Note: Descriptions are shown in the official language in which they were submitted.
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NTN-2 MEMBER OF TNF LIGAND FAMILY
All publications, patents and patent applications cited in this specification
are hereby incorporated by reference as if each individual publication, patent
or patent application was specifically and individually indicated to be
incorporated by reference.
INTRODUCTION
io
Field of the Invention
The field of this invention is polypeptide molecules which regulate cell
function, nucleic acid sequences encoding the polypeptides, and methods of
using the nucleic acid sequences and the polypeptides.
Background
-~-
Tumor necrosis factor-alpha (TNF-alpha) is a cytokine primarily produced
2o by activated macrophages. TNF-alpha stimulates T-cell and B-cell
proliferation and induces expression of adhesion molecules on endothelial
cells. This cytokine also plays an important role in host defense to
infection.
TNF-alpha activities are mediated through two distinct receptors, TNFR-p55
and TNFR-p75. These two receptors also mediate activities triggered by
soluble lymphotoxin-alpha (LT-alpha) secreted mainly by activated
lymphocytes. Specific stimulation of TNFR-p55 induces TNF activities such
as in vitro tumor cell cytotoxicity, expression of adhesion molecules on
endothelial cells and keratinocytes, activation of sphingomyelinase with
concomitant increases of ceramide, activation of NF-kappaB and induction
of manganese superoxide dismutase mRNA. Specific stimulation of TNFR-
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p75 results in proliferative response of mouse and human thymocytes and
cytoxic T cells, fibroblasts and natural killer cells and in GM-CSF secretion
in
PC60 cells.
TNF, especially in combination with gamma.-interferon (IFN-.gamma.), has
the ability to selectively kill or inhibit malignant cell lines that is
unmatched by any other combination of cytokines. Clinical trials in cancer
patients with TNF-.alpha. antitumor therapy have been disappointing,
however, because the toxic side effects of TNF have prevented obtaining
1o effective dose levels in man. These toxic side effects have been attributed
to TNF binding to the TNFR-p75 receptor while the cytotoxic activity on
malignant cells has been attributed to binding of TNF to the TNFR-p55
receptor.
SUMMARY OF THE INVENTION
The subject invention is a molecule that is homologous to tumor necrosis
factor (TNF). The invention provides methods and compositions relating
to the molecule, HUMAN NTN-2 polypeptide, and related nucleic acids.
Included are polypeptides comprising a HUMAN NTN-2-specific domain
and having HUMAN NTN-2 -specific activity. The polypeptides may be
produced recombinantly from transformed host cells with the subject
nucleic acids. The invention provides binding agents such as specific
antibodies, and methods of making and using the subject compositions in
diagnosis (e.g., genetic hybridization screens for HUMAN NTN-2
transcripts), therapy (e.g., gene therapy to modulate HUMAN NTN-2 gene
expression) and in the biopharmaceutical industry (e.g., reagents for
screening chemical libraries for lead pharmacological agents).
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Preferred uses for the subject HUMAN NTN-2 polypeptides include
modifying the physiology of a cell comprising an extracellular surface by
contacting the cell or medium surrounding the cell with an exogenous
HUMAN NTN-2 polypeptide under conditions whereby the added
polypeptide specifically interacts with a component of the medium and/or
the extracellular surface to effect a change in the physiology of the cell.
Also
preferred are methods for screening for biologically active agents, which
methods involve incubating a HUMAN NTN-2 polypeptide in the presence
of an extracellular HUMAN NTN-2 polypeptide-specific binding target and
1o a candidate agent, under conditions whereby, but for the presence of the
agent, the polypeptide specifically binds the binding target at a reference
affinity; detecting the binding affinity of the polypeptide to the binding
target
to determine an agent-biased affinity, wherein a difference between the
agent-biased affinity and the reference affinity indicates that the agent
modulates the binding of the polypeptide to the binding target.
Based upon its homology to TNF, it is expected that HUMAN NTN-2 will be
a mediator of immune regulation and inflammatory response, closely
linked to the development of disease. It may be useful for regulating
2o development, proliferation and death of cells of the lymphoid, hematopoitic
and other lineages. Also, HUMAN NTN-2 may be of use in the prevention
of septic shock, autoimmune disorders and graft-host disease. Furthermore,
HUMAN NTN-2 polypeptide may be used to identify its receptor.
BRIEF DESCRIPTION OF THE FIGURE
FIGURE 1 - Northern analysis of various human tissue specific RNAs using
a 608 nucleotide fragment of the HUMAN NTN-2 sequence as a probe.
Lanes 1 - 8 in order as follows: Heart, Brain, Placenta, Lung, Liver, Skeletal
3o Muscle, Kidney and Pancreas.
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DETAILED DESCRIPTION OF THE INVENTION
The invention provides HUMAN NTN-2 polypeptide which includes
natural HUMAN NTN-2 polypeptide and recombinant polypeptides
comprising a HUMAN NTN-2 amino acid sequence, or a functional
HUMAN NTN-2 polypeptide domain thereof having an assay-discernable
HUMAN NTN-2-specific activity. Accordingly, the polypeptides may be
deletion mutants of the disclosed natural HUMAN NTN-2 polypeptides
and may be provided as fusion products, e.g., with non- HUMAN NTN-2
to polypeptides. The subject HUMAN NTN-2 polypeptide domains have
HUMAN NTN-2-specific activity or function.
A number of applications for HUMAN NTN-2 are suggested from its
properties. HUMAN NTN-2, may be useful in the study and treatment of
conditions similar to those which are treated using TNF. Furthermore, the
HUMAN NTN-2 cDNA may be useful as a diagnostic tool, such as through
use of antibodies in assays for polypeptides in cell lines or use of
oligonucleotides as primers in a PCR test to amplify those with sequence
similarities to the oligonucleotide primer, and to see how much HUMAN
2o NTN-2 is present. The isolation of HUMAN NTN-2, of course, also
provides the key to isolate its putative receptor, other HUMAN NTN-2
binding polvpeptides, and/or study its antagonistic properties.
HUMAN NTN-2-specific activity or function may be determined by
convenient in vitro, cellbased, or in vivo assays - e.g., in vitro binding
assays, cell culture assays, in animals (e.g., immune response, gene therapy,
transgenics, etc.), etc. Binding assays encompass any assay where the specific
molecular interaction of a HUMAN NTN-2 polypeptide with a binding
target is evaluated. The binding target may be a natural binding target, or a
non-natural binding target such as a specific immune polypeptide such as an
antibody, or a HUMAN NTN-2 specific agent such as those identified in
assays described below.
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The claimed polypeptides may be isolated or pure - an "isolated" polypeptide
is one that is no longer accompanied by some of the material with which it
is associated in its natural state, and that preferably constitutes at least
about
0.5%, and more preferably at least about 5% by weight of the total
polypeptide in a given sample; a "pure" polypeptide constitutes at least
about 90%, and preferably at least about 99% by weight of the total
polypeptide in a given sample. The subject polypeptides and polypeptide
domains may be synthesized, produced by recombinant technology, or
purified from cells. A wide variety of molecular and biochemical methods
are available for biochemical synthesis, molecular expression and
purification of the subject compositions, see e.g., Molecular Cloning, A
Laboratory Manual (Sambrook, et al., Cold Spring Harbor Laboratory),
Current Protocols in Molecular Biology (Eds. Ausubel, et al., Greene Publ.
Assoc., Wiley-Interscience, NY).
The subject polypeptides find a wide variety of uses including use as
immunogens, targets in screening assays, bioactive reagents for modulating
cell growth, differentiation and/or function, etc. For example, the
invention provides methods for modifying the physiology of a cell
comprising an extracellular surface by contacting the cell or medium
surrounding the cell with an exogenous HUMAN NTN-2 polypeptide
under conditions whereby the added polypeptide specifically interacts with a
component of the medium and/or the extracellular surface to effect a
change in the physiology of the cell. According to these methods, the
extracellular surface includes plasma membrane-associated receptors; the
exogenous HUMAN NTN-2 refers to a polypeptide not made by the cell or,
if so, expressed at non-natural levels, times or physiologic locales; and
suitable media include in vitro culture media and physiological fluids such
as blood, synovial fluid, etc. The polypeptides may be may be introduced,
expressed, or repressed in specific populations of cells by any convenient
way such as microinjection, promoter-specific expression of recombinant
enzyme, targeted delivery of lipid vesicles, etc.
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The invention provides natural and non-natural HUMAN NTN-2-specific
binding agents, methods of identifying and making such agents, and their
use in diagnosis, therapy and pharmaceutical development. HUMAN
NTN-2-specific binding agents include HUMAN NTN-2-specific receptors,
such as somatically recombined protein receptors like specific antibodies or
T-cell antigen receptors (See, e.g., Harlow and Lane (1988) Antibodies, A
Laboratory Manual, Cold Spring Harbor Laboratory) and also includes other
natural binding agents identified with assays such as one-, two- and three-
hybrid screens, and non-natural binding agents identified in screens of
to chemical libraries such as described below. Agents of particular interest
modulate HUMAN NTN-2 function.
The invention provides HUMAN NTN-2 nucleic acids, which find a wide
variety of applications including use as translatable transcripts,
hybridization probes, PCR primers, diagnostic nucleic acids, etc., as well as
use in detecting the presence of HUMAN NTN-2 genes and gene transcripts
and in detecting or amplifying nucleic acids encoding additional HUMAN
NTN-2 homologs and structural analogs.
2o The subject nucleic acids are of synthetic/non-natural sequences and/or are
isolated, i.e., no longer accompanied by some of the material with which it is
associated in its natural state, preferably constituting at least about 0.5%,
more preferably at least about 5% by weight of total nucleic acid present in a
given fraction, and usually recombinant, meaning they comprise a non-
natural sequence or a natural sequence joined to nucleotides) other than
that which it is joined to on a natural chromosome. Nucleic acids
comprising the nucleotide sequence disclosed herein and fragments thereof,
contain such sequence or fragment at a terminus, immediately flanked by a
sequence other than that to which it is joined on a natural chromosome, or
flanked by a native flanking region fewer than 10 kb, preferably fewer than 2
kb, which is immediately flanked by a sequence other than that to which it is
joined on a natural chromosome. While the nucleic acids are usually RNA
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or DNA, it is often advantageous to use nucleic acids comprising other bases
or nucleotide analogs to provide modified stability, etc.
The amino acid sequences of the disclosed HUMAN NTN-2 polypeptide is
used to back translate HUMAN NTN-2 polypeptide-encoding nucleic acids
optimized for selected expression systems (Holler, et al. (1993) Gene 136: 323-
328; Martin, et al. (1995) Gene 154: 150-166) or used to generate degenerate
oligonucleotide primers and probes for use in the isolation of natural
HUMAN NTN-2 encoding nucleic acid sequences ("GCG" software, Genetics
Computer Group, Inc., Madison, WI). HUMAN NTN-2 encoding nucleic
acids may be part of expression vectors and may be incorporated into
recombinant host cells, e.g., for expression and screening, for transgenic
animals, for functional studies such as the efficacy of candidate drugs for
disease associated with HUMAN NTN-2 mediated signal transduction, etc.
i5 Expression systems are selected and/or tailored to effect HUMAN NTN-2
polypeptide structural and functional variants through alternative post-
translational processing.
The invention also provides for nucleic acid hybridization probes and
replication/amplification primers having a HUMAN NTN-2 cDNA specific
sequence and sufficient to effect specific hybridization with SEQ. LD. NO. 1.
Demonstrating specific hybridization generally requires stringent
conditions, for example, hybridizing in a buffer comprising 30% formamide
in 5 x SSPE (0.18 M NaCI, 0.01 M NaP04, pH7.7, 0.001 M EDTA) buffer at a
2~ temperature of 42°C and remaining bound when subject to washing at
42°C
with 0.2 x SSPE; preferably hybridizing in a buffer comprising 50%
formamide in 5 x SSPE buffer at a temperature of 42°C and remaining
bound
when subject to washing at 42°C with 0.2x SSPE buffer at 42°C.
HUMAN
NTN-2 cDNA homologs can also be distinguished from other polypeptides
3o using alignment algorithms, such as BLASTX (Altschul, et al. (1990) Basic
Local Alignment Search Tool, J. Mol. Biol. 215: 403-410).
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HUMAN NTN-2 hybridization probes find use in identifying wild-type and
mutant alleles in clinical and laboratory samples. Mutant alleles are used to
generate allele-specific oligonucleotide (ASO) probes for high-throughput
clinical diagnoses. HUMAN NTN-2 nucleic acids are also used to modulate
cellular expression or intracellular concentration or availability of active
HUMAN NTN-2. HUMAN NTN-2 inhibitory nucleic acids are typically
antisense - single stranded sequences comprising complements of the
disclosed natural HUMAN NTN-2 coding sequences. Antisense
modulation of the expression of a given HUMAN NTN-2 polypeptide may
to employ antisense nucleic acids operably linked to gene regulatory
sequences.
Cells are transfected with a vector comprising a HUMAN NTN-2 sequence
with a promoter sequence oriented such that transcription of the gene yields
an antisense transcript capable of binding to endogenous HUMAN NTN-2
encoding mRNA. Transcription of the antisense nucleic acid may be
constitutive or inducible and the vector may provide for stable
extrachromosomal maintenance or integration. Alternatively, single-
stranded antisense nucleic acids that bind to genomic DNA or mRNA
encoding a given HUMAN NTN-2 polypeptide may be administered to the
target cell, in or temporarily isolated from a host, at a concentration that
2o results in a substantial reduction in expression of the targeted
polypeptide.
An enhancement in HUMAN NTN-2 expression is effected by introducing
into the targeted cell type HUMAN NTN-2 nucleic acids which increase the
functional expression of the corresponding gene products. Such nucleic
acids may be HUMAN NTN-2 expression vectors, vectors which upregulate
the functional expression of an endogenous allele, or replacement vectors
for targeted correction of mutant alleles. Techniques for introducing the
nucleic acids into viable cells are known in the art and include retroviral-
based transfection, viral coat protein-liposome mediated transfection, etc.
3o The invention provides efficient methods of identifying agents, compounds
or lead compounds for agents active at the level of HUMAN NTN-2
modulatable cellular function. Generally, these screening methods involve
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assaying for compounds which modulate HUMAN NTN-2 interaction with
a natural HUMAN NTN-2 binding target. A wide variety of assays for
binding agents are provided including protein-protein binding assays,
immunoassays, cell based assays, etc. Preferred methods are amenable to
automated, cost-effective high throughput screening of chemical libraries
for lead compounds.
In vitro binding assays employ a mixture of components including a
HUMAN NTN-2 polypeptide, which may be part of a fusion product with
to another peptide or polypeptide, e.g., a tag for detection or anchoring,
etc.
The assay mixtures comprise a natural HUMAN NTN-2 binding target.
While native binding targets may be used, it is frequently preferred to use
portions thereof as long as the portion provides binding affinity and avidity
to the subject HUMAN NTN-2 conveniently measurable in the assay. The
assay mixture also comprises a candidate pharmacological agent. Candidate
agents encompass numerous chemical classes, though typically they are
organic compounds, preferably small organic compounds, and are obtained
from a wide variety of sources including libraries of synthetic or natural
compounds. A variety of other reagents such as salts, buffers, neutral
2o proteins, e.g., albumin, detergents, protease inhibitors, nuclease
inhibitors,
antimicrobial agents, etc., may also be included. The mixture components
can be added in any order that provides for the requisite bindings and
incubations may be performed at any temperature which facilitates optimal
binding. The mixture is incubated under conditions whereby, but for the
presence of the candidate pharmacological agent, the HUMAN NTN-2
specifically binds the cellular binding target, portion or analog with a
reference binding affinity. Incubation periods are chosen for optimal
binding but are also minimized to facilitate rapid, high throughput
screening.
After incubation, the agent-biased binding between the HUMAN NTN-2
and one or more binding targets is detected by any convenient way. For cell-
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free binding type assays, a separation step is often used to separate bound
from unbound components. Separation may be effected by precipitation,
immobilization, etc., followed by washing by, e.g., membrane filtration or
gel chromatography. For cell-free binding assays, one of the components
usually comprises or is coupled to a label. The label may provide for direct
detection as radioactivity, luminescence, optical or electron density, etc.,
or
indirect detection such as an epitope tag, an enzyme, etc. A variety of
methods may be used to detect the label depending on the nature of the label
and other assay components, e.g., through optical or electron density,
1o radiative emissions, nonradiative energy transfers, or indirectly detected
with antibody conjugates, etc. A difference in the binding affinity of the
HUMAN NTN-2 polypeptide to the target in the absence of the agent as
compared with the binding affinity in the presence of the agent indicates
that the agent modulates the binding of the HUMAN NTN-2 polypeptide to
the corresponding binding target. A difference, as used herein, is
statistically
significant and preferably represents at least a 50%, more preferably at least
a
90% difference.
The invention provides for a method for modifying the physiology of a cell
2o comprising an extracellular surface in contact with a medium, said method
comprising the step of contacting said medium with an exogenous
HUMAN NTN-2 polypeptide under conditions whereby said polypeptide
specifically interacts with at least one of a component of said medium and
said extracellular surface to effect a change in the physiology of said cell.
The invention further provides for a method for screening for biologically
active agents, said method comprising the steps of a) incubating a HUMAN
NTN-2 polypeptide in the presence of an extracellular HUMAN NTN-2
polypeptide specific binding target and a candidate agent, under conditions
3o whereby, but for the presence of said agent, said polypeptide specifically
binds said binding target at a reference affinity; b) detecting the binding
affinity of said polypeptide to said binding target to determine an agent-
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biased affinity, wherein a difference between the agent-biased affinity and
the reference affinity indicates that said agent modulates the binding of said
polypeptide to said binding target.
One embodiment of the invention is an isolated HUMAN NTN-2
polypeptide comprising the amino acid sequence as set forth herein or a
fragment thereof having HUMAN NTN-2-specific activity.
Another embodiment of the invention is a recombinant nucleic acid
1o encoding HUMAN NTN-2 polypeptide comprising the amino acid
sequence as set forth herein or a fragment thereof having HUMAN NTN-2-
specific activity.
Still another embodiment is an isolated nucleic acid comprising a
~5 nucleotide sequence as set forth herein or a fragment thereof having at
least
18 consecutive bases and sufficient to specifically hybridize with a nucleic
acid having the sequence of set forth herein in the presence of natural
HUMAN NTN-2 cDNA.
20 The present invention also provides for antibodies to the HUMAN NTN-2
polypeptide described herein which are useful for detection of the
polypeptide in, for example, diagnostic applications. For preparation of
monoclonal antibodies directed toward this HUMAN NTN-2 polypeptide,
any technique which provides for the production of antibody molecules by
25 continuous cell lines in culture may be used. For example, the hybridoma
technique originally developed by Kohler and Milstein (1975, Nature
26:495-497), as well as the trioma technique, the human B-cell hybridoma
technique (Kozbor et al., 1983, Immunology Today 4_:72), and the EBV-
hybridoma technique to produce human monoclonal antibodies (Cole et al.,
30 1985, in "Monoclonal Antibodies and Cancer Therapy," Alan R. Liss, Inc. pp.
77-96) and the like are within the scope of the present invention.
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The monoclonal antibodies for diagnostic or therapeutic use may be human
monoclonal antibodies or chimeric human-mouse (or other species)
monoclonal antibodies. Human monoclonal antibodies may be made by
any of numerous techniques known in the art (e.~,. Teng et al., 1983, Proc.
Natl. Acad. Sci. U.S.A. 80:7308-7312; Kozbor et al., 1983, Immunology Today
4:72-79; Olsson et al., 1982, Meth. Enzymol. 92:3-16). Chimeric antibody
molecules may be prepared containing a mouse antigen-binding domain
with human constant regions (Morrison et al., 1984, Proc. Natl. Acad. Sci.
U.S.A. 81:6851, Takeda et al., 1985, Nature 314:452).
Various procedures known in the art may be used for the production of
polyclonal antibodies to epitopes of the HUMAN NTN-2 polvpeptide
described herein. For the production of antibody, various host animals can
be immunized by injection with the HUMAN NTN-2 polypeptide, or a
fragment or derivative thereof, including but not limited to rabbits, mice
and rats. Various adjuvants may be used to increase the immunological
response, depending on the host species, and including but not limited to
Freund's (complete and incomplete), mineral gels such as aluminum
hydroxide, surface active substances such as lysolecithin, pluronic polyols,
2o polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,
dinitrophenol, and potentially useful human adjuvants such as BCG
(Bacille Calmette-Guerin) and Corvnebacterium parvum.
A molecular clone of an antibody to a selected HUMAN NTN-2 polypeptide
epitope can be prepared by known techniques. Recombinant DNA
methodology (see e.g., Maniatis et al., 1982, Molecular Cloning, A Laboratory
Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York)
may be used to construct nucleic acid sequences which encode a monoclonal
antibody molecule, or antigen binding region thereof.
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The present invention provides for antibody molecules as well as fragments
of such antibody molecules. Antibody fragments which contain the idiotype
of the molecule can be generated by known techniques. For example, such
fragments include but are not limited to: the F(ab')2 fragment which can be
produced by pepsin digestion of the antibody molecule; the Fab' fragments
which can be generated by reducing the disulfide bridges of the F(ab')2
fragment, and the Fab fragments which can be generated by treating the
antibody molecule with papain and a reducing agent. Antibody molecules
may be purified by known techniques, ~ immunoabsorption or
1o immunoaffinity chromatography, chromatographic methods such as HPLC
(high performance liquid chromatography), or a combination thereof.
The following examples are offered by way of illustration and not by way of
limitation.
EXAMPLE 1 - Cloning and Sequencing of Partial HUMAN NTN-2 Coding
a uence
Amino acid sequences of all the known human and mouse members of the
2o TNF family were used as tblastn queries to search the NIH EST database of
random fragments of mRNA sequences {Altschul, Stephen F., Warren Gish,
Webb Miller, Eugene W. Myers, and David J. Lipman (1990). Basic local
alignment search tool. J. Mol. Biol. 215:403-10). Each query generated a list
of hits, i.e. EST sequences with a substantial sequence similarity to the
query
sequence. Typically, the hits on top of the list corresponded to mRNA copies
of the query protein, followed by ESTs derived from other members of the
family and random-chance similarities.
A parser program was used to combine and sort all the hits from searches
with all the members of the family. This allowed rapid subtraction of all the
3o hits corresponding to known proteins. The remaining hits were analyzed
for conservation of sequence motifs characteristic for the family. Additional
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database searches were performed to identify overlapping ESTs. The partial
nucleotide and deduced amino acid sequence of Human NTN-2 was
determined as follows:
20 70 40 50
Seq IDtl ACT GGT TAC TTT TTT ATA TAT GGT CAG GTT TTA TAT ACT GAT AAG ACC TAC
GCC ATG
Seq ID~2 Thr Gly Tyr Phe Phe Ile Tyr Gly Gln Val Leu Tyr Thr Asp Lys Thr Tyr
Ala Met>
60 70 BO 90 100 110
GGA CAT CTA ATT CAG AGG NAG AAG GTC CAT GTC TTT GGG GAT GAA TTG AGT CTG GTG
Gly His Leu Ile Gln Arg Xxx Lys Val His Val Phe Gly Asp Glu Leu Ser Leu Val>
120 130 140 150 160 170
ACT TTG TTT CGA TGT ATT CAA AAT ATG CCT.GAA ACA CTA CCC AAT AAT TCC TGC TAT
Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu.Pro Asn Asn Ser Cys Tyr>
180 190 200 210 220
TCA GCT GGC ATT GCA AAA CTG GAA GAA GGA GAT GAA CTC CAA CTT GCA ATA CCA AGA
Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly Asp Glu Leu Gln Leu Ala Ile Pro Arg>
230 240 250 260 270 280
GAA AAT GCA CAA ATA TCA CTG GAT GGA GAT GTC ACA TTT TTT GGT GCA TTG AAA CTG
Glu Asn Ala Gln Ile Ser Leu Asp Gly Asp Val Thr Phe Phe Gly Ala Leu Lys Leu>
290
CTG TGA
Leu ~~~>
Using the nucleotide sequence of SEQ. LD. NO. 2 as a query, additional
database searches were performed to identify overlapping ESTs. Two
additional clones from the LM.A.G.E. consortium were discerned to contain
homologous sequence. These clones, GeneBank Accession Nos. AA166695
and T87299 were obtained from Research Genetics, Inc. (Huntsville, AL)
and sequenced using the ABI 373A DNA sequencer and Taq Dideoxy
Terminator Cycle Sequencing Kit (Applied Biosystems, Inc., Foster City, CA).
Alignment of the two additional clones with SEQ. LD. NO. 1 indicated a
total length of 680 nucleotides. Oligonucleotides were designed based on the
3o partial human sequence and used as primers for the reverse transcriptase
reaction and for PCR. A 608 nucleotide long sequence was obtained and
used as a probe to isolate the full length sequence as described below.
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EXAMPLE 2 - ISOLATION AND SEQUENCING OF FULL LENGTH
cDNA CLONE ENCODING HUMAN NTN-2
A human placenta cDNA library in lambda gt-10 was obtained from
Clontech Laboratories, Inc. (Palo Alto, CA). Plaques were plated at a density
of L25 x 106/20x20 cm plate, and replica filters taken following standard
procedures (Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd
Ed., page 8.4b, Cold Spring Harbor Laboratory, Cold Spring Harbor, New
l0 York). Filters were screened at normal stringency (2 x SSC, 65°C)
with a
probe corresponding to nucleotides 216 to 824 of the hNTN-2 sequence
shown in SEQ. LD. NO. 3. The probe was hybridized at 65°C in
hybridization
solution containing 0.5 mg/ml salmon sperm DNA to decrease non-specific
binding of the probe to the filter. Filters were washed in 2 x SSC at
65° C and
exposed overnight to X-ray film. Five positive clones were picked that
showed strong hybridization signals and also produced fragments when
PCR-amplified using oligos from the cDNA vector.
SeauT racing. of hNTN-2
The coding region from each of the five clones was sequenced using the ABI
373A DNA sequencer and Taq Dyedeoxy Terminator Cycle Sequencing Kit
(Applied Biosystems, Inc., Foster City, CA). The nucleotide and deduced
amino acid sequence of the full length hNTN-2 coding sequence obtained
from one of the clones is set forth as follows:
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20 30 40 50 60
$E0 ID x3 ATG GAT GAC TCC ACA GAA AGG GAC CAG TCA CGC C1T ACT TCT TGC C1'C AAG
AAA AGA GAA
SEO ID x4 Met Asp Asp Ser Thr Glu Arg Glu Gln Ser Arg Leu Thr Ser Cys Leu Lys
Lys Arg Glu>
70 80 90 100 110 120
GAA ATG AAA CIG AAG GAG TG'T GTT TCC ATC GTC CCA CGG AAG GAA AGC CCC TCT G2'C
CGA
Glu Met Lys Leu Lys Glu Cys Val Ser Ile Leu Pro Arg Lys Glu Ser Pro Ser Val
Arg>
5
130 140 150 160 170 180
. r . .
TCC TCC AAA GAC GGA AAG C1G C1G GCl' GCA ACC TTG CIG CTG GCA C2G CTG TCI' TGC
TGC
Ser Ser Lys Asp Gly Lys Leu Leu Ala Ala Thr Leu Leu Leu Ala Leu Leu Ser Cys
Cys>
190 200 210 220 230 240
. . . . ~
C'ir ACG GTG G1G TCT TTC TAC CAG GIG GCC GCC CI'G CAA GOG GAC CIG GCC AGC CfC
CGG
Leu Thr Val Val Ser Phe Tyr Gln Va1 Ala Ala Leu Gln Gly Asp Leu Ala Ser Leu
Arg>
I1 250 260 270 280 290 300
lU , . . . . .
GCA GAG CTG CAG GGC CAC CAC GCG GAG AAG CTG CCA GCA GGA GCA (GA GCC CCC AAG
GCC
Ala Glu Leu Gln Gly His His Ala Glu Lys.Leu Pro Ala Gly Ala Gly Ala Pro Lys
Ala>
310 320 330 340 350 360
~ . t ~ .
CSC C'1G GAG GAA GC'P CCA GCT G'I'C ACC GCG GGA CIG AAA A'1C TTT GAA CCA CCA
GCT CCA
Gly Leu Glu Glu Ala Pro Ala Val Thr Ala Gly Ixu Lys Ile Phe Glu Pro Pro Ala
Pro>
370 380 390 400 410 420
. , .
I5 GGA GAA GGC AAC TCC AGT CAG AAC AGC AGA AAT AAG CGT GCC GiT CAG OGT' CCA
GAA GAA
Gly Glu Gly Asn Ser Ser Gln Asn Ser Arg Asn Lys Arg Ala Val Gln Gly Pro Glu
Glu>
430 440 450 460 470 480
ACA GTC ACT CAA GAC TGC TIG CAA C1G ATT GCA GAC AGT GAA ACA CCA ACT ATA CAA
AAA
Thr Val Thr Gln Asp Cys Leu Gln Leu Ile Ala Asp Ser Glu Thr pro Thr Ile Gln
Lys>
490 500 510 520 530 540
. . a . . ,
OGP. TCT TAC ACA TTT GTT CCA TvG CIT CrC AGC TTT AAA AOG GGA AGT GCC CTA GAA
GAA
Gly Ser Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser Ala Leu Glu
Glu>
550 560 570 580 590 600
AAA GAG AAT AAA ATA TTG GTC AAA GAA ACT GGT TAC TTT TIT ATA TAT GGT CAG GIT
TT.~
Lys Glu Asn Lys Ile Leu Val Lys Glu Thr Gly Tyr Phe Phe Ile Tyr Gly Gln Val
Leu>
610 620 630 640 650 660
TAT ACT GAT AAG ACC TAC GCC ATG GGA CAT CTA ATT CAC AGG AAG AAG GTC CAT GTC
TTT
Tyr Thr Asp Lys Thr Tyr Ala Met Gly His Leu Ile Gln Arg Lys Lys Val His Val
Phe>
670 680 690 700 710 720
,
f~G GAT GAA T'iG AGf CTG GIG ACl' TTG TIT CGA TGT ATT CAA AAT A'1G CCT GAA ACA
CTA
Gly Asp Glu Leu Ser Leu Val Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Glu Thr
Leu>
730 740 750 760 770 780
v w . w . s
CCC AAT AAT TCC T~ TAT TCA CX."I' GGC ATT GCA AAA CPG GAA GAA GGA GAT GAA CTC
CAA
Pro Asn Asn Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly Asp Glu Leu
Gln>
790 B00 810 820 830 840
~ ~ ~ .
CTT GCA ATA CCA AGA GAA AAT GCA CAA ATA TCA CTG GAT GGA GAT GIC ACA TTT TTT
OGT
Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Leu ASp G1y Asp Val Thr Phe Phe
Gly>
850
GCA TTG AAA CTG C1G TGA
Ala Leu Lys Leu Leu "'>
16
SUBSTITUTE SHEET (RULE 26)
.~..
CA 02292835 1999-12-03
WO 98155621 PCT/US98/11294
EXAMPLE 3 - TISSUE SPECIFIC EXPRESSION OF hNTN-2
A fragment corresponding to nucleotides 216 to 824 of the hNTN-2 sequence
shown in SEQ. LD. NO. 3 was radiolabeled and utilized in Northern analysis
of various human tissue specific RNAs. The Northern blot containing
polyA+ RNA from several human tissues was obtained from Clontech
Laboratories, Inc. (Palo Alto, CA) and was hybridized at 65°C to
the
radiolabeled hNTN-2 probe in the presence of 0.5M NaP04 (pH 7), 1%
bovine serum albumin (Fraction V, Sigma), 7% SDS, 1 mM EDTA and 100
to ng/ml sonicated, denatured salmon sperm DNA. The filter was washed at
65°C with 2X SSC, 0.1% SDS and subjected to autoradiography for 16
hours
with one intensifying screen and X-ray film at -70°C.
The hNTN-2 probe hybridized strongly to a 2.7 kb transcript in human
heart, placenta, pancreas and lung tissue (Figure 1) and hybridized weakly to
RNA from brain and liver. Weaker levels of expression could also be found
in skeletal muscle and kidney. High expression of hNTN-2 in heart tissue
may suggest that the present invention may be utilized to treat heart disease.
Expression of hNTN-2 in lung and pancreas tissue may suggest that the
2o present invention may be utilized to treat lung and/or pancreas related
disorders.
Although the foregoing invention has been described in some detail by way
of illustration and example for purposes of clarity of understanding, it will
be readily apparent to those of ordinary skill in the art in light of the
teachings of this invention that certain changes and modifications may be
made thereto without departing from the spirit or scope of the appended
claims.
17
SUBSTITUTE SHEET (RULE 26)
