Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Glycoprotein – e.g. – mucins – proteoglycans – etc.
Reexamination Certificate
2002-07-03
2004-01-27
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Glycoprotein, e.g., mucins, proteoglycans, etc.
C530S350000, C530S857000, C514S008100, C435S069100, C435S070100
Reexamination Certificate
active
06683161
ABSTRACT:
FIELD OF THE INVENTION
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.
The invention further relates to screening methods for identifying agonists and antagonists of Ependymin activity.
BACKGROUND OF THE INVENTION
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.
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 clupeiforn 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).
Several lines of evidence have provided the basis for an understanding of the functional role(s) of the ependymins. Ca
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 Hoffmnan, supra). As a result, it is thought that ependymins may function in Ca
2+
homeostasis of the teleost piscine brain (Hoffman, W., supra).
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)).
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)).
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
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 Deposit Number 209464 on Nov. 14, 1997. The nucleotide sequence determined by sequencing the deposited Ependymin clone, which is shown in
FIGS. 1A
,
1
B, and
1
C (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 Deposit Number 209464, which molecules also can encode additional amino acids fused to the N-terminus of the Ependymin amino acid sequence.
The encoded polypeptide has a predicted leader sequence of 37 amino acids underlined in
FIGS. 1A
,
1
B, and
1
C; and the amino acid sequence of the predicted mature Ependymin protein is also shown in
FIGS. 1A
,
1
B, and
1
C, as amino acid residues 38-224 and as residues 1-187 in SEQ ID NO:2.
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
Ebner Reinhard
Ruben Steven M.
Human Genome Sciences Inc.
Human Genome Sciences Inc.
Low Christopher S. F.
Mitra Rita
LandOfFree
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