HK1117870A

HK1117870A - Neurotrophic factors

Info

Publication number: HK1117870A
Application number: HK08108333.1A
Authority: HK
Inventors: T‧E‧约翰森; N‧布洛姆; C‧汉森
Original assignee: 恩斯吉恩有限公司
Priority date: 1998-07-06
Filing date: 2008-07-28
Publication date: 2009-01-23

Description

Neurotrophic factors

The present application is a divisional application of an invention patent application having an application number of 99808289.9 and an application date of 5.7.1999, and entitled "neurotrophic factor".

Background

The present invention relates to neurotrophic factor polypeptides, nucleic acids encoding the neurotrophic factor polypeptides, and antibodies that specifically bind to neurotrophic factors.

Background

Neurotrophic factors are natural proteins that promote the survival of neuronal cells and tissues, maintain their phenotypic differentiation, prevent their degeneration and enhance their activity. Neurotrophic factors are isolated from neural tissue and non-neural tissue innervated by the nervous system and divided into functionally and structurally related groups, also known as families, superfamilies or subfamilies. The neurotrophic factor superfamily includes the fibroblast growth factor, neurotrophin and transforming growth factor-beta (TGF-beta) superfamily. Various neurotrophic factors can be distinguished by their physical structure, interaction with their complex receptors and their effect on various types of nerve cells. Classified in the TGF- β superfamily (Massague et al, Trends in Cell Biology, 1994, 4172-178) are glial Cell line-derived neurotrophic factor ligands ("GDNF"; WO93/06116, incorporated herein by reference) including GDNF, persephin ("PSP"; Milbrandt et al, neurons, 199820245-253, incorporated herein by reference) and neurturin ("NTN"; WO97/08196, incorporated herein by reference). Ligands of the GDNF subfamily are all capable of inducing signaling through RET receptor tyrosine kinases. The three ligands of the GDNF subfamily differ in their relative affinities for the neurotrophic receptor family, the GFR receptor.

Given the effects of neurotrophic factors on neuronal tissue, there remains a need to identify and characterize other neurotrophic factors for use in diagnosing and treating neurological diseases.

Brief description of the invention

The present invention relates to a novel neurotrophic factor, referred to herein as "neublastin", or "NBN". neublastin belongs to a member of the GDNF subfamily because it shares regions of homology with other GDNF ligands (see tables 3 and 4 below) and is able to interact with RET (see, for example, Airaksen et al, mol. cell. neuroscience, 199913313-325), which is a novel, unique neurotrophic factor. Unlike other GDNF ligands, neublastin exhibits high affinity for the GFR α 3-RET receptor complex and has a unique subregion in its amino acid sequence.

As used herein, "neublastin polypeptide" is a polypeptide having neurotrophic activity (as described in examples 6, 7, 8 and 9), which includes polypeptides having an amino acid sequence with at least 70% homology to the human "neublastin" polypeptide shown in SEQ ID NO: 2 AA_-95-AA₁₀₅SEQ ID NO: 2 AA₁-AA₁₀₅SEQ ID NO: AA of 4_-97-AA₁₄₀SEQ ID NO: AA of 4_-41-AA₁₄₀(pro), SEQ ID NO: AA of 4₁-AA₁₄₀SEQ ID NO: 9 AA_-80-AA₁₄₀("wild-type" prepro), SEQ ID NO: 9 AA_-41-AA₁₄₀(pro), SEQ ID NO: 5 AA₁-AA₁₄₀(mature 140AA), SEQ ID NO: AA of 6₁-AA₁₁₆(mature 116AA), seq id NO: AA of 7₁-AA₁₁₃(mature 113AA), SEQ ID NO: 10 AA₁-AA₁₄₀(mature 140AA), SEQ ID NO: 11 AA₁-AA₁₁₆(mature 116AA), SEQ ID NO: 12 AA₁-AA₁₁₃(mature 113 AA). In addition, the invention also includes polypeptides having an amino acid sequence that has at least 70% homology to a murine "neublastin" polypeptide as set forth in SEQ ID NO: AA of 16₁-AA₂₂₄。

The C-terminal sequence of the neublastin polypeptide identified above preferably has the sequence of SEQ id no: 2 AA₇₂-AA₁₀₅(i.e., AA of SEQ ID NO: 9)₁₀₇-AA₁₄₀) More preferably the amino acid sequence shown in SEQ ID NO: 2 AA₄₁-AA₁₀₅(i.e., AA of SEQ ID NO: 9)₇₆-AA₁₄₀) The amino acid sequence shown, or SEQ ID NO: AA of 16₁₉₁-AA₂₂₄The amino acid sequence shown.

In addition, it is preferred that neublastin polypeptides retain 7 conserved Cys residues, which are residues unique to the GDNF family and TGF- β superfamily.

Preferably, the neublastin polypeptide has a sequence identical to the sequence described above and SEQ ID NO: AA of 16₁-AA₂₂₄And most preferably greater than 95%, of the amino acid sequence of SEQ ID NO: 2 AA_-95-AA₁₀₅SEQ ID NO: 2 AA₁-AA₁₀₅And, SEQ ID NO: AA of 4_-97-AA₁₄₀，SEQ IDNO: AA of 4_-41-AA₁₄₀SEQ ID NO: AA of 4₁-AA₁₄₀SEQ ID NO: 9 AA_-80-AA₁₄₀("wild-type" prepro), SEQ id no: 9 AA_-41-AA₁₄₀(pro), SEQ ID NO: 5 AA₁-AA₁₄₀(mature 140AA), SEQ ID NO: AA of 6₁-AA₁₁₆(mature 116AA), SEQ ID NO: AA of 7₁-AA₁₁₃(mature 113AA), SEQ ID NO: 10 AA₁-AA₁₄₀(mature 140AA), SEQ ID NO: 11 AA₁-AA₁₁₆(mature 116AA), SEQ ID NO: 12 AA₁-AA₁₁₃(mature 113 AA).

As used herein, a "neublastin nucleic acid" is a polynucleotide that encodes a neublastin polypeptide. Thus, an isolated neublastin nucleic acid is a polynucleotide molecule having an open reading frame of nucleotide codons that, when exposed to the appropriate components required for translation, encodes a neublastin polypeptide. The neublastin nucleic acids of the invention may be RNA or DNA, such as genomic DNA, or DNA complementary to and/or transcribed from neublastin mRNA ("cDNA"). Accordingly, neublastin nucleic acids of the invention also include polynucleotide molecules that specifically hybridize under highly stringent hybridization conditions to polynucleotides encoding neublastin polypeptides. The invention also relates to nucleic acid primers, or fragments thereof, which can be used to identify, isolate and amplify polynucleotides encoding neublastin polypeptides. In certain embodiments of the invention, certain primers are neublastin-specific probes that can be used to hybridize to neublastin nucleic acids, but not to nucleic acids encoding other members of the GDNF family. "specific", "specificity" or "specifically" refers to the ability to hybridize to neublastin nucleic acid, but not to non-neublastin nucleic acid, including the ability to hybridize to unique nucleic acids encoding GDNF ligands (e.g., GDNF, persephin and neurturin).

In another embodiment, a neublastin nucleic acid of the invention is identified as a nucleic acid complementary to a polynucleotide encoding a neublastin polypeptide by having a complementary nucleic acid sequence, or demonstrating that it specifically hybridizes to a polynucleotide encoding neublastin under high stringency hybridization conditions. Specific neublastin nucleic acids include, but are not limited to, the nucleic acid sequences set forth herein and SEQ ID NOs: 1, SEQ ID NO: 3, SEQ ID NO: 8, SEQ ID NO: 13, SEQ id no: 14, SEQ ID NO: 15, SEQ ID NO: 29 and SEQ ID NO: 30 and primer seq id NO: 17-28, 31 and 32. The neublastin nucleic acids of the invention also include subregions or fragments unique to neublastin nucleic acids, including but not limited to the nucleic acid fragments shown in figure 8.

Neublastin polypeptides can be expressed using a neublastin nucleic acid of the invention, for example, by expressing a neublastin polypeptide in vitro, or by administering a neublastin nucleic acid to an animal for expression in vivo. neublastin nucleic acids may be included in a nucleic acid vector, such as an expression vector or a cloning vector. neublastin nucleic acids may, but need not, be maintained, replicated, transferred or expressed as part of a nucleic acid vector. Recombinant expression vectors containing neublastin polynucleotide sequences can be introduced into and/or maintained in cells. The host cell for the neublastin vector may be a prokaryotic cell. Alternatively, the neublastin nucleic acid may be introduced into a eukaryotic cell, such as a eukaryotic cell containing the appropriate elements responsible for post-translational processing of the polypeptide into a mature protein and/or for secretion of the polypeptide into the extracellular environment of the cell.

The invention also relates to neublastin neurotrophic factor neublastin. The neublastin may be in the form of a polypeptide, or a multimer of two or more neublastin polypeptides, such as a neublastin dimer. neublastin polypeptides are joined into multimers by intermolecular structural linkages well known to those skilled in the art, including, but not limited to, cysteine-cysteine interactions, sulfhydryl linkages, and non-covalent interactions. Particular neublastin polypeptides include, but are not limited to, the amino acid sequences disclosed herein and the amino acid sequences of SEQ ID NOs: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 16.

The neublastin polypeptides of the invention are useful for treating neuronal defects, including but not limited to damaged neurons and trauma neurons. Traumatic peripheral nerves include, but are not limited to, the myelinated or spinal nerves. neublastin polypeptides are useful for treating neurodegenerative diseases, such as cerebral ischemic neuronal injury; neurological disorders, such as peripheral neuropathy, Alzheimer's disease, Huntington's disease, Parkinson's disease, Amyotrophic Lateral Sclerosis (ALS). neublastin polypeptides may also be used to treat memory impairment, such as that associated with dementia.

Brief Description of Drawings

FIG. 1 is a schematic representation of³²Photographs of 2 Northern blots of P-labeled neublastin cDNA hybridizations comparing the relative expression levels of neublastin gene in multiple adult tissue types (a series) and multiple adult brain regions (B series).

FIG. 2 is a graph of³²A photograph of a Northern blot of P-labeled neublastin cDNA hybridization comparing the expression of neublastin cDNA in untransfected cell line HiB5, neublastin cDNA transfected cell line and GDNF-cDNA transfected cell line.

FIG. 3 is a photograph of 2 Western blots hybridized with neublastin-specific antibody Ab-2 (left blot; sequence A) or with neublastin-specific antibody Ab-1 (right blot; sequence B) comparing the expression levels of neublastin protein in untransfected HiB5 cells (lane 1) and a HiB5 cell line stably transfected with neublastin cDNA (lane 2).

Figure 4 is a graph showing the effect of neublastin on survival of cultured rat embryonic neurons, dopaminergic neurons, ventral midbrain neurons, and ChAT activity on cholinergic cranial neuromotor neurons in serum-free medium. In particular, FIG. 4A illustrates a dose-response curve for the effect of recombinant GDNF on ChAT activity (dpm/hr). FIG. 4B illustrates ChAT activity (dpm/hr) using diluted conditioned medium from neublastin-producing or GDNF-producing cells. Figure 4C illustrates the number of tyrosine hydroxylase immunoreactive cells per well.

FIG. 5 illustrates the effect of neublastin secreted by HiB5pUbi1zNBN22 cells on the function and survival of slice cultures of brain neurons in porcine embryonic dopaminergic ventral cerebellum co-cultured with HiB5pUbi1zNBN22 cells (neublastin) or HiB5 cells (control). Fig. 5A and 5B illustrate release of dopamine into the medium in DIV12[ dopamine (pmol/ml) -day 12] and DIV21[ dopamine (pmol/ml) -day 21], respectively. FIG. 5C illustrates the number of tyrosine hydroxylase immunoreactive cells [ TH-ir cells/culture ] in DIV21 slice cultures.

Figure 6 illustrates the effect of lentivirus-produced neublastin on nigral dopamine neurons in vivo.

Fig. 7 illustrates the genomic structure of a neublastin gene, including nucleic acid primers that can be used to identify the full-length neublastin gene and its spatial orientation relative to the genomic sequence (i.e., gene) encoding neublastin.

Figure 8 illustrates neublastin specific primers used to identify cDNA clones encoding human neublastin polypeptides that hybridize to nucleic acids encoding neublastin polypeptides, but not to nucleic acids encoding other known GDNF family members (i.e., GDNF, persephin, and neurturin).

Figure 9 illustrates the neurotrophic activity of polypeptides of the invention on dissociated rat lateral root ganglion cell cultures from different developmental stages compared to known neurotrophic factors [ 0: control experiment (lack of factor); 1: the presence of GDNF; 2: neurturin is present; 3: the presence of neublastin of the invention; e12: day 12 of the embryo; e16: day 16 of the embryo; p0: the day of birth; p7: postnatal day 7; p15: day 15 after birth ].

FIG. 10 shows the neublastin production by CHO cell lines.

FIG. 11 compares binding of neublastin and GDNF to GFR α -1 and GFR α -3 receptors.

FIG. 12 is a photograph of a Western blot showing the binding of R30 anti-peptide antibody and R31 anti-peptide antibody to neublastin.

FIG. 13 is a photograph of a gel showing extraction of neublastin by affinity binding to RETL 3-Ig.

FIG. 14 is a plasmid map of pET19b-Neublastin and the sequence of the synthetic gene of Neublastin.

FIG. 15 is a plasmid map of pMJB164-HisNeublastin and the sequence of HisNeublastin synthetic gene.

Detailed Description

Applicants have identified nucleic acids encoding novel neurotrophic factors (referred to herein as "neublastin" or "NBN"). neublastin belongs to the transforming growth factor-beta (TGF- β) superfamily of neurotrophic factors and is a member of the neurotrophic factor (GDNF) subclass among them derived from glial cell lines.

The cDNA encoding neublastin was initially identified as follows. Using the TBLASTN1.4.11 algorithm rule (Atschul et al, nucleic acids research, 1997, 253389-. AC005038 consists of 5 non-sequential sequence contigs of about 190,000bp, and AC005051 consists of 12 non-sequential sequence contigs of about 132,000 bp. The 290bp fragment identified in the 2 BAC clones was confirmed to have a region homologous but not identical to the cDNA coding region of the neurotrophic factor, human persephin.

2 Neublastin-specific PCR primers (an upper strand primer [ SEQ ID NO: 17] and a lower strand primer [ SEQ ID NO: 18]) were synthesized from the 290bp sequence. Screening of human fetal brain cDNA libraries, the initial screening included the use of 2 PCR primers [ SEQ ID NO: 17 and 18], screening a cDNA library "master plate" based on 96-well PCR, which consists of 500,000 cDNA clones containing about 5,000 clones per well. A second PCR-based screen was performed on a human fetal brain cDNA library "secondary plate" containing approximately 5,000 cloned E.coli glycerol stocks per well.

A 102bp fragment was identified when the primary and secondary plates were screened based on PCR [ SEQ id no: 13], positive cDNA clones (with 102bp fragments) were selected, plated on 2 plates containing LB/antibiotic and cultured overnight. A total of 96 bacterial colonies were selected from these plates and placed individually in wells of a new 96-well PCR plate, each well containing two PCR primers [ SEQ ID NO: 17 and 18] and the necessary PCR amplification reagents. PCR amplification was then performed and 96 independent PCR reactions were analyzed by 2% agarose gel electrophoresis. Positive colonies with clones containing 102bp fragments were then identified. Plasmid DNA was obtained from positive colonies containing the 102bp fragment and sequenced. Subsequent sequencing analysis revealed 861bp of full-length cDNA [ SEQ id no: 8] in the presence of a catalyst. In SEQ ID NO: the 663bp Open Reading Frame (ORF) or coding region (CDS) identified in 8 encodes a pre-pro-polypeptide (referred to as "pre-Neublastin pro") shown in SEQ id no: 9. according to SEQ ID NO: 9, 3 Neublastin polypeptide variants were identified, including:

(i) a 140AA polypeptide, referred to herein as NBN140, having the amino acid sequence of SEQ ID NO: 10;

(ii) a 116AA polypeptide, referred to herein as NBN116, having the amino acid sequence of SEQ ID NO: 11; and

(iii) a 113AA polypeptide, referred to herein as NBN113, having the amino acid sequence of SEQ ID NO: 12.

The complete cDNA sequence containing 782bp 5 'untranslated DNA, 663bp coding DNA and 447bp 3' untranslated DNA (1992 bp in total) has been presented to GenBank under accession number AF 120274.

The Neublastin coding sequence in the genome was identified as follows:

to clone the neublastin coding sequence in the genome, another set of primers was prepared, specifically including primer pair No. 1[ sense-SEQ ID NO: 23, antisense ═ SEQ ID NO: 24] and primer pair No. 2[ sense ═ SEQ ID NO: 25, antisense ═ SEQ ID NO: 26].

An 887bp DNA fragment was amplified from the human genomic DNA preparation by PCR using primer pair No. 2 and cloned into pCRII vector (Invitrogen) and then transformed into E.coli. The resulting plasmid was sequenced and a 861bp putative cDNA sequence (encoding a protein referred to herein as neublastin) was deduced (as shown in SEQ ID NO: 3). Similarly, 870bp DNA fragment was obtained by PCR of human genomic DNA using primer set No. 1. A42 bp region at the 3' end of the Open Reading Frame (ORF) was found in this fragment, which is additional to the 887bp sequence. The genomic structure of the neublastin gene can be deduced by comparing its nucleic acid sequences with those of other neurotrophic factors and mapping the exon-intron border sequences. This analysis demonstrated that the neublastin gene has at least two exons separated by a 70bp intron.

In addition, this sequence was used to screen for neublastin EST sequences in GenBank. 3 such sequences were identified with GenBank accession numbers AA844072, AA931637 and AA533512, indicating that neublastin nucleic acid is transcribed into mRNA.

The complete cDNA sequence obtained (AF120274) was compared with the genomic sequences of GenBank accession nos. AC005038 and AC005051, and the results showed that the neublastin gene consisted of at least 5 exons (including 3 coding exons) separated by 4 introns (see, for example, fig. 8). In summary, the exons have the deduced amino acid sequence of the full-length Neublastin polypeptide. It should also be noted that the 887bp fragment was found to contain the entire pro-neublastin coding region. Putative cDNA [ SEQ ID NO: 3] contains an Open Reading Frame (ORF) (181 amino acid residues) encoding pro-neublastin, which shows homology to 3 known human proteins-Persephin, Neurturin and GDNF.

Neublastin nucleic acids of the invention

In another aspect, the invention provides polynucleotides capable of expressing a polypeptide of the invention. Polynucleotides of the invention include DNA, cDNA and RNA sequences as well as antisense sequences, and include natural, synthetic and artificially engineered polynucleotides. The polynucleotides of the invention also include degenerate sequences resulting from the genetic code, but which still encode a neublastin polypeptide.

The term "polynucleotide" as defined herein refers to a polymeric form of nucleotides of at least 10 bases in length, preferably at least 15 bases in length. An "isolated polynucleotide" refers to a polynucleotide that is not immediately contiguous with both coding sequences, but is immediately contiguous with both coding sequences (one at the 5 'end and one at the 3' end) in the natural genome of the organism from which the polynucleotide is isolated. Thus, the term includes recombinant DNA which may be incorporated into an expression vector, into a self-replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which may be a separate molecule, such as a cDNA which is independent of other sequences.

The polynucleotides of the invention also include allelic variants and "mutant polynucleotides" having a nucleotide sequence that differs from the nucleotide sequences provided herein at one or more nucleotide positions.

In a preferred embodiment, the polynucleotide of the invention has a nucleic acid (DNA) sequence that hybridizes under at least moderate, moderate/high, or high stringency conditions as described in detail below with the nucleic acid sequence of seq id NO: 1, SEQ ID NO: 3, SEQ ID NO: 8, or SEQ ID NO: 15, its complementary strand, or a subsequence thereof.

In another preferred embodiment, the isolated polynucleotide of the invention has a nucleic acid (DNA) sequence that is identical to the sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 8, or SEQ id no: 15, preferably at least 80%, more preferably at least 90%, most preferably at least 95% homologous thereto.

In a most preferred embodiment, the polynucleotide has the sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 8, or the DNA sequence of SEQ ID NO: 15, or a polynucleotide sequence as set forth in seq id no.

The invention also provides novel primers and DNA sequences useful for identifying, isolating and amplifying neublastin polynucleotides encoding neublastin polypeptides or fragments thereof. The primer comprises SEQ ID NO: 17-28 and 31-32. In addition, the invention also provides neublastin DNA sequences generated by the primers, including SEQ ID NO: 13 and 14. In addition, the invention provides DNA sequences derived from the 3 'or 5' untranslated region ("UTR") in genomic DNA that flank the neublastin exon; the sequence can be used for identifying, separating and amplifying neublastin polynucleotide for coding neublastin polypeptide or its fragment.

The 3' UTR sequences of the present invention include the following:

SEQ ID NO: 1, nucleotide 721-865 (SEQ ID NO),

SEQ ID NO: nucleotide 718-861 of 3,

SEQ ID NO: nucleotide 718-861 of 8 (I),

SEQ ID NO: 15, and nucleotide 1647-2136, and

contiguous sequences from (i.e., contained within) 10 to 25 nucleotides in length in the above sequences (useful as primers).

The 5' UTR sequences of the present invention include the following:

SEQ ID NO: 1 from 1 to 10 nucleotides of (A),

SEQ ID NO: 8 from nucleotide 1 to nucleotide 57 of seq id no,

SEQ ID NO: 15 nucleotides 1-974, and

The polynucleotide of the present invention is preferably obtained by a cloning method, for example, "the latest molecular biology method" [ John Wiley & Sons Co.). In a preferred embodiment, the polynucleotides may be cloned from or generated on the basis of a human genomic DNA or cDNA library of the human brain.

Homology of DNA sequences

The DNA sequence homology referred to above can be determined as the degree of identity between two sequences, showing the difference between the first and second sequence. Homology may suitably be determined using computer programs known in the art, including, for example, GAP provided in the GCG package [ Needleman, S.B and Wunsch c.d., journal of molecular biology 1970, 48: 443-453]. DNA sequence comparisons were performed using GAPs with the following settings: GAP creation penalty of 5.0, GAP extension penalty of 0.3, and the analogous DNA sequence coding region mentioned above in association with SEQ ID NO: 1, or the CDS (coding) portion of the DNA sequence shown in SEQ id no: 3, or the CDS (coding) portion of the DNA sequence shown in SEQ ID NO: 8, or the CDS (coding) portion of the DNA sequence shown in SEQ ID NO: 15 preferably has a degree of identity of at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%.

The term "sequence identity" refers to the degree to which two polynucleotide sequences are identical on a nucleotide-by-nucleotide basis within a particular region of comparison. The term "percentage of sequence identity" is calculated by comparing two sequences that are most satisfactorily aligned within a region of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions within the region of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The term "substantially identical" as used herein refers to a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence having at least 80% sequence identity, preferably at least 85% identity, often 90 to 95% sequence identity, more often at least 99% sequence identity, to a reference sequence over a region of comparison.

Hybridization method

The polynucleotides of the invention have a nucleic acid sequence that hybridizes under at least moderate, moderate/high, or high stringency conditions as described in detail below with the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 8, or SEQ ID NO: 15, or its complementary strand, or a subsequence thereof.

Suitable experimental conditions for determining hybridization between a nucleotide probe and a homologous DNA or RNA sequence include: in advance in 5 XSSC [ sodium chloride/sodium citrate; see Sambrook et al; molecular cloning: a laboratory Manual, Cold spring harbor laboratory, Cold spring harbor, N.Y. 1989]The filters containing the DNA fragments or RNA to be hybridized are soaked for 10 minutes in 5 XSSC, 5 XDenhardt's solution [ see Sambrook et al, supra]0.5% SDS and 100. mu.g/ml denatured, sonicated salmon sperm DNA [ see Sambrook et al, supra]The prehybridization filter was neutralized, followed by hybridization at about 45 ℃ for 12 hours in the same solution containing 10ng/ml of probe, which was randomly-primed [ Feinberg A P ]&Vogelstein B; analytical biochemistry, 1983, 132, 6-13]And run through³²P-dCTP-tag (specific Activity > 1X 10)⁹cpm/. mu.g) of probe. The filters are then washed 2 times in 0.1 XSSC, 0.5% SDS at a temperature of at least 60 ℃ (medium stringency conditions), preferably at least 65 ℃ (medium/high stringency conditions), more preferably at least 70 ℃ (high stringency conditions), even more preferably at least 75 ℃ (very high stringency conditions) for up to 30 minutes. Molecules that hybridize to the oligonucleotide probe under these conditions can be detected using X-ray film.

Cloned polynucleotides

In particular, the isolated polynucleotide of the present invention may be a cloned polynucleotide. The term "cloned polynucleotide" as defined herein refers to a polynucleotide or DNA sequence cloned according to standard cloning methods most recently used in the field of genetic engineering, which can displace a DNA segment, in particular a cDNA, i.e.a cDNA produced from an RNA by the action of an enzyme, from its natural position to a different site where it can be regenerated.

Cloning can be accomplished by any suitable route, including, for example, reverse transcriptase techniques, PCR techniques, and the like, as well as excision and isolation of the desired DNA segment.

The cloned polynucleotides of the invention may also be referred to as "DNA constructs" or "isolated DNA sequences", in particular complementary DNA (cdna).

Biological source

The isolated polynucleotides of the invention may be obtained from any suitable source.

In a preferred embodiment, the polynucleotides of the invention are cloned from or generated on the basis of cDNA libraries comprising cDNA libraries obtained from, e.g., fetal or adult brain, especially forebrain, hindbrain, cortex, striatum, amygdala, cerebellum, caudate nucleus, corpus callosum, hippocampus, thalamic nucleus, subthalamic nucleus, bromonucleus, lenticular nucleus, substantia nigra, lateral root ganglion, trigeminal ganglion, superior mesenteric artery or thalamus; the spinal cord; a heart; a placenta; a lung; a liver; skeletal muscle; the kidney; a pancreas; a bowel; an eye; a retina; dental pulp; a hair follicle; the prostate; a pituitary; or the trachea.

Commercially available cDNA libraries from a variety of human and non-human tissues are available, for example, from Stratagene and Clontech. The isolated polynucleotides of the invention can be obtained by standard methods, such as those described in the working examples.

The neublastin polypeptide of the invention

As mentioned above, the term "neublastin polypeptide" as used herein is intended to meanNeurotrophically active polypeptides (as described in examples 6, 7, 8 and 9) including polypeptides having an amino acid sequence which has at least 70% homology to a "neublastin" polypeptide shown in SEQ ID NO: 2 AA_-95-AA₁₀₅SEQ ID NO: 2 AA₁-AA₁₀₅SEQ ID NO: AA of 4_-97-AA₁₄₀SEQ ID NO: AA of 4_-41-AA₁₄₀SEQ ID NO: AA of 4₁-AA₁₄₀SEQ ID NO: 9 AA_-80-AA₁₄₀("wild-type" prepro), SEQ ID NO: 9 AA_-41-AA₁₄₀(pro), SEQ ID NO: 5 AA₁-AA₁₄₀(mature 140AA), SEQ ID NO: AA of 6₁-AA₁₁₆(mature 116AA), SEQ ID NO: AA of 7₁-AA₁₁₃(mature 113AA), SEQ ID NO: 10 AA₁-AA₁₄₀(mature 140AA), SEQ ID NO: 11 AA₁-AA₁₁₆(mature 116AA), SEQ ID NO: 12 AA₁-AA₁₁₃(mature 113AA), SEQ ID NO: AA of 16₁-AA₂₂₄(murine prepro).

The C-terminal sequence of the neublastin polypeptide identified above preferably has the sequence of SEQ id no: 2 AA₇₂-AA₁₀₅(i.e., AA of SEQ ID NO: 9)₁₀₇-AA₁₄₀) More preferably the amino acid sequence shown in SEQ ID NO: 2 AA₄₁-AA₁₀₅(i.e., AA of SEQ ID NO: 9)₇₆-AA₁₄₀) The amino acid sequence shown.

Preferably, the neublastin polypeptide has an amino acid sequence with greater than 85%, most preferably greater than 95% homology to the sequence set forth above, i.e., SEQ ID NO: 2 AA_-95-AA₁₀₅SEQ ID NO: 2 AA₁-AA₁₀₅SEQ ID NO: AA of 4_-97-AA₁₄₀SEQ ID NO: AA of 4_-41-AA₁₄₀SEQ ID NO: AA of 4₁-AA₁₄₀SEQ ID NO: 9 AA_-80-AA₁₄₀("wild-type" prepro), SEQ ID NO: 9 AA_-41-AA₁₄₀(pro), SEQ ID NO: 5 AA₁-AA₁₄₀(mature 140AA), SEQ ID NO: AA of 6₁-AA₁₁₆(mature 116AA), SEQ ID NO: AA of 7₁-AA₁₁₃(mature 113AA), SEQ ID NO: 10 AA₁-AA₁₄₀(mature 140AA), SEQ ID NO: 11 AA₁-AA₁₁₆(mature 116AA), SEQ ID NO: 12 AA₁-AA₁₁₃(mature 113AA), SEQ ID NO: AA of 16₁-AA₂₂₄(murine prepro) any of the above polypeptides having the C-terminal sequence of the neublastin polypeptide identified above preferably has the amino acid sequence of SEQ ID NO: 2 AA₇₂-AA₁₀₅(i.e., AA of SEQ ID NO: 9)₁₀₇-AA₁₄₀) More preferably the amino acid sequence shown in SEQ ID NO: 2 AA₄₁-AA₁₀₅(i.e., AA of SEQ ID NO: 9)₇₆-AA₁₄₀) Or SEQ ID NO: AA of 16₁₉₁-AA₂₂₄The amino acid sequence shown.

In addition, the invention includes polypeptides having an amino acid sequence with at least 70% homology to a murine "neublastin" polypeptide as set forth in SEQ ID NO: AA of 16₁-AA₂₂₄。

In one embodiment, preferred polypeptides of the invention are prepro (shown in SEQ ID NO: 2, 4,9 and 16, respectively), pro (shown in AA of SEQ ID NO: 2, respectively) sequences of neublastin_-75-AA₁₀₅Or SEQ ID NO: AA of 4 and 9_-41-AA₁₄₀) And the mature sequence (shown in SEQ ID NO: 5,6, 7, 10, 11 or 12, preferably SEQ ID NO: 10, 11, 12).

The polypeptides of the invention include variant polypeptides. In the context of the present invention, the term "variant polypeptide" refers to a polypeptide (or protein) having an amino acid sequence which is identical to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 16 differ at one or more amino acid positions. Such variant polypeptides include modified polypeptides as described above, as well as conservative substitutions, splice variants, isoforms, homologs and polymorphisms from other species.

The term "conservative substitution" as defined herein denotes the replacement of an amino acid residue by another residue of similar biological properties. For example, conservative amino acid substitutions are expected to have little or no effect on biological activity, and are more desirable when less than 10% of the total number of polypeptide or protein residues are conservative substitutions. Conservative amino acid substitutions are preferably less than 5% change in the polypeptide or protein, and most preferably less than 2% change in the polypeptide or protein (e.g., when calculated based on NBN113, the most preferred conservative substitutions are less than 3 amino acid substitutions in the wild-type mature amino acid sequence). In a particularly preferred embodiment, there is only a single amino acid substitution in the mature sequence, wherein both the substituted and substituted amino acids are non-cyclic.

Other examples of particularly conservative substitutions include the substitution of a hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of a polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like.

The term conservative substitution also includes the substitution of a substituted amino acid residue for an unsubstituted parent amino acid residue, so long as an antibody raised against the substituted polypeptide is also immunoreactive with the unsubstituted polypeptide.

Modification of the original amino acid sequence results in a protein having substantially the same activity as the corresponding polypeptide without modification, and thus, the protein can be considered a functional analog of the parent protein. The modifications may be deliberate, e.g. by site-directed mutagenesis, or may be generated spontaneously, including splice variants, isoforms, homologues and polymorphisms from other species. The invention also includes the functional analogs.

In addition, modifications to the original amino acid sequence can result in a protein that does not retain the biological activity of the parent protein, including dominant negative forms and the like. Dominant negative proteins interfere with the wild-type protein by binding to, or otherwise sequestering, a modulator (e.g., an upstream or downstream component) that normally functionally interacts with the polypeptide. The invention also includes such dominant negative forms.

A "signal peptide" is a peptide sequence that allows a newly synthesized polypeptide that binds to the signal peptide to be localized to the Endoplasmic Reticulum (ER) for further post-translational processing and distribution.

For neublastin, as used herein, "heterologous signal peptide" refers to a non-human neublastin signal peptide, and generally refers to signal peptides of some mammalian proteins other than neublastin.

The skilled artisan will recognize that: cultured human cells can be genetically modified using human neublastin DNA sequences (cDNA or genomic DNA), or sequences that differ from human neublastin DNA by silent codon changes or codon changes that produce conservative amino acid substitutions, to allow the cells to overexpress and secrete enzymes.

The polypeptides of the invention also include chimeric polypeptides or cleavable fusion polypeptides in which another polypeptide is fused to the N-terminus or C-terminus of the polypeptide or fragment thereof. Chimeric polypeptides can be produced by fusing a nucleic acid sequence (or portion thereof) encoding another polypeptide to a nucleic acid sequence (or portion thereof) of the invention.

The techniques for producing chimeric polypeptides are standard techniques. Such techniques generally require that the sequences be linked in such a way that the two sequences are in the same reading frame and that expression of the fusion polypeptide is under the control of the same promoter and terminator.

The polypeptides of the invention also include truncated forms of the full-length neublastin molecule. In the truncated molecule, one or more amino acids are deleted from the N-terminus or C-terminus, preferably from the N-terminus.

Amino acid sequence homology

The degree of homology of a candidate polypeptide with the neublastin polypeptide of the invention is determined as the degree of identity between two amino acid sequences. A high level of sequence identity indicates that the first sequence is likely derived from the second sequence.

Homology can be determined by computer analysis, such as, but not limited to, the ClustalX computer sequence alignment program [ Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, & Higgins DG: ClustalX windows interface: a flexible strategy for multiple sequence alignments by means of a mass analysis tool; nucleic acid studies, 1997, 25 (24): 4876-82], default parameter values are suggested herein. Using this procedure, the mature portion of the polypeptide encoded by the analogous DNA sequence of the invention is identical to the mature portion of the polypeptide encoded by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 16 is at least 90%, more preferably at least 95%, and most preferably at least 98%.

Based on the results of homology assays, it was demonstrated that the polypeptides of the present invention belonging to the TGF- β superfamily are related to the GDNF subfamily, but that the polypeptides of the present invention are a distinctive member of this subfamily.

Biologically active polypeptides

The polypeptides of the invention may be provided in any biologically active form, including pre-pro-protein, mature protein, glycosylated protein, phosphorylated protein or any other post-translationally modified protein.

The polypeptide of the invention may in particular be an N-glycosylated polypeptide, preferably the polypeptide is glycosylated at the N-residues indicated in the sequence listing.

In a preferred embodiment, the polypeptide of the invention has the amino acid sequence of SEQ ID NO: 9, having a glycosylated asparagine residue at position 122; SEQ ID NO: 10, having a glycosylated asparagine residue at position 122; SEQ ID NO: 11, having a glycosylated asparagine residue at position 98; or SEQ ID NO: 12, having a glycosylated asparagine residue at position 95.

The present invention also includes neubIastin fusion proteins, such as Ig-fusion proteins, for example, see U.S. Pat. No. 5,434,131 (incorporated herein by reference).

In one embodiment, the present invention provides a polypeptide having the sequence of SEQ ID NO: 2, or a polypeptide having an amino acid sequence identical to SEQ ID NO: 2, preferably at least about 90%, more preferably at least about 98%, and most preferably at least about 99% homologous thereto.

In another embodiment, the present invention provides a polypeptide having the amino acid sequence of SEQ ID NO: 4, or a polypeptide having an amino acid sequence identical to SEQ ID NO: 4, preferably at least about 95%, more preferably at least about 98%, and most preferably at least about 99%.

In a third embodiment, the present invention provides a polypeptide having the sequence of SEQ ID NO: 5, or a polypeptide having an amino acid sequence identical to SEQ ID NO: 5, more preferably at least about 95%, and most preferably at least about 98% homologous to the sequence set forth in seq id no.

In a fourth embodiment, the present invention provides a polypeptide having the sequence of SEQ ID NO: 6, or a polypeptide having an amino acid sequence identical to SEQ ID NO: 6, more preferably at least about 95%, and most preferably at least about 98% homologous to the sequence set forth in seq id no.

In a fifth embodiment, the present invention provides a polypeptide having the sequence of SEQ ID NO: 7, or a polypeptide having an amino acid sequence identical to SEQ ID NO: 7, more preferably at least about 95%, and most preferably at least about 98% homologous to the sequence set forth in seq id no.

The neublastin polypeptides of the invention include allelic variants, such as SEQ ID NO: 5-7, wherein Xaa represents Asn or Thr and Yaa represents Ala or Pro.

In a sixth embodiment, the present invention provides a polypeptide having the sequence of SEQ ID NO: 9, or a polypeptide having an amino acid sequence identical to SEQ ID NO: 9, more preferably at least about 95%, and most preferably at least about 98% homologous to the sequence set forth in seq id no.

In a seventh embodiment, the present invention provides a polypeptide having the sequence of SEQ ID NO: 10, or a polypeptide having an amino acid sequence identical to SEQ ID NO: 10, more preferably at least about 90%, even more preferably at least about 95%, and most preferably at least about 98% homologous to the corresponding polypeptide.

In an eighth embodiment, the present invention provides a polypeptide having the sequence of SEQ ID NO: 11, or a polypeptide having an amino acid sequence identical to SEQ ID NO: 11, more preferably at least about 90%, most preferably at least about 95%, and most preferably at least about 98%.

In a ninth embodiment, the present invention provides a polypeptide having the sequence of SEQ ID NO: 12, or a polypeptide having an amino acid sequence identical to SEQ ID NO: 12, more preferably at least about 95%, and most preferably at least about 98% homologous thereto.

In a tenth embodiment, the present invention provides a polypeptide having the amino acid sequence of SEQ ID NO: 16, or a polypeptide having an amino acid sequence identical to SEQ ID NO: 16, more preferably at least about 95%, and most preferably at least about 98%, homologous to the sequence set forth in seq id No.16, said polypeptide being a pro-murine pre-neublastin.

In another embodiment, the polypeptides of the invention have a GDNF subfamily fingerprint, i.e., the underlined amino acid residues in table 3.

In another embodiment, the present invention provides a polypeptide encoded by a polynucleotide sequence that hybridizes under high stringency conditions with the nucleic acid sequence of SEQ ID NO: 1, its complementary strand, or a sub-sequence thereof. In a preferred embodiment, the polypeptide of the invention is encoded by a polynucleotide sequence that hybridizes with the polynucleotide sequence of seq id NO: 1 is at least 70% homologous. In a most preferred embodiment, the polypeptide of the invention consists of SEQ ID NO: 1, or a variant thereof.

In another embodiment, the present invention provides novel polypeptides encoded by a polynucleotide sequence that hybridizes under high stringency conditions with the nucleic acid sequence of SEQ ID NO: 3, its complementary strand, or a sub-sequence thereof. In a preferred embodiment, the polypeptide of the invention is encoded by a polynucleotide sequence that hybridizes to the nucleic acid sequence of SEQ ID NO: 3 is at least 70% homologous. In a most preferred embodiment, the polypeptide of the invention consists of SEQ ID NO: 3, or a polynucleotide sequence as set forth in figure 3.

In another embodiment, the present invention provides novel polypeptides encoded by a polynucleotide sequence that hybridizes under high stringency conditions with the nucleic acid sequence of SEQ ID NO: 8, its complementary strand, or a sub-sequence thereof. In a preferred embodiment, the polypeptide of the invention is encoded by a polynucleotide sequence that hybridizes to the nucleic acid sequence of SEQ ID NO: 8 is at least 70% homologous. In a most preferred embodiment, the polypeptide of the invention consists of SEQ ID NO: 8, or a variant thereof.

In another embodiment, the present invention provides novel polypeptides encoded by a polynucleotide sequence that hybridizes under high stringency conditions with the nucleic acid sequence of SEQ ID NO: 15, its complementary strand, or a sub-sequence thereof. In a preferred embodiment, the polypeptide of the invention is encoded by a polynucleotide sequence that hybridizes to the nucleic acid sequence of SEQ ID NO: 15 is at least 70% homologous. In a most preferred embodiment, the polypeptide of the invention consists of SEQ ID NO: 15, or a fragment thereof.

Biological source

The polypeptides of the invention may be isolated from mammalian cells, preferably from human cells or murine cells.

In a most preferred embodiment, the polypeptide of the invention is isolated from human heart tissue, human skeletal muscle, human pancreas, human brain tissue, in particular from the caudate nucleus or thalamus, or is obtainable from DNA of mammalian origin, as will be discussed in more detail below.

Neurotrophic activity

The neublastin polypeptides of the invention are useful for moderating metabolism, growth, differentiation or survival of neural or neuronal cells. In particular, neublastin polypeptides are useful for treating or ameliorating a disorder or disease in a living animal, such as a human, which disorder or disease is responsive to the activity of a neurotrophic agent. Such treatments and methods are described in more detail below.

Antibodies

Neublastin-specific antibodies can be generated using the neublastin polypeptides or polypeptide fragments of the invention. As used herein, a "neublastin-specific antibody" is an antibody, e.g., a polyclonal antibody or a monoclonal antibody, that immunoreacts with a neublastin polypeptide or polypeptide fragment, or specifically binds to an epitope of a neublastin polypeptide.

The preparation of polyclonal and monoclonal antibodies is well known in the art. In particular, polyclonal antibodies can be obtained as described in: for example, Green et al: "production of polyclonal antisera",immunochemical method(compiled by Manson); humana Press, 1992, p 1-5; coligan et al: "production of polyclonal antisera in rabbits, rats, mice and hamsters",new freeEpidemic disease Method1992, section 2.4.1; ed Harlow and David Lane (eds):"antibody: fruit of Chinese wolfberry Laboratory handbook "Cold spring harbor laboratory press, 1988; these methods are incorporated herein by reference. In particular, monoclonal antibodies can be obtained as described in: kohler&Milstein, nature, 1975, 256: 495; a color, an et al,novel immunological methods1992, sections 2.5.1-2.6.7; and Harlow et al,"antibody: laboratory manual "Cold spring harbor laboratory press, 1988, p 726; these methods are incorporated herein by reference.

Briefly, monoclonal antibodies can be obtained by injecting, for example, a mouse with a composition containing an antigen, confirming antibody production by removing a serum sample, removing a spleen, obtaining B lymphocytes, fusing the B lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones producing antibodies against the antigen, and isolating the antibodies from the hybridoma culture.

Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques including affinity chromatography using protein A Sepharose, size exclusion chromatography and ion exchange chromatography, see, for example, Coligan et al,novel immunological methods1992, sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3; and Barnes et al: "purification of immunoglobulin G (IgG)",molecular biological method(ii) a Humana Press, 1992, Vol.10, p 79-104. The polyclonal or monoclonal antibodies are optionally further purified by binding to and eluting from a matrix to which the antibody-producing polypeptide is bound.

Antibodies that bind to the neublastin polypeptides of the invention can be prepared using intact polypeptides or fragments containing the desired small peptide (as the immunizing antigen). The polypeptides used for immunizing animals may be obtained by recombinant DNA techniques or by chemical synthesis, optionally conjugated to a carrier protein. Commonly used carrier proteins chemically coupled to the peptides include Keyhole Limpet Hemocyanin (KLH), thyroglobulin, Bovine Serum Albumin (BSA), and tetanus toxoid. The coupled peptide may then be used to immunize an animal, which may be, inter alia, a mouse, rat, hamster, or rabbit.

In one embodiment, antibodies can be generated using the following peptides:

peptide 1: CRPTRYEAVSFMDVNST (amino acids 108-124 of SEQ ID NO: 9); or

Peptide 2: ALRPPPGSRPVSQPC (amino acids 93-107 of SEQ ID NO: 9). Methods of using these polypeptides to generate antibodies are described in example 10.

We also generated rabbit polyclonal antibodies against the following peptides:

peptide R27: GPGSRARAAGARGC (amino acids 30-43 of SEQ ID NO: 9);

peptide R28: LGHRSDELVRFRFC (amino acids 57-70 of SEQ ID NO: 9);

peptide R29: CRRARSPHDLSL (amino acids 74-85 of SEQ ID NO: 9);

peptide R30: LRPPPGSRPVSQPC (amino acids 94-107 of SEQ ID NO: 9);

peptide R31: STWRTVDRLSATAC (amino acids 123-136 of SEQ ID NO: 9).

In this set of peptides, only the peptides R30 and R31 relatively close to the C-terminus recognize proteins denatured under reducing conditions on Western blots.

As detailed below, we have also identified other neublastin-derived peptides derived from mature proteins, which, according to the known GDNF structure (eignebrot and Gerber,Nat.Struct.Biol.19974435-438), presumably they are surface exposed loops and can therefore be used for the production of antibodies:

region 1: CRLRSQLVPVRALGLGHRSDELVRFRFC (AA 43-70 of SEQ ID NO: 9);

region 2: CRRARSPHDLSLASLLGAGALRPPPGSRPVSQPC (AA 74-107 of SEQ ID NO: 9);

region 3: CRPTRYEAVSFMDVNSTWRTVDRLSATAC (AA 108-136 of SEQ ID NO: 9).

In another aspect of the invention, antibodies that specifically bind to neublastin or a neublastin-derived peptide can be used to detect the presence of said neublastin neurotrophic factor in a variety of media, and are particularly useful in diagnosing disorders or diseases associated with the neublastin molecules of the invention. Various detection methods including ELISA, RIA and FACS are known in the art.

The antibodies of the invention may also be used to block the action of neurotrophic factors, and in particular may be neutralizing antibodies.

Methods for producing the polypeptides of the invention

As described in detail below, cells containing a DNA sequence encoding a neublastin polypeptide of the invention are cultured under conditions that allow production of the polypeptide, followed by recovery of the polypeptide from the culture medium. When a cell needs to be genetically modified in order to produce a neublastin polypeptide, the cell may be modified by conventional methods or by gene activation.

DNA molecules containing neublastin cDNA or genomic DNA sequences can be included in expression constructs according to conventional methods and transfected into cells by standard methods including, but not limited to liposome-, Polybrene-or DEAE dextran-mediated transfection, electroporation, calcium phosphate precipitation, microinjection or velocity-driven microprojectile bombardment ("biolistics"). Alternatively, a system for delivering DNA via a viral vector may be used. Viruses known to be useful for gene transfer include adenovirus, adeno-associated virus, lentivirus, herpes virus, mumps virus, poliovirus, retrovirus, Sindbis virus and vaccinia virus, such as canarypox virus, and baculovirus infection of insect cells, particularly SfP9 insect cells.

Alternatively, cells can be modified using a gene activation ("GA") method, as described, for example, in U.S. Pat. nos. 5,733,761 and 5,750,376 (both incorporated herein by reference).

Thus, the term "genetically modified cell" as used herein includes cells which are capable of expressing a particular gene product following introduction of a DNA molecule encoding the gene product and/or regulatory elements controlling the expression of the coding sequence of the gene product. DNA molecules can be incorporated into specific genomic sites by gene-targeted introduction.

Recombinant expression vector

In another aspect of the invention, there is provided a recombinant expression vector comprising a polynucleotide of the invention. The recombinant expression vector of the invention may be any suitable eukaryotic expression vector. Preferred recombinant expression vectors are the ubiquitin promoter-containing vector pTEJ-8(Johansen TE, Schoeeller MS, Tolstoy S, Schwartz T;FEBS Lett1990267289-294) and derivatives thereof, such as pUbi 1Z. A preferred commercially available eukaryotic expression vector is, for example, vector pcDNA-3 (available from Invitrogen) containing a viral promoter. Another preferred expression vector uses the SV40 early promoter and the adenovirus major late promoter (derived from plasmid pAD 2. beta.; Norton and Coffin, molecular cell biology, 1985, 5, 281).

The invention also provides prokaryotic expression vectors and synthetic genes (syngene) whose codons are optimized for prokaryotic expression. Synthetic genes were constructed with lower GC content and preferred codons for bacteria (e.g., e.coli). The synthetic genes were cloned into two vectors, pET19b and pMJB164 (a derivative of pET19 b). The construction of pET19b is shown in FIG. 14. In this construct, the sequence encoding the mature domain of neublastin is fused directly to the initiating methionine. The construction of pMJB164 is shown in FIG. 15.

Production cell

In another aspect of the invention, there is provided a production cell which, following genetic manipulation, contains an isolated polynucleotide sequence of the invention, and/or a recombinant expression vector of the invention. In particular, the cells of the invention may be genetically manipulated to express, over-express or co-express the polypeptides of the invention transiently or stably. Methods for producing transient and stable expression are known in the art.

The polynucleotides of the present invention may be inserted into an expression vector, such as a plasmid, virus or other expression vector, operably linked to an expression control sequence in a specific linkage such that the coding sequence is expressed under conditions compatible with the expression control sequence. Suitable expression control sequences include promoters, enhancers, transcription terminators, start codons, splicing signals for introns, and stop codons, all of which are maintained in the correct reading frame for the polynucleotides of the invention to allow for proper translation of the mRNA. Expression control sequences also include other components, such as leader sequences and fusion partner sequences.

The promoter may in particular be a constitutive or inducible promoter. When cloning into bacterial systems, inducible promoters can be used, such as pL, plac, ptrp, ptac (ptrp-lac hybrid promoter) of bacteriophage lambda. When cloned into a mammalian system, promoters derived from the genome of mammalian cells, such as the ubiquitin, TK, or metallothionein promoters, or promoters derived from mammalian viruses, such as the retroviral long terminal repeat, the adenoviral late promoter, or the vaccinia 7.5K promoter, may be used. Promoters obtained by recombinant DNA or synthetic techniques may also be used to provide for transcription of the polynucleotides of the invention.

Suitable expression vectors typically contain an expression origin, a promoter and a specific gene capable of selecting transformed cells with an epitope, including the T7-based expression vector expressed in bacteria [ Rosenberg et al, Gene, 1987, 56, 125], pTEJ-8, pUbi1Z, pcDNA-3 and pMSXND expression vectors [ Lee and Nathans, J. Biochem., 19882633521 ] expressed in mammalian cells, baculovirus-derived vectors expressed in insect cells, and oocyte expression vector PTLN [ Lorenz C, Pusch M & Jentsch T J: heteromultimeric CLC chloride channels having novel properties; Natl.Acad.Sci.USA 19969313362-13366 ].

In a preferred embodiment, the cell of the invention is a eukaryotic cell, e.g. a mammalian cell, such as a human cell, an oocyte or a yeast cell. Cells of the invention include, but are not limited to, Human Embryonic Kidney (HEK) cells, such as HEK293 cells, BHK21 cells, Chinese Hamster Ovary (CHO) cells, Xenopus Laevis Oocytes (XLO). In another embodiment, the cell of the invention is a fungal cell, e.g., a filamentous fungal cell. In another preferred embodiment, the cell is an insect cell, most preferably an Sf9 cell. Another preferred mammalian cell of the invention is the PC12, HiB5, RN33b cell line and human neural progenitor cells. Most preferred are human cells.

Examples of primary or secondary cells include fibroblasts, epithelial cells (including breast and intestinal epithelial cells), endothelial cells, blood components (including lymphocytes and bone marrow cells), glial cells, hepatocytes, keratinocytes, myocytes, neural cells, or precursors of these cell types. Examples of immortalized human cell lines useful in the methods of the invention include, but are not limited to, Bowes melanoma cells (ATCC accession number CRL9607), Daudi cells (ATCC accession number CCL213), HeLa cells and derivatives of HeLa cells (ATCC accession numbers CCL2, CCL2.1 and CCL2.2), HL-60 cells (ATCC accession number CCL240), HT-1080 cells (ATCC accession number CCL121), Jurkat cells (ATCC accession number TIB152), KB cancer cells (ATCC accession number CCL17), K-562 leukemia cells (ATCC accession number CCL243), MCF-7 breast cancer cells (ATCC accession number BTH22), MOLT-4 cells (ATCC accession number 1582), Namalwa cells (ATCC accession number CRL1432), Raji cells (ATCC accession number CCL 36 86), RPMI 26 cells (ATCC accession number CCL155), ATCC-7 cells (ATCC accession number 1583), Blmalwa cell accession number CRL1432), and ovarian cancer cells (ATCC accession number CLVA 4), cancer research, 1988, 48, 5927-5932), and heterohybridoma cells produced by fusing a human cell with a cell of another species. Secondary human fibroblast cell lines such as WI-38(ATCC accession number CCL75) and MRC-5(ATCC accession number CCL171) may also be used.

When the cell of the invention is a eukaryotic cell, incorporation of the heterologous polynucleotide of the invention may be achieved in particular by infection (using viral vectors), transfection (using plasmid vectors), precipitation using calcium phosphate, microinjection, electroporation, lipofection, or other physico-chemical methods known in the art.

In a more preferred embodiment, an isolated polynucleotide sequence of the invention and/or a recombinant expression vector of the invention can be transfected into a mammalian host cell, neural progenitor cell, astrocyte, T-cell, hematopoietic stem cell, non-dividing cell, or brain endothelial cell, said cell containing at least one DNA molecule capable of mediating cellular immortalization and/or transformation.

Endogenous genes in host cells can be activated by introducing regulatory elements, in particular a promoter, which leads to transcription of the endogenous gene encoding the neublastin polypeptide of the invention.

Pharmaceutical composition

In another aspect of the invention, novel pharmaceutical compositions are provided which contain a therapeutically effective amount of a polypeptide of the invention.

For use in therapy, the polypeptides of the invention may be administered in any convenient form. In a preferred embodiment, the polypeptide of the invention is mixed with one or more adjuvants, excipients, carriers and/or diluents in a pharmaceutical composition, which can be prepared by one skilled in the art using conventional methods known in the art.

Such pharmaceutical compositions may contain a polypeptide of the invention or an antibody thereof. The composition may be administered alone or in combination with one or more other agents, drugs or hormones.

The pharmaceutical compositions of the present invention may be administered by any suitable route, including, but not limited to, orally, intravenously, intramuscularly, interarterial, intramedullary, intrathecally, intraventricularly, transdermally, subcutaneously, intraperitoneally, intranasally, antral, topically, sublingually, or rectally, buccally, intravaginally, intraorbitally, intracerebrally, intracranially, intraspinally, intraventricularly, intracisternally, intracapsularly, intrapulmonary, transmucosally, or via inhalation.

Methods, devices and pharmaceutical products for intrapulmonary delivery are described, for example, in U.S. Pat. Nos. 5,785,049, 5,780,019 and 5,775,320 (all incorporated herein by reference). Administration can be by periodic injection of boluses of the pharmaceutical product; the drug may also be administered more continuously by intravenous or intraperitoneal routes, from external (e.g., IV bags) or internal (e.g., bioerodible implants, bioartificial organs, or implanted neublastin producer cell colonies) drug libraries. See, for example, U.S. Pat. Nos. 4,407,957, 5,798,113 and 5,800,828 (all incorporated herein by reference). Methods and devices for intrapulmonary delivery are described, for example, in U.S. Pat. Nos. 5,654,007, 5,780,014 and 5,814,607 (all incorporated herein by reference).

In particular, neublastin of the invention may be administered using any suitable delivery means, including:

(a) pump (see for exampleMedicine treatment yearbook，27：912(1993)；Cancer treatment，41：1270(1993)；Cancer (carcinoma) Study of symptoms,44: 1698(1984), incorporated herein by reference),

(b) microencapsulation (see, for example, U.S. Pat. Nos. 4,352,883; 4,353,888; and 5,084,350; incorporated herein by reference),

(c) sustained release polymeric implants (see, e.g., Sabel, U.S. Pat. No. 4,883,666, incorporated herein by reference),

(d) macroencapsulation (see, for example, U.S. Pat. Nos. 5,284,761, 5,158,881, 4,976,859 and 4,968,733 and published PCT patent applications WO92/19195, WO95/05452, all incorporated herein by reference),

(e) naked or unencapsulated cell grafts for transplantation into the CNS (see for example U.S. Pat. Nos. 5,082,670 and 5,618,531, both incorporated herein by reference),

(f) subcutaneous, intravenous, intraarterial, intramuscular injection or injection to other appropriate sites; and

(g) orally administered in capsules, liquids, tablets, pills or extended release formulations.

In one embodiment of the invention, neublastin is delivered directly to the CNS, preferably to the ventricles, brain parenchyma, intrathecal space or other suitable CNS location, most preferably intrathecally.

In another preferred embodiment, we wish to administer the drug by subcutaneous injection, intravenous administration or intravenous perfusion systemic delivery.

Other useful parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, pump delivery, encapsulated cell delivery, liposome delivery, needle-delivered injection, needleless injection, nebulizers, aerosols, electroporation, and transdermal patches.

Additional details regarding formulation and administration techniques are found in the latest version of Remington's pharmaceutical science (Maack publishing company, Easton, PA).

The active ingredient is administered in one or several doses per day. Suitable dosages contemplated by the present invention range from 0.5ng neublastin per kg body weight to about 50 μ g/kg per administration and from about 1.0ng/kg to about 100 μ g/kg per day. The neublastin pharmaceutical composition should provide a local concentration of neurotrophic factor from about 5ng/ml cerebrospinal fluid ("CSF") to 25ng/ml CSF.

Of course, the dosage to be administered should be carefully adjusted to the desired result and the exact dosage should be determinable by the physician, depending on the age, weight and physical condition of the individual being treated, as well as the route of administration, dosage form and regimen.

In other embodiments, neublastin polypeptides of the invention can be administered by gene delivery using cell lines and vectors described in the "methods of treatment" section below. To generate such a therapeutic cell line, the polynucleotide of the invention may be inserted into an expression vector, such as a plasmid, virus or other expression vector, which polynucleotide is operably linked to an expression control sequence in a specific linkage such that the coding sequence is expressed under conditions compatible with the expression control sequence. Suitable expression control sequences include promoters, enhancers, transcription terminators, start codons, splicing signals for introns, and stop codons, all of which are maintained in the correct reading frame for the polynucleotides of the invention to allow for proper translation of the mRNA. Expression control sequences also include other components, such as leader sequences and fusion partner sequences.

The promoter may in particular be a constitutive or inducible promoter. Constitutive promoters may be synthetic, viral or promoters derived from the genome of mammalian cells, such as the human ubiquitin promoter. In a preferred embodiment, the therapeutic cell line is a human immortalized neural cell line expressing a polypeptide of the invention. For implantation, we wish to implant about 10⁵To 10¹⁰Individual cells, more preferably implanted 10⁶To about 10⁸And (4) cells.

Method of treatment

The present invention relates to polynucleotides and proteins, polypeptides, peptide fragments or derivatives derived therefrom, and antibodies directed against said proteins, peptides or derivatives, which are useful for treating or ameliorating a disorder or disease of a living animal body, including a human, which disorder or disease is responsive to the activity of a neurotrophic agent.

The polypeptides of the invention can be used directly to treat pathological processes that are responsive to neublastin polypeptides, for example, via injection, implantation or ingestion of a pharmaceutical composition.

The polynucleotides of the invention, including their complementary sequences, may be used to express the neurotrophic factors of the invention. This object is achieved by a cell line expressing such a protein, peptide or derivative of the invention, or by a viral vector encoding such a protein, peptide or derivative of the invention, or by a host cell expressing such a protein, peptide or derivative. These cells, vectors and compositions can be administered to a target area of treatment to affect the disease process responsive to the neublastin polypeptide.

Suitable expression vectors may be derived from lentiviruses, retroviruses, adenoviruses, herpesviruses or vaccinia viruses, or from a variety of bacterially produced plasmids, which may be used in vivo to deliver nucleotide sequences to whole organisms or target organs, tissues or cell populations. Other methods include, but are not limited to, lipofection, electroporation, transfection with carrier peptides containing nuclear or other localization signals, and gene delivery via sustained release systems. In another aspect of the invention, "antisense" nucleotide sequences complementary to the neublastin gene or a portion thereof may be used to inhibit or enhance expression of neublastin.

Another aspect of the invention relates to a method of treating or ameliorating a disorder or disease of a living animal body, including a human, which disorder or disease is responsive to the activity of a neurotrophic agent.

The disorder or disease may be, inter alia, damage to the nervous system caused by trauma, surgery, ischemia, infection, metabolic disease, nutritional deficiency, malignancy or a toxic agent and genetic or spontaneous processes.

The damage may occur particularly in sensory neurons or retinal ganglion cells, including neurons in the lateral root ganglion or in any tissue from the group consisting of: geniculate, lithologic and nodular ganglia; the auricular vestibular complex of the VIII cranial nerve; the ventral lateral pole of the upper mandibular lobe of the trigeminal ganglion; and the nuclei of the brain trigeminal nerve.

In a preferred embodiment of the method of the invention, the disease or disorder is a neurodegenerative disease involving damaged and injured neurons, such as traumatic damage to peripheral nerves, medulla and/or spinal cord, cerebral ischaemic neuronal damage, neuropathy (especially peripheral neuropathy), peripheral nerve trauma or trauma, ischaemic stroke, acute brain injury, acute spinal cord injury, nervous system tumours, multiple sclerosis, exposure to neurotoxins, metabolic diseases (such as diabetes or renal dysfunction) and damage caused by infectious agents, neurodegenerative disorders including Alzheimer's disease, Huntington's disease, Parkinson-Plus syndrome, progressive supranuclear palsy (Steele-Richardson-Olszewski syndrome), olivopontocerebellar atrophy (OPCA), Shy-Drager syndrome (multiple system atrophy), guamanian parkinsonian dementia complex, amyotrophic lateral sclerosis, or any other congenital or neurodegenerative disease, and memory impairment associated with dementia.

In preferred embodiments, we wish to treat neurons of the sensory and/or autonomic system. In another preferred embodiment, we wish to treat motor neuron diseases such as amyotrophic lateral sclerosis ("ALS") and spinal muscular atrophy. In another preferred embodiment, we wish to use the neublastin molecules of the invention to enhance nerve recovery following trauma. In one embodiment, we contemplate the use of a nerve guidance channel having a matrix containing a neublastin polypeptide. Such nerve guidance channels are disclosed, for example, in U.S. Pat. No. 5,834,029 (incorporated herein by reference).

In preferred embodiments, the polypeptides and nucleic acids of the invention (and pharmaceutical compositions containing them) can be used to treat peripheral neuropathy. Among the peripheral neuropathies that are contemplated for treatment with the molecules of the present invention are trauma-induced neuropathies, such as those caused by physical injury or disease state, physical injury to the brain, physical injury to the spinal cord, stroke associated with brain injury, and neurological disorders associated with neurodegeneration.

We also wish to treat neuropathy induced by chemotherapy (e.g. caused by delivery of chemotherapeutic agents such as taxol or cisplatin); toxin-induced neuropathy, drug-induced neuropathy, vitamin-deficiency-induced neuropathy; idiopathic and diabetic neuropathy, for example, U.S. patents 5,496,804 and 5,916,555 (both incorporated herein by reference).

We also contemplate the use of the neublastin nucleotides and polypeptides of the invention for the treatment of non-neuropathies, mono-polyneuropathy and multi-neuropathies, including axonal and demyelinating neuropathies.

In another preferred embodiment, the polypeptides and nucleic acids of the invention (and pharmaceutical compositions containing them) are useful in treating a variety of ocular diseases, including retinal photoreceptor loss in patients afflicted with macular degeneration, retinitis pigmentosa, glaucoma, and similar diseases.

It is another object of the present invention to provide a method for preventing degenerative changes associated with the above-mentioned diseases and disorders, which comprises implanting into the brain of a mammal a human vector or cell capable of producing a biologically active form of neublastin or a precursor of neublastin, i.e. a molecule susceptible to conversion by the mammal body into a biologically active form of neublastin, or alternatively, a cell secreting neublastin may be encapsulated within, for example, a semipermeable membrane.

Cells can be cultured in vitro for transplantation or implantation into the brain of a mammal, including a human.

In a preferred embodiment, the gene encoding the polypeptide of the invention may be transfected into a suitable cell line, for example an immortalised rat neural stem cell line, such as HiB5 and RN33b, or into a human immortalised neural progenitor cell line, using an expression vector such as that described in international patent application WO98/32869, and the resulting cell line implanted into the brain of a living body (including a human) to secrete the therapeutic polypeptide of the invention in the CNS.

Diagnostic and screening methods

Neublastin nucleic acids can be used to determine whether an individual is predisposed to a neurological disorder due to a defect in a neublastin gene, such as a defect in a neublastin allele, obtained, for example, by genetic inheritance, abnormal embryonic development, or acquired DNA damage. Such assays include, for example, detecting defects or point mutations within the neublastin gene, or detecting inheritance of gene defect causes with specific Restriction Fragment Length Polymorphisms (RFLP), detecting the presence or absence of a normal neublastin gene by hybridizing a nucleic acid sample from the patient with a nucleic acid probe specific for the neublastin gene, and assaying the ability of the probe to hybridize to the nucleic acid.

Neublastin nucleic acids are particularly useful as hybridization probes. Such hybridization assays can be used to detect, predict, diagnose or monitor conditions, disorders or diseases associated with abnormal levels of mRNA encoding neublastin protein. Neublastin nucleic acids can be used as "markers" for neublastin neurotrophic factor-dependent physiological processes. These processes include, but are not limited to, "normal" physiological processes (e.g., neuronal function) and pathological processes (e.g., neurodegenerative diseases). Identification of a particular patient subpopulation with abnormal (i.e., elevated or reduced) levels of neublastin protein and/or mRNA encoding neublastin has led to a new disease classification. An "abnormal level" as defined herein refers to: the level is increased or decreased relative to a control sample or a non-diseased individual as measured by a quantitative or qualitative method.

Neublastin nucleic acids and polypeptides of the invention may also be used to screen and identify neublastin analogs, including small molecule mimetics of neublastin. In one contemplated embodiment, the present invention provides a method of identifying a candidate compound that induces a neublastin-mediated biological effect, comprising the steps of: providing a test cell which is induced to express a detectable product when contacted with neublastin, exposing the cell to a candidate compound, and detecting the detectable product. Expression of the detectable product is an indication that the candidate compound is capable of inducing a neublastin-mediated biological effect.

In addition, the neublastin nucleic acids and polypeptides of the invention may be used in DNA or protein chips, or in computer programs to identify related novel gene sequences and proteins encoded thereby, including allelic variants and single nucleotide polymorphisms ("SNPs"). Such methods are described, for example, in U.S. Pat. nos. 5,795,716; 5,754,524, respectively; 5,733,729; 5,800,992; 5,445,934; 5,525,464 (all incorporated herein by reference).

Examples

Example 1: method for isolating neublastin nucleic acids

The method comprises the following steps: rapid screening of neublastin gene in human fetal brain cDNA

Through homology to human persephin, a 290bp fragment was identified in two high throughput genomic sequences (HGTS) submitted to GenBank (accession numbers AC005038 and AC 005051). Two neublastin specific primers were synthesized from the nucleic acid sequence of the 290bp fragment. neublastin upper strand primer ("nbnint. sense") has the sequence 5'-CCTGGCCAGCCTACTGGG-3' [ SEQ ID NO: 17]. The neublastin bottom strand primer ("nbnint. antisense") has the sequence 5'-AAGG AGACCGCTTCGTAGCG-3' [ SEQ ID NO: 18]. These primers were used to perform 96-well PCR reactions.

The 96-well master plate contains plasmid DNA of 500,000 cDNA clones, approximately 5000 clones per well. The 96-well sub-plate used E.coli DH10B glycerol stock containing 50 clones per well.

Neublastin nucleic acid was identified by 3 rounds of amplification using polymerase chain reaction ("PCR") techniques; amplification increases the number of nucleic acid copies in a sample.

Screening a main plate: human fetal brain cDNA master plates were screened with gene-specific primers to isolate human neublastin cDNA using the 96-well PCR screening technique described above.

30ng (30ng) of human fetal brain cDNA (6 ng/. mu.l; Origene Technologies) were obtained from the corresponding wells of the master plate and placed in a total volume of 25. mu.l of a solution containing the following reagents: 0.2mM of the above two gene-specific primers (i.e., NBNint. sense and NBNint. antisense), 1 × Standard PCR Buffer (Buffer V, Advanced Bio-technologies, UK), 0.2mM dNTP (Amersham-Pharmacia), 0.1M GC-Melt (Clontech Laboratories, USA) and 0.5 unit Taq DNA polymerase (5U/. mu.l; Advanced Biotechnologies, UK).

PCR thermocycling reactions were performed using the following protocol and conditions. The DNA was initially denatured at 94 ℃ for 3 minutes, then subjected to 35 cycles as follows: denaturation at 94 ℃ for 1 min, annealing at 55 ℃ for 1 min, first extension at 72 ℃ for 90 sec; final extension at 72 ℃ for 5 min. 96 individual PCR reaction products were analyzed by gel electrophoresis using a 2% agarose gel containing ethidium bromide dye. The 102bp positive PCR product from one well corresponds to a unique 96-well sub-plate.

The 102bp nucleic acid fragment has the following sequence [ SEQ ID NO: 13]

5′-CCTGGCCAGCCTACTGGGCGCCGGGGCCCTGCGACCGCCCCCGGGCTCCCGGCCCGTCAGCCAGCCCTGCTGCCGACCCACGCGCTACGAAGCGGTCTCCTT-3′

Secondary plate screening: mu.l of glycerol stock from the corresponding subplate wells was placed in a total volume of 25. mu.l and screened by PCR-mediated amplification for 96-well human fetal brain subplates, the solution containing: 0.2mM of the above two gene-specific primers, 1 × Standard PCR Buffer (Buffer V, Advanced Biotechnologies, UK), 0.2mM dNTP (Amersham-Pharmacia), 0.1M GC-Melt (Clontech Laboratories, USA) and 0.5 unit Taq DNA polymerase (5U/. mu.l; Advanced Biotechnologies, UK).

The PCR thermocycling conditions used were the same as those used for the master plate screening. Analysis of 96 independent PCR reactions on a 2% agarose gel containing ethidium bromide identified positive wells giving a 102bp PCR fragment.

Colony PCR: 1ml of glycerol stock from the wells of the positive subpanels was diluted 100-fold with Luria Broth (LB), then 1ml and 10ml of the above dilution were plated on 2 separate agar plates containing Luria Broth (LB) and 100. mu.g/ml carbenicillin, and the LB plates were incubated overnight at 30 ℃. From these plates, 96 colonies were picked and placed in wells of a new 96-well PCR plate in a final volume of 25. mu.l per well containing 0.2mM of the above two gene-specific primers, 1 × Standard PCR Buffer (Buffer V, Advanced Biotechnologies, UK), 0.2mM dNTP (Amersham-Pharmacia), 0.1M GC-Melt (Clontech laboratories, USA) and 0.5 units Taq DNA polymerase (5U/. mu.l; Advanced dBIotechnologies, UK).

The PCR thermocycling conditions used were the same as those used for the master plate screening. 96 independent PCR reactions were analyzed on a 2% agarose gel containing ethidium bromide, followed by the identification of positive colonies containing a 102bp fragment.

Sequencing of plasmid DNA prepared from this positive colony revealed 861bp of full-length cDNA [ SEQ ID NO: 8]. The cDNA encodes a pre-neublastin pro [ SEQ ID NO: 9]. Automated DNA sequencing was performed using BigDye ® terminator cycle sequencing kit (PE Applied Biosystems, USA). The gel was sequenced electrophoretically on ABI Prism 377(PE Applied Biosystems, USA).

The method 2 comprises the following steps: cloning of neublastin cDNA from human brain:

another method for amplifying full length neublastin cDNA or cDNA fragments can be performed by using RACE (rapid amplification of cDNA ends), neublastin-specific primers nbnint.sense and nbnint.antisense as described above, in combination with vector-specific or adaptor-specific primers, for example by using the Marathon cDNA amplification kit (Clontech Laboratories, USA, cat.no. k 1802-1).

The full-length neublastin cDNA was amplified using whole human brain Marathon-Ready cDNA (Clontech Laboratories, USA, Cat. No. 7400-1). The primers used for amplification included neublastin top primer 5'-ATGGAACTTGGACTT GG-3' [ SEQ id no: 19] ("nbnext. sense"), and neublastin bottom strand primer 5'-TCCATCACCCACCGG C-3' [ SEQ ID NO: 20 ("nbnext. antisense"), and an adaptor primer AP1 contained in the Marathon-Ready cDNA. Another upper strand primer, 5'-CTAGGAGCCCATGCCC-3' [ SEQ ID NO: 28]. Another lower strand primer 5'-GAGCGAGCCCTCAGCC-3' [ SEQ ID NO: 33]. Similarly, another lower strand primer, SEQ ID NO: 24 and 26.

The method 3 comprises the following steps: cloning of neublastin cDNA from human brain:

another method for cloning neublastin cDNA is by screening adult or fetal brain libraries with one or more neublastin probes as exemplified in figure 1. These libraries include: λ gt11 human brain (Clontech Laboratories, USA, Cat. No. HL3002b); or λ gt11 human fetal brain (Clontech Laboratories, USA, Cat. No. HL3002b).

The method 4 comprises the following steps: rapid screening of neublastin gene in mouse fetal cDNA

The protocol for rapid screening of neublastin gene was performed as follows. The 96-well master plate contains plasmid DNA of 500,000 cDNA clones, approximately 5000 clones per well. The 96-well sub-plate used E.coli glycerol stocks containing 50 clones per well. 3 rounds of PCR-mediated amplification were performed to identify the gene of interest (i.e., neublastin).

Screening a main plate: mouse neublastin cDNA was isolated by 96-well PCR screening of mouse fetal cDNA master plates using gene-specific primers. The following two primers were synthesized:

(1) neublastin C2 primer (nbnint. sense): 5'-GGCCACCGCTCCGACGAG-3' [ SEQ ID NO: 21 ]; and (2) neublastin C2as primer (nbnint. antisense): 5'-GGCGGTCCACGGTTCTCCAG-3' [ SEQ ID NO: 22]. By using these two gene-specific primers, a positive PCR product of 220bp was identified. The 220bp nucleic acid has the following sequence [ SEQ ID NO: 14]:

5′-GGCCACCGCTCCGACGAGCTGATACGTTTCCGCTTCTGCAGCGGCTCGTGCCGCCGAGCACGCTCCCAGCACGATCTCAGTCTGGCCAGCCTACTGGGCGCTGGGGCCCTACGGTCGCCTCCCGGGTCCCGGCCGATCAGCCAGCCCTGCTGCCGGCCCACTCGCTATGAGGCCGTCTCCTTCATGGACGTGAACAGCACCTGGAGAACCGTGGACCGCC-3′

then, 96-well PCR reaction was carried out as follows. 30ng of mouse fetal brain cDNA (6 ng/. mu.l; Origene Technologies) was obtained from the corresponding well of the master plate and placed in a total volume of 25. mu.l of a solution containing the following reagents: 0.2mM of the two gene-specific primers described above (i.e., the C2 primer (NBNint. sense) and the neublastin C2as primer (NBNint. antisense)), 1 × Standard PCR Buffer (Buffer V, Advanced Bio-technologies, UK), 0.2mM dNTP (Amersham-Pharmacia), 0.1M GC-Melt (Clontech Laboratories, USA) and 0.5 unit Taq DNA polymerase (5U/. mu.l; Advanced Biotechnologies, UK).

The following PCR thermal cycling conditions were used: the DNA was initially denatured at 94 ℃ for 3 minutes, then subjected to 35 cycles as follows: denaturation at 94 ℃ for 1 min, annealing at 55 ℃ for 1 min, and extension at 72 ℃ for 90 sec; final extension at 72 ℃ for 5 min. 96 independent PCR reactions were analyzed on a 2% agarose gel containing ethidium bromide dye. The resulting 220bp positive PCR product from one well corresponds to a unique 96-well sub-plate. 96 independent PCR reactions were analyzed on a 2% agarose gel containing ethidium bromide dye. The identified 220bp positive PCR product corresponds to a well unique to the 96-well sub-plate.

Secondary plate screening: mu.l of glycerol stock from the corresponding secondary plate wells was placed in a final total volume of 25. mu.l of a solution containing: 0.2mM of the above two gene-specific primers, 1 × Standard PCR Buffer (Buffer V, Advanced Biotechnologies, UK), 0.2mM dNTP (Amersham-Pharmacia), 0.1M GC-Melt (Clontech Laboratories, USA) and 0.5 unit Taq DNA polymerase (5U/. mu.l; Advanced Biotechnologies, UK). The PCR thermocycling conditions used were the same as those used for the master plate screening described above.

96 independent PCR reactions were analyzed on a 2% agarose gel containing ethidium bromide and positive wells producing a 220bp fragment were identified.

Colony PCR: 1ml of glycerol stock from the wells of the positive subplate was diluted 100-fold with Luria Broth (LB), and 1ml and 10ml of the above dilution were spread on 2 separate LB plates containing 100gg/ml carbenicillin and incubated overnight at 30 ℃. A total of 96 colonies were isolated and transferred to wells of a 96-well PCR plate in a final volume of 25. mu.l per well containing 0.2mM of the above two gene-specific primers, 1 × Standard PCR Buffer (Buffer V, Advanced Biotechnologies, UK), 0.2mM dNTP (Amersham-Pharmacia), 0.1M GC-Melt (Clontech Laboratories, USA) and 0.5 units Taq DNA polymerase (5U/. mu.l; Advanced Biotechnologies, UK).

The PCR thermocycling conditions used were the same as those used for the master plate screening. 96 independent PCR reactions were analyzed by gel electrophoresis on a 2% agarose gel containing ethidium bromide, and positive colonies were identified by the presence of a 220bp fragment.

Plasmid DNA was prepared from the positive colonies, and clones were subjected to automated DNA sequencing using BigDye ® terminator cycle sequencing kit with AmpliTaq DNA polymerase. The gel was sequenced by electrophoresis on ABIPrism 377(PE Applied Biosystems, USA). The resulting cloned sequence showed 2136bp of full-length cDNA [ SEQ ID NO: 15]. The cDNA comprises a cDNA having the sequence of SEQ ID NO: 16, which encodes a mouse pre-neublastin pro-polypeptide.

Example 2: cloning of genomic neublastin

As discussed above, applicants identified a 290bp nucleic acid fragment in two human BAC clones (accession numbers AC005038 and AC005051) registered to GenBank, which fragment has regions of homology to persephin and the flanking sequences of persephin. Applicants used the 861bp deduced sequence described above to design additional primers in order to clone nucleic acids encoding additional neublastin nucleic acids using Lasergene software (DNASAR). The neublastin gene was cloned by PCR reaction on genomic DNA using two primer pairs.

These two pairs of primers are as follows.

Primer pair No. 1:

5' CCA AgC CCA CCT ggg TgC CCT CTT TCT CC 3 (sense) [ SEQ id no: 23]

5' CAT CAC CCA Ccg gCA ggg gCC TCT Cag 3 (antisense) [ SEQ id no: 24].

Primer pair No. 2:

5' gAgCCCAtgCCCggCCTgATCTCAgCCCgAggACA 3 (sense) [ SEQ id no: 25]

5' CCCTggCTgAggCCgCTggCTAgTgggACTCTgC 3 (antisense) [ SEQ id no: 26].

A887 bp DNA fragment was amplified from a human genomic DNA preparation purchased from Clontech Laboratories (Cat. No.6550-1) using primer pair No. 1.

PCR protocol: expand with buffer 1 was used^TMPCR was performed by the High Fidelity PCR system (Boehringer Mannheim). To a PCR reaction mixture of a total volume of 50. mu.l were added 5% dimethyl sulfoxide (DMSO) and 17.5pmol each dNTP. The thermocycling reaction was pre-denaturation at 94 ℃ for 2 min, followed by 35 cycles of a two-step cycle of 94 ℃ for 10 sec and 68 ℃ for 1 min. The thermal cycle was terminated by incubation at 68 ℃ for 5 minutes. Thermal cycling was performed in a PTC-225 DNAEngine tetra thermal cycler (MJ Research, MA). The PCR products were analyzed by gel electrophoresis on 2% agarose (FMC) and then photographed.

An 887bp fragment amplified from human genomic DNA with primer pair No.1 was cloned into pCRII vector (Invitrogen) and transformed into XL 1-Blue competent E.coli cells (Stratagene). The resulting plasmid, designated neublastin-2, was sequenced using thermal sequencing enzyme (Amersham pharmacia Biotech). Sequencing products were analyzed by electrophoresis on an ALFExpress automatic sequencer (Amerer-sham Pharmacia Biotech).

Sequencing of the fragment obtained by PCR amplification of human genomic DNA using the second primer pair (primer pair No.1 above) revealed an additional 42bp region at the 3' primer end of the open reading frame. This full-length sequence was analyzed by comparing it to the nucleic acid sequences of other neurotrophic factors, and mapping exon-intron border sequences using a Gene-finding software program that identified appropriate splicing linkers and regions of high coding potential using Netgene and Gene Mark software (Brunak et al, J. mol. biol., 1991, 22049-65; Borodovsky et al, nucleic acids research, 1995, 23, 3554-62). The exon-intron border sequence was further confirmed by the cDNA obtained from the above rapid screen.

As shown in FIG. 7, the resulting neublastin gene has two exons separated by a 70bp intron. In summary, the exons have the deduced amino acid sequence of the full-length Neublastin polypeptide. Putative cDNA [ SEQ ID NO: 3] contains a sequence encoding 238 amino acid residues [ SEQ ID NO: 4] (ORF) open reading frame. The Neublastin-2 clone contains the entire coding sequence of the Neublastin pro-gene. The amino acid sequence encoded by this gene shows high homology with 3 proteins, presephin, neublastin and GDNF.

Example 3: expression of neublastin nucleic acids

The following techniques were used to detect neural and non-neural tissue in rodents and humans, as well as neublastin RNA expression in multiple developmental immature and adult stages.

Method for detecting Neublastin RNA expression using RT-PCR: according to SEQ ID NO: 1, the following primers were synthesized: (1) neublastin c2 primer: 5'-GGCCACCGCTCCGACGAG-3' [ SEQ ID NO: 21 ]; and (2) neublastin C2as primer: 5'-GGCGGTCCACGGTTCTCCAG-3' [ SEQ ID NO: 22]. DNA fragments were amplified by RT-PCR from adult and fetal whole brain mRNA using this set of primers, and one of the DNA fragments generated by this reaction was 220 bp. The 220bpDNA fragment was identified, and the results showed that the neublastin gene was expressed in adult and fetal brain tissues. These primers were also used to amplify a 220bp DNA fragment from genomic DNA.

Method for detecting Neublastin RNA expression by Northern blot hybridization:

PolyA with adult tissue⁺Northern blotting of RNA was purchased from the manufacturer (Clontech laboratories, USA) and used³²P-labeled neublastin cDNA was used as a probe to hybridize. Labeled neublastin cDNA was prepared according to the method described in example 1 above.

Preparing a probe: the neublastin nucleic acid DNA fragment (nucleotide 296. sub.819 of SEQ ID NO: 8) was labeled using the Rediprime II labeling kit (Amersham; Cat. No. RPN1633) for use as a hybridization probe according to the manufacturer's instructions. Briefly, DNA samples were diluted with 10mM TE buffer (10mM Tris-HCl, pH8.0, 1mM EDTA) to a concentration of 2.5-25 ng/45. mu.l. Then in a boiling water bathThe sample was heated to 95-100 ℃ for 5 minutes, placed on ice for 5 minutes to rapidly cool the sample, and centrifuged briefly to allow the contents to sink to the bottom of the reaction tube, thereby denaturing the DNA. Into the reaction tube were added all of the above denatured DNA and 5. mu.l of Redivue³²P]dCTP (Amersham Pharmacia Biotech Ltd), which reaction tube also contains buffer solution of dATP, dGTP, dTTP, Klenow enzyme without exonuclease in dried stabilized form and random primer. The solution was mixed by pipetting up and down 2 times, stirring the solution with a pipette, and incubating the reaction mixture at 37 ℃ for 10 minutes. The labeling reaction was stopped by adding 5. mu.l of 0.2M EDTA. For use as a hybridization probe, the labeled DNA is denatured into single strands by heating the DNA sample to 95-100 ℃ for 5 minutes, and then placing it on ice for 5 minutes to rapidly cool the DNA sample. Centrifuge the tube and mix the contents thoroughly. Finally, the single-stranded DNA probe was purified using the Nucleotide Removal kit (Qiagen).

The hybridization technology comprises the following steps: northern blots were prepared from the manufacturer ("multiple tissue Northern blots", Clontech Laboratories, USA, Cat. No.7760-1 and 7769-1), using neublastin prepared above according to the manufacturer's instructions³²P-labeled probes were hybridized to the blots. For hybridization, ExpressHyb solution (Clontech Laboratories, USA) and labeled probe at a concentration of about 3ng/ml were used. The ExpressHyb solution was heated to 68 ℃ and then stirred to dissolve any precipitate. Each Northern blot membrane (10X 10cm) was pre-hybridized for 30 minutes at 68 ℃ in at least 5ml of ExpressHyb solution in a Hybaid hybridization oven, according to the manufacturer's instructions. Standing at 95-100 deg.C for 2 min to allow neublastin³²The P-labeled probe was denatured and then rapidly cooled on ice. To 5ml of fresh ExpressHyb was added 14. mu.l of labeled probe and mixed thoroughly. The ExpressHyb solution used in the pre-hybridization was replaced by 5ml of fresh ExpressHyb solution containing the labeled probe uniformly distributed on the blotting membrane. The blot was incubated for 1 hour at 68 ℃ in a Hybaid hybridization oven. After incubation, the blot was washed and washed several times at low stringency (2 XSSC buffer containing 0.05% SDS, room temperature), followed by high stringency (containing 0.05% SDS)0.1 XSSC of 1% SDS, 50 ℃ C. [20 XSSC is 0.3M NaCl/0.3M sodium citrate, pH7.0]And (5) washing. The blot was exposed to Hyperfilm MP (Amersham pharmacia Biotech Ltd.) at-80 ℃ using an intensifying screen.

The results of the Northern blot hybridization experiments are shown in FIG. 1. FIGS. 1A (left) and 1B (right) are the same as those described in example 3³²polyA hybridized with P-labeled neublastin cDNA probe⁺RNAporter blot. The markers represent polynucleotides of 1.35 kilobase pairs ("kb"), 2.4kb, 4.4kb, 7.5kb, and 9.5kb in size. The membrane of FIG. 1A was prepared from mRNA extracted from various adult human tissues. From the results of Northern blot hybridization analysis, the applicant concluded that: neublastin mRNA is expressed in many adult tissues. The highest level of neublastin expression was detected in heart, skeletal muscle and pancreas. The membrane of FIG. 1B was prepared using RNA extracted from multiple regions of the adult brain. In the adult brain, expression levels were highest in the caudate nucleus and thalamus. In the brain, an mRNA transcript of approximately 5kb is the predominantly expressed form of neublastin mRNA.

A method for detecting neublastin RNA expression in a tissue using in situ hybridization:

neublastin RNA expression in animal tissues, e.g., rodent tissues, is assayed using neublastin antisense probes using the following technique.

Expression in mice:

preparing a tissue sample: pregnant mice were sacrificed by cervical dislocation on day 13.5 or 18.5 of pregnancy (B & K Universal, Stockholm, Sweden). Dissecting under sterile conditions, embryos are removed, immediately immersed in 0.1M phosphate buffered saline (PB) containing 4% paraformaldehyde ("PFA") for 24 to 30 hours, then removed from PFA and stored in PBs. The tissue for sectioning was prepared by immersing the tissue in a 30% sucrose solution, and then embedding the tissue in tissue tech (o.c. t.compound, sakura finetek USA, Torrance, CA). A series of 6 coronal or sagittal sections (12 μm each) were cut out on a cryostat and melt-fixed onto positively charged slides. After performing the same protocol as the embryonic stage, the head/brain of the neonate was fixed (P1, P7), adult brain tissue was dissected and immediately lyophilized in dry ice and sectioned on a cryostat without embedding.

Preparation of neublastin Riboprobe: an antisense neublastin RNA probe (hereinafter referred to as a neublastin riboprobe) was prepared as follows. Nucleotide 1109-1863 of mouse neublastin cDNA sequence [ SEQ ID NO: 15] subcloning into BlueScript vector (Stratagene). The resulting plasmid was cleaved into linear DNA using an EcoRI restriction endonuclease. EcoRI DNA fragments were transcribed in vitro using T3 RNA polymerase and digoxigenin ("DIG") RNA labeling kit according to the manufacturer's instructions (Boehringer Mannheim).

And (3) hybridization: cryostat sections were fixed in 4% PFA for 10 minutes, treated with 10mg/ml proteinase K for 5 minutes, dehydrated in 70% and 95% ethanol for 5 and 2 minutes, respectively, and then air dried. Hybridization buffer containing 1. mu.g/ml DIG-labeled probe (50% deionized formamide, 10% in 50% dextran sulfate solution, 1% Denhardt's solution, 250. mu.g/ml yeast tRNA, 0.3M NaCl, 20mM Tris-HCl (pH8), 5mM EDTA, 10mM NaPO. sup.₄1% sodium lauryl sarcosinate) was heated to 80 ℃ for 2 minutes and applied to the slices, which were then covered with parafilm and incubated at 55 ℃ for 16 to 18 hours.

The following day, sections were washed at 55 ℃ for 30 min under high stringency (2 XSSC with 50% formamide), then washed with RNase buffer and incubated with 20. mu.g/ml RNase A for 30 min at 37 ℃. To detect DIG-labeled probes, sections were preincubated for 1 hour in blocking solution (PBS containing 0.1% Tween-20 and 10% heat-inactivated goat serum) and then incubated overnight at 4 ℃ with alkaline phosphatase-conjugated anti-DIG antibody (Boehringer Mannheim) diluted 1: 5000. The following day, each section was washed 4 times for 2 hours with PBS containing 0.1% Tween-20, and then with NTMT buffer (100mM NaCl, 100mM Tris-HCl (pH9.5), 50mM MgCl₂0.1% tween-20) for 10 minutes each time 2 times. The sections were then incubated in BM-purple substrate containing 0.5mg/ml levamisole for 48 hours. Tong (Chinese character of 'tong')Color reactions were stopped by washing with PBS, sections were air dried and covered with envelopes with DPX (KEBO-lab, Sweden).

The results of the in situ hybridization reaction are shown in Table 1.

Table 1: expression of neublastin in mice

Structure of the product	E13.5	E18.5	P1	P7	Of adult origin
Structure of the product	E13.5	E18.5	P1	P7	Of adult origin	Forebrain	++
Anterior midbrain	-					Forebrain	++
Anterior midbrain	-					Lateral root ganglion	++
Spinal cord	+					Lateral root ganglion	++
Spinal cord	+					Retina		+++	+++	+
Smell ball		++	++	++		Retina		+++	+++	+
Smell ball		++	++	++		Dental pulp		++	++	+
Ganglion of trigeminal nerve		++	++	++		Dental pulp		++	++	+
Ganglion of trigeminal nerve		++	++	++		Striatum body		+	+	++
Cortex (cortex)		++	++	++	+	Striatum body		+	+	++
Cortex (cortex)		++	++	++	+	Tooth-shaped return ring				++	+

As shown in table 1, neublastin was expressed in the spinal cord and hindbrain at day 13.5 embryonic stage ("E13.5"), with only weak expression in the forebrain. Neublastin expression was also detected in developing retinal and sensory ganglia (lateral root ganglia and trigeminal ganglia (V)). In addition to the nervous system, weak signals were found in kidney, lung and intestine, indicating that neublastin is also expressed in these tissues.

Neublastin expression was most pronounced in the trigeminal ganglion (V) at day 18.5 embryonic stage ("E18.5"), and was also detected in the retina, striatum and cortex. In addition, expression was also observed in dental pulp.

Referring to table 1, neublastin expression increased progressively in the cortex, striatum and trigeminal ganglia (V) from the E18.5 time point to postnatal days 1 and 7. Neublastin expression was more pronounced in the outer cortex than in the inner cortex. At day P7, the same structure was expressed as at day P1, but additionally neublastin expression was found in the hippocampus, especially the dentate gyrus and cerebellum. In adult murine brains, neublastin is expressed abundantly in the dentate gyrus, whereas only very low or undetectable levels of neublastin expression were detected in other tissues tested.

Expression in rats:

the following experiment describes the hybridization of rat tissue with an alkaline phosphatase-labeled oligodeoxynucleotide neublastin antisense probe.

Preparing a tissue sample: after anaesthesia with pentobarbital, rat embryos were obtained from pregnant Wistar rats (molegaard Breeding, Denmark) (E14). Postnatal rats (P0, P7, adult) were killed by decapitation. Dissected brains and whole heads were immediately immersed in cold 0.9% NaCl, freshly frozen and cut into 20 μm (coronal or sagittal sections, 10 series) on a cryostat.

In situ hybridization: two series of sections were hybridized using an antisense Alkaline Phosphatase (AP) -conjugated oligodeoxynucleotide probe (5 '-NCAGGTGGTCCG TGGGGGGCGCCAAGACCGG-3' [ SEQ ID NO: 27], oligo. No.164675, DNA Technology, Denmark). The probe is similar to the probe shown in SEQ ID NO: 15, base 1140 to 1169 of the mouse neublastin cDNA.

Prior to hybridization, the sections were air-dried at room temperature, heated to 55 ℃ for 10 minutes, and then treated with 96% ethanol at 4 ℃ overnight. The sections were then air dried and incubated overnight at 39 ℃ in hybridization medium (5.0pmol probe/ml) (Finsen et al, neuroscience, 1992, 47, 105-113; West et al, J.Comp.Neurol, 1996, 370, 11-22).

Post-hybridization treatments included: washing with 1 XSSC (0.15M NaCl, 0.015M sodium citrate) at 55 ℃ for 4 times, 30 minutes each, followed by washing with Tris-HCl, pH9.5, 3 times, 10 minutes each, at room temperature, followed by the AP color developer. AP color reagent in-situ preparation, which contains nitro blue tetrazolium (NBT, Sigma), 5-bromine, 4-chlorine, 3-indole phosphoric acid (BCIP, Sigma) and Tris-HCl-MgCl₂Buffer, pH9.5(Finsen et al, neuroscience, 1992, 47, 105-. AP development was carried out in the dark for 48 hours at room temperature. The color reaction was stopped by rinsing the sections with distilled water. Sections were dehydrated in gradient acetone, softened with xylene-phenol wood oil (Allchem, UK), washed with xylene, and Eukitt (Bie) was used&Berntsen, Denmark) covered with an envelope.

Control reactions included (1) pre-treatment of sections with RNase A (50. mu.g/ml, Pharmacia, Sweden) prior to hybridization; (2) hybridizing the sections with a 100-fold excess of unlabeled probe; and (3) hybridizing the sections with only hybridization buffer. The results of the hybridization reaction are shown in Table 2.

Table 2: expression of neublastin in rats

Structure of the product	E14	P0/P1	P7	Of adult origin
Structure of the product	E14	P0/P1	P7	Of adult origin	Forebrain	++
Anterior midbrain	-				Forebrain	++
Anterior midbrain	-				Lateral root ganglion	++
Spinal cord	+				Lateral root ganglion	++
Spinal cord	+				Retina	+
Smell ball	(+)	++	++		Retina	+
Smell ball	(+)	++	++		Cerebellum		+	++	+
Ganglion of trigeminal nerve		++	++		Cerebellum		+	++	+
Ganglion of trigeminal nerve		++	++		Striatum body		+	+(+)
Cortex (cortex)	(+)	++	++	+	Striatum body		+	+(+)
Cortex (cortex)	(+)	++	++	+	Hippocampus japonicus		(+)	++	++

At day 14 embryonic stage (E14), neublastin is weakly expressed in the forebrain, hindbrain and spinal cord of rat embryos. Neublastin mRNA was also detected in the eye (retina), lateral root ganglia, trigeminal ganglia (V) and kidney, lung, heart, liver and intestine. Neublastin expression in the cortex and striatum was significant in neonatal (P0) rats. Neublastin expression was also detected in olfactory bulb and hippocampus. In 7-day-old (P7) rats, neublastin was expressed in the cortex, striatum, olfactory bulb and cerebellum. Significant signals were found in the hippocampus. In adult rats, very low or undetectable expression levels of neublastin were detected in most regions of the brain. A weak signal was detected in the thalamic nuclei and significant neublastin expression was detected in the hippocampus.

Example 4: neublastin polypeptides

In SEQ ID NO: the open reading frame or coding region (CDS) identified in 8 encodes a pre-pro-polypeptide (referred to as "pre-neublastin pro"). The amino acid sequence deduced from this open reading frame is shown in SEQ ID NO: 9, 3 neublastin polypeptide variants were identified, including:

(i) a polypeptide, referred to herein as NBN140, having the amino acid sequence of SEQ ID NO: 10;

(ii) a polypeptide, referred to herein as NBN116, having the amino acid sequence of SEQ ID NO: 11; and

(iii) a polypeptide, referred to herein as NBN113, having the amino acid sequence of SEQ ID NO: 12.

Similarly, according to SEQ ID NO: 3 encodes a polypeptide having the sequence of seq id NO: 4,3 neublastin variants were identified, including:

(i) has the sequence shown in SEQ ID NO: 5;

(ii) has the sequence shown in SEQ ID NO: 6; and

(iii) has the sequence shown in SEQ ID NO: 7.

Based on a Clustal W (1.75) based multiple sequence alignment, SEQ ID NO: 9 compared to the amino acid sequences of GDNF, persephin and neurturin. The comparative results are shown in Table 3.

Table 3: amino acid sequence comparison of neublastin with persephin, neurturin and GDNF

Neurturin-fullNeublastinPersephin-fullGDNF_HUMAN-fullNeurturin-fullNeublastinPersephin-fullGDNF_HUMAN-fullNeurturin-fullNeublastinPersephin-fullGDNF_HUMAN-fullNeurturin-fullNeublastinPersephin-fullGDNF_HUMAN-full

-----------MQRWKAAALASVLCSSVLSIWMCREGLLLSHRLGPAMELGLGGLSTLSHCPWPRRQPALWPTLAALALLSSVAEASLGSAPRSPAPREGPPP--------------------------MKLWDVVAVCLVLLHTASAFPLPAGKRPPEAPAEDRSLGRRRAPFALSSDSLVPLHRLPRTLDARIARLAQYRALLQGAPDAMELRELTPWAGRPPGPRRRAGPRRRVLASPAGHLPGGRTARWCSGRARRPPPQPSRPAPPPPAPPSALPRGGRAARAGGPG-MAVGKFLLGSLLLLSLQLGQGWGPDARGVPVADGEFSSEQVAKAGGTWLGTHRPLNMPEDYPDQFDDVMDFIQATIKRLKRSPDKQMAVLPRRERNRQAAAANPENSRGKGRARARLGARPCGLRELEVRVSELGLGYASDETVLFRYCAGACEA-AARVYDLGLRRSRARAAGARGCRLRSQLVPVRALGLGHRSDELVRFRFCSGSCRR-ARSPHDLSLASARLRRALSGPCQLWSLTLSVAELGLGYASEEKVIFRYCAGSCPRGARTQHGLALARRRGQRGKNRGCVLTAIHLNVTDLGLGYETKEELIFRYCSGSCDA-AETTYDKILKN* * : * ****: :.* : **:*:*:* * :. *LRQRRRLRRE---RVRAQPCCRPTAYEDEVSFLDAHSRYHTVHELSARECACV-LLGAGALRPPPGSRPVSQPCCRPTRYE-AVSFMDVNSTWRTVDRLSATACGCLGLQGQGRAHGG--------PCCRPTRYT-DVAFLDDRERWQRLPQLSAAACGCGGLSRNRRLVSD----KVGQACCRPIAFDDDLSFLDDNLVYHILRKHSAKRCGCI-* .**** : ::*:* . :: : . ** *.*

Denotes positions with a single fully conserved residue;

: indicating that one of the following "strong" groups is completely conserved: STA, NEQK, NHQK, NDEQ, QHRK MILV, MILF, HY, FYW;

indicates that one of the following "weaker" groups is completely conservative: -CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK, VLIM, HFY.

As can be seen from the amino acid sequence alignment shown in Table 3: neublastin has 7 conserved cysteine residues, which are located at positions conserved in the TGF- β superfamily. In one embodiment, preferred neublastin polypeptides contain (7) conserved cysteines as set forth in SEQ id no: 2, positions 8, 35, 39, 72, 73, 101 and 103, and in SEQ id no: 4 and 9, conserved positions are positions 43, 70, 74, 107, 108, 136 and 138. These 7 conserved cysteine residues are known to form 3 intramonomeric disulfide bonds in the TGF- β superfamily (e.g., disulfide bonds between cysteine residues 8-73, 35-101 and 39-103 in SEQ ID NO: 2, and cysteine residues 43-108, 70-136 and 74-138 in SEQ ID NOS: 4 and 9) and 1 intermonomic disulfide bond (disulfide bonds between cysteine residues 72-72 in SEQ ID NO: 2, and cysteine residues 107-107 in SEQ ID NOS: 4 and 9) which, together with the extended β chain region, constitute a conserved structural motif of the TGF- β superfamily, see, e.g., Daopin et al, proteins 1993, 17, 176-192.

Based on this sequence alignment, neublastin was shown to be a member of the GDNF subfamily of the neurotrophic factor (LGLGLG-FR (Y/F) CS GSC-QxCCRP-SAxxCGC, the underlined GDNF subfamily fingerprint in Table 3).

The homology of neublastin to other members of the GDNF family was calculated and the results are shown in table 4 below.

Table 4: homology of neublastin polypeptides to other members of the GDNF family

	Mature protein NBN140				Mature protein NBN113
	Mature protein NBN140				Mature protein NBN113					Homology of			Homology of full Length peptides	Homology of			Homology of full Length peptides
Neurotrophic factors	Identity of each other	Overlap (aa)	Strong homology	Identity of each other	Identity of each other	Overlap (aa)	Strong homology	Identity of each other		Homology of			Homology of full Length peptides	Homology of			Homology of full Length peptides
Neurotrophic factors	Identity of each other	Overlap (aa)	Strong homology	Identity of each other	Identity of each other	Overlap (aa)	Strong homology	Identity of each other	GDNFNTNPSPIHATGF-β2	34％(47/137)48％(61/127)44％(55/125)31％(25/81)23％(17/73)	1371271258173	48％(67/137)56％(72/127)56％(71/125)--	31.9％36.9％36.925.2％18.5％	36％(41/111)49％(56/114)45％(51/111)31％(25/81)23％(17/73)	1111141118173	52％(59/111)57％(66/114)57％(65/111)--	29.5％44.7％44.3％22.5％20.2％

GDNF ═ neurotrophic factor derived from glial cell line

NTN＝Neurturin

PSP＝Persephin

IHA-inhibin-alpha

TF-beta 2-transforming growth factor-beta 2

Strong homology indicates that one of the following "strong" groups is conserved: STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW.

Example 5: production of neublastin

We produced neublastin in both eukaryotic and prokaryotic cells as described below.

Expression vector: the full-length cDNA encoding neublastin was inserted into eukaryotic expression vector pUbi 1Z. The vector was generated by cloning the human UbC promoter into a modified form of pcDNA3.1/Zeo. Unmodified pcDNA3.1/Zeo is commercially available (Invitrogen). The modified pcDNA3.1/Zeo is smaller than the original vector because the ampicillin gene (positions 3933 to 5015) and the sequences from positions 2838 to 3134 have been removed. In the modified version of pcDNA3.1/Zeo, the CMV promoter was replaced by the UbC promoter from pTEJ-8(Johansen et al, FEBS Lett, 1990, 267, 289 294) to yield pUbi 1Z.

Mammalian cells express: the mammalian cell line HiB5, an immortalized rat neural cell line, was then transfected with pUbi1Z vector containing the neublastin coding sequence (Renfranz et al, cells, 1991, 66, 713-729). Several HiB5 cell lines stably expressing neublastin (as determined by RT-PCR) were established. In one such stable cell line, total RNA was combined with Northern blots³²P-labeled neublastin probe hybridization confirmed the expression of HiB5pUbi1zNBN 22. The results of these studies are shown in figure 2. HiB5pUbi1zNBN22 was then used as a source of neublastin to study the neurotrophic activity of neublastin.

FIG. 2 shows neublastin cDNA expression in HiB5pUbi1zNBN22 clone (i.e., using the invention as described above)³²P-labeled neublastin cDNA was hybridized to a Northern blot). Blots were prepared by extracting total RNA from untransfected HiB5 cells, HiB5pUbi1zNBN22 cells and HiB5pUbi1zGDNF14, respectively. As shown on the blot, the positions of the 28S and 18S rRNA bands correspond to 4.1kb and 1.9kb, respectively.

As shown in FIG. 3, antibodies raised against the neublastin-derived polypeptide also recognized approximately 13kD protein in conditioned medium from HiB5pUbi1zNBN22 clones, but not from untransfected HiB5 cells (see example 6).

Unmodified (i.e., not post-translationally modified) neublastin polypeptide NBN140[ SEQ ID NO: 10], NBN116[ SEQ ID NO: 11] and NBN113[ SEQ ID NO: 12] predicted molecular weights of 14.7kD, 12.4kD and 12.1kD, respectively.

The method comprises the following steps: total RNA (10. mu.g) from untransfected HiB5 cells and HiB5pUbi1zNBN22 clone was run on a 0.8% formaldehyde agarose gel and blotted onto a nylon membrane (Duralone, Stratagene) to prepare a Northern blot. The blot was aligned to 1.3kb as described in example 3³²P-labeled probes, which are labeled randomly over SEQ ID NO: 8 and further nucleotides from the 5 'UTR and 3' UTR of the neublastin cDNA. The blot was exposed to Hyperfilm MP (Amersham) using an intensifying screen at-80 ℃.

Conditioned media from HiB5pUbi1zNBN22 or untransfected HiB5 cells were incubated overnight in serum-free media supplemented with N2 supplement (Life Technologies; Cat. No.17502-048), concentrated, and separated on a 15% polyacrylamide gel (Amersham Pharmacia Biotech; Cat. No. 80-1262-01). The proteins were transferred to PVDF-membranes (Amersham Pharmacia Biotech; Cat. No. RPN-303F) and the non-specific protein-binding sites were blocked with PBS containing 0.1% Tween 20 and 5% skim milk. The membrane was incubated overnight with polyclonal neublastin antibody (1: 1000) followed by anti-rabbit IgG secondary antibody (Amersham Pharmacia Biotech; Cat. No. NA934) (1: 2000) conjugated to horseradish peroxidase. Immunostaining was observed using Enhanced Chemiluminescence (ECL) (Amersham Pharmacia Biotech; Cat. No. RPN2109) or ECL + (Amersham Pharmacia Biotech; Cat. No. RPN2132) according to the manufacturer's instructions (Amersham).

The results of these experiments are shown in figure 3. Fig. 3A and 3B show neublastin protein expression in transfected HiB5 cells. The overnight medium from untransfected HiB5 cells [ lane 1], or HiB5 clone stably transfected with neublastin cDNA [ lane 2] was concentrated as described above. The media was then analyzed by Western blotting using two different polyclonal antibodies Ab-1 and Ab-2 specific for neublastin as described in example 10. Both antibodies recognized a protein with a molecular weight of approximately 15kDa in the medium derived from transfected cells, whereas this protein was not found in untransfected HiB5 cells.

The cloned cDNA encoding neublastin may also be inserted into other eukaryotic expression vectors, such as eukaryotic expression vector TEJ-8(Johansen et al, FEBS Lett.1990, 267, 289-294) or pcDNA-3(Invitrogen), and the resulting expression plasmid transfected into any mammalian cell line, such as Chinese Hamster Ovary (CHO) cells, HEK293, COS, PC12 or RN33b cell lines or human neural stem cell lines. Neublastin protein is produced using a stable cell line expressing neublastin.

Expression in CHO cells

Construction of the plasmid DJC070.14To express Neublastin cDNA in chinese hamster ovary cells, a cDNA fragment encoding the human pre-Neublastin pro form was inserted into the mammalian expression vector pEAG347 to generate plasmid pjc070.14. pEAG347 contains the SV40 early and adenovirus major late promoters in tandem (derived from plasmid pAD 2. beta.; Norton and Coffin, molecular cell biology, 1985, 5, 281), the only NotI cloning site, followed by the SV40 late transcription terminator and polyA signals (from plasmid pCMV. beta.; MacGregor and Caskey, nucleic acids Res., 1989, 17, 2365). In addition, pEAG347 contains a plasmid backbone derived from pUC19 and dhfr derived from pSV2dhfr for MTX selection and amplification in transfected CHO cells.

Plasmid pJC070.14 was generated in two steps. First, the dna was purified using oligonucleotide KD2-8245 'AAGGAAAAAAGCGGCCGCCA TGGAACTTGG ACTTGGAGG 3' [ SEQ ID NO: 31], KD 2-8255 'TTTTTTCCTT GGCGG CCGCTCAGCCCAGGC AGCCGCAGG 3' [ SEQ ID NO: 32] and PFU polymerase, and isolating a fragment encoding the human pro-Neublastin form from plasmid pUbi1Z-NBN by polymerase chain reaction. This fragment was cloned into the Srf-1 site of pPCR-Script Amp SK (+) to generate plasmid pJC 069. In the second step, plasmid pJC069 was subjected to a partial Not-I digestion to generate a 685bp fragment (containing the neublastin gene) which was cloned into the Not-I site of plasmid pEAG347 to generate plasmid pJC070.14. Transcription of the neublastin gene in plasmid pJC070.14 is under the control of the adenovirus major late promoter.

Generation of a CHO cell line expressing neublastin200. mu.g of pJC070.14 were linearized by digestion with the restriction endonuclease Mlu-I. The DNA was extracted with phenol, chloroform, isoamyl alcohol (25: 24: 1) and precipitated with ethanol. The linearized DNA was resuspended in 20mM hepespH7.05, 137mM NaCl, 5mM KCl, 0.7mM Na₂HPO₄6mM glucose (HEBS), 4E7 CHO dukx B1 (dhfr) was introduced by electroporation (280V and 960. mu.F)^-) Cells (p 23). After electroporation, cells were returned to α + Modified Eagle's Medium (MEM) supplemented with 10% Fetal Bovine Serum (FBS) for 2 days of culture. The cells were then trypsinized and replated in α -MEM (lacking ribose and deoxyriboside) in 100mm plates (100,000 cells/dish) for 5 days with 10% dialyzed FBS. Subsequently, cells were divided at a density of 100,000 cells per 100mm dish and selected in 200nM methotrexate. Selecting resistant colonies, and scraping to 6-well culture plates; conditioned media from individual clones were screened using the test described below specific for neublastin. The 12 clones with the highest expression level of neublastin were scraped into T162 flasks and subsequently analyzed. As shown in FIG. 10, the CHO cell line produced 25 to 50ng/ml neublastin.

Ternary complex assay for neublastinWe analyzed the supernatant of CHO cell line cultures for the presence of neublastin using a modified version of the ternary complex assay described by Sanicola et al (Proc NatlAcad Sci USA 1997946238).

In this assay, the ability of GDNF-like molecules to mediate binding between the extracellular domain of RET and multiple co-receptors, GFR α 1, GFR α 2 and GFR α 3, can be assessed. Soluble forms of RET and co-receptors are produced as fusion proteins. Fusion proteins between the extracellular domain of rat RET and placental alkaline phosphatase (RET-AP) have been described, as well as fusion proteins between the extracellular domain of rat GFR α 1(WO 9744356; 1997, 11/27/1997, incorporated herein by reference) and the Fc domain of human IgG1(rGFR α 1-Ig) (Sanicola et al, Proc Natl Acad Sci USA 1997946238).

To generate a fusion protein between the extracellular domain of murine GFR α 3 and the Fc domain of human IgG1(mGFR α 3-Ig), a DNA fragment encoding amino acids 1-359 of murine RETL3 was ligated with a fragment containing the Fc domain of human IgG1 and cloned into expression vector pEAG347 to generate plasmid pGJ 144. Plasmid pGJ144 was transfected into Chinese Hamster Ovary (CHO) cells to generate a stable cell line capable of producing the fusion protein, which was purified on a protein A Sepharose immunoaffinity column using standard methods. Briefly, if GDNF-like molecules mediate the binding of co-receptors to RET in this assay, then the RET-AP fusion protein can be retained on the plate and the amount retained can be determined using a chemiluminescent substrate for alkaline phosphatase.

Dynex Microlite-1 ELISA plates (Dynex Technologies) were coated for 16 hours with 50mM bicarbonate/carbonate, pH9.6, containing 1. mu.g/ml goat antibody specific for human Fc. The ELISA plates were emptied and 300. mu.l TBS/0.5% Tween-20 (TBST) containing 1% I-block (Tropix) was added and left for 1 hour. After 3 washes with TBST, 100. mu.l of 1. mu.g/ml rGFR α 1-Ig or mGFR α 3-Ig diluted with conditioned medium from 293 EBNA cells expressing the RET-AP fusion gene was added to the wells. Then, 100. mu.l of conditioned medium from CHO neublastin clones were added to the upper wells of the vertical rows, each row of wells was serially diluted 2-fold down and incubated for 1.5 hours at room temperature. The ELISA plates were then washed 3 times with TBST, and washed with 200mM Tris, pH9.8, 10mM MgCl₂(CSPD buffer) 2 washes. The wash solution was then replaced with CSPD buffer containing 425. mu.M CSPD (Tropix) and 1mg/ml Sapphire chemiluminescence enhancer (Tropix), and incubated at room temperature for 30 minutes. Chemiluminescence output was measured using a Dynatech luminometer.

In the initial experiments, we investigated whether neubIastin produced by CHO cell lines could mediate binding of GFR α 1 or GFR α 3 to the extracellular domain of RET. As shown in fig. 11, conditioned medium from CHO cell clone #53 produced a strong signal in the ternary complex assay when the mGFR α 3-Ig fusion protein was included, but no signal was observed when the rGFR α 1-Ig fusion protein was included, indicating that neublastin bound GFR α 3 but not GFR α 1. This behavior clearly distinguishes neublastin from GDNF; as shown in FIG. 11, GDNF bound GFR α 1 but not GFR α 3. When conditioned medium or neat medium from the placental CHO cell line was tested, no signal was observed with either co-receptor fusion protein.

To quantify the expression level of neublastin in CHO cell lines, a standard curve was drawn using rGFR α 1-Ig and GDNF at a concentration starting at 1 ng/ml. Then using the standard curve to calculate neublastin concentrations of different CHO cell lines; the levels produced by the 5 CHO cell lines are shown in figure 10. Since this estimation is based on the untested assumption that the binding affinity between GDNF and GFR α 1 is similar to that between neublastin and GFR α 3, these levels are only approximate values.

Analysis of Neublastin from CHO cell line supernatants

To further analyze neublastin produced by CHO cell lines, proteins were extracted from the culture medium using GFR α 3-Ig fusion proteins and analyzed by Western blotting with polyclonal antibodies to the neublastin peptide.

In the first experiment neublastin was extracted with mGFR α 3-Ig bound to Sepharose beads. mGFR α 3-Ig was bound to Sepharose beads using conditions recommended by the manufacturer (Pharmacia). To 1.0ml of a sample of conditioned medium from a negative control CHO cell line or from the CHO cell line #16 producing neubIastin, 100 μ l of LmGFR α 3-Ig-Sepharose was added. The suspension was incubated on a shaking platform for 2 hours. Each suspension was centrifuged, the supernatant was removed, and then washed 3 times with 1.0ml of 10mM HEPES, 100mM NaCI, pH 7.5. Each resin was resuspended in 100. mu.l of 2 Xreduction sample buffer and heated to 100 ℃ for 5 minutes. Mu.l of sample buffer supernatant and 10. mu.l of molecular weight standards (FMC) were loaded onto each well of a 10-20% SDS-PAGE precast gel (Owl Scientific). The gel was run at 40mA constant current for 72 minutes.

For Western blot analysis, proteins were electroblotted onto nitrocellulose filters (Schleicher and Schuell) (400mA galvanostat, 45 min) in a buffer system of 10mM MCPS, 10% methanol, 0.05% SDS, pH11.2 in a Hofer Scientific apparatus. After transfer, the nitrocellulose filter was removed from the cassette and the molecular weight markers were visualized by staining with a 1% acetic acid solution containing 0.1% Ponceau S for 60 seconds. The membrane was cut into two sections and excess dye was removed by gentle agitation in distilled water. Membranes were blocked overnight at 4 ℃ with TBS containing 2% skim milk. Two pieces of membrane were incubated with two affinity purified anti-neublastin peptide antibodies (R30 and R31) each at a concentration of 1.0. mu.g/ml and dissolved in TBS containing 2% skim milk. The membrane was washed 3 times with TBS-Tween for 10 minutes each, and then incubated with goat anti-rabbit IgG-HRP conjugate (Biorad) diluted 1: 5000 for 30 minutes. The membranes were washed 3 times with TBS-Tween for 10 min each time and developed with ECL substrate (Amersham). As shown in figure 12, specific bands were detected in proteins extracted from neublastin-producing CHO cell lines (lanes 2 and 4) relative to the bands observed in proteins extracted from the negative control cell line with the two antibodies described above (lanes 1 and 3).

The lower band has a molecular weight of about 13kD and may represent the mature domain of neublastin, which is produced after cleavage of the pro-domain. The cleavage can be performed at 3 Arg- __ (e.g. -RXR ↓ -of the preproeublastin protein)_-) Any of the residues thereafter occur, thereby generating SEQ ID NOs: 10, 11 or 12, 140AA, 116AA or 113 AA. Unmodified (i.e., not post-translationally modified) neublastin polypeptide NBN140[ SEQ ID NO: 10]，NBN116[SEQ ID NO：11]And NBN113[ SEQ id no: 12]The predicted molecular weights of (A) are 14.7kD, 12.4kD and 12.1kD, respectively. Further analysis is required to confirm the identity of these bandsStructure and other neublastin specific bands.

In a second experiment, neublastin was extracted with hGFR α 3-Ig captured on ELISA plates. To generate a fusion protein between the extracellular domain of human GFR α 3(WO 97/44356; 1997, 11/27, incorporated herein by reference) and the Fc domain of human IgG1(hGFR α 3-Ig), a DNA fragment encoding amino acids 1-364 of human GFR α 3 was ligated with a fragment containing the Fc domain of human IgG1 and cloned into the expression vector CH269(Sanicola et al, Proc NatI Acad Sci USA 1997946238). The fusion protein encoded by this plasmid was transiently expressed in nuclear antigen (EBNA) cells encoded by 293-Epstein-Barr virus and purified on a protein A Sepharose immunoaffinity column using standard procedures.

6 wells of a 96-well plate were coated overnight at 4 ℃ with PBS (300 ml/well) containing 25mg/ml goat anti-human IgG (Fc. gamma. fragment specific; Jackson Immunoglucics). Wells were blocked with 400ml PBS containing 1% BSA for 1 hour at room temperature. PBST (PBS + 0.05% Tween 20) was washed 3 times, and 300ml hGFR α 3-Ig (10mg/ml in PBS with 0.1% BSA) was added to each well. The plates were incubated at room temperature for 1 hour and gently shaken (200 oscillations per minute) to allow maximum binding. The wells were then emptied and washed 3 more times with PBST. 250ml of conditioned medium from a negative control CHO cell line or from the neublastin producing CHO cell line #25 was added to each of the 3 wells. The plates were incubated at room temperature for 3 hours, gently shaken (300 oscillations per minute), and the wells washed 2 more times with PBST. To the first well 25ml of non-reducing Laemli loading buffer was added and the plate was shaken rapidly for 5 min to elute bound proteins (1300 rpm). The contents were transferred to the next well and the above process was repeated to elute the bound proteins in the second and third wells. After addition of b-mercaptoethanol (final concentration of 5%), the samples were boiled for 5 minutes and analyzed by SDS-PAGE on 10-20% polyacrylamide gels.

For Western blotting, proteins were transferred to nitrocellulose filters. The membranes were closed and probed with PBST containing 5% skim milk and washed with PBST. Neublastin was detected by electrochemiluminescence after reaction with polyclonal antibodies raised against two neublastin peptides (R30 and R31) (1ug/ml) followed by reaction with HRP-conjugated goat anti-rabbit antibody (BioRad). As shown in fig. 13, 5 neublastin specific bands were detected in proteins extracted from neublastin producing CHO cell line (lane 2). The lower two bands are very similar to the bands observed in fig. 12; the lower band may also represent the mature domain of neublastin, which is generated after cleavage of the pro-domain.

Subsequently, the band in FIG. 13 was analyzed (data not shown) and showed a size equivalent to the lowermost band in the gel of FIG. 13 after deglycosylation of the approximately 18kD band with PGNaseF. This indicates that glycosylated forms of neublastin protein can be produced in mammalian cells.

Expression of neublastin in E.coli

To express the neublastin gene in E.coli, synthetic genes were constructed with lower GC content and preferred E.coli codons. The synthetic genes were cloned into two vectors, pET19b and pMJB164 (a derivative of pET19 b). The construction of pET19b is shown in FIG. 14. In this construct, the sequence encoding the mature domain of neublastin (NBN113) is fused directly to the initiating methionine. The construction of pMJB164 is shown in FIG. 15. In this construct, the mature domain of neublastin is fused to a histidine tag (i.e., 10 histidines) separated by an enterokinase cleavage site. The histidine tag is preceded by the initial methionine.

Nucleotide sequence encoding neublastin in figure 14

ATGGCTGGAGGACCGGGATCTCGTGCTCGTGCAGCAGGAGCACGTGGCTGTCGTCTGCGTTCTCAACTAGTGCCGGTGCGTGCACTCGGACTGGGACACCGTTCCGACGAACTAGTACGTTTTCGTTTTTGTTCAGGATCTTGTCGTCGTGCACGTTCTCCGCATGATCTATCTCTAGCATCTCTACTAGGAGCCGGAGCACTAAGACCGCCGCCGGGATCTAGACCTGTATCTCAACCTTGTTGTAGACCTACTAGATACGAAGCAGTATCTTTCATGGACGTAAACTCTACATGGAGAACCGTAGATAGACTATCTGCAACCGCATGTGGCTGTCTAGGATGATAATAG[SEQ ID NO：29]

Nucleotide sequence encoding his-tagged neublastin in figure 15

ATGGGCCATCATCATCATCATCATCATCATCATCACTCGAGCGGCCATATCGACGACGACGACAAGGCTGGAGGACCGGGATCTCGTGCTCGTGCAGCAGGAGCACGTGGCTGTCGTCTGCGTTCTCAACTAGTGCCGGTGCGTGCACTCGGACTGGGACACCGTTCCGACGAACTAGTACGTTTTCGTTTTTGTTCAGGATCTTGTCGTCGTGCACGTTCTCCGCATGATCTATCTCTAGCATCTCTACTAGGAGCCGGAGCACTAAGACCGCCGCCGGGATCTAGACCTGTATCTCAACCTTGTTGTAGACCTACTAGATACGAAGCAGTATCTTTCATGGACGTAAACTCTACATGGAGAACCGTAGATAGACTATCTGCAACCGCATGTGGCTGTCTAGGATGATAATAG[SEQ ID NO：30]

Example 6: effect of neublastin on survival and ChAT Activity of embryonic dopaminergic neurons in rats

In this series of experiments, the effect exerted by the conditioned medium from neublastin producing HiB5pUbi1zNBN22 cells described above was evaluated.

Preparation of the culture: the ventral midbrain or spinal cord of rat E14 embryos was dissected in cold Hanks buffered saline (HBSS). The tissue blocks were incubated for 20 minutes at 37 ℃ in HBSS containing sterile filtered 0.1% trypsin (Worthington) and 0.05% DNase (Sigma). The tissue blocks were then washed 4 times with HBSS + 0.05% dnase and dissociated using a 1ml automatic pipette. The suspension was centrifuged at 600rpm for 5 minutes and the pellet was resuspended in serum conditioned medium (SCM; DMEM with 10% fetal bovine serum). Total cell number was estimated by trypan blue dye exclusion method at 100,000 cells/cm²(ii) was plated on 8-well channel slides (Nunc) coated with poly-L-lysine to assess dopaminergic neuron survival; or at 200,000 cells/cm²Was plated on 48-well plates (Nunc) to determine ChAT activity. At 37 ℃ in SCM at 5% CO₂/95％O₂And incubating the cells under an atmosphere of 95% humidity for 24 to 48 hours, and then replacing the original medium with serum-free medium (SFM) supplemented with neurotrophic factors.

Cells used to assess dopaminergic neuron survival were placed in SFM + trophic factors for 5 days, then fixed with 4% PFA for 5 minutes, and tyrosine hydroxylase was stained by immunohistochemistry.

Cells assayed for ChAT activity were placed in SFM for 3 days, then lysed with HBSS + 0.1% Triton X-100, and immediately frozen on dry ice until ChAT activity was assayed.

Adding nutritional factors: conditioned media were collected from untransfected HiB5 controls or HiB5 producing neublastin (HiB5pUbi1zNBN22) or GDNF (HiB5pUbi1 zGDNF-L17). HiB5pUbi1zNBN22 was shown to produce about 20ng GDNF/24 hr/10 by performing a GDNF-ELISA assay on conditioned media collected from cells⁵And (4) cells. Each cell line was incubated overnight with DMEM + 1% FCS, and the supernatant was removed and stored at-20 ℃ until use. When added to cells, the supernatant was diluted 50-fold with SFM. Each well was treated with HiB5 control supernatant (1: 50) + purified recombinant rat GDNF (0.03-10 ng/ml).

The results of these experiments are shown in figure 4. FIGS. 4A-4C show the effect of neublastin secreted by HiB5pUbi1zNBN22 cells on the survival of cultured rat embryonic neurons, dopaminergic neurons, anterior midbrain neurons and on ChAT activity in cholinergic cranial motor neurons in serum-free medium as described in example 5.1 above.

FIG. 4A illustrates a dose-response curve of the effect of recombinant GDNF on ChAT activity (dpm/hr) as determined in DIV5 in serum-free cultures initially established from E14 anterolateral midbrain [ i.e., HiB 5; GDNF 0.03 ng/ml; GDNF 0.1ng/ml; GDNF 0.3 ng/ml; GDNF1 ng/ml; GDNF 10 ng/ml; GDNF 100ng/ml ].

FIG. 4B illustrates ChAT activity (dpm/hr) as determined in DIV5 in serum-free cultures initially established from E14 antero-lateral midbrain. As shown in the figure, diluted conditioned medium from neublastin-producing HiB5pUbi1zNBN22 cells (neublastin) or GDNF-L17(GDNFL-17) cells producing GDNF [ i.e., neublastin 1: 10; neublastin 1: 50; GDNF L-171: 50 ].

FIG. 4C illustrates the number of tyrosine hydroxylase immunoreactive cells per well [ TH + cell number/well ] as determined in DIV7 in serum-free cultures initially established from E14 ventral mesencephalon. As shown in the figure, diluted conditioned medium from untransfected HiB5 cells (HiB5) or HiB5pUbi1zNBN22 cells producing neublastin (neublastin) or various concentrations of recombinant GDNF were added [ i.e., HiB 51: 10; HiB 51: 40; GDNF0.1 ng/ml; GDNF 10 ng/ml; GDNF 100ng/ml and neublastin 1: 40 ].

Conditioned medium from neublastin-transfected HiB5 cells diluted 1: 40 significantly increased the number of TH immunoreactive cells per well compared to control (untransfected) HiB5 cells at the same and lower dilutions (1: 10 and 1: 40) (see, e.g., FIG. 4B). The increase in TH-immunoreactive cells was comparable to that observed at the maximum GDNF concentration (10 ng/ml). This indicates that neublastin secreted into the culture medium has an effect on the survival of dopaminergic neuron populations derived from the rat embryo ventral midbrain. In contrast, unlike GDNF secreted by transfected HiB5 cells, conditioned medium from neublastin-transfected HiB5 cells had no effect on another neuronal population, cholinergic neurons, in the same cultures (see, e.g., fig. 4A).

Example 7: effect of neublastin on survival of thin-slice cultures of brain neurons in the foreside of porcine embryonic dopaminergic

In this experiment, the effect of co-cultured neublastin-producing HiB5pUbi1zNBN22 cells on thin-slice cultures of porcine embryonic ventral mesencephalon was investigated.

Preparation of the culture: the Ventral Midbrain (VM) was isolated from porcine embryos (E28; n ═ 12) under sterile conditions, cut into thin 400 μm sections, and placed in cold Gey's balanced salt solution (GIBCO) containing glucose (6.5 mg/ml). Tissue sections were cultured by an interfacial culture method, originally described by Stoppini et al [ L.Stoppini, P.A.Buchs, D.Muller, J.Neuroscientific methods, 1991, 37, 173-]Developed up。

Briefly, tissue sections were placed on a semi-permeable membrane (Millipore, 0.3 μm; 8 sections were placed on each membrane corresponding to one VM) and the membranes were inserted into serum-containing medium (Gibco BRL) in 6-well plates (Costar). Each well contained 1ml of medium (50% Optimem, 25% horse serum, 25% Hank's balanced salt solution (all from GIBCO)) supplemented with D-glucose to a final concentration of 25 mM. On day 0, 7000 transfected HiB5pUbi1zNBN22(neublastin) or 7000 untransfected HiB5 cells (controls) were seeded on each tissue section. First, HiB5 cells immortalized by the presence of a temperature-sensitive oncogene were proliferated by co-culturing in an incubator at 33 ℃ for 48 hours, and then transferred to an incubator at 37 ℃ to differentiate HiB5 cells. Medium was changed 2 times per week. Antimitotic agents and antibiotics are not used at any stage.

Determination of dopamine by HPLC: on days 12 and 21 in vitro, media were collected and analyzed for dopamine using HPLC with electrochemical detection (w.n.slooth, j.b.p.gramsbergen, journal of neuroscience methodology, 1995, 60, 141-49).

Tissue treatment and immunohistochemistry: on day 21, cultures were fixed for 60 minutes in phosphate buffer containing 4% paraformaldehyde. Dehydrated in 20% sucrose solution for 24 hours, frozen, and 20 μm sections (4 series) were cut on a cryostat and placed on gelatin coated microscope slides. Tyrosine Hydroxylase (TH) in one series of sections was immunostained. Briefly, sections were washed 3X 15 min with 0.05M Tris-buffered saline (TBS, pH7.4) containing 1% Triton X-100 and incubated for 30 min with TBS containing 10% fetal bovine serum (FBS, Life technologies). The tissues were then incubated with a 600-fold dilution of mouse anti-TH monoclonal antibody (Boehringer Mannheim) in TBS containing 10% FBS for 24 hours at 4 ℃. After washing with TBS containing 1% Triton X-100 for 3X 15 minutes, the sections were incubated for 60 minutes with biotinylated anti-mouse IgG antibody (Amersham) diluted 200-fold in TBS containing 10% FBS. The sections were then washed with TBS containing 1% Triton X-100 (3X 15 min) and washed with TBS containing 10%200-fold dilution of TBS in% FBS with streptavidin-peroxidase (Dako) was incubated for 60 min. After washing with TBS (3X 15 min), the reaction mixture was washed by washing with a solution containing 0.05% 3, 3-diaminobenzidine (Sigma) and 0.01% H₂O₂TBS treatment to observe bound antibodies. Finally, sections were dehydrated with ethanol, washed with xylene, and covered with envelopes in Eukitt.

Cell counting and morphometric analysis: immunoreactive TH-ir neurons were quantitated using bright field microscopy (Olympus). Only cells showing strong staining, with the cell structure remaining intact and clearly distinguishable nuclei were counted. The evaluation was performed on the basis of cell counts per 4 culture sections using a x 20-fold objective lens. The number of cells counted twice was corrected using the mean diameter of the nucleus of TH-ir neurons (6.6 ± 0.2 μm, n ═ 30) according to abercombie's formula (m.abercombie, anat.rec.194694239-47). The size of the nuclei was estimated using a neuronal tracing system (Neurolucida, Micro-BrightField).

The results of these experiments are shown in figure 5. FIGS. 5A-5C illustrate the effect of neublastin secreted by HiB5pUbi1zNBN22 cells on the function and survival of slice cultures of brain neurons in the dopaminergic ventral lateral foreside of porcine embryos co-cultured with HiB5pUbi1zNBN22 cells (neublastin) or HiB5 cells (control). Fig. 5A and 5B illustrate release of dopamine into the medium in DIV12[ dopamine (pmol/ml) -day 12] and DIV21[ dopamine (pmol/ml) -day 21], respectively. FIG. 5C illustrates the number of tyrosine hydroxylase immunoreactive cells [ TH-ir cells/culture ] per slice culture in DIV 21.

On day 12, HPLC analysis indicated: the medium obtained from the HiB5-neublastin co-culture contained 84% more dopamine than the medium obtained from the HiB5-C co-culture (fig. 5A). At day 21, the difference was 78% (FIG. 5B), and the cell count indicated that the HiB5-neublastin co-culture contained 66% more tyrosine hydroxylase immunoreactive neurons than the HiB5-C co-culture (p < 0.05) (FIG. 5C). This indicates that neublastin secreted by HiB5pUbi1zNBN22 clone has potential effect on the survival of porcine embryonic dopaminergic neurons.

Example 8: survival of lateral root ganglion cells in serum-free media

This example shows the neurotrophic activity of neublastin polypeptides compared to known neurotrophic factors.

Pregnant female mice were sacrificed by cervical dislocation and embryos were processed for culture as follows.

Lateral root ganglia were dissected from the indicated stage of C57/B16 mice (Mollegaard Breeding, Denmark) using electrolytically sharpened tungsten needles. Embryonic ganglia were incubated with calcium and magnesium free Hanks balanced salt solution containing 0.05% trypsin (Gibco/BRL) for 5 minutes at 37 ℃. The ganglia were treated with collagenase/dispase (1mg/ml) for 30 to 45 minutes, followed by trypsin/dnase (0.25%) for 15 minutes. After removal of the trypsin solution, the ganglia were washed 1 time with 10ml of DMEM containing 10% heat inactivated horse serum and gently triturated with a bead-fired Pasteur pipette to obtain a single cell suspension.

The cells were plated on 24-well plates (Nunc) previously coated with polyornithine (0.5mg/ml, overnight) and laminin (20mg/ml, 4 hours; Gibco/BRL) at 37 ℃ in humidified 5% CO₂Neurons were insulated in defined medium in an incubator consisting of Hams F14 supplemented with 2mM glutamine, 0.35% bovine serum albumin, 60ng/ml progesterone, 16mg/ml putrescine, 400ng/ml L-thyroxine, 38ng/ml sodium selenate, 340ng/ml triiodo-thyronine, 60mg/ml penicillin and 100mg/ml streptomycin.

After 48 hours of incubation, under phase contrast light microscopy, neurons were clearly identified due to their bipolar morphology. The percentage of survival of neurons in the absence or presence of trophic factors (added to the medium before seeding neurons at a concentration of 10ng/ml), or in conditioned medium from neublastin-producing HiB5pUbi1zNBN22 cells, was assessed by counting neurons in the wells at 48 hours.

The results of these experiments are shown in fig. 9, where:

0 represents a control experiment (lacking trophic factors);

1 represents the experiment in the presence of GDNF;

2 represents the experiment in the presence of Neurturin;

3 represents the experiment in the presence of Neublastin according to the invention;

e12 represents data from experiments performed on DRG cells isolated from day 12-embryos;

e16 represents data from experiments performed on DRG cells isolated from day 16-embryos;

p0 represents data from experiments performed on DRG cells isolated on the day of birth;

p7 represents data from experiments performed on DRG cells isolated from day 7 after birth; and

p15 represents data from experiments performed on DRG cells isolated from day 15 after birth.

These results clearly indicate that the neurotrophic factors of the present invention show activity comparable to or even superior to known neurotrophic factors.

Example 9: in vivo effects of neublastin on dopaminergic neurons of the substantia nigra

To test whether neublastin can protect mature nigral Dopamine (DA) neurons from 6-hydroxydopamine induced degradation, we utilized the parkinson's disease rat model (Sauer and Oertel, neuroscience, 1994, 59, 401-415) and lentiviral gene transfer of neublastin.

Lentivirus production: to generate the lentiviral transfer vector pHR' -neublastin encoding neublastin, the 1331bp BamHI fragment from neublastin cDNA was subcloned into the BamHI/BglII site of pSL301 (Invitrogen). Constructed therefromA1519 bp BamHI/XhoI fragment was excised in vivo and ligated to the BamHI/XhoI site of pHR' carrying a posttranslational fragment of woodchuck hepatitis virus (Zufferey R, Donello JE, Trono D, Hope TJ: regulatory elements post-transcriptional of woodchuck hepatitis virus enhance expression of transgenes delivered by retroviral vectors; J.Virol, 199973 (4): 2886-2892). To generate pHR-GDNF, the 701bpBamHI/XhoI fragment from pUbi1z-GDNF was ligated into the BamHI/XhoI site of pHR'.

Zufferey et al describe the generation of lentiviral vectors (Zufferey R, Nagy D, Mandel RJ, Naldini L, Trono D: multiple attenuated lentiviral vectors can be efficiently delivered in vivo; nat. Biotechnol, 1997, 15(9) 871-. Briefly, 293T cells were co-transfected with the transfer construct and the helper plasmid pr8.91 and pMDG. Virions released into the medium were collected at 48 and 72 hours post-transfection. To concentrate the virus, the medium was centrifuged at 141000g for 1.5 hours, and the pellet was dissolved in DMEM. The titer of the control carrying the Green Fluorescent Protein (GFP) gene in 283T cells was determined to be 10 by GFP fluorescence⁸Transformation Units (TU)/ml. The titer of the virions was determined using the RNA slot blotting technique (von Schwedler U, Song J, Aiken C, Trono D: Vif, crucial for DNA synthesis of human type I immunodeficiency Virus provirus in infected cells, J.Virol, 1993, 67(8) 4945-4955). In GDNF and neublastin supernatants, there were 10-fold fewer viral particles than in GFP supernatants.

Surgical plan: all animal-related work was performed according to the rules set out by the ethical committee for the use of animals at the laboratory of the Lund university.

A total of 21 adult female Sprague-Dawley rats (B & K Universal, Stockholm, Sweden) were used, illuminated at 12 hours: the rats were housed during the dark cycle without feeding and watering during the housing. Retrograde labeling and 6-OHDA lesions were performed 3 weeks prior to the lesions as described by Sauer and Oertel (Sauer and Oertel, neuroscience, 1994, 59, 401-415). Briefly, rats were injected bilaterally with 0.2. mu.l of a 2% retrotracked Fluoro-Gold (FG; Fluorochrome, Englewood, CO) solution (dissolved in 0.9% NaCl) under Equisthesin anesthesia (0.3ml/100 g). Injections were performed using a 2 μ l Hamilton syringe at the following coordinates: AP +0.5 mm; ML is + -3.4 mm from the anterior fontanel; DV-5.0 mm from dura mater, incisal line set at 0.0 mm. In addition, an additional 5 minutes of injection was performed at 0.05. mu.l/min before withdrawing the needle.

At 14 days after FG injection, animals received a total of 5 lentiviral vector deposits (1. mu.l/deposit) carrying the genes for Green Fluorescent Protein (GFP), neublastin or GDNF. The 4 deposits were injected into the striatum along two needle tracks at the following coordinates: AP +1.0mm, ML-2.6 mm, DV₁＝-5.0mm，DV₂-4.5mm and AP-0.0 mm, ML-3.7 mm, DV₁＝-5.0mm，DV₂-4.5 mm. At AP ═ 5.2mm, ML ═ 2.0mm, DV₁The deposit was injected at-6.3 mm onto the substantia nigra. The cutting line was set at-2.3 mm.

After 21 days of retrograde labeling and 7 days of lentivirus injection, the animals were re-anesthetized and a single deposit, namely 20 μ g of 6-OHDA (Sigma; calculated as free base and dissolved in 3 μ l of ice cold saline with 0.02% ascorbic acid) was injected into the right striatum at the same location as the FG deposit using a10 μ l Hamilton syringe. The injection rate was 1 μ l/min and was maintained for an additional 3 minutes before withdrawing the needle.

Tissue treatment: 21 days after injection of 6-OHDA, animals were deeply anesthetized with chloral hydrate, and saline (pH 7.4; room temperature) was perfused through the cardia for 1 minute, followed by perfusion of 200ml of ice-cold formaldehyde solution (0.1M phosphate buffer containing 4% paraformaldehyde, pH 7.4). Brains were dissected, fixed in the same fixative for 3 to 4 hours, and then transferred to 25% sucrose/0.1M phosphate buffer for 48 hours. Series of 5 40 μm sections through the striatum and Substantia Nigra (SN) were cut on an ice-cold microtome.

Quantitative assessment of dopaminergic neurons in SN: as described previously (Sauer and Oertel, neuroscience,1994, 59, 401-. Briefly, 3 serial sections were used, which were centered around the central terminal nuclear horizontal line of the accessory optic bundle (MTN; Paxinos and Watson (1997) panel-5.3), counting all labeled/stained neurons in the MTN lateral direction at 40-fold magnification (n-6-7/group). If FG-labeled neurons fluoresce brightly under surface illumination at a wavelength of 330nm, a neuronal profile is shown and at least one neural process is extended, i.e., these neurons are included.

On the injured side of animals receiving injection of lentivirus carrying GFP, the number of FG-positive substantia nigra neurons decreased to 18% of the intact side. In contrast, animals injected with lentivirus-neublastin showed near complete protection (89%) of the FG-positive substantia nigra neuron numbers. This was as effective as animals treated with lentivirus-GDNF, with 87% of retrogradely labeled neurons remaining on the injured side. This suggests that neublastin is a potent survival factor for damaged adult nigral dopamine neurons and is as potent as GDNF.

Figure 6 illustrates the in vivo effect of lentivirus-produced neublastin on nigral dopamine neurons. Neurons in the SN double dense layer of female Sprague Dawley rats were retrogradely labeled with Flunogold (FG), and 3 weeks later a single injection of the right striatum with 6-hydroxydopamine (6-OHDA). 1 week prior to injection of 6-OHDA, animals were injected with lentiviral vectors expressing neublastin [ neublastin ], GDNF [ GDNF ] or green fluorescent protein [ GFP ] as shown. The number of FG-labeled neurons on both sides of the striatum was determined 21 days after injection of 6-OHDA. The percentage of FG-labeled neurons in the lesion (right) side versus intact (left) side [% FG lesion/intact ] is shown for the striatum of 3 groups of animals.

Example 10: production of antibodies

To prepare anti-neublastin antibodies, the peptide 1: CRPTRYEAVSFMDVNST (amino acids 108 and 124 of SEQ ID NO: 9) or peptide 2: ALRPPPGSRPVSQPC (amino acids 93-107 of SEQ ID NO: 9) were used to immunize 2 rabbits, the peptides being conjugated to a carrier protein. 2 rabbits were immunized with each peptide at weeks 0,3, 6 and 10, and blood was collected at weeks 7 and 11. The second blood draw was affinity purified via a peptide affinity column. Depending on the peptide, the antibodies are referred to as Ab-1 and Ab-2.

Western blotting: 2X 10 in serum-free medium containing N2 supplement (GIBCO)⁶HiB5 cells stably transfected with neublastin cDNA (HiB5pUbi1zNBN22) or untransfected HiB5 cells were incubated overnight. The medium was concentrated on a small concentrator with a 5kDa cut-off membrane (Millipore, Bedford, Mass.), 5 × Laemmli sample buffer was added to the concentrated sample, heated to 95 ℃ for 5 minutes, the sample was separated by SDS polyacrylamide gel electrophoresis on a 15% acrylamide gel, and transferred to a PVDF-membrane. The residual protein-binding sites were blocked with PBS containing 5% skim milk and 0.1% Tween-20. The membrane was incubated overnight with neublastin antibody (1: 1000) followed by a secondary anti-rabbit or anti-mouse IgG antibody conjugated to horseradish peroxidase (1: 2000).

Immunostaining was visualized using enhanced chemiluminescence Plus (ECL +) according to the manufacturer's instructions (Amersham). The results of these experiments are shown in figure 3 and example 5.

Using standard techniques, we also generated rabbit polyclonal antibodies against the following peptides:

peptide R27: GPGSRARAAGARGC (amino acids 30-43 of SEQ ID NO: 9);

peptide R28: LGHRSDELVRFRFC (amino acids 57-70 of SEQ ID NO: 9);

peptide R29: CRRARSPHDLSL (amino acids 74-85 of SEQ ID NO: 9);

peptide R30: LRPPPGSRPVSQPC (amino acids 94-107 of SEQ ID NO: 9); and

peptide R31: STWRTVDRLSATAC (amino acids 123-136 of SEQ ID NO: 9).

Sequence listing

<160>33

<170>PatentIn Ver.2.0

<210>1

<211>865

<212>DNA

<213>Homo sapiens

<220>

<221>CDS

<222>(120)..(719)

<220>

<221>5′UTR

<222>(1)..(119)

<220>

<221>3′UTR

<222>(721)..(865)

<220>

<221> sig _ peptide

<222>(120)..(179)

<220>

<221> mat _ peptide

<222>(405)..(719)

<220>

<221> misc _ structure

<222>(661)..(663)

<223> CARBOYHD: glycosylated asparagine at Asn 87

<220>

<221> misc _ structure

<222>(426)..(623)

<223> DISULFID-Cys8-Cys73 disulfide bridge

<220>

<221> misc _ structure

<222>(507)..(707)

<223> DISULFID: cys35-Cys101 disulfide bridge

<220>

<221> misc _ structure

<222>(519)..(713)

<223> DISULFID: cys39-Cys103 disulfide bridge

<220>

<221> misc _ structure

<222>(616)..(619)

<223> DISULFID: cys72-Cys72 interchain disulfide bridge

<400>1

ctaggagccc atgcccggcc tgatctcagc ccgaggacag cccctccttg aggtccttcc 60

tccccaagcc cacctgggtg ccctctttct ccctgaggct ccacttggtc tctccgcgc 119

atg cct gcc ctg tgg ccc acc ctg gcc gct ctg gct ctg ctg agc agc 167

Met Pro Ala Leu Trp Pro Thr Leu Ala Ala Leu Ala Leu Leu Ser Ser

-95 -90 -85 -80

gtc gca gag gcc tcc ctg ggc tcc gcg ccc cgc agc cct gcc ccc cgc 215

Val Ala Glu Ala Ser Leu Gly Ser Ala Pro Arg Ser Pro Ala Pro Arg

-75 -70 -65

gaa ggc ccc ccg cct gtc ctg gcg tcc ccc gcc ggc cac ctg ccg ggg 263

Glu Gly Pro Pro Pro Val Leu Ala Ser Pro Ala Gly His Leu Pro Gly

-60 -55 -50

gga cgc acg gcc cgc tgg tgc agt gga aga gcc cgg cgg ccg cgc cgc 311

Gly Arg Thr Ala Arg Trp Cys Ser Gly Arg Ala Arg Arg Pro Arg Arg

-45 -40 -35

aga cac ttc tcg gcc cgc gcc ccc gcc gcc tgc acc ccc atc tgc tct 359

Arg His Phe Ser Ala Arg Ala Pro Ala Ala Cys Thr Pro Ile Cys Ser

-30 -25 -20

tcc ccg cgg gtc cgc gcg gcg cgg ctg ggg ggc cgg gca gcg cgc tcg 407

Ser Pro Arg Val Arg Ala Ala Arg Leu Gly Gly Arg Ala Ala Arg Ser

-15 -10 -5 -1 1

ggc agc ggg ggc gcg ggg tgc cgc ctg cgc tcg cag ctg gtg ccg gtg 455

Gly Ser Gly Gly Ala Gly Cys Arg Leu Arg Ser Gln Leu Val Pro Val

5 10 15

cgc gcg ctc ggc ctg ggc cac cgc tcc gac gag ctg gtg cgt ttc cgc 503

Arg Ala Leu Gly Leu Gly His Arg Ser Asp Glu Leu Val Arg Phe Arg

20 25 30

ttc tgc acc ggc tcc tgc ccg cgc gcg cgc tct cca cac gac ctc agc 551

Phe Cys Thr Gly Ser Cys Pro Arg Ala Arg Ser Pro His Asp Leu Ser

35 40 45

ctg gcc agc cta ctg ggc gcc ggg gcc ctg cga ccg Gcc ccg ggc tcc 599

Leu Ala Ser Leu Leu Gly Ala Gly Ala Leu Arg Pro Pro Pro Gly Ser

50 55 60 65

cgg ccc gtc agc cag ccc tgc tgc cga ccc acg cgc tac gaa gcg gtc 647

Arg Pro Val Ser Gln Pro Cys Cys Arg Pro Thr Arg Tyr Glu Ala Val

70 75 80

tcc ttc atg gac gtc aac agc acc tgg aga acc gtg gac cgc ctc tcc 695

Ser Phe Met Asp Val Asn Ser Thr Trp Arg Thr Val Asp Arg Leu Ser

85 90 95

gcc acc gcc tgc ggc tgc ctg ggc tgagggctcg ctccagggct ttgcagactg 749

Ala Thr Ala Cys Gly Cys Leu Gly

100 105

gacccttacc ggtggctctt cctgcctggg accctcccgc agagtcccac tagccagcgg 809

cctcagccag ggacgaaggc ctcaaagctg agaggcccct gccggtgggt gatgga 865

<210>2

<211>200

<212>PRT

<213>Homo sapiens

<400>2

Met Pro Ala Leu Trp Pro Thr Leu Ala Ala Leu Ala Leu Leu Ser Ser

-95 -90 -85 -80

Val Ala Glu Ala Ser Leu Gly Ser Ala Pro Arg Ser Pro Ala Pro Arg

-75 -70 -65

Glu Gly Pro Pro Pro Val Leu Ala Ser Pro Ala Gly His Leu Pro Gly

-60 -55 -50

Gly Arg Thr Ala Arg Trp Cys Ser Gly Arg Ala Arg Arg Pro Arg Arg

-45 -40 -35

Arg His Phe Ser Ala Arg Ala Pro Ala Ala Cys Thr Pro Ile Cys Ser

-30 -25 -20

Ser Pro Arg Val Arg Ala Ala Arg Leu Gly Gly Arg Ala Ala Arg Ser

-15 -10 -5 -1 1

Gly Ser Gly Gly Ala Gly Cys Arg Leu Arg Ser Gln Leu Val Pro Val

5 10 15

Arg Ala Leu Gly Leu Gly His Arg Ser Asp Glu Leu Val Arg Phe Arg

20 25 30

Phe Cys Thr Gly Ser Cys Pro Arg Ala Arg Ser Pro His Asp Leu Ser

35 40 45

Leu Ala Ser Leu Leu Gly Ala Gly Ala Leu Arg Pro Pro Pro Gly Ser

50 55 60 65

Arg Pro Val Ser Gln Pro Cys Cys Arg Pro Thr Arg Tyr Glu Ala Val

70 75 80

Ser Phe Met Asp Val Asn Ser Thr Trp Arg Thr Val Asp Arg Leu Ser

85 90 95

Ala Thr Ala Cys Gly Cys Leu Gly

100 105

<210>3

<211>861

<212>DNA

<213>Homo sapiens

<220>

<221>CDS

<222>(7)..(717)

<220>

<221>5′UTR

<222>(1)..(6)

<220>

<221>3′UTR

<222>(718)..(861)

<220>

<221> sig _ peptide

<222>(7)..(174)

<220>

<221> mat _ peptide

<222>(298)..(717)

<220>

<221> mat _ peptide

<222>(370)..(717)

<220>

<221> mat _ peptide

<222>(379)..(717)

<220>

<221> misc _ structure

<222>(661)..(663)

<223> CARBOYHD: glycosylated asparagine at Asn 122

<220>

<221> misc _ structure

<222>(424)..(621)

<223> DISULFID: cys43-Cys108 disulfide bridge

<220>

<221> misc _ structure

<222>(505)..(705)

<223> DISULFID: cys70-Cys136 disulfide bridge

<220>

<221> misc _ structure

<222>(517)..(711)

<223> DISULFID: cys74-Cys138 disulfide bridge

<220>

<221> misc _ structure

<222>(616)..(618)

<223> DISULFID: cys107-Cys107 interchain disulfide bridge

<400>3

gagccc atg ccc ggc ctg atc tca gcc cga gga cag ccc ctc ctt gag 48

Met Pro Gly Leu Ile Ser Ala Arg Gly Gln Pro Leu Leu Glu

-95 -90 -85

gtc ctt cct ccc caa gcc cac ctg ggt gcc ctc ttt ctc cct gag gct 96

Val Leu Pro Pro Gln Ala His Leu Gly Ala Leu Phe Leu Pro Glu Ala

-80 -75 -70

cca ctt ggt ctc tcc gcg cag cct gcc ctg tgg ccc acc ctg gcc gct 144

Pro Leu Gly Leu Ser Ala Gln Pro Ala Leu Trp Pro Thr Leu Ala Ala

-65 -60 -55

ctg gct ctg ctg agc agc gtc gca gag gcc tcc ctg ggc tcc gcg ccc 192

Leu Ala Leu Leu Ser Ser Val Ala Glu Ala Ser Leu Gly Ser Ala Pro

-50 -45 -40

cgc agc cct gcc ccc cgc gaa ggc ccc ccg cct gtc ctg gcg tcc ccc 240

Arg Ser Pro Ala Pro Arg Glu Gly Pro Pro Pro Val Leu Ala Ser Pro

-35 -30 -25 -20

gcc ggc cac ctg ccg ggg gga cgc acg gcc cgc tgg tgc agt gga aga 288

Ala Gly His Leu Pro Gly Gly Arg Thr Ala Arg Trp Cys Ser Gly Arg

-15 -10 -5

gcc cgg cgg ccg ccg ccg cag cct tct cgg ccc gcg ccc ccg ccg cct 336

Ala Arg Arg Pro Pro Pro Gln Pro Ser Arg Pro Ala Pro Pro Pro Pro

-1 1 5 10

gca ccc cca tct gct ctt ccc cgc ggg ggc cgc gcg gcg cgg gct ggg 384

Ala Pro Pro Ser Ala Leu Pro Arg Gly Gly Arg Ala Ala Arg Ala Gly

15 20 25

ggc ccg ggc aac cgc gct cgg gca gcg ggg gcg cgg ggc tgc cgc ctg 432

Gly Pro Gly Asn Arg Ala Arg Ala Ala Gly Ala Arg Gly Cys Arg Leu

30 35 40 45

cgc tcg cag ctg gtg ccg gtg cgc gcg ctc ggc ctg ggc cac cgc tcc 480

Arg Ser Gln Leu Val Pro Val Arg Ala Leu Gly Leu Gly His Arg Ser

50 55 60

gac gag ctg gtg cgt ttc cgc ttc tgc agc ggc tcc tgc cgc cgc gcg 528

Asp Glu Leu Val Arg Phe Arg Phe Cys Ser Gly Ser Cys Arg Arg Ala

65 70 75

cgc tct cca cac gac ctc agc ctg gcc agc cta ctg ggc gcc ggg gcc 576

Arg Ser Pro His Asp Leu Ser Leu Ala Ser Leu Leu Gly Ala Gly Ala

80 85 90

ctg cga ccg ccc ccg ggc tcc cgg ccc gtc agc cag ccc tgc tgc cga 624

Leu Arg Pro Pro Pro Gly Ser Arg Pro Val Ser Gln Pro Cys Cys Arg

95 100 105

ccc acg cgc tac gaa gcg gtc tcc ttc atg gac gtc aac agc acc tgg 672

Pro Thr Arg Tyr Glu Ala Val Ser Phe Met Asp Val Asn Ser Thr Trp

110 115 120 125

aga acc gtg gac cgc ctc tcc gcc aac ccc tgc ggc tgc ctg ggc 717

Arg Thr Val Asp Arg Leu Ser Ala Asn Pro Cys Gly Cys Leu Gly

130 135 140

tgagggctcg ctccagggct ttgcagactg gacccttacc ggtggctctt cctgcctggg 777

accctcccgc agagtcccac tagccagcgg cctcagccag ggacgaaggc ctcaaagctg 837

agaggcccct gccggtgggt gatg 86l

<210>4

<211>237

<212>PRT

<213>Homo sapiens

<400>4

Met Pro Gly Leu Ile Ser Ala Arg Gly Gln Pro Leu Leu Glu Val Leu

-95 -90 -85

Pro Pro Gln Ala His Leu Gly Ala Leu Phe Leu Pro Glu Ala Pro Leu

-80 -75 -70

Gly Leu Ser Ala Gln Pro Ala Leu Trp Pro Thr Leu Ala Ala Leu Ala

-65 -60 -55 -50

Leu Leu Ser Ser Val Ala Glu Ala Ser Leu Gly Ser Ala Pro Arg Ser

-45 -40 -35

Pro Ala Pro Arg Glu Gly Pro Pro Pro Val Leu Ala Ser Pro Ala Gly

-30 -25 -20

His Leu Pro Gly Gly Arg Thr Ala Arg Trp Cys Ser Gly Arg Ala Arg

-15 -10 -5

Arg Pro Pro Pro Gln Pro Ser Arg Pro Ala Pro Pro Pro Pro Ala Pro

-1 1 5 10 15

Pro Ser Ala Leu Pro Arg Gly Gly Arg Ala Ala Arg Ala Gly Gly Pro

20 25 30

Gly Asn Arg Ala Arg Ala Ala Gly Ala Arg Gly Cys Arg Leu Arg Ser

35 40 45

Gln Leu Val Pro Val Arg Ala Leu Gly Leu Gly His Arg Ser Asp Glu

50 55 60

Leu Val Arg Phe Arg Phe Cys Ser Gly Ser Cys Arg Arg Ala Arg Ser

65 70 75

Pro His Asp Leu Ser Leu Ala Ser Leu Leu Gly Ala Gly Ala Leu Arg

80 85 90 95

Pro Pro Pro Gly Ser Arg Pro Val Ser Gln Pro Cys Cys Arg Pro Thr

100 105 110

Arg Tyr Glu Ala Val Ser Phe Met Asp Val Asn Ser Thr Trp Arg Thr

115 120 125

Val Asp Arg Leu Ser Ala Asn Pro Cys Gly Cys Leu Gly

130 135 140

<210>5

<211>140

<212>PRT

<213>Homo sapiens

<220>

<223> wherein Xaa at position 134 represents Asn or Thr,

yaa at position 135 represents Ala or Pro

<400>5

Pro Pro Pro Gln Pro Ser Arg Pro Ala Pro Pro Pro Pro Ala Pro Pro

1 5 10 15

Ser Ala Leu Pro Arg Gly Gly Arg Ala Ala Arg Ala Gly Gly Pro Gly

20 25 30

Asn Arg Ala Arg Ala Ala Gly Ala Arg Gly Cys Arg Leu Arg Ser Gln

35 40 45

Leu Val Pro Val Arg Ala Leu Gly Leu Gly His Arg Ser Asp Glu Leu

50 55 60

Val Arg Phe Arg Phe Cys Ser Gly Ser Cys Arg Arg Ala Arg Ser Pro

65 70 75 80

His Asp Leu Ser Leu Ala Ser Leu Leu Gly Ala Gly Ala Leu Arg Pro

85 90 95

Pro Pro Gly Ser Arg Pro Val Ser Gln Pro Cys Cys Arg Pro Thr Arg

100 105 110

Tyr Glu Ala Val Ser Phe Met Asp Val Asn Ser Thr Trp Arg Thr Val

115 120 125

Asp Arg Leu Ser Ala Xaa Yaa Cys Gly Cys Leu Gly

130 135 140

<210>6

<211>116

<212>PRT

<213>Homo sapiens

<220>

<223> wherein Xaa at position 110 represents Asn or Thr,

yaa at position 111 represents Ala or Pro

<400>6

Ala Ala Arg Ala Gly Gly Pro Gly Asn Arg Ala Arg Ala Ala Gly Ala

1 5 10 15

Arg Gly Cys Arg Leu Arg Ser Gln Leu Val Pro Val Arg Ala Leu Gly

20 25 30

Leu Gly His Arg Ser Asp Glu Leu Val Arg Phe Arg Phe Cys Ser Gly

35 40 45

Ser Cys Arg Arg Ala Arg Ser Pro His Asp Leu Ser Leu Ala Ser Leu

50 55 60

Leu Gly Ala Gly Ala Leu Arg Pro Pro Pro Gly Ser Arg Pro Val Ser

65 70 75 80

Gln Pro Cys Cys Arg Pro Thr Arg Tyr Glu Ala Val Ser Phe Met Asp

85 90 95

Val Asn Ser Thr Trp Arg Thr Val Asp Arg Leu Ser Ala Xaa Yaa Cys

100 105 110

Gly Cys Leu Gly

115

<210>7

<211>113

<212>PRT

<213>Homo sapiens

<220>

<223> wherein Xaa at position 107 represents Asn or Thr,

yaa at position 108 represents Ala or Pro

<400>7

Ala Gly Gly Pro Gly Asn Arg Ala Arg Ala Ala Gly Ala Arg Gly Cys

1 5 10 15

Arg Leu Arg Ser Gln Leu Val Pro Val Arg Ala Leu Gly Leu Gly His

20 25 30

Arg Ser Asp Glu Leu Val Arg Phe Arg Phe Cys Ser Gly Ser Cys Arg

35 40 45

Arg Ala Arg Ser Pro His Asp Leu Ser Leu Ala Ser Leu Leu Gly Ala

50 55 60

Gly Ala Leu Arg Pro Pro Pro Gly Ser Arg Pro Val Ser Gln Pro Cys

65 70 75 80

Cys Arg Pro Thr Arg Tyr Glu Ala Val Ser Phe Met Asp Val Asn Ser

85 90 95

Thr Trp Arg Thr Val Asp Arg Leu Ser Ala Xaa Yaa Cys Gly Cys Leu

100 105 110

Gly

<210>8

<211>861

<212>DNA

<213>Homo sapiens

<220>

<221>CDS

<222>(58)..(717)

<220>

<221>5′UTR

<222>(1)..(57)

<220>

<221>3′UTR

<222>(718)..(861)

<220>

<221> sig _ peptide

<222>(58)..(174)

<220>

<221> mat _ peptide

<222>(298)..(717)

<220>

<221> mat _ peptide

<222>(370)..(717)

<220>

<221> mat _ peptide

<222>(379)..(717)

<220>

<221> misc _ structure

<222>(661)..(663)

<223> CARBOYHD: glycosylated asparagine at Asn 122

<220>

<221> misc _ structure

<222>(424)..(621)

<223> DISULFID: gly43-Gly108 disulfide bridge

<220>

<221> misc _ structure

<222>(505)..(705)

<223> DISULFID: gly70-Gly136 disulfide bridge

<220>

<221> misc _ structure

<222>(517)..(711)

<223> DISULFID: gly74-Gly138 disulfide bridge

<220>

<221> misc _ structure

<222>(616)..(618)

<223> DISULFID: gly107-Gly107 interchain disulfide bridge

<400>8

aggagggtgg gggaacagct caacaatggc tgatgggcgc tcctggtgtt gatagag 57

atg gaa ctt gga ctt gga ggc ctc tcc acg ctg tcc cac tgc ccc tgg 105

Met Glu Leu Gly Leu Gly Gly Leu Ser Thr Leu Ser His Cys Pro Trp

-80 -75 -70 -65

cct agg cgg cag cct gcc ctg tgg ccc acc ctg gcc gct ctg gct ctg 153

Pro Arg Arg Gln Pro Ala Leu Trp Pro Thr Leu Ala Ala Leu Ala Leu

-60 -55 -50

ctg agc agc gtc gca gag gcc tcc ctg ggc tcc gcg ccc cgc agc cct 201

Leu Ser Ser Val Ala Glu Ala Ser Leu Gly Ser Ala Pro Arg Ser Pro

-45 -40 -35

gcc ccc cgc gaa ggc ccc ccg cct gtc ctg gcg tcc ccc gcc ggc cac 249

Ala Pro Arg Glu Gly Pro Pro Pro Val Leu Ala Ser Pro Ala Gly His

-30 -25 -20

ctg ccg ggg gga cgc acg gcc cgc tgg tgc agt gga aga gcc cgg cgg 297

Leu Pro Gly Gly Arg Thr Ala Arg Trp Cys Ser Gly Arg Ala Arg Arg

-15 -10 -5 -1

ccg ccg ccg cag cct tct cgg ccc gcg ccc ccg ccg cct gca ccc cca 345

Pro Pro Pro Gln Pro Ser Arg Pro Ala Pro Pro Pro Pro Ala Pro Pro

1 5 10 15

tct gct ctt ccc cgc ggg ggc cgc gcg gcg cgg gct ggg ggc ccg ggc 393

Ser Ala Leu Pro Arg Gly Gly Arg Ala Ala Arg Ala Gly Gly Pro Gly

20 25 30

agc cgc gct cgg gca gcg ggg gcg cgg ggc tgc cgc ctg cgc tcg cag 441

Ser Arg Ala Arg Ala Ala Gly Ala Arg Gly Cys Arg Leu Arg Ser Gln

35 40 45

ctg gtg ccg gtg cgc gcg ctc ggc ctg ggc cac cgc tcc gac gag ctg 489

Leu Val Pro Val Arg Ala Leu Gly Leu Gly His Arg Ser Asp Glu Leu

50 55 60

gtg cgt ttc cgc ttc tgc agc ggc tcc tgc cgc cgc gcg cgc tct cca 537

Val Arg Phe Arg Phe Cys Ser Gly Ser Cys Arg Arg Ala Arg Ser Pro

65 70 75 80

cac gac ctc agc ctg gcc agc cta ctg ggc gcc ggg gcc ctg cga ccg 585

His Asp Leu Ser Leu Ala Ser Leu Leu Gly Ala Gly Ala Leu Arg Pro

85 90 95

ccc ccg ggc tcc cgg ccc gtc agc cag ccc tgc tgc cga ccc acg cgc 633

Pro Pro Gly Ser Arg Pro Val Ser Gln Pro Cys Cys Arg Pro Thr Arg

100 105 110

tac gaa gcg gtc tcc ttc atg gac gtc aac agc acc tgg aga acc gtg 681

Tyr Glu Ala Val Ser Phe Met Asp Val Asn Ser Thr Trp Arg Thr Val

115 120 125

gac cgc ctc tcc gcc acc gcc tgc ggc tgc ctg ggc tgagggctcg 727

Asp Arg Leu Ser Ala Thr Ala Cys Gly Cys Leu Gly

130 135 140

ctccagggct ttgcagactg gacccttacc ggtggctctt cctgcctggg accctcccgc 787

agagtcccac tagccagcgg cctcagccag ggacgaaggc ctcaaagctg agaggcccct 847

accggtgggt gatg 861

<210>9

<211>220

<212>PRT

<213>Homo sapiens

<400>9

Met Glu Leu Gly Leu Gly Gly Leu Ser Thr Leu Ser His Cys Pro Trp

-80 -75 -70 -65

Pro Arg Arg Gln Pro Ala Leu Trp Pro Thr Leu Ala Ala Leu Ala Leu

-60 -55 -50

Leu Ser Ser Val Ala Glu Ala Ser Leu Gly Ser Ala Pro Arg Ser Pro

-45 -40 -35

Ala Pro Arg Glu Gly Pro Pro Pro Val Leu Ala Ser Pro Ala Gly His

-30 -25 -20

Leu Pro Gly Gly Arg Thr Ala Arg Trp Cys Ser Gly Arg Ala Arg Arg

-15 -10 -5 -1

Pro Pro Pro Gln Pro Ser Arg Pro Ala Pro Pro Pro Pro Ala Pro Pro

1 5 10 15

Ser Ala Leu Pro Arg Gly Gly Arg Ala Ala Arg Ala Gly Gly Pro Gly

20 25 30

Ser Arg Ala Arg Ala Ala Gly Ala Arg Gly Cys Arg Leu Arg Ser Gln

35 40 45

Leu Val Pro Val Arg Ala Leu Gly Leu Gly His Arg Ser Asp Glu Leu

50 55 60

Val Arg Phe Arg Phe Cys Ser Gly Ser Cys Arg Arg Ala Arg Ser Pro

65 70 75 80

His Asp Leu Ser Leu Ala Ser Leu Leu Gly Ala Gly Ala Leu Arg Pro

85 90 95

Pro Pro Gly Ser Arg Pro Val Ser Gln Pro Cys Cys Arg Pro Thr Arg

100 105 110

Tyr Glu Ala Val Ser Phe Met Asp Val Asn Ser Thr Trp Arg Thr Val

115 120 125

Asp Arg Leu Ser Ala Thr Ala Cys Gly Cys Leu Gly

130 135 140

<210>10

<211>140

<212>PRT

<213>Homo sapiens

<220>

<221>CARBOHYD

<222>(122)

<223> glycosylated asparagine

<400>10

Pro Pro Pro Gln Pro Ser Arg Pro Ala Pro Pro Pro Pro Ala Pro Pro

1 5 10 15

Ser Ala Leu Pro Arg Gly Gly Arg Ala Ala Arg Ala Gly Gly Pro Gly

20 25 30

Ser Arg Ala Arg Ala Ala Gly Ala Arg Gly Cys Arg Leu Arg Ser Gln

35 40 45

Leu Val Pro Val Arg Ala Leu Gly Leu Gly His Arg Ser Asp Glu Leu

50 55 60

Val Arg Phe Arg Phe Cys Ser Gly Ser Cys Arg Arg Ala Arg Ser Pro

65 70 75 80

His Asp Leu Ser Leu Ala Ser Leu Leu Gly Ala Gly Ala Leu Arg Pro

85 90 95

Pro Pro Gly Ser Arg Pro Val Ser Gln Pro Cys Cys Arg Pro Thr Arg

100 105 110

Tyr Glu Ala Val Ser Phe Met Asp Val Asn Ser Thr Trp Arg Thr Val

115 120 125

Asp Arg Leu Ser Ala Thr Ala Cys Gly Cys Leu Gly

130 135 140

<210>11

<211>116

<212>PRT

<213>Homo sapiens

<220>

<221>CARBOHYD

<222>(98)

<223> glycosylated asparagine

<400>11

Ala Ala Arg Ala Gly Gly Pro Gly Ser Arg Ala Arg Ala Ala Gly Ala

1 5 10 15

Arg Gly Cys Arg Leu Arg Ser Gln Leu Val Pro Val Arg Ala Leu Gly

20 25 30

Leu Gly His Arg Ser Asp Glu Leu Val Arg Phe Arg Phe Cys Ser Gly

35 40 45

Ser Cys Arg Arg Ala Arg Ser Pro His Asp Leu Ser Leu Ala Ser Leu

50 55 60

Leu Gly Ala Gly Ala Leu Arg Pro Pro Pro Gly Ser Arg Pro Val Ser

65 70 75 80

Gln Pro Cys Cys Arg Pro Thr Arg Tyr Glu Ala Val Ser Phe Met Asp

85 90 95

Val Asn Ser Thr Trp Arg Thr Val Asp Arg Leu Ser Ala Thr Ala Cys

100 105 110

Gly Cys Leu Gly

115

<210>12

<211>113

<212>PRT

<213>Homo sapiens

<220>

<221>CARBOHYD

<222>(95)

<223> glycosylated asparagine

<400>12

Ala Gly Gly Pro Gly Ser Arg Ala Arg Ala Ala Gly Ala Arg Gly Cys

1 5 10 15

Arg Leu Arg Ser Gln Leu Val Pro Val Arg Ala Leu Gly Leu Gly His

20 25 30

Arg Ser Asp Glu Leu Val Arg Phe Arg Phe Cys Ser Gly Ser Cys Arg

35 40 45

Arg Ala Arg Ser Pro His Asp Leu Ser Leu Ala Ser Leu Leu Gly Ala

50 55 60

Gly Ala Leu Arg Pro Pro Pro Gly Ser Arg Pro Val Ser Gln Pro Cys

65 70 75 80

Cys Arg Pro Thr Arg Tyr Glu Ala Val Ser Phe Met Asp Val Asn Ser

85 90 95

Thr Trp Arg Thr Val Asp Arg Leu Ser Ala Thr Ala Cys Gly Cys Leu

100 105 110

Gly

<210>13

<211>102

<212>DNA

<213>Homo sapiens

<400>13

cctggccagc ctactgggcg ccggggccct gcgaccgccc ccgggctccc ggcccgtcag 60

ccagccctgc tgccgaccca cgcgctacga agcggtctcc tt 102

<210>14

<211>220

<212>DNA

<213>Murinae gen.sp.

<400>14

ggccaccgct ccgacgagct gatacgtttc cgcttctgca gcggctcgtg ccgccgagca 60

cgctcccagc acgatctcag tctggccagc ctactgggcg ctggggccct acggtcgcct 120

cccgggtccc ggccgatcag ccagccctgc tgccggccca ctcgctatga ggccgtctcc 180

ttcatggacg tgaacagcac ctggagaacc gtggaccgcc 220

<210>15

<211>2136

<212>DNA

<213>Murinae gen.sp.

<220>

<221>CDS

<222>(975)..(1646)

<400>15

gcggccgcga attcggcacg agggcgtctc gctgcagccc gcgatctcta ctctgcctcc 60

tggggtcttc tccaaatgtc tagcccccac ctagagggac ctagcctagc cagcggggac 120

cggatccgga gggtggagcg gccaggtgag ccctgaaagg tggggcgggg cgggggcgct 180

ctgggcccca ccccgggatc tggtgacgcc ggggctggaa tttgacaccg gacggcggcg 240

ggcaggaggc tgctgaggga tggagttggg ctcggccccc agatgcggcc cgcgggctct 300

gccagcaaca agtccctcgg gccccagccc tcgctgcgac tggggcttgg agccctgcac 360

ccaagggcac agaccggctg ccaaggcccc acttttaact aaaagaggcg ctgccaggtg 420

cacaactctg ggcatgatcc acttgagctt cgggggaaag cccagcactg gtcccaggag 480

aggcgcctag aaggacacgg accaggaccc ctttggtatg gagtgaacgc tgagcatgga 540

gtggaaggaa ctcaagttac tactttctcc aaccaccctg gtaccttcag ccctgaagta 600

cagagcagaa gggtcttaga agacaggacc acagctgtgt gagtctcccc cctgaggcct 660

tagacgatct ctgagctcag ctgagctttg tttgcccatc tggagaagtg agccattgat 720

tgaccttgtg gcatcgcgaa ggaacaggtc ctgccaagca cctaacacag agagcaaggt 780

tctccatcgc agctaccgct gctgagttga ctctagctac tccaacctcc tgggtcgctt 840

cgagagactg gagtggaagg aggaataccc caaaggataa ctaactcatc tttcagtttg 900

caagctgccg caggaagagg gtggggaaac gggtccacga aggcttctga tgggagcttc 960

tggagccgaa agct atg gaa ctg gga ctt gca gag cct act gca ttg tcc 1010

Met Glu Leu Gly Leu Ala Glu Pro Thr Ala Leu Ser

1 5 10

cac tgc ctc cgg cct agg tgg cag tca gcc tgg tgg cca aac cta gct 1058

His Cys Leu Arg Pro Arg Trp Gln Ser Ala Trp Trp Pro Thr Leu Ala

15 20 25

gtt cta gcc ctg ctg agc tgc gtc aca gaa gct tcc ctg gac cca atg 1106

Val Leu Ala Leu Leu Ser Cys Val Thr Glu Ala Ser Leu Asp Pro Met

30 35 40

tcc cgc agc ccc gcc gct cgc gac ggt ccc tca ccg gtc ttg gcg ccc 1154

Ser Arg Ser Pro Ala Ala Arg Asp Gly Pro Ser Pro Val Leu Ala Pro

45 50 55 60

ccc acg gac cac ctg cct ggg gga cac act gcg cat ttg tgc agc gaa 1202

Pro Thr Asp His Leu Pro Gly Gly His Thr Ala His Leu Cys Ser Glu

65 70 75

aga acc ctg cga ccc ccg cct cag tct cct cag ccc gca ccc ccg ccg 1250

Arg Thr Leu Arg Pro Pro Pro Gln Ser Pro Gln Pro Ala Pro Pro Pro

80 85 90

cct ggt ccc gcg ctc cag tct cct ccc gct gcg ctc cgc ggg gca cgc 1298

Pro Gly Pro Ala Lau Gln Ser Pro Pro Ala Ala Leu Arg Gly Ala Arg

95 100 105

gcg gcg cgt gca gga acc cgg agc agc cgc gca cgg acc aca gat gcg 1346

Ala Ala Arg Ala Gly Thr Arg Ser Ser Arg Ala Arg Thr Thr Asp Ala

110 115 120

cgc ggc tgc cgc ctg cgc tcg cag ctg gtg ccg gtg agc gcg ctc ggc 1394

Arg Gly Cys Arg Leu Arg Ser Gln Leu Val Pro Val Ser Ala Leu Gly

125 130 135 140

cta ggc cac agc tcc gac gag ctg ata cgt ttc cgc ttc tgc agc ggc 1442

Leu Gly His Ser Ser Asp Glu Leu Ile Arg Phe Arg Phe Cys Ser Gly

145 150 155

tcg tgc cgc cga gca cgc tcc cag cac gat ctc agt ctg gcc agc cta 1490

Ser Cys Arg Arg Ala Arg Ser Gln His Asp Leu Ser Lau Ala Ser Leu

160 165 170

ctg ggc gct ggg gcc cta cgg tcg cct ccc ggg tcc cgg ccg atc agc 1538

Leu Gly Ala Gly Ala Leu Arg Ser Pro Pro Gly Ser Arg Pro Ile Ser

175 180 185

cag ccc tgc tgc cgg ccc act cgc tat gag gcc gtc tcc ttc atg gac 1586

Gln Pro Cys Cys Arg Pro Thr Arg Tyr Glu Ala Val Ser Phe Met Asp

190 195 200

gtg aac agc acc tgg agg acc gtg gac cac ctc tcc gcc act gcc tgc 1634

Val Asn Ser Thr Trp Arg Thr Val Asp His Leu Ser Ala Thr Ala Cys

205 210 215 220

ggc tgt ctg ggc tgaggatgat ctatctccaa gcctttgcac actagaccca 1686

Gly Cys Leu Gly

tgtgttgccc tacctggaac agctccaccg ggcctcacta accaggagcc tcaactcagc 1746

aggatatgga ggctgcagag ctcaggcccc aggccggtga gtgacagacg tcgtcggcat 1806

gacagacaga gtgaaagatg tcggaaccac tgaccaacag tcccaagttg ttcatggatc 1866

ccagctctac agacaggaga aacctcagct aaagagaact cctctgggag aatccagaaa 1926

tggccctctg tcctggggaa tgaattttga agagatatat atacatatat acattgtagt 1986

cgcgttgctg gaccagcctg tgctgaaacc agtcccgtgt tcacttgtgg aagccgaagc 2046

cctatttatt atttctaaat tatttattta ctttgaaaaa aaacggccaa gtcggcctcc 2106

ctttagtgag ggttaatttg tgatcccggg 2136

<210>16

<211>224

<212>PRT

<213>Murinae gen.sp.

<400>16

Met Glu Leu Gly Leu Ala Glu Pro Thr Ala Leu Ser His Cys Leu Arg

1 5 10 15

Pro Arg Trp Gln Ser Ala Trp Trp Pro Thr Leu Ala Val Leu Ala Leu

20 25 30

Leu Ser Cys Val Thr Glu Ala Ser Leu Asp Pro Met Ser Arg Ser Pro

35 40 45

Ala Ala Arg Asp Gly Pro Ser Pro Val Leu Ala Pro Pro Thr Asp His

50 55 60

Leu Pro Gly Gly His Thr Ala His Leu Cys Ser Glu Arg Thr Leu Arg

65 70 75 80

Pro Pro Pro Gln Ser Pro Gln Pro Ala Pro Pro Pro Pro Gly Pro Ala

85 90 95

Leu Gln Ser Pro Pro Ala Ala Leu Arg Gly Ala Arg Ala Ala Arg Ala

100 105 110

Gly Thr Arg Ser Ser Arg Ala Arg Thr Thr Asp Ala Arg Gly Cys Arg

115 120 125

Leu Arg Ser Gln Leu Val Pro Val Ser Ala Leu Gly Leu Gly His Ser

130 135 140

Ser Asp Glu Leu Ile Arg Phe Arg Phe Cys Ser Gly Ser Cys Arg Arg

145 150 155 160

Ala Arg Ser Gln His Asp Leu Ser Leu Ala Ser Leu Leu Gly Ala Gly

165 170 175

Ala Leu Arg Ser Pro Pro Gly Ser Arg Pro Ile Ser Gln Pro Cys Cys

180 185 190

Arg Pro Thr Arg Tyr Glu Ala Val Ser Phe Met Asp Val Asn Ser Thr

195 200 205

Trp Arg Thr Val Asp His Leu Ser Ala Thr Ala Cys Gly Cys Leu Gly

210 215 220

<210>17

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PCR primer

<400>17

cctggccagc ctactggg 18

<210>18

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PCR primer

<400>18

aaggagaccg cttcgtagcg 20

<210>19

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PCR primer

<400>19

atggaacttg gacttgg 17

<210>20

<211>16

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PCR primer

<400>20

tccatcaccc accggc 16

<210>21

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PCR primer

<400>21

ggccaccgct ccgacgag 18

<210>22

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PCR primer

<400>22

ggcggtccac ggttctccag 20

<210>23

<211>29

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PCR primer

<400>23

ccaagcccac ctgggtgccc tctttctcc 29

<210>24

<211>27

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PCR primer

<400>24

catcacccac cggcaggggc ctctcag 27

<210>25

<211>35

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PCR primer

<400>25

gagcccatgc ccggcctgat ctcagcccga ggaca 35

<210>26

<211>34

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PCR primer

<400>26

ccctggctga ggccgctggc tagtgggact ctgc 34

<210>27

<211>31

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: hybridization of

<400>27

ncaggtggtc cgtggggggc gccaagaccg g 31

<210>28

<211>16

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PCR primer

<400>28

ctaggagccc atgccc 16

<210>29

<211>351

<212>DNA

<213>Homo sapiens

<400>29

atggctggag gaccgggatc tcgtgctcgt gcagcaggag cacgtggctg tcgtctgcgt 60

tctcaactag tgccggtgcg tgcactcgga ctgggacacc gttccgacga actagtacgt 120

tttcgttttt gttcaggatc ttgtcgtcgt gcacgttctc cgcatgatct atctctagca 180

tctctactag gagccggagc actaagaccg ccgccgggat ctagacctgt atctcaacct 240

tgttgtagac ctactagata cgaagcagta tctttcatgg acgtaaactc tacatggaga 300

accgtagata gactatctgc aaccgcatgt ggctgtctag gatgataata g 351

<210>30

<211>414

<212>DNA

<213>Homo sapiens

<400>30

atgggccatc atcatcatca tcatcatcat catcactcga gcggccatat cgacgacgac 60

gacaaggctg gaggaccggg atctcgtgct cgtgcagcag gagcacgtgg ctgtcgtctg 120

cgttctcaac tagtgccggt gcgtgcactc ggactgggac accgttccga cgaactagta 180

cgttttcgtt tttgttcagg atcttgtcgt cgtgcacgtt ctccgcatga tctatctcta 240

gcatctctac taggagccgg agcactaaga ccgccgccgg gatctagacc tgtatctcaa 300

ccttgttgta gacctactag atacgaagca gtatctttca tggacgtaaa ctctacatgg 360

agaaccgtag atagactatc tgcaaccgca tgtggctgtc taggatgata atag 414

<210>31

<211>39

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PCR primer

<400>31

aaggaaaaaa gcggccgcca tggaacttgg acttggagg 39

<210>32

<211>39

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PCR primer

<400>32

ttttttcctt ggcggccgct cagcccaggc agccgcagg 39

<210>33

<211>16

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PCR primer

<400>33

gagcgagccc tcagcc 16

Claims

1. An isolated Neublastin nucleic acid comprising a sequence selected from the group consisting of:

a) SEQ ID NO: 1;

b) a nucleic acid sequence comprising a nucleotide sequence encoding a polypeptide expressed as a sequence comprising SEQ ID NO: 2 AA₁-AA₁₀₅The open reading frame of a Neublastin polypeptide or a neurotrophic polypeptide derived therefrom, wherein said neurotrophic polypeptide is produced by adding, deleting and/or substituting less than 10.5 amino acids in the sequence of said Neublastin polypeptide or by conservative substitutions of less than 10% of the residues in the sequenceAnd from said Neublastin polypeptide, wherein said neurotrophic polypeptide has an amino acid sequence set forth in SEQ id no: 2 at positions 8, 35, 39, 72, 73, 101 and 103;

c) a nucleic acid capable of hybridizing to a nucleic acid having the sequence of SEQ id no: 1 or its complementary strand, and encodes a neurotrophic polypeptide.

2. The nucleic acid of claim 1, encoding a polypeptide that is SEQ ID NO: 2 AA₁-AA₁₀₅。

3. The nucleic acid of claim 1, wherein the encoded neurotrophic polypeptide comprises a GDNF subfamily motif: LGLGLGLG-FRxCxGxC-xxCCRP-SAxxCxC.

4. The nucleic acid of claim 1, wherein the encoded neurotrophic polypeptide is a member of the GDNF subfamily of neurotrophic factors.

5. An expression vector comprising the nucleic acid of any one of claims 1 to 4.

6. A cell transformed in vitro with a nucleic acid according to any one of claims 1 to 4 and/or an expression vector according to claim 5.

7. The cell of claim 6, wherein the cell is a eukaryotic cell.

8. The cell of claim 7, wherein the cell is selected from the group consisting of a mammalian cell and a fungal cell including a yeast cell.

9. The cell of claim 8, wherein the cell is selected from the group consisting of chinese hamster ovary cells, HEK293, COS, PC12, HiB5, RN33b, and human neural stem cells.

10. God managementA polypeptide comprising a polypeptide consisting of SEQ ID NO: 2 AA₁-AA₁₀₅A sequence derived from the amino acid sequence of SEQ ID NO, or a sequence derived from the amino acid sequence by addition, deletion and/or substitution of less than 10.5 amino acids, or a sequence encoded by the amino acid sequence of SEQ ID NO: 2 AA₁-AA₁₀₅A sequence derived from a constituent amino acid sequence having fewer than 10% of its residues conservatively substituted, or a polypeptide consisting of a sequence that hybridizes under high stringency solution hybridization conditions to a polypeptide having the sequence set forth in SEQ ID NO: 1 or a complementary strand thereof, or a nucleic acid encoding a specific hybridization,

wherein the neurotrophic polypeptide has an amino acid sequence set forth in SEQ ID NO: 2, positions 8, 35, 39, 72, 73, 101 and 103.

11. The neurotrophic polypeptide of claim 10, which comprises a GDNF subfamily motif: LGLGLGLG-FRxCxGxC-xxCCRP-SAxxCxC.

12. The neurotrophic polypeptide of claim 10, wherein said polypeptide is a member of the GDNF subfamily.

13. The neurotrophic polypeptide of claim 10, which comprises SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.

14. The polypeptide of any one of claims 10-13, wherein the polypeptide is glycosylated.

15. The polypeptide of claim 10, wherein the polypeptide is encoded by a nucleic acid of any one of claims 1 to 4.

16. A method of preparing a polypeptide according to any one of claims 10 to 15, said method comprising the step of expressing said polypeptide from a nucleic acid encoding said polypeptide.

17. The method of claim 16, comprising the step of culturing cells containing said Neublastin neurotrophic factor nucleic acid in a medium that permits production of said polypeptide.

18. The method of claim 17, further comprising the step of recovering the polypeptide from the culture medium.

19. A composition comprising the polypeptide of any one of claims 10-15 and a pharmaceutically acceptable carrier.

20. Use of a polypeptide according to any one of claims 10 to 15 in the manufacture of a medicament for the treatment of a neurodegenerative disease involving injured and traumatized neurons.

21. Use according to claim 20, wherein the disease is trauma of peripheral nerves, trauma of medullary and/or spinal nerves, cerebral ischemic neuronal damage, neuropathies and in particular peripheral neuropathies, alzheimer's disease, huntington's disease, parkinson's disease, amyotrophic lateral sclerosis or any other neurodegenerative disease, and memory impairment associated with dementia.

22. A method of producing a Neublastin polypeptide of any one of claims 10-15, said method comprising:

(a) introducing into a cell a polynucleotide encoding a Neublastin polypeptide according to any one of claims 10-15 or introducing into a cell, by homologous recombination, a regulatory sequence such that the regulatory sequence is capable of regulating the expression of an endogenous Neublastin gene, thereby producing a Neublastin producing cell;

(b) neublastin-producing cells are cultured under culture conditions that result in expression of the Neublastin polypeptide.

23. A synthetic gene encoding a Neublastin polypeptide, said synthetic gene comprising SEQ id no: 29 or 30.

24. A Neublastin polypeptide consisting of any one of the following sequences:

GPGSRARAAGARGC (AA 30-43 of SEQ ID NO: 9);

LGHRSDELVRFRFC (AA 57-70 of SEQ ID NO: 9);

CRRARSPHDLSL (AA 74-85 of SEQ ID NO: 9);

LRPPPGSRPVSQPC (AA 94-107 of SEQ ID NO: 9);

STWRTVDRLSATAC (AA 123-136 of SEQ ID NO: 9).

CRPTRYEAVSFMDVNST (AA 108-124 of SEQ ID NO: 9); or

ALRPPPGSRPVSQPC (AA 93-107 of SEQ ID NO: 9).

25. An antibody raised against any of the peptides of claim 24.