U.S. patent application number 10/403745 was filed with the patent office on 2003-09-04 for novel human lipase proteins, nucleic acids encoding them, and uses of both of these.
This patent application is currently assigned to Millennium Pharmaceuticals, Inc.. Invention is credited to Kapeller-Libermann, Rosana, Khodadoust, Mehran.
Application Number | 20030165975 10/403745 |
Document ID | / |
Family ID | 23627707 |
Filed Date | 2003-09-04 |
United States Patent
Application |
20030165975 |
Kind Code |
A1 |
Khodadoust, Mehran ; et
al. |
September 4, 2003 |
Novel human lipase proteins, nucleic acids encoding them, and uses
of both of these
Abstract
The invention provides isolated nucleic acids encoding human
lipase proteins and fragments, derivatives, and variants thereof.
These nucleic acids and proteins are useful for diagnosis,
prevention, and therapy of a number of human and other animal
disorders associated, for example, with aberrant lipid metabolism
or aberrant pancreatic activity. The invention also provides
antisense nucleic acid molecules, expression vectors containing the
nucleic acid molecules of the invention, host cells into which the
expression vectors have been introduced, and non-human transgenic
animals in which a nucleic acid molecule of the invention has been
introduced or disrupted. The invention still further provides
isolated polypeptides, fusion polypeptides, antigenic peptides, and
antibodies. Diagnostic, prognostic, screening, and therapeutic
methods involving use of compositions of the invention are also
provided. The nucleic acids and polypeptides of the present
invention are useful as modulating agents in regulating a variety
of cellular processes relating to mono-, di-, and triglyceride
metabolism and pancreatic function.
Inventors: |
Khodadoust, Mehran;
(Chestnut Hill, MA) ; Kapeller-Libermann, Rosana;
(Chestnut Hill, MA) |
Correspondence
Address: |
MILLENNIUM PHARMACEUTICALS, INC.
75 Sidney Street
Cambridge
MA
02139
US
|
Assignee: |
Millennium Pharmaceuticals,
Inc.
|
Family ID: |
23627707 |
Appl. No.: |
10/403745 |
Filed: |
March 31, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10403745 |
Mar 31, 2003 |
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09411132 |
Oct 1, 1999 |
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6558936 |
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Current U.S.
Class: |
435/6.18 ;
435/198; 435/320.1; 435/325; 435/69.1; 435/7.1; 530/388.26;
536/23.2 |
Current CPC
Class: |
C12N 9/20 20130101; A61K
48/00 20130101; A61K 38/00 20130101; C12Q 1/6883 20130101 |
Class at
Publication: |
435/6 ; 435/7.1;
435/69.1; 435/198; 435/320.1; 435/325; 536/23.2; 530/388.26 |
International
Class: |
C12Q 001/68; G01N
033/53; C07H 021/04; C12N 009/20; C12P 021/02; C12N 005/06 |
Claims
What is claimed is:
1. An isolated nucleic acid molecule selected from the group
consisting of: a) a nucleic acid molecule having a nucleotide
sequence which is at least 40% identical to the nucleotide sequence
of one of SEQ ID NOs: 1 and 2, or a complement thereof; b) a
nucleic acid molecule comprising a portion having a nucleotide
sequence identical to at least 56 consecutive nucleotide residues
of one of SEQ ID NOs: 1 and 2, or a complement thereof; c) a
nucleic acid molecule which encodes a polypeptide having the amino
acid sequence of SEQ ID NO: 3, or a complement thereof; d) a
nucleic acid molecule which encodes a fragment of a polypeptide
having the amino acid sequence of SEQ ID NO: 3, wherein the
fragment comprises at least 17 consecutive amino acid residues of
the polypeptide; and e) a nucleic acid molecule which encodes a
naturally-occurring allelic variant of a polypeptide having the
amino acid sequence of SEQ ID NO: 3, wherein the nucleic acid
molecule hybridizes under stringent conditions with a nucleic acid
molecule having of the nucleotide sequence of one of SEQ ID NOs: 1
and 2, or a complement thereof.
2. The isolated nucleic acid molecule of claim 1, which is selected
from the group consisting of: a) a nucleic acid having the
nucleotide sequence of one of SEQ ID NOs: 1 and 2, or a complement
thereof; and b) a nucleic acid molecule which encodes a polypeptide
having the amino acid sequence of SEQ ID NO: 3, or a complement
thereof.
3. The nucleic acid molecule of claim 1, further comprising a
vector nucleic acid sequence.
4. The nucleic acid molecule of claim 1 further comprising a
portion encoding a heterologous polypeptide.
5. A host cell which contains the nucleic acid molecule of claim
1.
6. The host cell of claim 5, wherein the host cell is a mammalian
host cell.
7. A non-human mammalian host cell containing the nucleic acid
molecule of claim 1.
8. An isolated polypeptide selected from the group consisting of:
a) a fragment of a first polypeptide having the amino acid sequence
of SEQ ID NO: 3, wherein the fragment comprises at least 17
contiguous amino acid residues of the first polypeptide; b) a
naturally-occurring allelic variant of a first polypeptide having
the amino acid sequence of SEQ ID NO: 3, wherein the first
polypeptide is encoded by a nucleic acid molecule which hybridizes
under stringent conditions with a nucleic acid molecule having of
the nucleotide sequence of one of SEQ ID NOs: 1 and 2, or a
complement thereof; c) a polypeptide encoded by a nucleic acid
molecule having a nucleotide sequence which is at least 40%
identical to one of SEQ ID NOs: 1 and 2, or a complement thereof;
and d) a polypeptide having the amino acid sequence of about
residues 18-467 of SEQ ID NO: 3.
9. The isolated polypeptide of claim 8 having the amino acid
sequence SEQ ID NO: 3.
10. The polypeptide of claim 8, wherein the amino acid sequence of
the polypeptide further comprises a heterologous amino acid
residue.
11. An antibody which selectively binds with the polypeptide of
claim 8.
12. A method for producing a polypeptide selected from the group
consisting of: a) a polypeptide having the amino acid sequence of
SEQ ID NO: 3; b) a polypeptide comprising a fragment of a first
polypeptide having the amino acid sequence of SEQ ID NO: 3, wherein
the fragment comprises at least 17 contiguous amino acid residues
of the first polypeptide; and c) a naturally-occurring allelic
variant of a first polypeptide having the amino acid sequence of
SEQ ID NO: 3, wherein the first polypeptide is encoded by a nucleic
acid molecule which hybridizes under stringent conditions with a
nucleic acid molecule having the nucleotide sequence of one of SEQ
ID NOs: 1 and 2, or a complement thereof; the method comprising
culturing the host cell of claim 5 under conditions in which the
nucleic acid molecule is expressed.
13. A method for detecting the presence of a polypeptide of claim 8
in a sample, the method comprising: a) contacting the sample with a
compound which selectively binds with a polypeptide of claim 8; and
b) determining whether the compound binds with the polypeptide in
the sample.
14. The method of claim 13, wherein the compound is an
antibody.
15. A kit comprising a compound which selectively binds with a
polypeptide of claim 8 and instructions for use.
16. A method for detecting the presence of a nucleic acid molecule
of claim 1 in a sample, the method comprising: a) contacting the
sample with a nucleic acid probe or primer which selectively
hybridizes with the nucleic acid molecule; and b) determining
whether the nucleic acid probe or primer binds with a nucleic acid
molecule in the sample.
17. The method of claim 16, wherein the sample comprises mRNA
molecules and is contacted with a nucleic acid probe.
18. A kit comprising a compound which selectively hybridizes with a
nucleic acid molecule of claim 1 and instructions for use.
19. A method for identifying a compound which binds with a
polypeptide of claim 8, the method comprising: a) contacting the
polypeptide, or a cell expressing the polypeptide with a test
compound; and b) determining whether the polypeptide binds with the
test compound.
20. The method of claim 19, wherein binding of the test compound
with the polypeptide is detected by a method selected from the
group consisting of: a) detection of binding by direct detection of
test compound/polypeptide binding; b) detection of binding using a
competition binding assay; c) detection of binding using an assay
for an activity characteristic of the polypeptide.
21. A method for modulating the activity of a polypeptide of claim
8, the method comprising contacting the polypeptide or a cell
expressing the polypeptide with a compound which binds with the
polypeptide in a sufficient concentration to modulate the activity
of the polypeptide.
22. A method for identifying a compound which modulates the
activity of a polypeptide of claim 8, the method comprising: a)
contacting the polypeptide with a test compound; and b) determining
the effect of the test compound on the activity of the polypeptide
to thereby identify a compound which modulates the activity of the
polypeptide.
23. An antibody substance which selectively binds with the
polypeptide of claim 8, wherein the antibody substance is made by
providing the polypeptide to an immunocompetent vertebrate and
thereafter harvesting blood or serum from the vertebrate.
24. A method of treating a patient afflicted with a disorder
associated with aberrant activity or expression of MLip-1 protein,
the method comprising administering to the patient a compound which
modulates the activity of the protein in an amount effective to
modulate the activity of the protein in the patient, whereby at
least one symptom of the disorder is alleviated.
25. A method of treating a patient afflicted with a disorder
associated with aberrant activity or expression of MLip-l protein,
the method comprising administering to the patient, in an amount
effective to modulate the activity of the protein in the patient, a
compound selected from the group consisting of i) the protein; ii)
a variant of the protein; iii) a nucleic acid encoding the protein;
and iv) an antisense nucleic acid which is capable of annealing
with either of an mRNA encoding the protein and a portion of a
genomic DNA encoding the protein, whereby at least one symptom of
the disorder is alleviated.
26. A method of diagnosing a disorder associated with aberrant
activity or expression of MLip-1 protein, the method being selected
from the group consisting of (i) a method comprising assessing the
level of expression of the gene encoding the protein in the patient
and comparing the level of expression of the gene with the normal
level of expression of the gene in a human not afflicted with the
disorder, whereby a difference between the level of expression of
the gene in the patient and the normal level is an indication that
the patient is afflicted with the disorder and (ii) a method
comprising comparing a sequence selected from the group consisting
of (a) the nucleotide sequence of a nucleic acid encoding MLip-1
and (b) the amino acid sequence of a portion of MLip-1 protein in a
sample obtained from a patient with the same sequence in a sample
obtained from a subject who is known not to be afflicted and not to
be predisposed to becoming afflicted with the disorder, whereby a
difference between the two sequences is an indication that the
patient is afflicted with the disorder.
27. A method of determining whether a patient is likely to become
afflicted with a disorder associated with aberrant activity or
expression of MLip-1 protein, the method comprising comparing a
sequence selected from the group consisting of (i) the nucleotide
sequence of a nucleic acid encoding MLip-1 and (ii) the amino acid
sequence of a portion of MLip-1 protein in a sample obtained from a
patient with the same sequence in a sample obtained from a subject
who is known not to be afflicted and not to be predisposed to
becoming afflicted with the disorder, whereby a difference between
the two sequences is an indication that the patient is likely to
become afflicted with the disorder.
Description
BACKGROUND OF THE INVENTION
[0001] Lipids are esters of long chain fatty acids (generally
C.sub.14 to C.sub.24 saturated and unsaturated fatty acids in
animal fats) and polyols such as glycerol, glycerol phosphates,
alkyl glyceryl ethers, glycerol phosphoryl-choline, glycerol
phosphoryl-serine, glycerol phosphoryl-ethanolamine, and the like.
Lipids, in the form of cell membranes and fats, for example,
constitute a significant proportion of animal body weight (e.g.,
about 5% to 25% of body weight in normal humans).
[0002] Lipids are not water-soluble, and generally do not cross
biological membranes efficiently by simple diffusion. Dietary
lipids are taken up primarily by hydrolysis of fatty acyl moieties
from their corresponding polyol moiety and diffusion of the two
moieties across the gut wall (although limited uptake of intact
lipids occurs). Following absorption, lipids are reformed by
reestablishment of ester bonds between polyol and fatty acyl
moieties, and lipids are delivered throughout the body in
esterified form (generally in lipoprotein-containing particles such
as chylomicrons, very low, intermediate, low, and high density
lipoprotein particles, and the like). Prior to uptake by cells
(either for storage or for metabolism), lipids must again be
hydrolyzed in order to facilitate passage across the cell membrane.
Thus, enzymes which catalyze formation and hydrolysis of the ester
bonds between fatty acyl moieties and polyol moieties of lipids
must be present at several physiological locations, and the
particular activities catalyzed by these enzymes (`lipases`) varies
depending on the physiological location and function of the
enzyme.
[0003] A number of lipase enzymes have been characterized in
various organisms, including in humans. However, it is far from
clear that all physiologically relevant lipases have been
discovered or characterized. The present invention provides novel
nucleotide and amino acid sequence information corresponding to one
or more human lipases.
SUMMARY OF THE INVENTION
[0004] The present invention is based, at least in part, on
discovery of human cDNA molecules which encode lipase proteins such
as the one herein designated MLip-1. These proteins catalyze
formation and cleavage of ester bonds between fatty acyl moieties
and glyceride moieties. MLip-1 protein, fragments thereof,
derivatives thereof, and variants thereof are collectively referred
to herein as polypeptides of the invention or proteins of the
invention. Nucleic acid molecules encoding polypeptides of the
invention (i.e., nucleic acids encoding MLip-1 protein, fragments
thereof, derivatives thereof, and variants thereof) are
collectively referred to as nucleic acids of the invention.
[0005] The nucleic acids and polypeptides of the present invention
are useful as modulating agents in regulating a variety of cellular
processes, particularly including processes which involve lipid
metabolism and pancreatic function. Accordingly, in one aspect, the
present invention provides isolated nucleic acid molecules encoding
a polypeptide of the invention or a biologically active portion
thereof. The present invention also provides nucleic acid molecules
which are suitable as primers or hybridization probes for detection
of nucleic acids encoding a polypeptide of the invention.
[0006] The invention also includes nucleic acid molecules which are
at least 40% (or, for example, 50%, 60%, 70%, 80%, 90%, 95%, or 98%
or more) identical to the nucleotide sequence of either of SEQ ID
NOs: 1 and 2, or a complement thereof.
[0007] The invention includes nucleic acid molecules which include
a fragment of at least 56 (or, for example, 58, 60, 70, 80, 100,
125, 150, 200, 250, 300, 350, 400, 450, 550, 650, 700, 800, 900,
1000, 1200, 1400, 1600, 1800, 2000, or 2352) consecutive nucleotide
residues of either of SEQ ID NOs: 1 and 2, or a complement
thereof.
[0008] The invention also includes nucleic acid molecules which
have a nucleotide sequence encoding a protein having an amino acid
sequence that is at least 50% (or, for example, 60%, 70%, 80%, 90%,
95%, or 98% or more) identical to all or residues about 18-467 of
the amino acid sequence SEQ ID NO: 3, or a complement thereof.
[0009] In certain embodiments, the nucleic acid molecules have the
nucleotide sequence of either of SEQ ID NOs: 1 and 2.
[0010] Also within the invention are nucleic acid molecules which
encode a fragment of a polypeptide having the amino acid sequence
of SEQ ID NO: 3, the fragment including at least 17 (or, for
example, 18, 20, 25, 30, 40, 50, 75, 100, 125, 150, 200, 250, 300,
400, or 467) consecutive amino acid residues of SEQ ID NO: 3.
[0011] The invention includes nucleic acid molecules which encode a
naturally-occurring allelic variant of a polypeptide having the
amino acid sequence of SEQ ID NO: 3, wherein the nucleic acid
molecule hybridizes under stringent conditions with a nucleic acid
molecule having a nucleic acid sequence comprising either of SEQ ID
NOs: 1 and 2, or a complement thereof.
[0012] The invention also includes nucleic acid molecules that
hybridize under stringent conditions with a nucleic acid molecule
having the nucleotide sequence of either of SEQ ID NOs: 1 and 2, or
a complement thereof. In other embodiments, the nucleic acid
molecules are at least 56 (or, for example, 58, 60, 70, 80, 100,
125, 150, 200, 250, 300, 350, 400, 450, 550, 650, 700, 800, 900,
1000, 1200, 1400, 1600, 1800, 2000, or 2352) nucleotides in length
and hybridize under stringent conditions with a nucleic acid
molecule having the nucleotide sequence of either of SEQ ID NOs: 1
and 2, or a complement thereof. In some embodiments, the isolated
nucleic acid molecules encode an immature or mature form of a
polypeptide of the invention. In other embodiments, the invention
provides an isolated nucleic acid molecule which is antisense with
respect to the coding strand of a nucleic acid of the
invention.
[0013] Another aspect of the invention provides vectors, e.g.,
recombinant expression vectors, comprising a nucleic acid molecule
of the invention. In a related aspect, the invention provides
isolated host cells, e.g., mammalian and non-mammalian cells,
containing such a vector or a nucleic acid of the invention. The
invention also provides methods for producing a polypeptide of the
invention by culturing, in a suitable medium, a host cell of the
invention containing a recombinant expression vector encoding a
polypeptide of the invention such that the polypeptide of the
invention is produced.
[0014] Another aspect of this invention includes isolated or
recombinant proteins and polypeptides of the invention. Isolated
polypeptides or proteins have an amino acid sequence that is at
least about 50% (or, for example, 60%, 75%, 90%, 95%, or 98% or
more) identical to all or a portion of the amino acid sequence of
SEQ ID NO: 3. Exemplary polypeptides of the invention include a
polypeptide having the amino acid sequence SEQ ID NO: 3, a
polypeptide having the amino acid sequence of only residues 1 to
about 17 of SEQ ID NO: 3 (i.e., the signal peptide of MLip-1), a
polypeptide having the amino acid sequence of about residues 18 to
467 of SEQ ID NO: 3 (i.e., mature MLip-1 protein), and a
polypeptide corresponding to a solvent-exposed portion of MLip-1
protein (e.g., about amino acid residues 80 to 105 of SEQ ID NO:
3).
[0015] Also within the invention are isolated polypeptides or
proteins which are encoded by a nucleic acid molecule having a
nucleotide sequence that is at least about 40% (or, for example,
50%, 75%, 85%, or 95% or more) identical to the nucleic acid
sequence of either of SEQ ID NOs: 1 and 2, and isolated
polypeptides or proteins which are encoded by a nucleic acid
molecule having a nucleotide sequence which hybridizes under
stringent hybridization conditions with a nucleic acid molecule
having the nucleotide sequence of either of SEQ ID NOs: 1 and
2.
[0016] Also within the invention are polypeptides which are
naturally-occurring allelic variants of a polypeptide that has the
amino acid sequence SEQ ID NO: 3, wherein the polypeptide is
encoded by a nucleic acid molecule which hybridizes under stringent
conditions with a nucleic acid molecule having the nucleotide
sequence of either of SEQ ID NOs: 1 and 2, or a complement
thereof.
[0017] In certain embodiments, proteins and polypeptides possess at
least one biological activity possessed by the corresponding
naturally-occurring human polypeptide. An activity or a biological
activity of a polypeptide of the invention refers to an activity
exerted by the polypeptide of the invention on a responsive cell,
on a portion of a cell (e.g., a cell membrane), on a cellular
nutrient (e.g., a triglyceride or other lipid), or on a cellular
metabolite or other product (e.g., cholesterol or membrane lipids).
Such activity can be assessed in vivo or in vitro, according to
standard techniques. MLip-1 polypeptides of the invention exhibit
lipase activity, and can be involved in a number of bodily
functions including, for example, dietary fat degradation and
absorption, cholesterol biosynthesis, and maintenance of plasma
lipid and lipoprotein levels. Such activities can, for example, be
an enzymatic activity exerted by a polypeptide of the invention on
another protein or on a non-protein substrate (e.g., on a
lipoprotein particle or a triglyceride).
[0018] By way of example, protein MLip-1, compounds which modulate
its activity, expression, or both, and compounds (e.g., antibodies)
which bind with MLip-l (collectively "MLip-1-related molecules")
exhibit the ability to affect growth, proliferation, survival,
differentiation, and activity of pancreatic tissue, in which MLip-1
is expressed. MLip-1-related molecules can be used to prevent,
diagnose, or treat disorders relating to inappropriate lipid
metabolism and aberrant pancreatic function. Exemplary disorders
for which MLip-1-related molecules are useful include diabetes,
obesity, nutritional disorders (e.g., lipid malabsorption and
malnutrition), metabolic disorders (particularly including lipid
metabolism anomalies such as hyperlipidemia of types I to V and
hypolipidemia), pancreatitis, obstruction of the pancreatic duct,
various lipidoses (e.g., Gaucher's disease and Niemann-Pick
disease), atherosclerosis, arteriosclerosis, coronary artery
disease, perforated peptic ulcer, abdominal lesions, intestinal
obstruction, peritonitis, and other diseases and disorders
associated with aberrant or physiologically inappropriate lipase
and lipase-like activity.
[0019] In one embodiment, a polypeptide of the invention has an
amino acid sequence that is sufficiently identical to an identified
domain of MLip-1 (e.g., a domain present at the surface of MLip-1
or the lipase domain described herein) that the polypeptide
exhibits an antigenic or enzymatic characteristic of MLip-1. Such
polypeptides comprise at least about 17 (18, 20, 25, 35, 50, 75,
100, 150, 200, 250, or 300 or more) amino acid residues, of which
at least about 65%, preferably at least about 75%, and more
preferably at least about 85%, 95%, or 98% are identical or similar
(representing conservative amino acid substitutions; i.e., between
amino acids having similar side chain moieties). Exemplary
antigenic and enzymatic characteristics of MLip-1 which are
exhibited by such polypeptides include lipase activity, ability to
bind with molecules (e.g., enzymatic substrates or cell-surface or
lipoprotein particle surface sites) with which MLip-1 is able to
bind, and ability to induce production of antibody substances
(e.g., free and cell-surface-bound immunoglobulins such as
antibodies and T cell receptors) which bind specifically with an
epitope which occurs at or near the surface of MLip-1 protein.
[0020] The polypeptides of the present invention, or biologically
active portions thereof, can be operably linked with a heterologous
amino acid sequence to form fusion proteins. In addition, one or
more polypeptides of the invention or biologically active portions
thereof can be incorporated into pharmaceutical compositions, which
can optionally include pharmaceutically acceptable carriers. Such
pharmaceutical compositions can be used to treat or prevent one or
more of the disorders identified herein.
[0021] The invention encompasses antibody substances that
specifically bind with a polypeptide of the invention including,
for example, MLip-1 protein and fragments thereof. Exemplary
antibody substances that are included within the scope of the
invention are monoclonal and polyclonal antibodies, antibody
fragments, single-chain antibodies, free and cell-surface-bound
antibodies, and T cell receptors. These antibody substances can be
made, for example, by providing the polypeptide of the invention to
an immunocompetent vertebrate and thereafter harvesting blood or
serum from the vertebrate. Antibody substances can, alternatively,
be generated by screening a library of phage (e.g., a filamentous
phage such as M13) which express one or more immunoglobulin
subunits (e.g., IgG heavy chains) on their surface to identify
phage particles which display a subunit which binds with MLip-1 or
an epitope thereof.
[0022] In another aspect, the present invention provides methods
for detecting activity or expression of a polypeptide of the
invention in a biological sample by contacting the biological
sample with an agent capable of detecting such activity (e.g., a
labeled substrate or another compound that can be detected after
being acted upon by an active polypeptide of the invention), with
an agent which binds specifically with a polypeptide of the
invention (e.g., an antibody substance of the invention), or with
an agent for detecting production of an RNA encoding a polypeptide
of the invention (e.g., a reverse transcriptase primer
complementary to a portion of an mRNA encoding the
polypeptide).
[0023] The present invention also provides diagnostic assays for
identifying the presence or absence of a genetic lesion or mutation
characterized by at least one of: (i) aberrant modification or
mutation of a gene encoding a polypeptide of the invention; (ii)
mis-regulation of a gene encoding a polypeptide of the invention;
and (iii) aberrant post-translational modification of a polypeptide
of the invention wherein a wild-type form of the gene encodes a
polypeptide which exhibits at least one activity of the polypeptide
of the invention. Such diagnostic assays include, for example, (i)
comparing the nucleotide sequence of all or part of a gene which
encodes a polypeptide of the invention and which is obtained from a
subject with the nucleotide sequence (or the corresponding part
thereof) of a gene obtained from a subject having a non-mutated
MLip-1 gene or one of SEQ ID NOs: 1 and 2; (ii) comparing the
presence or level in a sample obtained from a subject of a
polypeptide or polynucleotide corresponding to all or part of
MLip-1 with the presence or level in other samples (preferably
samples of the same type) obtained from one or more other subjects;
and (iii) determining whether a polypeptide or polynucleotide
corresponding to all or a part of MLip-1 that includes a sequence
corresponding to a post-translational modification site identified
herein, or determining whether a polypeptide of the invention is
modified at such a site.
[0024] In another aspect, the invention provides a method for
identifying a compound that modulates (i.e., inhibits or enhances)
the activity of or binds with a polypeptide of the invention. In
general, such methods entail measuring a biological activity of the
polypeptide in the presence and absence of a test compound and
identifying those compounds which alter the activity of the
polypeptide. Such methods can be performed in vitro or in vivo
(e.g., in an animal which naturally expresses the polypeptide or
nucleic acid or in an animal that has been modified such that it
artificially expresses the polypeptide or nucleic acid).
[0025] The invention also includes methods of identifying a
compound that modulates expression of a polypeptide or nucleic acid
of the invention by measuring expression of the polypeptide or
nucleic acid in the presence and absence of the compound.
[0026] In another aspect, the invention provides methods for
modulating activity of a polypeptide of the invention, the methods
comprising contacting a cell with an agent that modulates the
activity or expression of the polypeptide, such that activity or
expression in the cell is modulated (e.g., by contacting the cell
with a sufficient amount of the agent). In one embodiment, the
agent is an antibody that specifically binds with a polypeptide of
the invention. In another embodiment, the agent modulates
expression of a polypeptide of the invention by modulating
transcription, splicing, or translation of an RNA (e.g., a pre-mRNA
or an mRNA) encoding the polypeptide of the invention. In yet
another embodiment, the agent is a nucleic acid molecule having a
nucleotide sequence that is antisense with respect to the coding
strand of an RNA encoding a polypeptide of the invention. In still
other embodiments, the agent is a small molecule (e.g., a compound
having a molecular weight less than about 5,000) which modulates
activity or expression of a polypeptide or nucleic acid of the
invention.
[0027] In yet another aspect, the invention includes a method of
treating a patient afflicted with a disorder characterized by
aberrant activity of a polypeptide of the invention, or by aberrant
expression of a nucleic acid of the invention. The method comprises
administering to the patient an agent (e.g., a nucleic acid,
polypeptide, small molecule, antibody, or the like) in an amount
effective to modulate the activity of the polypeptide in the
patient or a to modulate the expression of the nucleic acid in the
patient. Following administration of the agent, at least one
symptom of the disorder is alleviated. In an alternative method of
treating a patient afflicted with a disorder associated with
aberrant activity or expression of MLip-1 protein, the method
comprises administering to the patient, in an amount effective to
modulate the level of activity of the protein in the patient, an
agent selected from the group consisting of i) a polypeptide of the
invention;
[0028] ii) a variant of a polypeptide of the invention;
[0029] iii) a nucleic acid encoding a polypeptide of the invention;
and
[0030] iv) an antisense nucleic acid which is capable of annealing
with either of an mRNA encoding a polypeptide of the invention and
a portion of a genomic DNA encoding a polypeptide of the
invention.
[0031] Following administration of the agent, at least one symptom
of the disorder is alleviated.
[0032] In still another aspect, the invention relates to a method
of diagnosing a disorder associated with aberrant expression of
MLip-1 protein in a patient. This method comprises assessing the
level of expression of the gene encoding the protein (e.g., by
assessing the quantity of a corresponding RNA, the quantity of a
corresponding protein, or the activity of a corresponding protein)
in the patient and comparing the level of expression of the gene
with the normal level of expression of the gene in a human not
afflicted with the disorder. A difference between the level of
expression of the gene in the patient and the normal level is an
indication that the patient is afflicted with the disorder.
[0033] The invention also includes a method of diagnosing a
disorder associated with expression of an aberrant or mutated
MLip-1 protein in a patient. This method can be performed by
comparing the nucleotide sequence of a nucleic acid encoding MLip-1
protein in a patient with a nucleotide sequence (e.g., one of SEQ
ID NOs: 1 and 2) encoding MLip-1 protein in a subject not afflicted
with the disorder. A difference between the two nucleotide
sequences is an indication that the patient is afflicted with the
disorder. This method can also be performed by comparing the amino
acid sequence of a portion (i.e., including all) of MLip-1 protein
in a sample obtained from the patient with the amino acid sequence
of the same portion of MLip-1 protein in a sample obtained from a
subject not afflicted with the disorder. A difference between the
two amino acid sequences is an indication that the patient is
afflicted with the disorder.
[0034] In yet another aspect, the invention relates to a method of
determining whether a patient is likely to become afflicted in the
future with a disorder associated with aberrant expression of
MLip-1 protein or with expression of an aberrant or mutated MLip-1
protein. In various embodiments, these prognostic methods comprise
(i) comparing the nucleotide sequence of a nucleic acid encoding
MLip-1 protein in a sample obtained from a patient with a
nucleotide sequence (e.g., one of SEQ ID NOs: 1 and 2) encoding
MLip-l protein in a subject known not to be afflicted and not to be
predisposed to becoming afflicted with the disorder or (ii)
comparing the amino acid sequence of all or a portion of MLip-1
protein obtained from a patient with the amino acid sequence (e.g.,
SEQ ID NO: 3) of MLip-1 protein obtained from a non-afflicted
subject.
[0035] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] FIG. 1, comprising FIGS. 1A and 1B, is an alignment of the
amino acid sequences of MLip-1 (SEQ ID NO: 3), hPL (SEQ ID NO: 4;
GenBank accession number M93283), hPLRP1 (SEQ ID NO: 5; GenBank
accession number M93284), and hPLRP2 (SEQ ID NO: 6; GenBank
accession number M93285) proteins made by the CLUSTAL method, using
DNAStar-Megalign software (PAM250 residue weight table and default
parameters).
[0037] FIG. 2 comprises FIGS. 2A, 2B, and 2C. The consensus
nucleotide sequence (SEQ ID NO: 1) of a cDNA encoding the human
MLip-1 protein described herein is listed in FIGS. 2A and 2B. The
amino acid sequence (SEQ ID NO: 3) of human MLip-1 protein is
listed in FIG. 2C.
[0038] FIG. 3 is an alignment of the amino acid sequences of MLip-1
(SEQ ID NO: 3), Mus musculus pancreatic lipase related protein 1
(MPLRP1; SEQ ID NO: 7; GenBank accession number G13108175), Rattus
norvegicus pancreatic lipase related protein 1 precursor (RPLRP1p;
SEQ ID NO: 8; GenBank accession number SP P54316), Canis familiaris
pancreatic triacylglycerol lipase precursor (CPTLp; SEQ ID NO: 9;
GenBank accession number GI 164048), and Canis familiaris
pancreatic lipase related protein 1 precursor (CPLRP1p; SEQ ID NO:
10; GenBank accession number SP P06857), the alignment made by the
CLUSTAL method using DNAStar-Megalign software (PAM250 residue
weight table and default parameters).
[0039] FIG. 4 is a hydrophilicity plot of human MLip-1 protein, in
which the locations of cysteine residues ("Cys") and potential
N-glycosylation sites ("Ngly") are indicated by vertical bars.
Portions of the plot situated above the horizontal line correspond
to hydrophobic regions of the protein, and portions of the plot
situated below the horizontal line correspond to hydrophilic
regions of the protein. The dashed vertical line indicates the
approximate location of the signal sequence cleavage site.
DETAILED DESCRIPTION OF THE INVENTION
[0040] The present invention is based, at least in part, on
identification of a human cDNA molecule which encodes a protein
herein designated MLip-1. MLip-1 is a lipase that is highly
expressed in pancreatic tissue. The invention includes MLip-1
protein, fragments, derivatives, and variants thereof (individually
and collectively, "polypeptides of the invention"), nucleic acids
encoding polypeptides of the invention, compounds (e.g., antibodies
and portions thereof and complementary polynucleotides) which bind
with one or more polypeptides or nucleic acids of the invention,
and compounds (e.g., small molecules) which modulate the activity,
expression, or both, of one or more polypeptides or nucleic acids
of the invention.
[0041] Certain characteristics of MLip-1 are now described.
[0042] Lipase MLip-1
[0043] A cDNA encoding at least a portion of human MLip-1 protein
was identified. MLip-1 protein is predicted by structural analysis
to be a secreted protein.
[0044] The full length of the cDNA encoding human MLip-1 (FIG. 2;
SEQ ID NO: 1) is 2352 nucleotide residues. The ORF of this cDNA,
nucleotide residues 125 to 1525 of SEQ ID NO: 1 (i.e., SEQ ID NO:
2), encodes a 467-amino acid residue immature protein (FIG. 2C; SEQ
ID NO: 3) which exhibits amino acid sequence homology with a number
of lipases and lipase-related proteins and which corresponds to an
approximately 449-amino acid residue mature protein. As indicated
in FIG. 4, the signal sequence of MLip-1 extends from amino acid
residue 1 to about residue 17 of SEQ ID NO: 3. This cleavage site
was predicted using the signal peptide prediction program SIGNALP
(Nielsen et al. (1997) Protein Engineering 10:1-6). It is
recognized that the carboxyl terminal boundary of the signal
sequence predicted using this program can be located one or two
residues from the residue identified above (i.e., from about
residue 15 to 19 of SEQ ID NO: 3). The signal sequence is normally
cleaved during processing of the mature protein, yielding secreted
mature MLip-1. However, it is recognized that MLip-1 protein can
persist, at least transiently, in a membrane-bound form in which
the signal sequence has not been cleaved. Mature MLip-1 protein can
be synthesized without the signal sequence polypeptide at the amino
terminus thereof, or it can be synthesized by generating immature
MLip-1 protein and cleaving the signal sequence therefrom.
[0045] MLip-1 proteins typically comprise a variety of potential
post-translational modification sites, such as those described
herein in Table 1, as predicted by computerized sequence analysis
of human MLip-1 protein using amino acid sequence comparison
software (comparing the amino acid sequence of MLip-1 with the
information in the PROSITE database {rel. 12.2; February, 1995} and
the Hidden Markov Models database {Rel. PFAM 3.3}). In certain
embodiments, a protein of the invention has at least 1, 2, 4, 6, 8,
10, or 15 or more of the post-translational modification sites
listed in Table I.
1TABLE I Amino Acid Type of Potential Modification Site Residues of
Amino Acid or Domain SEQ ID NO: 3 Sequence N-glycosylation site 74
to 77 NSST 125 to 128 NGSR 338 to 341 NGSH 412 to 415 NITS 439 to
442 NTSG N-myristoylation site 13 to 18 GTSRGK 31 to 36 GLPWTR 141
to 146 GAEVAY 170 to 175 GAHLAG 189 to 194 GLDPAG 231 to 236 GTIDAC
365 to 370 GSEVTQ 378 to 383 GGAIGK 397 to 402 GMTYTK 411 to 416
GNITSV Lipase serine active site 162 to 171 VHLIGHSLGA Lipase
domain 42 to 343 See FIG. 2 PLAT/LH2 domain 355 TO 467 See FIG.
2
[0046] MLip-1 protein comprises a lipase domain from about amino
acid residue 42 to about residue 343, including a conserved (among
lipases) active site serine residue at residue 168 of MLip-1. In
one embodiment, the protein of the invention has at least one
domain that is at least about 55%, preferably at least about 65%,
more preferably at least about 75%, yet more preferably at least
about 85%, and most preferably at least about 95% identical to this
lipase domain. Proteins of the invention also have a serine residue
at a position corresponding to serine-168 of MLip-1 although, of
course, the residue number at which the serine residue occurs can
vary, depending on the precise sequence of the protein. Lipase
domains occur in a variety of proteins involved in formation and
hydrolysis of one or more ester bonds of mono-, di-, and
tri-glycerides. Such proteins include, for example, pancreatic
lipases involved in dietary fat absorption, hepatic lipases
involved in cholesterol biosynthesis, lipoprotein lipases involved
in hydrolysis of lipids associated with chylomicrons and plasma
lipoprotein particles (e.g., very low, intermediate, low, and high
density lipoprotein particles), and gastric/lingual lipases
involved in initial degradation of dietary fats.
[0047] The amino acid sequence of nearly all lipase active sites
conforms to the following consensus sequence:
[0048] {L,I, or V}-X-{L,I,V,F, or Y}-{L,I,V,M,S, or T}-G-{H,Y,W, or
V}-S{acute over ()}-X-G-{G,S,T,A, or C}
[0049] wherein standard single amino acid codes are used (X being
any amino acid residue). The serine residue marked with an asterisk
is the active site residue. This consensus lipase serine active
site sequence occurs in the amino acid sequence of MLip-1, as
indicated in Table I.
[0050] Occurrence of a lipase domain, including a consensus lipase
active site, in the amino acid sequence of MLip-1 indicates that
MLip-1 is a lipase, or at least exhibits lipase or lipase-like
activity. MLip-1 is thus able to catalyze formation and breakage of
ester bonds that link one or more fatty acids to a glycerol moiety
such as glycerol, glycerol phosphates, alkyl glyceryl ethers,
glycerol phosphoryl-choline, glycerol phosphoryl-serine, glycerol
phosphoryl-ethanolamine, sphingolipids, cerebrosides, and the
like.
[0051] MLip-1 protein of the invention also contains a PLAT/LH2
domain (polycystin-1, lipoxygenase, alpha-toxin domain or
lipoxygenase homology domain). PLAT/LH2 domains occur in a variety
of membrane- and lipid-associated proteins, including many known
lipases, and mediate association of protein with membranes and
lipid vesicles (e.g., cell membranes and lipid globules that occur
in the digestive tract and blood stream). Occurrence of a PLAT/LH2
domain in MLip-1 is thus a further indication that this protein
exhibits lipase activity, particularly with regard to degradation
of extracellular lipids and generation and interconversion of
membrane-associated lipids.
[0052] MLip-1 protein exhibits amino acid sequence similarity to
human pancreatic proteins hPL (human pancreatic lipase), hPLRP1,
and hPLRP2 (human pancreatic lipase related proteins 1 and 2,
respectively; Giller et al., 1992, J. Biol. Chem. 267:16509-16516;
GenBank accession Nos. M93283, M93284, and M93285, respectively),
as indicated herein in FIGS. 1A and 1B. FIGS. 1A and 1B depict an
alignment of the amino acid sequences of human protein MLip-1 (SEQ
ID NO: 3) with the amino acid sequences of hPL (SEQ ID NO: 4),
hPLRP1 (SEQ ID NO: 5), and hPLRP2 (SEQ ID NO: 6). In this alignment
(PAM250 residue weight table), the amino acid sequence of MLip-1 is
revealed to be about 48% identical to the amino acid sequence of
hPL, about 47% identical to the amino acid sequence of hPLRP1, and
about 46% identical to the amino acid sequence of hPLRP2.
[0053] As described in the prior art, hPL, hPLRP1, and hPLRP2
appear to be secreted proteins (Giller et al., 1992, J. Biol. Chem.
267:16509-16516). The sequence similarity of MLip-1 with hPL,
hPLRP1, and hPLRP2 is a further indication that MLip-1 is a
secreted protein. When MLip-1 is secreted from an exocrine portion
of the pancreas, MLip-1 is able to catalyze conversion of dietary
fats (i.e., mono-, di-, and triglycerides) into compounds (e.g.,
fatty acids, glycerol moieties, and the like) than are more readily
absorbed by the body. When MLip-1 is secreted from a non-endocrine
portion of pancreatic or other tissue, it is capable of catalyzing
inter-conversion of fatty acids and mono-, di-, and tri-glycerides
(i.e., including phosphatides, phosphatidyl cholines, phosphatidyl
serines, phosphatidyl ethanolamines, and the like), thereby
modulating lipid metabolism of cells of pancreatic tissue and
tissues located in fluid communication with pancreatic tissue.
[0054] Protein MLip-1 also exhibits sequence similarity to several
non-human lipase-related proteins, as indicated in FIGS. 3A through
3C. These figures depict an alignment of the amino acid sequences
of human protein MLip-1 (SEQ ID NO: 3) with the amino acid
sequences of Mus musculus pancreatic lipase related protein 1
(Remington et al., 1999, Invest. Ophthalmol. Vis. Sci.
40:1081-1090; GenBank accession number AF061274; SEQ ID NO: 7),
Rattus norvegicus pancreatic lipase related protein 1 precursor
(Wicker-Planquart et al., 1992, FEBS Lett. 296:61-66; GenBank
accession number X61925; SEQ ID NO: 8), Canis familiaris pancreatic
triacylglycerol lipase precursor (Kerfelec et al., 1986, Pancreas
1:430-437; GenBank accession number M35302; SEQ ID NO: 9), and
Canis familiaris pancreatic lipase related protein 1 precursor
(Mickel et al., 1989, J. Biol. Chem. 264:12895-12901; SwissProt
accession number P06857; SEQ ID NO: 10). In this alignment (PAM250
residue weight table), the amino acid sequence of MLip-1 is
revealed to be about 49% identical to SEQ ID NO: 7, about 49%
identical to SEQ ID NO: 8, about 49% identical to SEQ ID NO: 9, and
about 49% identical to SEQ ID NO: 10. Similarity of MLip-1 to these
non-human lipase-related proteins is further evidence that MLip-1
exhibits lipase or lipase-like activity.
[0055] FIG. 4 depicts a hydrophilicity plot of protein MLip-1.
Relatively hydrophobic regions are above the dashed horizontal
line, and relatively hydrophilic regions are below the dashed
horizontal line. As described elsewhere herein, relatively
hydrophilic regions are generally located at or near the surface of
a protein, and are more frequently effective immunogenic epitopes
than are relatively hydrophobic regions. For example, the region of
human protein MLip-1 from about amino acid residue 95 to about
amino acid residue 105 appears to be located at or near the surface
of the protein, while the region from about amino acid residue 135
to about amino acid residue 150 appears not to be located at or
near the surface.
[0056] The predicted molecular weight of human protein MLip-1 is
about 52 kilodaltons prior to cleavage of the predicted signal
sequence, and about 50 kilodaltons after cleavage of the predicted
signal sequence.
[0057] Northern blot analysis of human adult and fetal tissues
indicated that mRNA corresponding to the cDNA encoding MLip-l is
expressed at detectable levels only in pancreas tissue.
[0058] Biological Function of Human MLip-1 Proteins, Nucleic Acids
Encoding Them, and Modulators of These Molecules
[0059] The observation that MLip-1 protein is expressed in
pancreatic tissue indicates that MLip-1 is a lipase involved in
aberrant and normal nutritional uptake and metabolism of lipids.
Thus, MLip-1 protein has a role in disorders which involve lipid
uptake and metabolism.
[0060] Occurrence of a lipase domain in protein MLip-1 is a further
indication that MLip-1 exhibits lipase activity and is involved in
disorders relating to lipid uptake and metabolism. Such disorders
include one or more of disorders which affect formation or
hydrolysis of ester bonds between fatty acyl moieties and glycerol
moieties (i.e., including glycerol, glycerol phosphates, alkyl
glyceryl ethers, glycerol phosphoryl-choline, glycerol
phosphoryl-serine, glycerol phosphoryl-ethanolamine, and the like),
disorders which affect serum levels of lipid-containing particles
(e.g., chylomicrons, lipoprotein particles, and the like), and
disorders which affect transmembrane transport of fatty acids.
Specific examples of such disorders include diabetes, obesity,
hyperlipidemia, hypolipidemia, and various lipidoses.
[0061] The observation that human protein MLip-1 shares sequence
homology with a number of other proteins involved in lipid
metabolism (e.g., various pancreatic lipases and pancreatic
lipase-related proteins) indicates that MLip-1 has activity
identical or analogous to the activity of one or more of those
proteins. Pancreatic lipases and lipase-related proteins are known
to be involved in a variety of physiological processes including,
for example, digestion of dietary lipids and normal pancreatic
function. Aberrant expression or activity of MLip-1 is thus
associated with lipid uptake disorders such as hyperlipidemia types
I, II, III, IV, and V, hypolipidemia, obesity, various lipidoses
(e.g., Gaucher's disease and Niemann-Pick disease), and linoleic
acid deficiency, with pancreas-associated disorders such as
pancreatitis, perforated peptic ulcer, abdominal lesions,
intestinal obstruction, and peritonitis, with nutritional disorders
such as lipid malabsorption and malnutrition, with atherosclerosis,
with arteriosclerosis, and with coronary artery disease, for
example.
[0062] Various aspects of the invention are described in further
detail in the following subsections.
[0063] I. Isolated Nucleic Acid Molecules
[0064] In one aspect,
[0065] the invention pertains to isolated nucleic acid molecules
that encode a polypeptide of the invention or a biologically active
portion thereof (e.g., mature human MLip-1), as well as nucleic
acid molecules sufficient for use as hybridization probes to
identify polynucleotides encoding a polypeptide of the invention
and fragments of such nucleic acid molecules suitable for use as
PCR primers for amplification or mutation (e.g., by site-directed
mutagenesis) of polynucleotides. As used herein, the term "nucleic
acid molecule" is intended to include DNA molecules (e.g.,
synthetic DNA, cDNA, or genomic DNA) and RNA molecules (e.g.,
pre-mRNA and mRNA) and analogs of the DNA or RNA generated using
nucleotide analogs. The nucleic acid molecule can be
single-stranded or double-stranded.
[0066] An "isolated" nucleic acid molecule is one which is
separated from other nucleic acid molecules which are present in
the natural source of the nucleic acid molecule. An isolated
nucleic acid molecule can be free or substantially free of
sequences (preferably protein-encoding sequences) which naturally
flank the nucleic acid (i.e., sequences located at the 5' and 3'
ends of the nucleic acid) in the genomic DNA of the organism from
which the nucleic acid is derived. For example, in various
embodiments, the isolated nucleic acid molecule can contain less
than about 5, 4, 3, 2, 1, 0.5, or 0.1 kilobase pairs of nucleotide
sequences which naturally flank the nucleic acid molecule in
genomic DNA of the cell from which the nucleic acid is derived.
Moreover, an "isolated" nucleic acid molecule, such as a cDNA
molecule, can be substantially free of other cellular material, or
culture medium when produced by recombinant techniques, or
substantially free of chemical precursors or other chemicals when
chemically synthesized.
[0067] A nucleic acid molecule of the present invention, e.g., a
nucleic acid molecule having the nucleotide sequence of all or a
portion of SEQ ID NOs: 1 and 2, or a complement thereof, or a
nucleic acid which has a nucleotide sequence comprising one of
these sequences, can be isolated using standard molecular biology
techniques and the sequence information provided herein. Using all
or a portion of the nucleic acid sequences of SEQ ID NO: 1 or 2 as
a hybridization probe, nucleic acid molecules of the invention can
be isolated using standard hybridization and cloning techniques
(e.g., as described in Sambrook et al., Eds., Molecular Cloning: A
Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
1989).
[0068] A nucleic acid molecule of the invention can be amplified
using cDNA, mRNA, or genomic DNA as a template and appropriate
oligonucleotide primers according to standard PCR amplification
techniques. The nucleic acid so amplified can be cloned into an
appropriate vector and characterized by DNA sequence analysis.
Furthermore, oligonucleotides corresponding to all or a portion of
a nucleic acid molecule of the invention can be prepared by
standard synthetic techniques, e.g., using an automated DNA
synthesizer.
[0069] In another embodiment, an isolated nucleic acid molecule of
the invention comprises a nucleic acid molecule which is a
complement of the nucleotide sequence of SEQ ID NO: 1 or 2, or a
portion thereof. A nucleic acid molecule which is complementary to
a given nucleotide sequence is one which is sufficiently
complementary to the given nucleotide sequence that it can
hybridize to the given nucleotide sequence, thereby forming a
stable duplex.
[0070] Moreover, nucleic acids of the invention can include a
portion of a nucleic acid sequence encoding a full length
polypeptide of the invention (i.e., MLip-1 protein). For example,
the portion can be a fragment which can be used as a probe or
primer for detecting or amplifying a portion of a nucleic acid that
shares homology with or is complementary to a nucleic acid encoding
MLip-1. Alternatively, the portion can be a fragment which encodes
a biologically active portion of a polypeptide of the invention,
including a fragment which can be transcribed, translated, or both,
to yield an active polypeptide of the invention.
[0071] The nucleotide sequence determined from cloning of the
MLip-1 gene enables generation of probes and primers designed for
use in identifying and cloning homologs from other mammals. The
probe or primer typically comprises a substantially purified
oligonucleotide. The oligonucleotide typically has at least one
region that hybridizes under stringent conditions to at least about
15, preferably about 25, more preferably about 50, 56, 58, 60, 70,
80, 100, 125, 150, 175, 200, 250, 300, 350, or 400 or more
consecutive nucleotides of the sense or anti-sense sequence of a
nucleic acid having the nucleic acid sequence of SEQ ID NO: 1 or 2,
or of a naturally-occurring mutant or variant of one of SEQ ID NOs:
1 and 2.
[0072] Probes based on the sequence of a nucleic acid molecule of
the invention can be used to detect transcripts or genomic
sequences encoding the same protein molecule encoded by a selected
nucleic acid molecule. The probe has a label attached thereto
(e.g., a radioisotope, a fluorescent compound, an enzyme, or an
enzyme co-factor). One or more such probes can be used as part of a
diagnostic test kit for identifying cells or tissues which
mis-express the protein, such as a kit for measuring levels of a
nucleic acid molecule encoding the protein in a sample of cells
from a subject, e.g., detecting mRNA levels or determining whether
a gene encoding the protein has been mutated or deleted.
[0073] A nucleic acid fragment encoding a biologically active
portion of a polypeptide of the invention can be prepared by
isolating a portion of one of SEQ ID NOs: 1 and 2, expressing the
encoded portion of the polypeptide (e.g., by recombinant expression
in vitro), and assessing the activity of the encoded portion of the
polypeptide. If the encoded portion exhibits lipase or lipase-like
activity, then the fragment encodes a biologically active portion
of a polypeptide of the invention.
[0074] The invention further encompasses nucleic acid molecules
that differ from the nucleotide sequence of SEQ ID NOs: 1 and 2 due
to degeneracy of the genetic code and thus encode the same protein
as that encoded by the nucleotide sequence of SEQ ID NO: 2.
[0075] In addition to the nucleotide sequences of SEQ ID NO: 2, it
will be appreciated by those skilled in the art that DNA sequence
polymorphisms that lead to changes in the amino acid sequence can
exist within a population (e.g., the human population or particular
groups, such as ethnic groups, within the human population). Such
genetic polymorphisms can exist among individuals within a
population due to natural allelic variation. An allele is one of a
group of genes which occur alternatively at a given genetic
locus.
[0076] As used herein, the phrase "allelic variant" refers to a
nucleotide sequence which occurs at a given locus or to a
polypeptide encoded by the nucleotide sequence. Such natural
allelic variations can typically result in from 0.1% to about 5%
variance in the nucleotide sequence of a given gene. Alternative
alleles can be identified by sequencing the gene of interest in a
number of different individuals. This can be readily carried out by
using hybridization probes to identify the same genetic locus in a
variety of individuals. Any and all such nucleotide variations and
resulting amino acid polymorphisms or variations that are the
result of natural allelic variation and that do not alter the
functional activity are intended to be within the scope of the
invention.
[0077] As used herein, the terms "gene" and "recombinant gene"
refer to nucleic acid molecules comprising an open reading frame
encoding a polypeptide of the invention.
[0078] Moreover, nucleic acid molecules encoding proteins of the
invention from other species (i.e., homologs), which have a
nucleotide sequence which differs from that of the human MLip-1
protein described herein are included within the scope of the
invention. Nucleic acid molecules corresponding to natural allelic
variants and homologs of a cDNA of the invention can be isolated
based on their identity to human nucleic acid molecules using
MLip-1 cDNA, or a portion thereof, as a hybridization probe
according to standard hybridization techniques under stringent
hybridization conditions. For example, a cDNA encoding one allelic
variant of a protein of the invention can be isolated based on its
hybridization with a nucleic acid molecule encoding a second
allelic variant of the protein.
[0079] Accordingly, in another embodiment, an isolated nucleic acid
molecule of the invention is at least 56 (or, for example, 58, 60,
70, 80, 100, 125, 150, 200, 250, 300, 350, 400, 450, 550, 650, 700,
800, 900, 1000, 1200, 1400, 1600, 1800, 2000, or 2352) nucleotides
in length and hybridizes under stringent conditions with the
nucleic acid molecule comprising the nucleotide sequence,
preferably the coding sequence, of SEQ ID NO: 1 or 2, or a
complement thereof. As used herein, the term "hybridizes under
stringent conditions" is intended to describe conditions for
hybridization and washing under which nucleotide sequences that are
at least 60% (65%, 70%, 75%, 80%, 85%, 90%, preferably 95% or more)
identical to each other typically remain hybridized to each other.
Such stringent conditions are known to those skilled in the art and
can be found in, for example, Current Protocols in Molecular
Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. A
preferred, non-limiting example of stringent hybridization
conditions is hybridization in 6.times.sodium chloride/sodium
citrate (SSC) at about 45.degree. C., followed by one or more
washes in 0.2.times.SSC, 0.1% SDS at a temperature of from about
50.degree. C. to 65.degree. C. Preferably, an isolated nucleic acid
molecule of the invention that hybridizes under stringent
conditions to the sequence of one of SEQ ID NOs: 1 and 2, or a
complement thereof, corresponds to a naturally-occurring nucleic
acid molecule. As used herein, a "naturally-occurring" nucleic acid
molecule refers to an RNA or DNA molecule having a nucleotide
sequence that occurs in nature (e.g., encodes a natural
protein).
[0080] To determine the percent identity of two amino acid
sequences or of two nucleic acids, the sequences are aligned for
optimal comparison purposes (e.g., gaps can be introduced in the
sequence of a first amino acid or nucleic acid sequence for optimal
alignment with a second amino or nucleic acid sequence). The amino
acid residues or nucleotides at corresponding amino acid positions
or nucleotide positions are then compared. When a position in the
first sequence is occupied by the same amino acid or nucleotide
residue as the corresponding position in the second sequence, then
the molecules are identical at that position. The percent identity
between the two sequences is a function of the number of identical
positions shared by the sequences (i.e., percent identity is equal
to the number of identical positions divided by the total number of
positions (e.g., overlapping positions) multiplied by 100). In one
embodiment, the two sequences are the same length, at least after
introducing gaps into one or both sequences.
[0081] Determination of percent identity between two sequences can
be accomplished using any of a number of mathematical algorithm. A
preferred, non-limiting example of a mathematical algorithm used
for comparison of two sequences is the algorithm of Karlin and
Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified
as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci USA
90:5873-5877. Such an algorithm is incorporated into the NBLAST and
XBLAST programs of Altschul, et al. (1990) J. Mol. Biol.
215:403-410. BLAST nucleotide searches can be performed with the
NBLAST program, score=100, wordlength=12 to obtain nucleotide
sequences homologous with a nucleic acid molecule of the invention.
BLAST protein searches can be performed using the XBLAST program,
score=50, wordlength=3 to obtain amino acid sequences homologous to
a protein molecules of the invention. To obtain gapped alignments
for comparison purposes, gapped BLAST analysis can be used as
described in Altschul et al. (1997) Nucleic Acids Res.
25:3389-3402. Alternatively, PSI-Blast can be used to perform an
iterated search which detects distant relationships between
molecules. Id. When using BLAST, gapped BLAST, and PSI-Blast
analyses, default parameters of the respective programs (e.g.,
XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.
Another preferred, non-limiting example of a mathematical algorithm
used for comparison of sequences is the algorithm of Myers and
Miller, (1988) CABIOS 4:11-17. Such an algorithm is incorporated
into the ALIGN program (version 2.0) which is part of the GCG
sequence alignment software package. When utilizing the ALIGN
program for comparing amino acid sequences, a PAM120 weight residue
table, a gap length penalty of 12, and a gap penalty of 4 can be
used. Yet another useful algorithm for identifying regions of local
sequence similarity and alignment is the FASTA algorithm as
described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA
85:2444-2448. When using the FASTA algorithm for comparing
nucleotide or amino acid sequences, a PAM120 weight residue table
can, for example, be used with a k-tuple value of 2.
[0082] The percent identity between two sequences can be determined
using techniques similar to those described above, with or without
allowing gaps. In calculating percent identity, only exact matches
are counted.
[0083] In addition to naturally-occurring allelic sequence variants
of a nucleic acid molecule of the invention that can exist in the
population, the skilled artisan will further appreciate that
changes can be introduced by mutation, thereby leading to changes
in the amino acid sequence of the encoded protein, without altering
the biological activity of the protein. For example, one can make
nucleotide substitutions leading to amino acid substitutions at
"non-essential" amino acid residues. A "non-essential" amino acid
residue is a residue that can be altered from the wild-type
sequence without altering the biological activity of MLip-1,
whereas an "essential" amino acid residue is required for
biological activity. For example, amino acid residues that are not
conserved or are only semi-conserved among homologs of various
species can be non-essential for activity and thus are likely
targets for alteration. Alternatively, amino acid residues that are
conserved among the homologs of various species (e.g., murine and
human) can be essential for activity and thus are not likely
targets for alteration.
[0084] Accordingly, another aspect of the invention pertains to
nucleic acid molecules encoding a polypeptide of the invention that
contain changes which alter amino acid residues that are not
essential for activity. Such polypeptides differ in amino acid
sequence from SEQ ID NO: 3, and yet retain biological activity. In
one embodiment, the isolated nucleic acid molecule has a nucleotide
sequence encoding a protein that includes an amino acid sequence
that is at least about 40% identical (or, for example, 50%, 60%,
70%, 80%, 90%, 95%, or 98% identical) to the amino acid sequence of
SEQ ID NO: 3.
[0085] An isolated nucleic acid molecule encoding a variant protein
can be created by introducing one or more nucleotide substitutions,
additions, or deletions into the nucleotide sequence of SEQ ID NO:
1 or 2, such that one or more amino acid residue substitutions,
additions, or deletions are introduced into the encoded protein.
Mutations can be introduced using standard techniques, such as
site-directed mutagenesis and PCR-mediated mutagenesis. Preferably,
conservative amino acid substitutions are made at one or more
predicted non-essential amino acid residues. A "conservative amino
acid substitution" is one in which the amino acid residue is
replaced with an amino acid residue having a similar side chain.
Families of amino acid residues having similar side chains have
been defined in the art. These families include amino acids with
basic side chains (e.g., lysine, arginine, histidine), acidic side
chains (e.g., aspartic acid, glutamic acid), non-charged polar side
chains (e.g., glycine, asparagine, glutamine, serine, threonine,
tyrosine, cysteine), non-polar side chains (e.g., alanine, valine,
leucine, isoleucine, proline, phenylalanine, methionine,
tryptophan), beta-branched side chains (e.g., threonine, valine,
isoleucine), and aromatic side chains (e.g., tyrosine,
phenylalanine, tryptophan, histidine). Alternatively, mutations can
be introduced randomly along all or part of the coding sequence,
such as by saturation mutagenesis, and the resultant mutants can be
screened for biological activity to identify mutants that retain
activity. Following mutagenesis, the encoded protein can be
expressed recombinantly and the activity of the protein can be
determined.
[0086] The present invention encompasses antisense nucleic acid
molecules, i.e., molecules which are complementary to a sense
nucleic acid encoding a polypeptide of the invention or a portion
thereof, such as nucleic acids complementary to the coding strand
of a double-stranded cDNA molecule or complementary to an mRNA
sequence. Accordingly, an antisense nucleic acid can hybridize with
a sense nucleic acid. The antisense nucleic acid can be
complementary to an entire coding strand, or to only a portion
thereof, e.g., all or part of the protein coding region (or open
reading frame). An antisense nucleic acid molecule can be antisense
with respect to all or part of a non-coding region of the coding
strand of a nucleotide sequence encoding a polypeptide of the
invention. The non-coding regions ("5' and 3' non-translated
regions") are the 5' and 3' sequences which flank the coding region
and which are not normally translated into amino acids.
[0087] An antisense oligonucleotide can be, for example, about 10,
15, 20, 25, 30, 35, 40, 45, 50, 60 or more nucleotides in length.
An antisense nucleic acid of the invention can be constructed using
chemical synthetic or enzymatic ligation methods known in the art.
For example, an antisense nucleic acid can be chemically
synthesized using naturally-occurring nucleotides or variously
modified nucleotides designed to increase the biological stability
of the molecules or to increase the physical stability of the
duplex formed between the antisense and sense nucleic acids.
Examples of such modified nucleotides are phosphorothioate
derivatives and acridine-substituted nucleotides. Examples of
modified nucleotides which can be used to generate the antisense
nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil,
5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,
5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylamino-me-
thyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine,
N.sub.6-isopentenyladenine, 1-methylguanine, 1-methylinosine,
2,2-dimethylguanine, 2-methyladenine, 2-methylguanine,
3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopenten- yladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methyl ester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine. Alternatively, the antisense nucleic acid can be
produced biologically using an expression vector into which a
nucleic acid has been sub-cloned in an antisense orientation (i.e.,
RNA transcribed from the inserted nucleic acid will have an
antisense orientation with respect to a target nucleic acid of
interest).
[0088] The antisense nucleic acid molecules of the invention are
typically administered to a subject or generated in situ such that
they hybridize with or bind with one or both of cellular mRNA and
genomic DNA encoding a selected polypeptide of the invention.
Hybridization of the antisense nucleic acid with the mRNA or
genomic DNA inhibits expression of the protein by inhibiting
translation or transcription, respectively. The hybridization can
occur by means of conventional nucleotide complementarity to form a
stable duplex, or, for example, in the case of an antisense nucleic
acid molecule which binds with DNA duplexes, by means of specific
interactions in the major groove of the double helix. An example of
a route of administration of antisense nucleic acid molecules of
the invention includes direct injection at a tissue site.
Alternatively, antisense nucleic acid molecules can be modified to
target selected cells and then administered systemically. For
example, for systemic administration, antisense molecules can be
modified such that they specifically bind with receptors or
antigens expressed on a selected cell surface, e.g., by linking the
antisense nucleic acid molecules to peptides or antibodies which
bind with cell surface receptors or antigens. The antisense nucleic
acid molecules can also be delivered to cells using vectors
described herein or other vectors. To achieve sufficient
intracellular concentrations of the antisense molecules, vector
constructs in which transcription of the antisense nucleic acid
molecule is placed under the control of a strong pol II or pol III
promoter are preferred.
[0089] An antisense nucleic acid molecule of the invention can be
an .alpha.-anomeric nucleic acid molecule. An a-anomeric nucleic
acid molecule forms specific double-stranded hybrids with
complementary RNA in which, contrary to the usual .alpha.-units,
the strands run parallel to each other (Gaultier et al. (1987)
Nucleic Acids Res. 15:6625-6641). The antisense nucleic acid
molecule can also comprise a 2'-o-methylribonucleotide (Inoue et
al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA
analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).
[0090] The invention also encompasses ribozymes. Ribozymes are
catalytic RNA molecules having ribonuclease activity. Ribozymes are
capable of cleaving a single-stranded nucleic acid, such as an
mRNA, which has a portion to which a portion of the ribozyme is
complementary. Thus, ribozymes (e.g., hammerhead ribozymes as
described in Haselhoff and Gerlach (1988) Nature 334:585-591) can
be used and catalytically cleave mRNA transcripts to thereby
inhibit translation of the protein encoded by the mRNA. A ribozyme
having specificity for a nucleic acid molecule encoding a
polypeptide of the invention can be designed based upon the
nucleotide sequence of a cDNA disclosed herein. For example, a
derivative of a Tetrahymena L-19 IVS RNA can be constructed in
which the nucleotide sequence of the active site of this ribozyme
is complementary to the portion of the mRNA to be cleaved, as
described in U.S. Pat. No. 4,987,071 and U.S. Pat. No. 5,116,742.
Alternatively, an mRNA encoding a polypeptide of the invention can
be used to select, from a pool of RNA molecules, a catalytic RNA
having a specific ribonuclease activity. See, e.g., Bartel and
Szostak (1993) Science 261:1411-1418.
[0091] The invention also encompasses nucleic acid molecules which
form triple helical structures. For example, expression of a
polypeptide of the invention can be inhibited by targeting
nucleotide sequences complementary to the regulatory region of the
gene encoding the polypeptide (e.g., the promoter or enhancer
region of a gene) to form triple helical structures that prevent
transcription of the gene in target cells. See generally Helene
(1991) Anticancer Drug Des. 6(6):569-84; Helene (1992) Ann. N.Y.
Acad. Sci. 660:27-36; and Maher (1992) Bioassays 14(12):807-15.
[0092] In various embodiments, the nucleic acid molecules of the
invention can be modified at the base moiety, sugar moiety, or
phosphate backbone to improve, e.g., stability, hybridization, or
solubility of the molecule. For example, the deoxyribose phosphate
backbone of the nucleic acids can be modified to generate peptide
nucleic acids (see Hyrup et al. (1996) Bioorganic & Medicinal
Chemistry 4(1): 5-23). As used herein, the terms "peptide nucleic
acids" or "PNAs" refer to nucleic acid mimics, e.g., DNA mimics, in
which the deoxyribose phosphate backbone is replaced by a
pseudopeptide backbone and only the four natural nucleobases are
retained. The neutral backbone of PNAs has been shown to enable
specific hybridization between the PNA and DNA or RNA under
conditions of low ionic strength. Synthesis of PNA oligomers can be
performed using standard solid phase peptide synthesis protocols,
as described (Hyrup et al. (1996), supra; Perry-O'Keefe et al.
(1996) Proc. Natl. Acad. Sci. USA 93: 14670-675).
[0093] PNAs can be used in therapeutic and diagnostic applications.
For example, PNAs can be used as antisense or antigene agents for
sequence-specific modulation of gene expression by, e.g., by
inducing transcription or translation arrest or by inhibiting
replication. PNAs can also be used, e.g., for analysis of single
base pair mutations in a gene by, e.g., PNA-directed PCR clamping;
as artificial restriction enzymes when used in combination with
other enzymes, e.g., S1 nucleases (Hyrup (1996), supra; or as
probes or primers for DNA sequence and hybridization (Hyrup (1996),
supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA 93:
14670-675).
[0094] In another embodiment, PNAs can be modified to enhance, for
example, their stability or cellular uptake by attaching lipophilic
or other helper groups to PNA, by formation of PNA-DNA chimeras, or
using liposomes or other drug delivery compositions known in the
art. For example, PNA-DNA chimeras can be generated which combine
the advantageous properties of PNA and DNA. Such chimeras allow DNA
recognition enzymes, e.g., RNASE H and DNA polymerases, to interact
with the DNA portion, while the PNA portion provides high binding
affinity and specificity. PNA-DNA chimeras can be made using
linkers of appropriate lengths, selected in terms of base stacking,
number of bonds between the nucleobases, and orientation, as
described (Hyrup (1996), supra). PNA-DNA chimeras can be
synthesized as described in Hyrup (1996), supra, and Finn et al.
(1996) Nucleic Acids Res. 24(17):3357-63. For example, a DNA chain
can be synthesized on a solid support using standard
phosphoramidite coupling chemistry and modified nucleoside analogs.
Compounds such as 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine
phosphoramidite can be used as a link between the PNA and the 5'
end of DNA (Mag et al. (1989) Nucleic Acids Res. 17:5973-88). PNA
monomers are then coupled in a step-wise manner to produce a
chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn
et al. (1996) Nucleic Acids Res. 24(17):3357-63). Alternatively,
chimeric molecules can be synthesized which have a 5' DNA segment
and a 3' PNA segment, as described (Peterser et al. (1975)
Bioorganic Med. Chem. Lett. 5:1119-11124).
[0095] In other embodiments, the oligonucleotide can include other
appended groups such as peptides (e.g., for targeting host cell
receptors in vivo), or agents which facilitate transport across the
cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad.
Sci. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad.
Sci. USA 84:648-652; PCT Publication number WO 88/09810) or the
blood-brain barrier (see, e.g., PCT Publication number WO
89/10134). In addition, oligonucleotides can be modified with
hybridization-triggered cleavage agents (see, e.g., Krol et al.
(1988) Bio/Techniques 6:958-976) or intercalating agents (see,
e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, the
oligonucleotide can be conjugated to another molecule, e.g., a
peptide, hybridization triggered cross-linking agent, transport
agent, hybridization-triggered cleavage agent, etc.
[0096] II. Isolated Proteins
[0097] In another aspect, the invention pertains to isolated
proteins, and biologically active portions thereof, as well as
polypeptide fragments suitable for use as immunogens to generate
antibodies which bind specifically with a polypeptide of the
invention. In one embodiment, the native polypeptide is isolated
from cells or tissue sources by an appropriate purification scheme
using standard protein purification techniques. In another
embodiment, polypeptides of the invention are produced by
recombinant DNA techniques. As an alternative to recombinant
expression, a polypeptide of the invention can be synthesized
chemically using standard peptide synthesis techniques.
[0098] An "isolated" or "purified" protein or biologically active
portion thereof is substantially free of cellular material or other
contaminating proteins which originate in the cell or tissue source
from which the protein is derived, or substantially free of
chemical precursors or other chemicals, when the polypeptide of the
invention is chemically synthesized. The language "substantially
free of cellular material" includes preparations of protein in
which the protein is separated from cellular components of the
cells from which it is isolated or recombinantly produced. Thus,
protein that is substantially free of cellular material includes
preparations of protein having less than about 30% (or, for
example, 20%, 10%, or 5%), by dry weight, heterologous protein
(also referred to herein as a "contaminating protein"). When the
protein or biologically active portion thereof is recombinantly
produced, it is also preferably substantially free of culture
medium, i.e., culture medium represents less than about 20%, 10%,
or 5% of the volume of the protein preparation. When the protein is
produced by chemical synthesis, it is preferably substantially free
of chemical precursors or other chemicals, i.e., it is separated
from chemical precursors or other chemicals which are involved in
the synthesis of the protein. Accordingly such preparations of the
protein have less than about 30% (or, for example, 20%, 10%, 5%),
by dry weight, chemical precursors or compounds other than the
polypeptide of interest.
[0099] Biologically active portions of a polypeptide of the
invention include polypeptides which have an amino acid sequence
sufficiently identical to or derived from the amino acid sequence
of MLip-1 protein (e.g., the amino acid sequence of SEQ ID NO: 3),
which include fewer amino acids than the full length protein, and
which exhibit at least one activity of the corresponding
full-length protein. Typically, biologically active portions
comprise a domain or motif which exhibits at least one activity
(e.g., specific binding capacity or catalytic capacity) of the
corresponding protein. A biologically active portion of a protein
of the invention can be a polypeptide which is, for example, 10,
17, 18, 25, 50, 100, 150, 200, or 300 or more amino acid residues
in length. Moreover, other biologically active portions, in which
other regions of the protein are deleted, can be prepared by
recombinant techniques and evaluated for one or more of the
functional activities of the native form of a polypeptide of the
invention.
[0100] Preferred polypeptides have the amino acid sequence SEQ ID
NO: 3. Other useful proteins have an amino acid sequence which is
substantially identical (e.g., at least about 40% or, for example,
50%, 60%, 70%, 80%, 90%, 95%, or 99%, identical) to SEQ ID NO: 3
and retain at least one activity of the corresponding
naturally-occurring protein, yet differ in amino acid sequence due
to natural allelic variation or mutagenesis.
[0101] In one embodiment, the invention includes a mutant
polypeptide that is a variant of a polypeptide of the invention and
can be assayed for: (1) the ability to form protein:protein
interactions with the polypeptide of the invention; (2) the ability
to bind a ligand of the polypeptide of the invention (e.g., a
triglyceride or other lipid); (3) the ability to catalyze a
chemical reaction (e.g. formation or breakage of acyl/glyceride
bonds) by which a protein of the invention is characterized (e.g.
lipase or lipase-like activity); or (4) the ability to modulate a
physiological activity of the protein, such as one of those
disclosed herein. Mutant polypeptides which exhibit one or more of
these activities are included in the invention, as are methods of
screening libraries of mutant polypeptides in order to identify
ones which exhibit such activities.
[0102] The invention also provides chimeric or fusion proteins. As
used herein, a "chimeric protein" or "fusion protein" comprises all
or part (preferably a biologically active part) of a polypeptide of
the invention operably linked with a heterologous polypeptide
(i.e., a polypeptide other than the same polypeptide of the
invention). Within the fusion protein, the term "operably linked"
is intended to indicate that the polypeptide of the invention and
the heterologous polypeptide are fused in-frame with each other.
The heterologous polypeptide can be fused with the amino-terminus
or the carboxyl-terminus of a polypeptide of the invention. Often,
in fusion expression vectors, a proteolytic cleavage site is
introduced at the junction of the two protein moieties to enable
separation of the recombinant protein from the fusion moiety
subsequent to purification of the fusion protein. Such enzymes, and
their cognate recognition sequences, include Factor Xa, thrombin,
and enterokinase.
[0103] One useful fusion protein is a GST fusion protein in which a
polypeptide of the invention is fused with the carboxyl terminus of
a GST sequence. Such fusion proteins can facilitate the
purification of a recombinant polypeptide of the invention.
[0104] In another embodiment, the fusion protein contains a
heterologous signal sequence at its amino terminus. For example,
the native signal sequence of a polypeptide of the invention can be
removed and replaced with a signal sequence from another protein.
For example, the gp67 secretory sequence of the baculovirus
envelope protein can be used as a heterologous signal sequence
(Current Protocols in Molecular Biology, Ausubel et al., eds., John
Wiley & Sons, 1992) in place of amino acid residues 1 to about
17 of SEQ ID NO: 3. Other examples of eukaryotic heterologous
signal sequences include the secretory sequences of melittin and
human placental alkaline phosphatase (Stratagene; La Jolla,
Calif.). In yet another example, useful prokaryotic heterologous
signal sequences include the phoA secretory signal (Sambrook et
al., supra) and the protein A secretory signal (Pharmacia Biotech;
Piscataway, N.J.).
[0105] In yet another embodiment, the fusion protein is an
immunoglobulin fusion protein in which all or part of a polypeptide
of the invention is fused with sequences derived from a member of
the immunoglobulin protein family. The immunoglobulin fusion
proteins of the invention can be incorporated into pharmaceutical
compositions and administered to a subject to inhibit an
interaction between a ligand (e.g., a soluble or membrane-bound
ligand) and a protein on the surface of a cell (e.g., a receptor),
to thereby suppress signal transduction in vivo. Inhibition of
ligand/receptor interaction can be useful therapeutically, for
example for treating pancreas-related disorders and for modulating
(e.g., promoting or inhibiting) lipid metabolism. Moreover, an
immunoglobulin fusion protein of the invention can be used as an
immunogen to produce antibodies directed against a polypeptide of
the invention in a subject, to purify a ligand of a polypeptide of
the invention, and in screening assays to identify a molecule which
inhibits interaction of a polypeptide of the invention with a
ligand thereof.
[0106] Chimeric and fusion proteins of the invention can be
produced using standard recombinant DNA techniques. In another
embodiment, the fusion gene can be synthesized using conventional
techniques including techniques which involve operation of an
automated DNA synthesizer. Alternatively, PCR amplification of gene
fragments can be performed using anchor primers which give rise to
complementary overhanging regions at the end of consecutive gene
fragments, which can subsequently be annealed and re-amplified to
generate a chimeric gene sequence (see, e.g., Ausubel et al.,
supra). Moreover, many expression vectors are commercially
available that encode a fusion protein moiety (e.g., a portion of a
GST protein). Through exercise of ordinary skill, a nucleic acid
encoding a polypeptide of the invention can be cloned into such an
expression vector in such a way that the fusion moiety is linked
in-frame with a polypeptide of the invention.
[0107] The present invention includes to variants of the
polypeptides of the invention. Exemplary variants have an altered
amino acid sequence and can function as either agonists (i.e.,
mimetics) or as antagonists of MLip-1. Variants can be generated by
mutagenesis, e.g., discrete point mutation or truncation. An
agonist can retain substantially the same, or a subset, of the
biological activities associated with the naturally-occurring form
of the protein. An antagonist of a protein can inhibit one or more
of the activities of the naturally-occurring form of the protein
by, for example, competitively binding a triglyceride and
inhibiting transmembrane transport thereof. Thus, specific
biological effects can be elicited by treatment with a variant of
limited function. Treatment of a subject with a variant having a
subset of the biological activities of the naturally-occurring form
of the protein can have fewer side effects in a subject, relative
to treatment with the naturally-occurring form of the protein.
[0108] Variants of a protein of the invention which function as
either agonists (mimetics) or as antagonists can be identified by
screening combinatorial libraries of mutants, e.g., truncation
mutants, of MLip-1 for agonist or antagonist activity. In one
embodiment, a variegated library of variants is generated by
combinatorial mutagenesis at the nucleic acid level and is encoded
by a variegated gene library. A variegated library of variants can
be produced by, for example, enzymatically ligating a mixture of
synthetic oligonucleotides into gene sequences such that a
degenerate set of potential protein sequences is expressible as
individual polypeptides, or alternatively, as a set of larger
fusion proteins (e.g., for phage display). There are a variety of
methods which can be used to produce libraries of potential
variants of the polypeptides of the invention from a degenerate
oligonucleotide sequence. Methods for synthesizing degenerate
oligonucleotides are known in the art (see, e.g., Narang (1983)
Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323;
Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic
Acid Res. 11:477). Methods for assessing transmembrane transport of
compounds such as triglycerides are known in the art.
[0109] In addition, libraries of fragments of the coding sequence
of a polypeptide of the invention can be used to generate a
variegated population of polypeptides for screening and subsequent
selection of variants. For example, a library of coding sequence
fragments can be generated by treating a double stranded PCR
fragment of the coding sequence of interest with a nuclease under
conditions wherein nicking occurs only about once per molecule,
denaturing the double stranded DNA, re-naturing the DNA to form
double stranded DNA which can include sense/antisense pairs from
different nicked products, removing single stranded portions from
reformed duplexes by treatment with S1 nuclease, and ligating the
resulting fragment library into an expression vector. Using this
method, an expression library can be derived which encodes amino
terminal and internal fragments of various sizes of MLip-1.
[0110] Several techniques are known in the art for screening gene
products of combinatorial libraries made by point mutations or
truncation, and for screening cDNA libraries for gene products
having a selected property. The most widely used techniques (which
are amenable to high throughput analysis) for screening large gene
libraries typically include cloning the library into replicable
expression vectors, transforming appropriate cells with the
resulting library of vectors, and expressing the combinatorial
genes under conditions in which detection of a desired activity
facilitates isolation of the vector encoding the gene, the product
of which was detected. Recursive ensemble mutagenesis (REM), a
technique which enhances the frequency of functional mutants in the
libraries, can be used in combination with one or more of the
screening assays described herein to identify variants of MLip-1
(Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815;
Delgrave et al. (1993) Protein Engineering 6(3):327-331).
[0111] III. Antibodies
[0112] An isolated polypeptide of the invention, or a fragment
thereof, can be used as an immunogen to generate antibodies and
other antibody substances using standard techniques for polyclonal
and monoclonal antibody preparation. Full-length MLip-1 can be used
or, alternatively, the invention provides antigenic peptide
fragments for use as immunogens. The antigenic peptide of a protein
of the invention comprises at least 8 (preferably 10, 15, 17, 18,
20, or 30 or more) amino acid residues of a protein having the
amino acid sequence SEQ ID NO: 3 at those residues, and encompasses
an epitope of the protein such that an antibody substance raised
against the peptide (i.e., a polypeptide which binds specifically
with the peptide) forms a specific immune complex with the
protein.
[0113] Preferred epitopes encompassed by the antigenic peptide are
regions that are located on the surface of the protein, e.g.,
hydrophilic regions. FIG. 4 is a hydrophobicity plot of MLip-1
protein. This plot or similar analyses (including a variety of
known computer-based algorithms for analyzing protein sequence
hydrophilicity/hydrophobicity) can be used to identify hydrophilic
regions.
[0114] An immunogen typically is used to prepare antibodies by
immunizing a suitable (i.e., immunocompetent) subject such as a
rabbit, goat, mouse, or other mammal or vertebrate. An appropriate
immunogenic preparation can contain, for example,
recombinantly-expressed or chemically-synthesized polypeptide. The
preparation can further include an adjuvant, such as Freund's
complete adjuvant, Freund's incomplete adjuvant, or a similar
immunostimulatory agent.
[0115] Accordingly, in one aspect, the invention pertains to
antibodies directed against a polypeptide of the invention. The
terms "antibody" and "antibody substance" as used interchangeably
herein refer to immunoglobulin molecules and immunologically active
portions of immunoglobulin molecules, i.e., molecules that contain
an antigen binding site which specifically binds an antigen, such
as a polypeptide of the invention. A molecule which specifically
binds with a polypeptide of the invention is a molecule which binds
the polypeptide, but does not substantially bind other molecules in
a sample, e.g., a biological sample which naturally contains the
polypeptide. Examples of immunologically active portions of
immunoglobulin molecules include F(ab) and F(ab').sub.2 fragments
which can be generated by treating the antibody with an enzyme such
as pepsin. The invention thus includes, for example, T cell
receptors and polyclonal and monoclonal antibodies which bind
specifically with MLip-1 protein or a fragment or variant thereof.
The term "monoclonal antibody" or "monoclonal antibody
composition", as used herein, refers to a population of antibody
molecules that contain only one species of an antigen binding site
capable of immunoreacting with a particular epitope.
[0116] Polyclonal antibodies can be prepared as described above, by
immunizing a suitable subject with a polypeptide of the invention
as an immunogen. The antibody titer in the immunized subject can be
monitored over time by standard techniques, such as using an enzyme
linked immunosorbent assay (ELISA) involving an immobilized
polypeptide. If desired, the antibody molecules can be harvested or
isolated from the subject (e.g., from the blood or serum of the
subject) and further purified by well-known techniques, such as
protein A chromatography to obtain the IgG fraction. At an
appropriate time following immunization, e.g., when the specific
antibody titers are highest, antibody-producing cells can be
obtained from the subject and used to prepare monoclonal antibodies
by standard techniques, such as the hybridoma technique originally
described by Kohler and Milstein (1975) Nature 256:495-497, the
human B cell hybridoma technique (Kozbor et al. (1983) Immunol.
Today 4:72), the EBV-hybridoma technique (Cole et al. (1985),
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.
77-96), or one of several known trioma techniques. The technology
for producing hybridomas is well known (see generally Current
Protocols in Immunology (1994) Coligan et al. (eds.) John Wiley
& Sons, Inc., New York, N.Y.). Hybridoma cells producing a
monoclonal antibody of the invention are detected by screening the
hybridoma culture supernatants for antibodies that bind the
polypeptide of interest, e.g., using a standard ELISA assay.
[0117] As an alternative to preparing monoclonal antibody-secreting
hybridomas, a monoclonal antibody directed against a polypeptide of
the invention can be identified and isolated by screening a
recombinant combinatorial immunoglobulin library (e.g., an antibody
phage display library) with the polypeptide of interest. Kits for
generating and screening phage display libraries are commercially
available (e.g., the Pharmacia Recombinant Phage Antibody System,
Catalog number 27-9400-01; and the Stratagene SurfZAP Phage Display
Kit, Catalog number 240612). Additionally, examples of methods and
reagents particularly amenable for use in generating and screening
antibody display library can be found in, for example, U.S. Pat.
No. 5,223,409; PCT Publication number WO 92/18619; PCT Publication
number WO 91/17271; PCT Publication number WO 92/20791; PCT
Publication number WO 92/15679; PCT Publication number WO 93/01288;
PCT Publication number WO 92/01047; PCT Publication number WO
92/09690; PCT Publication number WO 90/02809; Fuchs et al. (1991)
Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod.
Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281;
Griffiths et al. (1993) EMBO J. 12:725-734.
[0118] Additionally, recombinant antibodies, such as chimeric and
humanized monoclonal antibodies, comprising both human and
non-human portions, which can be made using standard recombinant
DNA techniques, are within the scope of the invention. Such
chimeric and humanized monoclonal antibodies can be produced by
recombinant DNA techniques known in the art, for example using
methods described in PCT Publication number WO 87/02671; European
Patent Application 184,187; European Patent Application 171,496;
European Patent Application 173,494; PCT Publication number WO
86/01533; U.S. Pat. No. 4,816,567; European Patent Application
125,023; Better et al. (1988) Science 240:1041-1043; Liu et al.
(1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987)
J. Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci.
USA 84:214-218; Nishimura et al. (1987) Cancer Res. 47:999-1005;
Wood et al. (1985) Nature 314:446-449; and Shaw et al. (1988) J.
Natl. Cancer Inst. 80:1553-1559); Morrison (1985) Science
229:1202-1207; Oi et al. (1986) Bio/Techniques 4:214; U.S. Pat. No.
5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan et al.
(1988) Science 239:1534; and Beidler et al. (1988) J. Immunol.
141:4053-4060.
[0119] Completely human antibodies are particularly desirable for
therapeutic treatment of human patients. Such antibodies can be
produced using transgenic mice which are incapable of expressing
endogenous immunoglobulin heavy and light chains genes, but which
can express human heavy and light chain genes. The transgenic mice
are immunized in the normal fashion with a selected antigen, e.g.,
all or a portion of MLip-1 protein. Monoclonal antibodies directed
against the antigen can be obtained using conventional hybridoma
technology. The human immunoglobulin transgenes harbored by the
transgenic mice rearrange during B cell differentiation, and
subsequently undergo class switching and somatic mutation. Thus,
using such a technique, it is possible to produce therapeutically
useful IgG, IgA, and IgE antibodies. For an overview of this
technology for producing human antibodies, see Lonberg and Huszar
(1995, Int. Rev. Immunol. 13:65-93). For a detailed discussion of
this technology for producing human antibodies and human monoclonal
antibodies and protocols for producing such antibodies, see, e.g.,
U.S. Pat. No. 5,625,126; U.S. Pat. No. 5,633,425; U.S. Pat. No.
5,569,825; U.S. Pat. No. 5,661,016; and U.S. Pat. No. 5,545,806. In
addition, companies such as Abgenix, Inc. (Freemont, Calif.), can
be engaged to provide human antibodies directed against a selected
antigen using technology similar to that described above.
[0120] Completely human antibodies which recognize a selected
epitope can be generated using a technique designated as "guided
selection." In this approach, a selected non-human monoclonal
antibody, e.g., a murine antibody, is used to guide selection of a
completely human antibody recognizing the same epitope (Jespers et
al. (1994) Bio/technology 12:899-903).
[0121] An antibody which binds specifically with a polypeptide of
the invention (e.g., a monoclonal antibody) can be used to isolate
the polypeptide by standard techniques, such as affinity
chromatography or immunoprecipitation. Moreover, such an antibody
can be used to detect the protein (e.g., in a cellular lysate or
cell supernatant) in order to evaluate the abundance and pattern of
expression of the polypeptide. The antibodies can also be used
diagnostically to monitor protein levels in tissue as part of a
clinical testing procedure, e.g., to determine the efficacy of a
given treatment regimen. Detection can be facilitated by coupling
the antibody with a detectable substance. Examples of detectable
substances include various enzymes, prosthetic groups, fluorescent
materials, luminescent materials, bioluminescent materials, and
radioactive materials. Examples of suitable enzymes include
horseradish peroxidase, alkaline phosphatase, .beta.-galactosidase,
or acetylcholinesterase. Examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin. Examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride and
phycoerythrin. An example of a luminescent material includes
luminol. Examples of bioluminescent materials include luciferase,
luciferin, green fluorescent protein, and aequorin. Examples of
suitable radioactive material include .sup.125I, .sup.131I,
.sup.35S and .sup.3H.
[0122] IV. Recombinant Expression Vectors and Host Cells
[0123] In another aspect, the invention pertains to vectors,
preferably expression vectors which comprise a nucleic acid
encoding a polypeptide of the invention. As used herein, the term
"vector" refers to a nucleic acid molecule capable of transporting
another nucleic acid with which it has been linked. One type of
vector is a "plasmid", which refers to a circular, double stranded
DNA loop into which additional DNA segments can be ligated. Another
type of vector is a virus vector, wherein additional DNA segments
can be ligated into the viral genome. Certain vectors are capable
of autonomous replication in a host cell into which they are
introduced (e.g., some virus vectors, bacterial vectors having a
bacterial origin of replication, and episomal mammalian vectors).
Other vectors (e.g., other virus vectors and non-episomal mammalian
vectors) are integrated into the genome of a host cell upon
introduction into the host cell and are replicated along with the
host genome. Moreover, certain vectors, namely expression vectors,
are capable of directing expression of genes or protein-coding
sequences with which they are operably linked. Expression vectors
useful in recombinant DNA techniques are often in the form of
plasmids. However, the invention includes such other forms of
expression vectors as virus vectors (e.g., replication defective
retroviruses, adenoviruses, and adeno-associated viruses) and
linear DNA vectors, which serve analogous functions.
[0124] The recombinant expression vectors of the invention comprise
a nucleic acid of the invention in a form suitable for expression
of the nucleic acid in a host cell. This means that the recombinant
expression vectors include one or more regulatory sequences
operably linked with the nucleic acid sequence to be expressed. The
choice of regulatory sequence can depend on the host cells to be
used for expression. Within a recombinant expression vector,
"operably linked" is intended to mean that the nucleotide sequence
of interest is covalently bonded with the regulatory sequence(s) in
a manner which allows expression of the nucleotide sequence (e.g.,
in an in vitro transcription/translation system or in a host cell).
The term "regulatory sequence" includes promoters, enhancers and
other expression control elements (e.g., polyadenylation signals).
Such regulatory sequences are described, for example, in Goeddel,
Gene Expression Technology: Methods in Enzymology 185, Academic
Press, San Diego, Calif. (1990). Regulatory sequences include those
which direct constitutive expression of a nucleotide sequence in
many types of host cell and those which direct expression of the
nucleotide sequence only in certain host cells (e.g.,
tissue-specific regulatory sequences). Design of the expression
vector can depend on such factors as the identity of the host cell
to be transformed, the level of expression of protein that is
desired, and the like. The expression vectors of the invention can
be introduced into host cells to produce proteins or peptides
encoded by nucleic acids, including fusion proteins or peptides, as
described herein.
[0125] The recombinant expression vectors of the invention can be
designed for expression of a polypeptide of the invention in
prokaryotic (e.g., E. coli) or eukaryotic cells (e.g., insect cells
{e.g., using a baculovirus expression vector}, yeast cells or
mammalian cells). Suitable host cells are discussed further in
Goeddel, supra. Alternatively, the recombinant expression vector
can be transcribed and translated in vitro, for example using T7
promoter regulatory sequences and T7 polymerase. Suitable in vitro
transcription/translation methods and kits are known in the
art.
[0126] Expression of proteins in prokaryotes is most often
performed in E. coli using vectors which contain constitutive or
inducible promoters that direct expression of either fusion or
non-fusion proteins.
[0127] Typical fusion expression vectors include pGEX (Pharmacia
Biotech Inc; Smith and Johnson (1988) Gene 67:31-40), pMAL (New
England Biolabs, Beverly, Mass.), and pRIT5 (Pharmacia, Piscataway,
N.J.) which fuse glutathione S-transferase (GST), maltose E binding
protein, or protein A, respectively, with the target recombinant
protein.
[0128] Examples of suitable inducible non-fusion E. coli expression
vectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pET
11d (Studier et al., Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, Calif. (1990) 60-89).
Gene expression from the pTrc vector relies on host RNA polymerase
transcription from a hybrid trp-lac fusion promoter. Gene
expression from the pET 11d vector relies on transcription from a
T7 gn10-lac fusion promoter mediated by a co-expressed viral RNA
polymerase (T7 gn1). This viral polymerase is supplied by host
strains BL21(DE3) or HMS174(DE3) from a resident .lambda. prophage
which harbors a T7 gn1 gene under the transcriptional control of a
lacUV 5 promoter.
[0129] One strategy for maximizing recombinant protein expression
in E. coli is to express the protein in a host bacterium that has
an impaired capacity to proteolytically cleave the recombinant
protein (Gottesman, Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, Calif. (1990) 119-128).
Another strategy is to alter the nucleic acid sequence of the
nucleic acid to be inserted into an expression vector so that the
individual codons for each amino acid are those preferentially
utilized in E. coli (Wada et al. (1992) Nucleic Acids Res.
20:2111-2118). Such alteration of nucleic acid sequences of the
invention can be done using standard DNA synthesis techniques.
[0130] In another embodiment, the expression vector is a yeast
expression vector. Examples of vectors for expression in yeast S.
cerevisiae include pYepSec1 (Baldari et al. (1987) EMBO J.
6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943),
pJRY88 (Schultz et al. (1987) Gene 54:113-123), pYES2 (Invitrogen
Corporation, San Diego, Calif.), and pPicZ (Invitrogen Corp, San
Diego, Calif.).
[0131] Alternatively, the expression vector can be a baculovirus
expression vector. Baculovirus vectors which are useful for
expression of proteins in cultured insect cells (e.g., Sf 9 cells)
include the pAc series (Smith et al. (1983) Mol. Cell Biol.
3:2156-2165) and the pVL series (Lucklow and Summers (1989)
Virology 170:31-39) of vectors.
[0132] In yet another embodiment, a nucleic acid of the invention
is expressed in mammalian cells using a mammalian expression
vector. Examples of mammalian expression vectors include pCDM8
(Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO
J. 6:187-195). When used in mammalian cells, control of expression
vector functions can be mediated by viral regulatory elements. For
example, commonly used promoters are derived from polyoma virus,
adenovirus 2, cytomegalovirus, and simian virus 40. For other
suitable expression systems for both prokaryotic and eukaryotic
cells, see chapters 16 and 17 of Sambrook et al., supra.
[0133] In another embodiment, the recombinant mammalian expression
vector is capable of directing expression of the nucleic acid
preferentially in a particular cell type. The vector comprises a
tissue-specific regulatory elements are used to express the nucleic
acid). Tissue-specific regulatory element operably linked with the
nucleic acid. Non-limiting examples of suitable tissue-specific
promoters include the albumin promoter (liver-specific; Pinkert et
al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters
(Calame and Eaton (1988) Adv. Immunol. 43:235-275), including
promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J.
8:729-733) and immunoglobulins (Banerji et al. (1983) Cell
33:729-740; Queen and Baltimore (1983) Cell 33:741-748),
neuron-specific promoters (e.g., the neurofilament promoter; Byrne
and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477),
pancreas-specific promoters (Edlund et al. (1985) Science
230:912-916), and mammary gland-specific promoters (e.g., milk whey
promoter; U.S. Pat. No. 4,873,316 and European Application
Publication number 264,166). Vectors which comprise a
developmentally-regulated promoters are also included, for example
the murine hox promoters (Kessel and Gruss (1990) Science
249:374-379) and the .alpha.-fetoprotein promoter (Campes and
Tilghman (1989) Genes Dev. 3:537-546).
[0134] The invention also includes a recombinant expression vector
comprising a DNA molecule of the invention cloned into an
expression vector in an antisense orientation. That is, the DNA
molecule is operably linked with a regulatory sequence in a manner
that enables expression (by transcription of the DNA molecule) of
an RNA molecule which is antisense with respect to an mRNA encoding
a polypeptide of the invention. Regulatory sequences operably
linked with a nucleic acid cloned in an antisense orientation can
be selected which direct continuous expression of the antisense RNA
molecule in a variety of cell types. For example, viral promoters,
enhancers, regulatory sequences, and combinations of these can be
selected which direct constitutive, tissue-specific or cell
type-specific expression of antisense RNA. An antisense expression
vector can be in the form of a recombinant plasmid, phagemid, or
attenuated virus from which antisense nucleic acids are produced
under the control of a high efficiency regulatory region. The
activity of such a region can be determined by the cell type into
which the vector is introduced. For a discussion of regulation of
gene expression using antisense genes, see Weintraub et al.
(Reviews--Trends in Genetics, Vol. 1(1) 1986).
[0135] In another aspect, the invention pertains to host cells into
which a recombinant expression vector of the invention has been
introduced. The terms "host cell" and "recombinant host cell" are
used interchangeably herein. It is understood that such terms refer
not only to particular subject cells, but to the progeny or
potential progeny of such cells as well. Because certain
modifications can occur in succeeding generations, due to mutation
or environmental influences for example, such progeny will not, in
some instances, be identical to the parent cells, but are
nevertheless included within the scope of the invention. The host
cell can be any prokaryotic (e.g., E. coli) or eukaryotic cell
(e.g., insect cells, yeast, or mammalian cells).
[0136] Vector DNA can be introduced into prokaryotic or eukaryotic
cells using conventional transformation or transfection techniques.
As used herein, the terms "transformation" and "transfection" are
intended to refer to a variety of art-recognized techniques for
introducing a foreign nucleic acid into a host cell. Such methods
include, for example, calcium phosphate or calcium chloride
co-precipitation, DEAE-dextran-mediated transfection, lipofection,
and electroporation. Suitable methods for transforming or
transfecting host cells are described in Sambrook, et al. (supra),
and other laboratory manuals.
[0137] For stable transfection of mammalian cells, it is known that
as few as a small fraction of cells integrate foreign DNA into
their genome, depending on, for example, the identity of the cells
and the expression vector and transfection technique used. In order
to identify and select these integrants, a gene that encodes a
selectable marker (e.g., an antibiotic resistance marker) can be
introduced into the host cells along with the gene of interest.
Preferred selectable markers include those which confer resistance
to drugs, such as G418, hygromycin, and methotrexate. Cells stably
transfected with the introduced nucleic acid can be identified by
drug selection (e.g., cells that have incorporated the selectable
marker gene will survive, while other cells die).
[0138] A host cell of the invention, such as a prokaryotic or
eukaryotic host cell in culture, can be used to produce a
polypeptide of the invention. Accordingly, the invention further
provides methods for producing a polypeptide of the invention using
host cells of the invention. In one embodiment, the method
comprises culturing the host cell of invention (into which a
recombinant expression vector encoding a polypeptide of the
invention has been introduced) in a suitable medium, so that the
polypeptide is produced by the cell. In another embodiment, the
method further comprises isolating the polypeptide from the culture
medium or the host cell.
[0139] V. Transgenics
[0140] Host cells of the invention can also be used to produce
non-human transgenic animals. For example, in one embodiment, a
host cell of the invention is a fertilized oocyte or an embryonic
stem cell into which a nucleic acid encoding a polypeptide of the
invention has been introduced. Such host cells can then be used to
generate a non-human transgenic animal into the genome of which an
exogenous sequence encoding a polypeptide of the invention has been
introduced. These host cells can, alternatively, be used to
generate a homologous recombinant animal in which an endogenous
nucleic acid encoding a polypeptide of the invention is altered.
Such animals are useful for studying the function, the activity, or
both, of the polypeptide and are also useful for identifying and
evaluating modulators of polypeptide activity. As used herein, a
"transgenic animal" is a non-human animal, preferably a mammal,
more preferably a rodent such as a rat or mouse, in which one or
more of the cells of the animal includes a transgene. Other
examples of transgenic animals include non-human primates, sheep,
dogs, cows, goats, chickens, amphibians, etc. A transgene is
exogenous DNA which is integrated into the genome of a cell from
which a transgenic animal develops and which remains in the genome
of the mature animal. The transgene directs expression of an
encoded gene product in one or more cell types or tissues of the
transgenic animal. As used herein, a "homologous recombinant
animal" is a non-human animal, preferably a mammal, more preferably
a mouse, in which an endogenous gene has been altered by homologous
recombination between the endogenous gene and an exogenous DNA
molecule introduced into a cell (e.g., an embryonic cell) of the
animal prior to development of the animal.
[0141] A transgenic animal of the invention can be created by
introducing a nucleic acid encoding a polypeptide of the invention
(or a homologue thereof) into the male pronucleus of a fertilized
oocyte (e.g., by microinjection, retroviral infection, and
development of the oocyte in a pseudopregnant female foster
animal). Intronic sequences and polyadenylation signals can be
included in the transgene in order to increase the efficiency of
expression of the transgene. One or more tissue-specific regulatory
sequences can be operably linked with the transgene to direct
expression of the polypeptide of the invention to particular cells.
Methods for generating transgenic animals by embryo manipulation
and microinjection, particularly animals such as mice, have become
conventional in the art and are described, for example, in U.S.
Pat. Nos. 4,736,866 and 4,870,009, U.S. Pat. No. 4,873,191 and in
Hogan, Manipulating the Mouse Embryo, (Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods
are used for production of other transgenic animals. A transgenic
founder animal can be identified based upon the presence of the
transgene in its genome, expression of mRNA encoding the transgene
in tissues or cells of the animals, or both. A transgenic founder
animal can be used to breed additional animals which harbor the
transgene. Moreover, transgenic animals harboring the transgene can
be bred with other transgenic animals harboring the same or other
transgenes.
[0142] To generate a homologous recombinant animal, a vector is
prepared which contains a nucleic acid of the invention (i.e.,
encoding at least a portion of MLip-1). A deletion, addition, or
substitution can be introduced into the nucleic acid to alter
expression of the nucleic acid or a property (e.g., tissue level or
activity) of the encoded polypeptide. For example, the vector can
be designed such that, upon homologous recombination, the
endogenous nucleic acid is functionally disrupted (i.e., no longer
encodes a functional protein). Such vectors are colloquially
referred to as "knock-out" vectors, and animals generated using
such vectors are designated "knock-out" animals. Alternatively, the
vector can be designed such that, upon homologous recombination,
the endogenous gene is mutated or otherwise altered but still
encodes functional protein (e.g., the upstream regulatory region
can be altered to affect expression of the endogenous protein). In
the homologous recombination vector, the altered portion of the
gene is flanked at its 5' and 3' ends by additional nucleic acid of
the gene to permit homologous recombination to occur between the
exogenous gene carried by the vector and an endogenous nucleic acid
(e.g. an endogenous gene) in an embryonic stem cell. The additional
flanking nucleic acid sequences are of sufficient length for
successful homologous recombination with the endogenous gene.
Typically, several kilobases of flanking DNA (both at the 5' and 3'
ends) are included in the vector (see, e.g., Thomas and Capecchi
(1987) Cell 51:503 for a description of homologous recombination
vectors). The vector is introduced into an embryonic stem cell line
(e.g., by electroporation) and cells in which the introduced gene
has homologously recombined with the endogenous gene are selected
(see, e.g., Li et al. (1992) Cell 69:915). Selected cells are
injected into a blastocyst of an animal (e.g., a mouse) to form
aggregation chimeras (see, e.g., Bradley in Teratocarcinomas and
Embryonic Stem Cells: A Practical Approach, Robertson, ed. (IRL,
Oxford, 1987) pp. 113-152). A chimeric embryo can be implanted into
a suitable pseudopregnant female foster animal, and the resulting
embryo can be carried to term by the foster animal. Progeny
harboring the homologously recombined DNA in their germ cells can
be used to breed animals in which all cells of the animal contain
the homologously recombined DNA (i.e., by germline transmission of
the transgene). Methods for constructing homologous recombination
vectors and homologous recombinant animals are described further in
Bradley (1991) Current Opinion in Bio/Technology 2:823-829 and in
PCT publications WO 90/11354, WO 91/01140, WO 92/0968, and WO
93/04169.
[0143] In another embodiment, transgenic non-human animals are
generated in which the transgene comprises a system for regulating
expression of the transgene. An example of such a system is the
cre/loxP recombinase system of bacteriophage P1. For a description
of the cre/loxP recombinase system, see, e.g., Lakso et al. (1992)
Proc. Natl. Acad. Sci. USA 89:6232-6236. Another example of a
recombinase system is the FLP recombinase system of Saccharomyces
cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355). If a
cre/loxP recombinase system is used to regulate expression of the
transgene, animals containing transgenes encoding both the Cre
recombinase and a selected protein are required. Such animals can
be provided by constructing "double" transgenic animals, e.g., by
mating two transgenic animals, one containing a transgene encoding
a selected protein and the other containing a transgene encoding a
recombinase.
[0144] Clones of non-human transgenic animals described herein can
be produced, for example, according to the methods described in
Wilmut et al. (1997) Nature 385:810-813 and PCT Publication numbers
WO 97/07668 and WO 97/07669.
[0145] VI. Pharmaceutical Compositions
[0146] The nucleic acids, polypeptides, antibodies, vectors, and
host cells (also referred to herein as "active agents") of the
invention can be incorporated into pharmaceutical compositions
suitable for administration to a patient. Such compositions
typically comprise the agent and a pharmaceutically acceptable
carrier. As used herein the language "pharmaceutically acceptable
carrier" is intended to include any and all solvents, dispersion
media, coatings, anti-bacterial and anti-fungal agents, isotonic
and absorption delaying agents, and the like, that are compatible
with pharmaceutical administration. The use of such media and
agents for pharmaceutically active substances is well known in the
art. Except insofar as any conventional media or agent is
incompatible with an active agent of the invention, use thereof in
the pharmaceutical compositions of the invention is contemplated.
Supplementary active compounds can also be incorporated into the
compositions.
[0147] The invention includes methods for preparing pharmaceutical
compositions for modulating expression, activity, or level of
activity (e.g., in a tissue or body fluid) of a polypeptide or
nucleic acid of the invention. Such methods comprise formulating a
pharmaceutically acceptable carrier with an agent which modulates
expression, activity, or activity level of a polypeptide or nucleic
acid of the invention. Such compositions can further include
additional active agents. Thus, the invention further includes
methods for preparing a pharmaceutical composition by formulating a
pharmaceutically acceptable carrier with an agent that modulates
expression or activity of a polypeptide or nucleic acid of the
invention and one or more additional active compounds.
[0148] The agent which modulates expression, activity, or activity
level can, for example, be a small molecule. For example, such
small molecules include peptides, peptidomimetics, amino acids,
amino acid analogs, polynucleotides, polynucleotide analogs,
nucleotides, nucleotide analogs, organic or inorganic compounds
(i.e., including heteroorganic and organometallic compounds) having
a molecular weight less than about 10,000 grams per mole, organic
or inorganic compounds having a molecular weight less than about
5,000 grams per mole, organic or inorganic compounds having a
molecular weight less than about 1,000 grams per mole, organic or
inorganic compounds having a molecular weight less than about 500
grams per mole, and salts, esters, and other pharmaceutically
acceptable forms of such compounds.
[0149] It is understood that appropriate doses of small molecule
agents, protein or polypeptide agents, antibody substances, and
other active agents of the invention depends upon a number of
factors within the ken of the ordinarily skilled physician,
veterinarian, or researcher. The dose(s) of these agents will vary,
for example, depending upon the identity, size, and condition of
the subject or sample being treated, further depending upon the
route by which the composition is to be administered, if
applicable, and the effect which the practitioner desires the agent
to have upon the nucleic acid or polypeptide of the invention.
Exemplary doses of a small molecule include milligram or microgram
amounts per kilogram of subject weight or sample weight (e.g.,
about 1 nanogram per kilogram to about 500 milligrams per kilogram,
about 100 micrograms per kilogram to about 5 milligrams per
kilogram, or about 1 microgram per kilogram to about 50 micrograms
per kilogram).
[0150] Exemplary doses of a protein or polypeptide include gram,
milligram, or microgram amounts per kilogram of subject or sample
weight (e.g., about 1 microgram per kilogram to about 5 grams per
kilogram, about 100 micrograms per kilogram to about 500 milligrams
per kilogram, or about 1 milligram per kilogram to about 50
milligrams per kilogram). For antibodies, the preferred dosage is
about 0.1 milligrams per kilogram to 100 milligrams per kilogram of
body weight (generally about 10 milligrams per kilogram to 20
milligrams per kilogram). If the antibody is to act in the brain, a
dosage of about 50 milligrams per kilogram to 100 milligrams per
kilogram is usually appropriate. Generally, partially human
antibodies and fully human antibodies have a longer half-life
within the human body than other antibodies. Accordingly, lower
dosages and less frequent administration are often possible.
Modifications such as lipidation can be used to stabilize
antibodies and to enhance uptake and tissue penetration (e.g., into
the brain). A method for lipidation of antibodies is described by
Cruikshank et al. (1997, J. Acquired Immune Deficiency Syndromes
and Human Retrovirology 14:193).
[0151] It is furthermore understood that appropriate doses of one
of these agents depend upon the potency of the agent with respect
to the expression or activity to be modulated. Such appropriate
doses can be determined using the assays described herein. When one
or more of these agents is to be administered to an animal (e.g., a
human) in order to modulate expression or activity of a polypeptide
or nucleic acid of the invention, a physician, veterinarian, or
researcher can, for example, prescribe a relatively low dose at
first, subsequently increasing the dose until an appropriate
response is obtained. In addition, it is understood that the
specific dose level for any particular animal subject will depend
upon a variety of factors including the activity of the specific
agent employed, the age, body weight, general health, gender, and
diet of the subject, the time of administration, the route of
administration, the rate of excretion, any drug combination, and
the degree of expression or activity to be modulated.
[0152] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include parenteral, e.g.,
intravenous, intradermal, subcutaneous, oral (e.g., inhalation or
ingestion), transdermal (topical), transmucosal, and rectal
administration. Solutions or suspensions used for parenteral,
intradermal, or subcutaneous application can include the following
components: a sterile diluent such as water for injection, saline
solution, fixed oils, polyethylene glycols, glycerine, propylene
glycol, or other synthetic solvents; antibacterial agents such as
benzyl alcohol or methyl parabens; antioxidants such as ascorbic
acid or sodium bisulfite; chelating agents such as
ethylenediamine-tetraacetic acid; buffers such as acetates,
citrates, or phosphates; and agents for adjustment of tonicity such
as sodium chloride or dextrose. pH can be adjusted using acids or
bases, such as hydrochloric acid or sodium hydroxide. The
parenteral preparation can be enclosed in ampules, disposable
syringes, or multiple dose vials made of glass or plastic.
[0153] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (i.e., where the agent is water
soluble) or dispersions and sterile powders for the extemporaneous
preparation of sterile injectable solutions or dispersions. For
intravenous administration, suitable carriers include physiological
saline, bacteriostatic water, Cremophor EL.TM. (BASF; Parsippany,
N.J.), and phosphate buffered saline (PBS). In each instance, the
composition should be sterile and should be fluid to the extent
that easy syringability exists. It should also be stable under the
conditions of manufacture and storage and preferably includes a
preservative to prevent contamination by microorganisms such as
bacteria and fungi. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol, a polyol (e.g.,
glycerol, propylene glycol, liquid polyethylene glycol, or the
like), and suitable mixtures thereof. The proper fluidity can be
maintained, for example, by the use of a coating such as lecithin,
by the maintenance of the required particle size in the case of
dispersion and by the use of surfactants. Growth or survival of
microorganisms can be prevented by including one or more
anti-bacterial and anti-fungal agents (e.g., parabens,
chlorobutanol, phenol, ascorbic acid, thimerosal, or the like) in
the composition. Prolonged absorption of the injectable
compositions can be achieved by including in the composition an
agent which delays absorption, such as aluminum monostearate or
gelatin.
[0154] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., a polypeptide or antibody)
in the required amount in an appropriate solvent with one or a
combination of ingredients enumerated above, as required, followed
by filtered sterilization. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle which
contains a basic dispersion medium, and then incorporating other
ingredients such as one or more of those enumerated above. In the
case of sterile powders for preparation of sterile injectable
solutions, preferred methods of preparation include vacuum drying
and freeze-drying. Each of these methods yields a powder comprising
the active ingredient and any additional desired ingredient from a
previously sterile-filtered solution thereof.
[0155] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Oral compositions can also be prepared in a fluid carrier for use,
for example, as a mouthwash, wherein the compound in the fluid
carrier is applied orally and swished and expectorated or
swallowed.
[0156] Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches, and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose; a disintegrating agent such as
alginic acid, Primogel.TM., or corn starch; a lubricant such as
magnesium stearate or Sterotes.TM.; a glidant such as colloidal
silicon dioxide; a sweetening agent such as sucrose or saccharin;
or a flavoring agent such as peppermint, methyl salicylate, or
orange flavoring.
[0157] For administration by inhalation, the compounds can be
delivered in the form of a dispersed powder or an aerosol spray
from a pressurized container or dispenser which contains a suitable
propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
[0158] Systemic administration can also be achieved using
transmucosal or transdermal delivery methods. For transmucosal or
transdermal administration, penetrants appropriate to the barrier
to be permeated are used in the formulation. Such penetrants are
generally known in the art and include, for example, detergents,
bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished using nasal sprays, swabs,
suppositories, or other intranasal dosage forms or applicators. For
transdermal administration, the active compounds can be formulated
as ointments, salves, gels, creams, wound dressings, patches, or
the like.
[0159] The compounds can also be prepared in the form of
suppositories (e.g., using conventional suppository bases such as
cocoa butter and other glycerides) or retention enemas for rectal
delivery.
[0160] In one embodiment, the active compounds are prepared using
carriers that protect the compound against rapid elimination from
the body, such as a controlled release formulation. Exemplary
controlled release formulations include implants and
microencapsulated delivery systems. Biodegradable, biocompatible
polymers can be used in such implants and systems, including, for
example, ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, and polylactic acid. Methods for
preparation of such formulations are known in the art. The
materials can also be obtained from commercial entities such as
Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal
suspensions (including liposomes having monoclonal antibodies
incorporated therein or at the liposomal surface) can be used as
pharmaceutically acceptable carriers in the pharmaceutical
compositions of the invention. These can be prepared according to
methods known to those skilled in the art, for example, as
described in U.S. Pat. No. 4,522,811.
[0161] Oral or parenteral compositions in dosage unit form are
preferred for ease of administration and uniformity of dosage.
"Dosage unit form," as used herein, refers to physically discrete
units suitable for administration as complete, individual dosages
for the subject to be treated. Each unit contains a pre-selected
quantity of active agent of the invention in association with a
suitable pharmaceutically acceptable carrier, wherein the quantity
is calculated to produce a desired therapeutic effect. The quantity
of the active agent and the form of the dosage unit are dictated by
and directly dependent on the unique characteristics of the active
agent, the particular therapeutic effect to be achieved, and the
limitations inherent in the art of compounding such an active agent
for the treatment of individuals.
[0162] Nucleic acid molecules of the invention can be inserted into
vectors and used as gene therapy vectors. Gene therapy vectors can
be delivered to a subject by, for example, intravenous injection,
local administration (U.S. Pat. No. 5,328,470), or by stereotactic
injection (see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA
91:3054-3057). The pharmaceutical preparation of the gene therapy
vector can include the gene therapy vector in an acceptable
diluent, or can comprise a slow release matrix in which the gene
delivery vehicle is embedded. Alternatively, where the complete
gene delivery vector can be produced intact from recombinant cells
(e.g., as with retroviral vectors) the pharmaceutical preparation
can include one or more cells which produce the gene delivery
system.
[0163] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0164] VII. Uses and Methods of the Invention
[0165] Nucleic acids, polypeptides, small molecules, and antibodies
described herein can be used in one or more of the following
methods: a) screening assays; b) detection assays (e.g.,
chromosomal mapping, tissue typing, and forensic biology assays);
c) predictive medicine (e.g., diagnostic assays, prognostic assays,
monitoring of clinical trials, and pharmacogenomic applications);
and d) methods of treatment (e.g., therapeutic and prophylactic
methods). For example, polypeptides of the invention can to used
for all of the purposes identified herein in portions of the
disclosure relating to individual types of protein of the invention
(e.g., MLip-1 proteins and derivatives, fragments, and variants
thereof; i.e., "MLip-1-related polypeptides"). Isolated nucleic
acids of the invention and nucleic acids encoding MLip-1-related
polypeptides can be used to express polypeptides (e.g., using a
recombinant expression vector in a host cell for gene therapy
applications), to detect mRNA (e.g., in a biological sample) or a
genetic lesion, and to modulate the level of activity of a
polypeptide of the invention in a cell or tissue. In addition, the
polypeptides of the invention can be used to screen drugs or
compounds which modulate activity or expression of MLip-1 and to
treat disorders characterized by insufficient or excessive
production of MLip-1 or production of a form of MLip-1 which has
decreased or aberrant activity compared to the wild type
protein.
[0166] This invention includes novel pharmacological agents
identified by the above-described screening assays and uses of such
agents for treatments as described herein.
[0167] A. Screening Assays
[0168] The invention provides a method (also referred to herein as
a "screening assay") for identifying modulators, i.e., candidate or
test compounds or agents (e.g., peptides, peptidomimetics, antibody
substances, small molecules, or other drugs) which bind with MLip-1
or another polypeptide of the invention, or have a stimulatory or
inhibitory effect on, for example, expression or activity of MLip-1
or another polypeptide of the invention.
[0169] In one embodiment, the invention provides assays for
screening candidate or test compounds which bind with or modulate
activity of MLip-1 protein or a biologically active portion
thereof. The test compounds of the present invention can be
obtained using any of numerous approaches known in combinatorial
library methods known in the art. Known types of combinatorial
libraries include: biological libraries; spatially addressable
parallel solid phase or solution phase libraries; synthetic library
methods requiring deconvolution; the "one-bead one-compound"
library method; and synthetic library methods using affinity
chromatography selection. The biological library approach is
generally limited to peptide libraries, while the other four
approaches are applicable to peptide, non-peptide oligomer, or
small molecule libraries of compounds (Lam (1997) Anticancer Drug
Des. 12:145).
[0170] Examples of methods for making molecular libraries are found
in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.
Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA
91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et
al. (1993) Science 261:1303; Carrell et al. (1994) Angew. Chem.
Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed.
Engl. 33:2061; and Gallop et al. (1994) J. Med. Chem. 37:1233.
[0171] Libraries of compounds can be presented in solution (e.g.,
Houghten (1992) Bio/Techniques 13:412-421), or on beads (Lam (1991)
Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556),
bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat. Nos.
5,571,698; 5,403,484; and 5,223,409), plasmids (Cull et al. (1992)
Proc. Natl. Acad. Sci. USA 89:1865-1869), or phage (Scott and Smith
(1990) Science 249:386-390; Devlin (1990) Science 249:404-406;
Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87:6378-6382; and
Felici (1991) J. Mol. Biol. 222:301-310).
[0172] In one embodiment, the invention includes a cell-based assay
involving a cell which expresses a membrane-bound form of a
polypeptide of the invention, or a biologically active portion
thereof, on the cell surface. The cell is contacted with a test
compound, and the ability of the test compound to bind with the
polypeptide is determined. The cell, for example, can be a yeast
cell or a cell of mammalian origin. Determining the ability of the
test compound to bind with the polypeptide can be accomplished, for
example, by coupling the test compound with a radioisotope or
enzymatic label such that binding of the test compound with the
polypeptide or biologically active portion thereof can be
determined by detecting the labeled compound in a complex. For
example, test compounds can be labeled with .sup.125I, .sup.35S,
.sup.14C, or .sup.3H, either directly or indirectly, and the
radioisotope detected by direct counting of radio-emission or by
scintillation counting. Alternatively, test compounds can be
enzymatically labeled with, for example, horseradish peroxidase,
alkaline phosphatase, or luciferase, and the enzymatic label
detected by determination of conversion of an appropriate substrate
to product. In a preferred screening method, a cell which expresses
a cell-bound form of a polypeptide of the invention (e.g., MLip-1
protein lacking a signal sequence cleavage site), or a biologically
active portion thereof, on its surface is contacted with a compound
which is known to bind with or known to be a substrate for the
polypeptide in an assay mixture. The assay mixture is contacted
with a test compound, and the ability of the test compound to
interact with the polypeptide is determined. Ability of the test
compound to interact with the polypeptide can be assessed by
determining the ability of the test compound to preferentially bind
with the polypeptide relative to the known compound or by
determining the ability of the test compound to catalyze conversion
of the known compound to a different compound.
[0173] In another embodiment, the assay involves assessment of an
activity characteristic of a polypeptide of the invention, wherein
binding of the test compound with the polypeptide or biologically
active portion thereof alters (i.e., increases or decreases) the
activity of the polypeptide. For example, the method described in
Giller et al. (1992, J. Biol. Chem. 267:16509-16516) or any other
known method for evaluating lipase activity (e.g., the
LIPASE-PS.TM. kit, Sigma Chemical Co., St. Louis, Mo.) may be used
to assess lipase activity in a cell expressing a nucleic acid
encoding a nucleic acid of the invention or in a medium in which
the cell is grown. In this assay, a test cell which expresses a
nucleic acid encoding a polypeptide of the invention (i.e., in
either a membrane-bound or a secreted form) is contacted with a
fluid containing a labeled lipase substrate (e.g., a tritiated
triglyceride or the Sigma Lipase-PS.TM. substrate reagent), and
release of the label from the substrate is assessed. For example,
cultured HeLa cells can be transfected with a recombinant Vaccinia
virus vector comprising a nucleic acid encoding a polypeptide of
the invention. A tritiated triglyceride is added to the medium, and
the medium containing the labeled compound is rinsed from the cells
after a selected amount of time. The tritium content of the cells
(i.e., corresponding to uptake by the cell of a tritiated fatty
acid) or the tritiated fatty acid (or glycerol compound, depending
on the site of tritiation) is assessed using, for example, a liquid
chromatography device coupled with a scintillation counter. The
skilled artisan will understand how this assay can be modified to
accommodate particular test cells, nucleic acid vectors, and
particular mono-, di-, and tri-glycerides, as well as lipids
derived from various glycerol compounds (e.g., glycerol, glycerol
phosphates, alkyl glyceryl ethers, glycerol phosphoryl-choline,
glycerol phosphoryl-serine, glycerol phosphoryl-ethanolamine, and
the like).
[0174] In another embodiment, an assay is a cell-based assay
comprising contacting a cell expressing a cell membrane-bound form
of a polypeptide of the invention (e.g. MLip-1 protein lacking a
signal sequence cleavage site), with a test compound and
determining the ability of the test compound to modulate (e.g.,
stimulate or inhibit) the activity of the polypeptide or
biologically active portion thereof. Determining the ability of the
test compound to modulate the activity of the polypeptide or
biologically active portion thereof can be accomplished, for
example, by determining the ability of the polypeptide to bind with
or interact with a target molecule or to transport lipids or fatty
acyl moieties across the cytoplasmic membrane or to incorporate
them into the membrane. An analogous cell-free assay may be
performed using a mature MLip-1 protein or another non-cell-bound
form of a polypeptide of the invention, wherein the ability of the
polypeptide to catalyze formation or hydrolysis of ester bonds
between a glycerol moiety and a fatty acyl moiety is assessed.
[0175] Determining the ability of a polypeptide of the invention to
bind with or interact with a target molecule can be accomplished by
one of the methods described above for determining direct binding.
As used herein, a "target molecule" is a molecule with which a
selected polypeptide (e.g., mature MLip-1 protein) binds or
interacts with in nature, for example, a molecule on the surface of
a cell which expresses the selected protein, a molecule on the
surface of a second cell, a molecule in the extracellular milieu,
or a molecule associated with a plasma lipoprotein particle In yet
another embodiment, a screening assay of the present invention is a
cell-free assay comprising contacting a polypeptide of the
invention (e.g., MLip-1 protein or a biologically active portion
thereof) with a test compound and determining the ability of the
test compound to bind with the polypeptide. Binding of the test
compound with the polypeptide can be determined either directly or
indirectly, as described above. In one embodiment, the assay
includes contacting the polypeptide with a known compound which
binds the polypeptide to form an assay mixture, contacting the
assay mixture with a test compound, and determining the ability of
the test compound to interact with the polypeptide. Ability of the
test compound to interact with the polypeptide comprises
determining the ability of the test compound to preferentially bind
with the polypeptide, relative to the ability of the known compound
to bind therewith.
[0176] In one or more embodiments of the above assay methods of the
present invention, it can be desirable to immobilize either a
polypeptide of the invention or a target molecule thereof in order
to facilitate separation of complexed from non-complexed forms of
either the polypeptide or the target molecule. Immobilization of
assay components also facilitates automation of the assay. Binding
of a test compound with the polypeptide, or interaction of the
polypeptide with a target molecule in the presence and absence of a
test compound, can be accomplished in any vessel suitable for
containing the reactants. Examples of such vessels include
microtiter plates, test tubes, and micro-centrifuge tubes. In one
embodiment, a fusion protein can be provided which has a domain
that facilitates binding of the protein with a matrix. For example,
glutathione-S-transferase fusion proteins can be adsorbed onto
glutathione Sepharose.TM. beads (Sigma Chemical; St. Louis, Mo.) or
glutathione-derivatized microtiter plates. Such fusion proteins can
be combined with the test compound, and the mixture is incubated
under conditions conducive to protein-ligand complex formation or
protein activity (e.g., at physiological conditions for salt and
pH). Following incubation, the beads or microtiter plate wells are
washed to remove any non-bound components and one or both of
complex formation and lipase activity is measured either directly
or indirectly, for example, as described above. Alternatively, the
complexes can be dissociated from the matrix, and the level of
binding or activity of the polypeptide of the invention can be
determined using standard techniques.
[0177] Other techniques for immobilizing a protein on a matrix can
be used in the screening assays of the invention. For example, a
polypeptide of the invention can be immobilized utilizing
conjugation of biotin and streptavidin. Biotinylated polypeptides
can be prepared from biotin-NHS (N-hydroxy-succinimide) using
techniques well known in the art (e.g., biotinylation kit, Pierce
Chemicals; Rockford, Ill.), and immobilized in the wells of
streptavidin-coated 96 well plates (Pierce Chemical).
Alternatively, antibodies reactive with polypeptides but which do
not interfere with binding of the polypeptides to a target molecule
(e.g., an enzymatic substrate such as a triacylglyceride) can be
derivatized to the wells of the plate, and non-bound polypeptide of
the invention can be trapped in the wells by antibody conjugation.
Methods for detecting such complexes, in addition to those
described above for the GST-immobilized complexes, include
immunodetection of complexes using antibodies reactive with the
polypeptide of the invention or target molecule, as well as
enzyme-linked assays which rely on detecting an enzymatic activity
associated with the polypeptide of the invention (e.g., hydrolysis
of a labeled triacylglyceride).
[0178] In another embodiment, modulators of expression of a
polypeptide of the invention are identified in a method in which a
cell is contacted with a test compound and expression of mRNA or
encoding MLip-1 or MLip-1 protein in the cell is determined. The
level of expression of MLip-1 mRNA or protein in the presence of
the test compound is compared with the level of expression of the
mRNA or protein in the absence of the test compound. The test
compound can then be identified as a modulator of expression of
MLip-1 based on this comparison. For example, when expression of
MLip-1 mRNA or protein is greater (i.e., statistically
significantly greater) in the presence of the test compound than in
its absence, the test compound is identified as a stimulator of
MLip-1 mRNA or protein expression. Alternatively, when expression
of MLip-1 mRNA or protein is less (i.e., statistically
significantly less) in the presence of the test compound than in
its absence, the test compound is identified as an inhibitor of
MLip-1 mRNA or protein expression. The level of MLip-1 mRNA or
protein expression in the cells can be determined by methods
described herein.
[0179] In yet another aspect of the invention, a polypeptide of the
invention can be used as a "bait protein" in a two-hybrid assay or
three hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et
al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem.
268:12046-12054; Bartel et al. (1993) Bio/Techniques 14:920-924;
Iwabuchi et al. (1993) Oncogene 8:1693-1696; and PCT Publication
number WO 94/10300), to identify other proteins, which bind with or
interact with the polypeptide of the invention and modulate
activity of the polypeptide. Such binding proteins are also likely
to be involved in the propagation of signals by the polypeptide as,
for example, upstream or downstream elements of a signaling pathway
involving the polypeptide.
[0180] This invention further pertains to novel agents identified
by the above-described screening assays and uses thereof for
treatments as described herein.
[0181] B. Detection Assays
[0182] Portions or fragments of the nucleic acids identified herein
(including entire nucleic acids, i.e. wherein the portion is the
entirety of a nucleic acid) can be used in numerous ways as
polynucleotide reagents. For example, such nucleic acids can be
used to: (i) map the MLip-1 gene on a chromosome and, thus, locate
MLip-1 gene regions associated with genetic disease; (ii) identify
an individual from a minute biological sample (tissue typing); and
(iii) aid in forensic identification of a biological sample. These
applications are described in the subsections below.
[0183] 1. Chromosome Mapping
[0184] All or a portion of a nucleic acid encoding MLip-1 (e.g. a
nucleic acid having a nucleotide sequence consisting of all or a
portion of SEQ ID NO: 1 or 2) can be used to map the location of
the gene on a chromosome. Mapping of the sequence to a chromosome
can be used to associate MLip-1 with one or more diseases.
[0185] Briefly, the MLip-1 gene can be mapped to a chromosome by
preparing PCR primers (preferably 15 to 25 nucleotide residues in
length) from the sequence of a nucleic acid of the invention.
Computer analysis of the sequence of a nucleic acid of the
invention can be used to rapidly select primers that do not span
more than one exon in the genomic DNA, as these could complicate
the amplification process. The primers can then be used for PCR
screening of somatic cell hybrids containing individual human
chromosomes. Only hybrids containing the human gene corresponding
to the gene sequences will yield an amplified fragment. For a
review of this technique, see D'Eustachio et al. (1983, Science
220:919-924).
[0186] PCR mapping of somatic cell hybrids is a rapid procedure for
assigning a particular sequence to a particular chromosome. Three
or more sequences can be assigned per day using a single thermal
cycler. Using the nucleotide sequences of one or more of the
nucleic acids of the invention to design oligonucleotide primers,
sub-localization can be achieved using panels of fragments obtained
from specific chromosomes. Other mapping strategies which can be
used to map a gene to its chromosome include in situ hybridization
(described in Fan et al. (1990) Proc. Natl. Acad. Sci. USA
87:6223-27), pre-screening using labeled flow-sorted chromosomes,
and pre-selection by hybridization to chromosome specific cDNA
libraries. Fluorescence in situ hybridization (FISH) of a DNA
sequence with a metaphase chromosomal spread can be used to provide
a precise chromosomal location in one step. For a review of this
technique, see Verma et al. ("Human Chromosomes: A Manual of Basic
Techniques", Pergamon Press, New York, 1988).
[0187] Reagents for chromosome mapping can be used individually to
mark a single chromosome or a single site on a chromosome.
Alternatively, panels of reagents can be used for marking multiple
sites or multiple chromosomes. Reagents corresponding to non-coding
regions of the genes are preferred for mapping purposes. Coding
sequences are more likely to be conserved within gene families,
thus increasing the chance of cross hybridizations during
chromosomal mapping.
[0188] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data. (Such data are found, for
example, in V. McKusick, Mendelian Inheritance in Man, available
on-line through Johns Hopkins University Welch Medical Library).
The relationship between genes and disease, mapped to the same
chromosomal region, can then be identified using linkage analysis
(i.e., analysis of co-inheritance of physically adjacent genes),
described in, e.g., Egeland et al. (1987) Nature 325:783-787.
[0189] Moreover, differences in genomic nucleotide sequences
between individuals afflicted and not afflicted with a disease
associated with the MLip-1 gene can be determined. If a mutation is
observed in some or all of the afflicted individuals but not in any
non-afflicted individuals, then the mutation is likely to be a
causative agent of the disease, or at least strongly associated
with occurrence of the disease. Comparison of afflicted and
non-afflicted individuals generally involves first looking for
structural alterations in chromosomes obtained from patients of the
two groups. Exemplary structural alterations include deletions and
translocations that are visible from chromosome spreads or
detectable using PCR amplification of all or part of the MLip-1
gene. Ultimately, complete sequencing of genes from several
individuals can be performed to confirm the presence of a mutation
and to distinguish mutations from polymorphisms.
[0190] 2. Tissue Typing
[0191] Nucleotide sequences of the nucleic acids of the invention
can be used to identify individuals from minute biological samples.
The United States military, for example, is considering the use of
restriction fragment length polymorphism (RFLP) for identification
of its personnel. In this technique, an individual's genomic DNA is
digested with one or more restriction enzymes, and probed by
Southern blotting to yield individually unique bands which can be
used for identification. This method does not exhibit the current
limitations of the "dog tags" identification system, in which
identification devices can be lost, switched, or stolen, making
positive identification difficult. The sequences of the nucleic
acids of the invention are useful as additional DNA markers for
RFLP (described in U.S. Pat. No. 5,272,057).
[0192] Furthermore, the sequences of the nucleic acids of the
invention can be used to provide an alternative technique which
determines the DNA sequence of selected portions of an individual's
genome. Thus, the nucleic acid sequences described herein can be
used to prepare pairs of PCR primers for amplification of at least
a portion of a human genome. These primer pairs can be used to
amplify an individual's DNA and subsequently sequence it.
[0193] Panels of corresponding DNA sequences obtained from
individuals, prepared in this manner, can provide unique individual
identifications, as each individual will have a unique set of such
DNA sequences, owing in part to allelic differences. The sequences
of the nucleic acids of the invention can be used to obtain such
identification sequences from individuals and from tissue. The
nucleotide sequences of the invention uniquely represent portions
of the human genome. Allelic variation occurs to some degree in the
coding regions of these sequences, and to a greater degree in the
non-coding regions. It is estimated that allelic variation between
individual humans occurs with a frequency of about once every 500
base pairs. Each of the sequences described herein can be used as a
standard against which DNA obtained from an individual can be
compared for identification purposes. Because greater numbers of
polymorphisms occur in the non-coding regions, fewer sequences are
necessary to differentiate individuals. Non-coding portions of SEQ
ID NO: 1 can provide positive individual identification with a
panel of about 10 to 1,000 primers each of which yields a
non-coding amplified sequence of 100 bases. If a predicted coding
sequence, such as one in SEQ ID NO: 2 is used, a more appropriate
number of primers for positive individual identification would be
about 500-2,000.
[0194] If a panel of nucleic acid reagents having the nucleotide
sequences described herein is used to generate a unique
identification database for an individual, then the same reagents
can be used later to identify tissue from that individual. Using
the unique identification database, positive identification of the
individual, living or dead, can be made using extremely small
tissue samples.
[0195] 3. Use of Partial Gene Sequences in Forensic Biology
[0196] DNA-based identification techniques can be used in forensic
biology. Forensic biology is a scientific field employing genetic
typing of biological evidence recovered at a crime scene as a means
for positively identifying, for example, a perpetrator of a crime.
To make such an identification, PCR technology can be used to
amplify nucleic acid obtained from very small biological samples
such as tissues (e.g., hair or skin) or body fluids (e.g., blood,
saliva, or semen) recovered at a crime scene. The amplified
sequence can be compared with a standard, allowing identification
of the origin of the biological sample.
[0197] The nucleotide sequences of the present invention can be
used to generate polynucleotide reagents (e.g., PCR primers)
targeted to specific loci (e.g., the MLip-1 gene) in the human
genome, which can enhance the reliability of DNA-based forensic
identifications by, for example, providing another "identification
marker" (i.e., another DNA sequence that is unique to a particular
individual). As mentioned above, nucleotide sequence information
can be used for identification as an accurate alternative to
patterns formed by restriction enzyme generated fragments.
Sequences corresponding to non-coding regions are particularly
appropriate for this use, because greater numbers of polymorphisms
and mutations occur in non-coding regions than in coding regions,
making it easier to differentiate individuals using this technique.
Examples of polynucleotide reagents include nucleic acids of the
invention such as MLip-1 gene fragments derived from non-coding
regions of the gene and having a length of at least 20 or 30
bases.
[0198] Nucleotide sequences described herein can further be used to
generate polynucleotide reagents, e.g., labeled or label-able
probes which can be used in, for example, in situ hybridization
techniques, to identify a specific tissue, e.g., pancreas tissue.
This can be very useful in cases where a forensic pathologist is
presented with a tissue of unknown origin. Panels of such probes
can be used to identify tissue by species and/or by organ type.
[0199] C. Predictive Medicine
[0200] The present invention also pertains to the field of
predictive medicine, in which diagnostic assays, prognostic assays,
pharmacogenomics, and monitoring clinical trails are used for
prognostic (predictive) purposes to treat an individual
prophylactically. Accordingly, one aspect of the present invention
relates to diagnostic assays for assessing expression of a
polypeptide or nucleic acid of the invention. Such assays can also
be used to assess activity of a polypeptide of the invention in the
context of a biological sample (e.g., blood, serum, cells, tissue).
Each of these diagnostic assays is useful for determining whether
an individual is afflicted with a disease or disorder associated
with aberrant expression or activity of MLip-1 protein, or is at
risk of developing such a disorder. By way of example, mutations in
a gene corresponding to a nucleic acid of the invention can be
assayed in a biological sample. Such assays can be used for
prognostic or predictive purpose to treat an individual prior to
onset of a disorder characterized by or associated with aberrant
expression or activity of MLip-1 protein.
[0201] Another aspect of the invention provides methods for
assessing expression of a nucleic acid or polypeptide of the
invention and methods for assessing activity of a polypeptide of
the invention in an individual. These assays can be used to select
appropriate therapeutic or prophylactic agents for that individual.
A selection process of this sort is referred to herein and in the
art as "pharmacogenomics." Pharmacogenomics enables selection of
agents (e.g., drugs) for therapeutic or prophylactic treatment of
an individual based on the genotype of the individual. In these
methods, the genotype of the individual is examined to determine
the ability of the individual to respond favorably to
administration of a particular agent.
[0202] Yet another aspect of the invention pertains to monitoring
the influence of one or more agents (e.g., drugs or other
compounds) on expression or activity of a polypeptide of the
invention in clinical trials. These and other methods are described
in further detail in the following sections.
[0203] 1. Diagnostic Assays
[0204] An exemplary method for detecting the presence or absence of
a polypeptide or nucleic acid of the invention in a biological
sample involves obtaining a biological sample from a subject and
contacting the biological sample with a compound or an agent
capable of detecting the polypeptide or nucleic acid (e.g., mRNA,
genomic DNA) such that the presence of the polypeptide or nucleic
acid is detected in the biological sample. A preferred agent for
detecting mRNA or genomic DNA encoding MLip-1 protein is a labeled
polynucleotide probe capable of hybridizing with mRNA or genomic
DNA encoding the polypeptide. The nucleic acid probe can be, for
example, a full-length cDNA, such as the nucleic acid of SEQ ID NO:
1 or a portion thereof, such as an oligonucleotide of at least 15,
30, 50, 56, 100, 250 or 500 nucleotides in length which
specifically hybridizes under stringent conditions with an mRNA or
genomic DNA encoding MLip-l protein. Other suitable probes for use
in the diagnostic assays of the invention are described herein.
[0205] A preferred agent for detecting a polypeptide of the
invention is an antibody capable of binding specifically with the
polypeptide, such as an antibody substance comprising a detectable
label. Antibodies can be polyclonal, or more preferably,
monoclonal. An intact antibody, a fragment of an antibody (e.g., a
Fab or F(ab').sub.2 fragment), a T cell receptor or fragment
thereof, or another immunoglobulin which binds specifically with an
epitope of MLip-1 can be used. The term "labeled", with regard to
the antibody substance, encompasses direct labeling of the antibody
substance by coupling (i.e., physically linking) a detectable
substance with the antibody substance, as well as indirect labeling
of the antibody substance. Examples of indirect labeling include
detection of a primary antibody using a fluorescently labeled
secondary antibody (i.e., one which binds specifically with the
first antibody or with antibodies of the same type). The term
"biological sample" is intended to include tissues, cells, and
biological fluids isolated from a subject, as well as tissues,
cells, and fluids present within a subject. That is, the detection
method of the invention can be used to detect mRNA, protein, or
genomic DNA in a biological sample in vitro as well as in vivo. For
example, in vitro techniques for detection of mRNA include Northern
hybridization and in situ hybridizations. In vitro techniques for
detection of a polypeptide of the invention include enzyme linked
immunosorbent assay (ELISA), Western blotting, immunoprecipitation,
and immunofluorescence detection. In vitro techniques for detection
of genomic DNA include, for example, Southern hybridization.
Furthermore, in vivo techniques for detection of a polypeptide of
the invention include introducing into a subject a labeled antibody
directed against the polypeptide. For example, the antibody can be
labeled with a radioactive marker, the presence and location of
which can be detected in a subject by standard imaging techniques
involving, for example, imaging using a `gamma camera` detector of
gamma radiation.
[0206] In one embodiment, the biological sample contains protein
molecules obtained from a subject. Alternatively, the biological
sample can contain mRNA molecules obtained from a subject or
genomic DNA molecules obtained from the subject. A preferred
biological sample is a tissue (e.g., a pancreatic tissue) sample or
a body fluid sample (e.g. gastric juice) isolated by conventional
means from a subject.
[0207] In another embodiment, the methods of the invention involve
obtaining a control biological sample from a control subject,
contacting the control sample with a compound or agent capable of
detecting a polypeptide of the invention, or an mRNA or genomic DNA
encoding a polypeptide of the invention, such that the presence of
the polypeptide or an mRNA or genomic DNA encoding such a
polypeptide is detected in the biological sample. The presence of
the polypeptide, mRNA, or genomic DNA encoding the polypeptide in
the control sample can be compared with the presence of the
polypeptide or mRNA or genomic DNA encoding the polypeptide in the
test sample. This method is useful, for example, for comparing
levels of protein and RNA in a sample obtained from a patient who
is suspected of being afflicted with an MLip-1 associated disorder
with the corresponding levels in a normal (i.e., non-afflicted)
patient. This method is also useful for detecting the presence of a
normal or mutant allele of the MLip-1 gene in a patient.
[0208] The invention also encompasses kits for detecting the
presence of a polypeptide or nucleic acid of the invention in a
biological sample. Such kits can be used to determine if a subject
is afflicted with or is at increased risk of developing a disorder
associated with aberrant expression of MLip-1 protein. For example,
the kit can comprise a labeled compound or agent capable of
detecting a polypeptide or nucleic acid of the invention in a
biological sample and means for determining the amount of the
labeled compound that interacts with the polypeptide or nucleic
acid in the sample (e.g., an antibody which binds the polypeptide
or an oligonucleotide probe which binds with DNA or mRNA encoding
the polypeptide). Kits can include instructions for assessing
whether a subject is suffering from or is at risk of developing a
disorder associated with aberrant expression of MLip-1 protein if
the amount of the polypeptide or mRNA encoding the polypeptide is
above or below a normal level.
[0209] For antibody-based kits, the kit can comprise, for example:
(1) a first antibody (e.g., attached to a solid support) which
binds with a polypeptide of the invention; and (2) a second,
different antibody which binds with either the polypeptide or the
first antibody and is conjugated with a detectable agent.
[0210] For oligonucleotide-based kits, the kit can comprise, for
example: (1) an oligonucleotide (e.g., a detectably labeled
oligonucleotide) which hybridizes with a nucleic acid of the
invention or (2) a pair of primers useful for amplifying a nucleic
acid of the invention. The kit can also comprise, e.g., a buffering
agent, a preservative, or a protein stabilizing agent. The kit can
also comprise components necessary for detecting the detectable
agent (e.g., an enzyme, a substrate, a scintillation cocktail,
etc.). The kit can also contain one or more control samples which
can be assayed and compared with results obtained using the test
sample. Each component of the kit can be enclosed within an
individual container and all of the various containers can be
within a single package, optionally together with instructions for
assessing whether the subject is suffering from or is at risk of
developing a disorder associated with aberrant expression or
activity of MLip-1 protein.
[0211] 2. Prognostic Assays
[0212] The methods described herein can be used as diagnostic or
prognostic assays to identify subjects at risk of developing a
disease or disorder associated with aberrant expression or activity
of MLip-1 protein. Thus, the present invention provides a method in
which a biological sample is obtained from a subject and a
polypeptide or nucleic acid (e.g., mRNA or genomic DNA) of the
invention is detected in the sample. The presence of an aberrant
MLip-1 polypeptide or nucleic acid or detection of an aberrant
level of MLip-1 expression or activity is an indication that the
subject is predisposed to become afflicted with (i.e., is at an
increased risk of developing) an MLip-1-associated disorder
[0213] Furthermore, the prognostic assays described herein can be
used to determine whether a subject can be administered an agent
(e.g., an agonist, antagonist, peptidomimetic, protein, peptide,
antibody substance, nucleic acid, small molecule, or other drug
candidate) to treat an MLip-1-associated disease or disorder prior
to onset or worsening of the disease or disorder or a symptom
thereof. For example, such methods can be used to determine whether
a subject can be effectively treated using a specific agent or
class of agents (e.g., agents of a type which decrease MLip-1
activity).
[0214] The methods of the invention can be used to detect genetic
lesions or mutations in an MLip-1 gene, thereby determining if a
subject having the lesioned gene is at risk for developing a
disorder characterized by aberrant expression or activity of MLip-1
protein. In preferred embodiments, the methods include detecting,
in a sample of cells obtained from the subject, the presence or
absence of a genetic lesion or mutation characterized by at least
one of an alteration affecting the integrity of a gene encoding the
polypeptide of the invention and mis-expression of a gene encoding
a polypeptide of the invention. For example, such genetic lesions
or mutations can be detected by assessing the existence of one or
more of: 1) a deletion of one or more nucleotide residues from the
MLip-1 gene; 2) an addition of one or more nucleotide residues to
the MLip-1 gene; 3) a substitution of one or more nucleotide
residues of the MLip-1 gene; 4) a chromosomal rearrangement of the
MLip-1 gene; 5) an alteration in the level of a messenger RNA
transcript of the MLip-1 gene; 6) an aberrant modification of the
MLip-1 gene, such as a modification of the methylation pattern of
the gene; 7) the presence of a non-wild type splicing pattern of a
messenger RNA transcript of the MLip-1 gene; 8) the presence of a
non-wild type level of MLip-1 protein; 9) an allelic loss of the
MLip-1 gene; and 10) an inappropriate post-translational
modification of the protein encoded by the MLip-1 gene. As
described herein, for example, there are a large number of assay
techniques known in the art which can be used for detecting lesions
in a gene.
[0215] In certain embodiments, detection of the lesion involves the
use of a probe/primer in a polymerase chain reaction (see, e.g.,
U.S. Pat. Nos. 4,683,195 and 4,683,202). Exemplary methods of this
type include anchor PCR, RACE PCR, or, alternatively, a ligation
chain reaction (LCR; see, e.g., Landegran et al. (1988) Science
241:1077-1080; and Nakazawa et al. (1994) Proc. Natl. Acad. Sci.
USA 91:360-364). LCR can be particularly useful for detecting point
mutations in a gene (see, e.g., Abravaya et al. (1995) Nucleic
Acids Res. 23:675-682). Detection of an MLip-1 gene lesion can
involve collecting a sample of cells from a patient, isolating a
nucleic acid (e.g., genomic, mRNA, or both) from cells of the
sample, contacting the nucleic acid with one or more primers which
specifically hybridize with the MLip-1 gene under conditions such
that hybridization and amplification of the gene (if it is present)
occurs, and detecting the presence or absence of an amplification
product. Alternatively, the size of the amplification product can
be assessed and compared with the length of the corresponding
amplification product in a control sample. PCR, LCR, or both, can
be desirable to use as a preliminary amplification step in
conjunction with any of the other techniques used for detecting
mutations described herein.
[0216] Alternative amplification methods include: self-sustained
sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci.
USA 87:1874-1878), transcriptional amplification system (Kwoh, et
al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta
Replicase (Lizardi et al. (1988) Bio/Technology 6:1197), or any
other nucleic acid amplification method, which can be followed by
detection of the amplified portion using techniques known in the
art. Detection methods are preferably able to detect nucleic acid
molecules present in very low numbers.
[0217] In an alternative embodiment, mutations in the MLip-1 gene
of a cell are identified by one or more alterations in restriction
enzyme cleavage patterns of a nucleic acid obtained from the cell.
For example, sample and control DNA is isolated, (optionally)
amplified, digested with one or more restriction endonucleases, and
fragment length sizes are determined by gel electrophoresis and
compared. Differences in fragment length sizes between sample and
control DNA digestion mixtures indicates that one or more mutations
have occurred in the sample DNA. Use of sequence specific ribozymes
(see, e.g., U.S. Pat. No. 5,498,531) can be used to score nucleic
acids for the presence of specific mutations by assessing the
presence or absence of a ribozyme cleavage site in a sample nucleic
acid.
[0218] In other embodiments, genetic mutations are identified by
hybridizing sample and control nucleic acids, e.g., DNA or RNA,
with high density arrays containing hundreds or thousands of
oligonucleotides probes (Cronin et al. (1996) Human Mutation
7:244-255; Kozal et al. (1996) Nature Medicine 2:753-759). For
example, genetic mutations can be identified using two-dimensional
arrays of polynucleotides containing light-generated DNA probes as
described in Cronin et al., supra. Briefly, a first array of probes
comprising sequential overlapping probes differing in frame by a
single nucleotide residue can be used to scan long stretches of DNA
in a sample. Nucleotide sequence differences between the sample
nucleic acid and the sequences of first array probes are detectable
as less stringent hybridization between the sample nucleic acid and
portions of the array. This step allows the identification of point
mutations. This step is followed by hybridization of the sample
nucleic acid with a second oligonucleotide array that allows the
characterization of specific mutations. These second arrays are
smaller, specialized probe arrays complementary to all variants or
mutations detected. Each mutation array is composed of parallel
probe sets, one complementary to the wild-type gene and the other
complementary to the mutant gene.
[0219] In yet another embodiment, any of a variety of sequencing
reactions known in the art is used to directly sequence all or a
portion of the MLip-l gene and detect mutations by comparing the
sequence of the sample nucleic acids with the corresponding
wild-type (i.e., normal, control) sequence. Examples of sequencing
reactions include those based on techniques developed by Maxim and
Gilbert (1977, Proc. Natl. Acad. Sci. USA 74:560) or Sanger (1977,
Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplated that
any of a variety of automated sequencing procedures can be utilized
when performing the diagnostic assays (e.g., (1995) Bio/Techniques
19:448), including sequencing by mass spectrometry (see, e.g., PCT
Publication number WO 94/16101; Cohen et al. (1996) Adv.
Chromatogr. 36:127-162; and Griffin et al. (1993) Appl. Biochem.
Biotechnol. 38:147-159).
[0220] Other methods for detecting mutations in the MLip-1 gene
include methods in which protection from cleavage agents is used to
detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (e.g.,
Myers et al. (1985) Science 230:1242). In general, the technique of
mismatch cleavage entails generating heteroduplexes formed by
hybridizing (preferably labeled) RNA or DNA containing the
wild-type sequence with RNA or DNA obtained from a sample such as a
patient sample. The resulting double-stranded duplexes are treated
with an agent which cleaves single-stranded regions of the duplex.
Single stranded regions exist at sites of base pair mismatches
between the control and sample strands. For example, RNA/DNA
duplexes can be treated with RNase to digest mismatched regions,
and DNA/DNA hybrids can be treated with S1 nuclease to digest
mismatched regions.
[0221] In other embodiments, either DNA/DNA or RNA/DNA duplexes can
be treated with hydroxylamine or osmium tetroxide and with
piperidine in order to digest mismatched regions. After digestion
of the mismatched regions, the resulting material is then separated
(e.g., by size on denaturing polyacrylamide gels), and the
fragments are analyzed to determine the site of mutation. See,
e.g., Cotton et al. (1988) Proc. Natl. Acad. Sci. USA 85:4397;
Saleeba et al. (1992) Methods Enzymol. 217:286-295. In a preferred
embodiment, the control DNA or RNA can be labeled for
detection.
[0222] In still another embodiment, the mismatch cleavage reaction
employs one or more proteins that recognize mismatched base pairs
in double-stranded DNA (e.g., DNA mismatch repair enzymes) in
defined systems for detecting and mapping point mutations in cDNAs
obtained from samples of cells. For example, mutY enzyme of E. coli
cleaves A residues at G/A mismatches, and thymidine DNA glycosylase
from HeLa cells cleaves T residues at G/T mismatches (Hsu et al.
(1994) Carcinogenesis 15:1657-1662). According to an exemplary
embodiment, a probe based on a selected sequence, e.g., the
wild-type sequence of MLip-1, is hybridized with a cDNA or other
DNA product from a sample. The resulting duplex is treated with a
DNA mismatch repair enzyme, and the cleavage products, if any, are
detected using known electrophoresis or other polynucleotide
separation protocols. See, e.g., U.S. Pat. No. 5,459,039.
[0223] In other embodiments, alterations in electrophoretic
mobility are used to identify mutations in genes. For example,
single strand conformation polymorphism (SSCP) can be used to
detect differences in electrophoretic mobility between mutant and
wild type nucleic acids, as described (Orita et al. (1989) Proc.
Natl. Acad. Sci. USA 86:2766; see also Cotton (1993) Mutat. Res.
285:125-144; Hayashi (1992) Genet. Anal. Tech. Appl. 9:73-79).
Single-stranded DNA fragments of sample and control nucleic acids
are denatured and allowed to re-nature. Secondary structure of
single-stranded nucleic acids varies according to their sequences,
and the resulting alteration in electrophoretic mobility enables
detection of sequence differences of as few as one nucleotide
residue. The DNA fragments can be labeled or detected with labeled
probes. The sensitivity of the assay can be enhanced using RNA
(rather than DNA), because the secondary structure of RNA is more
sensitive to sequence changes than that of DNA. In a preferred
embodiment, heteroduplex analysis is used to separate double
stranded heteroduplex molecules on the basis of changes in
electrophoretic mobility, as described (Keen et al. (1991) Trends
Genet. 7:5).
[0224] In yet another embodiment, the movement of mutant or
wild-type fragments in polyacrylamide gels containing a gradient of
denaturant is assayed using denaturing gradient gel electrophoresis
(DGGE; Myers et al. (1985) Nature 313:495). When DGGE is used as
the method of analysis, DNA is modified to insure that it does not
completely denature, for example by adding a `GC clamp` of
approximately 40 base pairs of high-melting GC-rich DNA by PCR. In
another embodiment, a temperature gradient is used in place of a
denaturant gradient to identify differences in the mobility of
control and sample DNA, as described (Rosenbaum and Reissner (1987)
Biophys. Chem. 265:12753).
[0225] Examples of other techniques for detecting point mutations
include, but are not limited to, selective oligonucleotide
hybridization, selective amplification, and selective primer
extension. For example, oligonucleotide primers can be prepared in
which the known mutation is located centrally. The primers are
hybridized with target DNA under conditions which permit
hybridization only if a perfect match is found (Saiki et al. (1986)
Nature 324:163); Saiki et al. (1989) Proc. Natl. Acad. Sci. USA
86:6230). Such allele specific oligonucleotides can be hybridized
with PCR-amplified target DNA or with one of a number of mutant
sequences when the oligonucleotides are attached to the hybridizing
membrane and hybridized with labeled target DNA.
[0226] Alternatively, allele specific amplification (ASA)
technology can be used to detect mutations in the MLip-1 gene.
Oligonucleotides used as primers for ASA have an allele-specific
(e.g. mutant allele-specific) sequence situated at the central
portion of one or more primers. Amplification thus depends on
hybridization of the primer(s) with the specific allele, as
described (Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448).
Alternatively, ASA can be performed using an allele-specific
sequence at the extreme 3' end of one primer such that, under
appropriate conditions, mismatching prevents or reduces polymerase
extension (Prossner (1993) Tibtech 11:238). It can be desirable to
introduce a novel restriction site in the region of an MLip-1
mutation to facilitate cleavage-based detection (Gasparini et al.
(1992) Mol. Cell Probes 6:1). Amplification can be performed using
Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci.
USA 88:189). In such cases, ligation will occur only if there is a
perfect match at the 3' end of the 5' sequence, thus making it
possible to detect the presence of a known mutation at a specific
site by looking for the presence or absence of amplification.
[0227] The methods described herein can be performed, for example,
using pre-packaged diagnostic kits comprising at least one probe
nucleic acid or antibody reagent described herein, which can be
used (e.g., in clinical settings) to diagnose patients exhibiting
symptoms or a family history of a disease or illness involving
aberrant activity or expression of the MLip-1 gene. Furthermore,
any cell type or tissue (e.g., a pancreatic tissue) in which MLip-1
protein is expressed can be utilized in the prognostic assays
described herein.
[0228] 3. Pharmacogenomics
[0229] Agents or modulators which have a stimulatory or inhibitory
effect on activity or expression of MLip-1 protein, for example as
identified by a screening assay described herein can be
administered to patients to treat (prophylactically or
therapeutically) disorders associated with aberrant activity or
expression of MLip-1 protein. In conjunction with such treatment,
pharmacogenomics of an individual (i.e., the relationship between
the individual's genotype and the individual's response to a
foreign compound or drug) can be considered. In general, two types
of pharmacogenomic conditions can be differentiated. Genetic
conditions transmitted as a single factor altering the way drugs
act on the body are referred to as conditions which effect "altered
drug action." Differences in metabolism of therapeutics can lead to
toxicity, reduced therapeutic effectiveness, or therapeutic failure
by altering the relation between dose and blood concentration of
the pharmacologically active agent. For example,
glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common
inherited enzymopathy in which the main clinical complication is
hemolysis after ingestion of oxidant drugs (anti-malarials,
sulfonamides, analgesics, nitrofurans) and consumption of fava
beans.
[0230] Pharmacogenomic analysis of individuals permits selection of
effective agents (e.g., drugs) for prophylactic or therapeutic
treatments, based on the individual's genotype. Such
pharmacogenomics can be used to determine appropriate dosages and
therapeutic regimens for individuals. Accordingly, the activity of
MLip-1 protein, expression of a nucleic acid encoding MLip-1
protein, or mutation content of the MLip-1 gene in an individual
can be assessed in the presence of a variety of agents in order to
select appropriate agent(s) for therapeutic or prophylactic
treatment of the individual.
[0231] As an illustrative embodiment, the activity of drug
metabolizing enzymes is a major determinant of both the intensity
and duration of drug action. The discovery of genetic polymorphisms
of drug metabolizing enzymes (e.g., N-acetyltransferase 2 {NAT 2}
and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an
explanation of why some patients do not obtain expected drug
effects or exhibit exaggerated drug responses or serious toxicity
following administration of a the standard and safe dose of a drug.
These polymorphisms occur as two phenotypes in the population,
namely the extensive metabolizer (EM) and poor metabolizer (PM)
phenotypes. The prevalence of PM varies among different
populations. For example, the gene encoding CYP2D6 is highly
polymorphic, and several mutations have been identified in PM, all
of which result in absence of functional CYP2D6. Poor metabolizers
of CYP2D6 and CYP2C 19 frequently experience exaggerated drug
responses and side effects when they receive standard doses of
drugs. If a metabolite is the active therapeutic moiety, a PM will
exhibit no therapeutic response, as demonstrated for the analgesic
effect of codeine mediated by CYP2D6-catalyzed generation of its
metabolite, morphine. At the other extreme are the so called
ultra-rapid metabolizers who do not respond to standard doses.
Recently, the molecular basis of ultra-rapid metabolism has been
determined to be CYP2D6 gene amplification.
[0232] Thus, the activity of a polypeptide of the invention,
expression of a nucleic acid encoding the polypeptide, or mutation
content of a gene encoding the polypeptide in an individual can be
determined to thereby select appropriate agent(s) for therapeutic
or prophylactic treatment of the individual. In addition,
pharmacogenomic analysis can be used to predict an individual's
drug responsiveness phenotype. This knowledge, when applied to
dosing or drug selection, can avoid adverse reactions or
therapeutic failure. Therapeutic or prophylactic efficiency can be
thereby improved.
[0233] 4. Monitoring of Effects During Clinical Trials
[0234] Monitoring the influence of agents (e.g., drug compounds) on
expression or activity of MLip-1 protein (e.g., ability to modulate
transmembrane transport of a fatty acyl moiety of a lipid or to
hydrolyze or form ester bonds of a lipid) can be applied not only
in basic drug screening, but also in clinical trials. For example,
the effectiveness of an agent, as determined by a screening assay
as described herein, to increase MLip-1 gene expression, protein
level, or protein activity, can be monitored in clinical trials of
subjects exhibiting decreased gene expression, protein level, or
protein activity. Alternatively, the effectiveness of an agent, as
determined by a screening assay, to decrease gene expression,
protein level, or protein activity, can be monitored in clinical
trials of subjects exhibiting increased gene expression, protein
level, or protein activity.
[0235] For example, ability of an agent (e.g., compound, drug or
small molecule) to modulate activity or expression of MLip-1
protein (e.g., as identified in a screening assay described herein)
can be identified. Thus, in order to study the effect of agents on
disorders relating to aberrant lipid metabolism, for example, in a
clinical trial, cells can be isolated and RNA prepared and analyzed
to determine the levels of expression of the MLip-1 gene or of
another gene implicated in the disorder. The levels of gene
expression (i.e., a gene expression pattern) can be quantified by
Northern blot analysis or RT-PCR, as described herein, or
alternatively by assessing the amount of MLip-1 protein produced,
by one of the methods as described herein. The gene expression
pattern can serve as a marker, indicative of the physiological
response of the cells to the agent. Accordingly, this response
marker can be assessed before, and at various points during,
treatment of the individual with the agent in order to examine the
effectiveness of the trial and, if desired, the necessity of
altering the trial.
[0236] In one embodiment, the present invention includes a method
for monitoring the effectiveness of treatment of a subject with an
agent (e.g., an agonist, antagonist, peptidomimetic, protein,
peptide, antibody substance, nucleic acid, small molecule, or other
drug candidate identified by the screening assays described herein)
comprising (i) obtaining a pre-administration sample from a subject
prior to administration of the agent; (ii) detecting the level of a
polypeptide or nucleic acid of the invention in the
pre-administration sample; (iii) obtaining one or more
post-administration samples from the subject; (iv) detecting the
level the of the polypeptide or nucleic acid of the invention in
the post-administration samples; (v) comparing the levels of the
polypeptide or nucleic acid in the pre- and post-administration
samples; and (vi) altering administration of the agent to the
subject accordingly. For example, increased administration of the
agent can be desirable to increase or decrease MLip-1 expression or
activity (i.e., to increase the effectiveness of the agent).
[0237] C. Methods of Treatment
[0238] The present invention provides both prophylactic and
therapeutic methods of treating a subject at risk for developing,
susceptible to, or afflicted with a disorder associated with
aberrant expression or activity of MLip-1 protein. Such disorders
are described elsewhere in this disclosure.
[0239] 1. Prophylactic Methods
[0240] In one aspect, the invention provides a method for
preventing or inhibiting a disease or disorder associated with
aberrant expression or activity of MLip-1 protein in a subject. The
method comprises administering to the subject an agent that
modulates expression or at least one activity of MLip-1 protein.
Subjects at risk for a disease which is caused or contributed to by
aberrant expression or activity of a polypeptide can be identified
by, for example, any or a combination of diagnostic or prognostic
assays as described herein. Administration of a prophylactic agent
can occur prior to the manifestation of symptoms characteristic of
the aberrance, such that a disease or disorder is prevented,
inhibited in its progression, or inhibited in the severity of the
disease or disorder. Depending on the type of aberrance, for
example, an agonist or antagonist agent can be used for treating
the subject. Selection of an appropriate agent can be made based on
screening assays described herein.
[0241] 2. Therapeutic Methods
[0242] In another aspect, the invention pertains to methods of
modulating expression or activity of MLip-1 protein for therapeutic
purposes. The modulatory method of the invention involves
contacting a cell with an agent that modulates expression of MLip-1
protein or modulates one or more of the activities of MLip-1
protein. An agent that modulates expression or activity can be an
agent as described herein, such as a nucleic acid or polypeptide of
the invention, a peptidomimetic, an antibody substance, or a small
molecule. In one embodiment, the agent stimulates one or more of
the biological activities of MLip-1 protein. In another embodiment,
the agent inhibits one or more biological activities of MLip-1
protein. Examples of such inhibitory agents include antisense
nucleic acid molecules and antibodies. These modulatory methods can
be performed in vitro (e.g., by culturing a cell with the agent)
or, alternatively, in vivo (e.g., by administering the agent to a
subject). As such, the present invention provides methods of
treating an individual afflicted with a disease or disorder
characterized by aberrant expression or activity of MLip-1 protein.
In one embodiment, the method involves administering an agent
(e.g., an agent identified by a screening assay described herein),
or combination of agents that modulates (e.g., up-regulates or
down-regulates) MLip-1 expression or activity. In another
embodiment, the method involves administering a polypeptide of the
invention, or a nucleic acid of the invention, as therapy to
compensate for reduced or aberrant expression or activity of the
polypeptide.
[0243] Stimulation of MLip-1 activity is desirable in situations in
which activity or expression is abnormally low or down-regulated
and/or in which increased activity is likely to have a beneficial
effect, e.g., in pancreatic insufficiency disorders. Conversely,
inhibition of MLip-1 activity is desirable in situations in which
activity or expression is abnormally high or up-regulated and/or in
which decreased activity is likely to have a beneficial effect.
[0244] The contents of all references, patents, and published
patent applications cited in this disclosure are incorporated by
reference.
[0245] Equivalents
[0246] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are encompassed by the following claims.
Sequence CWU 1
1
10 1 2352 DNA Homo sapiens unsure (2159) unsure (2307) unsure
(2313) 1 ggaattcccg ggtcgaccca cgcgtccgca ttgtgaggaa aaccacttag
tattttatag 60 tgaggtgact ttacaagtaa agatcttcaa gaagattttt
atgtgattta aaaaatcagc 120 ttagatgctt ggaatttgga ttgttgcatt
cttgttcttt ggcacatcaa gaggaaaaga 180 agtttgctat gaaaggttag
ggtgtttcaa agatggttta ccatggacca ggactttctc 240 aacagagttg
gtaggtttac cctggtctcc agagaagata aacactcgtt tcctgctcta 300
cactatacac aatcccaatg cctatcagga gatcagtgcg gttaattctt caactatcca
360 agcctcatat tttggaacag acaagatcac ccgtatcaac atagctggat
ggaaaacaga 420 tggcaaatgg cagagagaca tgtgcaatgt gttgctacag
ctggaagata taaattgcat 480 taatttagat tggatcaacg gttcacggga
atacatccat gctgtaaaca atctccgtgt 540 tgttggtgct gaggtggctt
attttattga tgttctcatg aaaaaatttg aatattcccc 600 ttctaaagtg
cacttgattg gccacagctt gggagcacac ctggctgggg aagctgggtc 660
aaggatacca ggccttggaa gaataactgg gttggaccca gctgggccat ttttccacaa
720 cactccaaag gaagtcaggc tagacccctc ggatgccaac tttgttgacg
ttattcatac 780 aaatgcagct cgcatcctct ttgagcttgg tgttggaacc
attgatgctt gtggtcatct 840 tgacttttac ccaaatggag ggaagcacat
gccaggatgt gaagacttaa ttacaccttt 900 actgaaattt aacttcaatg
cttacaaaaa agaaatggct tccttctttg actgtaacca 960 tgcccgaagt
tatcaatttt atgctgaaag cattcttaat cctgatgcat ttattgctta 1020
tccttgtaga tcctacacat cttttaaagc aggaaattgc ttcttttgtt ccaaagaagg
1080 ttgcccaaca atgggtcatt ttgctgatag atttcacttc aaaaatatga
agactaatgg 1140 atcacattat tttttaaaca cagggtccct ttccccattt
gcccgttgga ggcacaaatt 1200 gtctgttaaa ctcagtggaa gcgaagtcac
tcaaggaact gtctttcttc gtgtaggcgg 1260 ggcaattggg aaaactgggg
agtttgccat tgtcagtgga aaacttgagc caggcatgac 1320 ttacacaaaa
ttaatcgatg cagaggttaa cgttggaaac attacaagtg ttcagttcat 1380
ctggaaaaaa catttgtttg aagattctca gaataagttg ggagcagaaa tggtgataaa
1440 tacatctggg aaatatggat ataaatctac cttctgtagc caagacatta
tgggacctaa 1500 tattctccag aacctgaaac catgctaatc tcagatacag
tcttgatgga tttctttagt 1560 aggagcaatg aagaaaagtg tctccttcca
cctggcatcc agaccaaatt tgacccttgt 1620 aaatgactta gtcatttaca
agggtcttac tcagagtcaa gtacgggttt gctttttttc 1680 tgtgtagaat
gttcatctaa ctgcacctta aaaacacact gaaccctggg acaaaagata 1740
attactatga tctgtaggaa tctggatatc attgacaaaa tagagctgtt ttggaatttt
1800 cctgaataag aggaggtgat gcaaatgtat gttgagtgta taaactcact
ggacaaaagt 1860 aagcctctgg cttgctgagt ttttgaagta tattttcagg
tataataatc attgttctaa 1920 aattatataa aactatttgt tatgttgtta
aatcttgctg agacaaatta tgactatagt 1980 gcatgatata tagtagatta
taaccttgtg ggttgatgtg tctatctagt aataataaaa 2040 actaatgaga
tggcactagt atttccaagg tgttccttgg tgttcagggt gtgcccaaga 2100
gagattttgg agcttatctg ttatgtgttc atcagttagc aatgggacct gaagttcanc
2160 aacccagggt atagccccct tcctccaaag tccctgccac aggagaatta
ctcctctctc 2220 tgggtcttga atgctctatg gtgaatttgt atttagcctc
aaggcagcat ttcatttgta 2280 aagcacttgg gtaacccttt gttcttncaa
tancaatatt ataatattta aatatgaaaa 2340 aaaaaaaaaa aa 2352 2 1401 DNA
Homo sapiens 2 atgcttggaa tttggattgt tgcattcttg ttctttggca
catcaagagg aaaagaagtt 60 tgctatgaaa ggttagggtg tttcaaagat
ggtttaccat ggaccaggac tttctcaaca 120 gagttggtag gtttaccctg
gtctccagag aagataaaca ctcgtttcct gctctacact 180 atacacaatc
ccaatgccta tcaggagatc agtgcggtta attcttcaac tatccaagcc 240
tcatattttg gaacagacaa gatcacccgt atcaacatag ctggatggaa aacagatggc
300 aaatggcaga gagacatgtg caatgtgttg ctacagctgg aagatataaa
ttgcattaat 360 ttagattgga tcaacggttc acgggaatac atccatgctg
taaacaatct ccgtgttgtt 420 ggtgctgagg tggcttattt tattgatgtt
ctcatgaaaa aatttgaata ttccccttct 480 aaagtgcact tgattggcca
cagcttggga gcacacctgg ctggggaagc tgggtcaagg 540 ataccaggcc
ttggaagaat aactgggttg gacccagctg ggccattttt ccacaacact 600
ccaaaggaag tcaggctaga cccctcggat gccaactttg ttgacgttat tcatacaaat
660 gcagctcgca tcctctttga gcttggtgtt ggaaccattg atgcttgtgg
tcatcttgac 720 ttttacccaa atggagggaa gcacatgcca ggatgtgaag
acttaattac acctttactg 780 aaatttaact tcaatgctta caaaaaagaa
atggcttcct tctttgactg taaccatgcc 840 cgaagttatc aattttatgc
tgaaagcatt cttaatcctg atgcatttat tgcttatcct 900 tgtagatcct
acacatcttt taaagcagga aattgcttct tttgttccaa agaaggttgc 960
ccaacaatgg gtcattttgc tgatagattt cacttcaaaa atatgaagac taatggatca
1020 cattattttt taaacacagg gtccctttcc ccatttgccc gttggaggca
caaattgtct 1080 gttaaactca gtggaagcga agtcactcaa ggaactgtct
ttcttcgtgt aggcggggca 1140 attgggaaaa ctggggagtt tgccattgtc
agtggaaaac ttgagccagg catgacttac 1200 acaaaattaa tcgatgcaga
ggttaacgtt ggaaacatta caagtgttca gttcatctgg 1260 aaaaaacatt
tgtttgaaga ttctcagaat aagttgggag cagaaatggt gataaataca 1320
tctgggaaat atggatataa atctaccttc tgtagccaag acattatggg acctaatatt
1380 ctccagaacc tgaaaccatg c 1401 3 467 PRT Homo sapiens 3 Met Leu
Gly Ile Trp Ile Val Ala Phe Leu Phe Phe Gly Thr Ser Arg 1 5 10 15
Gly Lys Glu Val Cys Tyr Glu Arg Leu Gly Cys Phe Lys Asp Gly Leu 20
25 30 Pro Trp Thr Arg Thr Phe Ser Thr Glu Leu Val Gly Leu Pro Trp
Ser 35 40 45 Pro Glu Lys Ile Asn Thr Arg Phe Leu Leu Tyr Thr Ile
His Asn Pro 50 55 60 Asn Ala Tyr Gln Glu Ile Ser Ala Val Asn Ser
Ser Thr Ile Gln Ala 65 70 75 80 Ser Tyr Phe Gly Thr Asp Lys Ile Thr
Arg Ile Asn Ile Ala Gly Trp 85 90 95 Lys Thr Asp Gly Lys Trp Gln
Arg Asp Met Cys Asn Val Leu Leu Gln 100 105 110 Leu Glu Asp Ile Asn
Cys Ile Asn Leu Asp Trp Ile Asn Gly Ser Arg 115 120 125 Glu Tyr Ile
His Ala Val Asn Asn Leu Arg Val Val Gly Ala Glu Val 130 135 140 Ala
Tyr Phe Ile Asp Val Leu Met Lys Lys Phe Glu Tyr Ser Pro Ser 145 150
155 160 Lys Val His Leu Ile Gly His Ser Leu Gly Ala His Leu Ala Gly
Glu 165 170 175 Ala Gly Ser Arg Ile Pro Gly Leu Gly Arg Ile Thr Gly
Leu Asp Pro 180 185 190 Ala Gly Pro Phe Phe His Asn Thr Pro Lys Glu
Val Arg Leu Asp Pro 195 200 205 Ser Asp Ala Asn Phe Val Asp Val Ile
His Thr Asn Ala Ala Arg Ile 210 215 220 Leu Phe Glu Leu Gly Val Gly
Thr Ile Asp Ala Cys Gly His Leu Asp 225 230 235 240 Phe Tyr Pro Asn
Gly Gly Lys His Met Pro Gly Cys Glu Asp Leu Ile 245 250 255 Thr Pro
Leu Leu Lys Phe Asn Phe Asn Ala Tyr Lys Lys Glu Met Ala 260 265 270
Ser Phe Phe Asp Cys Asn His Ala Arg Ser Tyr Gln Phe Tyr Ala Glu 275
280 285 Ser Ile Leu Asn Pro Asp Ala Phe Ile Ala Tyr Pro Cys Arg Ser
Tyr 290 295 300 Thr Ser Phe Lys Ala Gly Asn Cys Phe Phe Cys Ser Lys
Glu Gly Cys 305 310 315 320 Pro Thr Met Gly His Phe Ala Asp Arg Phe
His Phe Lys Asn Met Lys 325 330 335 Thr Asn Gly Ser His Tyr Phe Leu
Asn Thr Gly Ser Leu Ser Pro Phe 340 345 350 Ala Arg Trp Arg His Lys
Leu Ser Val Lys Leu Ser Gly Ser Glu Val 355 360 365 Thr Gln Gly Thr
Val Phe Leu Arg Val Gly Gly Ala Ile Gly Lys Thr 370 375 380 Gly Glu
Phe Ala Ile Val Ser Gly Lys Leu Glu Pro Gly Met Thr Tyr 385 390 395
400 Thr Lys Leu Ile Asp Ala Glu Val Asn Val Gly Asn Ile Thr Ser Val
405 410 415 Gln Phe Ile Trp Lys Lys His Leu Phe Glu Asp Ser Gln Asn
Lys Leu 420 425 430 Gly Ala Glu Met Val Ile Asn Thr Ser Gly Lys Tyr
Gly Tyr Lys Ser 435 440 445 Thr Phe Cys Ser Gln Asp Ile Met Gly Pro
Asn Ile Leu Gln Asn Leu 450 455 460 Lys Pro Cys 465 4 467 PRT Homo
sapiens 4 Met Leu Ile Phe Trp Thr Ile Thr Leu Phe Leu Leu Gly Ala
Ala Lys 1 5 10 15 Gly Lys Glu Val Cys Tyr Glu Asp Leu Gly Cys Phe
Ser Asp Thr Glu 20 25 30 Pro Trp Gly Gly Thr Ala Ile Arg Pro Leu
Lys Ile Leu Pro Trp Ser 35 40 45 Pro Glu Lys Ile Gly Thr Arg Phe
Leu Leu Tyr Thr Asn Glu Asn Pro 50 55 60 Asn Asn Phe Gln Ile Leu
Leu Leu Ser Asp Pro Ser Thr Ile Glu Ala 65 70 75 80 Ser Asn Phe Gln
Met Asp Arg Lys Thr Arg Phe Ile Ile His Gly Phe 85 90 95 Ile Asp
Lys Gly Asp Glu Ser Trp Val Thr Asp Met Cys Lys Lys Leu 100 105 110
Phe Glu Val Glu Glu Val Asn Cys Ile Cys Val Asp Trp Lys Lys Gly 115
120 125 Ser Gln Ala Thr Tyr Thr Gln Ala Ala Asn Asn Val Arg Val Val
Gly 130 135 140 Ala Gln Val Ala Gln Met Leu Asp Ile Leu Leu Thr Glu
Tyr Ser Tyr 145 150 155 160 Pro Pro Ser Lys Val His Leu Ile Gly His
Ser Leu Gly Ala His Val 165 170 175 Ala Gly Glu Ala Gly Ser Lys Thr
Pro Gly Leu Ser Arg Ile Thr Gly 180 185 190 Leu Asp Pro Val Glu Ala
Ser Phe Glu Ser Thr Pro Glu Glu Val Arg 195 200 205 Leu Asp Pro Ser
Asp Ala Asp Phe Val Asp Val Ile His Thr Asp Ala 210 215 220 Ala Pro
Leu Ile Pro Phe Leu Gly Phe Gly Thr Asn Gln Gln Met Gly 225 230 235
240 His Leu Asp Phe Phe Pro Asn Gly Gly Glu Ser Met Pro Gly Cys Lys
245 250 255 Lys Asn Ala Leu Ser Gln Ile Val Asp Leu Asp Gly Ile Trp
Ala Gly 260 265 270 Thr Arg Asp Phe Val Ala Cys Asn His Leu Arg Ser
Tyr Lys Tyr Tyr 275 280 285 Leu Glu Ser Ile Leu Asn Pro Asp Gly Phe
Ala Ala Tyr Pro Cys Thr 290 295 300 Ser Tyr Lys Ser Phe Glu Ser Asp
Lys Cys Phe Pro Cys Pro Asp Gln 305 310 315 320 Gly Cys Pro Gln Met
Gly His Tyr Ala Asp Lys Phe Ala Gly Arg Thr 325 330 335 Ser Glu Glu
Gln Gln Lys Phe Phe Leu Asn Thr Gly Glu Ala Ser Asn 340 345 350 Phe
Ala Arg Trp Arg Tyr Gly Val Ser Ile Thr Leu Ser Gly Arg Thr 355 360
365 Ala Thr Gly Gln Ile Lys Val Ala Leu Phe Gly Asn Lys Gly Asn Thr
370 375 380 His Gln Tyr Ser Ile Phe Arg Gly Ile Leu Lys Pro Gly Ser
Thr His 385 390 395 400 Ser Tyr Glu Phe Asp Ala Lys Leu Asp Val Gly
Thr Ile Glu Lys Val 405 410 415 Lys Phe Leu Trp Asn Asn Asn Val Ile
Asn Pro Thr Leu Pro Lys Val 420 425 430 Gly Ala Thr Lys Ile Thr Val
Gln Lys Gly Glu Glu Lys Thr Val Tyr 435 440 445 Asn Phe Cys Ser Glu
Asp Thr Val Arg Glu Asp Thr Leu Leu Thr Leu 450 455 460 Thr Pro Cys
465 5 469 PRT Homo sapiens 5 Met Leu Pro Pro Trp Thr Leu Gly Leu
Leu Leu Leu Ala Thr Val Arg 1 5 10 15 Gly Lys Glu Val Cys Tyr Gly
Gln Leu Gly Cys Phe Ser Asp Glu Lys 20 25 30 Pro Trp Ala Gly Thr
Leu Gln Arg Pro Val Lys Leu Leu Pro Trp Ser 35 40 45 Pro Glu Asp
Ile Asp Thr Arg Phe Leu Leu Tyr Thr Asn Glu Asn Pro 50 55 60 Asn
Asn Phe Gln Leu Ile Thr Gly Thr Glu Pro Asp Thr Ile Glu Ala 65 70
75 80 Ser Asn Phe Gln Leu Asp Arg Lys Thr Arg Phe Ile Ile His Gly
Phe 85 90 95 Leu Asp Lys Ala Glu Asp Ser Trp Pro Ser Asp Met Cys
Lys Lys Met 100 105 110 Phe Glu Val Glu Lys Val Asn Cys Ile Cys Val
Asp Trp Arg His Gly 115 120 125 Ser Arg Ala Met Tyr Thr Gln Ala Val
Gln Asn Ile Arg Val Val Gly 130 135 140 Ala Glu Thr Ala Phe Leu Ile
Gln Ala Leu Ser Thr Gln Leu Gly Tyr 145 150 155 160 Ser Leu Glu Asp
Val His Val Ile Gly His Ser Leu Gly Ala His Thr 165 170 175 Ala Ala
Glu Ala Gly Arg Arg Leu Gly Gly Arg Val Gly Arg Ile Thr 180 185 190
Gly Leu Asp Pro Ala Gly Pro Cys Phe Gln Asp Glu Pro Glu Glu Val 195
200 205 Arg Leu Asp Pro Ser Asp Ala Val Phe Val Asp Val Ile His Thr
Asp 210 215 220 Ser Ser Pro Ile Val Pro Ser Leu Gly Phe Gly Met Ser
Gln Lys Val 225 230 235 240 Gly His Leu Asp Phe Phe Pro Asn Gly Gly
Lys Glu Met Pro Gly Cys 245 250 255 Lys Lys Asn Val Leu Ser Thr Ile
Thr Asp Ile Asp Gly Ile Trp Glu 260 265 270 Gly Ile Gly Gly Phe Val
Ser Cys Asn His Leu Arg Ser Phe Glu Tyr 275 280 285 Tyr Ser Ser Ser
Val Leu Asn Pro Asp Gly Phe Leu Gly Tyr Pro Cys 290 295 300 Ala Ser
Tyr Asp Glu Phe Gln Glu Ser Lys Cys Phe Pro Cys Pro Ala 305 310 315
320 Glu Gly Cys Pro Lys Met Gly His Tyr Ala Asp Gln Phe Lys Gly Lys
325 330 335 Thr Ser Ala Val Glu Gln Thr Phe Phe Leu Asn Thr Gly Glu
Ser Gly 340 345 350 Asn Phe Thr Ser Trp Arg Tyr Lys Val Ser Val Thr
Leu Ser Gly Lys 355 360 365 Glu Lys Val Asn Gly Tyr Ile Arg Ile Ala
Leu Tyr Gly Ser Asn Glu 370 375 380 Asn Ser Lys Gln Tyr Glu Ile Phe
Lys Gly Ser Leu Lys Pro Asp Ala 385 390 395 400 Ser His Thr Cys Ala
Ile Asp Val Asp Phe Asn Val Gly Lys Ile Gln 405 410 415 Lys Val Lys
Phe Leu Trp Asn Lys Arg Gly Ile Asn Leu Ser Glu Pro 420 425 430 Lys
Leu Gly Ala Ser Gln Ile Thr Val Gln Ser Gly Glu Asp Gly Thr 435 440
445 Glu Tyr Asn Phe Cys Ser Ser Asp Thr Val Glu Glu Asn Val Leu Gln
450 455 460 Ser Leu Tyr Pro Cys 465 6 465 PRT Homo sapiens 6 Met
Leu Pro Leu Trp Thr Leu Ser Leu Leu Leu Gly Ala Val Ala Gly 1 5 10
15 Lys Glu Val Cys Tyr Glu Arg Leu Gly Cys Phe Ser Asp Asp Ser Pro
20 25 30 Trp Ser Gly Ile Thr Glu Arg Pro Leu His Ile Leu Pro Trp
Ser Pro 35 40 45 Lys Asp Val Asn Thr Arg Phe Leu Leu Tyr Thr Asn
Glu Asn Pro Asn 50 55 60 Asn Phe Gln Glu Val Ala Ala Asp Ser Ser
Ser Ile Ser Gly Ser Asn 65 70 75 80 Phe Lys Thr Asn Arg Lys Thr Arg
Phe Ile Ile His Gly Phe Ile Asp 85 90 95 Lys Gly Glu Glu Asn Trp
Leu Ala Asn Val Cys Lys Asn Leu Phe Lys 100 105 110 Val Glu Ser Val
Asn Cys Ile Cys Val Asp Trp Lys Gly Gly Ser Arg 115 120 125 Thr Gly
Tyr Thr Gln Ala Ser Gln Asn Ile Arg Ile Val Gly Ala Glu 130 135 140
Val Ala Tyr Phe Val Glu Phe Leu Gln Ser Ala Phe Gly Tyr Ser Pro 145
150 155 160 Ser Asn Val His Val Ile Gly His Ser Leu Gly Ala His Ala
Ala Gly 165 170 175 Glu Ala Gly Arg Arg Thr Asn Gly Thr Ile Gly Arg
Ile Thr Gly Leu 180 185 190 Asp Pro Ala Glu Pro Cys Phe Gln Gly Thr
Pro Glu Leu Val Arg Leu 195 200 205 Asp Pro Ser Asp Ala Lys Phe Val
Asp Val Ile His Thr Asp Gly Ala 210 215 220 Pro Ile Val Pro Asn Leu
Gly Phe Gly Met Ser Gln Val Val Gly His 225 230 235 240 Leu Asp Phe
Phe Pro Asn Gly Gly Val Glu Met Pro Gly Cys Lys Lys 245 250 255 Asn
Ile Leu Ser Gln Ile Val Asp Ile Asp Gly Ile Trp Glu Gly Thr 260 265
270 Arg Asp Phe Ala Ala Cys Asn His Leu Arg Ser Tyr Lys Tyr Tyr Thr
275 280 285 Asp Ser Ile Val Asn Pro Asp Gly Phe Ala Gly Phe Pro Cys
Ala Ser 290 295 300 Tyr Asn Val Phe Thr Ala Asn Lys Cys Phe Pro Cys
Pro Ser Gly Gly 305 310 315 320 Cys Pro Gln Met Gly His Tyr Ala Asp
Arg Tyr Pro Gly Lys Thr Asn 325 330 335 Asp Val Gly Gln Lys Phe Tyr
Leu Asp Thr Gly Asp Ala Ser Asn Phe 340 345 350 Ala Arg Trp Arg Tyr
Lys Val Ser Val Thr Leu Ser Gly Lys Lys Val 355 360 365 Thr Gly His
Ile Leu Val Ser Leu Phe Gly Asn Lys Gly Asn Ser Lys 370 375 380 Gln
Tyr Glu Ile Phe Lys Gly Thr Leu Lys Pro Asp Ser Thr His Ser 385 390
395 400 Asn Glu Phe Asp Ser Asp
Val Asp Val Gly Asp Leu Gln Met Val Lys 405 410 415 Phe Ile Trp Tyr
Asn Asn Val Ile Asn Pro Thr Leu Pro Arg Val Gly 420 425 430 Ala Ser
Lys Ile Ile Val Glu Thr Asn Val Gly Lys Gln Phe Asn Phe 435 440 445
Cys Ser Pro Glu Thr Val Arg Glu Glu Val Leu Leu Thr Leu Thr Pro 450
455 460 Cys 465 7 473 PRT Mus musculus 7 Met Leu Ile Leu Trp Thr
Ile Pro Leu Phe Leu Leu Gly Ala Ala Gln 1 5 10 15 Gly Lys Glu Val
Cys Tyr Asp Asn Leu Gly Cys Phe Ser Asp Ala Glu 20 25 30 Pro Trp
Ala Gly Thr Ala Ile Arg Pro Leu Lys Leu Leu Pro Trp Ser 35 40 45
Pro Glu Lys Ile Asn Thr Arg Phe Leu Leu Tyr Thr Asn Glu Asn Pro 50
55 60 Thr Ala Phe Gln Thr Leu Gln Leu Ser Asp Pro Ser Thr Ile Glu
Ala 65 70 75 80 Ser Asn Phe Gln Val Ala Arg Lys Thr Arg Phe Ile Ile
His Gly Phe 85 90 95 Ile Asp Lys Gly Glu Glu Asn Trp Val Val Asp
Met Cys Lys Asn Met 100 105 110 Phe Gln Val Glu Glu Val Asn Cys Ile
Cys Val Asp Trp Lys Arg Gly 115 120 125 Ser Gln Thr Thr Tyr Thr Gln
Ala Ala Asn Asn Val Arg Val Val Gly 130 135 140 Ala Gln Val Ala Gln
Met Ile Asp Ile Leu Val Arg Asn Phe Asn Tyr 145 150 155 160 Ser Ala
Ser Lys Val His Leu Ile Gly His Ser Leu Gly Ala His Val 165 170 175
Ala Gly Glu Ala Gly Ser Arg Thr Pro Gly Leu Gly Arg Ile Thr Gly 180
185 190 Leu Asp Pro Val Glu Ala Asn Phe Glu Gly Thr Pro Glu Glu Val
Arg 195 200 205 Leu Asp Pro Ser Asp Ala Asp Phe Val Asp Val Ile His
Thr Asp Ala 210 215 220 Ala Pro Leu Ile Pro Phe Leu Gly Phe Gly Thr
Asn Gln Met Val Gly 225 230 235 240 His Phe Asp Phe Phe Pro Asn Gly
Gly Gln Tyr Met Pro Gly Cys Lys 245 250 255 Lys Asn Ala Leu Ser Gln
Ile Val Asp Ile Asp Gly Ile Trp Ser Gly 260 265 270 Thr Arg Asp Phe
Val Ala Cys Asn His Leu Arg Ser Tyr Lys Tyr Tyr 275 280 285 Leu Glu
Ser Ile Leu Asn Pro Asp Gly Phe Ala Ala Tyr Pro Cys Ala 290 295 300
Ser Tyr Arg Asp Phe Glu Ser Asn Lys Cys Phe Pro Cys Pro Asp Gln 305
310 315 320 Gly Cys Pro Gln Met Gly His Tyr Ala Asp Lys Phe Ala Asn
Asn Thr 325 330 335 Ser Val Glu Pro Gln Lys Phe Phe Leu Asn Thr Gly
Glu Ala Lys Asn 340 345 350 Phe Ala Arg Trp Arg Tyr Arg Val Ser Leu
Thr Phe Ser Gly Arg Thr 355 360 365 Val Thr Gly Gln Val Lys Val Ser
Leu Phe Gly Ser Asn Gly Asn Thr 370 375 380 Arg Gln Cys Asp Ile Phe
Arg Gly Ile Ile Lys Pro Gly Ala Thr His 385 390 395 400 Ser Asn Glu
Phe Asp Ala Lys Leu Asp Val Gly Thr Ile Glu Lys Val 405 410 415 Lys
Phe Leu Trp Asn Asn His Val Val Asn Pro Ser Phe Pro Lys Val 420 425
430 Gly Ala Ala Lys Ile Thr Val Gln Lys Gly Glu Glu Arg Thr Glu His
435 440 445 Asn Phe Cys Ser Glu Glu Thr Val Arg Glu Asp Ile Leu Leu
Thr Leu 450 455 460 Leu Pro Cys Lys Thr Ser Asp Thr Met 465 470 8
473 PRT Rattus norvegicus 8 Met Leu Thr Leu Trp Thr Val Ser Leu Phe
Leu Leu Gly Ala Ala Gln 1 5 10 15 Gly Lys Glu Val Cys Tyr Asp Asn
Leu Gly Cys Phe Ser Asp Ala Glu 20 25 30 Pro Trp Ala Gly Thr Ala
Ile Arg Pro Leu Lys Leu Leu Pro Trp Ser 35 40 45 Pro Glu Lys Ile
Asn Thr Arg Phe Leu Leu Tyr Thr Asn Glu Asn Pro 50 55 60 Thr Ala
Phe Gln Thr Leu Gln Leu Ser Asp Pro Leu Thr Ile Gly Ala 65 70 75 80
Ser Asn Phe Gln Val Ala Arg Lys Thr Arg Phe Ile Ile His Gly Phe 85
90 95 Ile Asp Lys Gly Glu Glu Asn Trp Val Val Asp Met Cys Lys Asn
Met 100 105 110 Phe Gln Val Glu Glu Val Asn Cys Ile Cys Val Asp Trp
Lys Lys Gly 115 120 125 Ser Gln Thr Thr Tyr Thr Gln Ala Ala Asn Asn
Val Arg Val Val Gly 130 135 140 Ala Gln Val Ala Gln Met Ile Asp Ile
Leu Val Lys Asn Tyr Ser Tyr 145 150 155 160 Ser Pro Ser Lys Val His
Leu Ile Gly His Ser Leu Gly Ala His Val 165 170 175 Ala Gly Glu Ala
Gly Ser Arg Thr Pro Gly Leu Gly Arg Ile Thr Gly 180 185 190 Leu Asp
Pro Val Glu Ala Asn Phe Glu Gly Thr Pro Glu Glu Val Arg 195 200 205
Leu Asp Pro Ser Asp Ala Asp Phe Val Asp Val Ile His Thr Asp Ala 210
215 220 Ala Pro Leu Ile Pro Phe Leu Gly Phe Gly Thr Asn Gln Met Ser
Gly 225 230 235 240 His Leu Asp Phe Phe Pro Asn Gly Gly Gln Ser Met
Pro Gly Cys Lys 245 250 255 Lys Asn Ala Leu Ser Gln Ile Val Asp Ile
Asp Gly Ile Trp Ser Gly 260 265 270 Thr Arg Asp Phe Val Ala Cys Asn
His Leu Arg Ser Tyr Lys Tyr Tyr 275 280 285 Leu Glu Ser Ile Leu Asn
Pro Asp Gly Phe Ala Ala Tyr Pro Cys Ala 290 295 300 Ser Tyr Lys Asp
Phe Glu Ser Asn Lys Cys Phe Pro Cys Pro Asp Gln 305 310 315 320 Gly
Cys Pro Gln Met Gly His Tyr Ala Asp Lys Phe Ala Gly Lys Ser 325 330
335 Gly Asp Glu Pro Gln Lys Phe Phe Leu Asn Thr Gly Glu Ala Lys Asn
340 345 350 Phe Ala Arg Trp Arg Tyr Arg Val Ser Leu Ile Leu Ser Gly
Arg Met 355 360 365 Val Thr Gly Gln Val Lys Val Ala Leu Phe Gly Ser
Lys Gly Asn Thr 370 375 380 Arg Gln Tyr Asp Ile Phe Arg Gly Ile Ile
Lys Pro Gly Ala Thr His 385 390 395 400 Ser Ser Glu Phe Asp Ala Lys
Leu Asp Val Gly Thr Ile Glu Lys Val 405 410 415 Lys Phe Leu Trp Asn
Asn Gln Val Ile Asn Pro Ser Phe Pro Lys Val 420 425 430 Gly Ala Ala
Lys Ile Thr Val Gln Lys Gly Glu Glu Arg Thr Glu Tyr 435 440 445 Asn
Phe Cys Ser Glu Glu Thr Val Arg Glu Asp Thr Leu Leu Thr Leu 450 455
460 Leu Pro Cys Glu Thr Ser Asp Thr Val 465 470 9 467 PRT Canis
familiaris 9 Met Val Ser Ile Trp Thr Ile Ala Leu Phe Leu Leu Gly
Ala Ala Lys 1 5 10 15 Ala Lys Glu Val Cys Tyr Glu Gln Ile Gly Cys
Phe Ser Asp Ala Glu 20 25 30 Pro Trp Ala Gly Thr Ala Ile Arg Pro
Leu Lys Val Leu Pro Trp Ser 35 40 45 Pro Glu Arg Ile Gly Thr Arg
Phe Leu Leu Tyr Thr Asn Lys Asn Pro 50 55 60 Asn Asn Phe Gln Thr
Leu Leu Pro Ser Asp Pro Ser Thr Ile Glu Ala 65 70 75 80 Ser Asn Phe
Gln Thr Asp Lys Lys Thr Arg Phe Thr Ile His Gly Phe 85 90 95 Ile
Asn Lys Gly Glu Glu Asn Trp Leu Leu Asp Met Cys Lys Asn Met 100 105
110 Phe Lys Val Glu Glu Val Asn Cys Ile Cys Val Asp Trp Lys Lys Gly
115 120 125 Ser Gln Thr Ser Tyr Thr Gln Ala Ala Asn Asn Val Arg Val
Val Gly 130 135 140 Ala Gln Val Ala Gln Met Leu Ser Met Leu Ser Ala
Asn Tyr Ser Tyr 145 150 155 160 Ser Pro Ser Gln Val Gln Leu Ile Gly
His Ser Leu Gly Ala His Val 165 170 175 Ala Gly Glu Ala Gly Ser Arg
Thr Pro Gly Leu Gly Arg Ile Thr Gly 180 185 190 Leu Asp Pro Val Glu
Ala Ser Phe Gln Gly Thr Pro Glu Glu Val Arg 195 200 205 Leu Asp Pro
Thr Asp Ala Asp Phe Val Asp Val Ile His Thr Asp Ala 210 215 220 Ala
Pro Leu Ile Pro Phe Leu Gly Phe Gly Thr Ser Gln Gln Met Gly 225 230
235 240 His Leu Asp Phe Phe Pro Asn Gly Gly Glu Glu Met Pro Gly Cys
Lys 245 250 255 Lys Asn Ala Leu Ser Gln Ile Val Asn Leu Asp Gly Ile
Trp Glu Gly 260 265 270 Thr Arg Asp Phe Val Ala Cys Asn His Leu Arg
Ser Tyr Lys Tyr Tyr 275 280 285 Ser Glu Ser Ile Leu Asn Pro Asp Gly
Phe Ala Ser Tyr Pro Cys Ala 290 295 300 Ser Tyr Arg Ala Phe Glu Ser
Asn Lys Cys Phe Pro Cys Pro Asp Gln 305 310 315 320 Gly Cys Pro Gln
Met Gly His Tyr Ala Asp Lys Phe Ala Val Lys Thr 325 330 335 Ser Asp
Glu Thr Gln Lys Tyr Phe Leu Asn Thr Gly Asp Ser Ser Asn 340 345 350
Phe Ala Arg Trp Arg Tyr Gly Val Ser Ile Thr Leu Ser Gly Lys Arg 355
360 365 Ala Thr Gly Gln Ala Lys Val Ala Leu Phe Gly Ser Lys Gly Asn
Thr 370 375 380 His Gln Phe Asn Ile Phe Lys Gly Ile Leu Lys Pro Gly
Ser Thr His 385 390 395 400 Ser Asn Glu Phe Asp Ala Lys Leu Asp Val
Gly Thr Ile Glu Lys Val 405 410 415 Lys Phe Leu Trp Asn Asn Asn Val
Val Asn Pro Thr Phe Pro Lys Val 420 425 430 Gly Ala Ala Lys Ile Thr
Val Gln Lys Gly Glu Glu Lys Thr Val His 435 440 445 Ser Phe Cys Ser
Glu Ser Thr Val Arg Glu Asp Val Leu Leu Thr Leu 450 455 460 Thr Pro
Cys 465 10 467 PRT Canis familiaris 10 Met Val Ser Ile Trp Thr Ile
Ala Leu Phe Leu Leu Gly Ala Ala Lys 1 5 10 15 Ala Lys Glu Val Cys
Tyr Glu Gln Ile Gly Cys Phe Ser Asp Ala Glu 20 25 30 Pro Trp Ala
Gly Thr Ala Ile Arg Pro Leu Lys Val Leu Pro Trp Ser 35 40 45 Pro
Glu Arg Ile Gly Thr Arg Phe Leu Leu Tyr Thr Asn Lys Asn Pro 50 55
60 Asn Asn Phe Gln Thr Leu Leu Pro Ser Asp Pro Ser Thr Ile Glu Ala
65 70 75 80 Ser Asn Phe Gln Thr Asp Lys Lys Thr Arg Phe Ile Ile His
Gly Phe 85 90 95 Ile Asp Lys Gly Glu Glu Asn Trp Leu Leu Asp Met
Cys Lys Asn Met 100 105 110 Phe Lys Val Glu Glu Val Asn Cys Ile Cys
Val Asp Trp Lys Lys Gly 115 120 125 Ser Gln Thr Ser Tyr Thr Gln Ala
Ala Asn Asn Val Arg Val Val Gly 130 135 140 Ala Gln Val Ala Gln Met
Leu Ser Met Leu Ser Ala Asn Tyr Ser Tyr 145 150 155 160 Ser Pro Ser
Gln Val Gln Leu Ile Gly His Ser Leu Gly Ala His Val 165 170 175 Ala
Gly Glu Ala Gly Ser Arg Thr Pro Gly Leu Gly Arg Ile Thr Gly 180 185
190 Leu Asp Pro Val Glu Ala Ser Phe Gln Gly Thr Pro Glu Glu Val Arg
195 200 205 Leu Asp Pro Thr Asp Ala Asp Phe Val Asp Val Ile His Thr
Asp Ala 210 215 220 Ala Pro Leu Ile Pro Phe Leu Gly Phe Gly Thr Ser
Gln Gln Met Gly 225 230 235 240 His Leu Asp Phe Phe Pro Asn Gly Gly
Glu Glu Met Pro Gly Cys Lys 245 250 255 Lys Asn Ala Leu Ser Gln Ile
Val Asp Leu Asp Gly Ile Trp Glu Gly 260 265 270 Thr Arg Asp Phe Val
Ala Cys Asn His Leu Arg Ser Tyr Lys Tyr Tyr 275 280 285 Ser Glu Ser
Ile Leu Asn Pro Asp Gly Phe Ala Ser Tyr Pro Cys Ala 290 295 300 Ser
Tyr Arg Ala Phe Glu Ser Asn Lys Cys Phe Pro Cys Pro Asp Gln 305 310
315 320 Gly Cys Pro Gln Met Gly His Tyr Ala Asp Lys Phe Ala Val Lys
Thr 325 330 335 Ser Asp Glu Thr Gln Lys Tyr Phe Leu Asn Thr Gly Asp
Ser Ser Asn 340 345 350 Phe Ala Arg Trp Arg Tyr Gly Val Ser Ile Thr
Leu Ser Gly Lys Arg 355 360 365 Ala Thr Gly Gln Ala Lys Val Ala Leu
Phe Gly Ser Lys Gly Asn Thr 370 375 380 His Gln Phe Asn Ile Phe Lys
Gly Ile Leu Lys Pro Gly Ser Thr His 385 390 395 400 Ser Asn Glu Phe
Asp Ala Lys Leu Asp Val Gly Thr Ile Glu Lys Val 405 410 415 Lys Phe
Leu Trp Asn Asn Asn Val Val Asn Pro Thr Phe Pro Lys Val 420 425 430
Gly Ala Ala Lys Ile Thr Val Gln Lys Gly Glu Glu Lys Thr Val His 435
440 445 Ser Phe Cys Ser Glu Ser Thr Val Arg Glu Asp Val Leu Leu Thr
Leu 450 455 460 Thr Pro Cys 465
* * * * *
References