termination codon and polyadenylation
signal arranged so that a cDNA can be conveniently placed under
expression control of the CMV promoter and operably linked to the
SV40 intron and the polyadenylation signal by means of restriction
sites in the polylinker. The HA tag corresponds to an epitope
derived from the influenza hemagglutinin protein described by
Wilson and colleagues (Cell 37:767 (1984)). The fusion of the HA
tag to the target protein allows easy detection and recovery of the
recombinant protein with an antibody that recognizes the HA
epitope. pcDNAIII contains, in addition, the selectable neomycin
marker.
[0231] A DNA fragment encoding the complete Ependymin polypeptide
is cloned into the polylinker region of the vector so that
recombinant protein expression is directed by the CMV promoter. The
plasmid construction strategy is as follows. The Ependymin cDNA of
the deposited clone is amplified using primers that contain
convenient restriction sites, much as described above for
construction of vectors for expression of Ependymin in E. coli.
Suitable primers include the following, which are used in this
example. The 5' primer, containing the underlined Asp 718 site, a
Kozak sequence, an AUG start codon, a sequence, and 24 nucleotides
of the 5' coding region of the complete Ependymin polypeptide, has
the following sequence: 5' GAT CGC GGT ACC GCC ATC ATG CCA GGA CGC
GCT CCC CTC CGC 3' (SEQ ID NO:12). The 3' primer, containing the
underlined Bam HI and 20 of nucleotides complementary to the 3'
coding sequence immediately before the stop codon, has the
following sequence: 5' GAT CGC GGA TCC TCA CCA GGA GCA GTC TTC GC
3' (SEQ ID NO:13).
[0232] The PCR amplified DNA fragment and the vector, pcDNAI/Amp,
are digested with Asp 718 and Bam HI and then ligated. The ligation
mixture is transformed into E. coli strain SURE (STRATAGENE.TM.
Cloning Systems, La Jolla, Calif. 92037), and the transformed
culture is plated on ampicillin media plates which then are
incubated to allow growth of ampicillin resistant colonies. Plasmid
DNA is isolated from resistant colonies and examined by restriction
analysis or other means for the presence of the fragment encoding
the complete Ependymin polypeptide.
[0233] For expression of recombinant Ependymin, COS cells are
transfected with an expression vector, as described above, using
DEAE-dextran, as described, for instance, by Sambrook and coworkers
(Molecular Cloning: a Laboratory Manual, Cold Spring Laboratory
Press, Cold Spring Harbor, N.Y. (1989)). Cells are incubated under
conditions for expression of Ependymin by the vector.
[0234] Expression of the Ependymin-HA fusion protein is detected by
radiolabeling and immunoprecipitation, using methods described in,
for example Harlow and colleagues (Antibodies: A Laboratory Manual,
2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y. (1988)). To this end, two days after transfection, the cells
are labeled by incubation in media containing .sup.35S-cysteine for
8 hours. The cells and the media are collected, and the cells are
washed and the lysed with detergent-containing RIPA buffer: 150 mM
NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5,
as described by Wilson and colleagues (supra). Proteins are
precipitated from the cell lysate and from the culture media using
an HA-specific monoclonal antibody. The precipitated proteins then
are analyzed by SDS-PAGE and autoradiography. An expression product
of the expected size is seen in the cell lysate, which is not seen
in negative controls.
Example 3(b)
Cloning and Expression in CHO Cells
[0235] The vector pC4 is used for the expression of Ependymin
polypeptide. Plasmid pC4 is a derivative of the plasmid pSV2-dhfr
(ATCC.TM. Accession No. 37146). The plasmid contains the mouse DHFR
gene under control of the SV40 early promoter. Chinese hamster
ovary--or other cells lacking dihydrofolate activity that are
transfected with these plasmids can be selected by growing the
cells in a selective medium (alpha minus MEM, Life
Technologies.TM.) supplemented with the chemotherapeutic agent
methotrexate. The amplification of the DHFR genes in cells
resistant to methotrexate (MTX) has been well documented (see,
e.g., Alt, F. W., et al., J. Biol. Chem. 253:1357-1370 (1978);
Hamlin, J. L. and Ma, C. Biochem. et Biophys. Acta, 1097:107-143
(1990); Page, M. J. and Sydenham, M. A. Biotechnology 9:64-68
(1991)). Cells grown in increasing concentrations of MTX develop
resistance to the drug by overproducing the target enzyme, DHFR, as
a result of amplification of the DHFR gene. If a second gene is
linked to the DHFR gene, it is usually co-amplified and
over-expressed. It is known in the art that this approach may be
used to develop cell lines carrying more than 1,000 copies of the
amplified gene(s). Subsequently, when the methotrexate is
withdrawn, cell lines are obtained which contain the amplified gene
integrated into one or more chromosome(s) of the host cell.
[0236] Plasmid pC4 contains for expressing the gene of interest the
strong promoter of the long terminal repeat (LTR) of the Rouse
Sarcoma Virus (Cullen, et al., Mol. Cell. Biol. 5:438-447 (1985))
plus a fragment isolated from the enhancer of the immediate early
gene of human cytomegalovirus (CMV; Boshart, et al., Cell
41:521-530 (1985)). Downstream of the promoter are the following
single restriction enzyme cleavage sites that allow the integration
of the genes: Bam HI, Xba I, and Asp 718. Behind these cloning
sites the plasmid contains the 3' intron and polyadenylation site
of the rat preproinsulin gene. Other high efficiency promoters can
also be used for the expression, e.g., the human .beta.-actin
promoter, the SV40 early or late promoters or the long terminal
repeats from other retroviruses, e.g., HIV and HTLVI. Clontech's
Tet-Off and Tet-On gene expression systems and similar systems can
be used to express the Ependymin polypeptide in a regulated way in
mammalian cells (Gossen, M., and Bujard, H. Proc. Natl. Acad. Sci.
USA 89:5547-5551 (1992)). For the polyadenylation of the mRNA other
signals, e.g., from the human growth hormone or globin genes can be
used as well. Stable cell lines carrying a gene of interest
integrated into the chromosomes can also be selected upon
co-transfection with a selectable marker such as gpt, G418 or
hygromycin. It is advantageous to use more than one selectable
marker in the beginning, e.g., G418 plus methotrexate.
[0237] The plasmid pC4 is digested with the restriction enzymes Xba
I and Asp 718 and then dephosphorylated using calf intestinal
phosphates by procedures known in the art. The vector is then
isolated from a 1% agarose gel.
[0238] The DNA sequence encoding the complete Ependymin polypeptide
is amplified using PCR oligonucleotide primers corresponding to the
5' and 3' sequences of the desired portion of the gene. The 5'
primer containing the underlined Xba I site, a Kozak sequence (in
italics), an AUG start codon, and 30 nucleotides of the 5' coding
region of the complete Ependymin polypeptide, has the following
sequence: 5' GAT CGC TCT AGA TCC GCC ACC ATG CCA GGA CGC GCT CCC
CTC CGC ACC GTC 3' (SEQ ID NO:14). The 3' primer, containing the
underlined Asp 718 and 30 of nucleotides complementary to the 3'
coding sequence immediately before the stop codon as shown in FIGS.
1A, 1B, and 1C (SEQ ID NO:1), has the sequence shown in the above
example as SEQ ID NO:11.
[0239] The amplified fragment is digested with the endonucleases
Xba I and Asp 718 and then purified again on a 1% agarose gel. The
isolated fragment and the dephosphorylated vector are then ligated
with T4 DNA ligase. E. coli HB101 or XL-1 Blue cells are then
transformed and bacteria are identified that contain the fragment
inserted into plasmid pC4 using, for instance, restriction enzyme
analysis.
[0240] Chinese hamster ovary cells lacking an active DHFR gene are
used for transfection. Five .mu.g of the expression plasmid pC4 is
cotransfected with 0.5 .mu.g of the plasmid pSVneo using
LIPOFECTIN.TM. (Felgner, et al., supra). The plasmid pSV2-neo
contains a dominant selectable marker, the neo gene from Tn5
encoding an enzyme that confers resistance to a group of
antibiotics including G418. The cells are seeded in alpha minus MEM
supplemented with 1 mg/ml G418. After 2 days, the cells are
trypsinized and seeded in hybridoma cloning plates (Greiner,
Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/ml
of metothrexate plus 1 mg/ml G418. After about 10-14 days single
clones are trypsinized and then seeded in 6-well petri dishes or 10
ml flasks using different concentrations of methotrexate (50 nM,
100 nM, 200 nM, 400 nM, 800 nM). Clones growing at the highest
concentrations of methotrexate are then transferred to new 6-well
plates containing even higher concentrations of methotrexate (1
.mu.M, 2 .mu.M, 5 .mu.M, 10 mM, 20 mM). The same procedure is
repeated until clones are obtained which grow at a concentration of
100-200 .mu.M. Expression of the desired gene product is analyzed,
for instance, by SDS-PAGE and Western blot or by reversed phase
HPLC analysis.
Example 4
Tissue Distribution of Ependymin mRNA Expression
[0241] Northern blot analysis is carried out to examine Ependymin
gene expression in human tissues, using methods described by, among
others, Sambrook and colleagues (supra). A cDNA probe containing
the entire nucleotide sequence of the Ependymin protein (SEQ ID
NO:1) is labeled with .sup.32P using the Rediprime.TM. DNA labeling
system (Amersham Life Science), according to manufacturer's
instructions. After labeling, the probe is purified using a CHROMA
SPIN-100.TM. column (Clontech Laboratories, Inc.), according to
manufacturer's protocol number PT1200-1. The purified labeled probe
is then used to examine various human tissues for Ependymin
mRNA.
[0242] Multiple Tissue Northern (MTN) blots containing various
human tissues (H) or human immune system tissues (IM) are obtained
from CLONTECH.TM. and are examined with the labeled probe using
ExpressHyb.TM. hybridization solution (CLONTECH.TM.) according to
manufacturer's protocol number PT1190-1. Following hybridization
and washing, the blots are mounted and exposed to film at
-70.degree. C. overnight, and films developed according to standard
procedures.
[0243] It will be clear that the invention may be practiced
otherwise than as particularly described in the foregoing
description and examples. Numerous modifications and variations of
the present invention are possible in light of the above teachings
and, therefore, are within the scope of the appended claims.
[0244] The entire disclosure of all publications (including
patents, patent applications, journal articles, laboratory manuals,
books, or other documents) cited herein are hereby incorporated by
reference.
[0245] Further, the Sequence Listing submitted herewith, and the
Sequence Listing submitted with U.S. application Ser. No.
09/229,583, filed on Jan. 13, 1999, the Sequence Listing submitted
with U.S. Provisional Application Ser. No. 60/071,330, filed on
Jan. 14, 1998, and the Sequence Listing submitted with U.S.
Provisional Application Ser. No. 60/075,278, filed on Feb. 19, 1998
(to both of which the present application claims benefit of the
filing date under 35 U.S.C. .sctn. 119(e)), in both computer and
paper forms are hereby incorporated by reference in their
entireties.
* * * * *