U.S. patent application number 10/242499 was filed with the patent office on 2003-06-12 for novel human purinergic p2u receptor.
This patent application is currently assigned to Incyte Genomics, Inc.. Invention is credited to Au-Young, Janice, Coleman, Roger, Guegler, Karl J., Stuart, Susan G..
Application Number | 20030109674 10/242499 |
Document ID | / |
Family ID | 23823188 |
Filed Date | 2003-06-12 |
United States Patent
Application |
20030109674 |
Kind Code |
A1 |
Coleman, Roger ; et
al. |
June 12, 2003 |
Novel human purinergic P2U receptor
Abstract
The present invention provides nucleotide and amino acid
sequences that identify and encode a novel purinergic P.sub.U2
receptor (PNR) expressed in human placenta. The present invention
also provides for antisense molecules to the nucleotide sequences
which encode PNR, expression vectors for the production of purified
PNR, antibodies capable of binding specifically to PNR,
hybridization probes or oligonucleotides for the detection of
PNR-encoding nucleotide sequences, genetically engineered host
cells for the expression of PNR, and diagnostic tests based on
PNR-encoding nucleic acid molecules or antibodies produced against
the polypeptide PNR.
Inventors: |
Coleman, Roger; (Sunnyvale,
CA) ; Au-Young, Janice; (Brisbane, CA) ;
Stuart, Susan G.; (Montara, CA) ; Guegler, Karl
J.; (Menlo park, CA) |
Correspondence
Address: |
INCYTE GENOMICS, INC.
3160 PORTER DRIVE
PALO ALTO
CA
94304
US
|
Assignee: |
Incyte Genomics, Inc.
Palo Alto
CA
|
Family ID: |
23823188 |
Appl. No.: |
10/242499 |
Filed: |
September 11, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10242499 |
Sep 11, 2002 |
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09102710 |
Jun 22, 1998 |
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6479630 |
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09102710 |
Jun 22, 1998 |
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08459046 |
Jun 2, 1995 |
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6008039 |
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Current U.S.
Class: |
530/350 ;
435/320.1; 435/325; 435/6.16; 435/69.1; 435/70.21; 530/388.22;
536/23.5 |
Current CPC
Class: |
C12N 9/6472 20130101;
C12N 15/1096 20130101; A61P 43/00 20180101; A61K 38/00 20130101;
C07K 14/705 20130101; C07K 2319/00 20130101; A61K 48/00 20130101;
A61P 9/12 20180101; C07K 14/47 20130101; A61P 35/00 20180101 |
Class at
Publication: |
530/350 ;
530/388.22; 536/23.5; 435/6; 435/69.1; 435/70.21; 435/320.1;
435/325; 514/12 |
International
Class: |
C12Q 001/68; C12P
021/02; C12N 005/06; C07K 014/705; C07K 016/28; C07H 021/04; A61K
038/17 |
Claims
What is claimed is:
1. An isolated polypeptide selected from the group consisting of:
a) a polypeptide comprising an amino acid sequence of SEQ ID NO:2,
b) a polypeptide comprising a naturally occurring amino acid
sequence at least 90% identical to an amino acid sequence of SEQ ID
NO:2, c) a biologically active fragment of a polypeptide having an
amino acid sequence of SEQ ID NO:2, and d) an immunogenic fragment
of a polypeptide having an amino acid sequence of SEQ ID NO:2.
2. An isolated polypeptide of claim 1, comprising an amino acid
sequence of SEQ ID NO:2.
3. An isolated polynucleotide encoding a polypeptide of claim
1.
4. An isolated polynucleotide encoding a polypeptide of claim
2.
5. An isolated polynucleotide of claim 4, having a sequence of SEQ
ID NO:1.
6. A recombinant polynucleotide comprising a promoter sequence
operably linked to a polynucleotide of claim 3.
7. A cell transformed with a recombinant polynucleotide of claim
6.
8. A transgenic organism comprising a recombinant polynucleotide of
claim 6.
9. A method of producing a polypeptide of claim 1, the method
comprising: a) culturing a cell under conditions suitable for
expression of the polypeptide, wherein said cell is transformed
with a recombinant polynucleotide, and said recombinant
polynucleotide comprises a promoter sequence operably linked to a
polynucleotide encoding the polypeptide of claim 1, and b)
recovering the polypeptide so expressed.
10. A method of claim 9, wherein the polypeptide comprises the
amino acid sequence of SEQ ID NO:2.
11. An isolated antibody which specifically binds to a polypeptide
of claim 1.
12. An isolated polynucleotide selected from the group consisting
of:: a) a polynucleotide comprising a polynucleotide sequence of
SEQ ID NO: 1, b) a polynucleotide comprising a naturally occurring
polynucleotide sequence at least 90% identical to a polynucleotide
sequence of SEQ ID NO: 1, c) a polynucleotide complementary to a
polynucleotide of a), d) a polynucleotide complementary to a
polynucleotide of b) and e) an RNA equivalent of a)-d).
13. An isolated polynucleotide comprising at least 60 contiguous
nucleotides of a polynucleotide of claim 12.
14. A method of detecting a target polynucleotide in a sample, said
target polynucleotide having a sequence of a polynucleotide of
claim 12, the method comprising: a) hybridizing the sample with a
probe comprising at least 20 contiguous nucleotides comprising a
sequence complementary to said target polynucleotide in the sample,
and which probe specifically hybridizes to said target
polynucleotide, under conditions whereby a hybridization complex is
formed between said probe and said target polynucleotide or
fragments thereof, and b) detecting the presence or absence of said
hybridization complex, and, optionally, if present, the amount
thereof.
15. A method of claim 14, wherein the probe comprises at least 60
contiguous nucleotides.
16. A method of detecting a target polynucleotide in a sample, said
target polynucleotide having a sequence of a polynucleotide of
claim 12, the method comprising: a) amplifying said target
polynucleotide or fragment thereof using polymerase chain reaction
amplification, and b) detecting the presence or absence of said
amplified target polynucleotide or fragment thereof, and,
optionally, if present, the amount thereof.
17. A composition comprising a polypeptide of claim 1 and a
pharmaceutically acceptable excipient.
18. A composition of claim 17, wherein the polypeptide comprises an
amino acid sequence of SEQ ID NO:2.
19. A method for treating a disease or condition associated with
decreased expression of functional PNR, comprising administering to
a patient in need of such treatment the composition of claim
17.
20. A method of screening a compound for effectiveness as an
agonist of a polypeptide of claim 1, the method comprising: a)
exposing a sample comprising a polypeptide of claim 1 to a
compound, and b) detecting agonist activity in the sample.
21. A composition comprising an agonist compound identified by a
method of claim 20 and a pharmaceutically acceptable excipient.
22. A method for treating a disease or condition associated with
decreased expression of functional PNR, comprising administering to
a patient in need of such treatment a composition of claim 21.
23. A method of screening a compound for effectiveness as an
antagonist of a polypeptide of claim 1, the method comprising: a)
exposing a sample comprising a polypeptide of claim 1 to a
compound, and b) detecting antagonist activity in the sample.
24. A composition comprising an antagonist compound identified by a
method of claim 23 and a pharmaceutically acceptable excipient.
25. A method for treating a disease or condition associated with
overexpression of functional PNR, comprising administering to a
patient in need of such treatment a composition of claim 24.
26. A method of screening for a compound that specifically binds to
the polypeptide of claim 1, the method comprising: a) combining the
polypeptide of claim 1 with at least one test compound under
suitable conditions, and b) detecting binding of the polypeptide of
claim 1 to the test compound, thereby identifying a compound that
specifically binds to the polypeptide of claim 1.
27. A method of screening for a compound that modulates the
activity of the polypeptide of claim 1, said method comprising: a)
combining the polypeptide of claim 1 with at least one test
compound under conditions permissive for the activity of the
polypeptide of claim 1, b) assessing the activity of the
polypeptide of claim 1 in the presence of the test compound, and c)
comparing the activity of the polypeptide of claim 1 in the
presence of the test compound with the activity of the polypeptide
of claim 1 in the absence of the test compound, wherein a change in
the activity of the polypeptide of claim 1 in the presence of the
test compound is indicative of a compound that modulates the
activity of the polypeptide of claim 1.
28. A method of screening a compound for effectiveness in altering
expression of a target polynucleotide, wherein said target
polynucleotide comprises a polynucleotide sequence of claim 5, the
method comprising: a) exposing a sample comprising the target
polynucleotide to a compound, under conditions suitable for the
expression of the target polynucleotide, b) detecting altered
expression of the target polynucleotide, and c) comparing the
expression of the target polynucleotide in the presence of varying
amounts of the compound and in the absence of the compound.
29. A method of assessing toxicity of a test compound, the method
comprising: a) treating a biological sample containing nucleic
acids with the test compound, b) hybridizing the nucleic acids of
the treated biological sample with a probe comprising at least 20
contiguous nucleotides of a polynucleotide of claim 12 under
conditions whereby a specific hybridization complex is formed
between said probe and a target polynucleotide in the biological
sample, said target polynucleotide comprising a polynucleotide
sequence of a polynucleotide of claim 12 or fragment thereof, c)
quantifying the amount of hybridization complex, and d) comparing
the amount of hybridization complex in the treated biological
sample with the amount of hybridization complex in an untreated
biological sample, wherein a difference in the amount of
hybridization complex in the treated biological sample is
indicative of toxicity of the test compound.
30. A diagnostic test for a condition or disease associated with
the expression of PNR in a biological sample, the method
comprising: a) combining the biological sample with an antibody of
claim 11, under conditions suitable for the antibody to bind the
polypeptide and form an antibody:polypeptide complex, and b)
detecting the complex, wherein the presence of the complex
correlates with the presence of the polypeptide in the biological
sample.
31. The antibody of claim 11, wherein the antibody is: a) a
chimeric antibody, b) a single chain antibody, c) a Fab fragment,
d) a F(ab').sub.2 fragment, or e) a humanized antibody.
32. A composition comprising an antibody of claim 11 and an
acceptable excipient.
33. A method of diagnosing a condition or disease associated with
the expression of PNR in a subject, comprising administering to
said subject an effective amount of the composition of claim
32.
34. A composition of claim 32, wherein the antibody is labeled.
35. A method of diagnosing a condition or disease associated with
the expression of PNR in a subject, comprising administering to
said subject an effective amount of the composition of claim
34.
36. A method of preparing a polyclonal antibody with the
specificity of the antibody of claim 11, the method comprising: a)
immunizing an animal with a polypeptide consisting of an amino acid
sequence of SEQ ID NO:2, or an immunogenic fragment thereof, under
conditions to elicit an antibody response, b) isolating antibodies
from said animal, and c) screening the isolated antibodies with the
polypeptide, thereby identifying a polyclonal antibody which binds
specifically to a polypeptide comprising an amino acid sequence of
SEQ ID NO:2.
37. An polyclonal antibody produced by a method of claim 36.
38. A composition comprising the polyclonal antibody of claim 37
and a suitable carrier.
39. A method of making a monoclonal antibody with the specificity
of the antibody of claim 11, the method comprising: a) immunizing
an animal with a polypeptide consisting of an amino acid sequence
of SEQ ID NO:2, or an immunogenic fragment thereof, under
conditions to elicit an antibody response, b) isolating antibody
producing cells from the animal, c) fusing the antibody producing
cells with immortalized cells to form monoclonal antibody-producing
hybridoma cells, d) culturing the hybridoma cells, and e) isolating
from the culture monoclonal antibody which binds specifically to a
polypeptide comprising an amino acid sequence of SEQ ID NO:2.
40. A monoclonal antibody produced by a method of claim 39.
41. A composition comprising the monoclonal antibody of claim 40
and a suitable carrier.
42. The antibody of claim 11, wherein the antibody is produced by
screening a Fab expression library.
43. The antibody of claim 11, wherein the antibody is produced by
screening a recombinant immunoglobulin library.
44. A method of detecting a polypeptide comprising an amino acid
sequence of SEQ ID NO:2 in a sample, the method comprising: a)
incubating the antibody of claim 11 with a sample under conditions
to allow specific binding of the antibody and the polypeptide, and
b) detecting specific binding, wherein specific binding indicates
the presence of a polypeptide comprising an amino acid sequence of
SEQ ID NO:2 in the sample.
45. A method of purifying a polypeptide comprising an amino acid
sequence of SEQ ID NO:2 from a sample, the method comprising: a)
incubating the antibody of claim 11 with a sample under conditions
to allow specific binding of the antibody and the polypeptide, and
b) separating the antibody from the sample and obtaining the
purified polypeptide comprising an amino acid sequence of SEQ ID
NO:2.
46. A microarray wherein at least one element of the microarray is
a polynucleotide of claim 12.
47. A method of generating an expression profile of a sample which
contains polynucleotides,, the method comprising: a) labeling the
polynucleotides of the sample, b) contacting the elements of the
microarray of claim 46 with the labeled polynucleotides of the
sample under conditions suitable for the formation of a
hybridization complex, and c) quantifying the expression of the
polynucleotides in the sample.
48. An array comprising different nucleotide molecules affixed in
distinct physical locations on a solid substrate, wherein at least
one of said nucleotide molecules comprises a first oligonucleotide
or polynucleotide sequence specifically hybridizable with at least
30 contiguous nucleotides of a target polynucleotide, and wherein
said target polynucleotide is a polynucleotide of claim 12.
49. An array of claim 48, wherein said first oligonucleotide or
polynucleotide sequence is completely complementary to at least 30
contiguous nucleotides of said target polynucleotide.
50. An array of claim 48, wherein said first oligonucleotide or
polynucleotide sequence is completely complementary to at least 60
contiguous nucleotides of said target polynucleotide.
51. An array of claim 48, wherein said first oligonucleotide or
polynucleotide sequence is completely complementary to said target
polynucleotide.
52. An array of claim 48, which is a microarray.
53. An array of claim 48, further comprising said target
polynucleotide hybridized to a nucleotide molecule comprising said
first oligonucleotide or polynucleotide sequence.
54. An array of claim 48, wherein a linker joins at least one of
said nucleotide molecules to said solid substrate.
55. An array of claim 48, wherein each distinct physical location
on the substrate contains multiple nucleotide molecules, and the
multiple nucleotide molecules at any single distinct physical
location have the same sequence, and each distinct physical
location on the substrate contains nucleotide molecules having a
sequence which differs from the sequence of nucleotide molecules at
another distinct physical location on the substrate.
Description
[0001] This application is a divisional application of U.S.
application Ser. No. 09/102,710, filed Jun. 22, 1998 entitled A
NOVEL HUMAN PURINERGIC P.sub.2U RECEPTOR which is a divisional
application of U.S. application Ser. No. 08/459,046, filed Jun. 2,
1995, now U.S. Pat. No. 6,008,039, entitled POLYNUCLEOTIDE ENDING A
NOVEL PURINERGIC P.sub.2U RECEPTOR all of which applications and
patents are hereby expressly incorporated by reference.
FIELD OF THE INVENTION
[0002] This invention relates to nucleic acid and amino acid
sequences of a novel human purinergic p.sub.2u receptor, and to the
use of these sequences in the diagnosis, treatment, and prevention
of activated or inflamed cells and/or tissues.
BACKGROUND OF THE INVENTION
[0003] Purinergic Receptor
[0004] The purinergic P.sub.2U or nucleotide receptor is an
integral part of the plasmalemma of various mammalian cell types.
The P.sub.U2 receptor described in this application is most similar
to a G-protein coupled surface receptor from rat. These receptors
are associated with cells such as neutrophils, endothelial cells,
and fibroblasts in the immune, neural, muscular, pulmonary and
vascular systems. P.sub.2U receptors stimulate phosphoinositide
metabolism and the release of intracellular Ca.sup.++ in the
presence of extracellular nucleotides, particularly UTP or ATP. In
macrophages, Mg.sup.++ inhibits the response of P.sub.2U to ATP
(Alonso-Torre SR and A Trautmann (1994) J Biol Chem 268:18640-47);
and in lung epithelial cells, stimulation of the P.sub.2U receptor
by nucleotides modulates chloride secretion. P.sub.2 receptors have
a very low affinity for adenosine and are not activated by the
methylxanthine antagonists, caffeine and theophylline.
[0005] The P.sub.2U receptor is in the P.sub.2 receptor family for
which the common structural features have been described: 1) seven
hydrophobic domains, 2) consensus N-linked glycosylation sequences
near the amino terminus, 3) a number of residues common to G-
protein coupled receptors (asn.sup.51, asp.sup.79, cys.sup.106, and
cys.sup.183), and 4) potential phosphorylation sites in the third
intracellular and carboxyterminal domains (Parr CE et al (1994)
Proc Natl Acad Sci 91:3275-79).
[0006] In addition to P.sub.2U, there are four other P.sub.2
receptor subtypes. The P.sub.2X receptor mediates smooth muscle
response following sympathetic nerve stimulation and contains an
intrinsic cation channel. The P.sub.2Y receptor is found in smooth
muscle and vascular tissue where it induces vasodilation in
response to nitric oxide. The P.sub.2Z receptor is found primarily
on mast or other immune cells, and when activated by ATP, it
appears to cause cell permeabilization. The P.sub.2T receptor,
which is only found on platelets, inhibits adenylate cyclase and
stimulates the release of intracellular calcium ions. In contrast,
P1 receptors are stimulated by adenosine rather than
nucleotides.
[0007] The G-protein coupled receptors (T7G) characteristically
contain seven hydrophobic domains which span the plasma membrane
and form a bundle of antiparallel a helices. These transmembrane
segments are designated by roman numerals and account for many of
the structural and functional features of the receptor. In most
cases, the bundle of helices forms a binding pocket; however, the
binding site for bulky molecules includes the extracellular
N-terminal segment or one or more of the three extracellular loops.
Binding may also alter the receptor's intracellular configuration
(Watson S and Arkinstall S (1994) The G-Protein Linked Receptor
Facts Book, Academic Press, San Diego Calif.).
[0008] The activated receptor interacts with an intracellular
G-protein complex which mediates further intracellular signalling
activities, generally the production of second messengers such as
cyclic AMP (cAMP), phospholipase C, inositol triphosphate, or ion
channel proteins. Coupling to G-proteins involves a variable
sequence in the C-terminal 10-20 amino acids of the third internal
loop between the transmembrane segments V and VI and the
intracellular segment immediately C-terminal to transmembrane
segment VII. Interaction with Gq also requires the N-terminal 10-20
amino acids of the third internal loop.
[0009] Both structural and functional features of T7Gs allow their
classification into five categories: .beta.-type, muscarinic-type,
neurokinin-type, nonneurokinin-type, and miscellaneous (Bolander FF
(1994) Molecular Endocrinology, Academic Press, San Diego Calif.);
each of which are discussed below. P.sub.2U is a .beta.-type
receptor and has structural features shared with .beta.-adrenergic,
a-adrenergic, histamine, dopamine, and serotonin receptors. These
receptors have a short N-terminus with two conserved
N-glycosylation sites, a moderately short third internal loop, and
a long C-terminus containing a Ser/Thr-rich region. All adrenergic
receptors elevate cAMP or intracellular calcium.
[0010] The novel purinergic receptor which is the subject of this
patent application was identified among the cDNAs derived from a
placental library. Incyte Clone 179696 is a novel homolog of
RNU09402, a G-protein coupled surface receptor from rat (Rice WR et
al (1995) Am J Respir Cell Molec Biol 12:27-32). Purinergic
receptors of the placenta are likely found on immune or vascular
cells and appear to play an important role in signal transduction
and other specialized functions of the placenta as briefly
described below.
[0011] Placenta
[0012] The placenta is a thickened discoid temporary organ that
acts as the site of interchange of substances between the maternal
and fetal bloodstreams. Such substances include oxygen, nutrients,
hormones, excretory products, humoral antibodies (immunoglobulin G,
IgG), drugs, viruses, or any other chemical or infectious agent
that may be present in the maternal circulation.
[0013] The placenta consists of a fetal part derived from the
chorion, one of the extraembryonic surrounding membranes of the
conceptus and of a maternal part (decidua basalis) derived from the
region of endometrium that underlies the implantation site. The
placenta is thus the only organ composed of cells derived from two
individuals. The boundary between maternal and fetal tissues is
marked by extracellular products of necrosis referred to as
fibrinoid. The anatomy of the human placenta is discussed in detail
in Benirschke and Kaufmann, (1992) Pathology of the Human Placenta,
Springer-Verlag, New York City, pp 542-635.
[0014] Development
[0015] The late blastocyst consists of an inner cell mass that
gives rise to the embryo and an outer, single layer of trophoblast
cells that encloses the blastocyst cavity. Following implantation,
trophoblasts become highly invasive, erode and attach to the
secretory endometrium. This invasive process involves
matrix-degrading metalloproteinases (MMPs) and tissue inhibitors of
metalloproteinases (TIMPs), adhesion receptors and their
extracellular ligands, and the class I human leukocyte antigen-G
(HLA-G) molecule. The invasive process is reviewed in Fisher and
Damsky (1993 Semin Cell Biol 4(3): 183-188) and in Graham and Lala
(1992 Biochem Cell Biol 70:867-874).
[0016] Trophoblasts give rise to two layers. The inner layer is
composed of individual cells, cytotrophoblasts, which have high
proliferative potential. The outer layer is composed of syncytial
cells, syncytiotrophoblasts, which invade the endometrium and
become surrounded by cavernous spaces (lacunae) filled with
maternal blood. Finger-like extensions of the cytotrophoblasts grow
into these protrusions and act as primary placental villi. The
capillaries found in this tissue are a part of the embryonic
circulation. Tufted extensions of part of the chorion or chorionic
villi are associated with the decidua basalis and develop into the
large, elaborately branched outgrowths of the villous chorion. The
syncytiotrophoblasts remain until the end of pregnancy, but by the
fifth month of gestation, most of the cytotrophoblasts begin to
fuse with the syncytiotrophoblast. The few remaining
cytotrophoblasts form a discontinuous basal layer.
[0017] Chorion
[0018] The chorion or fetal part of the placenta has a chorionic
plate at the point where the chorionic villi arise. The finger-like
villi extend into the endometrial lacuna which are filled with
maternal blood released under pressure from the endometrial spiral
arteries. A connective tissue core in which the fetal blood vessels
develop is derived from extraembryonic mesenchyme surrounded by
syncytiotrophoblast and cytotrophoblast cell layers.
[0019] During pregnancy, surface area of the villi increases
dramatically. The surfaces of the villi are active in the exchange
of substances between fetal and maternal circulatory systems.
Receptors within the apical microvilli facilitate transport of
glucose, amino acids, and IgG from mother to fetus. The mechanism
for IgG movement is similar to that of IgA across epithelia. The
transport of various materials, particularly nutrients, by the
placenta is reviewed in Smith et al (1992 Ann Rev Nutrition
12:183-206) and Schneider (1991 Reprod Fertil Dev 3:345-353). The
placenta is more than a simple conduit for nutrients; it engages in
considerable metabolic activity contributing to the quality and
quantity of nutrients supplied to the fetus (cf. Hay (1991)
Diabetes 40S:44-50).
[0020] Although the villi express foreign paternal as well as
maternal antigens and a maternal immune response would be expected
against the fetal "allograft", the fetus is not usually rejected.
The type of Fetal factors such as major histocompatibility complex
(MHC) I (but not MHC II) and low antigen density and maternal
response (suppressor cells and molecules) all contribute to a
complex and unique tolerance. The absence of MHC II may be
particularly significant, since MHC II has been implicated in the
rejection of organ allografts.
[0021] Decidua Basalis
[0022] The function of the endometrium is to support the
implantation and development of the embryo. During each menstrual
cycle, the most superficial layer or functionalis, undergoes
dramatic changes in preparation for these events. During
proliferative phase in the first half of the cycle, rising estrogen
levels stimulate the division of epithelial and stromal cells in
the functionalis. The uterine lining is ready by the time of
ovulation at day 14.
[0023] During the secretory phase in the second half of the cycle,
endometrial cells differentiate in response to rising levels of
progesterone. Beginning as early as day 15, glycogen appears in the
basal region of the epithelial cells and displaces the nuclei. By
day 18, the glycogen is dispersed, the nuclei have returned to a
basal portion of the cell, Golgi are prominent apically, and
secretion is maximal. Concurrently, the nuclear envelope indents to
form a channel system associated with the nucleolus. This system is
believed to facilitate a rapid transfer of ribosomal components
between the nucleus and the cytoplasm. Uterine secretions contain
significant amounts of glucose and specific glycoproteins such as
PP14 which may confer immunosuppression in preparation for contact
with the "foreign" embryo.
[0024] Implantation induces a decidual response that is
characterized by pronounced changes in the endometrial stroma.
Fibroblast-like cells transform into large, active decidual cells
that become an important component of the decidua basalis.
Predecidual cells, which appear in the endometrial stroma during
the fourth week of every menstrual cycle, form a cuff around small
vessels in the stroma. The vessels become more permeable as
menstruation or placental development approaches.
[0025] The predecidual cells appear to limit embryo invasion, play
a role in embryo nutrition, and protect fetal tissue from
rejection. These cells produce prolactin (and possibly relaxin),
secrete prostaglandins, and have receptors for both estrogen and
progesterone. The effects of estrogen and progesterone on the
endometrium, both during the cycle and following implantation, are
complemented and implemented by a variety of growth factors.
Insulin-like growth factors (IGFS) have a major role in the
stimulation of endometrial cell division. With rising levels of
progesterone after ovulation, IGF-binding proteins, including the
placental protein PP12 synthesized by the predecidual cells, are
secreted. IGF-binding proteins reduce the availability of IGFs and
thus play a role in the shift from a proliferative to a secretory
endometrium.
[0026] The decidua basalis supplies arterial blood to and receives
venous blood from the lacunae situated between the villi. Although
the maternal blood vessels are open during implantation, the fetal
vessels remain intact. Fetal and maternal blood do not mix, except
on rare occasions at the end of pregnancy. During this period when
the cytotrophoblast is no longer continuous and the capillaries of
the villi are very close to the surface, a very slight exchange of
blood may occur. At that time, the walls of the fetal capillaries
are separated from the matemal blood only by the
syncytiotrophoblast.
[0027] During pregnancy, cells from the connective tissue stroma of
the decidua basalis and a lesser number of cells from the decidua
parietalis and decidua capsularis form decidual cells. These large,
slightly basophilic cells have many profiles of rough endoplasmic
reticulum, long mitochondria, and membrane-limited granules
contained in club-shaped projections of the cell surface. Decidual
cells are more numerous during the first half of pregnancy, contain
a nucleus with a prominent nucleolus, and secrete prolactin which
is similar to pituitary prolactin.
[0028] At the end of a full-term pregnancy, the placenta has the
shape of a thick disk. The umbilical cord usually arises from the
center of the placenta and connects the circulation of the fetus
with the fetal placental circulation. Fetal venous blood reaches
the placenta through the two umbilical arteries which branch and
ultimately give rise to the vessels of the chorionic villi. In
these villi, the fetal blood receives oxygen, loses its CO.sub.2
and returns to the fetus through the umbilical vein. Although the
chorionic villi are submerged in maternal blood, the fetal
placental blood is isolated by the structures that form the
placental barrier--the endothelium and basal lamina of the fetal
capillaries; the mesenchyme in the villus interior; the basal
lamina of the trophoblast; the cytotrophoblast, during the first
half of pregnancy; and the syncytiotrophoblast.
[0029] The placenta is permeable to several substances and normally
transfers oxygen, water, electrolytes, carbohydrates, lipids,
proteins, vitamins, hormones, antibodies, and some drugs from the
maternal to the fetal circulation. Carbon dioxide, water, hormones,
and residual products of metabolism are transferred from fetal
blood to maternal blood. The complexity of this bidirectional
transport reflects the function of the placental layers as the
equivalent of three organ systems--respiratory, gastrointestinal,
and urinary. The mechanism of transport is extremely varied,
ranging from simple diffusion of gases to many types of
receptor-mediated transport including the active transport of amino
acids and a special shuttle mechanism for IgG. IgG is the only
immunoglobulin which crosses the placental barrier, enters fetal
circulation, and protects the newborn against infection. Makiya and
Stignrand (1992 Clin Chem 38:2543-45) suggest that placental
alkaline phosphatase binds the Fc portion of IgG and acts as the
placental IgG receptor.
[0030] Maternal Immunologic Tolerance of Fetal Tissue
[0031] Villi expressing foreign (paternal) antigens are exposed
directly to maternal blood. Even though a maternal immune response
occurs, fetal tissue is not typically rejected. Low expression of
MHC I, absence of MHC II, and suppression of maternal response
contribute to this unique tolerance. The trophoblast which is the
true allograft and comes in contact with maternal blood, does not
express classical MHC antigens. Occasionally, maternal IgG may harm
the fetus relative to Rhesus factor (Rh) or maternal immune
thrombocytopenic purpura.
[0032] An understanding of how the trophoblast/fetus escapes
rejection might allow development of rational strategies for
combating pregnancy disorders, such as preeclampsia or intrauterine
growth retardation, having an immunological basis. The
fetal-maternal immune interaction is reviewed in Herrera-Gonzalez
and Dresser (1993 Dev Comp Immunol 17:1-18).
[0033] Placental Hormones
[0034] Soon after implantation, fetal villi begin to control
maternal physiology creating an optimal environment for fetal
development. Immediately after implantation, the
syncytiotrophoblast synthesizes human chorionic gonadotropin (HCG),
a glycoprotein hormone that mimics the effects of luteinizing
hormone (LH) through the first few months of gestation. HCG has a
subunit identical to that of LH and follicle-stimulating hormone
(FSH). LH acts on and maintains the corpus luteum by stimulating
estrogen and progesterone synthesis.
[0035] Beginning at about eight weeks into gestation, the
syncytiotrophoblast assumes the role of the corpus luteum and
begins to secrete estrogen and progesterone. The steroid hormones
progesterone and estrogen are made by both kinds of trophoblast,
but estrogen production requires the metabolic cooperation of the
fetal adrenal cortex and liver. The syncytiotrophoblast which
continues to produce these hormones throughout gestation utilizes
both maternal and fetal androgen precursors to form estrogens and
massive amounts are released into the maternal bloodstream.
[0036] Placental progesterone is synthesized from cholesterol
obtained primarily from circulating low-density lipoprotein (LDL).
Membranes of the microvilli provide surface area for LDL receptors.
LDL is initially shuttled into lysosomes and cholesterol is
released by the action of acid hydrolases. Then the cholesterol is
transported to mitochondria where it is acted upon by enzyme
complexes within the tubular cristae.
[0037] The syncytiotrophoblast is also the chief source of human
chorionic somatomammotropin (HCS), a glycoprotein hormone with both
lactogenic and growth-promoting activity. HCS is similar to growth
hormone and has effects on maternal carbohydrate, fat, and protein
metabolism. As maternal utilization of fatty acids increases,
available glucose is reserved for the fetus. HCS has its major
effect, in conjunction with prolactin, on development of the
mammary gland.
[0038] Cytotrophoblasts produce significant amounts of
platelet-derived growth factor-beta (PDGF-8) as well as the PDGF-a
and -.beta. receptors (Holmgren et al (1992) Growth Factors
6:219-231). PDGF may play a role in cytotrophoblast proliferation.
The action of various cytokines on the placenta is reviewed in
Mitchell et al (1993 Placenta 14:249-275) and Rutanen (1993 Ann Med
25:343-347).
[0039] Pathology of the Placenta
[0040] Preeclampsia, now referred to as "pregnancy-induced
hypertension" (PIH), deserves special note. Common in pregnancy,
preeclampsia is characterized by sudden development of
hypertension, edema, and proteinuria. More severe toxemia or
eclampsia includes convulsions and coma which may jeopardize both
mother and fetus. The pathological changes of the placenta found in
PIH are decidual arteriolopathy, infarcts, abruptio placenta, and
Tenney-Parker changes.
[0041] The principal cause of preeclampsia is still unknown
although it is certain that the disease relates to the presence of
placental tissue, since the delivery of the placenta (or
hydatidiform mole) ends the disease process. An obliterative
thickening of arterial walls and a reduced number of small arteries
in the villi have been observed and may explain the increase in
vascular resistance in PIH. Another cause of uneven blood flow may
be vasoconstriction. While the blood levels of the vasoconstrictor,
angiotensin II, are not increased, uterine vascular responsiveness
is greatly increased. Vasoconstriction may be induced by a
reduction of unopposed thromboxane and angiotensin II. Reduced
oxygen tension in the maternal blood supplied to the intervillous
lacunae may also play a role.
[0042] Many types of infections by viruses, bacteria, mycoplasmas,
or parasites cause pathological changes in the placenta. Infections
may ascend from the endocervical canal, or they may reach the
placenta through the maternal blood. Rarely are they acquired by
amniocentesis, chorionic villus sampling, amnioscopy, percutaneous
umbilical blood sampling, or intrauterine fetal transfusions. Some
infections cause gross and microscopic changes of the placenta,
while others leave few characteristic or specifically recognizable
traces.
[0043] Other disorders of the placenta include, but are not limited
to, abruptio placentae; placenta previa; placental or maternal
floor infarction; placenta accreta, increta, and percreta;
extrachorial placentas; chorangioma; chorangiosis; chronic
villitis; placental villous edema; widespread fibrosis of the
terminal villi; intervillous thrombi; hemorrhagic endovasculitis;
erythroblastosis fetalis; and nonimmune fetal hydrops. The
pathology of the human placenta and decidua is discussed in
Benirschke and Kaufmann, (1992) Pathology of the Human Placenta,
Springer-Verlag, New York City pp. 542-635, and in Naeye (1992),
Disorders of the Placenta, Fetus, and Neonate: Diagnosis and
Clinical Significance, Mosby Year Book, St. Louis Mo.
SUMMARY OF THE INVENTION
[0044] The subject invention provides a unique nucleotide sequence
which encodes a novel human purinergic P.sub.2U receptor (PNR).
Incyte Clone No 179696 was used to identify and clone the full
length cDNA (pnr) from the placenta cDNA library.
[0045] The invention also comprises the use of this PNR or its
variants to intercede in physiologic or pathologic conditions and
include diagnosis or therapy of activated or inflamed cells and/or
tissues with pnr nucleic acids, fragments or oligomers thereof.
Aspects of the invention include the antisense DNA of pnr; cloning
or expression vectors containing pnr; host cells or organisms
transformed with expression vectors containing pnr; a method for
the production and recovery of purified PNR from host cells;
purified protein, PNR, which can be used to generate antibodies for
diagnosis or therapy of activated or inflamed cells and/or
tissues.
DESCRIPTION OF THE FIGURES
[0046] FIG. 1 shows the nucleotide (SEQ ID NO:1) and amino acid
(SEQ ID NO:2) alignments of the consensus sequence for PNR. The
primers XLR (278-298) and XLF (587-610) for full length cloning are
shown as arrows.
[0047] FIGS. 2A, 2B, 2C and 2D display the amino acid alignment of
PNR (SEQ ID NO:2) with rat purinergic receptor (RNU09402; SEQ ID
NO:3). The residues by which the P.sub.2U receptor is defined
(e.g., asn.sup.44, asp.sup.72, cys.sup.99, and cys.sup.177 of SEQ
ID NO:2) are shown (Parr et al, supra).
DETAILED DESCRIPTION OF THE INVENTION
[0048] Definitions
[0049] As used herein, PNR, refers to purinergic receptor homologs,
naturally occurring PNRs and active fragments thereof, which are
encoded by mRNAs transcribed from the cDNA (pnr) of Seq ID No
1.
[0050] "Active" refers to those forms of PNR which retain the
biologic and/or immunologic activities of any naturally occurring
PNR.
[0051] "Naturally occurring PNR" refers to PNRs produced by human
cells that have not been genetically engineered and specifically
contemplates various PNRs arising from post-translational
modifications of the polypeptide including but not limited to
acetylation, carboxylation, glycosylation, phosphorylation,
lipidation and acylation.
[0052] "Derivative" refers to PNRs chemically modified by such
techniques as ubiquitination, labeling (e.g., with radionuclides,
various enzymes, etc.), pegylation (derivatization with
polyethylene glycol), and insertion or substitution by chemical
synthesis of amino acids such as ornithine, which do not normally
occur in human proteins.
[0053] "Recombinant variant" refers to any polypeptide differing
from naturally occurring PNRs by amino acid insertions, deletions,
and substitutions, created using recombinant DNA techniques.
Guidance in determining which amino acid residues may be replaced,
added or deleted without abolishing activities of interest, such as
normal signal transduction, may be found by comparing the sequence
of the particular PNR with that of homologous peptides and
minimizing the number of amino acid sequence changes made in highly
conserved regions.
[0054] Preferably, amino acid "substitutions" are the result of
replacing one amino acid with another amino acid having similar
structural and/or chemical properties, such as the replacement of a
leucine with an isoleucine or valine, an aspartate with a
glutamate, or a threonine with a serine, ie, conservative
replacements. "Insertions" or "deletions" are typically in the
range of about 1 to 5 amino acids. The variation allowed may be
experimentally determined by producing the peptide synthetically or
by systematically making insertions, deletions, or substitutions of
nucleotides in a pnr molecule using recombinant DNA techniques and
assaying the expressed, recombinant variants for activity.
[0055] Where desired, a "signal or leader sequence" can direct the
polypeptide through the membrane of a cell. Such a sequence may be
naturally present on the polypeptides of the present invention or
provided from heterologous sources by recombinant DNA
techniques.
[0056] A polypeptide "fragment," "portion," or "segment" is a
stretch of amino acid residues of at least about 5 amino acids,
often at least about 7 amino acids, typically at least about 9 to
13 amino acids, and, in various embodiments, at least about 17 or
more amino acids. To be active, any PNR peptide must have
sufficient length to display biologic and/or immunologic
activity.
[0057] An "oligonucleotide" or polynucleotide "fragment",
"portion","probe" or "segment" is a stretch of nucleotide residues
which is long enough to use in polymerase chain reaction (PCR) or
various hybridization procedures. Oligonucleotides are prepared
based on the cDNA sequence which encodes PNR provided by the
present invention and are used to amplify, or simply reveal,
related RNA or DNA molecules. Oligonucleotides comprise portions of
the DNA sequence having at least about 10 nucleotides and as many
as about 35 nucleotides, preferably about 25 nucleotides. Nucleic
acid probes comprise portions of pnr sequence having fewer
nucleotides than about 6 kb, preferably fewer than about 1 kb.
After appropriate testing to eliminate false positives, both
oligonucleotides and nucleic acid probes may be used to determine
whether mRNAs encoding PNR are present in a cell or tissue or to
isolate similar natural nucleic acid sequences from chromosomal DNA
as described by Walsh PS et al (1992, PCR Methods Appl
1:241-50).
[0058] Probes may be derived from naturally occurring or
recombinant single- or double-stranded nucleic acids or be
chemically synthesized. They may be labeled by nick translation,
Klenow fill-in reaction, PCR or other methods well known in the
art. Probes of the present invention, their preparation and/or
labeling are elaborated in Sambrook J et al (1989) Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold
Spring Harbor N.Y.; or Ausubel FM et al (1989) Current Protocols in
Molecular Biology, John Wiley & Sons, New York City, both
incorporated herein by reference.
[0059] Recombinant variants encoding T7Gs may be synthesized or
selected by making use of the "redundancy" in the genetic code.
Various codon substitutions, such as the silent changes which
produce specific restriction sites, may be introduced to optimize
cloning into a plasmid or viral vector or to increase expression in
a particular prokaryotic or eukaryotic system. Codon usage-specific
mutations may also be introduced or chimeras containing the domains
of related peptides added to test or modify the properties of any
part of the polypeptide, particularly to change ligand-binding
affinities, interchain affinities, or degradation/turnover
rate.
Detailed Description of the Invention
[0060] The present invention provides a unique nucleotide sequence
identifying a novel homolog of the human purinergic receptor which
was first identified in a human placenta cDNA library. The sequence
for pnr is shown in SEQ ID No 1 and is homologous to the GenBank
sequence, RNU09402 (Rice et al, supra). Because P.sub.2U is
specifically expressed in cells active in immunity, the nucleic
acid (pnr), polypeptide (PNR) and antibodies to PNR are useful in
investigations of and interventions in the normal and abnormal
physiologic and pathologic processes which comprise the placenta's
role in immunity. Therefore, an assay for upregulated expression of
PNR can accelerate diagnosis and proper treatment of conditions
caused by abnormal signal transduction due to systemic and local
infections, traumatic and other tissue damage, hereditary or
environmental diseases associated with hypertension, carcinomas,
cystic fibrosis, and other physiologic or pathologic problems.
[0061] The nucleotide sequence encoding PNR (or its complement) has
numerous other applications in techniques known to those skilled in
the art of molecular biology. These techniques include use as
hybridization probes for Southerns or northerns, use as oligomers
for PCR, use for chromosomal and gene mapping, use in the
recombinant production of PNR, use in generation of anti-sense DNA
or RNA, their chemical analogs and the like, and use in production
of chimeric molecules for selecting agonists, inhibitors or
antagonists for design of domain-specific therapeutic molecules.
Uses of the nucleotides encoding PNR disclosed herein are exemplary
of known techniques and are not intended to limit their use in any
technique known to a person of ordinary skill in the art.
Furthermore, the nucleotide sequences disclosed herein may be used
in molecular biology techniques that have not yet been developed,
provided the new techniques rely on properties of nucleotide
sequences that are currently known, e.g., the triplet genetic code,
specific base pair interactions, etc.
[0062] It will be appreciated by those skilled in the art that as a
result of the degeneracy of the genetic code, a multitude of
PNR-encoding nucleotide sequences, some bearing minimal homology to
the nucleotide sequence of any known and naturally occurring gene
may be produced. The invention has specifically contemplated each
and every possible variation of nucleotide sequence that could be
made by selecting combinations based on possible codon choices.
These combinations are made in accordance with the standard triplet
genetic code as applied to the nucleotide sequence of naturally
occurring PNR, and all such variations are to be considered as
being specifically disclosed.
[0063] Although nucleotide sequences which encode PNR and its
variants are preferably capable of hybridizing to the nucleotide
sequence of the naturally occurring PNR gene under stringent
conditions, it may be advantageous to produce nucleotide sequences
encoding PNR or its derivatives possessing a substantially
different codon usage. Codons can be selected to increase the rate
at which expression of the peptide occurs in a particular
prokaryotic or eukaryotic expression host in accordance with the
frequency with which particular codons are utilized by the host.
Other reasons for substantially altering the nucleotide sequence
encoding PNR and its derivatives without altering the encoded amino
acid sequence include the production of RNA transcripts having more
desirable properties, such as a greater half-life, than transcripts
produced from the naturally occurring sequence.
[0064] The nucleotide sequence encoding PNR may be joined to a
variety of other nucleotide sequences by means of well established
recombinant DNA techniques (cf Sambrook J et al, supra). Useful
nucleotide sequences for joining to pnr include an assortment of
cloning vectors--plasmids, cosmids, lambda phage derivatives,
phagemids, and the like--that are well known in the art and may be
chosen for such characteristics as the size insert they can
accommodate, their international utility, their fidelity, etc.
Other vectors of interest include expression vectors, replication
vectors, probe generation vectors, sequencing vectors, YAC and BAC
mapping vectors , and the like. In general, these vectors may
contain an origin of replication functional in at least one
organism, convenient restriction endonuclease sensitive sites, and
selectable markers for the host cell.
[0065] Another aspect of the subject invention is to provide for
pnr-specific nucleic acid hybridization probes capable of
hybridizing with naturally occurring nucleotide sequences encoding
PNR. Such probes may also be used for the detection of PNR-encoding
sequences and should preferably contain at least 50% of the
nucleotides from any particular domain of interest from this pnr
encoding sequence. The hybridization probes of the subject
invention may be derived from the nucleotide sequence of the SEQ ID
NO 1 or from genomic sequence including promoter, enhancer elements
and introns of the respective naturally occurring pnr.
Hybridization probes may be labeled by a variety of reporter
groups, including radionuclides such as .sup.32P or .sup.35S, or
enzymatic labels such as alkaline phosphatase coupled to the probe
via avidin/biotin coupling systems, and the like.
[0066] PCR, as described in U.S. Pat. Nos. 4,683,195; 4,800,195;
and 4,965,188, provides additional uses for oligonucleotides based
upon the nucleotide sequences which encode PNR. Such probes used in
PCR may be of recombinant origin, may be chemically synthesized, or
may be a mixture of both and comprise a discrete nucleotide
sequence for diagnostic use or a degenerate pool of possible
sequences for identification of closely related P.sub.U2 or related
T7G sequences.
[0067] Full length genes may be cloned from known sequence using a
new method which employs XL-PCR (Perkin-Elmer, Foster City, Calif.)
to amplify long pieces of DNA. This method was developed to allow a
single researcher to process multiple genes (up to 20 or more) at a
time and to obtain an extended (possibly full-length) sequence
within 6-10 days. It replaces current methods which use labelled
probes to screen libraries and allow one researcher to process only
about 3-5 genes in 14-40 days.
[0068] In the first step, which can be performed in about two days,
primers are designed and synthesized based on a known partial
sequence. In step 2, which takes about six to eight hours, the
sequence is extended by PCR amplification of a selected library.
Steps 3 and 4, which take about one day, are purification of the
amplified cDNA and its ligation into an appropriate vector. Step 5,
which takes about one day, involves transforming and growing up
host bacteria. In step 6, which takes approximately five hours, PCR
is used to screen bacterial clones for extended sequence. The final
steps, which take about one day, involve the preparation and
sequencing of selected clones. If the full length cDNA has not been
obtained, the entire procedure is repeated using either the
original library or some other preferred library. The preferred
library may be one that has been size-selected to include only
larger cDNAs or may consist of single or combined commercially
available libraries, eg. lung, liver, heart and brain from
Gibco/BRL (Gaithersburg Md.). The cDNA library may have been
prepared with oligo dT or random primers. The advantage of using
random primed libraries is that they will have more sequences which
contain 5' ends of genes. A randomly primed library may be
particularly useful if an oligo dT library does not yield a
complete gene. Obviously, the larger the protein, the less likely
it is that the complete gene will be found in a single plasmid.
[0069] Other means for producing hybridization probes for closely
related sequences include the cloning of nucleic acid sequences
encoding PNR or its derivatives into vectors for the production of
mRNA probes. Such vectors are known in the art and are commercially
available and may be used to synthesize RNA probes in vitro by
means of the addition of the appropriate RNA polymerase as T7 or
SP6 RNA polymerase and the appropriate labeled nucleotides.
[0070] It is now possible to produce a DNA sequence, or portions
thereof, encoding PNR and/or its derivatives entirely by synthetic
chemistry. Such molecules can be inserted into any of the many
available vectors using reagents and methods that are known in the
art at the time of the filing of this application. Moreover,
synthetic chemistry may be used to introduce mutations into the pnr
sequences or any portion thereof.
[0071] The nucleotide sequence can be used to develop an assay to
detect activation, inflammation, or disease associated with
abnormal levels of PNR expression. The nucleotide sequence can be
labeled by methods known in the art and added to a fluid or tissue
sample from a patient. After an incubation period sufficient to
effect hybridization, the sample is washed with a compatible fluid
which contains a visible marker, a dye or other appropriate
molecule(s), if the nucleotide has been labeled with an enzyme.
After the compatible fluid is rinsed off, the dye is quantitated
and compared with a standard. If the amount of dye is significantly
elevated (or lowered, as the case may be), the nucleotide sequence
has hybridized with the sample, and the assay indicates an abnormal
condition such as inflammation or disease.
[0072] The nucleotide sequence for pnr can be used to construct
hybridization probes for mapping that T7G gene. The nucleotide
sequence provided herein may be mapped to a chromosome and specific
regions of a chromosome using well known genetic and/or chromosomal
mapping techniques. These techniques include in situ hybridization,
linkage analysis against known chromosomal markers, hybridization
screening with libraries or flow-sorted chromosomal preparations
specific to known chromosomes, and the like. The technique of
fluorescent in situ hybridization of chromosome spreads has been
described, among other places, in Verma et al (1988) Human
Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York
City.
[0073] Fluorescent in situ hybridization of chromosomal
preparations and other physical chromosome mapping techniques may
be correlated with additional genetic map data. Examples of genetic
map data can be found in the 1994 Genome Issue of Science (265:1981
f). Correlation between the location of pnr on a physical
chromosomal map and a specific disease (or predisposition to a
specific disease) can help delimit the region of DNA associated
with that genetic disease. The nucleotide sequence of the subject
invention may be used to detect differences in gene sequence
between normal and carrier or affected individuals.
[0074] The nucleotide sequence encoding PNR may be used to produce
purified PNR using well known methods of recombinant DNA
technology. Among the many publications that teach methods for the
expression of genes after they have been isolated is Goeddel (1990)
Gene Expression Technology, Methods and Enzymology, Vol 185,
Academic Press, SanDiego Calif. PNR may be expressed in a variety
of host cells, either prokaryotic or eukaryotic. Host cells may be
from the same species in which pnr nucleotide sequences are
endogenous or from a different species. Advantages of producing PNR
by recombinant DNA technology include obtaining adequate amounts of
the protein for purification and the availability of simplified
purification procedures.
[0075] Cells transformed with DNA encoding PNR may be cultured
under conditions suitable for the expression of PNR and recovery of
the protein from the cell culture. PNR produced by a recombinant
cell may be secreted or may be contained intracellularly depending
on the particular genetic construction used. In general, it is more
convenient to prepare recombinant proteins in secreted form.
Purification steps vary with the production process and the
particular protein produced.
[0076] Various methods for the isolation of PNR polypeptide may be
accomplished by procedures well known in the art. For example, such
a polypeptide may be purified by immunoaffinity chromatography by
employing the antibodies provided by the present invention. Various
other methods of protein purification well known in the art include
those described in Deutscher M (1990) Methods in Enzymology, Vol
182, Academic Press, San Diego Calif.; and in Scopes R (1982)
Protein Purification: Principles and Practice, Springer-Verlag, New
York City, both incorporated herein by reference.
[0077] In addition to recombinant production, fragments of PNR may
be produced by direct peptide synthesis using solid-phase
techniques (cf Stewart et al (1969) Solid-Phase Peptide Synthesis,
WH Freeman Co, San Francisco Calif.; Merrifield J (1963) J Am Chem
Soc 85:2149-2154). In vitro protein synthesis may be performed
using manual techniques or by automation. Automated synthesis may
be achieved, for example, using Applied Biosystems 431A Peptide
Synthesizer (ABI, Foster City, Calif.) in accordance with the
instructions provided by the manufacturer. Various fragments of PNR
may be chemically synthesized separately and combined using
chemical methods to produce the full length molecule.
[0078] PNR for antibody induction does not require biological
activity; however, the protein must be immunogenic. Peptides used
to induce specific antibodies may have an amino acid sequence
consisting of at least five amino acids, preferably at least 10
amino acids. They should mimic a structural portion of the amino
acid sequence of the protein and may contain the entire amino acid
sequence of a single domain of PNR. Short stretches of PNR amino
acids may be fused with those of another protein such as keyhole
limpet hemocyanin, and antibody produced against the fusion
protein.
[0079] Antibodies specific for PNR may be produced by inoculation
of an appropriate animal with the polypeptide or an antigenic
fragment. An antibody is specific for PNR if it is produced against
an epitope of the polypeptide and binds to at least part of the
natural or recombinant protein. Antibody production includes not
only the stimulation of an immune response by injection into
animals, but also analogous steps in the production of synthetic
antibodies or other specific-binding molecules such as the
screening of recombinant immunoglobulin libraries (cf Orlandi R et
al (1989) PNAS 86:3833-37, or Huse WD et al (1989) Science
256:1275-81) or the in vitro stimulation of lymphocyte populations.
Current technology (Winter G and Milstein C (1991) Nature
349:293-99) provides for a number of highly specific binding
reagents based on the principles of antibody formation. These
techniques may be adapted to produce molecules specifically binding
particular domains of PNR.
[0080] An additional embodiment of the subject invention is the use
of PNR specific antibodies or the like as bioactive agents to treat
abnormal signal transduction associated with systemic and local
infections, traumatic and other tissue damage, hereditary or
environmental diseases associated with hypertension, carcinomas,
cystic fibrosis, and other physiologic or pathologic problems.
[0081] Bioactive compositions comprising agonists, antagonists, or
inhibitors of PNR may be administered in a suitable therapeutic
dose determined by any of several methodologies including clinical
studies on mammalian species to determine maximum tolerable dose
and on normal human subjects to determine safe dosage.
Additionally, the bioactive agent may be complexed with a variety
of well established compounds or compositions which enhance
stability or pharmacological properties such as half-life. It is
contemplated that a therapeutic, bioactive composition may be
delivered by intravenous infusion into the bloodstream or any other
effective means which could be used for treatment.
[0082] The examples below are provided to describe the subject
invention. These examples are provided by way of illustration and
are not included for the purpose of limiting the invention.
EXAMPLES
I Isolation of mRNA and Construction of the cDNA Library
[0083] Placental tissue was obtained from a term pregnancy (40
weeks gestation) of a male neonate delivered by Caesarean section.
The tissue was flash frozen, ground in a mortar and pestle, and
lyzed immediately in buffer containing guanidinium isothiocyanate.
Lysis was followed by several phenol chloroform extractions and
ethanol precipitation. Poly A.sup.+ RNA was isolated using
biotinylated oligo d(T) primer and streptavidin coupled to a
paramagnetic particle (Promega Corp, Madison Wis.) and sent to
Stratagene (La Jolla Calif.).
[0084] Stratagene prepared the cDNA library using oligo d(T)
priming. Synthetic adapter oligonucleotides were ligated onto the
cDNA molecules enabling them to be inserted into the Uni-ZAP.TM.
vector system (Stratagene). This allowed high efficiency
unidirectional (sense orientation) lambda library construction and
the convenience of a plasmid system with blue/white color selection
to detect clones with cDNA insertions.
[0085] The quality of the cDNA library was screened using DNA
probes, and then, the pBluescript.RTM. phagemid (Stratagene) was
excised. This phagemid allows the use of a plasmid system for easy
insert characterization, sequencing, site-directed mutagenesis, the
creation of unidirectional deletions and expression of fusion
polypeptides. Subsequently, the custom-constructed library phage
particles were infected into E. coli host strain XL1-Blue.RTM.
(Stratagene). The high transformation efficiency of this bacterial
strain increases the probability that the cDNA library will contain
rare, under-represented clones. Alternative unidirectional vectors
might include, but are not limited to, pcDNAI (Invitrogen, San
Diego Calif.) and pSHlox-1 (Novagen, Madison Wis.).
II Isolation of cDNA Clones
[0086] The phagemid forms of individual cDNA clones were obtained
by the in vivo excision process, in which the host bacterial strain
was co-infected with both the library phage and an f1 helper phage.
Polypeptides or enzymes derived from both the library-containing
phage and the helper phage nicked the DNA, initiated new DNA
synthesis from defined sequences on the target DNA, and created a
smaller, single stranded circular phagemid DNA molecule that
included all DNA sequences of the pBluescript phagemid and the cDNA
insert. The phagemid DNA was released from the cells and purified
and used to reinfect fresh host cells (SOLR, Stratagene) where
double-stranded phagemid DNA was produced. Because the phagemid
carries the gene for b-lactamase, the newly transformed bacteria
were selected on medium containing ampicillin.
[0087] A alternative phagemid purification procedure uses the
QIAWELL-8 Plasmid Purification System from the QIAGEN.RTM. DNA
Purification System (QIAGEN Inc, Chatsworth Calif.). This product
provides a convenient, rapid and reliable high-throughput method
for lysing the bacterial cells and isolating highly purified
phagemid DNA using QIAGEN anion-exchange resin particles with
EMPORE.TM. membrane technology from 3M in a multiwell format. The
DNA was eluted from the purification resin and prepared for DNA
sequencing and other analytical manipulations.
III Sequencing of cDNA Clones
[0088] The cDNA inserts from random isolates of the placenta
library were sequenced in part. Methods for DNA sequencing are well
known in the art. Conventional enzymatic methods employed DNA
polymerase Klenow fragment, SEQUENASE.RTM. (US Biochemical Corp,
Cleveland Ohio) or Taq polymerase to extend DNA chains from an
oligonucleotide primer annealed to the DNA template of interest.
Methods have been developed for the use of both single- and double-
stranded templates. The chain termination reaction products were
electrophoresed on urea-acrylamide gels and detected either by
autoradiography (for radionuclide-labeled precursors) or by
fluorescence (for fluorescent-labeled precursors). Recent
improvements in mechanized reaction preparation, sequencing and
analysis using the fluorescent detection method have permitted
expansion in the number of sequences that can be determined per day
using machines such as the Catalyst 800 and the Applied Biosystems
377 or 373 DNA sequencers.
IV Homology Searching of cDNA Clones and Deduced Proteins
[0089] Each sequence so obtained was compared to sequences in
GenBank using a search algorithm developed by Applied Biosystems
and incorporated into the INHERIT.TM. 670 Sequence Analysis System.
In this algorithm, Pattern Specification Language (developed by TRW
Inc., Los Angeles Calif.) was used to determine regions of
homology. The three parameters that determine how the sequence
comparisons run were window size, window offset, and error
tolerance. Using a combination of these three parameters, the DNA
database was searched for sequences containing regions of homology
to the query sequence, and the appropriate sequences were scored
with an initial value. Subsequently, these homologous regions were
examined using dot matrix homology plots to distinguish regions of
homology from chance matches. Smith-Waterman alignments were used
to display the results of the homology search.
[0090] Peptide and protein sequence homologies were ascertained
using the INHERIT.TM. 670 Sequence Analysis System in a way similar
to that used in DNA sequence homologies. Pattern Specification
Language and parameter windows were used to search protein
databases for sequences containing regions of homology which were
scored with an initial value. Dot-matrix homology plots were
examined to distinguish regions of significant homology from chance
matches.
[0091] Alternatively, BLAST, which stands for Basic Local Alignment
Search Tool, is used to search for local sequence alignments
(Altschul SF (1993) J Mol Evol 36:290-300; Altschul, SF et al
(1990) J Mol Biol 215:403-10). BLAST produces alignments of both
nucleotide and amino acid sequences to determine sequence
similarity. Because of the local nature of the alignments, BLAST is
especially useful in determining exact matches or in identifying
homologs. Whereas it is ideal for matches which do not contain
gaps, it is inappropriate for performing motif-style searching. The
fundamental unit of BLAST algorithm output is the High-scoring
Segment Pair (HSP).
[0092] An HSP consists of two sequence fragments of arbitrary but
equal lengths whose alignment is locally maximal and for which the
alignment score meets or exceeds a threshold or cutoff score set by
the user. The BLAST approach is to look for HSPs between a query
sequence and a database sequence, to evaluate the statistical
significance of any matches found, and to report only those matches
which satisfy the user-selected threshold of significance. The
parameter E establishes the statistically significant threshold for
reporting database sequence matches. E is interpreted as the upper
bound of the expected frequency of chance occurrence of an HSP (or
set of HSPs) within the context of the entire database search. Any
database sequence whose match satisfies E is reported in the
program output.
V Identification, Full Length Cloning, Sequencing and
Translation
[0093] Analysis of INHERIT.TM. results from randomly picked and
sequenced portions of clones from placenta library identified
Incyte 179696 as a homolog of the purinergic receptor RNU09402. The
cDNA insert comprising Incyte 179696 was fully sequenced and used
as the basis for cloning the full length cDNA.
[0094] The cDNA of Incyte 179696 was extended to full length using
a modified XL-PCR (Perkin Elmer) procedure. Primers were designed
based on known sequence; one primer was synthesized to initiate
extension in the antisense direction (XLR) and the other to extend
sequence in the sense direction (XLS or XLF). The primers allowed
the sequence to be extended "outward" generating amplicons
containing new, unknown nucleotide sequence for the genes of
interest. The primers were designed using Oligo 4.0 (National
Biosciences Inc, Plymouth Minn.). In general, primers should be
22-30 nucleotides in length, have a GC content of 50% or more, and
anneal to the target sequence at temperatures about
68.degree.-72.degree. C. Any stretch of nucleotides which would
result in hairpin structures and primer-primer dimerizations were
avoided.
[0095] The placenta cDNA library was used as a template, and XLR
and XLS primers were used to amplify sequences containing the gene
of interest. The enzymes of the XL-PCR kit were found to provide
high fidelity in the amplification providing kit instructions were
followed. In the extension of P.sub.2U sequence, 25 pMol of each
primer and a thoroughly premixed enzyme solution were effective in
obtaining the extended sequence.
[0096] Amplification was conducted using the MJ PTC200 (MJ
Research, Watertown Mass.) and the following parameters:
1 Step 1 94.degree. C. for 60 sec (initial denaturation) Step 2
94.degree. C. for 15 sec Step 3 65.degree. C. for 1 min Step 4
68.degree. C. for 7 min Step 5 Repeat step 2-4 for 15 additional
times Step 6 94.degree. C. for 15 sec Step 7 65.degree. C. for 1
min Step 8 68.degree. C. for 7 min + 15 sec/cycle Step 9 Repeat
step 6-8 for 11 additional times Step 10 72.degree. C. for 8 min
Step 11 4.degree. C. (and holding)
[0097] At the end of 28 cycles, 50 .mu.l of the reaction mix was
removed; and the remaining reaction mix was run for an additional
10 cycles as outlined below:
2 Step 1 94.degree. C. for 15 sec Step 2 65.degree. C. for 1 min
Step 3 68.degree. C. for (10 min + 15 sec)/cycle Step 4 Repeat step
1-3 for 9 additional times Step 5 72.degree. C. for 10 min
[0098] A 5-10 .mu.l aliquot of the reaction mixture was analyzed on
a mini-gel to determine successful reactions. Although all extended
cDNA species potentally contained the full length gene, some of the
largest products were selected and separated from template by
electrophoresis on a low concentration (about 0.6-0.8%) agarose
gel. The bands representing the gene of interest were cut out of
the gel and purified using a method like the QIAQuick.TM. gel
extraction kit (QIAGEN Inc, Chatsworth Calif.). Klenow enzyme was
used to convert eventual overhangs into blunt ends to facilitate
religation and cloning of the products.
[0099] After ethanol precipitation, the products were redissolved
in 13 .mu.l of ligation buffer. Then, 1 .mu.l T4-DNA ligase (15
units) and 1 .mu.l T4 polynucleotide kinase were added, and the
mixture was incubated at room temperature for 2-3 hours or
overnight at 16.degree. C. Competent E. coli cells (in 40 .mu.l of
appropriate media) were transformed with 3 .mu.l of ligation
mixture and cultured in 80 .mu.l of SOC medium. After incubation
for one hour at 37.degree. C., the whole transformation mixture was
plated on LB-agar containing 2.times.carbenicillin. The following
day, 12 colonies were randomly picked from each plate and cultured
in 150 .mu.l of liquid LB/carbenicillin medium placed in an
individual well of an appropriate, commercially-available, sterile
96-well microtiter plate. The following day, 5 .mu.l of each
overnight culture was tranferred into a non-sterile 96-well plate
and after dilution 1:10 with water, 5 .mu.l of each sample was
transferred into a PCR array.
[0100] For PCR amplification, 15 .mu.l of PCR mix
(1.33.times.concentrated containing 0.75 units of Taq polymerase, a
vector primer and one or both of the gene specific primers used for
the extension reaction) were added to each well. Amplification was
performed using the following conditions:
3 Step 1 94.degree. C. for 60 sec Step 2 94.degree. C. for 20 sec
Step 3 55.degree. C. for 30 sec Step 4 72.degree. C. for 90 sec
Step 5 Repeat steps 2-4 for an additional 29 times Step 6
72.degree. C. for 180 sec Step 7 4.degree. C. (and holding)
[0101] Aliquots of these PCR reactions were run on agarose gels
together with molecular weight markers. The sizes of the PCR
products were compared to the original partial cDNAs, and
appropriate clones were selected, ligated into plasmid and
sequenced.
[0102] The cDNA (SEQ ID NO 1) and amino acid (SEQ ID NO 2)
sequences for human PNR are shown in FIG. 1. When the translation
of the sequence was searched against protein databases such as
SwissProt and PIR, no exact matches were found. FIG. 2 shows the
comparison of the human PNR sequence with that of the rat
purinergic sequence, RNU09402.
VI Antisense analysis
[0103] Knowledge of the correct, complete cDNA sequence of PNR
enables its use as a tool for antisense technology in the
investigation of gene function. Oligonucleotides, cDNA or genomic
fragments comprising the antisense strand of pnr can be used either
in vitro or in vivo to inhibit expression of the mRNA. Such
technology is now well known in the art, and antisense molecules
can be designed at various locations along the nucleotide
sequences. By treatment of cells or whole test animals with such
antisense sequences, the gene of interest can be effectively turned
off. Frequently, the function of the gene can be ascertained by
observing behavior at the intracellular, cellular, tissue or
organismal level (eg, lethality, loss of differentiated function,
changes in morphology, etc).
[0104] In addition to using sequences constructed to interrupt
transcription of a particular open reading frame, modifications of
gene expression can be obtained by designing antisense sequences to
intron regions, promoter/enhancer elements, or even to trans-acting
regulatory genes. Similarly, inhibition can be achieved using
Hogeboom base-pairing methodology, also known as "triple helix"
base pairing.
VII Expression of PNR
[0105] Expression of pnr may be accomplished by subcloning the
cDNAs into appropriate expression vectors and transfecting the
vectors into analogous expression hosts. In this particular case,
the cloning vector previously used for the generation of the cDNA
library also provides for direct expression of pnr sequences in E.
coli. Upstream of the cloning site, this vector contains a promoter
for .beta.-galactosidase, followed by sequence containing the
amino-terminal Met and the subsequent 7 residues of
.beta.-galactosidase. Immediately following these eight residues is
an engineered bacteriophage promoter useful for artificial priming
and transcription and a number of unique restriction sites,
including Eco RI, for cloning.
[0106] Induction of the isolated, transfected bacterial strain with
IPTG using standard methods will produce a fusion protein
corresponding to the first seven residues of .beta.-galactosidase,
about 15 residues of "linker", and the peptide encoded within the
cDNA. Since cDNA clone inserts are generated by an essentially
random process, there is one chance in three that the included cDNA
will lie in the correct frame for proper translation. If the cDNA
is not in the proper reading frame, it can be obtained by deletion
or insertion of the appropriate number of bases by well known
methods including in vitro mutagenesis, digestion with exonuclease
III or mung bean nuclease, or the inclusion of an oligonucleotide
linker of appropriate length.
[0107] The pnr cDNA can be shuttled into other vectors known to be
useful for expression of protein in specific hosts. Oligonucleotide
primers containing cloning sites as well as a segment of DNA (about
25 bases) sufficient to hybridize to stretches at both ends of the
target cDNA can be synthesized chemically by standard methods.
These primers can then used to amplify the desired gene segment by
PCR. The resulting gene segment can be digested with appropriate
restriction enzymes under standard conditions and isolated by gel
electrophoresis. Alternately, similar gene segments can be produced
by digestion of the cDNA with appropriate restriction enzymes.
Using appropriate primers, segments of coding sequence from more
than one gene can be ligated together and cloned in appropriate
vectors. It is possible to optimize expression by construction of
such chimeric sequences.
[0108] Suitable expression hosts for such chimeric molecules
include, but are not limited to, mammalian cells such as Chinese
Hamster Ovary (CHO) and human 293 cells, insect cells such as Sf9
cells, yeast cells such as Saccharomyces cerevisiae, and bacteria
such as E. coli. For each of these cell systems, a useful
expression vector may also include an origin of replication to
allow propagation in bacteria and a selectable marker such as the
.beta.-lactamase antibiotic resistance gene to allow plasmid
selection in bacteria. In addition, the vector may include a second
selectable marker such as the neomycin phosphotransferase gene to
allow selection in transfected eukaryotic host cells. Vectors for
use in eukaryotic expression hosts may require RNA processing
elements such as 3' polyadenylation sequences if such are not part
of the cDNA of interest.
[0109] Additionally, the vector may contain promoters or enhancers
which increase gene expression. Such promoters are host specific
and include MMTV, SV40, and metallothionine promoters for CHO
cells; trp, lac, tac and T7 promoters for bacterial hosts; and
alpha factor, alcohol oxidase and PGH promoters for yeast.
Transcription enhancers, such as the rous sarcoma virus enhancer,
may be used in mammalian host cells. Once homogeneous cultures of
recombinant cells are obtained through standard culture methods,
large quantities of recombinantly produced PNR can be recovered
from the conditioned medium and analyzed using chromatographic
methods known in the art.
VIII Isolation of Recombinant PNR
[0110] PNR may be expressed as a chimeric protein with one or more
additional polypeptide domains added to facilitate protein
purification. Such purification facilitating domains include, but
are not limited to, metal chelating peptides such as
histidine-tryptophan modules that allow purification on immobilized
metals, protein A domains that allow purification on immobilized
immunoglobulin, and the domain utilized in the FLAGS
extension/affinity purification system (Immunex Corp, Seattle
Wash.). The inclusion of a cleavable linker sequence such as Factor
XA or enterokinase (Invitrogen, San Diego Calif.) between the
purification domain and the pnr sequence may be useful to
facilitate expression of PNR.
IX Testing of P.sub.2U Receptors
[0111] The procedures for testing purinergic receptors were
published by Erb et al (1993, Proc Natl Acad Sci 90:10449-53). The
function of PNRs can easily be tested in cultured K562 human
leukemia cells because these cells lack P.sub.2U receptors. K562
cells are transfected with expression vectors containing pnr and
loaded with fura-a, fluorescent probe for Ca.sup.++. Activation of
properly assembled and functional P.sub.2U receptors with
extracellular UTP or ATP mobilizes intracellular Ca.sup.++ which
reacts with fura-a and is measured spectrofluorometrically. In
addition these procedures can be used to define the affinity and
effective concentration of those extracellular nucleotides which
activate such receptors. Likewise, chimeric receptors--combining
extracellular receptive sequences of any newly discovered T7G--with
the transmembrane and intracellular segments of a known molecule
such as pnr are useful in defining potential ligands for the new
molecule.
[0112] Chimeric or modified P.sub.2U receptors containing
substitutions in the transmembrane or intracellular regions may be
activated using UTP and the resulting biological activity assessed.
Once function is established, the amino- or carboxy-terminal
residues are useful in testing antagonists or inhibitors of
intracellular Ca.sup.++ release or phosphoinositide metabolism.
X Production of PNR Specific Antibodies
[0113] Two approaches are utilized to raise antibodies to PNR, and
each approach is useful for generating either polyclonal or
monoclonal antibodies. In one approach, denatured protein from
reverse phase HPLC separation is obtained in quantities up to 75
mg. This denatured protein can be used to immunize mice or rabbits
using standard protocols; about 100 micrograms are adequate for
immunization of a mouse, while up to 1 mg might be used to immunize
a rabbit. For identifying mouse hybridomas, the denatured protein
can be radioiodinated and used to screen potential murine B-cell
hybridomas for those which produce antibody. This procedure
requires only small quantities of protein, such that 20 mg would be
sufficient for labeling and screening of several thousand
clones.
[0114] In the second approach, the amino acid sequence of an
appropriate PNR domain, as deduced from translation of the cDNA, is
analyzed to determine regions of high immunogenicity. Oligopeptides
comprising appropriate hydrophilic regions, as illustrated in FIG.
3, are synthesized and used in suitable immunization protocols to
raise antibodies. Analysis to select appropriate epitopes is
described by Ausubel FM et al (supra). The optimal amino acid
sequences for immunization are usually at the C-terminus, the
N-terminus and those intervening, hydrophilic regions of the
polypeptide which are likely to be exposed to the external
environment when the protein is in its natural conformation.
[0115] Typically, selected peptides, about 15 residues in length,
are synthesized using an Applied Biosystems Peptide Synthesizer
Model 431A using fmoc-chemistry and coupled to keyhole limpet
hemocyanin (KLH; Sigma, St Louis Mo.) by reaction with
M-maleimidobenzoyl-N- hydroxysuccinimide ester (MBS; cf. Ausubel FM
et al, supra). If necessary, a cysteine may be introduced at the
N-terminus of the peptide to permit coupling to KLH. Rabbits are
immunized with the peptide-KLH complex in complete Freund's
adjuvant. The resulting antisera are tested for antipeptide
activity by binding the peptide to plastic, blocking with 1% bovine
serum albumin, reacting with antisera, washing and reacting with
labeled (radioactive or fluorescent), affinity purified, specific
goat anti-rabbit IgG.
[0116] Hybridomas may also be prepared and screened using standard
techniques. Hybridomas of interest are detected by screening with
labeled PNR to identify those fusions producing the monoclonal
antibody with the desired specificity. In a typical protocol, wells
of plates (FAST; Becton-Dickinson, Palo Alto Calif.) are coated
during incubation with affinity purified, specific
rabbit-anti-mouse (or suitable anti-species Ig) antibodies at 10
mg/ml. The coated wells are blocked with 1% BSA, washed and
incubated with supernatants from hybridomas. After washing the
wells are incubated with labeled PNR at 1 mg/ml. Supernatants with
specific antibodies bind more labeled PNR than is detectable in the
background. Then clones producing specific antibodies are expanded
and subjected to two cycles of cloning at limiting dilution. Cloned
hybridomas are injected into pristane-treated mice to produce
ascites, and monoclonal antibody is purified from mouse ascitic
fluid by affinity chromatography on Protein A. Monoclonal
antibodies with affinities of at least 10e8 Me-1, preferably 10e9
to 10e10 or stronger, will typically be made by standard procedures
as described in Harlow and Lane (1988) Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.;
and in Goding (1986) Monoclonal Antibodies: Principles and
Practice, Academic Press, New York City, both incorporated herein
by reference.
XI Diagnostic Test Using PNR Specific Antibodies
[0117] Particular PNR antibodies are useful for investigating
signal transduction and the diagnosis of infectious or hereditary
conditions which are characterized by differences in the amount or
distribution of PNR or downstream products of an active signalling
cascade. Since PNR was found in a human placenta library, it
appears to be upregulated in cell types mainly involved in immune
protection or defense.
[0118] Diagnostic tests for PNR include methods utilizing antibody
and a label to detect PNR in human body fluids, membranes, cells,
tissues or extracts of such. The polypeptides and antibodies of the
present invention may be used with or without modification.
Frequently, the polypeptides and antibodies will be labeled by
joining them, either covalently or noncovalently, with a substance
which provides for a detectable signal. A wide variety of labels
and conjugation techniques are known and have been reported
extensively in both the scientific and patent literature. Suitable
labels include radionuclides, enzymes, substrates, cofactors,
inhibitors, fluorescent agents, chemiluminescent agents, magnetic
particles and the like. Patents teaching the use of such labels
include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;
4,277,437; 4,275,149; and 4,366,241. Also, recombinant
immunoglobulins may be produced as shown in U.S. Pat. No.
4,816,567, incorporated herein by reference.
[0119] A variety of protocols for measuring soluble or
membrane-bound PNR, using either polyclonal or monoclonal
antibodies specific for the protein, are known in the art. Examples
include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay
(RIA) and fluorescent activated cell sorting (FACS). A two-site
monoclonal-based immunoassay utilizing monoclonal antibodies
reactive to two non-interfering epitopes on PNR is preferred, but a
competitive binding assay may be employed. These assays are
described, among other places, in Maddox, Del. et al (1983, J Exp
Med 158:1211f).
XII Purification of Native PNR Using Specific Antibodies
[0120] Native or recombinant PNR can be purified by immunoaffinity
chromatography using antibodies specific for PNR. In general, an
immunoaffinity column is constructed by covalently coupling the
anti-PNR antibody to an activated chromatographic resin.
[0121] Polyclonal immunoglobulins are prepared from immune sera
either by precipitation with ammonium sulfate or by purification on
immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway,
N.J.). Likewise, monoclonal antibodies are prepared from mouse
ascites fluid by ammonium sulfate precipitation or chromatography
on immobilized Protein A. Partially purified immunoglobulin is
covalently attached to a chromatographic resin such as
CnBr-activated Sepharose (Pharmacia, Piscataway N.J.). The antibody
is coupled to the resin, the resin is blocked, and the derivative
resin is washed according to the manufacturer's instructions.
[0122] Such immunoaffinity columns may be utilized in the
purification of PNR by preparing a fraction from cells containing
PNR in a soluble form. This preparation may be derived by
solubilization of whole cells or of a subcellular fraction obtained
via differential centrifugation (with or without addition of
detergent) or by other methods well known in the art.
Alternatively, soluble PNR containing a signal sequence may be
secreted in useful quantity into the medium in which the cells are
grown.
[0123] A soluble PNR-containing preparation is passed over the
immunoaffinity column, and the column is washed under conditions
that allow the preferential absorbance of PNR (eg, high ionic
strength buffers in the presence of detergent). Then, the column is
eluted under conditions that disrupt antibody/PNR binding (eg, a
buffer of pH 2-3 or a high concentration of a chaotrope such as
urea or thiocyanate ion), and PNR is collected.
XIII Drug Screening
[0124] This invention is particularly useful for screening
therapeutic compounds by using PNR or binding fragments thereof in
any of a variety of drug screening techniques. The polypeptide or
fragment employed in such a test may either be free in solution,
affixed to a solid support, borne on a cell surface or located
intracellularly. One method of drug screening utilizes eukaryotic
or prokaryotic host cells which are stably transformed with
recombinant nucleic acids expressing the polypeptide or fragment.
Drugs are screened against such transformed cells in competitive
binding assays. Such cells, either in viable or fixed form, can be
used for standard binding assays. One may measure, for example, the
formation of complexes between PNR and the agent being tested.
Alternatively, one can examine the diminution in complex formation
between PNR and a receptor caused by the agent being tested.
[0125] Thus, the present invention provides methods of screening
for drugs or any other agents which can affect signal transduction.
These methods, well known in the art, comprise contacting such an
agent with PNR polypeptide or a fragment thereof and assaying (i)
for the presence of a complex between the agent and the PNR
polypeptide or fragment, or (ii) for the presence of a complex
between the PNR polypeptide or fragment and the cell. In such
competitive binding assays, the PNR polypeptide or fragment is
typically labeled. After suitable incubation, free PNR polypeptide
or fragment is separated from that present in bound form, and the
amount of free or uncomplexed label is a measure of the ability of
the particular agent to bind to PNR or to interfere with the PNR
and agent complex.
[0126] Another technique for drug screening provides high
throughput screening for compounds having suitable binding affinity
to the PNR polypeptides and is described in detail in European
Patent Application 84/03564, published on Sep. 13, 1984,
incorporated herein by reference. Briefly stated, large numbers of
different small peptide test compounds are synthesized on a solid
substrate, such as plastic pins or some other surface. The peptide
test compounds are reacted with PNR polypeptide and washed. Bound
PNR polypeptide is then detected by methods well known in the art.
Purified PNR can also be coated directly onto plates for use in the
aforementioned drug screening techniques. In addition,
non-neutralizing antibodies can be used to capture the peptide and
immobilize it on the solid support.
[0127] This invention also contemplates the use of competitive drug
screening assays in which neutralizing antibodies capable of
binding PNR specifically compete with a test compound for binding
to PNR polypeptides or fragments thereof. In this manner, the
antibodies can be used to detect the presence of any peptide which
shares one or more antigenic determinants with PNR.
XIV Rational Drug Design
[0128] The goal of rational drug design is to produce structural
analogs of biologically active polypeptides of interest or of small
molecules with which they interact, eg, agonists, antagonists, or
inhibitors. Any of these examples can be used to fashion drugs
which are more active or stable forms of the polypeptide or which
enhance or interfere with the function of a polypeptide in vivo
(cf. Hodgson J (1991) Bio/Technology 9:19-21, incorporated herein
by reference).
[0129] In one approach, the three-dimensional structure of a
protein of interest, or of a protein-inhibitor complex, is
determined by x-ray crystallography, by computer modeling or, most
typically, by a combination of the two approaches. Both the shape
and charges of the polypeptide must be ascertained to elucidate the
structure and to determine active site(s) of the molecule. Less
often, useful information regarding the structure of a polypeptide
may be gained by modeling based on the structure of homologous
proteins. In both cases, relevant structural information is used to
design efficient inhibitors. Useful examples of rational drug
design may include molecules which have improved activity or
stability as shown by Braxton S and Wells J A (1992, Biochemistry
31:7796-7801) or which act as inhibitors, agonists, or antagonists
of native peptides as shown by Athauda S B et al (1993 J Biochem
113:742-46), incorporated herein by reference.
[0130] It is also possible to isolate a target-specific antibody,
selected by functional assay, as described above, and then to solve
its crystal structure. This approach, in principle, yields a
pharmacore upon which subsequent drug design can be based. It is
possible to bypass protein crystallography altogether by generating
anti-idiotypic antibodies (anti-ids) to a functional,
pharmacologically active antibody. As a mirror image of a mirror
image, the binding site of the anti-ids is expected to be an analog
of the original receptor. The anti-id can then be used to identify
and isolate peptides from banks of chemically or biologically
produced peptides. The isolated peptides then act as the
pharmacore.
[0131] By virtue of the present invention, sufficient amount of
polypeptide may be made available to perform such analytical
studies as X-ray crystallography. In addition, knowledge of the PNR
amino acid sequence provided herein will provide guidance to those
employing computer modeling techniques in place of or in addition
to x-ray crystallography.
XV Identification of Other Members of the Signal Transduction
Complex
[0132] The inventive purified PNR is a research tool for
identification, characterization and purification of interacting G
or other signal transduction pathway proteins. Radioactive labels
are incorporated into a selected PNR domain by various methods
known in the art and used in vitro to capture interacting
molecules. A preferred method involves labeling the primary amino
groups in PNR with .sup.125I Bolton-Hunter reagent (Bolton, A E and
Hunter, W M (1973) Biochem J 133: 529). This reagent has been used
to label various molecules without concomitant loss of biological
activity (Hebert Calif. et al (1991) J Biol Chem 266: 18989; McColl
S et al (1993) J Immunol 150:4550-4555). Membrane-bound molecules
are incubated with the labeled PNR molecules, washed to removed
unbound molecules, and the PNR complex is quantified. Data obtained
using different concentrations of PNR are used to calculate values
for the number, affinity, and association of PNR complex.
[0133] Labeled PNR is also useful as a reagent for the purification
of molecules with which PNR interacts. In one embodiment of
affinity purification, PNR is covalently coupled to a
chromatography column. Cells and their membranes are extracted, PNR
is removed and various PNR-free subcomponents are passed over the
column. Molecules bind to the column by virtue of their PNR
affinity. The PNR-complex is recovered from the column,
dissociated, and subjected to N-terminal protein sequencing. This
amino acid sequence is then used to identify the captured molecule
or to design degenerate oligonucleotide probes for cloning its gene
from an appropriate cDNA library.
[0134] In another alternate method, antibodies are raised against
PNR, specifically monoclonal antibodies. The monoclonal antibodies
are screened to identify those which inhibit the binding of labeled
PNR. These monoclonal antibodies are then used in affinity
purification or expression cloning of associated molecules.
[0135] Other soluble binding molecules are identified in a similar
manner. Labeled PNR is incubated with extracts or other appropriate
materials derived from mast cells and putative target cells. After
incubation, PNR complexes (which are larger than the lone PNR
molecule) are identified by a sizing technique such as size
exclusion chromatography or density gradient centrifugation and are
purified by methods known in the art. The soluble binding
protein(s) are subjected to N-terminal sequencing to obtain
information sufficient for database identification, if the soluble
protein is known, or for cloning, if the soluble protein is
unknown.
XVI Administration of Antibodies, Inhibitors, or Antagonists of
PNR
[0136] Antibodies, inhibitors, or antagonists of PNR (or other
molecules to limit signal transduction, LST), can provide different
effects when administered therapeutically. LSTs will be formulated
in a nontoxic, inert, pharmaceutically acceptable aqueous carrier
medium preferably at a pH of about 5 to 8, more preferably 6 to 8,
although the pH may vary according to the characteristics of the
antibody, inhibitor, or antagonist being formulated and the
condition to be treated. Characteristics of LSTs include solubility
of the molecule, half-life and antigenicity/immunogenicity; these
and other characteristics may aid in defining an effective carrier.
Native human proteins are preferred as LSTs, but organic or
synthetic molecules resulting from drug screens may be equally
effective in particular situations.
[0137] LSTs may be delivered by known routes of administration
including but not limited to topical creams and gels; transmucosal
spray and aerosol; transdermal patch and bandage; injectable,
intravenous and lavage formulations; and orally administered
liquids and pills particularly formulated to resist stomach acid
and enzymes. The particular formulation, exact dosage, and route of
administration will be determined by the attending physician and
will vary according to each specific situation.
[0138] Such determinations are made by considering multiple
variables such as the condition to be treated, the LST to be
administered, and the pharmacokinetic profile of the particular
LST. Additional factors which may be taken into account include
disease state (e.g. severity) of the patient, age, weight, gender,
diet, time and frequency of adminis-tration, drug combination,
reaction sensitivities, and tolerance/response to therapy. Long
acting LST formulations might be administered every 3 to 4 days,
every week, or once every two weeks depending on half-life and
clearance rate of the particular LST.
[0139] Normal dosage amounts may vary from 0.1 to 100,000
micrograms, up to a total dose of about 1 g, depending upon the
route of administration. Guidance as to particular dosages and
methods of delivery is provided in the literature. See U.S. Pat.
No. 4,657,760; 5,206,344; or 5,225,212. Those skilled in the art
will employ different formulations for different LSTs.
Administration to particular cell types will necessitate different
methods of delivery, ie. vascular endothelial cells versus glial
cells.
[0140] It is contemplated that abnormal signal transduction and the
conditions or diseases which trigger such activity may precipitate
damage that is treatable with LSTs. These conditions or diseases
may be specifically diagnosed by the tests discussed above, and
such testing should be performed in suspected cases of systemic and
local infections, traumatic and other tissue damage, hereditary or
environmental diseases associated with hypertension, carcinomas,
cystic fibrosis, and other physiologic or pathologic problems.
[0141] All publications and patents mentioned in the above
specification are herein incorporated by reference. Various
modifications and variations of the described method and system of
the invention will be apparent to those skilled in the art without
departing from the scope and spirit of the invention. Although the
invention has been described in connection with specific preferred
embodiments, it should be understood that the invention as claimed
should not be unduly limited to such specific embodiments. Indeed,
various modifications of the above-described modes for carrying out
the invention which are obvious to those skilled in the field of
molecular biology or related fields are intended to be within the
scope of the following claims.
Sequence CWU 1
1
3 1 984 DNA Homo sapiens misc_feature Incyte ID No 179696CB1 1
atggaatggg acaatggcac agaccaggct ctgggcttgc cacccaccac ctgtgtctac
60 cgcgagaact tcaagcaact gctgctccca cctgtgtatt cggcggtgct
ggcgcctgcc 120 ctcccgctga acatctgtgt cattacccag atctgcacgt
cccgccgggc cctgacccgc 180 acggccgtgt acaccctaaa ccttgctctg
cctgacctgc tatatgcctg ctccctgccc 240 ctgctcatct acaactatgc
ccaaggtgat cactggccct ttggcgactt cgcctgccgc 300 ctggtccgct
tcctcttcta tgccaacctg cacgggagga tcctcttcct cacctgcatc 360
agcttccagc gctacctggg catctgccac ccgctggccc cctggcacaa acgtgggggc
420 cgccgggctg cctggctagt gtgtgtagcc gtgtggctgg ccgtgacaac
ccagtgcctg 480 cccacagcca tcttcgctgc cacaggcatc cagcgtaacc
gcactgtctg ttatgacctc 540 agcccgcctg ccctggccac ccactatatg
ccctatggga tggctctcac tgtcatcggc 600 ttcctgctgc cctttgctgc
cctgctggcc tgctactgtc tcctggcctg ccgcctgtgc 660 cgccaggatg
gcccggcaga gcctgtggcc caggagcggc gtggcaaggc ggcccgcatg 720
gccgtggtgg tggctgctgt ctttggcatc agcttcctgc cttttcacat caccaagaca
780 gcctacctgg cagtgcgctc gacgccgggc gtcccctgca ctgtattgga
ggcctttgca 840 gcggcctaca aaggcacgcg gccgtttgcc agtgccaaca
gcgtgctgga ccccatcctc 900 ttctacttca cccagaagaa gttccgccgg
cgaccacatg agctcctaca gaaactcaca 960 gacaaatggc agaggcaggg tcgc 984
2 328 PRT Homo sapiens misc_feature Incyte ID No 179696CD1 2 Met
Glu Trp Asp Asn Gly Thr Asp Gln Ala Leu Gly Leu Pro Pro 1 5 10 15
Thr Thr Cys Val Tyr Arg Glu Asn Phe Lys Gln Leu Leu Leu Pro 20 25
30 Pro Val Tyr Ser Ala Val Leu Ala Pro Ala Leu Pro Leu Asn Ile 35
40 45 Cys Val Ile Thr Gln Ile Cys Thr Ser Arg Arg Ala Leu Thr Arg
50 55 60 Thr Ala Val Tyr Thr Leu Asn Leu Ala Leu Pro Asp Leu Leu
Tyr 65 70 75 Ala Cys Ser Leu Pro Leu Leu Ile Tyr Asn Tyr Ala Gln
Gly Asp 80 85 90 His Trp Pro Phe Gly Asp Phe Ala Cys Arg Leu Val
Arg Phe Leu 95 100 105 Phe Tyr Ala Asn Leu His Gly Arg Ile Leu Phe
Leu Thr Cys Ile 110 115 120 Ser Phe Gln Arg Tyr Leu Gly Ile Cys His
Pro Leu Ala Pro Trp 125 130 135 His Lys Arg Gly Gly Arg Arg Ala Ala
Trp Leu Val Cys Val Ala 140 145 150 Val Trp Leu Ala Val Thr Thr Gln
Cys Leu Pro Thr Ala Ile Phe 155 160 165 Ala Ala Thr Gly Ile Gln Arg
Asn Arg Thr Val Cys Tyr Asp Leu 170 175 180 Ser Pro Pro Ala Leu Ala
Thr His Tyr Met Pro Tyr Gly Met Ala 185 190 195 Leu Thr Val Ile Gly
Phe Leu Leu Pro Phe Ala Ala Leu Leu Ala 200 205 210 Cys Tyr Cys Leu
Leu Ala Cys Arg Leu Cys Arg Gln Asp Gly Pro 215 220 225 Ala Glu Pro
Val Ala Gln Glu Arg Arg Gly Lys Ala Ala Arg Met 230 235 240 Ala Val
Val Val Ala Ala Val Phe Gly Ile Ser Phe Leu Pro Phe 245 250 255 His
Ile Thr Lys Thr Ala Tyr Leu Ala Val Arg Ser Thr Pro Gly 260 265 270
Val Pro Cys Thr Val Leu Glu Ala Phe Ala Ala Ala Tyr Lys Gly 275 280
285 Thr Arg Pro Phe Ala Ser Ala Asn Ser Val Leu Asp Pro Ile Leu 290
295 300 Phe Tyr Phe Thr Gln Lys Lys Phe Arg Arg Arg Pro His Glu Leu
305 310 315 Leu Gln Lys Leu Thr Asp Lys Trp Gln Arg Gln Gly Arg 320
325 3 374 PRT Rattus norvegius misc_feature RNU09402 3 Met Ala Ala
Gly Leu Asp Ser Trp Asn Ser Thr Ile Asn Gly Thr 1 5 10 15 Trp Glu
Gly Asp Glu Leu Gly Tyr Lys Cys Arg Phe Asn Glu Asp 20 25 30 Phe
Lys Tyr Val Leu Leu Pro Val Ser Tyr Gly Val Val Cys Val 35 40 45
Leu Gly Leu Cys Leu Asn Val Val Ala Leu Tyr Ile Phe Leu Cys 50 55
60 Arg Leu Lys Thr Trp Asn Ala Ser Thr Thr Tyr Met Phe His Leu 65
70 75 Ala Val Ser Asp Ser Leu Tyr Ala Ala Ser Leu Pro Leu Leu Val
80 85 90 Tyr Tyr Tyr Ala Gln Gly Asp His Trp Pro Phe Ser Thr Val
Leu 95 100 105 Cys Lys Leu Val Arg Phe Leu Phe Tyr Thr Asn Leu Tyr
Cys Ser 110 115 120 Ile Leu Phe Leu Thr Cys Ile Ser Val His Arg Ser
Leu Gly Val 125 130 135 Leu Arg Pro Leu His Ser Leu Arg Trp Gly His
Ala Arg Tyr Ala 140 145 150 Arg Arg Val Ala Ala Val Val Trp Val Leu
Val Leu Ala Cys Gln 155 160 165 Thr Pro Val Leu Tyr Phe Val Thr Thr
Ser Val Arg Gly Thr Arg 170 175 180 Ile Thr Cys His Asp Thr Ser Asp
Arg Glu Leu Phe Ser His Phe 185 190 195 Val Ala Tyr Ser Ser Val Met
Leu Gly Leu Leu Phe Ala Val Pro 200 205 210 Phe Ser Ile Ile Leu Val
Cys Tyr Val Leu Met Ala Arg Arg Leu 215 220 225 Leu Lys Pro Ala Tyr
Gly Thr Thr Gly Leu Pro Arg Ala Lys Arg 230 235 240 Lys Ser Val Arg
Thr Ile Ala Leu Val Leu Ala Val Phe Ala Leu 245 250 255 Cys Phe Leu
Pro Phe His Val Thr Arg Thr Leu Tyr Tyr Ser Phe 260 265 270 Arg Ser
Leu Asp Leu Ser Cys His Thr Leu Asn Ala Ile Asn Met 275 280 285 Ala
Tyr Lys Ile Thr Arg Pro Leu Ala Ser Ala Asn Ser Cys Leu 290 295 300
Asp Pro Val Leu Tyr Phe Leu Ala Gly Gln Arg Leu Val Arg Phe 305 310
315 Ala Arg Asp Ala Lys Pro Ala Thr Glu Pro Thr Pro Ser Pro Gln 320
325 330 Ala Arg Arg Lys Leu Gly Leu His Arg Pro Asn Arg Thr Asp Thr
335 340 345 Val Arg Lys Asp Leu Ser Ile Ser Ser Asp Asp Ser Arg Arg
Thr 350 355 360 Glu Ser Thr Pro Ala Gly Ser Glu Thr Lys Asp Ile Arg
Leu 365 370
* * * * *