U.S. patent application number 10/016481 was filed with the patent office on 2002-08-22 for prokineticin polypeptides, related compositions and methods.
Invention is credited to Ehlert, Frederick J., Zhou, Qun-Yong.
Application Number | 20020115610 10/016481 |
Document ID | / |
Family ID | 22928483 |
Filed Date | 2002-08-22 |
United States Patent
Application |
20020115610 |
Kind Code |
A1 |
Zhou, Qun-Yong ; et
al. |
August 22, 2002 |
Prokineticin polypeptides, related compositions and methods
Abstract
The invention provides isolated polypeptides that stimulate
gastrointestinal smooth muscle contraction, including human
prokineticin 1 and human prokineticin 2 polypeptides, and
functional fragments and modifications thereof. Also provided are
methods of stimulating gastrointestinal smooth muscle contraction
in a mammal, by administering to the mammal an effective amount of
a prokineticin polypeptide. The invention also provides nucleic
acid molecules encoding a prokineticin polypeptide, and antibodies
that selectively bind a prokineticin polypeptide. Further provided
are methods of identifying a prokineticin receptor ligand, agonist
or antagonist.
Inventors: |
Zhou, Qun-Yong; (Irvine,
CA) ; Ehlert, Frederick J.; (Irvine, CA) |
Correspondence
Address: |
CAMPBELL & FLORES LLP
4370 LA JOLLA VILLAGE DRIVE
7TH FLOOR
SAN DIEGO
CA
92122
US
|
Family ID: |
22928483 |
Appl. No.: |
10/016481 |
Filed: |
November 1, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60245882 |
Nov 3, 2000 |
|
|
|
Current U.S.
Class: |
435/194 ;
514/12.1; 530/324 |
Current CPC
Class: |
C07K 14/47 20130101;
A61K 39/00 20130101; A61P 35/00 20180101; A61P 31/00 20180101; A61P
1/00 20180101; A61P 29/00 20180101; A61K 38/00 20130101 |
Class at
Publication: |
514/12 ;
530/324 |
International
Class: |
A61K 038/17; C07K
014/575 |
Claims
What is claimed is:
1. An isolated polypeptide that stimulates gastrointestinal smooth
muscle contraction, comprising an amino acid sequence at least 80%
identical to the sequence of human prokineticin 1 (SEQ ID NO:3),
said sequence comprising the N-terminal 6 amino acids of SEQ ID
NO:3, the 10 conserved cysteine residues of SEQ ID NO:3, and from 0
to 9 of the 9 C-terminal amino acids of SEQ ID NO:3.
2. The isolated polypeptide of claim 1, wherein amino acid residues
that differ from residues in SEQ ID NO:3 are conservative
substitutions thereof.
3. The isolated polypeptide of claim 1, wherein amino acid residues
that differ from residues in SEQ ID NO:3 consist of the
corresponding residues from SEQ ID NO:6.
4. The isolated polypeptide of claim 3, comprising SEQ ID
NO:13.
5. The isolated polypeptide of claim 1, comprising amino acids 1-77
of SEQ ID NO:3.
6. The isolated polypeptide of claim 1, comprising SEQ ID NO:3.
7. The isolated polypeptide of claim 1, comprising a 6XHis tag.
8. The isolated polypeptide of claim 1, which is detectably
labeled.
9. An isolated peptide comprising at least 10 contiguous amino
acids of SEQ ID NO:3, wherein said peptide is immunogenic.
10. A pharmaceutical composition, comprising the isolated
polypeptide of claim 1 and a pharmaceutically acceptable
carrier.
11. A method of stimulating gastrointestinal smooth muscle
contraction in a mammal, comprising administering to said mammal an
effective amount of the polypeptide of claim 1.
12. A nucleic acid molecule encoding the polypeptide of claim
1.
13. An expression vector containing the nucleic acid molecule of
claim 12 operatively linked to a promoter of gene expression.
14. A host cell comprising the expression vector of claim 13.
15. A method of preparing the isolated polypeptide of claim 1,
comprising culturing the host cell of claim 14 so as to express
said polypeptide, substantially purifying said polypeptide, and
refolding said polypeptide.
16. An antibody that selectively binds the polypeptide of claim
1.
17. An isolated polypeptide that stimulates gastrointestinal smooth
muscle contraction, comprising an amino acid sequence at least 80%
identical to the sequence of human prokineticin 2 (SEQ ID NO:6),
said sequence comprising the N-terminal 6 amino acids of SEQ ID
NO:6, the 10 conserved cysteine residues of SEQ ID NO:6, and from 0
to 4 of the 4 C-terminal amino acids of SEQ ID NO:6.
18. The isolated polypeptide of claim 17, wherein amino acid
residues that differ from residues in SEQ ID NO:6 are conservative
substitutions thereof.
19. The isolated polypeptide of claim 17, wherein amino acid
residues that differ from residues in SEQ ID NO:6 consist of the
corresponding residues from SEQ ID NO:3.
20. The isolated polypeptide of claim 19, comprising SEQ ID
NO:14.
21. The isolated polypeptide of claim 17, comprising amino acids
1-77 of SEQ ID NO:6.
22. The isolated polypeptide of claim 17, comprising SEQ ID
NO:6.
23. The isolated polypeptide of claim 17, comprising a 6XHis
tag.
24. The isolated polypeptide of claim 17, which is detectably
labeled.
25. An isolated peptide comprising at least 10 contiguous amino
acids of SEQ ID NO:6, wherein said peptide is immunogenic.
26. A pharmaceutical composition, comprising the isolated
polypeptide of claim 17 and a pharmaceutically acceptable
carrier.
27. A method of stimulating gastrointestinal smooth muscle
contraction in a mammal, comprising administering to said mammal an
effective amount of the polypeptide of claim 17.
28. A nucleic acid molecule encoding the polypeptide of claim
17.
29. An expression vector containing the nucleic acid molecule of
claim 17 operatively linked to a promoter of gene expression.
30. A host cell comprising the expression vector of claim 29.
31. A method of preparing the isolated polypeptide of claim 17,
comprising culturing the host cell of claim 30 so as to express
said polypeptide, substantially purifying said polypeptide, and
refolding said polypeptide.
32. An antibody that selectively binds the polypeptide of claim
17.
33. A method of identifying a prokineticin receptor ligand,
comprising contacting a preparation comprising prokineticin
receptor with one or more candidate compounds, and identifying a
compound that specifically binds to said receptor, said compound
being characterized as a prokineticin receptor ligand.
34. The method of claim 33, wherein said preparation is an
intestinal smooth muscle preparation or membrane preparation
thereof.
35. The method of claim 33, wherein said preparation is a cell line
or membrane preparation thereof.
36. The method of claim 35, wherein said cell line is M2A7 (ATCC
CRL-2500).
37. The method of claim 33, wherein the ability of said ligand to
selectively agonize or antagonize prokineticin receptor signaling
is further determined.
38. The method of claim 37, wherein said signaling is determined in
a cell line.
39. The method of claim 38, wherein said cell line is M2A7 (ATCC
CRL-2500).
40. The method of claim 37, wherein said signaling is determined by
monitoring calcium mobilization.
41. The method of claim 33, wherein the ability of said ligand to
modulate smooth muscle contractility is further determined.
42. A method of identifying a prokineticin receptor agonist,
comprising contacting a preparation comprising a prokineticin
receptor with one or more candidate compounds, and identifying a
compound that selectively promotes production of a prokineticin
receptor signal, said compound being characterized as a
prokineticin receptor agonist.
43. The method of claim 42, wherein said preparation is a cell
line.
44. The method of claim 43, wherein said cell line is M2A7 (ATCC
CRL-2500).
45. The method of claim 42, wherein said signaling is determined by
monitoring calcium mobilization.
46. The method of claim 42, wherein the ability of said agonist to
modulate smooth muscle contractility is further determined.
47. A method of identifying a prokineticin receptor antagonist,
comprising contacting a preparation comprising a prokineticin
receptor with one or more candidate compounds in the presence of a
prokineticin, and identifying a compound that selectively inhibits
production of a prokineticin receptor signal, said compound being
characterized as a prokineticin receptor antagonist.
48. The method of claim 47, wherein said prokineticin comprises an
amino acid sequence selected from the group consisting of amino
acids 1-77 of SEQ ID NOS:3 and amino acids 1-77 of SEQ ID NO:6.
49. The method of claim 47, wherein said preparation is a cell
line.
50. The method of claim 49, wherein said cell line is M2A7 (ATCC
CRL-2500).
51. The method of claim 47, wherein said signaling is determined by
monitoring calcium mobilization.
52. The method of claim 47, wherein the ability of said antagonist
to modulate smooth muscle contractility is further determined.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/245,882, filed Nov. 3, 2000, which is
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] The main function of gastrointestinal (GI) smooth muscle is
to mix and propel intralumenal contents, which enables efficient
digestion of food, progressive absorption of nutrients, and
eventual evacuation of residual components. The activity of GI
smooth muscle is regulated by intrinsic and extrinsic neural
signals, including classical neurotransmitters, co-existing
neuropeptides, and circulating peptide hormones. In addition, a
number of humoral agents including histamine, serotonin, and
adenosine that are produced by nonneural GI cells also influence
the activity of smooth muscle cells.
[0003] A number of clinical conditions are associated with altered
GI motility, including irritable bowel syndrome, diabetic
gastroparesis, postoperational ileus, chronic constipation,
gastrointestinal reflux disease, chronic diarrhea, infectious
diseases, malabsorptive disorders, inflammatory bowel disorders,
and intestinal cancers. The identification of regulators of
gastrointestinal motility should facilitate the development of
novel therapeutics for disorders that involve impaired or enhanced
gastrointestinal motility.
[0004] Two potential regulators of gastrointestinal motility have
recently been identified. Mamba intestinal toxin (MITl), a small
protein that potently stimulates the contraction of guinea-pig
ileum, has been purified from mamba snake venom (Schweitz et al.,
Toxicon 28:847-856 (1990) and Schweitz et al., FEBS Letters
461:183-188 (1999)). Recently, a protein of similar size and having
greater than 40% identity with MITl, including all 10 conserved
cysteines, has been purified from frog skin secretions (Mollay et
al., Eur. J. Pharmacol. 374:189-196 (1999)). The frog protein,
named Bv8, was also found to potently stimulate the contraction of
GI smooth muscle.
[0005] Methods of recombinantly preparing these snake and frog
polypeptides, or of recombinantly preparing other polypeptides
containing 10 cysteines, have not previously been described,
limiting the utility of these regulators for therapeutic use.
Additionally, snake and frog polypeptides could elicit antibodies
if administered to mammals that would likely reduce their efficacy
as therapeutics.
[0006] Accordingly, there exists a need to identify endogenous
human polypeptides that stimulate or inhibit gastrointestinal
motility, and to develop methods of preparing these compounds
recombinantly as therapeutics. There also exists a need to identify
small molecule agonists and antagonists of endogenous
gastrointestinal regulators that can be used therapeutically. The
present invention satisfies this need, and provides related
advantages as well.
SUMMARY OF THE INVENTION
[0007] The invention provides isolated polypeptides that stimulate
gastrointestinal smooth muscle contraction. In one embodiment, the
polypeptide contains an amino acid sequence at least 80% identical
to the sequence of human prokineticin 1 (SEQ ID NO:3), wherein the
sequence contains the N-terminal 6 amino acids of SEQ ID NO:3, the
10 conserved cysteine residues of SEQ ID NO:3, and from 0 to 9 of
the 9 C-terminal amino acids of SEQ ID NO:3. In another embodiment,
the polypeptide containis an amino acid sequence at least 80%
identical to the sequence of human prokineticin 2 (SEQ ID NO:6),
wherein the sequence contains the N-terminal 6 amino acids of SEQ
ID NO:6, the 10 conserved cysteine residues of SEQ ID NO:6, and
from 0 to 4 of the 4 C-terminal amino acids of SEQ ID NO:6.
[0008] Also provided are methods of stimulating gastrointestinal
smooth muscle contraction in a mammal, by administering to the
mammal an effective amount of a prokineticin polypeptide.
[0009] The invention also provides nucleic acid molecules encoding
a prokineticin polypeptide.
[0010] Further provided are antibodies that selectively bind a
prokineticin polypeptide.
[0011] The invention also provides methods of identifying a
prokineticin receptor ligand, by contacting a preparation
containing prokineticin receptor with one or more candidate
compounds, and identifying a compound that specifically binds to
the receptor. Such a compound is characterized as a prokineticin
receptor ligand.
[0012] Also provided are methods of identifying a prokineticin
receptor agonist, by contacting a preparation containing a
prokineticin receptor with one or more candidate compounds, and
identifying a compound that selectively promotes production of a
prokineticin receptor signal. Such a compound is characterized as a
prokineticin receptor agonist.
[0013] Further provided are methods of identifying a prokineticin
receptor antagonist, by contacting a preparation containing a
prokineticin receptor with one or more candidate compounds in the
presence of a prokineticin, and identifying a compound that
selectively inhibits production of a prokineticin receptor signal.
Such a compound is characterized as a prokineticin receptor
antagonist.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 shows the amino acid sequences of A) prokineticin 1
precursor (SEQ ID NO:2); B) prokineticin 2 precursor (SEQ ID NO:4);
C) frog BV8 (SEQ ID NO:11) and D) partial sequence of MIT 1 (SEQ ID
NO:12). Ten conservative cysteine residues are marked (*). Signal
peptides are underlined. The arrow indicates an intron splice
site.
[0015] FIG. 2 shows the expression pattern of prokineticins. A
human RNA master blot was probed with A) prokineticin 1 and B)
prokineticin 2 cDNA. FIG. 2C show the blot diagram indicating the
RNA sources for each dot. FIG. 2D shows a Northern blot analysis
with prokineticin 1. Each lane contains RNA from different brain
tissues as indicated: 1. Cerebellum; 2. Cerebral cortex; 3.
Medulla; 4. Spinal cord; 5. Occipital pole; 6. Frontal lobe; 7.
Temporal lobe; 8. Putamen; 9, Amygdala; 10. Caudate nucleus; 11.
Corpus callosum; 12. Hippocampus; 13. Whole brain; 14. Substanti
anigra.; 15. Subthalamic nucleus; 16. Thalamus.
[0016] FIG. 3 shows the production and purification of human
prokineticins: A) SDS-PAGE (18%) of prokineticin samples stained
with Coomassie blue G-250. Lane 1, molecular weight standards; lane
2, whole bacterial lysate after induction; lane 3, Ni-NTA affinity
chromatography-purified prokineticin; lane 4, Factor Xa digested
prokineticin; lane 5, refolded prokineticin after HPLC
purification. Each lane was loaded with 10-15 .mu.g total protein.
B) Reverse phase HPLC separation of refolded protein mixture. Peak
2 contains refolded prokineticin. C) Electrospray mass spectrum of
refolded prokineticin 1.
[0017] FIG. 4 shows the effects of prokineticins on the
contractility of guinea-pig ileal longitudinal smooth muscle. The
contractile responses to prokineticin 1 (2 nM) were measured in
ileum in the absence (A) and in the presence of tetrodotoxin (0.1
.mu.M; B) and verapamil (1 .mu.M; C). FIG. 4D shows the
concentration-response relationship for the contractile effects of
prokineticins. Results are given as percentage of maximum
contractility. Data are from three independent experiments.
Contractile effects of oxotremorine-M in ileum in the absence (E)
and in the presence of verapamil (1 .mu.M; F) are also shown.
Arrows indicate when drugs were added.
[0018] FIG. 5A shows Scatchard analysis of the specific binding of
.sup.125I-prokineticin 1 to guinea pig ileal membrane. Figure 5B
shows the inhibition of binding of .sup.125I-prokineticin 1 (20 pM)
by different concentrations of unlabeled prokineticin 1 (filled
squares) and unlabeled prokineticin 2 (open squares). Open circles
show displacement of .sup.125I -prokineticin 1 (20 pM) with
different concentrations of GTP.UPSILON.S.
[0019] FIG. 6 shows a schematic diagram of chimeras constructed
between prokineticin 1 and prokineticin 2, designated chimera 12
(SEQ ID NO:13) and chimera 21 (SEQ ID NO:14).
[0020] FIG. 7 shows functional characterization of chimeric
prokineticins. FIG. 7A shows a dose-response curve of chimeric and
wild type prokineticins assayed for their ability to contract
guinea-pig ileum. FIG. 7B shows time constants of chimeric and wild
type prokineticins. The time constant indicates the time elapsed
from peak contraction to midway contraction (half way from peak to
sustained plateau contraction). After normalizing against the
oxotremorine M-induced contraction, the peak and sustained plateau
contraction elicited by prokineticins are about 80% and 40%,
respectively. The midway contraction is thus about 60% of maximum
contraction.
[0021] FIG. 8A shows the effect of administration of various doses
of prokineticin 1 as an IV bolus on contractions in guinea pig
ileum in vivo. FIG. 8B shows the contractile response to 1000 ng/kg
of prokineticin 1.
[0022] FIG. 9 shows calcium mobilization, as determined in a FLIPR
assay, elicited in HEK293 or M2A7 cells by the indicated
concentrations of prokineticin 1 or prokineticin 2.
DETAILED DESCRIPTION OF THE INVENTION
[0023] The invention provides an isolated prokineticin polypeptide
that is able to stimulate gastrointestinal (GI) smooth muscle
contraction. The prokineticin polypeptides of the invention can be
used, for example, in therapeutic methods to treat disorders
involving impaired GI motility. Such polypeptides can also be used,
for example, in screening methods to identify prokineticin receptor
ligands, including receptor agonists and antagonists, which can be
used therapeutically to treat disorders involving impaired or
enhanced GI motility.
[0024] As used herein, the term "prokineticin polypeptide" refers
to a polypeptide comprising the amino acid sequence of human
prokineticin 1 shown as the non-underlined sequence in FIG. 1A (SEQ
ID NO:3), or comprising the amino acid sequence of human
prokineticin 2 shown as the non-underlined sequence in FIG. 1B (SEQ
ID NO:6); and to a polypeptide containing minor modifications to
SEQ ID NOS:3 or 6 that has GI smooth muscle contractile activity;
and to a fragment of the reference polypeptide that has GI smooth
muscle contractile activity.
[0025] As used herein, the terms "comprising," "having,"
"encoding," and "containing," and derivatives of these terms, are
intended to be open-ended. The term "consisting" is intended to be
closed-ended.
[0026] As used herein, the term "minor modification" to the
sequences designated SEQ ID NOS:3 or 6 refers to one or more
additions, deletions or substitutions compared with the recited
amino acid sequence; one or more chemical or enzymatic
modifications to the polypeptide; or substitution of one or more
L-configuration amino acids with corresponding D-configuration
amino acids. Such modifications can be advantageous, for example,
in enhancing the stability, expression, bioactivity, or receptor
affinity of the polypeptide, or for facilitating its identification
or purification.
[0027] The GI smooth muscle contractile activity of a modified
polypeptide can be determined by ex vivo or in vivo methods known
in the art, such as the ex vivo and in vivo guinea pig ileal
bioassays described in the Example, to confirm that it has GI
smooth muscle contractile activity. Suitable assays for determining
GI smooth muscle contractile activity can alternatively be
performed using other GI smooth muscle tissue that responds to
prokineticin 1 or 2, such as fundic muscle strip or proximal colon
(see Example). Likewise, suitable assays can be performed using
other mammals, including, for example, mice, rats, cats, dogs,
sheep, goats, pigs, cows and primates.
[0028] A modified prokineticin polypeptide that elicits GI smooth
muscle contractile activity can elicit at least 10%, 25%, 50%, 75%,
100% or more of the maximal GI smooth muscle contraction of human
prokineticin 1 or 2, under the same conditions. A modified
prokineticin polypeptide that elicits GI smooth muscle contractile
activity can be less potent, similarly potent, or more potent than
human prokineticin 1 or 2, under the same conditions. For example,
a modified polypeptide can have an EC.sub.50 that is 5-fold,
10-fold, 50-fold or 100-fold higher or lower than the EC.sub.50 for
human prokineticin 1 or 2. A modified prokineticin polypeptide that
elicits GI smooth muscle contractile activity can also elicit
contractions for the same duration or for a longer or shorter
duration than human prokineticin 1 or 2, under the same
conditions.
[0029] A chimeric polypeptide encoded by exons 1 and 2 of
prokineticin 1 and exon 3 of prokineticin 2, designated chimera 12
(SEQ ID NO:13) (see FIG. 6) is an example of a modified
prokineticin that elicits ileal contractions with a similar potency
as prokineticins 1 or 2 (see FIG. 7A), but which causes prolonged
contractions in comparison with prokineticins 1 or 2 (see FIG.
7B).
[0030] A chimeric polypeptide encoded by exons 1 and 2 of
prokineticin 2, and exon 3 of prokineticin 1, designated chimera 21
(SEQ ID NO:13) (see FIG. 6) is an example of a modified
prokineticin that elicits ileal contractions with an 8-fold lower
potency than prokineticins 1 or 2 (see FIG. 7A), and which causes
prolonged contractions in comparison with prokineticins 1 or 2 (see
FIG. 7B).
[0031] Modifications to the amino acid sequence designated SEQ ID
NOS:3 or 6 can be randomly generated, such as by random insertions,
deletions or substitutions of nucleotides in a nucleic acid
molecule encoding SEQ ID NOS:3 or 6. Alternatively, modifications
can be directed, such as by site-directed mutagenesis of a nucleic
acid molecule encoding SEQ ID NOS:3 or 6.
[0032] Computer programs known in the art can provide guidance in
predicting which amino acid residues can be modified without
abolishing the function of the polypeptide (see, for example,
Eroshkin et al., Comput. Appl. Biosci. 9:491-497 (1993)).
[0033] Furthermore, guidance in modifying amino acid residues of
SEQ ID NOS:3 or 6 while retaining activity can be provided by
comparison of SEQ ID NOS:3 or 6 with the sequence of their
mammalian homologs, such as homologs in non-human primates, mouse,
rat, rabbit, bovine, porcine, ovine, canine or feline species, as
well as sequences of their homologs in non-mammalian vertebrates,
including frog Bv8 polypeptide (SEQ ID NO:11) and snake MIT1
polypeptide (SEQ ID NO:12) (see FIG. 1). It is well known in the
art that evolutionarily conserved amino acid residues and domains
are more likely to be important for maintaining biological activity
than less well-conserved residues and domains. Thus, it would be
expected that substituting a residue that is highly conserved among
mammalian prokineticins, frog Bv8 polypeptide and snake MIT1
polypeptide, such as the N-terminal sequence, or any of the 10
cysteines, would likely be detrimental to activity, whereas
substitution of less highly conserved residues, such as the
C-terminal residues, is likely to be tolerated.
[0034] As described in the Example, retention of the six N-terminal
residues (AVITGA) of prokineticin, without addition, deletion or
substitution, is apparently required to retain smooth muscle
contractile activity (see Table 1). However, modifications of the
AVITGA sequence can result in polypeptides that do not exhibit GI
smooth muscle contractile activity, but that antagonize the smooth
muscle contractile activity of wild-type prokineticins. The
N-terminal mutants designated SEQ ID NOS:16 and 18 are examples of
modified prokineticin polypeptides with antagonistic activity.
[0035] The N-terminal domain of prokineticins, while apparently
required for GI smooth muscle contractile activity, is not
sufficient for GI smooth muscle contractile activity. Specifically,
a prokineticin N-terminal peptide (SEQ ID NO:19), or a polypeptide
with the cysteine-rich domain of prokineticin substituted with the
cysteine-rich domain of either co-lipase or dickkopf4, did not
exhibit smooth muscle contractile activity.
[0036] The cysteine-rich domain of prokineticins was shown to also
be required for GI smooth muscle contractile activity (see Table
1). Specifically, correct cysteine pairing was shown to be required
in order to retain activity, as substitutions at either of two
cysteines abolished activity.
[0037] The sequence C-terminal to the final cysteine is presumably
not required, as evidenced by GI smooth muscle contractile activity
of prokineticin polyepeptides with a 6XHis tag inserted at this
position. Additionally, as evidenced by similar activities of
prokineticins 1 and 2, despite only being 44% identical, amino acid
sequence substitutions at positions other than the N-terminus and
conserved cysteine residues are well tolerated.
[0038] Substitutions to the amino acid sequences designated SEQ ID
NOS:3 or 6 can either be conservative or non-conservative.
Conservative amino acid substitutions include, but are not limited
to, substitution of an apolar amino acid with another apolar amino
acid (such as replacement of leucine with an isoleucine, valine,
alanine, proline, tryptophan, phenylalanine or methionine);
substitution of a charged amino acid with a similarly charged amino
acid (such as replacement of a glutamic acid with an aspartic acid,
or replacement of an arginine with a lysine or histidine);
substitution of an uncharged polar amino acid with another
uncharged polar amino acid (such as replacement of a serine with a
glycine, threonine, tyrosine, cysteine, asparagine or glutamine);
or substitution of a residue with a different functional group with
a residue of similar size and shape (such as replacement of a
serine with an alanine; an arginine with a methionine; or a
tyrosine with a phenylalanine).
[0039] Specifically contemplated substitutions to the amino acid
sequences designated SEQ ID NOS:3 and 6 include replacement of
residues from wild-type prokineticin 1 with residues from
prokineticin 2, and vice versa. The replacements can be of single
residues, multiple residues throughout the polypeptide, or multiple
contiguous residues. Because chimeras between SEQ ID NOS:3 and 6,
namely SEQ ID NOS:13 and 14, were demonstrated to exhibit prolonged
contractile activity in comparison with wild-type prokineticins, it
is contemplated that substituted prokineticins can be potent
therapeutics in vivo.
[0040] Additions to the amino acid sequence designated SEQ ID NOS:3
or 6 include, but are not limited to, the addition of "tag"
sequences, which are preferably added at the C terminus. Such tag
sequence include, for example, epitope tags, histidine tags,
glutathione-S-transferase (GST), and the like, or sorting
sequences. Such additional sequences can be used, for example, to
facilitate recombinant expression, purification or characterization
of a prokineticin. Exemplary polypeptides containing additions to
the sequences designated SEQ ID NOS:3 or 6 are the active
prokineticins prepared as described in the Example by the insertion
of a 6XHis-tag after the C-terminal cysteine.
[0041] Deletions to the amino acid sequences designated SEQ ID
NOS:3 or 6 include, but are not limited to, deletion of one or more
residues at the C-termini that are not highly conserved among the
active polypeptides shown in FIG. 1. Deleted sequences can
optionally be replaced by tag sequences, as described
previously.
[0042] Chemical and enzymatic modifications to the polypeptide
containing the amino acid sequence designated SEQ ID NOS:3 or 6
include, but are not limited to the following: replacement of
hydrogen by an alkyl, acyl, or amino group; esterification of a
carboxyl group with a suitable alkyl or aryl moiety; alkylation of
a hydroxyl group to form an ether derivative; phosphorylation or
dephosphorylation of a serine, threonine or tyrosine residue; or N-
or O-linked glycosylation.
[0043] As used herein, the term "isolated" indicates that the
molecule is altered by the hand of man from how it is found in its
natural environment. Preferably, an "isolated" prokineticin
polypeptide can be a "substantially purified" molecule, that is at
least 60%, 70%, 80%, 90 or 95% free from cellular components with
which it is naturally associated. An isolated polypeptide can be in
any form, such as in a buffered solution, a suspension, a
lyophilized powder, recombinantly expressed in a heterologous cell,
bound to a receptor or attached to a solid support.
[0044] The invention provides isolated polypeptides that stimulate
gastrointestinal smooth muscle contraction. In one embodiment, the
polypeptide contains an amino acid sequence at least 50% identical
to the sequence of human prokineticin 1 (SEQ ID NO:3), and
including the N-terminal 6 amino acids of SEQ ID NO:3, the 10
conserved cysteine residues of SEQ ID NO:3, and from 0 to 9 of the
9 C-terminal amino acids of SEQ ID NO:3. The encoded polypeptide
can thus have at least 60%, 65%, 70%, 75% identity, including at
least 80%, 85%, 90%, 95%, 96%, 98%, 99% or greater identity to SEQ
ID NO:3. An exemplary polypeptide contains the amino acid sequence
designated SEQ ID NO:3, or amino acids 1-77 thereof.
[0045] In one embodiment, the isolated polypeptide does not contain
the amino acid sequence NNFGNGRQERRKRKRSKRKKE (SEQ ID NO:7). In
another embodiment, the isolated polypeptide does not contain the
amino acid sequence SHVANGRQERRRAKRRKRKKE (SEQ ID NO:8).
[0046] In another embodiment, the polypeptide contains an amino
acid sequence at least 50% identical to the sequence of human
prokineticin 2 (SEQ ID NO:6), and including the N-terminal 6 amino
acids of SEQ ID NO:6, the 10 conserved cysteine residues of SEQ ID
NO:6, and from 0 to 4 of the 4 C-terminal amino acids of SEQ ID
NO:6. The encoded polypeptide can thus have at least 60%, 65%, 70%,
75% identity, including at least 80%, 85%, 90%, 95%, 96%, 98%, 99%
or greater identity to SEQ ID NO:6. An exemplary polypeptide
contains the amino acid sequence designated SEQ ID NO:6, or amino
acids 1-77 thereof.
[0047] As used herein, the term "percent identity" with respect to
two molecules is intended to refer to the number of identical
nucleotide or amino acid residues between the aligned portions of
two sequences, expressed as a percentage of the total number of
aligned residues, as determined by comparing the entire sequences
using an optimized manual alignment or computer alignment, such as
a BLAST 2.0 alignment (Tatusova et al., FEMS Microbiol Lett.
174:247-250 (1999)).
[0048] For certain applications, such as in the screening methods
disclosed herein, a prokineticin polypeptide can be labeled with a
detectable moiety, such as a radiolabel, a fluorochrome, a
ferromagnetic substance, a luminescent tag or a detectable binding
agent such as biotin. Other suitable labeled moieties are well
known in the art.
[0049] The invention also provides methods for preparing an
isolated prokineticin polypeptide that is able to stimulate GI
smooth muscle contraction, by culturing host cells (described
below) so as to express a recombinant prokineticin polypeptide, and
refolding the polypeptide under conditions that minimize protein
aggregation.
[0050] Recombinant expression of polypeptides containing multiple
cysteine residues often results in the incorrect formation of
inter- and intra-molecular disulfide bonds, which leads to the
production of inactive, aggregated bacterial proteins. As disclosed
herein, these problems can be overcome using conditions that
minimize protein aggregation during refolding of the expressed
polypeptide. Exemplary conditions that minimize protein aggregation
are described in the Example, and differ from conventional
conditions for preparing recombinant protein by including one or
more of the following refolding conditions: 1) keeping protein
concentration low (e.g. about 100 .mu.g/ml); 2) dialysing, rather
than diluting, the peptides to remove denaturing agent; 3) omitting
oxidants from buffers; 4) maintaining high concentrations of urea
in all buffers; 5) maintaining high concentrations of glycerol
(e.g. at least about 10%) in buffers; and 6) keeping peptides and
buffers at low temperature (e.g. about 4.degree. C.). Of these
conditions, it is contemplated that low protein concentration (i.e.
less than about 250 .mu.g/ml, preferably less than 200 .mu.g/ml,
150 .mu.g/ml, 100 .mu.g/ml, or 50 .mu.g/ml) and high urea
concentration (e.g. at least about 1.5M, such as about 2M, 4M, 6M,
8M or higher) are the most important factors in successful
refolding of active prokineticins.
[0051] It is expected that the same or similar conditions as those
described herein can be used to recombinantly express and refold
other polypeptides containing multiple cysteines, including
dickkopf, co-lipase, MIT-1 and Bv8, so as to isolate a biologically
active polypeptide.
[0052] In a preferred method for preparing an isolated prokineticin
polypeptide that is able to stimulate GI smooth muscle contraction,
a prokineticin polypeptide is recombinantly expressed in bacteria
as a fusion protein (e.g. as a GST fusion) containing a tag (e.g. a
6XHis tag), and partially purified by affinity isolation (e.g. on a
nickel column). The fused polypeptide is then cleaved so as to
remove the heterologous protein (e.g. using protease factor Xa
cleavage between GST and prokineticin), and the prokineticin
polypeptide refolded under conditions described above to minimize
protein aggregation. To obtain more highly purified polypeptide,
the polypeptide can further be purified by column chromatography
(e.g. reverse-phase HPLC). Those skilled in the art recognize that
modification to these preferred methods for recombinantly
expressing, refolding and purifying active prokineticin
polypeptides can readily be determined, such as employing
alternative heterologous sequences, cleavable sequences, tags, host
cells and buffer conditions.
[0053] Alternatively, an isolated prokineticin polypeptide can be
prepared by biochemical procedures. As disclosed herein,
prokineticins 1 and 2 are expressed in a variety of human tissues
(see Example, and particularly FIG. 2). Therefore, an isolated
prokineticin polypeptide can be isolated from tissues or cells that
normally express these polypeptides, by biochemical procedures
routinely used in the art, including membrane fractionation,
chromatography, electrophoresis and ligand affinity methods, or
using immunoaffinity methods with the prokineticin antibodies
described herein. Following biochemical isolation, an inactive
prokineticin can be refolded by the methods described above to
restore activity.
[0054] Likewise, an isolated prokineticin polypeptide can be
prepared by chemical synthesis procedures known in the art.
Following chemical synthesis, an inactive prokineticin can be
refolded by the methods described herein to restore activity.
[0055] If desired, such as to optimize their functional activity,
selectivity, stability or bioavailability, chemically synthesized
polypeptides can be modified to include D-stereoisomers,
non-naturally occurring amino acids, and amino acid analogs and
mimetics. Examples of modified amino acids and their uses are
presented in Sawyer, Peptide Based Drug Design, ACS, Washington
(1995) and Gross and Meienhofer, The Peptides: Analysis, Synthesis,
Biology, Academic Press, Inc., New York (1983). For certain
applications, it can also be useful to incorporate one or more
detectably labeled amino acids into a chemically synthesized
polypeptide or peptide, such as radiolabeled or fluorescently
labeled amino acids.
[0056] The invention also provides isolated peptides containing, or
consisting of, at least 10 contiguous amino acids of the amino acid
sequences designated SEQ ID NOS:3 or 6 which can, but need not, be
able to stimulate gastrointestinal (GI) smooth muscle contraction.
Such isolated peptides are useful, for example, in preparing and
purifying prokineticin antibodies of the invention. Such peptides
can also act as antagonists to block signaling through a
prokineticin receptor, and thus can be used in therapeutic and
screening methods. An isolated prokineticin peptide can thus
contain, or consist of, at least 12, 15, 20, 25 or more contiguous
amino acids of SEQ ID NOS:3 or 6, including at least, or not more
than, 30, 40, 50, 60, 70, 80, 81 or 86 contiguous amino acids.
[0057] In one embodiment, an isolated prokineticin peptide
contains, or consists of, at least 10 contiguous residues from
within amino acid residues 6 and 48 of SEQ ID NO:3. In another
embodiment, an isolated prokineticin peptide contains, or consists
of, at least 10 contiguous residues from within amino acid residues
6 and 48 of SEQ ID NO:6.
[0058] An isolated peptide containing at least 10 contiguous amino
acids of SEQ ID NOS:3 or 6 can be immunogenic. As used herein, the
term "immunogenic" refers to a peptide that either is capable of
inducing prokineticin-specific antibodies, or is capable of
competing with prokineticin-specific antibodies for binding to a
prokineticin. Peptides that are likely to be immunogenic can be
predicted using methods and algorithms known in the art and
described, for example, by Irnaten et al., Protein Eng. 11:949-955
(1998), and Savoie et al., Pac. Symp. Biocomput. 1999:182-189
(1999). The immunogenicity of the peptides of the invention can be
confirmed by methods known in the art.
[0059] The isolated prokineticin polypeptide and peptides of the
invention can optionally be conjugated to a carrier, such as KLH,
serum albumin, tetanus toxoid and the like, using standard linking
techniques, to enhance their immunogenicity. Additionally or
alternatively, the isolated polypeptides and peptides can be
formulated with an adjuvant known in the art, such as Freund's
complete or incomplete adjuvant.
[0060] An isolated prokineticin peptide of at least 10 contiguous
residues can conveniently be prepared by chemical synthesis, or by
chemical or enzymatic digestion of longer peptides, prepared as
described above. An isolated prokineticin peptide of at least 10
contiguous residues can also be prepared recombinantly, such as
fused to a protein tag. Those skilled in the art can determine an
appropriate method of preparing an isolated prokineticin peptide,
depending on its size, sequence, and intended application.
[0061] The invention also provides an isolated nucleic acid
molecule encoding a prokineticin polypeptide that is able to
stimulate GI smooth muscle contraction. The invention nucleic acid
molecules are suitable for a variety of screening, therapeutic and
diagnostic applications. For example, an invention nucleic acid
molecule can be expressed in vitro and the encoded prokineticin
polypeptide isolated. An invention nucleic acid molecule can also
be expressed in vivo, to restore normal prokineticin activity in
patients, or expressed in an antisense orientation to block
prokineticin expression in patients in need thereof. Additionally,
the invention nucleic acid molecules can be used as probes or
primers to identify and isolate prokineticin-encoding nucleic acid
molecules from other species, or to identify structurally related
molecules. Such probes and primers are also useful diagnostically
to determine normal and abnormal expression of prokineticin in
human tissues, and thus to predict susceptibility to conditions
associated with altered prokineticin expression.
[0062] As used herein, the term "isolated nucleic acid molecule" is
intended to mean that the nucleic acid molecule is altered, by the
hand of man, from how it is found in its natural environment. For
example, an isolated nucleic acid molecule can be a molecule
operatively linked to an exogenous nucleic acid sequence. An
isolated nucleic acid molecule can also be a molecule removed from
some or all of its normal flanking nucleic acid sequences.
[0063] An isolated molecule can alternatively, or additionally, be
a "substantially pure" molecule, in that the molecule is at least
60%, 70%, 80%, 90 or 95% free from cellular components with which
it is naturally associated. An isolated nucleic acid molecule can
be in any form, such as in a buffered solution, a suspension, a
lyophilized powder, attached to a solid support (e.g. as a
component of a DNA array), or in a cell.
[0064] As used herein, the term "nucleic acid molecule" refers to a
polynucleotide of natural or synthetic origin, which can be single-
or double-stranded, can correspond to genomic DNA, cDNA or RNA, and
can represent either the sense or antisense strand or both.
[0065] The term "nucleic acid molecule" is intended to include
nucleic acid molecules that contain one or more non-natural
nucleotides, such as nucleotides having modifications to the base,
the sugar, or the phosphate portion, or having one or more
non-natural linkages, such as phosphothioate linkages. Such
modifications can be advantageous in increasing the stability of
the nucleic acid molecule, particularly when used in hybridization
applications.
[0066] Furthermore, the term "nucleic acid molecule" is intended to
include nucleic acid molecules modified to contain a detectable
moiety, such as a radiolabel, a fluorochrome, a ferromagnetic
substance, a luminescent tag or a detectable binding agent such as
biotin. Nucleic acid molecules containing such moieties are useful
as probes for detecting the presence or expression of prokineticin
nucleic acid molecule.
[0067] Prokineticin polypeptides that are able to stimulate GI
smooth muscle contraction have been described above. Accordingly,
it is routine for those skilled in the art to prepare isolated
nucleic acid molecules encoding such polypeptides. Exemplary
isolated nucleic acid molecules encoding a prokineticin polypeptide
that is able to stimulate GI smooth muscle contraction contains, or
consists of, a) the nucleotide sequences designated SEQ ID NOS:1 or
4; b) the portion of the nucleotide sequences designated SEQ ID
NOS:1 or 4 that encodes SEQ ID NOS:3 or 6 (i.e. nucleotides 55-370
of the nucleotide sequence designated SEQ ID NO:1 and nucleotides
10-334 of the nucleotide sequence designated SEQ ID NO:4); c) a
nucleotide sequence that encodes an active modification or active
fragment of SEQ ID NOS:3 or 6; and d) a sequence that is degenerate
with respect to either a), b) or c).
[0068] In one embodiment, the isolated nucleic acid molecule does
not encode the amino acid sequence NNFGNGRQERRKRKRSKRKKE (SEQ ID
NO:7). In another embodiment, the isolated nucleic acid molecule
does not encode the amino acid sequence SHVANGRQERRRAKRRKRKKE(SEQ
ID NO:8). In yet another embodiment, an isolated nucleic acid
molecule encoding a prokineticin polypeptide excludes naturally
occuring signal polypeptides, such as nucleic acid molecules
encoding the underlined portions of the amino acid sequences shown
in FIG. 1A and 1B (MRGATRVSIMLLLVTVSDC (SEQ ID NO:9) and
MRSLCCAPLLLLLLLPLLLTPPAGDA (SEQ ID NO:10)).
[0069] In certain embodiments, an isolated nucleic acid molecule
encoding a prokineticin polypeptide specifically excludes nucleic
acid molecules having the exact sequence of genomic fragments ESTs
and cDNAs whose sequences are compiled in publically available
databases, such as GenBank Accession Nos. AI277349, AA883760,
AQ426386, AC068519, AC026973, AL358215 and AL390797 or sequences
which encode amino acid sequences having GenBank Accession Nos.
AF182066, AF182064, AF182069 and AF182065.
[0070] In one embodiment, an isolated nucleic acid molecule
encoding a prokineticin polypeptide excludes mammalian sequences
present in the GenBank database that contain sequences which do not
encode SEQ ID NOS:3 and 6 (e.g. nucleic acid molecules that encode
5' and 3' untranslated regions, introns or other exons present on
chromosomes 1 or 3).
[0071] The invention further provides an isolated nucleic acid
molecule encoding a prokineticin polypeptide that is able to
stimulate GI smooth muscle contraction, wherein the nucleic acid
molecule is operatively linked to a promoter of gene expression. As
used herein, the term "operatively linked" is intended to mean that
the nucleic acid molecule is positioned with respect to either the
endogenous promoter, or a heterologous promoter, in such a manner
that the promoter will direct the transcription of RNA using the
nucleic acid molecule as a template.
[0072] Methods for operatively linking a nucleic acid to a
heterologous promoter are well known in the art and include, for
example, cloning the nucleic acid into a vector containing the
desired promoter, or appending the promoter to a nucleic acid
sequence using PCR. A nucleic acid molecule operatively linked to a
promoter of RNA transcription can be used to express prokineticin
transcripts and polypeptides in a desired host cell or in vitro
transcription-translation system.
[0073] The choice of promoter to operatively link to an invention
nucleic acid molecule will depend on the intended application, and
can be determined by those skilled in the art. For example, if a
particular gene product may be detrimental to a particular host
cell, it may be desirable to link the invention nucleic acid
molecule to a regulated promoter, such that gene expression can be
turned on or off. Alternatively, it may be preferred to have
expression driven by either a weak or strong constitutive promoter.
Exemplary promoters suitable for mammalian cell systems include,
for example, the SV40 early promoter, the cytomegalovirus (CMV)
promoter, the mouse mammary tumor virus (MMTV) steroid-inducible
promoter, and the Moloney murine leukemia virus (MMLV) promoter.
Exemplary promoters suitable for bacterial cell systems include,
for example, T7, T3, SP6, lac and trp promoters.
[0074] The invention further provides a vector containing an
isolated nucleic acid molecule encoding a prokineticin polypeptide.
Exemplary vectors include vectors derived from a virus, such as a
bacteriophage, a baculovirus or a retrovirus, and vectors derived
from bacteria or a combination of bacterial sequences and sequences
from other organisms, such as a cosmid or a plasmid. The vectors of
the invention will generally contain elements such as an origin of
replication compatible with the intended host cells; transcription
termination and RNA processing signals; one or more selectable
markers compatible with the intended host cells; and one or more
multiple cloning sites. Optionally, the vector will further contain
sequences encoding tag sequences, such as GST tags, and/or a
protease cleavage site, such as a Factor Xa site, which facilitate
expression and purification of the encoded polypeptide.
[0075] The choice of particular elements to include in a vector
will depend on factors such as the intended host cells; the insert
size; whether expression of the inserted sequence is desired; the
desired copy number of the vector; the desired selection system,
and the like. The factors involved in ensuring compatibility
between a host cell and a vector for different applications are
well known in the art.
[0076] In applications in which the vectors are to be used for
recombinant expression of the encoded polypeptide, the isolated
nucleic acid molecules will generally be operatively linked to a
promoter of gene expression, as described above, which may be
present in the vector or in the inserted nucleic acid molecule. An
exemplary vector suitable for fusion protein expression in
bacterial cells is the pGEX-3.times. vector (Amersham Pharmacia
Biotech, Piscataway, N.J.).
[0077] Also provided are cells containing an isolated nucleic acid
molecule encoding a prokineticin polypeptide. The isolated nucleic
acid molecule will generally be contained within a vector. The
isolated nucleic acid molecule can be maintained episomally, or
incorporated into the host cell genome.
[0078] The cells of the invention can be used, for example, for
molecular biology applications such as expansion, subcloning or
modification of the isolated nucleic acid molecule. For such
applications, bacterial cells, such as laboratory strains of E.
coli, are useful, and expression of the encoded polypeptide is not
required.
[0079] The cells of the invention can also advantageously be used
to recombinantly express and isolate the encoded polypeptide. For
such applications bacterial cells (e.g. E. coli), insect cells
(e.g. Drosophila), yeast cells (e.g. S. cerevisiae, S. pombe, or
Pichia pastoris), and vertebrate cells (e.g. mammalian primary
cells and established cell lines; and amphibian cells, such as
Xenopus embryos and oocytes). An exemplary cell suitable for
recombinantly expressing prokineticin polypeptides is an E. coli
BL21 cell.
[0080] The invention further provides isolated polynucleotides that
contain at least 20 contiguous nucleotides from SEQ ID NOS:1 or 4,
such as portions of SEQ ID NOS:1 or 4 that encode SEQ ID NOS:2, 3,
5 or 6, or from the complement thereof. The polynucleotides of the
invention are thus of sufficient length to be useful as sequencing
primers, PCR primers and hybridization probes to detect or isolate
nucleic acid molecules encoding prokineticin polypeptides, and are
also useful as therapeutic antisense reagents to inhibit
prokineticin expression. The polynucleotides of the invention can,
but need not, encode prokineticin polypeptides that are able to
stimulate GI smooth muscle contraction. Those skilled in the art
can determine the appropriate length and sequence of a
polynucleotide of the invention for a particular application.
[0081] As used herein, the term "polynucleotide" refers to a
nucleic acid molecule that contains at least 20 contiguous
nucleotides from the reference sequence and which may, but need
not, encode a functional polypeptide. Thus, a polynucleotide of the
invention can contain at least 20, 22 or 25 contiguous nucleotides,
such as at least, or not more than, 30, 40, 50, 60, 70, 80, 90,
100, 125, 150, 175, 200, 250, or 300 contiguous nucleotides from
SEQ ID NOS:1 or 4, or from their complement. A polynucleotide of
the invention does not consist of the exact sequence of an EST
present in publically available databases, including the sequences
designated by GenBank Accession Nos. AI277349, AA883760, AQ426386,
AC068519, AC026973, AL358215 and AL390797 or sequences which encode
amino acid sequences having GenBank Accession Nos. AF182066,
AF182064, AF182069 and AF182065.
[0082] For certain applications, such as for detecting prokineticin
expression in a sample, it is desirable to use isolated
polynucleotide molecules of the invention that specifically
hybridize to a nucleic acid molecule encoding a prokineticin. The
term "specifically hybridize" refers to the ability of a nucleic
acid molecule to hybridize, under stringent hybridization
conditions as described below, to a nucleic acid molecule that
encodes a prokineticin, without hybridizing to a substantial extent
under the same conditions with nucleic acid molecules that do not
encode a prokineticin, such as unrelated molecules that
fortuitously contain short regions of identity with a prokineticin.
Thus, a nucleic acid molecule that "specifically hybridizes" is of
a sufficient length and contains sufficient distinguishing sequence
from a prokineticin for use in expression analysis, such as tissue
blots and Northern blots (see FIG. 2).
[0083] As used herein, the term "stringent conditions" refers to
conditions equivalent to hybridization of a filter-bound nucleic
acid molecule to a nucleic acid in a solution containing 50%
formamide, 5.times.Denhart's solution, 5.times.SSC, 0.2% SDS at
42.degree. C., followed by washing the filter in 0.1.times.SSC and
0.1% SDS at 65.degree. C. twice for 30 minutes. Equivalent
conditions to the stringent conditions set forth above are well
known in the art, and are described, for example in Sambrook et
al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory, New York (1992).
[0084] The invention further provides a kit containing a pair of
polynucleotides of the invention packaged together, either in a
single container or separate containers. The pair of
polynucleotides are preferably suitable for use in polymerase chain
reaction (PCR) applications. Thus, the pair of polynucleotides can
be used to detect or quantitate normal or abnormal expression of a
nucleic acid molecule encoding a prokineticin. The pair of
polynucleotides can also be used to amplify a nucleic acid molecule
encoding a prokineticin, or any portion thereof, for sequencing,
subcloning or for preparing sequence modifications. The kit can
further contain written instructions for use of the pair of
polynucleotides in PCR applications, or solutions and buffers
suitable for such applications.
[0085] The isolated prokineticin nucleic acid molecules of the
invention can be prepared by methods known in the art. An exemplary
method for preparing an isolated prokineticin nucleic acid molecule
involves amplification of the nucleic acid molecule using
prokineticin-specific primers and the polymerase chain reaction
(PCR). Using PCR, a prokineticin nucleic acid molecule having any
desired boundaries can be amplified exponentially starting from
only a few DNA or RNA molecules, such as from a single cell. PCR
methods, including methods of isolating homologs of a given nucleic
acid molecule in other species using degenerate primers, are well
known in the art.
[0086] Alternatively, an isolated prokineticin nucleic acid
molecule can be prepared by screening a library, such as a genomic
library, cDNA library or expression library, with a detectable
prokineticin nucleic acid molecule or with an antibody. Human
libraries, and libraries from a large variety of mammalian species,
are commercially available or can be produced from species or cells
of interest. The library clones identified as containing a
prokineticin nucleic acid molecule can be isolated, subcloned or
sequenced by routine methods.
[0087] Furthermore, an isolated prokineticin nucleic acid molecule
can be prepared by direct synthetic methods. For example, a single
stranded nucleic acid molecule can be chemically synthesized in one
piece, or in several pieces, by automated synthesis methods known
in the art. The complementary strand can likewise be synthesized in
one or more pieces, and a double-stranded molecule made by
annealing the complementary strands. Direct synthesis is
particularly advantageous for producing relatively short molecules,
such as probes and primers, and also for producing nucleic acid
molecules containing modified nucleotides or linkages.
[0088] The invention also provides an antibody specific for a
prokineticin polypeptide or peptide, such as an antibody specific
for a polypeptide having the amino acid sequence of SEQ ID NOS:3 or
6. Also provided is an antibody specific for an isolated
immunogenic peptide that contains at least 10 contiguous amino
acids of SEQ ID NOS:3 or 6.
[0089] The antibodies of the invention can be used, for example, to
detect prokineticin expression in research and diagnostic
applications. Such antibodies are also useful for identifying
nucleic acid molecules that encode prokineticin polypeptides
present in mammalian expression libraries, and for purifying
prokineticin polypeptides by immunoaffinity methods. Furthermore,
such antibodies can be administered therapeutically to bind to and
block the activity of prokineticin, such as in applications in
which it is desirable to inhibit GI smooth muscle contractions.
[0090] The term "antibody," as used herein, is intended to include
molecules having specific binding activity for a prokineticin
peptide or polypeptide of at least about 1.times.10.sup.5 M.sup.-1,
preferably at least 1.times.10.sup.7 M.sup.-1, more preferably at
least 1.times.10.sup.9 M.sup.-1. The term "antibody" includes both
polyclonal and monoclonal antibodies, as well as antigen binding
fragments of such antibodies (e.g. Fab, F(ab').sub.2, Fd and Fv
fragments and the like). In addition, the term "antibody" is
intended to encompass non-naturally occurring antibodies,
including, for example, single chain antibodies, chimeric
antibodies, bifunctional antibodies, CDR-grafted antibodies and
humanized antibodies, as well as antigen-binding fragments
thereof.
[0091] Methods of preparing and isolating antibodies, including
polyclonal and monoclonal antibodies, using peptide and polypeptide
immunogens, are well known in the art and are described, for
example, in Harlow and Lane, Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory Press (1988). Non-naturally occurring
antibodies can be constructed using solid phase peptide synthesis,
can be produced recombinantly or can be obtained, for example, by
screening combinatorial libraries consisting of variable heavy
chains and variable light chains. Such methods are described, for
example, in Huse et al. Science 246:1275-1281 (1989); Winter and
Harris, Immunol. Today 14:243-246 (1993); Ward et al., Nature
341:544-546 (1989); Hilyard et al., Protein Engineering: A
Practical approach (IRL Press 1992); and Borrabeck, Antibody
Engineering, 2d ed. (Oxford University Press 1995).
[0092] The invention provides a method of identifying a
prokineticin receptor ligand. The method is practiced by contacting
a preparation containing prokineticin receptor with one or more
candidate compounds, and identifying a candidate compound that
specifically binds the receptor. Such a compound is characterized
as a prokineticin receptor ligand.
[0093] The term "ligand," as used herein, includes compounds that
bind to the prokineticin receptor at the same or different site as
prokineticin.
[0094] As used herein, the term "candidate compound" refers to any
biological or chemical compound. For example, a candidate compound
can be a naturally occurring macromolecule, such as a polypeptide,
nucleic acid, carbohydrate, lipid, or any combination thereof. A
candidate compound also can be a partially or completely synthetic
derivative, analog or mimetic of such a macromolecule, or a small
organic molecule prepared by combinatorial chemistry methods. If
desired in a particular assay format, a candidate compound can be
detectably labeled or attached to a solid support.
[0095] Methods for preparing large libraries of compounds,
including simple or complex organic molecules, metal-containing
compounds, carbohydrates, peptides, proteins, peptidomimetics,
glycoproteins, lipoproteins, nucleic acids, antibodies, and the
like, are well known in the art and are described, for example, in
Huse, U.S. Pat. No. 5,264,563; Francis et al., Curr. Opin. Chem.
Biol. 2:422-428 (1998); Tietze et al., Curr. Biol., 2:363-371
(1998); Sofia, Mol. Divers. 3:75-94 (1998); Eichler et al., Med.
Res. Rev. 15:481-496 (1995); and the like. Libraries containing
large numbers of natural and synthetic compounds also can be
obtained from commercial sources.
[0096] The number of different candidate compounds to test in the
methods of the invention will depend on the application of the
method. For example, one or a small number of candidate compounds
can be advantageous in manual screening procedures, or when it is
desired to compare efficacy among several predicted ligands,
agonists or antagonists. However, it will be appreciated that the
larger the number of candidate compounds, the greater the
likelihood of identifying a compound having the desired activity in
a screening assay. Additionally, large numbers of compounds can be
processed in high-throughput automated screening assays. Therefore,
"one or more candidate compounds" can be, for example, 2 or more,
such as 5, 10, 15, 20, 50 or 100 or more different compounds, such
as greater than about 10.sup.3, 10.sup.5 or 10.sup.7 different
compounds.
[0097] A suitable preparation for a identifying a prokineticin
receptor ligand can employ a tissue, cell, cell membrane, or
purified prokineticin receptor, so long as the preparation contains
a prokineticin receptor in a suitable conformation for binding
prokineticin with a similar affinity and specificity as a
prokineticin receptor expressed on GI smooth muscle tissues.
[0098] In one embodiment, the preparation is an intestinal smooth
muscle preparation, such as a mammalian ileal, fundic muscle or
proximal colon preparation, or membrane preparation thereof. A
suitable intestinal smooth muscle preparation is a guinea pig ileal
preparation prepared by the methods described in the Example.
[0099] In another embodiment, the preparation is a cell line that
expresses prokineticin receptor, or membrane preparation thereof. A
cell line that expresses prokineticin receptor can be identified by
methods known in the art, such as the competitive binding assays
described in the Example. An exemplary cell line that expresses
prokineticin receptor is the melanoma cell line M2A7 (available
from American Type Culture Collection as ATCC CRL-2500). Other cell
lines that express prokineticin receptor include M2 melanoma cells
(Cunningham et al., Science 255;325-327 (1992)) and RC-4B/C
pituitary tumor cells (ATCC CRL-1903).
[0100] A suitable control cell line that does not express
prokineticin receptor is HEK293 (available from American Type
Culture Collection as CRL-1573). Other control cell include COS-7,
COS-1, Ltk-, NIH3T3, C6, NS10Y and HT-29 cells.
[0101] Appropriate assays to identify receptor ligands are known in
the art. Such assays can involve directly determining binding of
the candidate compound to the receptor preparation. Direct assays
are suitable when an appropriate control preparation is available
that does not contain the prokineticin receptor. Such assays can
also involve determining the ability of the candidate compound to
compete with a prokineticin polypeptide for binding to the receptor
preparation. Competition assays can be performed by detectably
labeling a candidate compound and competing the compound with an
unlabeled prokineticin polypeptide, or competing an unlabeled
candidate compound with a detectably labeled prokineticin
polypeptide.
[0102] As used herein, the term "detectably labeled" refers to
derivation with, or conjugation to, a moiety that is detectable by
any analytical means. An exemplary detectable moiety is a
radioisotope (e.g. .sup.14C, .sup.131I, .sup.32P or .sup.3H),
fluorochrome (e.g. fluoroscein, green fluorescent protein),
ferromagnetic substance, or luminescent substance. Methods of
detectably labeling organic and inorganic compounds with such
moieties are well known in the art.
[0103] An exemplary competitive binding assay suitable for
detecting a prokineticin receptor ligand is described in the
Example, below. Other suitable receptor binding assays, including
high-throughput assays, are described, for example, in
Mellentin-Micelotti et al., Anal. Biochem. 272:P182-190 (1999);
Zuck et al., Proc. Natl. Acad. Sci. USA 96:11122-11127 (1999); and
Zhang et al., Anal. Biochem. 268;134-142 (1999).
[0104] Other suitable assays for detecting binding include, for
example, scintillation proximity assays (SPA) (Alouani, Methods
Mol. Biol. 138:135-41 (2000)), UV or chemical cross-linking (Fancy,
Curr. Opin. Chem. Biol. 4:28-33 (2000)), competition binding assays
(Yamamura et al., Methods in Neurotransmitter Receptor Analysis,
Raven Press, New York, 1990), biomolecular interaction analysis
(BIA) such as surface plasmon resonance (SPR) (Weinberger et al.,
Pharmacogenomics 1:395-416 (2000)), mass spectrometry (MS)
(McLafferty et al., Science 284:1289-1290 (1999) and Degterev, et
al., Nature Cell Biology 3:173-182 (2001)), nuclear magnetic
resonance (NMR) (Shuker et al., Science 274:1531-1534 (1996),
Hajduk et al., J. Med. Chem. 42:2315-2317 (1999), and Chen and
Shapiro, Anal. Chem. 71:669A-675A (1999)), and fluorescence
polarization assays (FPA) (Degterev et al., supra, 2001). An
appropriate binding assay can be chosen depending on the nature and
purity of the receptor preparation and the number and nature of the
candidate compounds.
[0105] A compound that is determined to be a prokineticin receptor
ligand can further be tested to determine whether it is an agonist
or antagonist of prokineticin receptor. Likewise, a compound that
is determined to be a prokineticin receptor ligand can further be
tested to determine whether it modulates, either positively or
negatively, GI smooth muscle contractility, using an in vitro or in
vivo assay known in the art, such as the assays described
herein.
[0106] The invention further provides a method of identifying a
prokineticin receptor agonist. The method is practiced by
contacting a preparation containing a prokineticin receptor with
one or more candidate compounds, and identifying a compound that
selectively promotes production of a prokineticin receptor signal.
Such a compound is characterized as a prokineticin receptor
agonist.
[0107] The invention also provides a method of identifying a
prokineticin receptor antagonist. The method is practiced by
contacting a preparation containing a prokineticin receptor with
one or more candidate compounds in the presence of a prokineticin,
and identifying a compound that selectively inhibits production of
a prokineticin receptor signal. Such a compound is characterized as
a prokineticin receptor antagonist. Using the invention method,
prokineticin mutants designated SEQ ID NOS:16 and 18 were
identified as prokineticin receptor antagonists.
[0108] The invention methods can be performed in the presence of a
suitable concentration of a prokineticin, such as within 10-fold of
its EC.sub.50. Thus, an agonist that competes with prokineticin for
signaling through the prokineticin receptor, or indirectly
potentiates the signaling activity of prokineticin, can be readily
identified. Likewise, an antagonist that prevents prokineticin from
binding the prokineticin receptor, or indirectly decreases the
signaling activity of prokineticin, can also be identified.
[0109] As used herein, the term "prokineticin receptor agonist"
refers to a molecule that selectively activates or increases normal
signal transduction through the prokineticin receptor. As used
herein, the term "prokineticin receptor antagonist" refers to a
compound that selectively inhibits or decreases normal signal
transduction through the prokineticin receptor.
[0110] For therapeutic applications, a prokineticin receptor
agonist preferably has an EC.sub.50, and a prokineticin receptor
antagonist preferably has an IC.sub.50, of less than about
10.sup.-7 M, such as less than 10.sup.-8 M, and more preferably
less than 10.sup.-9 or 10.sup.-10 M. However, depending on the
stability, selectivity and toxicity of the compound, a prokineticin
receptor agonist with a higher EC.sub.50, or a prokineticin
receptor antagonist with a higher IC.sub.50, can also be useful
therapeutically.
[0111] As described herein, the endogenous prokineticin receptor
appears to be a G-protein coupled receptor. Signaling through the
prokineticin receptor promotes intracellular calcium ion
mobilization, suggesting that the prokineticin receptor normally
couples to G.alpha.q-containing G proteins. Therefore, signaling
through the prokineticin receptor can be detected by any assay
known in the art that detects intracellular calcium ion
mobilization. Such an assay can be performed in the presence or
absence of a prokineticin.
[0112] A suitable preparation for detecting calcium ion
mobilization can be a tissue or cell line expressing the
prokineticin receptor, such as an intestinal smooth muscle
preparation, or the M2A7 cell line.
[0113] Calcium ion mobilization can conveniently be measured using
detectably labeled Ca.sup.2+ ion indicators, such as fluorescently
labeled or radiolabeled indicators, and suitable detection systems.
Exemplary Ca.sup.2+ ion indicators include FLUO-3 AM, FLUO-4 AM,
FURA-2, INDO-1, FURA RED, CALCIUM GREEN, CALCIUM ORANGE, CALCIUM
CRIMSON, BTC, and OREGON GREEN BAPTA (see, for example, Grynkiewitz
et al., J. Biol. Chem. 260:3440-3450 (1985); Sullivan et al., in
Calcium Signal Protocol, Methods in Molecular Biology 114: 125-133,
Edited by David G. Lambert, Human Press, Totowa, N.J. (1999);
Miyawaki et al., Proc. Natl. Acad. Sci. USA 96:2135-2140 (1999);
and Coward et al., Analyt. Biochem. 270:242-248 (1999)). A suitable
detection system for monitoring calcium ion mobilization is the
FLIPR (Fluorometric Imaging Plate Reader) system available from
Molecular Devices.
[0114] The specificity of G.alpha. subunits for cell-surface
receptors is determined by the C-terminal five amino acids of the
G.alpha.. Thus, a variety of signal transduction pathways can be
assayed to determine transduction of a G-protein coupled signal by
a prokineticin receptor, by recombinantly expressing a chimeric
G.alpha. containing the five C-terminal residues of a G.alpha.
known or predicted to couple to ADP-glucose receptor (such as
G.alpha. q or a promiscuous G.alpha. such as G.alpha.16), with the
remainder of the protein corresponding to a G.alpha. that couples
to the signal transduction pathway to be assayed (e.g. G.alpha.s,
to assay increased cAMP production, or G.alpha.q to assay
intracellular Ca.sup.2+ mobilization). Based on the known sequences
of G.alpha. subunits, nucleic acid molecules encoding chimeric
G.alpha. can be constructed and expressed by methods known in the
art and described, for example, in Conklin et al., Nature
363:274-276 (1993), and Komatsuzaki et al., FEBS Letters
406:165-170 (1995).
[0115] Thus, depending on the G.alpha. subunit endogenously or
recombinantly expressed in the assay system, prokineticin receptor
signals that can be determined include, but are not limited to,
calcium ion mobilization; increased or decreased production or
liberation of arachidonic acid, acetylcholine, diacylglycerol,
cGMP, cAMP, inositol phosphate and ions; altered cell membrane
potential; GTP hydrolysis; influx or efflux of amino acids;
increased or decreased phosphorylation of intracellular proteins;
and activation of transcription of an endogenous gene or
promoter-reporter construct downstream of any of the
above-described second messenger pathways.
[0116] Suitable assays for detecting agonistic and antagonistic
activity of G protein coupled receptors, including high-throughput
signaling assays, are well known in the art and reviewed, for
example, in reviewed, for example, in Tate et al., Trends in
Biotech. 14:426-430 (1996).
[0117] Assay methods for identifying compounds that selectively
bind to or modulate signaling through a prokineticin receptor (e.g.
ligands, agonists and antagonists) generally involve comparison to
a control. One type of a "control" is a preparation that is treated
identically to the test preparation, except the control is not
exposed to the candidate compound. Another type of "control" is a
preparation that is similar to the test preparation, except that
the control preparation does not express the receptor, or has been
modified so as not to respond selectively to prokineticin. In this
situation, the response of the test preparation to a candidate
compound is compared to the response (or lack of response) of the
control preparation to the same compound under substantially the
same reaction conditions.
[0118] A compound that is determined to be a prokineticin receptor
agonist or antagonist can further be tested to determine whether it
modulates, either positively or negatively, GI smooth muscle
contractility, using an in vitro or in vivo assay known in the art,
such as the assays described herein.
[0119] The invention also provides compositions suitable for use in
assays to identify prokineticin ligands, agonists and antagonists.
Suitable compositions contain a cell or tissue containing a
prokineticin receptor and a prokineticin polypeptide, which
optionally can be detectably labeled. An exemplary composition
comprises a GI smooth muscle preparation, such as an ileal smooth
muscle preparation. A further exemplary composition comprises a
cell line, such as M2A7.
[0120] The prokineticin polypeptides described herein, as well as
prokineticin ligands, agonists and antagonists identified by the
described screening methods, are potential therapeutic compounds
that can be administered to individuals with conditions associated
with abnormal gastrointestinal motility, or other conditions
associated with altered expression, or activity of a prokineticin
or its receptor. The invention compounds can be formulated and
administered in a manner and in an amount appropriate for the
condition to be treated; the weight, gender, age and health of the
individual; the biochemical nature, bioactivity, bioavailability
and side effects of the particular compound; and in a manner
compatible with concurrent treatment regimens. An appropriate
amount and formulation for a particular therapeutic application in
humans can be extrapolated based on the activity of the compound in
the in vitro binding and signaling assays described herein, or from
recognized animal models of the particular disorder.
[0121] The total amount of therapeutic compound can be administered
as a single dose or by infusion over a relatively short period of
time, or can be administered in multiple doses administered over a
more prolonged period of time. Additionally, the compound can be
administered in a slow-release matrice, which can be implanted for
systemic delivery at or near the site of the target tissue.
Contemplated matrices useful for controlled release of therapeutic
compounds are well known in the art, and include materials such as
DepoFoam.TM., biopolymers, micropumps, and the like.
[0122] The therapeutic compounds can be administered to a mammal by
routes known in the art including, for example, intravenously,
intramuscularly, subcutaneously, intraorbitally, intracapsularly,
intraperitoneally, intracisternally, intra-articularly,
intracerebrally, orally, intravaginally, rectally, topically,
intranasally, or transdermally. Preferred routes for human
administration are oral and intravenous administration, with oral
routes particularly preferred.
[0123] Preferably, the therapeutic compounds are administered to a
mammal as a pharmaceutical composition comprising the compound and
a pharmaceutically acceptable carrier. The choice of
pharmaceutically acceptable carrier depends on the route of
administration of the compound and on its particular physical and
chemical characteristics. Pharmaceutically acceptable carriers are
well known in the art and include sterile aqueous solvents such as
physiologically buffered saline, and other solvents or vehicles
such as glycols, glycerol, oils such as olive oil and injectable
organic esters. A pharmaceutically acceptable carrier can further
contain physiologically acceptable compounds that stabilize the
compound, increase its solubility, or increase its absorption. Such
physiologically acceptable compounds include carbohydrates such as
glucose, sucrose or dextrans; antioxidants, such as ascorbic acid
or glutathione; chelating agents; and low molecular weight
proteins.
[0124] For applications that require the compounds and compositions
to cross the blood-brain barrier, or to cross cell membranes,
formulations that increase the lipophilicity of the compound are
particularly desirable. For example, the compounds of the invention
can be incorporated into liposomes (Gregoriadis, Liposome
Technology, Vols. I to III, 2nd ed. (CRC Press, Boca Raton Fla.
(1993)). Liposomes, which consist of phospholipids or other lipids,
are nontoxic, physiologically acceptable and metabolizable carriers
that are relatively simple to make and administer.
[0125] In one embodiment, a pharmaceutical composition containing a
prokineticin polypeptide or a prokineticin agonist is administered
to a mammal in an effective amount to stimulate gastrointestinal
motility. Impaired GI motility is a common clinical manifestation
of a variety of disorders, including irritable bowel syndrome,
diabetic gastroparesis, postoperational ileus, chronic
constipation, and gastrointestinal reflux disease, and the
compositions of the invention can thus be used to ameliorate the
symptoms of such disorders.
[0126] In another embodiment, a pharmaceutical composition
containing a prokineticin antagonist is administered to a mammal in
an effective amount to inhibit gastrointestinal motility. Enhanced
GI motility is associated with diarrhea, which is a common symptom
of infectious diseases, malabsorptive disorders, inflammatory bowel
disorders, and intestinal cancers, and antagonistic compositions of
the invention can thus be used to ameliorate the symptoms of such
disorders.
[0127] Injection of Bv8 or MIT1 into the brain ventricles of rats
leads to hyperalgesia (Mollay et al., Eur J Pharmacol. 374:189-196
(1999)). Therefore, prokineticin antagonists (e.g. prokineticin
antibodies, mutant polypeptides comprising SEQ ID NOS:16 or 18, and
other compounds determined by the methods described herein) can be
administered to a mammal in an effective amount to act as an
analgesic (pain killer).
[0128] Those skilled in the art can determine other conditions for
which it is appropriate to administer a pharmaceutical composition
of the invention, and can monitor the safety and efficacy of the
therapy.
[0129] Preferably, the mammal administered a pharmaceutical
composition of the invention is a human, but for certain
applications the mammal can alternatively be a veterinary animal or
a research animal. For example, in preclinical studies, the methods
of the invention can be practiced with animals that serve as
credible models of human disease, such as non-human primates, pigs,
dogs, cats, and rodents (e.g. rats, mice and guinea pigs). Those
skilled in the art understand which animals serve as appropriate
models for a human disease of interest.
[0130] The following examples are intended to illustrate but not
limit the present invention.
EXAMPLE I
Identification, Preparation and Characterization of Prokineticins 1
and 2
[0131] This example shows the cloning, recombinant expression,
purification and biological activities of human prokineticins 1 and
2, as well as modifications thereof.
Materials and Methods
RNA Blot
[0132] Human multiple tissue RNA blots containing normalized
samples of polyA RNA were used as described by the manufacture's
instructions (Clontech). The blots were probed with random
primer-labeled probes (nucleotides 1-550 and 1-1178 for
prokineticin 1 and prokineticin 2 cDNAs), and signals were
visualized by exposing to Kodak XAR film.
Production, Refolding and Purification of Recombinant
Prokineticins
[0133] The coding sequences for mature prokineticins were cloned
into prokacyotic expression vector pGEX-3X (Pharmacia). The extra
nucleotides between the factor Xa protease digestion site of GST
(Glutathione-s-transferase) tag and mature prokineticins were
removed by site-directed mutagenesis and confirmed by sequencing.
To facilitate protein purification, a 6XHis-tag was added to the
C-terminus so that the fusion proteins could be purified with
Ni-NTA affinity chromatography (Qiagen).
[0134] The method for production of fusion proteins is as follows.
The E. coli cells (BL21) were grown to OD 0.8 and induced with 600
nM IPTG for 2 hours at 37.degree. C. The cells were then pelleted,
washed, and lysed with buffer A (6 M guanidine hydrochloride, 100
mM NaH.sub.2PO.sub.4 and 10 mM Tris, pH 8.0). Fusion proteins were
allowed to bind to Ni-NTA beads and then washed extensively with
buffer C (8 M urea, 100 mM NaH.sub.2PO.sub.4, and 10 mM Tris, pH
6.3) and buffer D (8M urea, 100 mM NaH.sub.2PO.sub.4, and 10 mM
Tris, pH 5.9). Fusion protein-bound beads were equilibrated with
factor Xa digestion buffer (50 mM Tris, 150 nM NaCl, and 1 mM CaCl,
pH 7.5). Factor Xa digestion was performed overnight at room
temperature with 10 ng/.mu. fusion protein. Cleaved GST tag was
then washed away with buffer D. Mature prokineticins were then
eluted with buffer E (8 M urea, 100 mM NaH.sub.2PO.sub.4, and 10 mM
Tris, pH 4.5). Fractions were analyzed by SDS-PAGE. The pooled
recombinant prokineticins were then refolded as follows. Proteins
were diluted to 100 .mu.g/ml with buffer E, and dialyzed against
renaturing buffer (4 M urea, 5 mM cysteine, 0.02% Tween-20, 10%
glycerol, 10 mM Tris, 150 mM NaCI, 100 mM NaH.sub.2PO.sub.4, pH
8.3). New renaturing buffer (same component except 2 M urea) was
then added, and dialysis was continued for four more days with at
least one more change of renaturing buffer. The refolded protein
was then desalted with a spin column (Qiagen) and analyzed by
receptor binding or bioassay. The final purification was performed
with reverse phase-HPLC (LKB). Functional proteins were eluted with
0.08% trifluoroacetic acid and 10-50% acetonitrile gradient. The
elution of protein was monitored at 206 nm. Trifluoroacetic acid
and acetonitrile were then evaporated by lyophilization.
Mass Spectrometry
[0135] The electrospray ionization mass spectrometry was performed
with a 6.5 T HiResESI Fourier Transform mass spectrometer (IonSpec,
Irvine, Calif.) as previously described (Li et al., Anal. Chem.
66:2077-2083 (1994)). Protein eluted from RP-HPLC was lyophilized
and dissolved in nanopure water and then diluted to a concentration
of 1 nM with methanol-water- acetic acid (49.5%:49.5%:1%, v/v/v).
100 .mu.l of sample was infused.
Measurement of Smooth Muscle Contraction in Isolated Organ
Preparations
[0136] Guinea pigs were euthanized with CO.sub.2, and a section of
ileum (2-3 cm) approximately 10 cm rostral to the cecum was
removed. The ileum was washed clean with Krebs-Ringer bicarbonate
(KRB) buffer (124 mM NaCl, 5 mM KCl, 1.3 mM MgSO.sub.4, 26 nM
NaHCO.sub.3, 1.2 mM KH.sub.2PO.sub.4, 1.8 mM CaCl, and 10 mM
glucose) and mounted longitudinally in an organ bath containing KRB
buffer. Isometric contractions were measured with a
force-displacement transducer and polygraph as described previously
(Thomas et al., Biochem. Pharmacol. 51:779-788 (1993)). The ileum
was allowed to incubate for 1 hr, and then three test doses of the
muscarinic agonist, oxotremorine-M, were added to ensure that the
contractions were reproducible and of sufficient magnitude. The
ileum was washed and allowed to rest for 5 min between each test
dose. The longitudinal fundic strip and the zig-zag tracheal
preparation were prepared as described previously (15). Isolated
colon (proximal and distal) was prepared as described (Sawyer et
al., J. Pharmacol. Exp. Ther. 284:269-277 (1998)). Aorta and
femoral artery were taken from adult rats. A 10 ml bath was used
for aorta and femoral artery experiments. Tension was recorded on a
Grass polygraph with initial preloads of 0.5 g for intestinal
tissues and tracheal preparations and 2 g for aorta and femoral
artery.
Iodination
[0137] Prokineticin 1 was iodinated by the iodogen method as
described (Fraker and Speck, Biochem. Biophys. Res. Commun.
80:849-857 (1978)). Briefly, refolded prokineticin 1 (7.5 .mu.g)
was incubated with 50 .mu.g of iodogen in 50 .mu.L of 0.5 M PBS
buffer, pH 7.2 for 15 minutes at room temperature. The reaction was
stopped by removal of the mixture from the iodogen tube and placing
it in a microfuge tube with 100 .mu.L of PBS containing 1 mM NaI.
Following the addition of 100 .mu.L of PBS with 1 mM NaI and 0.1%
BSA, the free iodine was removed by gel filtration on Bio-Gel P2
and the radioactivity was counted. Assuming all the radioactivity
was incorporated into 6.0 .mu.g prokineticin 1 recovered (80%
recovery rate), specific radioactivity was calculated as 819
cpm/fmol or 372 Ci/mole.
Receptor Binding
[0138] Membranes were prepared from guinea pig ileum as described
(Li et al., Mol. Pharmacol. 57:446-452 (2000)), except additional
steps of differential centrifugation (800 g, 10,000 g, 100,000 g,
4.degree. C., 20 min each) were applied to reduce the background
binding. Incubation was performed in 4 ml in 20 mM Tris-HCl pH 7.4
buffer containing 0.1% BSA at room temperature. For saturation
binding, 1.5-200 pM of labeled prokineticin 1 was used.
Non-specific binding was defined in the presence of 20 nM unlabeled
prokineticin 1. For displacement experiments, unlabeled protein was
pre-incubated with membrane in 3 ml total reaction volume for 1 hr,
then .sup.125I-prokineticin 1 (20 pM) was added. The membrane was
incubated for an additional 3 hrs at room temperature. The binding
mixture was filtered through GF-C glass filters and washed with 10
ml of 20 mM Tris-HCI, pH 7.4. Radioactivity retained on filters was
counted in gamma counter. The data were analyzed with the LIGAND
program.
Results
Identification and Analysis of Two Mammalian Homologues for Frog
Bv8 and Snake MIT1
[0139] In an effort to identify mammalian homologues of frog Bv8
and snake MIT1, multiple databases (EST and HGTS) were searched
using the BLAST 2.1 algorithm (Altschul et al. Nucleic Acids Res.
25:3389-3400 (1997)), with their protein sequences as queries. A
search of the EST database revealed the presence of two human EST
sequences (ai277349 and aa883760). Sequence analysis of these two
EST clones revealed that aa 883760 encodes a predicted protein
(Heijne Nucleic Acids Res. 14:4683-4690 (1986)) with a signal
peptide of 19 amino acids and a mature protein of 86 amino acids.
Clone ai277349 was found to be a partial cDNA. Full-length sequence
for EST clone ai277349, cloned by 5' RACE with human brain cDNA as
template, was found to contain a signal peptide of 27 amino acids
and a mature protein of 81 amino acids (FIG. 1). These proteins
were respectively named as prokineticin 1 and prokineticin 2 (see
below).
[0140] Sequence analysis reveals that prokineticin 1 and 2 have
about 44% amino acid identity, including ten conserved cysteines.
Both prokineticins possess about 43% identity with frog Bv8 and
snake MIT1. Interestingly, the N-terminal sequences before the
first cysteine (AVITGA) is completely conserved among all species
(FIG. 1), suggesting the functional significance of this region.
Preliminary analysis of the mouse prokineticin 1 gene indicates
that the N-terminal sequence AVITG is derived from the first exon
that also contains the signal peptide sequence, whereas the
cysteine-rich sequences are from other exon(s).
Prokineticins are Expressed in Various Adult and Embryonic
Tissues
[0141] As an initial survey of prokineticin expression, a human
masterr blot was probed using fragments of human prokineticin
cDNAS. Both prokineticins were widely expressed in various adult
tissues, with a generally higher expression level of prokineticin 1
compared to prokineticin 2 (FIG. 2A, 2B). The exception was found
in GI tract, liver and spleen, whereas prokineticin 2 expression
seemed comparable to that of prokineticin 1. The highest level of
prokineticin 1 expression is found in testis and placenta. Among
human fetal tissues, all showed a similar level of expression,
again with an expression level of prokineticin 1 higher than that
of prokineticin 2.
[0142] The expression of prokineticins in human brain was further
examined by Northern blot analysis. FIG. 2D showed that
prokineticin 1 mRNA size is about 1.5 kb with the highest
expression in the putamen, thalamus, temporal lobe, and corpus
callosum. Prokineticin 2 expression in human brain was undetectable
(data not shown).
Production, Refolding and Purification of Human Prokineticins
[0143] As the N-terminal sequences were completely conserved (FIG.
1), recombinant proteins with authentic N-terminal residue were
produced first as GST-fusion proteins, followed by digestion with
protease factor Xa to remove the GST tag. FIG. 3 shows that a
protein with correct molecular weight was produced by factor Xa
digestion.
[0144] Bioassay with guinea-pig ileum preparations revealed the
unfolded recombinant proteins were inactive. As NMR examination
indicated that 10 cysteines of MIT1 are formed into 5 disulfide
bonds (Boisbouvier et al., J. Mol. Biol. 283:205-219 (1998)) and
these 10 cysteines are all conserved in human prokineticins, it was
considered that these disulfide bonds were probably essential for
protein bioactivities. Thus considerable effort was devoted to
ensure proper disulfide bond formation (out of 945 possible
combinations).
[0145] Initial refolding in a single dilution into refolding buffer
was unsuccessful, as almost all recombinant proteins were
precipitated, probably due to the formation of inter-molecular
disulfide bonds. A series of modifications to control protein
aggregation and to slow disulfide bond formation were then adopted.
These modifications included: 1) reduction of protein concentration
to 100 .mu.g/ml or less to favor forming intra- but not
inter-molecular disulfide bonds; 2) refolding proteins by dialysis
method instead of direct dilution; 3) using higher levels of urea
(4 M and then 2 M) in all dialysis buffers; 4) omitting oxidants
cystine or oxidized glutathione from redox pairs, leaving only 5 mM
cysteine or 3 mM reduced glutathione; 5) adding glycerol to further
reduce protein aggregation; 6) cooling proteins and buffers to
4.degree. C. before initiating the refolding process. These
carefully controlled steps allowed the successful refolding of
recombinant prokineticins with minimal protein aggregation.
[0146] The refolded proteins were finally purified by RP-HPLC (FIG.
3A, lane 5). Mass spectrometry confirmed the formation of five
disulfide bonds in refolded recombinant prokineticin 1. The
molecular weight of 6XHis-tagged prokineticin 1, determined with a
Fourier transform mass spectrometer, was found to be 10480.30 Da
(FIG. 3C). As the calculated molecular weight with all ten
cysteines present in reduced form was 10490.20, five pairs of
disulfide bonds were clearly formed.
Refolded Recombinant Prokineticins Potently Contract
Gastrointestinal Smooth Muscle
[0147] The refolded recombinant prokineticins were then tested on
isolated smooth muscle preparations. FIG. 4 shows that both
recombinant prokineticin 1 and prokineticin 2 potently stimulated
the contraction of guinea-pig ileum longitudinal muscle with ED50
values of about 0.46 and 0.90 nM, respectively. Prokineticin 1 (5
nM) also stimulated the contraction of fundic muscle strip and
proximal colon, but had no effect on distal colon (25 nM, data not
shown). Recombinant prokineticin 1 (25 nM) also had no effect on
other smooth muscle tissues, including aorta and femoral artery,
trachea and gallbladder. Thus, the contractile effect of
prokineticins appears to be specific for GI smooth muscle.
[0148] To probe the possible signaling mechanisms of prokineticins,
a number of kinase and ion channel inhibitors were tested.
Tetrodotoxin (TTX), which is known to block nerve action potential
propagation, had no effect on prokineticin 1-stimulated ileum
longitudinal muscle contraction (FIG. 4B), indicating that
prokineticin 1 acts directly on the smooth muscle. The contractile
mechanism of prokineticin was further investigated with a number of
compounds, including the protein kinase C inhibitor calphostin C (1
.mu.M), the phospholipase A2 inhibitor 7,
7-dimethyl-(5Z,8Z)-eicosa-dienoic acid (10 .mu.M), the tyrosine
kinase inhibitor genistein (5 .mu.M), the MEK inhibitor PD 098059
(10 .mu.M) and L-type calcium channel blocker verapamil. Only
verapamil was effective, with 1 .mu.M completely inhibiting the
contractile effect of 2 nM prokineticin I (FIG. 4C). The same
concentration of verapamil also completely blocked the contractile
action of 100 nM oxotremorine-M (FIG. 4F). This result indicates
that, like muscarinic M3 receptor mediated contraction of the ileum
(Eglen et al., Pharmacol. Rev. 48:531-565 (1996) and Ehlert et al.,
Muscurinic Receptors and Gastrointestinal Smooth Muscle, ed. Eglen,
CRC Press, pgs 92-147 (1997)), calcium entry via the voltage-gated
calcium channel is an essential component of prokineticin
signaling.
Bioactivities of Prokineticins are Mediated by Membrane
Receptors
[0149] The potent contractile action of recombinant prokineticins
on guinea-pig GI smooth muscle and the inhibitory effect of the
calcium channel blocker verapamil suggest a receptor-mediated
mechanism for prokineticins. To provide direct evidence that
prokineticins are interacting with selective membrane receptors,
recombinant prokineticin was labeled with .sup.125I and receptor
binding experiments were carried out.
[0150] Scatchard analysis indicated that the specific binding of
prokineticin 1 was best fitted with two-site model (F=38.78,
P<0.001 versus one site model; FIG. 5A). The high- and
low-affinity constants (K.sub.d) were 5.0.+-.0.8 pM and 227.+-.63
pM (n=3), respectively. The B.sub.max for high- and low-affinity
sites were 7.8.+-.1.2 and 26.4.+-.8.4 fmol/mg of protein,
respectively (n=3). Competition experiments revealed that the
specific binding was displaced by recombinant prokineticin 1. The
displacement curves were also best fitted with two-site model (with
K.sub.i of 8.0.+-.3.9 pM, and 1.50.+-.0.9 nM, n=3 for high- and
low-affinity sites, respectively) (FIG. 5B). FIG. 5B also shows
that prokineticin 2 displaced labeled prokineticin 1 with similar
affinity (K.sub.i of 4.2 pM for high affinity and 1.22 nM for low
affinity site, average of two experiments).
[0151] Because agonist binding to many G protein-coupled receptors
is inhibited by GTP, it was investigated whether GTP.UPSILON.S had
any effect on specific .sup.125I-labeled prokineticin 1 binding. As
shown in FIG. 5B, GTP.UPSILON.S caused a concentration-dependent
inhibition of .sup.125I-prokineticin 1 binding. At the highest
concentration tested (10 .mu.M), GTP.UPSILON.S displaced 85% of the
specific prokineticin binding to ileal membranes. These results
suggest that prokineticin receptor(s) belong to the G
protein-coupled receptor family.
Stability of Prokineticins
[0152] Experiments were also performed to determine the half-life
of prokineticins. The half-life of intravenously injected iodinated
human prokineticin 1 was approximately 3 hours, compared to 10 min
for motilin, a small peptide that also increases GI motility. A
reasonably long half-life in the blood circulation is crucial for
achieving therapeutic effect. Therefore, prokineticins are likely
to be effective as therapeutics.
Structure/Activity Relationship Studies of Prokineticins
[0153] Sequence analysis indicated that prokineticins may contain
two functional domains, namely the short N-terminus and the
cysteine-rich C-terminus. As the N-terminal sequences preceding the
first cysteine are completely conserved among prokineticins (FIG.
1), it was predicted that this region has functional
importance.
[0154] In addition to prokineticins, the ten-cysteine motif is also
found in a number of secreted proteins, including colipase, a
cofactor for intestinal lipid digestive enzyme lipase, and
dickkopfs, a family of proteins that have an important role in
early embryonic development.
[0155] A number of N-terminal substitution, deletion, and insertion
mutants were constructed, and recombinant, refolded proteins
produced. Bioassays with ileal smooth muscle preparations revealed
that these mutant proteins at concentrations up to 250 nM are not
able to elicit contractions (Table 1). However, an N-terminal
deletion mutant (SEQ ID NO:16) and an N-terminal insertion mutant
(SEQ ID NO:18) were able to weakly antagonize the contractile
effect of prokineticin 1. Therefore, N-terminal variants of
prokineticins, such as SEQ ID NOS:16 and 18, are potential
therapeutics for inhibiting GI contractility.
1 TABLE 1 Contrac- Antago- tile nistic Polypeptide Activity
Activity Wild Type AVITGA [Prokineticin 1] + - (SEQ ID NO:3)
Insertion GILAVITGA [Prokineticin 1] - NO (SEQ ID NO:15) Deletion
VITGA [Prokineticin 1] - + (SEQ ID NO:16) Substitution AAAAAA
[Prokineticin 1] - NO (SEQ ID NO:17) Insertion MAVITGA
[Prokineticin 1] - + (SEQ ID NO:18) Chimera AVITGA [Co-lipase] - -
Chimera AVITGA [dickkopf4] - NO peptide AVITGACERDVQCG - - (SEQ ID
NO:19) Cys mutation AVITGA [Prokineticin 1] 18S - - Cys mutation
AVITGA [Prokineticin 1] 60R - -
[0156] Chimeric recombinant proteins containing N-terminal
sequences from prokineticin 1 and the C-terminal ten-cysteine
domain from either colipase or Dickkopf 4 were also constructed.
These two chimeric recombinant proteins were non-functional when
tested with ileal smooth muscle preparation at concentrations up to
250 nM. Also tested was an N-terminal peptide (SEQ ID NO:19), which
also was non-functional.
[0157] These results indicate that both N-terminal conserved
sequence and C-terminal cysteine-rich domain are essential for the
contractile activity of prokineticins.
Chimeric Prokineticins
[0158] A search of the draft human genome database with
prokineticin cDNAs as queries revealed that genes encoding
prokineticin 1 and 2 are composed of three exons. The signaling
peptide and N-terminal conserved AVITG sequence are encoded in the
first exon, while the cysteine-rich domain is encoded by exons 2
and 3. The 21 amino acid insertion of prokineticin 2 is encoded by
an alternatively spliced mini-exon. To explore the functional
difference of prokineticin 1 and 2, chimeric polypeptides were made
with their exons 3 swapped (see FIG. 6). The chimeric polypeptides
were designated chimera 12 (SEQ ID NO:13) and chimera 21 (SEQ ID
NO:14), designating the swapped exons, as shown in FIG. 6.
[0159] Functional assays of refolded chimeric prokineticins 12 and
21 indicated that both of these chimeric polypeptides are active in
contracting GI smooth muscle (FIG. 7A). However, the EC.sub.50 for
the chimeric prokineticin 21 (SEQ ID NO:14) was about 8-fold higher
than prokineticin 1 or prokineticin 2. Additionally, although the
peak contractions were not affected, chimeric prokineticin
polypeptides resulted in prolonged contraction of ileal strips
(FIG. 7B). For wild type prokineticins, the time constants to
midway contraction (half way from peak contraction to sustained
plateau) were about 15 mins. In contrast, for the chimeric
polypeptides, these time constants were prolonged to about 40
mins.
[0160] These results suggest that the chimeric prokineticins
interact slightly differently with the receptor than wild type
prokineticins, and cause less pronounced tachyphylaxis. Thus, the
chimeric prokineticins (SEQ ID NOS:13 and 14) may have more potent
pharmacological activity in vivo than wild-type prokineticins.
Effects of Prokineticin on Guinea Pig Ileum Smooth Muscle In
Vivo.
[0161] To monitor the effects of prokineticin on the contraction of
ileal smooth muscle in vivo, extraluminal force transducers were
implanted on the serosal surface of the guinea pig ileum.
Recombinant prokineticin 1 was then administered as a bolus into
the jugular vein over a 10-second period. As shown in FIG. 8, an
intravenous bolus of prokineticin 1 contracts guinea pig ileal
smooth muscle in a dose-dependent manner. The threshold dose of
prokineticin 1 is about 0.03 .mu.g/kg, and a dose of 30 .mu.g/kg
produces the maximum effect.
[0162] Therefore, prokineticins, demonstrated above to be able to
contract ileal smooth muscle in ex vivo preparations, are also
effective in vivo.
Prokineticin Signal Transduction
[0163] To probe the potential signaling mechanisms of
prokineticins, cell lines were identified that express prokineticin
receptor endogenously. Over twenty cell lines were screened for
binding to iodinated prokineticin 1. One cell line, M2A7 melanoma
cells (ATCC CRL-2500; Cunningham et al., Science 255;325-327
(1992)), clearly displayed specific binding, with a receptor level
of about 150 fmole/mg protein. Other cell lines that specifically
bound prokineticin included M2 melanoma cells (Cunningham et al.,
Science 255;325-327 (1992)) and RC-4B/C pituitary tumor cells (ATCC
CRL-1903). Cell lines that did not bind prokineticin included
HEK293, COS-7, COS-1, Ltk-, NIH3T3, C6, NS10Y and HT-29 cells.
[0164] To assess signaling in M2A7 cells, cytosolic calcium was
measured by fura-3 fluorescence using a FLIPR system (Fluorometric
Imaging Plate Reader; Molecular Devices). Cells were suspended in
HEPES medium and incubated with 2 .mu.M of fura-3 AM for 20 min at
31.degree. C. The cells were then centrifuged, washed, resuspended
in fura-3-free medium and seeded into 96 wells at 4.times.10.sup.4
cells per well. The cells were loaded with Fluo-3 AM (Molecular
Probes) in standard buffer solution (130 mM NaCl,2 mM CaCl.sub.2, 5
mM KCl, 10 mM glucose, 0.45 mM KH.sub.2PO.sub.4, 0.4 mM
Na.sub.2HPO.sub.4, 8 mM MgSO.sub.4, 4.2 mM NaHCO.sub.3, 20 mM HEPES
and 10 .mu.M probenecid) with 0.1% fetal bovine serum for 1 h at
37.degree. C., then washed with standard buffer solution. Transient
changes in [Ca.sub.2+ ].sub.i evoked by prokineticin (0.01, 0.1,
0.3, 1, 3, 10, 100 nM) were monitored using the FLIPR system in
96-well plates at 488 nm for 210 s.
[0165] As shown in FIG. 9, prokineticins can mobilize calcium in
M2A7 melanoma cells, with EC.sub.50 of about 12 and 21 nM for
recombinant prokineticin 1 and prokineticin 2, respectively. The
signaling is specific, as there was no response in HEK 293 cells.
The calcium signaling mobilized by prokineticins is comparable to
calcium signal activated by control MCH (melanin-concentrating
hormone) receptor SLC1 (Saito et al., Nature 400:265-269 (1999)).
The calcium signals elicited by prokineticins are more much robust
than the modest calcium signal induced by activation of a typical
receptor tyrosine kinase. This result is consistent with the
observation, described above, that the tyrosine protein kinase
inhibitor genistein (5 mM) had no effect on the contractile
activity of prokineticin on ileal smooth muscle.
[0166] These results indicate that the prokineticin receptor(s)
is/are likely to be GPCR(s), and to signal through G.alpha.q.
Discussion
[0167] The results described above establish the existence of
mammalian homologues of frog BV8 and snake MIT1. To reflect their
potent and specific effects on GI smooth muscle, these proteins
have been named prokineticins. Their high potency in specifically
stimulating the contraction of guinea-pig ileum smooth muscle but
not other smooth muscles including aorta, femoral artery, trachea,
and gallbladder indicate that prokineticins may be important
endogenous regulators of GI motility. Prokineticins may regulate GI
smooth muscle as neurocrine signaling molecules, or circulating
hormones, or paracrine humoral agents. Since prokineticins are also
widely expressed outside the GI system, it is possible that
prokineticins may be released from remote organs and regulate GI
activity. In this respect, it has also been determined that
prokineticins are resistant to protease treatment, which supports
their potential long-range and long-term effects.
[0168] The molecular size and the processing of prokineticins
distinguish them from typical neuropeptides, and indicate they are
more similar to cytokines. As one mechanism for eliminating
pathogenic organisms is to enhance motility and push the offending
organisms out of the GI tract, prokineticins may also be part of
defending immune response, i.e. functioning as inflammatory
cytokines that increase the GI motility.
[0169] The high potency of recombinant prokineticins on GI
contractility suggests that prokineticins probably interact with
cell surface receptor(s). This conclusion is reinforced by the
receptor binding experiments described above, which demonstrate a
saturably high affinity for the iodinated recombinant prokineticin.
Moreover, the observation that 10 .mu.M GTP.UPSILON.S can displace
almost all of the specific binding indicates the involvement of G
protein in prokineticin receptor signaling. Furthermore, the
inhibitory effect of the calcium channel blocker verapamil on the
contractile effect of prokineticin is consistent with a
receptor-mediated mechanism for prokineticins, and also suggests a
similar signaling mechanism of prokineticins as those of the M3
muscarinic and motilin receptor in contracting GI smooth muscle:
calcium entry via voltage-gated calcium channel is an essential
component. Thus, prokineticin receptor most likely is a G protein
coupled receptor.
[0170] However, alternative interpretations are possible. For
instance, prokineticins may cause smooth muscle contraction by
directly activating non-selective cation ion channels, or blocking
inhibitory potassium channels on GI smooth muscle cells.
[0171] Sequence analysis indicates that prokineticins may contain
two functional domains: the short N-terminus and the cysteine-rich
C-terminus. Since the N-terminal sequences preceding the first
cysteine are completely conserved among prokineticins (FIG. 1),
this region is likely to have functional importance. In addition to
prokineticins and their isoforms from other species, a similar
ten-cysteine motif is also found in a number of other secreted
proteins, including colipase, a cofactor for intestinal lipid
digestive enzyme lipase (van Tilbeurgh et al., Nature 359:159-162
(1992)) and dikkopfs, a family of proteins that have important
roles in early embryonic development (Glinka et al., Nature
391:357-362 (1998) and Aravind et al., Curr. Biol. 8:R477-478
(1998)). Interestingly, dickkopfs actually possess two ten-cysteine
domains that have mirror symmetry. X-ray crystallography and
solution structural analysis have demonstrated that MIT1 is formed
of five pairs of disulfide bonds and folded into a structure
similar to colipase (Boisbouvier et al., J. Mol. Biol. 283:205-219
(1998)).
[0172] Successful refolding of proteins with five pairs of
disulfide bonds has not hitherto been accomplished in vitro.
Refolding of proteins with more than three pairs of disulfide bonds
is still regarded as challenging and difficult (Georgiou et al.,
Curr. Opin. Biotechnol. 7:190-197 (1996) and Lihe et al., Curr.
Opin. Biotechnol. 9:497-501 (1998)). The expression of such
disulfide bond-rich proteins in E. coli often results in no
formation of disulfide bonds, or more probably the formation of
incorrect intramolecular or intermolecular disulfide bonds. These
events routinely lead to production of inactive recombinant
proteins and their aggregation in bacterial inclusion bodies.
[0173] In this study, a slow exchange method was utilized to refold
prokineticins that have five pairs of disulfide bonds. A number of
factors eventually contributed to the successful refolding of
prokineticins: 1) a slow rate of removal of denaturing agent; 2)
using only reducing agents in the redox refolding mixture, allowing
slow formation of disulfide bonds; 3) low temperature; 4) high
concentration of urea and glycerol in dialyzing buffer to prevent
protein aggregation; 5) low concentration of recombinant protein to
favor forming intra- but not inter-molecular disulfide bonds. These
refolding conditions can be used to design protocols for refolding
other recombinant proteins that possess multiple disulfide
bonds.
[0174] In summary, cDNAs encoding two prokineticins have been
described. Refolded recombinant prokineticins potently and
specifically stimulate the contraction of GI smooth muscle. As
impaired GI motility is a very common clinical manifestation in
many common disorders including irritable bowel syndrome, diabetic
gastroparesis, postoperational ileus, chronic constipation, and
gastroesophageal reflux disease (Longo et al., Dis Colon Rectum
36:696-708 (1993); Tonini, Pharmacol. Res. 33:217-226 (1996);
Samsom and Smout, Dig Dis. 15:263-274 (1998); Achem and Robinson,
Dig Dis. 16:38-46 (1998) and Briejer et al., Trends Pharmacol Sci.
20:1-3 (1999)), the discovery of endogenous regulators of GI smooth
muscle should facilitate the development of novel therapeutics for
such disorders that will benefit from altered GI motility.
[0175] All journal article, reference and patent citations provided
above, in parentheses or otherwise, whether previously stated or
not, are incorporated herein by reference in their entirety.
[0176] Although the invention has been described with reference to
the examples provided above, it should be understood that various
modifications can be made without departing from the spirit of the
invention.
Sequence CWU 1
1
19 1 1377 DNA Homo sapiens CDS (55)...(369) 1 ggggaagcga gaggcatcta
agcaggcagt gttttgcctt caccccaagt gacc atg 57 Met 1 aga ggt gcc acg
cga gtc tca atc atg ctc ctc cta gta act gtg tct 105 Arg Gly Ala Thr
Arg Val Ser Ile Met Leu Leu Leu Val Thr Val Ser 5 10 15 gac tgt gct
gtg atc aca ggg gcc tgt gag cgg gat gtc cag tgt ggg 153 Asp Cys Ala
Val Ile Thr Gly Ala Cys Glu Arg Asp Val Gln Cys Gly 20 25 30 gca
ggc acc tgc tgt gcc atc agc ctg tgg ctt cga ggg ctg cgg atg 201 Ala
Gly Thr Cys Cys Ala Ile Ser Leu Trp Leu Arg Gly Leu Arg Met 35 40
45 tgc acc ccg ctg ggg cgg gaa ggc gag gag tgc cac ccc ggc agc cac
249 Cys Thr Pro Leu Gly Arg Glu Gly Glu Glu Cys His Pro Gly Ser His
50 55 60 65 aag gtc ccc ttc ttc agg aaa cgc aag cac cac acc tgt cct
tgc ttg 297 Lys Val Pro Phe Phe Arg Lys Arg Lys His His Thr Cys Pro
Cys Leu 70 75 80 ccc aac ctg ctg tgc tcc agg ttc ccg gac ggc agg
tac cgc tgc tcc 345 Pro Asn Leu Leu Cys Ser Arg Phe Pro Asp Gly Arg
Tyr Arg Cys Ser 85 90 95 atg gac ttg aag aac atc aat ttt taggcgcttg
cctggtctca ggatacccac 399 Met Asp Leu Lys Asn Ile Asn Phe 100 105
catccttttc tgagcacagc ctggattttt atttctgcca tgaaacccag ctcccatgac
459 tctcccagtc cctacactga ctaccctgat ctctcttgtc tagtacgcac
atatgcacac 519 aggcagacat acctcccatc atgacatggt ccccaggctg
gcctgaggat gtcacagctt 579 gaggctgtgg tgtgaaaggt ggccagcctg
gttctcttcc ctgctcaggc tgccagagag 639 gtggtaaatg gcagaaagga
cattccccct cccctcccca ggtgacctgc tctctttcct 699 gggccctgcc
cctctcccca catgtatccc tcggtctgaa ttagacattc ctgggcacag 759
gctcttgggt gcattgctca gagtcccagg tcctggcctg accctcaggc ccttcacgtg
819 aggtctgtga ggaccaattt gtgggtagtt catcttccct cgattggtta
actccttagt 879 ttcagaccac agactcaaga ttggctcttc ccagagggca
gcagacagtc accccaaggc 939 aggtgtaggg agcccaggga ggccaatcag
ccccctgaag actctggtcc cagtcagcct 999 gtggcttgtg gcctgtgacc
tgtgaccttc tgccagaatt gtcatgcctc tgaggccccc 1059 tcttaccaca
ctttaccagt taaccactga agcccccaat tcccacagct tttccattaa 1119
aatgcaaatg gtggtggttc aatctaatct gatattgaca tattagaagg caattagggt
1179 gtttccttaa acaactcctt tccaaggatc agccctgaga gcaggttggt
gactttgagg 1239 agggcagtcc tctgtccaga ttggggtggg agcaagggac
agggagcagg gcaggggctg 1299 aaaggggcac tgattcagac cagggaggca
actacacacc aacctgctgg ctttagaata 1359 aaagcaccaa ctgaactg 1377 2
105 PRT Homo sapiens 2 Met Arg Gly Ala Thr Arg Val Ser Ile Met Leu
Leu Leu Val Thr Val 1 5 10 15 Ser Asp Cys Ala Val Ile Thr Gly Ala
Cys Glu Arg Asp Val Gln Cys 20 25 30 Gly Ala Gly Thr Cys Cys Ala
Ile Ser Leu Trp Leu Arg Gly Leu Arg 35 40 45 Met Cys Thr Pro Leu
Gly Arg Glu Gly Glu Glu Cys His Pro Gly Ser 50 55 60 His Lys Val
Pro Phe Phe Arg Lys Arg Lys His His Thr Cys Pro Cys 65 70 75 80 Leu
Pro Asn Leu Leu Cys Ser Arg Phe Pro Asp Gly Arg Tyr Arg Cys 85 90
95 Ser Met Asp Leu Lys Asn Ile Asn Phe 100 105 3 86 PRT Homo
sapiens 3 Ala Val Ile Thr Gly Ala Cys Glu Arg Asp Val Gln Cys Gly
Ala Gly 1 5 10 15 Thr Cys Cys Ala Ile Ser Leu Trp Leu Arg Gly Leu
Arg Met Cys Thr 20 25 30 Pro Leu Gly Arg Glu Gly Glu Glu Cys His
Pro Gly Ser His Lys Val 35 40 45 Pro Phe Phe Arg Lys Arg Lys His
His Thr Cys Pro Cys Leu Pro Asn 50 55 60 Leu Leu Cys Ser Arg Phe
Pro Asp Gly Arg Tyr Arg Cys Ser Met Asp 65 70 75 80 Leu Lys Asn Ile
Asn Phe 85 4 1406 DNA Homo sapiens CDS (10)...(333) 4 gagggcgcc atg
agg agc ctg tgc tgc gcc cca ctc ctg ctc ctc ttg ctg 51 Met Arg Ser
Leu Cys Cys Ala Pro Leu Leu Leu Leu Leu Leu 1 5 10 ctg ccg ccg ctg
ctg ctc acg ccc cgc gct ggg gac gcc gcc gtg atc 99 Leu Pro Pro Leu
Leu Leu Thr Pro Arg Ala Gly Asp Ala Ala Val Ile 15 20 25 30 acc ggg
gct tgt gac aag gac tcc caa tgt ggt gga ggc atg tgc tgt 147 Thr Gly
Ala Cys Asp Lys Asp Ser Gln Cys Gly Gly Gly Met Cys Cys 35 40 45
gct gtc agt atc tgg gtc aag agc ata agg att tgc aca cct atg ggc 195
Ala Val Ser Ile Trp Val Lys Ser Ile Arg Ile Cys Thr Pro Met Gly 50
55 60 aaa ctg gga gac agc tgc cat cca ctg act cgt aaa gtt cca ttt
ttt 243 Lys Leu Gly Asp Ser Cys His Pro Leu Thr Arg Lys Val Pro Phe
Phe 65 70 75 ggg cgg agg atg cat cac act tgc cca tgt ctg cca ggc
ttg gcc tgt 291 Gly Arg Arg Met His His Thr Cys Pro Cys Leu Pro Gly
Leu Ala Cys 80 85 90 tta cgg act tca ttt aac cga ttt att tgt tta
gcc caa aag 333 Leu Arg Thr Ser Phe Asn Arg Phe Ile Cys Leu Ala Gln
Lys 95 100 105 taatcgctct ggagtagaaa ccaaatgtga atagccacat
cttacctgta aagtcttact 393 tgtgattgtg ccaaacaaaa aatgtgccag
aaagaaatgc tcttgcttcc tcaactttcc 453 aagtaacatt tttatctttg
atttgtaaat gatttttttt ttttttttta tcgaaagaga 513 attttacttt
tggatagaaa tatgaagtgt aaggcattat ggaactggtt cttatttccc 573
tgtttgtgtt ttggtttgat ttggcttttt tcttaaatgt caaaaacgta cccattttca
633 caaaaatgag gaaaataaga atttgatatt ttgttagaaa aacttttttt
tttttttctc 693 accaccccaa gccccatttg tgccctgccg cacaaataca
cctacagctt ttggtccctt 753 gcctcttcca cctcaaagaa tttcaaggct
cttaccttac tttatttttg tccatttctc 813 ttccctcctc ttgcatttta
aagtggaggg tttgtctctt tgagtttgat ggcagaatca 873 ctgatgggaa
tccagctttt tgctggcatt taaatagtga aaagagtgta tatgtgaact 933
tgacactcca aactcctgtc atggcacgga agctaggagt gctgctggac ccttcctaaa
993 cctgtcactc aagaggactt cagctctgct gttgggctgg tgtgtggaca
gaaggaatgg 1053 aaagccaaat taatttagtc cagatttcta ggtttgggtt
tttctaaaaa taaaagatta 1113 catttacttc ttttactttt tataaagttt
tttttcctta gtctcctact tagagatatt 1173 ctagaaaatg tcacttgaag
aggaagtatt tattttaatc tggcacaaca ctaattacca 1233 tttttaaagc
ggtattaagt tgtaatttaa accttgtttg taactgaaag gtcgattgta 1293
atggattgcc gtttgtacct gtatcagtat tgctgtgtaa aaattctgta tcagaataat
1353 aacagtactg tatatcattt gatttatttt aatattatat ccttattttt gtc
1406 5 108 PRT Homo sapiens 5 Met Arg Ser Leu Cys Cys Ala Pro Leu
Leu Leu Leu Leu Leu Leu Pro 1 5 10 15 Pro Leu Leu Leu Thr Pro Arg
Ala Gly Asp Ala Ala Val Ile Thr Gly 20 25 30 Ala Cys Asp Lys Asp
Ser Gln Cys Gly Gly Gly Met Cys Cys Ala Val 35 40 45 Ser Ile Trp
Val Lys Ser Ile Arg Ile Cys Thr Pro Met Gly Lys Leu 50 55 60 Gly
Asp Ser Cys His Pro Leu Thr Arg Lys Val Pro Phe Phe Gly Arg 65 70
75 80 Arg Met His His Thr Cys Pro Cys Leu Pro Gly Leu Ala Cys Leu
Arg 85 90 95 Thr Ser Phe Asn Arg Phe Ile Cys Leu Ala Gln Lys 100
105 6 81 PRT Homo sapiens 6 Ala Val Ile Thr Gly Ala Cys Asp Lys Asp
Ser Gln Cys Gly Gly Gly 1 5 10 15 Met Cys Cys Ala Val Ser Ile Trp
Val Lys Ser Ile Arg Ile Cys Thr 20 25 30 Pro Met Gly Lys Leu Gly
Asp Ser Cys His Pro Leu Thr Arg Lys Val 35 40 45 Pro Phe Phe Gly
Arg Arg Met His His Thr Cys Pro Cys Leu Pro Gly 50 55 60 Leu Ala
Cys Leu Arg Thr Ser Phe Asn Arg Phe Ile Cys Leu Ala Gln 65 70 75 80
Lys 7 21 PRT Homo sapiens 7 Asn Asn Phe Gly Asn Gly Arg Gln Glu Arg
Arg Lys Arg Lys Arg Ser 1 5 10 15 Lys Arg Lys Lys Glu 20 8 21 PRT
Homo sapiens 8 Ser His Val Ala Asn Gly Arg Gln Glu Arg Arg Arg Ala
Lys Arg Arg 1 5 10 15 Lys Arg Lys Lys Glu 20 9 19 PRT Homo sapiens
9 Met Arg Gly Ala Thr Arg Val Ser Ile Met Leu Leu Leu Val Thr Val 1
5 10 15 Ser Asp Cys 10 26 PRT Homo sapiens 10 Met Arg Ser Leu Cys
Cys Ala Pro Leu Leu Leu Leu Leu Leu Leu Pro 1 5 10 15 Leu Leu Leu
Thr Pro Pro Ala Gly Asp Ala 20 25 11 96 PRT Bombina variegata 11
Met Lys Cys Phe Ala Gln Ile Val Val Leu Leu Leu Val Ile Ala Phe 1 5
10 15 Ser His Gly Ala Val Ile Thr Gly Ala Cys Asp Lys Asp Val Gln
Cys 20 25 30 Gly Ser Gly Thr Cys Cys Ala Ala Ser Ala Trp Ser Arg
Asn Ile Arg 35 40 45 Phe Cys Ile Pro Leu Gly Asn Ser Gly Glu Asp
Cys His Pro Ala Ser 50 55 60 His Lys Val Pro Tyr Asp Gly Lys Arg
Leu Ser Ser Leu Cys Pro Cys 65 70 75 80 Lys Ser Gly Leu Thr Cys Ser
Lys Ser Gly Glu Lys Phe Lys Cys Ser 85 90 95 12 81 PRT Dendroaspis
polylepis polylepis 12 Ala Val Ile Thr Gly Ala Cys Glu Arg Asp Leu
Gln Cys Gly Lys Gly 1 5 10 15 Thr Cys Cys Ala Val Ser Leu Trp Ile
Lys Ser Val Arg Val Cys Thr 20 25 30 Pro Val Gly Thr Ser Gly Glu
Asp Cys His Pro Ala Ser His Lys Ile 35 40 45 Pro Phe Ser Gly Gln
Arg Lys Met His His Thr Cys Pro Cys Ala Pro 50 55 60 Asn Leu Ala
Cys Val Gln Thr Ser Pro Lys Lys Phe Lys Cys Leu Ser 65 70 75 80 Lys
13 81 PRT Artificial Sequence synthetic construct 13 Ala Val Ile
Thr Gly Ala Cys Glu Arg Asp Val Gln Cys Gly Ala Gly 1 5 10 15 Thr
Cys Cys Ala Ile Ser Leu Trp Leu Arg Gly Leu Arg Met Cys Thr 20 25
30 Pro Leu Gly Arg Glu Gly Glu Glu Cys His Pro Gly Ser His Lys Val
35 40 45 Pro Phe Phe Gly Arg Arg Met His His Thr Cys Pro Cys Leu
Pro Gly 50 55 60 Leu Ala Cys Leu Arg Thr Ser Phe Asn Arg Phe Ile
Cys Leu Ala Gln 65 70 75 80 Lys 14 86 PRT Artificial Sequence
synthetic construct 14 Ala Val Ile Thr Gly Ala Cys Asp Lys Asp Ser
Gln Cys Gly Gly Gly 1 5 10 15 Met Cys Cys Ala Val Ser Ile Trp Val
Lys Ser Ile Arg Ile Cys Thr 20 25 30 Pro Met Gly Lys Leu Gly Asp
Ser Cys His Pro Leu Thr Arg Lys Val 35 40 45 Pro Phe Phe Arg Lys
Arg Lys His His Thr Cys Pro Cys Leu Pro Asn 50 55 60 Leu Leu Cys
Ser Arg Phe Pro Asp Gly Arg Tyr Arg Cys Ser Met Asp 65 70 75 80 Leu
Lys Asn Ile Asn Phe 85 15 89 PRT Artificial Sequence synthetic
construct 15 Gly Ile Leu Ala Val Ile Thr Gly Ala Cys Glu Arg Asp
Val Gln Cys 1 5 10 15 Gly Ala Gly Thr Cys Cys Ala Ile Ser Leu Trp
Leu Arg Gly Leu Arg 20 25 30 Met Cys Thr Pro Leu Gly Arg Glu Gly
Glu Glu Cys His Pro Gly Ser 35 40 45 His Lys Val Pro Phe Phe Arg
Lys Arg Lys His His Thr Cys Pro Cys 50 55 60 Leu Pro Asn Leu Leu
Cys Ser Arg Phe Pro Asp Gly Arg Tyr Arg Cys 65 70 75 80 Ser Met Asp
Leu Lys Asn Ile Asn Phe 85 16 85 PRT Artificial Sequence synthetic
construct 16 Val Ile Thr Gly Ala Cys Glu Arg Asp Val Gln Cys Gly
Ala Gly Thr 1 5 10 15 Cys Cys Ala Ile Ser Leu Trp Leu Arg Gly Leu
Arg Met Cys Thr Pro 20 25 30 Leu Gly Arg Glu Gly Glu Glu Cys His
Pro Gly Ser His Lys Val Pro 35 40 45 Phe Phe Arg Lys Arg Lys His
His Thr Cys Pro Cys Leu Pro Asn Leu 50 55 60 Leu Cys Ser Arg Phe
Pro Asp Gly Arg Tyr Arg Cys Ser Met Asp Leu 65 70 75 80 Lys Asn Ile
Asn Phe 85 17 86 PRT Artificial Sequence synthetic construct 17 Ala
Ala Ala Ala Ala Ala Cys Glu Arg Asp Val Gln Cys Gly Ala Gly 1 5 10
15 Thr Cys Cys Ala Ile Ser Leu Trp Leu Arg Gly Leu Arg Met Cys Thr
20 25 30 Pro Leu Gly Arg Glu Gly Glu Glu Cys His Pro Gly Ser His
Lys Val 35 40 45 Pro Phe Phe Arg Lys Arg Lys His His Thr Cys Pro
Cys Leu Pro Asn 50 55 60 Leu Leu Cys Ser Arg Phe Pro Asp Gly Arg
Tyr Arg Cys Ser Met Asp 65 70 75 80 Leu Lys Asn Ile Asn Phe 85 18
87 PRT Artificial Sequence synthetic construct 18 Met Ala Val Ile
Thr Gly Ala Cys Glu Arg Asp Val Gln Cys Gly Ala 1 5 10 15 Gly Thr
Cys Cys Ala Ile Ser Leu Trp Leu Arg Gly Leu Arg Met Cys 20 25 30
Thr Pro Leu Gly Arg Glu Gly Glu Glu Cys His Pro Gly Ser His Lys 35
40 45 Val Pro Phe Phe Arg Lys Arg Lys His His Thr Cys Pro Cys Leu
Pro 50 55 60 Asn Leu Leu Cys Ser Arg Phe Pro Asp Gly Arg Tyr Arg
Cys Ser Met 65 70 75 80 Asp Leu Lys Asn Ile Asn Phe 85 19 14 PRT
Artificial Sequence synthetic construct 19 Ala Val Ile Thr Gly Ala
Cys Glu Arg Asp Val Gln Cys Gly 1 5 10
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