Title: Lipoxygenase proteins
and polynucleotides encoding the same
United States Patent: 7,144,730
Issued: December 5, 2006
Inventors: Turner, Jr.; C.
Alexander (The Woodlands, TX), Zambrowicz; Brian (The Woodlands, TX),
Nehls; Michael (Stockdorf, DE), Friedrich; Glenn (Houston, TX), Sands;
Arthur T. (The Woodlands, TX)
Assignee: Lexicon Genetics
Incorporated (The Woodlands, TX)
Filed: April 23, 2003
The nucleotide and corresponding amino
acid sequences are reported for a novel class of mammalian lipoxygenase
proteins. The novel lipoxygenase encoding polynucleotides were obtained
from human gene trap clones and human cDNA libraries.
SUMMARY OF THE
The present invention relates to the
discovery, identification, and characterization of nucleotides that encode
novel human lipoxygenase proteins, and the corresponding amino acid
sequences of these proteins. The novel human proteins (NHPs) described for
the first time herein share structural similarity with animal and plant
lipoxygenase proteins. As such, the novel genes represent a new class of
lipoxygenase proteins with a range of homologues and orthologs that
transcend a broad range of phyla and species.
The invention comprises (a) polypeptides with SEQ ID NOS:2, 4, 6, 8, 10,
12, 14, 16, 18, 20, 22, 24, 26, and 28; (b) homologues and allelic
variants of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and
28; (c) fragments of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,
24, 26, and 28 of any size, for example, from 4 amino acids to less than
the full-length of a polypeptide of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16,
18, 20, 22, 24, 26, or 28 and any number between; (d) fragments of SEQ ID
NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28 that correspond
to a functional domain (for example, a catalytic domain, a signal
sequence, a ligand binding domain, a regulatory domain, etc.); (e) fusion
proteins comprising a polypeptide sequence of any one of (a) through (d);
(f) mutant polypeptides (including engineered and naturally occurring
mutants) comprising a polypeptide sequence of any one of (a) through (d),
including, but not limited to, deletion mutants, insertion mutants,
substitution mutants, and mutant polypeptides in which all or a part of at
least one of the domains is deleted or altered (e.g., a mutant of the
active site with altered substrate specificity).
The invention further comprises (g) polynucleotides with SEQ ID NOS:1, 3,
5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, and 29; (h) polynucleotides
encoding any one of the polypeptides of the invention including, but not
limited to, polypeptides specifically described in (a) through (f) above;
(i) polynucleotides capable of hybridizing to a second polynucleotide that
is complementary to a polynucleotide described in (g) and/or (h) above
under conditions of low, medium, or high stringency; (j) oligonucleotides
corresponding to a segment of a polynucleotide described in (g) through (i)
above and such oligonucleotides having any size from 2 nucleotides through
less than the full-length polynucleotide and any length inbetween.
In certain embodiments, the novel human nucleic acid sequences described
herein, encode proteins/open reading frames (ORFs) of 711, 489, 556, 334,
615, 460, 291, 69, 139, 195, 110, 867, 645, and 771 amino acids in length
(see SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28
The invention further comprises antibodies to any one of the polypeptides
or polynucleotides of the invention. The invention also comprises host
cells that are engineered to contain and/or express any one of the
polynucleotides and/or polypeptides of the invention.
The invention also comprises agonists and antagonists of the described
NHPs, including small molecules, large molecules, mutant NHPs, or portions
thereof that compete with native NHP, and antibodies. The invention
further comprises nucleotide sequences that can be used to inhibit the
expression of the described NHPs (e.g., antisense and ribozyme
oligonucleotides and/or polynucleotides, and gene or regulatory sequence
replacement constructs) or to enhance the expression of the described NHP
genes (e.g., expression constructs that place the described gene under the
control of a strong promoter system), and transgenic animals that express
a NHP transgene, or "knock-outs" (which can be conditional) that do not
express functional NHP.
Further, the present invention also relates to methods for identifying
compounds that modulate, i.e., act as agonists or antagonists of, NHP
expression and/or NHP product activity that utilize purified preparations
of the described NHPs and/or NHP product, or cells expressing the same.
Such compounds can be used as therapeutic agents for the treatment of any
of a wide variety of symptoms associated with biological disorders or
OF THE INVENTION
Lipoxygenases oxidize, or oxygenate,
lipids to produce leukotrienes. Depending on the leukotriene synthesized,
a wide variety of biological functions can be affected. Typically,
leukotrienes will bind cognate receptors an trigger a biological effect
(such as, for example, signal transduction). Interfering with lipoxygenase
activity ultimately effects leukotriene production and downstream
leukotriene-mediated processes. Alternatively, enhancing lipoxygenase
activity in vivo, can boost the effects/activity levels the corresponding
biological processes. Various lipoxygenase activities can be found in a
variety of cells and tissues in both animals and plants. Three predominant
types of lipoxygenases include the 5-, 12-, and 15-lipoxygenases, and each
type of lipoxygenase can have additional forms depending upon the tissues
or cells in which they are expressed.
The 5-, 12-, and 15-lipoxygenases, and the leukotrienes they produce, have
been implicated with a variety of diseases and disorders. Given that
leukotrienes can modulate inflammatory reactions, they have been
associated with a spectrum of mammalian diseases including, but not
limited to, asthma, eye diseases, anaphylaxis, lung disease, hematological
disorders, infectious diseases, granulomatosis, abscess, pacreatitis,
prostatitis, hepatitis, atherosclerosis, heart disease, graft rejection,
thrombosis, restenosis, ulcers, kidney disease, hypertension, dermatoses,
cramping, autoimmune disorders (lupus, scleroderma, Crohn's disease,
rheumatoid arthritis, etc.), granulomatosis, hyperproliferative diseases,
cancer, nausea, headache, metastases, inflammatory bowel disorder,
allergy, cancer, arthritis, eczema, melanoma, erythema, acne, psoriasis,
shingles, infectious disease, and diabetes. Accordingly, one embodiment of
the present invention are processes for identifying compounds useful for
the treatment of one or more of the above diseases and disorders that
include the use of one or more of the described lipoxygenase-like genes,
proteins, or a novel portion thereof.
Given the biological importance of lipoxygenases, the genes encoding such
proteins (and the proteins encoded thereby as well as inhibitors thereof)
have been subjected to intense scientific/commercial scrutiny (see, for
example, U.S. Pat. Nos. 5,036,105, 5,162,365, 5,504,097, 5,066,679,
5,830,453, 4,761,403, 5,589,506, 5,026,729, and 5,861,268) (all of which
are herein incorporated by reference in their entirety).
The presently described NHPs share significant similarity with previously
described human lipoxygenases. Expression studies using RT-PCR detect NHP
transcripts in, inter alia, neural tissue (i.e., brain, spinal cord,
etc.), skin, testis, prostate, adrenal gland, cervix, salivary gland,
pancreas, heart, lymphoid cells (lymph node, spleen, thymus), and mammary
glands. Northern analysis showed a predominant signal in testis, with less
predominant, but longer, transcripts detectable in testis, lymph node, and
spinal cord. A full length cDNA of a NHP coding region (with 5' and 3'
extensions) was isolated from a human brain cDNA library (Edge BioSystems,
Gaithersburg, Md.) and sequenced (SEQ ID NO: 29). A possible murine
ortholog of the described NHPs is predominantly expressed in skin (Kinzig
et al., 1999, Genomics 58:158 164).
The invention encompasses the use of the described NHP nucleotides, NHPs
and peptides, as well as antibodies, preferably monoclonal antibodies, or
binding fragments, domains, or fusion proteins thereof, or anti-idiotypic
variants derived therefrom, that bind NHPs, other antagonists that inhibit
binding activity or expression, or agonists that activate NHP activity or
increase NHP expression, in the diagnosis and/or treatment of disease.
In particular, the invention described in the subsections below
encompasses NHP polypeptides or peptides corresponding to functional
domains of NHPs, mutated, truncated or deleted NHPs (e.g., NHPs missing
one or more functional domains or portions thereof), NHP fusion proteins
(e.g., a NHP or a functional domain of a NHP fused to an unrelated protein
or peptide such as an immunoglobulin constant region, i.e., IgFc),
nucleotide sequences encoding such products, and host cell expression
systems that can produce such NHP products.
The invention also encompasses antibodies and anti-idiotypic antibodies
(including Fab fragments), antagonists and agonists of the NHP, as well as
compounds or nucleotide constructs that inhibit expression of a NHP gene
(transcription factor inhibitors, antisense and ribozyme molecules, or
gene or regulatory sequence replacement constructs), or promote expression
of NHP (e.g., expression constructs in which NHP coding sequences are
operatively associated with expression control elements such as promoters,
promoter/enhancers, etc.). The invention also relates to host cells and
animals genetically engineered to express the NHPs (or mutant variants
thereof) or to inhibit or "knock-out" expression of an animal homolog of
an endogenous NHP gene.
The NHPs or peptides, NHP fusion proteins, NHP nucleotide sequences,
antibodies, antagonists and agonists can be useful for the detection of
mutant NHPs or inappropriately expressed NHPs for the diagnosis of
disease. The NHP proteins or peptides, NHP fusion proteins, NHP nucleotide
sequences, host cell expression systems, antibodies, antagonists, agonists
and genetically engineered cells and animals can be used for screening for
drugs (or high throughput screening of combinatorial libraries) effective
in the treatment of the symptomatic or phenotypic manifestations of
perturbing the normal function of NHP in the body.
The use of engineered host cells and/or animals offers an advantage in
that such systems allow for both the identification of compounds that
interact with an NHP, and also provide information regarding the
biological significance of the NHP.
Finally, NHP products (especially soluble derivatives such as peptides
corresponding to the NHP), and NHP fusion protein products (such as NHP-Ig
fusion proteins, i.e., fusions of a NHP, or a domain of a NHP, to an IgFc),
antibodies and anti-idiotypic antibodies (including Fab fragments),
antagonists or agonists (including compounds that modulate signal
transduction which may act on downstream targets in a NHP-associated
leukotriene pathway) can be used to directly treat diseases or disorders.
Nucleotide constructs encoding such NHP products can be delivered to host
cells that subsequently express the products in vivo; these genetically
engineered cells function as "bioreactors" in the body delivering a
continuous supply of a NHP, a NHP peptide, or a NHP fusion protein to the
body. Nucleotide constructs encoding functional NHPs, mutant NHPs, as well
as antisense and ribozyme molecules can also be used in "gene therapy"
approaches for the modulation of NHP expression. Thus, the invention also
encompasses pharmaceutical formulations and methods for treating
Claim 1 of 3 Claims
1. An isolated polynucleotide
comprising a first polynucleotide capable of hybridizing to a second
polynucleotide in 0.5 M NaHPO.sub.4, 7% sodium dodecyl sulfate (SDS), 1 mM
EDTA at 65.degree. C. with washing in 0.1.times.SSC/0.1% SDS at 68.degree.
C., wherein the first polynucleotide encodes a polypeptide having
lipoxygenase activity and wherein the second polynucleotide is complementary
to the entire sequence of a polynucleotide with the sequence of SEQ ID NO:1.
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