Note: Descriptions are shown in the official language in which they were submitted.
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NOVEL OR~,'HAN RECEPTOR
This International Application claims priority of U.S. Provisional
Application Serial No. 60/054,869 filed August 6, 1997. Throughout this
application various publications are referenced. The disclosures of these
publications in their entireties are hereby incorporated by reference into
this
application.
INTRODUCTION
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. The present
invention provides for novel orphan receptor molecules, their use and
assay systems useful for identifying novel ligands that interact with these
receptors.
BACKGROUND OF THE INVENTION
2o The tumor necrosis factor receptor (TNFR) superfamily consists mostly of
transmembrane proteins that elicit signal transduction in a variety of cells.
Tumor necrosis factor-alpha (TNF-alpha) is a cytokine primarily produced
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.
TNT-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
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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-
p75 results in proliferative response of mouse and human thymocytes and
cytotoxic T cells, fibroblasts and natural killer cells and in GM-CSF
secretion
in PC60 cells.
The identification of a new member of the TNFR superfamily that regulates
to bone resorption was recently reported. The newly identified protein was
termed Osteoprotegerin (OPG) and was postulated to act as a humoral
regulator of bone resorption by blocking the differentiation of osteoclasts,
the cells responsible for bone resorption. (Simonet, W.S., et al., 1997, Cell
89:
309-319; International Publication Number WO 97/ 23614 published 3 July
1997 in the name of Amgen, Inc.). However, relatively little is known about
the soluble factors that act physiologically to regulate osteoclast
development.
Novel receptor molecules are often identified and isolated by searching for
2o additional members of known families of receptors using, for example, PCR-
based screens or computer searches of EST databases involving known
regions of homology among the family members. (See, for example,
Maisonpierre, et al., 1993, Oncogene 8: 1631-1637). Isolation of such so
called
"orphan" receptors, for which no ligand is known, and subsequent
determination of the tissues in which such receptors are expressed, provides
insight into the regulation of the growth, proliferation and regeneration of
cells in target tissues. Further, such receptors may be used to isolate their
cognate ligands, which may then be used to regulate the survival, growth
and regeneration of cells expressing the receptor. Alternatively, in the case
of soluble receptors, the receptor itself can behave as a ligand.
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SUMMARY OF THE INVENTION
The present invention provides for a novel orphan human receptor,
termed HUMAN NTR-1, which is expressed in the pancreas, skeletal
muscle, and fetal and adult hearts. The protein is related to osteoprotegerin
(OPG) and to tumor necrosis factor receptor (TNFR). The present invention
further provides for an isolated nucleic acid molecule encoding HUMAN
NTR-1. Based upon its homology to osteoprotegerin, it is expected that
HUMAN NTR-1 will be involved in the regulation of bone mass, and may
1o be useful for regulating development, proliferation and death of osteoblast
or osteoclast cells or for regulating muscle metabolism and may be
implicated in diseases or disorders of muscle.
The present invention also provides for a protein or polypeptide that
comprises the extracellular domain of HUMAN NTR-2 as well as the
nucleic acid which encodes such extracellular domain. The invention
further provides for vectors comprising an isolated nucleic acid molecule
encoding HUMAN NTR-1 or its extracellular domain, which can be used to
express HUMAN NTR-1 or its extracellular domain in bacteria, yeast, insect
or mammalian cells, preferably COS or CHO cells.
The invention further provides for use of the HUMAN NTR-1 receptor or
its extracellular or intracellular domain in screening for drugs that interact
with HUMAN NTR-1. Novel agents that bind to the receptors) described
herein may mediate survival and differentiation in cells naturally
expressing the receptor, but also may confer survival and proliferation
when used to treat cells engineered to express the receptor. In particular
embodiments, the extracellular domain (soluble receptor) of HUMAN NTR-
1 is utilized in screens for cognate ligands.
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Preferred uses for the subject HUMAN NTR-1 polypeptides include
screening for agents that bind to the receptor polypeptides. The agents may
be biologically active agents (agonists), which activate the HUMAN NTR-1
receptor or they may bind and block activation of the receptor (antagonists).
Screening methods include incubating a HUMAN NTR-1 polypeptide in the
presence of an extracellular HUMAN NTR-1 polypeptide-specific binding
target and 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
to 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.
The invention also provides for a nucleic acid probe capable of hybridizing
i5 with a sequence included within the nucleic acid sequence encoding
HUMAN NTR-1 useful for the detection of NTR-1 expressing tissue in
humans and animals.
The invention further provides for antibodies directed to HUMAN NTR-1.
The present invention also has diagnostic and therapeutic utilities. In
particular embodiments of the invention, methods of detecting aberrancies
in the function or expression of the receptor described herein may be used in
the diagnosis of disorders. In other embodiments, manipulation of the
receptor or agonists or antagonists which bind this receptor may be used in
the treatment of diseases. In further embodiments, the extracellular domain
of the receptor is utilized as a blocking agent which blocks the binding of
receptor to its target.
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In a further embodiment of the invention, patients who suffer from an
excess of NTR-1 may be treated by administering an effective amount of
anti-sense RNA or anti-sense oligodeoxyribonucleotides corresponding to
the HUMAN NTR-1 gene coding region, thereby decreasing expression of
NTR-1.
DETAILED DESCRIPTIOI~OF THE INVENTION
The invention provides HUMAN NTR-1 polypeptide which includes
isolated HUMAN NTR-1 polypeptide and recombinant polypeptides
comprising a HUMAN NTR-1 amino acid sequence, or a functional
HUMAN NTR-1 polypeptide domain thereof having an assay-discernable
HUMAN NTR-1-specific activity. Accordingly, the polypeptides may be
deletion mutants of the disclosed HUMAN NTR-1 polypeptides and may be
provided as fusion products, e.g., with non - HUMAN NTR-1 polypeptides.
The subject HUMAN NTR-1 polypeptide domains have HUMAN NTR-1-
specific activity or function.
A number of applications for HUMAN NTR-1 are suggested from its
2o properties. HUMAN NTR-1, may be useful in the study and treatment of
conditions similar to those which are treated using TNF. Furthermore, the
HUMAN NTR-1 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
NTR-1 is present. The isolation of HUMAN NTR-1, of course, also provides
the-key to isolate its putative ligand, other HUMAN NTR-1 binding
polypeptides, and/or study its properties.
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HUMAN NTR-1-specific activity or function may be determined by
convenient in vi r , cell based, or inin vivo assays - e.g., 'n vi 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 NTR-I 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 NTR-1 specific agent such as those identified in
assays described below.
to
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
25 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
2o 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 polypepddes 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
3o comprising an extracellular surface by contacting the cell or medium
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surrounding the cell with an exogenous HUMAN NTR-1 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 NTR-1 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.
The invention provides HUMAN NTR-1-specific binding agents, methods
of identifying and making such agents, and their use in diagnosis, therapy
and pharmaceutical development. HUMAN NTR-1-specific binding agents
include HUMAN NTR-1-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
2o Harbor Laboratory) and also includes other binding agents identified with
assays such as one-, two- and three-hybrid screens, and non-natural binding
agents identified in screens of chemical libraries such as described below.
Agents of particular interest modulate HUMAN NTR-1 function.
The invention provides HUMAN NTR-1 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 NTR-1 genes and gene transcripts
and in detecting or amplifying nucleic acids encoding additional HUMAN
3o NTR-1 homologs and structural analogs.
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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
l0 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
or DNA, it is often advantageous to use nucleic acids comprising other bases
i5 or nucleotide analogs to provide modified stability, etc.
The amino acid sequences of the disclosed HUMAN NTR-1 polypeptide is
used to back translate HUMAN NTR-1 polypeptide-encoding nucleic acids
optimized for selected expression systems (Holler, et al. (1993) Gene 136: 323-
20 328; 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 NTR-1 encoding nucleic acid sequences ("GCG" software, Genetics
Computer Group, Inc., Madison, WI). HUMAN NTR-1 encoding nucleic
acids may be part of expression vectors and may be incorporated into
25 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 NTR-1 mediated signal transduction, etc.
Expression systems are selected and/or tailored to effect HUMAN NTR-1
polypeptide structural and functional variants through alternative post-
30 translational processing.
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The invention also provides for nucleic acid hybridization probes and
replication/amplification primers having a HUMAN NTR-1 cDNA specific
sequence and sufficient to effect specific hybridization with SEQ. 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
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
NTR-1 cDNA homologs can also be distinguished from other polypeptides
using alignment algorithms, such as BLASTX (Altschul, et al. (1990) Basic
Local Alignment Search Tool, J. Mol. Biol. 215: 403-410).
~5 HUMAN NTR-1 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 NTR-1 nucleic acids are also used to modulate
cellular expression or intracellular concentration or availability of active
2o HUMAN NTR-1. HUMAN NTR-1 inhibitory nucleic acids are typically
antisense - single stranded sequences comprising complements of the
disclosed HUMAN NTR-1 coding sequences. Antisense modulation of the
expression of a given HUMAN NTR-1 polypeptide may employ antisense
nucleic acids operably linked to gene regulatory sequences. Cells are
25 transfected with a vector comprising a HUMAN NTR-1 sequence with a
promoter sequence oriented such that transcription of the gene yields an
anti~ense transcript capable of binding to endogenous HUMAN NTR-1
encoding mRNA. Transcription of the antisense nucleic acid may be
constitutive or inducible and the vector may provide for stable
3o extrachromosomal maintenance or integration. Alternatively, single-
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stranded antisense nucleic acids that bind to genomic DNA or mRNA
encoding a given HUMAN NTR-1 polypeptide may be administered to the
target cell, in or temporarily isolated from a host, at a concentration that
results in a substantial reduction in expression of the targeted polypeptide.
An enhancement in HUMAN NTR-1 expression is effected by introducing
into the targeted cell type HUMAN NTR-1 nucleic acids which increase the
functional expression of the corresponding gene products. Such nucleic
acids may be HUMAN NTR-1 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.
The invention provides efficient methods of identifying agents, compounds
or lead compounds for agents active at the level of HUMAN NTR-1
modulatable cellular function. Generally, these screening methods involve
assaying for compounds which modulate the interaction of HUMAN NTR-
1 with a natural HUMAN NTR-1 binding target. A wide variety of assays
for binding agents are provided including protein-protein binding assays,
2o 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 NTR-1 polypeptide, which may be part of a fusion product with
another peptide or polypeptide, e.g., a tag for detection or anchoring, etc.
The~assay mixtures comprise a natural HUMAN NTR-1 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
3o to the subject HUMAN NTR-1 conveniently measurable in the assay. The
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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
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
1o binding. The mixture is incubated under conditions whereby, but for the
presence of the candidate pharmacological agent, the HUMAN NTR-1
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 NTR-1 and
one or more binding targets is detected by any convenient way. For cell-free
binding type assays, a separation step is often used to separate bound from
2o 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,
radiative emissions, nonradiative energy transfers, or indirectly detected
with antibody conjugates, etc. A difference in the binding affinity of the
3o HUMAN NTR-1 polypeptide to the target in the absence of the agent as
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compared with the binding affinity in the presence of the agent indicates
that the agent modulates the binding of the HUMAN NTR-1 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
comprising an extracellular surface in contact with a medium, said method
comprising the step of contacting said medium with an exogenous HUMAN
1o NTR-1 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
NTR-1 polypeptide in the presence of an extracellular HUMAN NTR-1
polypeptide specific binding target and a candidate agent, under conditions
whereby, but for the presence of said agent, said polypeptide specifically
binds said binding target at a reference affinity; b) detecting the binding
2o affinity of said polypeptide to said binding target to determine an agent-
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 NTR-1
polypeptide comprising the amino acid sequence as set forth herein or a
fragment thereof having HUMAN NTR-1-specific activity.
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Another embodiment of the invention is a recombinant nucleic acid
encoding HUMAN NTR-1 polypeptide comprising the amino acid sequence
as set forth herein or a fragment thereof having HUMAN NTR-1-specific
activity.
Still another embodiment is an isolated nucleic acid comprising a
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
1o HUMAN NTR-1 cDNA.
The present invention also provides for antibodies to the HUMAN NTR-1
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 NTR-1 polypeptide,
any technique which provides for the production of antibody molecules by
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
2o technique (Kozbor et al., 1983, Immunology Today 4_:72), and the EBV-
hybridoma technique to produce human monoclonal antibodies (Cole et al.,
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.
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 (~., 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
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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 NTR-1 polypeptide
described herein. For the production of antibody, various host animals can
be immunized by injection with the HUMAN NTR-1 polypeptide, or a
fragment or derivative thereof, including but not limited to rabbits, mice
1o 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,
polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,
dinitrophenol, and potentially useful human adjuvants such as BCG
(Bacille Calmette-Guerin) and Cor~mebacterium~arvum.
A molecular clone of an antibody to a selected HUMAN NTR-1 polypeptide
epitope can be prepared by known techniques. Recombinant DNA
2o 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.
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
3o which can be generated by reducing the disulfide bridges of the F(ab')2
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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, g_g_, immunoabsorption or
immunoaffinity chromatography, chromatographic methods such as HPLC
(high performance liquid chromatography), or a combination thereof.
The following example is offered by way of illustration and not by way of
limitation.
1o EXAMPLE 1' Cloning and Seauencin~ of HUMAN NTR-1 gene
Amino acid sequences of known human and mouse members of the 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
2o 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
hits corresponding to known proteins. The remaining hits were analyzed
for conservation of sequence motifs characteristic for the family. Additional
database searches were performed to identify overlapping ESTs. Two
human cDNA clones, LD. Nos. 366305 (the '305 clone) and 592256 (the '256
clone) from the LM.A.G.E. consortium, were discerned to contain
homologous sequence. These clones, GeneBank Accession Nos. AA025672
3o and AA155646, were obtained from Research Genetics, Inc. (Huntsville, AL}
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and sequenced using the ABI 373A DNA sequencer and Taq Dideoxy
Terminator Cycle Sequencing Kit (Applied Biosystems, Inc., Foster City, CA).
The '305 clone contained a stop codon and poly-A tail and thus was
determined to encode the 3' end of the molecule. The '256 clone aligned
with the 5' end of the '305 clone over an approximately 300 nucleotide
stretch. Together, the overlapping clones encode a polypeptide of
approximately 220 amino acids, but was missing about 80 amino acids from
the 5' end as compared to Osteoprotegerin. Further, since there was no
o coding sequence for a predicted signal peptide or for an initial methionine,
the 5' end of the '256 clone was deemed incomplete. The 5' RACE procedure
was then used to obtain a nucleotide sequence encoding 63 missing amino
acids including the signal peptide. Using PCR primers flanking the coding
sequence, a single fragment was constructed that contained the entire coding
i5 sequence. The sequence of HUMAN NTR-1 was then further confirmed by
sequencing. The nucleotide and deduced amino acid sequence of HUMAN
NTR-1 is set forth herein. Northern analysis revealed HUMAN NTR-1
transcript in adult lung, skeletal muscle, kidney, placenta and pancreas as
well as in fetal heart and stomach.
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.
The present invention is not to be limited in scope by the specific
embodiments described herein. Indeed, various modifications of the
3o invention in addition to those described herein will become apparent to
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those skilled in the art from the foregoing description and accompanying
figures. Such modifications are intended to fall within the scope of the
appended claims.
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Human NTR-1 Sequence
i0_ a0 30 40 50 60
SCq ID~'1 ATG AG<i GCG CTG GAG GGG CCA GGC CTCi TCG CTQ CTG TGC CTQ GTti TT(i
GC(t CTO CCT GCC
Seq 10#2 Met Arg Ala Leu Glu Gly Pro Gly Leu Ser Leu Leu Cys Leu Val Leu Ala
Leu Pro Ala
70 80 90 100 iio iao
CTG CTG CCG GTCi CC(i GCT OTA 'CGC GGA GTG GG GAA ACA CCC ACC TAC CCC TCiCi
CGG GAC
Leu Leu Pro Val Pro Ala Val Arg Gly Val Ala Glu Thr Pro Thr Tyr Pro Trp Arg
Asp
130 140 150 160 170 180
GG GAG AG GGG' GAO CGO CTG GTO TGC GøC CAG TCiC CCC CG GGC ACC TTT GTG GG CGG
Ala Glu Thr Gly Glu Arg Leu Vai Cys Ala Gln Cys Pro Pro Gly Thr Phe Val Gln
Arg
i90 a00 aio aao a3o aao
CCG TGC CGC CGA GAC AGC CCC ACCT ACG TGT GGC CCG T(iT CCA CCG CGC GC TAC ACG
GG
Pro Cys Arg Arg Asp Ser Pro Thr Thr Cys Gly Pro Cys Pro Pro Arg His Tyr Thr
Gln
a5o aso a7o aso ago 300
TTC TGG AAC TAC CTG C~1G CGC TGC CGC TAC TGC AAC GTC CTC TGC GGG GACi CGT GAG
GAG
Phe Trp Asn Tyr Leu Glu Arg Cys Arg Tyr Gys Asn Val Leu Cys Gly Glu Arg Glu
Glu
310 3a0 330 340 350 ~ 360
GAG GG CGG GCT TGC GC GCC ACC CAC AAC CGT GCC TGC CGC TGC CGC ACC GGC TTC TTC
Glu Ala Arg.Ala Cys His Ala Thr His Asn Arg Ala Cys Arg Cys Arg Thr ply Phe
Phe
370 380 390 400 4i0 4a0
GCG GC GCT GGT TTC TGC TTG CiAG GC GG TCG TGT CCA CCT GGT GCCvGGC GTfi ATT GCC
Ala His Ala Gly Phe Cys Leu Glu His Ala Ser Cys Pro Pro Gly Ala Gly Val Ile
Ala
430 440 450 460 470 480
CCG GGC ACC CCC AGC CAG AAC ACO GG TGC CAG CCC TGC CCC CG GGC ACC TTC TG C~C
Pro Gly Thr Pro-Ser Gin Asn Thr Gln Cys Gln Pro Cys Pro Pro Gly Thr Phe Ser
Ala
490 500 510 5a0 530 540
AGC AGC TCC AGC TG GAG CAG TGC CAG CCC GC CGC AAC TGC ACG GCC CTti GGC CTG GCC
Ser Ser Ser Ser Ser Glu Oln Cys Gin Pro His Arg Asn Cys Thr Ala Leu Gly Leu
Ala
550 560 570 580 590 600
CTC A71~' GTG CG GGC TCT TCC TCC CAT GAC ACC CTG TGC ACC AGC'TGC ACT CGC TTC
CCC
Leu Asn Val Pro Gly Ser Ser Ser Hfs Asp Thr Leu Cys Thr Ser Cys Thr Oily Phe
Pro
610 6a0 630 640 650 660
CTC AGC A~ AGG GTA CG GGA GCT GAG GAG TGT GAG CGT GCC GTC ATC GAC TTT GTG GCT
Leu Ser Thr Arg Val Pro Oly Ala Glu Glu Cys Giu Arg Ala Val Ile Asp Phe Val
Ala
670 . 680 690 700 710 720
T'm GG GAC ATC TCC ATC AAG AGG CT<3 GO CGG CTG CTG CAG GCC CTC CAG GCC CCG GAG
Phe Gln Asp Ile Ser Ile Lys Arg Leu Gln Arg Leu Leu Gln Ala Leu Glu Ala Pro
Glu
730 740 750 760 770 780
GGC TGG GGT CCG ACA CCA AGG GCG GGC CGC GCG GCC TTG CAG CTC AAG CTG CGT CGG
CGG
Gly Trp Gly Pro Thr Pro Arg Ala Gly Arg Ala Ala Leu Gln Leu Lys Leu Arg Arg
Arg
790 800 810 820 830 840
CTC ACG GAG CTC CTG GGG GCG CAG GAC GGG GCG CTG CTG GTG CGG CTG CTG CAG GCG
CTG
Leu Thr Glu Leu Leu Gly Ala Gln Asp Gly Ala Leu Leu Val Arg Leu Leu Gln Ala
Leu
850 860 870 880 890 900
CGC CiTG GCC AGG ATG CCC GGG CTG GAG CGG AGC CTC CGT GAG CGC TTC CTC CCT GTG
CAC
Arg Val Ala Arg Met Pro Gly Leu Glu Arg Ser Val Arg Glu Arg Phe Leu Pro Val
His
TGA
~~~
