U.S. patent application number 13/946907 was filed with the patent office on 2014-05-15 for compositions for diagnosis and therapy of diseases associated with aberrant expression of futrins (r-spondins) and/or wnt.
This patent application is currently assigned to Deutsches Krebsforschungszentrum. The applicant listed for this patent is Deutsches Krebsforschungszentrum. Invention is credited to Andrey Glinka, Olga Kazanskaya, Christof Niehrs, Wei Wu.
Application Number | 20140134703 13/946907 |
Document ID | / |
Family ID | 34486059 |
Filed Date | 2014-05-15 |
United States Patent
Application |
20140134703 |
Kind Code |
A1 |
Niehrs; Christof ; et
al. |
May 15, 2014 |
Compositions for Diagnosis and Therapy of Diseases Associated with
Aberrant Expression of Futrins (R-Spondins) and/or Wnt
Abstract
The present invention relates to a composition useful for the
diagnosis of diseases associated with aberrant expression of the
genes encoding the secreted proteins Futrin 1, 2, 3 and/or 4
(=R-Spondin 2, 3, 1 and 4, respectively), e.g. in connection with
tumors or diseases of the muscle, kidneys or bones. The present
invention also relates to a pharmaceutical composition containing a
compound which is capable of modifying (a) the expression of the
gene encoding Futrin 1, 2, 3 and/or 4 or (b) the activity of the
Futrin 1, 2, 3 and/or 4 protein.
Inventors: |
Niehrs; Christof;
(Heidelberg, DE) ; Wu; Wei; (Beijing, CN) ;
Glinka; Andrey; (Edingen-Neckarhausen, DE) ;
Kazanskaya; Olga; (Heidelberg, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Deutsches Krebsforschungszentrum |
Heidelberg |
|
DE |
|
|
Assignee: |
Deutsches
Krebsforschungszentrum
Heidelberg
DE
|
Family ID: |
34486059 |
Appl. No.: |
13/946907 |
Filed: |
July 19, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13931481 |
Jun 28, 2013 |
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13946907 |
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10575217 |
May 7, 2007 |
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PCT/EP04/11269 |
Oct 8, 2004 |
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13931481 |
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Current U.S.
Class: |
435/188 ;
530/387.3; 530/387.9; 530/391.3 |
Current CPC
Class: |
C07K 16/18 20130101;
C07K 2317/54 20130101; G01N 33/574 20130101; C07K 16/22 20130101;
G01N 33/57492 20130101; C12Q 1/6883 20130101; C07K 2317/24
20130101; C07K 2317/55 20130101; C12Q 1/6897 20130101; C12Q
2600/136 20130101; C12Q 2600/158 20130101; G01N 33/5041 20130101;
C07K 16/30 20130101; G01N 2500/10 20130101 |
Class at
Publication: |
435/188 ;
530/387.9; 530/387.3; 530/391.3 |
International
Class: |
C07K 16/22 20060101
C07K016/22 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 10, 2003 |
EP |
03023000.7 |
Claims
1-20. (canceled)
21. An isolated antibody, that binds the Futrin 2 polypeptide of
SEQ ID NO:27, wherein the antibody inhibits wnt signaling.
22. The antibody of claim 21, wherein the antibody is a monoclonal
antibody.
23. The antibody of claim 22, wherein the antibody is a chimeric
antibody or humanized antibody.
24. The antibody of claim 23, wherein the antibody is an antibody
fragment.
25. The antibody of claim 24, wherein the antibody fragment is a
Fab fragment or F(ab').sub.2.
26. The antibody of claim 21, wherein the antibody binds furin-like
domain 1 and/or furin-like domain 2 of SEQ ID NO:27.
27. The antibody of claim 26, wherein the antibody is a monoclonal
antibody.
28. The antibody of claim 27, wherein the antibody is a chimeric
antibody or humanized antibody.
29. The antibody of claim 28, wherein the antibody is an antibody
fragment.
30. The antibody of claim 29, wherein the antibody fragment is a
Fab fragment or F(ab').sub.2.
31. The antibody of claim 21, wherein the antibody reduces wnt
reporter signaling in a mammalian cell line cultured in the
presence of Futrin 2 by at least 40%.
32. The antibody of claim 31, wherein the antibody is a monoclonal
antibody.
33. The antibody of claim 32, wherein the antibody is a chimeric
antibody or humanized antibody.
34. The antibody of claim 33, wherein the antibody is an antibody
fragment.
35. The antibody of claim 34, wherein the antibody fragment is a
Fab fragment or F(ab').sub.2.
36. The antibody of claim 26, wherein the antibody reduces wnt
reporter signaling in a mammalian cell line cultured in the
presence of Futrin 2 by at least 40%.
37. The antibody of claim 36, wherein the antibody is a monoclonal
antibody.
38. The antibody of claim 37, wherein the antibody is a chimeric
antibody or humanized antibody.
39. The antibody of claim 38, wherein the antibody is an antibody
fragment.
40. The antibody of claim 39, wherein the antibody fragment is a
Fab fragment or F(ab').sub.2.
41. The antibody of claim 22, wherein the antibody is labeled with
a detectable label.
42. The antibody of claim 41, wherein the detectable label is a
radioisotope or enzyme label.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 13/931,481, filed Jun. 28, 2013, which is a divisional of U.S.
application Ser. No. 10/575,217, filed Apr. 10, 2006, the contents
of both of which are hereby incorporated by reference, which is a
national stage application of International Application No.
PCT/EP2004/011269 having an international filing date of Oct. 8,
2004 and which claims benefit under 35 U.S.C. .sctn.119 to European
Patent Application No. 0302300.7 filed Oct. 10, 2003.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Jan. 8, 2014, is named P5660D2US_Sequence_Listing.txt and is
31,550 bytes in size.
BACKGROUND
[0003] The present invention relates to compositions useful for the
diagnosis and therapy of diseases associated with aberrant
expression of the genes encoding the proteins Futrin 1, 2, 3 and/or
4 (=R-Spondin 2, 3, 1 and 4, respectively). These diseases include
tumors of e.g. the breast, ovary, liver, uterus, cervix, colon,
lung, ovary, rectum, testis, pancreas, bones and skin, as well as
diseases involving muscle, bone, lipid and glucose metabolism, and
obesity. The present invention also relates to a pharmaceutical
composition containing a compound which is capable of modifying (a)
the expression of the gene encoding Futrin 1, 2, 3 and/or 4 or (b)
the activity of Futrin 1, 2, 3 and/or 4.
[0004] The Wnt signal cascade plays a crucial role as regards
regulation of survival, proliferation and differentiation of cells
during embryogenesis, and in the adult as shown, e.g., in
Drosophila, Xenopus and mice (Nusse and Varmus, Cell 69 (1992),
1073-1087). Wnt-genes encode secretory glycoproteins which activate
a well characterized signal cascade via a Wnt receptor called
"frizzled".
[0005] The Wnt signalling cascade and its components also play an
important role in various diseases which makes it desirable to
modulate its activity:
i) Cancer
[0006] Tumorigenesis represents a complex multistage process in
which genetic changes and environmental factors are thought to
deregulate the cellular processes that control cell proliferation
and differentiation. Several studies indicate that an aberrant Wnt
signal cascade is involved in the development of colon cancer,
breast cancer and melanoma (Pfeifer, Science, 2 75 (1997),
1752-1753; Polakis, Genes Dev. 14 (2000), 1837-1851). The first
gene encoding a protein of the Wnt signal cascade, int-1, was
isolated from mouse mammary tumor virus (MMTV) and it could be
shown that it is an oncogene. It is thus well established that an
aberrant regulation of the activity of Wnt and/or components of the
Wnt signal cascade downstream of the Wnt signal, e.g., beta-catenin
and APC, are involved in tumorigenesis.
ii) Bone Disease
[0007] Wnt signals promote bone formation (e.g. Yang, Development,
130(2003), 1003-15; Fischer, J. Biol. Chem. 277 (2002)
30870-30878). Consistent with this notion, a gain-of-function
mutation of the Wnt receptor LRP5 causes high bone disease (Boyden,
et al., 346 (2002) N Engl J Med, 1513-21.; Little, et al., 70
(2002) Am J Hum Genet, 11-9.). Conversely, inactivating mutations
in LRP5 leads to osteoporosis-pseudoglioma syndrome in humans
(Kato, et al., 157 (2002) J Cell Biol, 303-14.; Gong, et al., 107
(2001) Cell, 513-23.). iii) Eye Disease Inactivating mutation in
the Wnt receptor LRP5 lead to pseudoglioma in humans and eye
malformations in mice (Kato, et al., 157 (2002) J Cell Biol
303-314; Gong, et al., 107 (2001) Cell, 513-523).
iv) Kidney
[0008] Aberrant Wnt signalling is involved in renal fibrosis
(Surendran, Am J Physiol Renal Physiol 282 (2002) 431-441) and
polycystic kidney disease (Saadi-Kheddouci, Oncogene 20 (2001)
5972-5981).
v) Lipid and Glucose Metabolism, Obesity
[0009] Deficiency of the Wnt receptor LRP5 in mice leads to
increased plasma cholesterol levels in mice fed a high-fat diet,
because of the decreased hepatic clearance of chylomicron remnants.
In addition, when fed a normal diet, LRP5-deficient mice show a
markedly impaired glucose tolerance (Fujino, et al., 100 (2003)
Proc Natl Acad Sci USA, 229-234.) Administration of the LRP5
antagonist Dkk1 to mice reduces glucose uptake in various cell line
and decreases fat deposition (WO 02/066509).
[0010] It is thus clear from the above that the Wnt signalling
pathway is involved in a variety of human diseases. Yet, means for
the therapy or diagnosis of diseases associated with a
dis-regulated Wnt signal cascade are insufficiently available.
Thus, the use of reliable diagnostic molecular markers would be
helpful for an understanding of the molecular basis of diseases
associated with an aberrant Wnt signal cascade. It can be expected
that such markers are also useful for therapy and for the
development of novel therapeutic avenues for treatment of Wnt
signal cascade dependent diseases, as detailed above.
[0011] Thus, the technical problem underlying the present invention
is to provide means for diagnosis and therapy of diseases
associated with an aberrant Wnt signal cascade.
[0012] The solution to said technical problem is achieved by
providing the embodiments characterized in the claims.
[0013] During the experiments resulting in the present invention
four genes, futrin 1, 2, 3 and 4, could be identified the products
of which are modulators of the Wnt pathway. Futrin 2 was previously
identified as hPWTSR (Chen et al., 29 (2002), Mol. Biol. Rep.
287-292), a protein of before unknown role or function, expressed
in numerous cell types. Further, human Futrin 1, 2, 3, and 4 were
described as Stem Cell Growth Factor-Like Polypeptides, which are
able to promote proliferation of hematopoietic stem cells
(WO-A-01/77169; WO-A-01/07611).
SUMMARY
[0014] In the present invention the following is shown for the
first time: 1) Futrins enhance Wnt signalling and this is of
physiological relevance since inhibition of Futrin 1 or 2 results
in inhibition of the Wnt signal cascade (Wnt/(.beta.-catenin
signalling). These data show that Futrins can be regarded as Wnt
modulators. Futrin 1 (Rspo2) is coexpressed with and positively
regulated by Wnt signals and synergizes with Wnt to activate
.beta.-catenin. Analysis of functional interaction with components
of the Wnt/.beta.-catenin pathway suggests that Rspo2 functions
extracellularly at the level of receptor ligand interaction.
Antisense Morpholino experiments in Xenopus embryos and RNAi
experiments in HeLa cells revealed that Rspo2 is required for
Wnt/.beta.-catenin signalling. In Xenopus embryos depleted of Rspo2
the muscle markers myoD and myf5 fail to be activated and later
muscle development is impaired. The results indicate that Rspo2 is
a novel activator of the Wnt/.beta.-catenin cascade. Thus, Futrins
like Rspo2 (Futrin 1) are useful for the diagnosis and the
development of therapies for Wnt-LRP mediated diseases, including
but not limited to tumor suppression, bone formation, cholesterol
and glucose metabolism (including diabetes), obesity, kidney
disease and eye disease. 2.) Since the data obtained show that
Futrin 1 is required for muscle formation Futrin 1 is useful for
the diagnosis and the development of therapies for muscle related
diseases, including muscle regeneration. 3.) The data show that
Futrins are aberrantly expressed in a variety of human tumors.
Thus, Futrins are useful for tumor diagnosis and the development of
cancer therapies. For example it has been found out that in most of
the tumors the expression of Futrins 1-3 is dramatically decreased
(colon, stomach, lung, rectum tumors for Futrin 1, breast, ovary,
bladder, uterus, cervix, rectum tumors for Futrin 2, uterus and
cervix tumors for Futrin 3). In a few cases the expression of
Futrin 1-3 is upregulated (one case of stomach tumor for Futrin 1
and 2, ovary tumor for Futrin 3). Futrin 4 shows very low level of
expression in most of the tissues studied, except ovary.
[0015] Thus, the inhibition of the Wnt signal cascade by inhibiting
the expression/activity of Futrins or by stimulating the
expression/activity of the Futrins will have a therapeutic effect.
Likewise, the activation of the Wnt signal cascade by decreasing
the expression of Futrin and/or by repressing the activity of the
polypeptide itself will have a therapeutic effect.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1: Identification of Xenopus Futrin 1 (Rspo2).
[0017] TOP-FLASH reporter, vector DNA from pools of 250 clones each
and pCSfrizzled8 were co-transfected. Luciferase reporter assays in
293T cells were carried out in 96 well plates in duplicates as
described (Wu et al., Curr Biol 10 (2000), 1611-1614). Luciferase
activity was normalized against cotransfected Renilla-luc activity
using a commercial kit (Clonetech). RLU: relative luciferase
units.
[0018] FIG. 2A-F: Futrin 1 (Rspo2) Promotes Wnt/.beta.-Catenin
Signaling.
[0019] (FIG. 2A) Identification of Xenopus Rspo2 by expression
screening. An expression screen was carried out in 293T cells
transfected with a Wnt-responsive reporter (TOPFLASH) and plasmid
DNA pools from a Xenopus eye cDNA library. RLU: relative light
units. (FIG. 2B) The C-terminus of Rspo2 mediates cell surface
retention. Myc-tagged Rspo2 (wt) or Rspo2.DELTA.C (.DELTA.C) were
transfected in 293T cells, and cell lysate and medium were analysed
by Western blot. Co, untransfected cells. (FIG. 2C) Rspo2 activates
Wnt/.beta.-catenin signalling. TOPFLASH luciferase reporter assays
were carried out in 293T cells with the following transfected DNAs:
mouse Wnt1 (Wnt), 5 ng; mouse frizzled8(fz), 1 ng; Xenopus Rspo2
(Rspo2), 0.1, 0.3, 0.9 and 2.7 ng. (FIG. 2D) Rspo2 enhances Wnt3a
signalling. Mouse Wnt3a, Xenopus Rspo2.DELTA.C (Rspo2.DELTA.C) or
mock-conditioned media were added to 293T cells followed by
TOPFLASH luciferase reporter assays. (FIG. 2E) Rspo2 stabilizes
.beta.-catenin. (top) 293T cells were treated with Rspo2.DELTA.C,
Wnt3a or mock-conditioned media for 1 or 4 hours as indicated.
Cytosolic fractions were subjected to Western blot and probed for
.beta.-catenin and .alpha.-tubulin (loading control). (bottom)
immunohistochemical staining of .beta.-catenin in SHEP cells after
3 hour treatment with the conditioned media indicated. Arrowheads
indicate nuclear .beta.-catenin. The percentage of cells showing
the represented staining is 90% (Co), 85% (Rspo2), 80% (Wnt3a) and
90% (Wnt3a+Rspo2). (FIG. 2F) Domain analysis of Rspo2. (top)
schematic drawing of Xenopus Rspo2 and deletion constructs, sp,
signal peptide; FU1, 2, furin-like domains; TSP1, thrombospondin
type 1 domain; C, positively charged C-terminus. (bottom). TOPFLASH
luciferase reporter assays were carried out in 293T cells with the
indieated constructs. Equal protein production was confirmed by
Western blot (data not shown).
[0020] FIG. 3A-D: Multisequence Nucleic Acid Alignment of cDNAs
Encoding Human Futrin 1 (SEQ ID NO:21) Futrin 2 (SEQ ID NO:33),
Futrin 3 (SEQ ID NO:20) and Futrin 4 (SEQ ID NO:23) and Xenopus
Futrin 1 (SEQ ID NO:24).
[0021] Identical nucleotides are highlighted in grey. All nucleic
acid sequences begin with the translation initiator ATG codon
indicated with an asterisk.
[0022] FIG. 4: Multisequence Amino Acid Alignment of Human Futrin 1
(SEQ ID NO:26), Futrin 2 (SEQ ID NO:27), Futrin 3 (SEQ ID NO:34)
and Futrin 4 (SEQ ID NO:28) Deduced from Human cDNAs (See FIG.
3).
[0023] Identical amino acids are highlighted in grey, similar amino
acids are in dotted box.
[0024] FIG. 5: Futrins Promote Wnt Signaling.
[0025] Cotransfection experiments in 293T cells. Wnt-responsive
luciferase reporter assays were performed in 96 well plates in
triplicates as described (Wu et al., Curr Biol 10 (2000),
1611-1614). Luciferase activity was normalized against Renilla
activity using a commercial kit (Clonetech). Wnt1=mouse Wnt1,
fz8=mouse frizzled8, Futrin1=xenopus Futrin 1, Wnt3A=mouse Wnt3A,
(0.1 ng) in A indicates amount of plasmid DNA transfected per well,
RLU: relative luciferase units.
[0026] FIG. 6A-C: Sequence Comparison of Human Rspo Proteins
(hR-Spondin 1 to 4).
[0027] (FIG. 6A) Alignment of human (h) Rspo proteins (SEQ ID
NOS:29-32, respectively, in order of appearance) (corresponding to
the alignments of human Fut1-4 in FIG. 4 except that different
designations are used). The signal peptide, furin-like domains and
thrombospondin type 1 domain are underlined and conserved amino
acids are shown in grey. (FIG. 6B, FIG. 6C) Rspo homology matrix
showing overview of amino acid identity in % between human Rspo
proteins (FIG. 6B) and between Xenopus (X) or mouse (m) Rspo2 and
human Rspo proteins, respectively (FIG. 6C).
[0028] FIG. 7: Human Futrin 1 and 2 are Required for Wnt
Signaling.
[0029] Hela cells were transfected in 24-well plates with the Wnt
reporter 7LEF-Rev-fosLuc, pRL-TK and pSuper plasmids that produce
either siRNA against human Futrin1 and 2 or a nonsense control.
Three days after transfection, mouse Wnt3A conditioned medium was
added to the culture to stimulate Wnt signalling. 24 hours later,
luciferase activity was determined.
[0030] FIG. 8A-M: Expression Analysis of Xenopus and Mouse
R-Spondins.
[0031] (FIG. 8A) Rspo2 expression during Xenopus development at the
indieated embryonic stages analysed by RT-PCR. Histone H4 was used
for normalization. -RT, minus reverse transcription control. (FIG.
8B-H) Xenopus whole mount in situ hybridizations of the indicated
genes. (FIG. 8B) Stage 11 embryo, dorso-vegetal view; dbl, dorsal
blastoporal lip. (FIG. 8C) Stage 12 embryo, dorsal view with
anterior up. An anterior neural expression domain is indicated by
arrowhead. (FIG. 8D) Stage 15 embryo, dorsal view with anterior up.
(FIG. 8E) Stage 14 embryo, dorsal view with anterior up. (FIG.
8F-G) Tailbud stage embryos; ba, branchial arches; cm, cranial
musculature; di, diencephalon; dnt, dorsal neural tube; mhb,
midbrain-hindbrain boundary; ov, otic vesicle; pn, pronephros; pdm,
proctodeum; s, somites; tb, tailbud mesoder. Inset in (F) shows a
transverse section at the level indicated by arrowhead, showing
expression in dorsal neural tube and in the dorsal- and
ventral-most parts of the somites. (FIG. 8H) Dissected Xenopus
tadpole brain (lateral view) showing expression in diencephalons
(di) and zona limitans intrathala ica (zli), where sonic hedgehog
is expressed (inset). dt, vt, dorsal and ventral thalamus,
respectively, sc, spinal cord; tel, telencephalon; (FIG. 8I-M)
Mouse whole mount in situ hybridizations of the indicated genes.
(FIG. 8I) limb buds of day 12.5 mouse embryos. AER, apical
ectodermal ridge. (FIG. 8J) Day 7.5 mouse embryo showing Rspo3
expression in the primitive streak. (FIG. 8K-M) Day 9.5 mouse
embryos. di, diencephalon; dnt, dorsal neural tube; inet,
metencephalon; tel, telencephalon.
[0032] FIG. 9A-E: Regulation of Xenopus R-Spondins by Wnt
Signaling.
[0033] (FIG. 9A) Comparison of XRspo2, XWnt8 and XWnt3a expression
pattern in early neurula embryos by whole mount in situ
hybridization. Dorsal view, anterior up. (FIG. 9B) (top) Diagram of
experiment. Four cell stage embryos were injected with 50 pg
pCS-ppl (preprolactin), pCSXWnt8 or pCS-.beta.-catenin into each
blastomere, DMZ5 were dissected, cultured until stage 11 equivalent
and analyzed by RT-PCR. (Bottom) RT-PCR analysis of the indicated
genes. -RT, minus reverse transcription control. (FIG. 9C-E) Four
cell stage embryos were injected with 50 pg pCS-Wnt8, pCS-Wizt3a or
pCS-ppl into one blastomere and fixed at stage 11 for in situ
hybridisation with XRspo2.
[0034] FIG. 10A-C: Futrin 2 is Required for Muscle Formation.
[0035] FIG. 10AA-FIG. 10AL. Depletion of Futrin 1 protein causes
downregulation of early muscle markers and muscle defects. (FIG.
10AA, FIG. 10AC, FIG. 10AE, FIG. 10AG, FIG. 10AI, FIG. 10AK)
Embryos injected with control morpholino oligo (5 ng), with Fut1-Mo
(5 ng). Oligos were mixed with lineage tracer mRNA (LacZ) (blue
staining) in (FIG. 10AC, FIG. 10AD, FIG. 10AE, FIG. 10AF, FIG.
10AG, FIG. 10AH) or preprolactin (ppl) RNA, visualised by in situ
hybridisation (red staining) (FIG. 10AA, FIG. 10AB, FIG. 10AJ, FIG.
10AK, FIG. 10AL). In situ hybridization with probes to Xmyf5 (FIG.
10AA, FIG. 10AB), XmyoD (FIG. 10AC, FIG. 10AD), Xbra (FIG. 10AE,
FIG. 10AF), Xnot (FIG. 10AG, FIG. 10AH) and muscle actin (FIG.
10AI-L) (magenta staining). Embryos in FIG. 10AK and FIG. 10AL are
cut transversally. Note reduction of muscle in FIG. 10AL (right
side). FIG. 10B. Fut1-Mo act specifically to inhibit translation of
their cognate DNA constructs when overexpressed in embryos. mRNA
(C-terminal myc tagged fut1) was injected equatorially in both
blastomeres at 2-cell stage embryos. The same embryos were then
injected at the 8-cell stage with 5 ng of Fut1-Mo (lane3) or
control morpholino (lane2) in all vegetal blastomeres and harvested
at stage 11. Tagged Futrin1 protein was then visualised with a-myc
antibody. FIG. 10C. Rescue of muscle marker reduction caused by
FutI-Mo by coinjection of XFut1 mRNA containing point mutations in
the 5' region corresponding to morpholino oligo. Embryos were
coinjected with 5 ng of FutI-Mo (1, 2) with 50 pg of ppl or XFut1
mRNA radially in one blastomere at 4-cell stage.
[0036] Expression of MyoD (1, 3) and Myf5 (2, 4) were analysed by
in situ hybridization. All embryos were grouped into 3 classes
(examples for MyoD are shown on the bottom of the figure): embryos
with expression level on the injected side from 1-30% (class A);
30-70% (class B), and 70-100% (class C) from normal level. Bars
represent the percentage of the embryos corresponding to type A, B
or C. (n for Fut-Mo+ppl: 57 embryos for MyoD, 45 embryos for Myf5;
n for Fut-Mo+mRNA Ffut1: 39 embryos for MyoD and 41 embryo for
Myf5).
[0037] FIG. 11A-N: Rspo2 Promotes Neural and Muscle Differentiation
in Xenopus.
[0038] (FIG. 11A) Four-cell stage embryos were injected animally
with 100 pg Rspo2, 100 pg Xwnt8, or 200 pg .beta.-catenin mRNA in
each blastomere. At stage 8, animal caps were dissected, cultured
until stage 18 equivalent and analyzed for expression of the
indicated marker genes, -RT, minus reverse transcription control.
(FIG. 11B) Two-cell stage embryos were injected with 100 pg Rspo2
or preprolactin (ppl) mRNA in each blastomere. Note ectopic cement
glands (arrowhead) and shortened body axis in Rspo2 injected
embryo. (FIG. 11C-J) Whole mount in situ hybridisations of the
indicated genes. Eight-cell stage embryos were injected with 100 pg
of Rspo2 or ppl RNAs as indicated into one animal blastomere. LacZ
mRNA was coinjected as lineage tracer in all panels except FIG.
11C, FIG. 11G and FIG. 11H. (FIG. 11C-H) Stage 15 neurulae in
anterior view. (FIG. 11I-J) Stage 11 gastrulae in vegetal view,
dorsal up. (FIG. 11K-N) Rspo2 promotes muscle formation. (FIG. 11K)
Diagram of the experiments. Four-cell stage embryos were injected
with 50 pg plasmid DNA constructs in all blastomeres, the indicated
fragments were explanted at stage 10.5, cultured, and processed for
whole mount in situ hybridisation (FIG. 11M) of RT-PCR (FIG. 11N).
(FIG. 11L) Stage 40 equivalent VMZ of LMZ explants. Note tail-like
structures in VMZ5 from Rspo2 injected embryos. (FIG. 11M) In situ
hybridization of stage 25 VMZ5 for muscle actin. (FIG. 11N) RT-PCR
analysis for the indicated genes in stage 25 equivalent VMZ and
stage 11 equivalent DMZ explants. Xdd I, dominant negative. Xenopus
dishevelled, ; Co, preprolactin.
[0039] FIG. 12A-D: Rspo2 Interferes with BMP-4, Activin and Nodal
but not with FGF in Xenopus.
[0040] (FIG. 12A-D) Four-cell stage embryos were injected animally
with indicated RNAs. At stage 8, animal caps were dissected,
cultured until stage 10 equivalent and analyzed for expression of
Vent2 (FIG. 12A) or Xbra (FIG. 12B-D). -RT, minus reverse
transcription control. Embryos injected with 100 pg preprolactin
(ppl) were used as control. Amounts of mRNAs used: 100 pg Rspo2, 50
pg or 250 pg of BMP-4, 50 pg of activin, 25 or 100 pg of Wnt8, 200
pg of .beta.-catenin, 50 or 100 pg of XnrI, 2 or 20 pg of FGF8.
[0041] FIG. 13A-D: Xenopus Rspo2 is Required for Muscle
Formation.
[0042] (FIG. 13A) Rspo2Mo specifically inhibits translation of its
cognate DNA. Top, Diagram of the experiment. Two-cell stage Xenopus
embryos were injected with 100 pg Myc-tagged Rspo2 mRNA in the
animal region, at 8-cell stage, the same embryos were then injected
with 5 ng of Rspo2Mo of CoMo in all animal blastomeres, harvested
at stage 11 and processed for Western blot analysis of Myc-tagged
Rspo2 and .alpha.-tubulin. (FIG. 13Ba-l) Depletion of Rspo2 protein
causes muscle defects and down-regulation of myogeneic markers.
Four-cell stage embryos were injected equatorially into one
blastomere with 5 ng control morpholino oligonucleotides (CoMo) or
Rspo2Mo as indicated together with 50 pg ppl mRNA or 50 pg LacZ RNA
as lineage tracer and analysed at tailbud of gastrula stage by in
situ hybridization for the indicated genes. In (FIG. 13Ba-f) double
in situ hybridization for gene of interest (dark blue) and for ppl
(red) was used, (FIG. 13Ba-d) Stage 25 embryos. (FIG. 13Ba-b):
myotomes, visualised by muscle actin expression show malformations
an Rspo2Mo injected side (FIG. 13Bb). (FIG. 13Bc-d) Transversal
section at the trunk level showing reduced muscle volume in (FIG.
13Bd). (FIG. 13Be-h): myf5 and myoD expression (dark blue) is
down-regulated in the Rspo2Mo injected region (red in FIG. 13Be,
FIG. 13Bf of light blue in FIG. 13Bg, FIG. 13Bh). (FIG. 13Bi-l):
Xbra and Xnot2 expression (dark blue) is not affected in the region
of Rspo2Mo injections (light blue). (FIG. 13C) Rspo2Mo acts
specifically. Rescue of myf5 reduction by co-injected Rspo2 mRNA.
Four cell stage embryos were injected in one blastomere with 5 ng
of control morpholino (CoMo), Rspo2Mo, 50 pg ppl mRNA of 50 pg
Rspo2 mRNA containing mismatches to Rspo2Mo. Expression of myf5 was
analysed by in situ hybridization at gastrula stage. The percentage
of embryos with, strongly affected (A), moderately reduced (B) and
normal myf5 expression (C) as displayed in the representative
embryos is indicated. Standard deviation was calculated from three
independent experiments. (FIG. 13Da-d) Rspo2Mo Blocks Wnt
signalling upstream of dishevel led during muscle formation.
Four-cell stage embryos were radially injected with 5 ng of Rspo2Mo
or CoMo, 50 pg pCS-XWnt8, pCS-dishevel led (Xdsh), pCS-dominant
negative GSK-36 (dnGSK) or pCS-.beta.-catenin. At stage 10.5 DMZ5
(FIG. 13Da) or VMZ5 (FIG. 13Db) or LMZ5 (FIG. 13Dc-d) were
explanted and cultured until stage 11 (FIG. 13Da, FIG. 13Dc) or 25
(FIG. 13Db, FIG. 13Dd) for RT-PCR analysis of the genes indicated.
-RT: minus reverse transcription control.
[0043] FIG. 14A-D: R-Spondins are Required for Wnt Signalling in
HeLa Cells.
[0044] (FIG. 14A) RT-PCR analysis showing differential expression
of human Rspo1-4 in HeLa and 293T cell lines. Actin was used for
normalisation. (FIG. 14B) Specificity of siRNAs. 293T cells were
cotransfected with pSUPER-Rspo2 or -3 (siRNA), FLAG-tagged Rspo3
and GFP (transfection control). Expression of Rspo3 and GFP were
analyzed by Western blot. (FIG. 14C-D) R-spondins are required for
Wnt/.beta.-catenin signalling in HeLa cells. Wnt luciferase
reporter assay in HeLa cells cotransfected with the indicated
constructs. Wnt3a was added as conditioned medium. RLU, relative
light units.
[0045] FIG. 15A-E: Futrin Expression is Deregulated in Various
Human Tumors.
[0046] (FIG. 15A-E) The expression of Futrin 1, 2, 3 and -4 or
ubiquitin (to show equal loading) was analysed by radioactive
hybridisation on arrayed mRNAs (Clontech, Cancer Profiling Array
II) from normal and cancerous tissue samples from different
patients. Abbreviations N, normal tissues; T, tumor tissues.
[0047] FIG. 16: Expression of Human R-Spondins in Tumor
Samples.
[0048] Dot blot analysis of human Rspo1, 2 and 3 in normal and
tumour samples. The same CDNA samples (Cancer Profiling Array II,
Clontech) were hybridized with human Rspo1, 2, 3 and ubiquitin
probes, (top) The Cancer Profiling Array contains pairs of cDNA5
from tumor and corresponding normal tissue samples from individual
patients and spotted side-by-side as indicated. Organ abbreviations
are: Br, breast; ov, ovary; co, colon; st, stomach; lu, lungs;
kidney; bi, bleeder, vu, vulva; pr, prostate; tr, trachea; liv,
liver; ut, uterus; ce, cervix; re, rectum; th, thyroid gland; te,
testis; sk, skin; sin, small intestine, pa, pancreas.
[0049] FIG. 17A-C: Interaction of R-Spondins with Components of the
Wnt/.beta.-Catenin Signalling Pathway.
[0050] (FIG. 17A-C) Wnt luciferase reporter assay in 293T cells
co-transfected with the indicated constructs. Results are indicated
as fold-stimulation over reporter alone. DNA doses were 5 ng Wnt1,
1 ng Fz8, 3 ng LRP6, 10 ng axin, 10 ng dntcf3, 5 ng dkk1, 10 ng
Xdsh, 1 ng Wnt3a.
DETAILED DESCRIPTION
[0051] The present invention relates to a diagnostic composition
comprising: [0052] (a) at least one nucleic acid molecule
comprising the nucleotide sequence encoding Futrin 1, 2, 3 or 4 as
depicted in FIG. 3; and/or [0053] (b) at least one polypeptide
molecule comprising the amino acid sequence encoding Futrin 1,2,3
or 4 as depicted in FIG. 4 or 6a; and/or [0054] (c) at least one
nucleic acid molecule the complementary strand of which hybridizes
to a nucleic acid molecule of (a) and which encodes a polypeptide
with the biological activity of Futrin 1, 2, 3 or 4; and/or [0055]
(d) at least one fragment of (a), (b) or (c) having the biological
activity of Futrin 1, 2, 3 or 4; [0056] (e) at least one nucleic
acid molecule the sequence of which differs from the sequence of
the nucleic acid molecule of (a), (c) or (d) due to the degeneracy
of the genetic code, and/or [0057] (f) at least one ligand capable
of specifically binding to the molecule of (a), (b), (c), (d) or
(e).
[0058] As used herein the term "polypeptide" not only refers to
polypeptids encoded by the nucleotide/amino acid sequences as
depicted in FIGS. 3 and/or 4 but also to polypeptides differing in
amino acid sequence due to insertion, deletion and/or substitution
of one ore more amino acids and showing at least one biological
activity of a Futrin, e.g. the ability to promote Wnt signalling.
Preferably, the related nucleic acids and/or polypeptides are
nucleic acids and/or polypeptides the sequence of which shows an
identity of at least 40%, in particular an identity of at least
65%, preferably of at least 80% and, particularly preferred, of at
least 90% to the amino acid sequences of the polypeptides encoded
by the nucleotide sequences shown in FIG. 3.
[0059] The nucleic acid molecules useful as probes can be both DNA
and RNA molecules, preferably they are single-stranded DNA
molecules. They can be isolated from natural sources or can be
synthesized according to know methods.
[0060] As a hybridization probe nucleic acid molecules can be used,
for example, that have a nucleotide sequence which is exactly or
basically complementary to a nucleotide sequence as depicted in
FIGS. 3 and 4 or 6a, respectively, or parts of these sequences. The
fragments used as hybridization probe can be synthetic fragments
that were produced by means of conventional synthetic methods.
[0061] As used herein, the term "hybridizing" relates to
hybridization under conventional hybridization conditions,
preferably under stringent conditions as described, for example, in
Sambrook et al., Molecular Cloning, A Laboratory Manual 2nd edition
(1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y. However, in certain cases, a hybridizing nucleic acid molecule
can also be detected at lower stringency hybridization conditions.
Changes in the stringency of hybridization and signal detection are
primarily accomplished through the manipulation of formamide
concentration (lower percentages of formamide result in lowered
stringency, salt conditions, or temperature. For example, lower
stringency conditions include an overnight incubation at 37.degree.
C. in a solution comprising 6.times.SSPE (20.times.SSPE=3M NaCl;
9.2M NaH.sub.2PO.sub.4; 0.02M EDTA, pH7.4), 0.5% SDS, 30%
formamide, 100 .mu.g/ml salmon sperm blocking DNA, following by
washes at 50.degree. C. with 1.times.SSPE, 0.1% SDS. In addition,
to achieve even lower stringency, washes performed following
stringent hybridization can be done at higher salt concentrations
(e.g. 5.times.SSC). Variations in the above conditions may be
accomplished through the inclusion and/or substitution of alternate
blocking reagents used to suppress background in hybridization
experiments. The inclusion of specific blocking reagents may
require modification of the hybridization conditions described
above, due to problems with compatibility.
[0062] The term "ligand" as used herein refers to any molecule
which is capable of specifically binding to Futrin 1, 2, 3, or 4,
thus allowing to determine the level of receptor molecules.
Examples of such molecules include antibodies, oligonucleotides,
proteins or small molecules. The molecule can be the natural ligand
of Futrins, or can be closely related to said ligand, e.g., a
fragment of the ligand, or a natural substrate, a structural or
functional mimetic; see, e.g., Coligan, Current Protocols in
Immunology 1(2) (1991); Chapter 5. In either case, the molecule can
be isolated or rationally designed using known techniques; see also
infra.
[0063] Preferably, the ligand is an antibody. The term "antibody",
preferably, relates to antibodies which consist essentially of
pooled monoclonal antibodies with different epitopic specifities,
as well as distinct monoclonal antibody preparations. Monoclonal
antibodies are made from an antigen containing Futrin 1, 2, 3, or 4
or fragments thereof by methods well known to those skilled in the
art (see, e.g., Kohler et al., Nature 256 (1975), 495). As used
herein, the term "antibody" (Ab) or "monoclonal antibody" (Mab) is
meant to include intact molecules as well as antibody fragments
(such as, for example, Fab and F(ab') 2 fragments) which are
capable of specifically binding to Futrin 1, 2, 3 and/or 4. Fab and
f(ab')2 fragments lack the Fc fragment of intact antibody, clear
more rapidly from the circulation, and may have less non-specific
tissue binding than an intact antibody. (Wahl et al., J. Nucl. Med.
24: 316-325 (1983)). Thus, these fragments are preferred, as well
as the products of a FAB or other immunoglobulin expression
library. Moreover, antibodies of the present invention include
chimerical, single chain, and humanized antibodies.
[0064] For certain purposes, e.g. diagnostic methods, the nucleic
acid molecule used as probe or the ligand, e.g., antibody, can be
detectably labeled, for example, with a radioisotope, a
bioluminescent compound, a chemiluminescent compound, a fluorescent
compound, a metal chelate, or an enzyme.
[0065] The nucleic acid molecules can be used, for example, as
probes or primers in the diagnostic assays described below and
allow, e.g., the analysis of the expression of Futrin 1, 2, 3, or
-4 by determining the mRNA level or the determination of mutations
within the coding region or regulatory regions leading to
polypeptide molecules with altered, e.g. destroyed, activity, or
leading to altered expression. Preferably, the nucleic acid
molecules are oligonucleotides having a length of at least 10, in
particular of at least 15 and particularly preferred of at least 50
nucleotides. These nucleic acid molecules of the invention can also
be used, for example, as primers for a PCR reaction.
[0066] The present invention also relates to the use of a nucleic
acid molecule or ligand as defined above for the preparation of a
diagnostic composition for the diagnosis of a disease associated
with (a) aberrant expression of Futrin 1, 2, 3, or -4 and/or (b)
aberrant activity of a Futrin 1, 2, 3, or -4 polypeptide.
[0067] In a preferred embodiment, the target to which the nucleic
acid molecule hybridizes is an mRNA.
[0068] The present invention also provides a method of diagnosing a
disease associated with (a) aberrant expression of Futrin 1, 2, 3,
or -4 and/or (b) aberrant activities or amounts of a Futrin 1, 2,
3, or -4 polypeptide in a subject comprising: [0069] (a)
determining (a) the amount of expression of Futrin 1, 2, 3, or -4
and/or (b) the amount of biologically active Futrin 1, 2, 3 and/or
4 polypeptide in a biological sample; and [0070] (b) diagnosing a
disease associated with (a) aberrant expression of Futrin 1, 2, 3
and/or 4 and/or (b) aberrant activities or amounts of a Futrin 1,
2, 3 and/or 4 polypeptide or a risk for the development of such
disease based on an altered amount of expression of Futrin 1, 2, 3
and/or 4 and/or (b) altered activities or amounts of biologically
active Futrin 1, 2, 3 and/or 4 polypeptide compared to a
control.
[0071] Suitable assay formats are well known to the person skilled
in the art and, in addition, described below. Suitable positive
control samples expressing human Futrin proteins are, e.g., HEK 293
cells.
[0072] The Futrin 1, 2, 3, or 4 polypeptide or the corresponding
mRNA, e.g. in biological fluids or tissues, may be detected
directly in situ, e.g. by in situ hybridization or it may be
isolated from other cell components by common methods known to
those skilled in the art before contacting with a probe. Detection
methods include Northern Blot analysis, RNase protection, in situ
methods, e.g. in situ hybridization, in vitro amplification methods
(PCR, LCR, QRNA replicase or RNA-transcription/amplification (TAS,
3SR), reverse dot blot disclosed in EP-B1 O 237 362), immunoassays,
Western Blot and other detection assays that are known to those
skilled in the art.
[0073] The probe (e.g. a specific antibody or specific
oligonucleotide) of the diagnostic composition can be detectably
labeled. In a preferred embodiment, said diagnostic composition
contains an anti-Futrin 1, 2, 3 and/or 4 antibody and allows said
diagnosis, e.g., by ELISA and contains the antibody bound to a
solid support, for example, a polystyrene microtiter dish or
nitrocellulose paper, using techniques known in the art.
Alternatively, said diagnostic compositions are based on a RIA and
contain said antibody marked with a radioactive isotope. Suitable
antibody assay labels are known in the art and include enzyme
labels, such as, glucose oxidase, and radioisotopes, such as iodine
(.sup.125I, .sup.121I), carbon (.sup.14C), sulfur (.sup.35S),
tritium (.sup.3H), indium (.sup.112In), and technetium rhodamine,
and biotin. In addition to assaying Futrin levels in a biological
sample, the polypeptide can also be detected in vivo by imaging.
Antibody labels or markers for in vivo imaging of protein include
those detectable by X-radiography, NMR or ESR. For X-radiography,
suitable labels include radioisotopes such as barium or cesium,
which emit detectable radiation but are not overtly harmful to the
subject. Suitable markers for NMR and ESR include those with a
detectable characteristic spin, such as deuterium, which may be
incorporated into the antibody by labeling of nutrients for the
relevant hybridoma. A protein-specific antibody or antibody
fragment which has been labeled with an appropriate detectable
imaging moiety, such as a radioisotope (for example, .sup.131I,
.sup.112In, .sup.99mTc), a radio-opaque substance, or a material
detectable by nuclear magnetic resonance, is introduced (for
example, parenterally, subcutaneously, or intraperitoneally) into
the mammal. It will be understood in the art that the size of the
subject and the imaging system used will determine the quantity of
imaging moiety needed to produce diagnostic images. In the case of
a radioisotope moiety, for a human subject, the quantity of
radioactivity injected will normally range from about 5 to 20
millicuries of .sup.99mTc. The labeled antibody or antibody
fragment will then preferentially accumulate at the location of
cells which contain the specific Futrin polypeptide. In vivo tumor
imaging is, e.g., described in S. W. Burchiel et al.,
"Immunopharmacokinetics of Radiolabeled Antibodies and Their
Fragments". (Chapter 13 in Tumor Imaging: The Radiochemical
Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson
Publishing Inc. (1982)).
[0074] In a further aspect, the present invention, relates to a
method for identifying a binding partner to a Futrin 1, 2, 3 and/or
-4 polypeptide comprising: [0075] (a) contacting-said polypeptide
with a compound to be screened; and [0076] (b) determining whether
the compound effects an activity of the polypeptide.
[0077] The invention also includes a method of identifying
compounds which bind to a Futrin 1, 2, 3 and/or 4 polypeptide
comprising the steps of: [0078] (a) incubating a candidate binding
compound with said polypeptide; and [0079] (b) determining if
binding has occurred.
[0080] Futrin 1, 2, 3 or -4 polypeptides may be used to screen for
proteins or other compounds that bind to Futrin 1, 2, 3 or -4 or
for proteins or other compounds to which Futrin1, 2, 3 and/or 4
bind. The binding of Futrin1, 2, 3 or -4 and the molecule may
activate (agonist), increase, inhibit (antagonist), or decrease
activity of Futrin 1, 2, 3 or 4 or the molecule bound. Examples of
such molecules include antibodies, oligonucleotides, proteins
(e.g., ligands), or small molecules.
[0081] Preferably, the molecule is closely related to the natural
ligand of Futrin 1, 2, 3 or -4, e.g., a fragment of the ligand, or
a natural substrate, a ligand, a structural or functional mimetic;
see, e.g., Coligan, Current Protocols in Immunology 1(2) (1991);
Chapter 5.
[0082] Preferably, the screening for these molecules involves
producing appropriate cells which express Futrin 1, 2, 3 and/or 4
either as a secreted protein or on the cell membrane. Preferred
cells include cells from mammals, yeast, Drosophila, or E. coli.
Cells expressing Futrin 1, 2, 3 and/or -4 (or cell membrane
containing the expressed polypeptide) are then preferably contacted
with a test compound potentially containing the molecule to observe
binding, stimulation, or inhibition of activity of Futrin 1, 2, 3
and/or -4.
[0083] The assay may simply test binding of a candidate compound to
Futrin 1, 2, 3 and/or 4, wherein binding is detected by a label, or
in an assay involving competition with a labeled competitor.
Further, the assay may test whether the candidate compound results
in a signal generated by binding to Futrin 1, 2, 3 and/or 4.
Suitable assays to analyze the activity of Futrin 1, 2, 3 and/or 4
include Wnt-inducible luciferase reporter assays in transfected HEK
293 cells, where Futrin 1, 2, 3 and/or 4 synergizes with Wnt to
enhance a Wnt-induced signal, such as is shown in FIG. 4.
[0084] Alternatively, the assay can be carried out using cell-free
preparations, polypeptide/molecule affixed to a solid support,
chemical libraries, or natural product mixtures. The assay may also
simply comprise the steps of mixing a candidate compound with a
solution containing Futrin 1, 2, 3 and/or 4, measuring
Futrin/molecule activity or binding, and comparing the
Futrin/molecule activity or binding to a standard.
[0085] Preferably, an ELISA assay can measure Futrin 1, 2, 3 and/or
4 level or activity in a sample (e.g., biological sample) using a
monoclonal or polyclonal antibody. The antibody can measure Futrin
1, 2, 3 and/or 4 level or activity by either binding, directly or
indirectly, to Futrin 1, 2, 3 and/or 4 or by competing with Futrin
1, 2, 3 and/or 4 for a substrate. All of these above assays can be
used as diagnostic or prognostic markers. The molecules discovered
using these assays can be used to treat disease or to bring about a
particular result in a patient (e.g., elimination of a tumor,
support of regenerative processes etc.) by modulating, preferably
activating the Futrin 1, 2, 3 and/or 4 molecule. Moreover, the
assays can discover agents which may inhibit or enhance the
production of Futrin 1, 2, 3 and/or 4 from suitably manipulated
cells or tissues.
[0086] Moreover, the invention includes a method of identifying
activators/agonists or inhibitors/antagonists of a Futrin 1, 2, 3
and/or 4 polypeptide comprising the steps of: [0087] (a) incubating
a candidate compound with said polypeptide; [0088] (b) assaying a
biological activity, and [0089] (c) determining if a biological
activity of said polypeptide has been altered.
[0090] Suitable assays to analyze the activity of Futrin 1, 2, 3
and/or 4 include Wnt-inducible luciferase reporter assays in
transfected HEK 293 cells, where Futrin 1, 2, 3 and/or 4 synergizes
with Wnt to enhance a Wnt-induced signal, such as is shown in FIG.
4.
[0091] In a further embodiment, the present invention relates to
method of identifying and obtaining a drug candidate for therapy of
diseases associated with (a) aberrant expression of Futrin 1, 2, 3
and/or 4 and/or (b) aberrant activities or amounts of a Futrin 1,
2, 3 and/or 4 polypeptide comprising the steps of [0092] (a)
contacting a Futrin 1, 2, 3 and/or 4 polypeptide or a cell
expressing said polypeptide, and optionally the corresponding
ligand (s), in the presence of components capable of providing a
detectable signal in response to binding to said drug candidate to
be screened; and [0093] (b) detecting presence or absence of a
signal or increase of the signal generated, wherein the presence or
increase of the signal is indicative for a putative drug.
[0094] Suitable assays to analyze the activity of Futrin 1, 2, 3
and/or 4 include Wnt-inducible luciferase reporter assays in
transfected HEK 293 cells, where Futrin 1, 2, 3 and/or 4 synergizes
with Wnt to enhance a Wnt-induced signal, such as is shown in FIG.
4.
[0095] The drug candidate may be a single compound or a plurality
of compounds. The term "plurality of compounds" in a method of the
invention is to be understood as a plurality of substances which
may or may not be identical.
[0096] Said compound or plurality of compounds may be chemically
synthesized or microbiologically produced and/or comprised in, for
example, samples, e.g., cell extracts from, e.g., plants, animals
or microorganisms. Furthermore, said compound(s) may be known in
the art but hitherto not known to be capable of suppressing or
activating Futrin1, 2, 3 and/or 4 polypeptides. The reaction
mixture may be a cell free extract or may comprise a cell or tissue
culture. Suitable set ups for the method of the invention are known
to the person skilled in the art and are, for example, generally
described in Alberts et al., Molecular Biology of the Cell, third
edition (1994) and in the appended examples. The plurality of
compounds may be, e.g., added to the reaction mixture, culture
medium, injected into a cell or otherwise applied to a transgenic
animal. The cell or tissue that may be employed in the method of
the invention preferably is a host cell, mammalian cell or
non-human transgenic animal.
[0097] If a sample containing a compound or a plurality of
compounds is identified in the method of the invention, then it is
either possible to isolate the compound from the original sample
identified as containing the compound capable of suppressing or
activating a Futrin 1, 2, 3 and/or 4 polypeptide, or one can
further subdivide the original sample, for example, if it consists
of a plurality of different compounds, so as to reduce the number
of different substances per sample and repeat the method with the
subdivisions of the original sample. Depending on the complexity of
the samples, the steps described above can be performed several
times, preferably until the sample identified according to the
method of the invention only comprises a limited number of or only
one substance(s). Preferably said sample comprises substances of
similar chemical and/or physical properties, and most preferably
said substances are identical.
[0098] Several methods are known to the person skilled in the art
for producing and screening large libraries to identify compounds
having specific affinity for a target. These methods include the
phage-display method in which randomized peptides are displayed
from phage and screened by affinity chromatography to an
immobilized receptor; see, e.g., WO 91/17271, WO 92/01047, U.S.
Pat. No. 5,223,409. In another approach, combinatorial libraries of
polymers immobilized on a chip are synthesized using
photolithography; see, e.g., U.S. Pat. No. 5,143,854, WO 90/15070
and WO 92/10092. The immobilized polymers are contacted with a
labeled receptor and scanned for label to identify polymers binding
to the receptor. The synthesis and screening of peptide libraries
on continuous cellulose membrane supports that can be used for
identifying binding ligands of the Futrin1, 2, 3 and/or -4
polypeptides and, thus, possible inhibitors and activators is
described, for example, in Kramer, Methods Mol. Biol. 87 (1998),
25-39. This method can also be used, for example, for determining
the binding sites and the recognition motifs in the Futrin 1, 2, 3
and/or -4 polypeptide. In like manner, the substrate specificity of
the DnaK chaperon was determined and the contact sites between
human interleukin-6 and its receptor; see Rudiger, EMBO J. 16
(1997), 1501-1507 and Weiergraber, FEBS Lett. 379 (1996), 122-126,
respectively. Furthermore, the above-mentioned methods can be used
for the construction of binding supertopes derived from the Futrin
1, 2, 3 or 4 polypeptide. A similar approach was successfully
described for peptide antigens of the anti-p24 (HIV-1) monoclonal
antibody; see Kramer, Cell 91 (1997), 799-809. A general route to
fingerprint analyses of peptide-antibody interactions using the
clustered amino acid peptide library was described in Kramer, Mol.
Immunol. 32 (1995), 459-465. In addition, antagonists of a Futrin
1, 2, 3 and/or 4 polypeptide can be derived and identified from
monoclonal antibodies that specifically react with a Futrin 1, 2, 3
and/or 4 polypeptide in accordance with the methods as described in
Doring, Mol. Immunol. 31 (1994), 1059-1067.
[0099] All these methods can be used in accordance with the present
invention to identify activators/agonists and
inhibitors/antagonists of a Futrin 1, 2, 3 and/or 4
polypeptide.
[0100] Various sources for the basic structure of such an activator
or inhibitor can be employed and comprise, for example, mimetic
analogs of a Futrin 1, 2, 3 and/or 4 polypeptide. Mimetic analogs
of a Futrin 1, 2, 3 and/or 4 polypeptide or biologically active
fragments thereof can be generated by, for example, substituting
the amino acids that are expected to be essential for the
biological activity with, e.g., stereoisomers, i.e. D-amino acids;
see e.g., Tsukida, J. Med. Chem. 40 (1997), 3534-3541. Furthermore,
in case fragments are used for the design of biologically active
analogs pro-mimetic components can be incorporated into a peptide
to reestablish at least some of the conformational properties that
may have been lost upon removal of part of the original
polypeptide; see, e.g., Nachman, Regul. Pept. 57 (1995), 359-370.
Furthermore, a Futrin 1, 2, 3 and/or 4 polypeptide can be used to
identify synthetic chemical peptide mimetics that bind to or can
function as a ligand, substrate or binding partner of said
polypeptide(s) as effectively as does the natural polypeptide; see,
e.g., Engleman, J. Clin. Invest. 99 (1997), 2284-2292. For example,
folding simulations and computer redesign of structural motifs of a
Futrin 1, 2, 3 and/or 4 polypeptide can be performed using
appropriate computer programs (Olszewski, Proteins 25 (1996),
286-299; Hoffman, Comput. Appl. Biosci. 11 (1995), 675-679).
Computer modeling of protein folding can be used for the
conformational and energetic analysis of detailed peptide and
protein models (Monge, J. Mol. Biol. 247 (1995), 995-1012; Renouf,
Adv. Exp. Med. Biol. 376 (1995), 37-45). In particular, the
appropriate programs can be used for the identification of
interactive sites of a Futrin 1, 2, 3 and/or 4 polypeptide and its
ligand or other interacting proteins by computer assistant searches
for complementary peptide sequences (Fassina, Immunomethods 5
(1994), 114-120. Further appropriate computer systems for the
design of protein and peptides are described in the prior art, for
example in Berry, Biochem. Soc. Trans. 22 (1994), 1033-1036; Wodak,
Ann. N. Y. Acad. Sci. 501 (1987), 1-13; Pabo, Biochemistry 25
(1986), 5987-5991. The results obtained from the above-described
computer analysis can be used for, e.g., the preparation of peptide
mimetics of a Futrin 1, 2, 3 and/or 4 polypeptide or fragments
thereof. Such pseudopeptide analogues of the natural amino acid
sequence of the protein may very efficiently mimic the parent
protein (Benkirane, J. Biol. Chem. 271 (1996), 33218-33224). For
example, incorporation of easily available achiral w-amino acid
residues into a Futrin 1, 2, 3 or -4 polypeptide or a fragment
thereof results in the substitution of amide bonds by polymethylene
units of an aliphatic chain, thereby providing a convenient
strategy for constructing a peptide mimetic (Banerjee, Biopolymers
39 (1996), 769-777). Superactive peptidomimetic analogues of small
peptide hormones in other systems are described in the prior art
(Zhang, Biochem. Biophys. Res. Commun. 224 (1996), 327-331).
Appropriate peptide mimetics of a Futrin 1, 2, 3 and/or 4
polypeptide can also be identified by the synthesis of peptide
mimetic combinatorial libraries through successive amide alkylation
and testing the resulting compounds, e.g., for their binding and
immunological properties. Methods for the generation and use of
peptidomimetic combinatorial libraries are described in the prior
art, for example in Ostresh, Methods in Enzymology 267 (1996),
220-234 and Dorner, Bioorg. Med. Chem. 4 (1996), 709-715.
Furthermore, a three-dimensional and/or crystallographic structure
of a Futrin 1, 2, 3 and/or 4 polypeptide can be used for the design
of peptide mimetic inhibitors of the biological activity of the
polypeptide (Rose, Biochemistry 35 (1996), 12933-12944; Rutenber,
Bioorg. Med. Chem. 4 (1996), 1545-1558).
[0101] It is also well known to the person skilled in the art, that
it is possible to design, synthesize and evaluate mimetics of small
organic compounds that, for example, can act as a substrate or
ligand to a Futrin 1, 2, 3 and/or 4 polypeptide. For example, it
has been described that D-glucose mimetics of hapalosin exhibited
similar efficiency as hapalosin in antagonizing multidrug
resistance assistance-associated protein in cytotoxicity; see Dinh,
J. Med. Chem. 41 (1998), 981-987.
[0102] The nucleic acid molecule encoding a Futrin 1, 2, 3 and/or 4
polypeptide can also serve as a target for activators and
inhibitors. Activators may comprise, for example, proteins that
bind to the mRNA of a gene encoding a Futrin 1, 2, 3 and/or 4
polypeptide, thereby stabilizing the native conformation of the
mRNA and facilitating transcription and/or translation, e.g., in
like manner as Tat protein acts on HIV-RNA. Furthermore, methods
are described in the literature for identifying nucleic acid
molecules such as an RNA fragment that mimics the structure of a
defined or undefined target RNA molecule to which a compound binds
inside of a cell resulting in retardation of cell growth or cell
death; see, e.g., WO 98/18947 and references cited therein. These
nucleic acid molecules can be used for identifying unknown
compounds of pharmaceutical interest, and for identifying unknown
RNA targets for use in treating a disease. These methods and
compositions can be used in screening for novel or for identifying
compounds useful to alter expression levels of polypeptids encoded
by a nucleic acid molecule. Alternatively, for example, the
conformational structure of the RNA fragment which mimics the
binding site can be employed in rational drug design to modify
known drugs to make them bind more avidly to the target. One such
methodology is nuclear magnetic resonance (NMR), which is useful to
identify drug and RNA conformational structures. Still other
methods are, for example, the drug design methods as described in
WO 95/35367, U.S. Pat. No. 5,322,933, where the crystal structure
of the RNA fragment can be deduced and computer programs are
utilized to design novel binding compounds.
[0103] The compounds which can be tested and identified according
to a method of the invention may be expression libraries, e.g.,
cDNA expression libraries, peptides, proteins, nucleic acids,
antibodies, small organic compounds, hormones, peptidomimetics,
PNA5 or the like (Milner, Nature Medicine 1 (1995), 879-880; Hupp,
Cell 83 (1995), 237-245; Gibbs, Cell 79 (1994), 193-198 and
references cited supra). Furthermore, genes encoding a putative
regulator of a Futrin 1, 2, 3 and/or 4 polypeptide and/or which
exert their effects up- or downstream a Futrin 1, 2, 3 and/or 4
polypeptide may be identified using, for example, insertion
mutagenesis using, for example, gene targeting vectors known in the
art. Said compounds can also be functional derivatives or analogues
of known inhibitors or activators. Such useful compounds can be for
example transacting factors which bind to a Futrin 1, 2, 3 and/or 4
polypeptide or regulatory sequences of the gene encoding it.
Identification of transacting factors can be carried out using
standard methods in the art (see, e.g., Sambrook, supra). To
determine whether a protein binds to the protein itself or
regulatory sequences, standard native gel-shift analyses can be
carried out. In order to identify a transacting factor which binds
to the protein or regulatory sequence, the protein or regulatory
sequence can be used as an affinity reagent in standard protein
purification methods, or as a probe for screening an expression
library. The identification of nucleic acid molecules which encode
polypeptides which interact with a Futrin 1, 2, 3 and/or 4
polypeptide described above can also be achieved, for example, as
described in Scofield (Science 274 (1996), 2063-2065) by use of the
so-called yeast "two-hybrid system". In this system the Futrin1, 2,
3 or 4 polypeptide or a smaller part thereof is linked to the
DNA-binding domain of the GAL4 transcription factor. A yeast strain
expressing this fusion polypeptide and comprising a lacZ reporter
gene driven by an appropriate promoter, which is recognized by the
GAL4 transcription factor, is transformed with a library of cDNA5
which will express plant proteins or peptides thereof fused to an
activation domain. Thus, if a peptide encoded by one of the cDNA5
is able to interact with the fusion peptide comprising a peptide of
a Futrin 1, 2, 3 and/or 4 polypeptide, the complex is able to
direct expression of the reporter gene. In this way the nucleic
acid molecules encoding Futrin1, 2, 3 and 4, respectively, and the
encoded peptide can be used to identify peptides and proteins
interacting with a Futrin1, 2, 3 and/or 4 polypeptide.
[0104] Once the transacting factor is identified, modulation of its
binding to or regulation of expression of a Futrin 1, 2, 3 and/or 4
polypeptide can be pursued, beginning with, for example, screening
for inhibitors against the binding of the transacting factor to a
Futrin1, 2, 3 or 4 polypeptide. Activation or repression of a
Futrin 1, 2, 3 and/or 4 polypeptide could then be achieved in
animals by applying the transacting factor (or its inhibitor) or
the gene encoding it, e.g. in an expression vector. In addition, if
the active form of the transacting factor is a dimer,
dominant-negative mutants of the transacting factor could be made
in order to inhibit its activity. Furthermore, upon identification
of the transacting factor, further components in the signal cascade
leading to activation (e.g. signal transduction) or repression of a
gene involved in the control of a Futrin 1, 2, 3 and/or 4
polypeptide then can be identified. Modulation of the activities of
these components can then be pursued, in order to develop
additional drugs and methods for modulating the metabolism of
protein degradation in animals. Thus, the present invention also
relates to the use of the two-hybrid system as defined above for
the identification of activators or inhibitors of a Futrin 1, 2, 3
and/or 4 polypeptide.
[0105] The compounds isolated by the above methods also serve as
lead compounds for the development of analog compounds. The analogs
should have a stabilized electronic configuration and molecular
conformation that allows key functional groups to be presented to a
Futrin 1, 2, 3 and/or 4 polypeptide or its ligand in substantially
the same way as the lead compound. In particular, the analog
compounds have spatial electronic properties which are comparable
to the binding region, but can be smaller molecules than the lead
compound, frequently having a molecular weight below about 2 kD and
preferably below about 1 kD. Identification of analog compounds can
be performed through use of techniques such as self-consistent
field (SCF) analysis, configuration interaction (CI) analysis, and
normal mode dynamics analysis. Computer programs for implementing
these techniques are available; e.g., Rein, Computer-Assisted
Modeling of Receptor-Ligand Interactions (Alan Liss, New York,
1989). Methods for the preparation of chemical derivatives and
analogues are well known to those skilled in the art and are
described in, for example, Beilstein, Handbook of Organic
Chemistry, Springer edition New York Inc., 175 Fifth Avenue, New
York, N.Y. 10010 U.S.A. and Organic Synthesis, Wiley, New York,
USA. Furthermore, said derivatives and analogues can be tested for
their effects according to methods known in the art; see also
supra. Furthermore, peptidomimetics and/or computer aided design of
appropriate derivatives and analogues can be used, for example,
according to the methods described above.
[0106] Once the described compound has been identified and
obtained, it is preferably provided in a therapeutically acceptable
form.
[0107] Accordingly, the present invention also relates to a
pharmaceutical composition comprising a nucleic acid molecule
encoding a Futrin 1, 2, 3 and/or 4 polypeptide, a Futrin 1, 2, 3
and/or 4 polypeptide itself, recombinant vector (for examples, see
below), antibody, activator/agonist, inhibitor/antagonist and/or
binding partner of a Futrin 1, 2, 3 and/or 4 polypeptide and a
pharmaceutically acceptable excipient, diluent or carrier.
[0108] Preferably, for therapeutic purposes, the Futrin 1, 2, 3
and/or 4 polypeptide is recombinantly produced by use of the
nucleic acid sequences shown in FIGS. 1 and 2. Suitable vectors for
recombinant expression are known to the person skilled in the art.
Preferably, they are plasmids, cosmids, viruses, bacteriophages and
other vectors usually used in the field of genetic engineering.
Vectors suitable for use in the present invention include, but are
not limited to the T7-based expression vector for expression in
mammalian cells and baculovirus-derived vectors for expression in
insect cells. Preferably, the nucleic acid molecule of the
invention is operatively linked to the regulatory elements in the
recombinant vector of the invention that guarantee the
transcription and synthesis of an mRNA in prokryotic and/or
eukaryotic cells that can be translated. The nucleotide sequence to
be transcribed can be operably linked to a promoter like a T7,
metallothionein I or polyhedrin promoter. The host cells used for
recombinant expression are prokaryotic or eukaryotic cells, for
example mammalian cells, bacterial cells, insect cells or yeast
cells. The polypeptide is isolated from the cultivated cells and/or
the culture medium. Isolation and purification of the recombinantly
produced polypeptide may be carried out by conventional means
including preparative chromatography and affinity and immunological
separations using, e.g., an anti-Futrin 1, 2, 3 or -4 antibody, or,
e.g., can be substantially purified by the one-step method
described in Smith and Johnson, Gene 67; 31-40 (1988).
[0109] Examples of suitable pharmaceutical carriers etc. are well
known in the art and include phosphate buffered saline solutions,
water, emulsions, such as oil/water emulsions, various types of
wetting agents, sterile solutions etc. Such carriers can be
formulated by conventional methods and can be administered to the
subject at a suitable dose. Administration of the suitable
compositions may be effected by different ways, e.g. by
intravenous, intraperetoneal, subcutaneous, intramuscular, topical
or intradermal administration. The route of administration, of
course, depends on the nature of the disease and the kind of
compound contained in the pharmaceutical composition. The dosage
regimen will be determined by the attending physician and other
clinical factors. As is well known in the medical arts, dosages for
any one patient depends on many factors, including the patient's
size, body surface area, age, sex, the particular compound to be
administered, time and route of administration, the kind and stage
of the disease, e.g., tumor, general health and other drugs being
administered concurrently.
[0110] The delivery of the nucleic acid molecules encoding a Futrin
1, 2, 3 and/or 4 polypeptide can be achieved by direct application
or, preferably, by using a recombinant expression vector such as a
chimeric virus containing these compounds or a colloidal dispersion
system. Direct application to the target site can be performed,
e.g., by ballistic delivery, as a colloidal dispersion system or by
catheter to a site in artery. The colloidal dispersion systems
which can be used for delivery of the above nucleic acid molecules
include macromolecule complexes, nanocapsules, microspheres, beads
and lipid-based systems including oil-in-water emulsions (mixed),
micelles, liposomes and lipoplexes, The preferred colloidal system
is a liposome. Organ-specific or cell-specific liposomes can be
used in order to achieve delivery only to the desired tissue. The
targeting of liposomes can be carried out by the person skilled in
the art by applying commonly known methods. This targeting includes
passive targeting (utilizing the natural tendency of the liposomes
to distribute to cells of the RES in organs which contain
sinusoidal capillaries) or active targeting (for example by
coupling the liposome to a specific ligand, e.g., an antibody, a
receptor, sugar, glycolipid, protein etc., by well known methods).
In the present invention monoclonal antibodies are preferably used
to target liposomes to specific tissues, e.g. tumor tissue, via
specific cell-surface ligands.
[0111] Preferred recombinant vectors useful for gene therapy are
viral vectors, e.g. adenovirus, herpes virus, vaccinia, or, more
preferably, an RNA virus such as a retrovirus. Even more
preferably, the retroviral vector is a derivative of a murine or
avian retrovirus. Examples of such retroviral vectors which can be
used in the present invention are: Moloney murine leukemia virus
(MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary
tumor virus (MuMTV) and Rous sarcoma virus (RSV). Most preferably,
a non-human primate retroviral vector is employed, such as the
gibbon ape leukemia virus (GaLV), providing a broader host range
compared to murine vectors. Since recombinant retroviruses are
defective, assistance is required in order to produce infectious
particles. Such assistance can be provided, e.g., by using helper
cell lines that contain plasmids encoding all of the structural
genes of the retrovirus under the control of regulatory sequences
within the LTR. Suitable helper cell lines are well known to those
skilled in the art. Said vectors can additionally contain a gene
encoding a selectable marker so that the transduced cells can be
identified. Moreover, the retroviral vectors can be modified in
such a way that they become target specific. This can be achieved,
e.g., by inserting a polynucleotide encoding a sugar, a glycolipid,
or a protein, preferably an antibody. Those skilled in the art know
additional methods for generating target specific vectors. Further
suitable vectors and methods for in vitro- or in vivo-gene therapy
are described in the literature and are known to the persons
skilled in the art; see, e.g., WO 94/29469 or WO 97/00957.
[0112] In order to achieve expression only in the tar-get organ,
e.g., a tumor to be treated, the nucleic acid molecules encoding a
Futrin 1, 2, 3 and/or 4 polypeptide can be linked to a tissue
specific promoter and used for gene therapy. Such promoters are
well known to those skilled in the art (see e.g. Zimmermann et al.,
(1994) Neuron 12, 11-24; Vidal et al.; (1990) EMBO J. 9, 833-840;
Mayford et al., (1995), Cell 81 891-904; Pinkert et al., (1987)
Genes & Dev. 1, 268-76).
[0113] The present invention also relates to the use of the above
compounds of the invention for the preparation of a pharmaceutical
composition for treatment of a disease associated with (a) aberrant
expression of Futrin 1, 2, 3 and/or 4 and/or genes involved into
the Wnt signal cascade, and/or (b) aberrant activities or amounts
of a Futrin 1, 2, 3 and/or 4 and/or a polypeptide involved into the
Wnt signal cascade. In a preferred embodiment, said disease is a
kidney, bone or muscle disease or tumor, preferably breast cancer,
a colon carcinoma or a melanoma.
[0114] Finally, the present invention relates to the use of a
nucleotide molecule encoding a polypeptide having a biological
activity of Futrin 1, 2, 3 and/or 4, a Futrin 1, 2, 3 and/or 4
polypeptide, an activator/agonist of a Futrin 1, 2, 3 and/or 4
polypeptide or binding partner of said polypeptide (s) for the
preparation of a pharmaceutical composition for inhibiting the Wnt
signal cascade which might be useful for supporting regenerative
processes in a patient, e.g. growth of tissue like muscle, bone,
hair, etc.
[0115] The following examples illustrate the invention.
Example 1
Materials and Methods
(A) Isolation of Futrins (=R-spondins) and Constructs
[0116] A Xenopus adult eye cDNA library in pCS2+ was used to
prepare pools of about 250 colonies. Plasmid DNA from each pool was
transiently transfected into 293T cells together with the Wnt
receptor frizzled8, the Wnt reporter TOPFLASH (Korinek et al.,
Science 275 (1997), 1784-7) and pRL-TK (Promega) using FuGENE6
(Roche) transfection reagent. Luciferase assay was carried out 24
hours after transfection. A positive clone was isolated from the
pool by sib selection. Human Rspo2 and 3 cDNA5 were obtained from
RZPD. Fragments of hRspo1 and 4 were RT-PCR amplified from mRNA of
293T cells and used as hybridization probes. Full length mouse
Rspo1 and 2 were isolated from a mouse embryonic day 13.5 cDNA
library. The sequence of X. tropicalis Rspo3 was obtained from
Sanger Institute database and a cDNA fragment was cloned by RT-PCR
from X. tropicalis embryos. C-terminally Myc or FLAGtagged
constructs and all deletion constructs were created by PCR. Xenopus
Rspo2.DELTA.C was cloned by deleting the last 37 amino acids. The
Rspo3 cDNA5 were cloned in pCS2+ and Bluescript vectors for use in
gene expression and as probes, respectively.
(B) Cell Culture, Recombinant Proteins and Luciferase Reporter
Assays
[0117] HEK293T, SHEP and HeLa cell lines were maintained in DMEM,
10% FCS and 10% CO.sub.2. Xenopus Rspo2.DELTA.C conditioned medium
was produced by transient transfection in 293T cells. Mouse Wnt3a
conditioned medium was produced from mouse L cells stably
transfected with Wnt3a (ATCC#CRL-2647) (Shibamoto et al., Genes
Cells 3 (1998), 659-670). Luciferase reporter assays in 293T cells
were carried out in 96-well plates as described (Wu et al., Curr.
Biol. 10 (2000), 1611-1614). Luciferase reporter assays in HeLa
cells were carried out in 24 well plates in triplicates using
Lipofectamine Plus transfection reagent (Invitrogen). Per well a
total of 400 ng DNA were transfected, including 80 ng 7lef-fos-Luc
(Novak et al., PNAS 95 (1998), 4374-4379), 10 ng pRL-TK, 10 ng
mouse frizzled8, 2 ng mouse lef1 and 300 ng pSuper plasmid DNAs. 3
days after transfection, either mouse Wnt3a conditioned medium or
medium containing 30 mM LICl was added to stimulate Wnt signalling.
24 hours later, luciferase activity was determined using the Dual
luciferase system (Promega).
(C) Embryos, Explants, In Situ Hybridization and RNA Synthesis
[0118] In vitro fertilisation, embryo culture, staging,
microinjection and culture of Xenopus embryo explants were carried
out as described (Gawantka et al., EMBO J. 14 (1995), 6268-79).
Double- and single-labeling whole-mount in situ hybridization was
carried out according to (Bradley et al., Development 122 (1996),
2739-50). A PCR fragment of tropicalis Rspo3 cDNA was used for in
situ hybridization on Xenopus laevis embryos. For vibratome
sectioning, embryos were placed in embedding medium (0.4% gelatine,
30% albumin, 20% sucrose in PBS) and mounted in the presence of 2%
glutaraldehyde. Sectioning was carried out using a VT100E vibratome
(Leica). Brains of 4day Xenopus embryos were excised in 1.times.
Barth solution and fixed for in situ hybridisation. Whole-mount in
situ hybridisation of mouse embryos was performed according to
previously described procedures (Koop et al., Mech. Dev. 59 (1996),
73-78). Preparation of mRNA for Xenopus injections was carried out
using the MegaScript in vitro transcription kit (Ambion), according
to the manufacturer's instructions.
(D) Morpholino Antisense Oligonucleotides and siRNA Constructs
[0119] The 5' nucleotide sequence of an additional (pseudo-) allele
for Xenopus Rspo2 gene was obtained using 5' RACE (GeneRacer kit,
Invitrogen). Based on these sequences, an antisense morpholino
oligonucleotide targeting both pseudoalleles around the ATG start
codon was designed (Rspo2Mo): GCCGTCCAAATGCAGTTTCAAC (SEQ ID NO:1).
pSuper constructs producing siRNA against human Rspo 2, 3 or a
non-sense control were made according to Brummelkamp et al.,
Science 296 (2002), 550-3. The sequences are: human Rspo2,
TCCCATTTGCAAGGGTTGT (SEQ ID NO:2); human Rspo3, AGCTGACTGTGATACCTGT
(SEQ ID NO:3); nonsense control, ACTACCGTTGTTATAGGTG (SEQ ID
NO:4).
(E) Immunohistochemistry, Western Blot and Dot Blot Analysis
[0120] Immunohistochemistry to detect .beta.-catenin in SHEP cells
was carried out according to (Scheiffele et al., J. Cell. Biol. 140
(1998), 795-806) using anti-.beta.-catenin antibody (Transduction
laboratories, Newington). For detection of tagged Rspo proteins or
loading controls on Western blot, anti-Myc (clone 9E10), anti-FLAG
(M2, SIGMA) monoclonal antibodies, chick anti-GFP (Chemicon,
Hampshire) and mouse anti-a-tubulin (SIGMA) antibodies were used.
Chemiluminescence detection (SuperSignal.RTM. solution, Pierce) was
carried out according to the manufacturer's instructions after
incubation of blots with anti-mouse IgG-HRP (Pierce). For Rspo
expression analysis in tumour samples the Cancer Profiling Array II
(Clontech, Palo Alto) was used and hybridization was carried out
according the manufacturer's instructions.
(F) RT-PCR
[0121] RT-PCR assays were carried out as described (Dosch et al.,
Development 124 (1997), 2325-34; Glinka et al., Nature 389 (1997),
517-519); additional primers were: Xenopus Rspo2 (forward,
GAATGCCCAGAAGGATTTGC (SEQ ID NO:5); reverse, GGGATGGTGTCTTTTGCTGG
(SEQ ID NO:6)); Xenopus Rspo3 (forward, GAAGCAAATTGGAGTCTGTCG (SEQ
ID NO:7); reverse, GATTGTTCTCAAACCCTTCAGG (SEQ ID NO:8)); human
Rspo1 (forward, ACAGACACAAGACACACACGC (SEQ ID NO:9); reverse,
TGTCTTCTGGTGGCCTCAG (SEQ ID NO:10)); human Rspo2 (forward,
CCGAGCCCCAGATATGAAC (SEQ ID NO:11); reverse, TGACCAACTTCACATCCTTCC
(SEQ ID NO:12)); human Rspo3 (forward, AGGGACTGAAACACGGGTC (SEQ ID
NO:13); reverse, TGTCTTCTGGTGGCCTCAG (SEQ ID NO:14)); human Rspo4
(forward, AAGCTGGGACACAGCACAG (SEQ ID NO:15); reverse,
GAAGCCTTGGAGCCTTGTC (SEQ ID NO:16)).
Example 2
Isolation of a cDNA Encoding Xenopus Futrin 1
[0122] A Xenopus adult eye cDNA library in the expression vector
pCS2+ was used to prepare pools of about 250 colonies, and plasmid
DNA from each pool was transiently transfected into 293T cells
together with the Wnt receptor frizzled8, the Wnt reporter
TOP-FLASH (Korinek et al. Science 275 (1997) 1784-1787) and
Renilla-luciferase for normalization, in 96-well plates using
FuGENE 6 (Roche, Basel). After 24 hours relative luciferase
activity was determined. One pool yielding a signal above
background was identified (FIGS. 1 and 2A) and a gene harboring
this activity was isolated from the pool by sib selection.
Sequencing analysis showed it represents Xenopus futrin 1. Database
searches revealed four closely related genes in human, called
hfutrin 1, 2, 3 and 4 (FIGS. 3A-D and 4 or 6A).
[0123] Xenopus futrin 1 (Rspo2) is predicted to encode a secreted
protein with 243 amino acids (mature protein) and an isoelectric
point of 9.8. All R-spondins contain an N-terminal signal peptide
(SP), two furin-like domains (FU), one thrombospondin type1 domain
(TSP1) and a C-terminal low complexity region enriched with
positively charged amino acids (C) (FIG. 2F). The furin-like
cysteine rich domain is found in e.g. furin-like endoproteases, EGF
receptor and insulin receptor. In signal transduction by certain
receptor tyrosine kinases furin repeats are required for receptor
aggregation. TSP1 repeats are found in Thrombospondin and other
extracellular matrix proteins like Mindin, F-spondin, SCO-spondin
as well as in a number of proteins involved in the complement
cascade. Proteins containing TSP1 repeats are involved in cell-cell
interaction, inhibition of angiogenesis and apoptosis (Adams and
Tucker, Dev. Dyn. 218 (2000), 280-99).
[0124] While Xenopus Rspo2 contains a predicted N-terminal signal
peptide, secreted protein is almost undetectable in the medium of
transiently transfected 293T cells. Since the C-terminus is
enriched with basic amino acids, which promotes cell surface
retention a C-terminally truncated protein was tested.
Rspo2.DELTA.C is effectively secreted into the medium from 293T
cells (FIG. 2B) and is functionally active (FIG. 2F). Since all
R-spondins share the basic C-terminus and since there are no
obvious ER- or Golgi retention signals, this suggests that the
proteins are normally associated with the cell surface.
Example 3
Futrins Promote Wnt Signalling
[0125] Xenopus Futrin1 and human Futrin1, 2, and 3 are able to
stimulate Wnt-responsive reporter expression in HEK 293T cells when
provided by transient transfection (FIGS. 2C and 5A). In addition,
they are able to enhance reporter expression induced by Wnt (mouse
Wnt1/3A) synergistically (FIGS. 2D and 5B). Cotransfection
experiments in HEK239T cells were carried out with the indicated
genes and the Wnt reporter TOP-FLASH (Korinek et al. Science 275
(1997) 1784-1787) and Renilla-luciferase for normalization, in
96-well plates using FuGENE 6 (Roche). After 24 hours relative
luciferase activity was determined.
[0126] All tested members of the Rspo family (e.g. murine Rspo1-3,
human Rspo2, 3) show equivalent effects (FIG. 6A and data not
shown). Rspo2 signalling is sensitive to the Wnt/.beta.-catenin
pathway inhibitors dominant negative TCF and dickkopf1 (FIG. 6B). A
synergistic signalling effect is observed when Rspo2 is
cotransfected with extracellular but not with intracellular
components of the Wnt/.beta.-catenin pathway (FIG. 6C). The
greatest cooperation is reproducibly seen between Rspo2 and Wnts,
either using conditioned media (FIG. 2D) or following
co-transfection (FIG. 6C).
[0127] A hallmark of Wnt/.beta.-catenin signalling activation is
the cytosolic accumulation of .beta.-catenin due to its
stabilisation. Treatment of 293T cells with Wnt3a conditioned
medium induces cytosolic .beta.-catenin after 1 hour and while
recombinant Rspo2.DELTA.C alone is not able to stabilize
.beta.-catenin during this interval, it strongly enhances activity
of Wnt3a to do so (FIG. 2E, top). After 4 hours treatment, both
Wnt3a and Rspo2.DELTA.C conditioned media are able to induce
.beta.-catenin accumulation to similar levels (FIG. 2E, top).
.beta.-catenin is known to enter the nuclei in response to Wnt
stimulation and activate gene expression together with Lef/Tcf
transcription factors. Treatment of SHEP cells by either Wnt3a or
Rspo2.DELTA.C conditioned media weakly induces .beta.-catenin
nuclear localization, while co-treatment with Wnt3a and
Rspo2.DELTA.C strongly enhances nucleae accumulation (FIG. 2E,
bottom). It can be concluded that R-spondins represent a novel
family of secreted proteins capable promoting Wnt/.beta.-catenin
signalling.
[0128] To functionally study its signalling domains, serial
deletions of Xenopus Rspo2 (FIG. 2F) were created. As discussed,
the basic C-terminus can be removed without loss of activity and
this is also true for the TSP1 domain (FIG. 2F). However, the
furin-like domains are necessary for Wnt/.beta.-catenin signalling
since deletion of either the furin-1 or -2 domains abolishes the
activity in reporter assays (FIG. 2F).
Example 4
Futrins are Required for Full Wnt Signalling
[0129] To test the requirement of Futrins in Wnt signalling, siRNA
mediated gene knock-out was utilized (Brummelkamp et al., Science.
2002, 296 (5567): 550-3). Hela cells were transfected using
Lipofectamine Plus with 80 ng Wnt reporter 7LEF-Rev-fosLuc, 10 ng
pRL-TK (Promega) and 300 ng pSuper constructs (Brummelkamp et al.)
that produce either siRNA against human Futrin1 and 2, or a
nonsense control. 7LEF-Rev-fosLuc reporter construct containing
seven LEF binding sites in front of minimal fos promoter followed
by firefly luciferase ORF was kindly provided by R. Grosschedl
(Howard Hughes Medical Institute). pSuper constructs contain
19-nucleotide sequences from human Futrin1 (sequence:
TCCCATTTGCAAGGGTTGT (SEQ ID NO:17)), human Futrin1 (sequence:
AGCTGACTGTGATACCTGT (SEQ ID NO:18)) or control nonsense sequence
(ACTACCGTTGTTATAGGTG (SEQ ID NO:19)).
[0130] One day after transfection medium was changed from 10% to
0.5% FCS. Three days after transfection, mouse Wnt3A conditioned
medium or control medium from 293 cells was added to the culture to
stimulate Wnt signalling. 24 hours later, luciferase activity was
determined. As shown in FIG. 7, Hela cells show reduced levels of
Futrin 1 and 2, and Wnt signalling dropped by 50%, indicating that
Futrins are required for full Wnt signalling. This effect can be
efficiently rescued by 5 ng recombinant mouse Futrin 1, attesting
its specificity. Data are normalized to Renilla luciferase
activity.
Example 5
Expression of R-Spondin Genes in Xenopus and Mouse Embryos
[0131] In Xenopus embryos, no maternal Rspo2 RNA is detected by
RT-PCR. Its zygotic expression starts at early gastrula stage and
remains constant throughout neurulation and organogenesis (FIG.
8A). By whole-mount in situ hybridization, weak expression of Rspo2
is observed throughout the ectoderm of early gastrulae (not shown).
During gastrulation strong expression is detected in the marginal
zone in both deep and superficial layers, but is excluded from the
Spemann organizer (FIG. 8B). At late gastrula stage Rspo2
expression persists in lateral plate mesoderm and becomes
detectable in trie anterior neural plate (FIG. 8C). At stage 15
expression is seen in two longitudinal stripes along the neural
plate (future roof plate), in the anterior neural plate and in
lateral and posterior mesoderm (FIG. 8D). Expression of Rspo2 at
tailbud stage (FIG. 8F) is restricted to several regions of the
brain, including diencephalon and midbrain-hindbrain boundary,
pronephros and dorsal neural tube. Expression is also detected in
the dorsal and ventral-most portions of somites, the dorsal fin and
the proctodeum. Rspo2 expression in the brain of late tadpoles is
mainly restricted to diencephalon, including the zona limitans
intrathalamica (zli) (FIG. 8H).
[0132] Xenopus Rspo3 expression is related to that of Rspo2. It is
first detected at gastrula stage (not shown) and in neurulae it is
expressed in the anterior border of the neural plate and posterior
mesoderm (FIG. 8E). At tail bud stage, it is coexpressed with Rspo2
in the central nervous system but shows additional expression in
branchial arches and the tailbud (FIG. 8G).
[0133] In mouse, Rspo3 transcripts are detected by in situ
hybridization at day 7.5 in the primitive streak (FIG. 8J) while
expression of Rspo1 and 2 is not detectable. At day 9.5, Rspo1-3
show differential expression in various neural and mesodermal
derivatives (FIG. 8K-M), mainly along dorsal neural tube (Rspo1 and
3), diencephalon (Rspo1, 2, 3), somites (Rspo3) and tailbud
mesoderm (Rspo3). In limb buds all three genes show prominent
differential expression (FIG. 8I), particularly, in the
morphogenetically active region such as the apical ectodermal ridge
(AER) (Rspo2).
Example 6
R-Spondins are Co-Expressed with and Regulated by Wnts
[0134] R-spondins not only show functional interaction with Wnt
signalling, but also co-expression with Wnt genes in many regions
during Xenopus and mouse embryonic development. In gastrula
mesoderm of both Xenopus and mouse, Rspo2 and -3 are co-expressed
with XWnt8 and mWnt3, respectively. Similarly, at later stages,
R-spondin family members are widely co-expressed with a number of
Wnt genes e.g. in midbrain-hindbrain boundary, dorsal neural tube
and limb bud and tail bud. A direct comparison between the
expression patterns of Xenopus Rspo2 and Wnt8 and Wnt3a shows a
large overlap (FIG. 9A).
[0135] Indeed, Wnts are able to induce Rspo expression since
Xenopus embryos injected with pCS-Wnt8 or pCS-.beta.-catenin DNA
upregulate both Rspo2 and Rspo3 by RT-PCR (FIG. 9B).
[0136] Likewise, embryos injected with pCS-Wnt8 or pCS-Wnt3a DNA
show ectopic Rspo2 expression by in situ hybridization (FIG. 8C-E).
The results indicate that the observed co-expression is due to
regulation of R-spondins by Wnts. This is consistent with the
observation that Rspo1 expression is reduced in dorsal spinal cord
of Wnt1 or Wnt3a knockout mouse embryos.
Example 7
Futrin 1 (R-Spondin 2) Activates Neural Markers and Regulates
(Promotes) Muscle Formation
[0137] Loss-of-function analysis in Xenopus embryos show that
Futrin 1 is required for muscle formation. Injections of antisence
morpholino oligo against futrin 1 (FutI-Mo) cause downregulation of
early muscle markers MyoD anf Myf5 and induce muscle defects (FIG.
10A). The specificity of this effect was documented, first, by the
ability of Fut1-Mo to inhibit translation of the cognate DNA
construct when overexpressed in embryos (FIG. 10B) and, second, by
the ability of XFut1mRNA to rescue the effect of Fut1-Mo (FIG.
10C).
[0138] When the Wnt/.beta.-catenin pathway is overactivated in
Xenopus embryos a variety of responses are observed: i) mRNA
injection of pathway activators typically induces secondary
embryonic axes in whole embryos and anterior neural markers in
animal caps and whole embryos; ii) DNA injection of pathway
activators driving expression after MBT, posteriorizes the central
nervous system (CNS). To test if Rspo2 is able to mimic any of
these effects synthetic mRNA was microinjected in Xenopus embryos.
However, Rspo2 mRNA injection does not induce secondary axes in
whole embryos and injection of pCS-Rspo2 DNA does not posteriorize
CNS, since heads are normal sized, Otx2 expression is expanded and
en2 unaffected (FIG. 11D, H).
[0139] In animal caps Rspo2 induces the pan-neural markers NCAM and
N-tubulin and the anterior neural marker, Otx2, as do Xwnt.beta.
and .beta.-catenin (FIG. 11A). Similarly, in whole embryos injected
with Rspo2 mRNA, ectopic cement glands and lateral expansion of the
neural plate are observed at the injected side, as shown by
expression of neural markers Sox3, Otx2, not2, en2 and Rx1 (FIGS.
11B-H). Ectopic Otx2 expression is observed already in gastrula
stage embryos (FIGS. 11I+J). These results are consistent with the
ability of Wnt/.beta.-catenin signalling to block BMP4 expression
and thereby to activate neural development.
[0140] To further analyze the mRNA overexpression effect of Rspo2
an BMP4, Activin, Nodal and FGF signaling, animal cap assays were
carried out and tested for the induction of target genes by three
growth factor signals (FIG. 12). Rspo2 expectedly blocks BMP4
mediated Xvent2 expression but surprisingly also inhibits Activin
and Nodal mediated Xbra induction (FIG. 12A-C). This is unlike
XWnt8 and .beta.-catenin mRNA injections, which do not affect Xbra
induction by Activin (FIG. 12B). In contrast to signaling by three
TGF-.beta. type growth factors, FGF8 induced Xbra expression is
unaffected by overexpressed Rspo2 (FIG. 12D). It can be concluded
that Rspo2 overexpression in addition to activating the
Wnt/.beta.-catenin pathway, is also able to interfere with
signaling by three TGF-.beta. family growth factors.
[0141] Another well known effect of zygotic Wnt/.beta.-catenin
signalling is its ability to promote myogenesis. For example, XWnt8
can induce muscle formation in ventral mesodermal cells. When
ventral marginal zones (VMZs) from Rspo2 injected embryos are
dissected and cultured until stage 40, they elongate, form tail
like structures, and are contractile. This phenotype is
indistinguishable from control lateral marginal zone explants
(LMZ5), which typically differentiate muscle (FIG. 4K-L). In situ
hybridization confirmed induction of muscle actin in both Wnt8 and
Rspo2 injected VMZs but not in control (preprolactin-injected) VMZs
(FIG. 11M). Since R-spondins are able to enhance Wnt signalling in
293T cells, their cooperation in Xenopus myogenesis was tested.
Myf5 is a myogenic marker characteristically expressed in lateral
mesoderm. However, it is excluded from dorsal mesoderm, and dorsal
marginal zone (DMZ) explants express only little myf5 as determined
by RT-PCR (FIG. 11N). In DMZ5 from embryos injected with DNA
constructs driving post MBT expression, myf5 is weakly induced by
Rspo2, moderately by Wnt8 and strongly by their combination (FIG.
11N, top). The myogenesis promoting effect of Rspo2 is repressed by
dominant negative dishevelled (Xddl), dkkl and GSK-3.beta., which
all block Wnt signalling (FIG. 11N, bottom). In summary, the
results suggest that Rspo2 can promote myogenesis via the
Wnt/.beta.-catenin pathway.
Example 8
R-Spondin2 is Required for Wnt/.beta.-Catenin-Mediated
Myogenesis
[0142] To investigate the physiological role of Rspo2 during
Xenopus embryogenesis, antisense morpholino oligonucleotides were
injected (Rspo2Mo). The ability of Rspo2Mo to block Rspo2 protein
production is demonstrated by Western blot (FIG. 13A). Injection of
this Morpholino into one dorso-animal blastomere at eight cell
stage results in eye defects, although the expression of early eye-
(Rx1, Pax6), anterior neural (Otx2) and pan-neural markers (Sox3)
is not obviously affected (not shown).
[0143] Equatorial Injection of Rspo2Mo in one blastomere at eight
cell stage leads to muscle defects at the injected side (FIG. 13B).
In situ hybridisation for muscle actin and transverse trunk
sections show that Rspo2Mo injection causes disorganized somites
(panels a-b) and reduced myotomes (panels c-d). Lineage tracing
experiments showed that Rspo2Mo injected cells contribute to
lateral plate mesoderm instead of somites or undergo cell death
(data not shown). At gastrula stage expression of the myogenic
markers myf5 and myoD is strongly down-regulated at the Rspo2Mo
injected side (panels e-h), while the pan-mesodermal marker Xhra
and the organizer marker Xnot2 are unaffected (panels i-i).
[0144] To test the specificity of Rspo2Mo, rescue experiments were
performed by co-injecting Rspo2Mo together with a Rspo2 RNA, in
which six non-coding nucleotides were mutated so that it would not
be targeted by this Morpholino. Expression of myf5 (FIG. 13C) and
myoD (data not shown) is effectively rescued by this mutant Rspo2
RNA, indicating that the effect of Rspo2Mo an myogenesis is
specific.
[0145] Rspo2Mo was used as a tool to examine the epistatic position
of Rspo in the Wnt/.beta.-catenin pathway. As read out for
Wnt/.beta.-catenin signalling the expression of myf5 and muscle
actin in marginal zone explants was used (FIG. 13D). In DMZ and VMZ
explants, Wnt8 DNA induces myf5 and muscle actin and this induction
is significantly blocked by Rspo2Mo (FIG. 13D, panels a-b, lanes
3-4). However, myf5 and muscle actin induction by intracellular
activators of the Wnt pathway like dishevelled, dnGSK-3.beta. and
.beta.-catenin is unaffected by Rspo2Mo (a-b, lanes 5-10). In LMZ
explants, Rspo2Mo Injection down-regulates endogenous myf5 and
muscle actin expression (FIG. 13D, panels c-d, lanes 1-2). In DNA
coinjections this effect is rescued effectively by
.beta.-catenin--but only poorly by XWnt8 (lanes 3-6). The residual
effect of XWnt8 is likely due to its non-cell autonomous action an
cells that did not receive Rspo2Mo. Taken together these results
indicate that Rspo2 affects the Wnt/.beta.-catenin pathway at the
level or upstream of dishevelled.
Example 9
R-Spondins are Required for Wnt/.beta.-Catenin Signalling in HeLa
Cells
[0146] Next the requirement of Rspo2 for Wnt signalling was tested
in mammalian cells using siRNA. Since there are four R-spondins
with apparently redundant function, HeLa cells were selected, that
only express Rspo3 and very weakly Rspo2 (FIG. 14A). Various other
cell lines, such as 293T cells, express all four genes,
complicating a siRNA approach.
[0147] siRNA mediated gene knock-down was carried out by
transfecting pSUPER constructs (Brummelkamp et al., Science 296
(2002), 3286-3305) to produce siRNAs targeted against Rspo2 and -3
(siRNA Rspo 2, 3). As control, a non-sense siRNA was used. To test
the efficiency of siRNA, FLAGtagged human Rspo3 was co-transfected
with siRNAs and its production was repressed by siRNA Rspo3 but not
siRNA Rspo2 (FIG. 14B).
[0148] In Wnt-reporter assays, both siRNA Rspo2 and -3 decreased
Wnt3a-induced luciferase activity compared to control siRNA (FIG.
14C). When siRNA Rspo2 and -3 are co-transfected, reporter activity
drops to 40%. This effect is specific since the decreased Wnt
signalling can be rescued by co-transfected mouse Rspo2, which is
not targeted by siRNAs against human R-spondins (FIG. 14C).
Furthermore, unlike Wnt3a-induced reporter activity, siRNA Rspo2+3
do not affect Li+-induced Wnt/.beta.-catenin signalling (FIG. 14D).
Since Li+ acts by inhibiting GSK3.beta. activity, this is again
consistent with Rspo2 acting at the level or upstream of
dishevelled. conclude that in HeLa cells R-spondins are required
for full Wnt/.beta.-catenin signalling.
Example 10
Futrin Expression is Deregulated in Human Tumors
[0149] Misregulation of Wnt/.beta.-catenin signalling is implicated
in tumorigenesis, e.g., colon cancer, breast cancer and melanoma
(Barker et al., 2000; Bienz and Clevers, 2000; Polakis, 2000).
Since R-spondins (futrins) promote Wnt/.beta.-catenin signalling
they may also play a role in tumorigenesis. Thus, the expression of
Futrin 1-4 in various normal and cancerous human tissues was
studied using radioactive hybridisation on Clontech Cancer
Profiling Array II. Hybridization with ubiquitin probe was used for
normalisation. The results show that expression of Futrins 1-3 is
dramatically deregulated in cancerous human tissues (FIG. 15). In
most of the tumors the expression of Futrins 1-3 is dramatically
decreased (colon, stomach, lung, rectum tumors for Futrin 1,
breast, ovary, bladder, uterus, cervix, rectum tumors for Futrin 2,
uterus and cervix tumors for Futrin 3). In a few cases the
expression of Futrin 1-3 is upregulated (one case of stomach tumor
for Futrin 1 and 2, ovary tumor for Futrin 3). Futrin 4 shows very
low level of expression in most of the tissues studied, except
ovary. Further results are shown in FIG. 16 also showing that
HRspo1 is weakly expressed in adult organs. It shows upregulation
in ovary tumours from two patients and in one stomach tumour
sample. HRspo2 is expressed in organs of endodermal origin,
including colon, rectum, small intestine and lung. Its expression
decreased in corresponding tumour samples. HRspo3 is expressed
widely and decreases in many tumour samples. In general, the
expression of hRspo1-3 is deregulated in a number of tumours, while
hRspo4 expression is very weak both in human adult organs and
tumours (data not shown).
Sequence CWU 1
1
32122DNAArtificial SequenceSynthetic construct 1gccgtccaaa
tgcagtttca ac 22219DNAArtificial SequenceSynthetic Construct
2tcccatttgc aagggttgt 19319DNAArtificial SequenceSynthetic
Construct 3agctgactgt gatacctgt 19419DNAArtificial
SequenceSynthetic Construct 4actaccgttg ttataggtg
19520DNAArtificial SequenceSynthetic construct 5gaatgcccag
aaggatttgc 20620DNAArtificial SequenceSynthetic Construct
6gggatggtgt cttttgctgg 20721DNAArtificial SequenceSynthetic
Construct 7gaagcaaatt ggagtctgtc g 21822DNAArtificial
SequenceSynthetic Construct 8gattgttctc aaacccttca gg
22921DNAArtificial SequenceSynthetic Construct 9acagacacaa
gacacacacg c 211019DNAArtificial SequenceSynthetic Construct
10tgtcttctgg tggcctcag 191119DNAArtificial SequenceSynthetic
Construct 11ccgagcccca gatatgaac 191221DNAArtificial
SequenceSynthetic Construct 12tgaccaactt cacatccttc c
211319DNAArtificial SequenceSynthetic Construct 13agggactgaa
acacgggtc 191419DNAArtificial SequenceSynthetic construct
14tgtcttctgg tggcctcag 191519DNAArtificialSynthetic Construct
15aagctgggac acagcacag 191619DNAArtificial SequenceSynthetic
Construct 16gaagccttgg agccttgtc 191719DNAArtificialSynthetic
Construct 17tcccatttgc aagggttgt 191819DNAArtificial
SequenceSynthetic Construct 18agctgactgt gatacctgt
191919DNAArtificial SequenceSynthetic construct 19actaccgttg
ttataggtg 1920843DNAHomo sapiens 20atgcggcttg ggctgtgtgt ggtggccctg
gttctgagct ggacgcacct caccatcagc 60agccggggga tcaaggggaa aaggcagagg
cggatcagtg ccgaggggag ccaggcctgt 120gccaaaggct gtgagctctg
ctctgaagtc aacggctgcc tcaagtgctc acccaagctg 180ttcatcctgc
tggagaggaa cgacatccgc caggtgggcg tctgcttgcc gtcctgccca
240cctggatact tcgacgcccg caaccccgac atgaacaagt gcatcaattc
ctctgcagta 300cctgcagctc taggccaggg tcctgccctc catgtagaat
gcaagatcga gcactgtgag 360gcctgcttca gccataactt ctgcaccaag
tgtaaggagg gcttgtacct gcacaagggc 420cgctgctatc cagcttgtcc
cgagggctcc tcagctgcca atggcaccat ggagtgcagt 480agtcctgcgc
aatgtgaaat gagcgagtgg tctccgtggg ggccctgctc caagaagcag
540cagctctgtg gtttccggag gggctccgag gagcggacac gcagggtgct
acatgcccct 600gtgggggacc atgctgcctg ctctgacacc aaggagaccc
ggaggtgcac agtgaggaga 660gtgccgtgtc ctgaggggca gaagaggagg
aagggaggcc agggccggcg ggagaatgcc 720aacaggaacc tggccaggaa
ggagagcaag gaggcgggtg ctggctctcg aagacgcaag 780gggcagcaac
agcagcagca gcaagggaca gtggggccac tcacatctgc agggcctgcc 840tag
84321732DNAHomo sapiens 21atgcagtttc gccttttctc ctttgccctc
atcattctga actgcatgga ttacagccac 60tgccaaggca accgatggag acgcagtaag
cgagctagtt atgtatcaaa tcccatttgc 120aagggttgtt tgtcttgttc
aaaggacaat gggtgtagcc gatgtcaaca gaagttgttc 180ttcttccttc
gaagagaagg gatgcgccag tatggagagt gcctgcattc ctgcccatcc
240gggtactatg gacaccgagc cccagatatg aacagatgtg caagatgcag
aatagaaaac 300tgtgattctt gctttagcaa agacttttgt accaagtgca
aagtaggctt ttatttgcat 360agaggccgtt gctttgatga atgtccagat
ggttttgcac cattagaaga aaccatggaa 420tgtgtggaag gatgtgaagt
tggtcattgg agcgaatggg gaacttgtag cagaaataat 480cgcacatgtg
gatttaaatg gggtctggaa accagaacac ggcaaattgt taaaaagcca
540gtgaaagaca caataccgtg tccaaccatt gctgaatcca ggagatgcaa
gatgacaatg 600aggcattgtc caggagggaa gagaacacca aaggcgaagg
agaagaggaa caagaaaaag 660aaaaggaagc tgatagaaag ggcccaggag
caacacagcg tcttcctagc tacagacaga 720gctaaccaat aa 73222819DNAHomo
sapiens 22atgcacttgc gactgatttc ttggcttttt atcattttga actttatgga
atacatcggc 60agccaaaacg cctcccgggg aaggcgccag cgaagaatgc atcctaacgt
tagtcaaggc 120tgccaaggag gctgtgcaac atgctcagat tacaatggat
gtttgtcatg taagcccaga 180ctattttttg ctctggaaag aattggcatg
aagcagattg gagtatgtct ctcttcatgt 240ccaagtggat attatggaac
tcgatatcca gatataaata agtgtacaaa atgcaaagct 300gactgtgata
cctgtttcaa caaaaatttc tgcacaaaat gtaaaagtgg attttactta
360caccttggaa agtgccttga caattgccca gaagggttgg aagccaacaa
ccatactatg 420gagtgtgtca gtattgtgca ctgtgaggtc agtgaatgga
atccttggag tccatgcacg 480aagaagggaa aaacatgtgg cttcaaaaga
gggactgaaa cacgggtccg agaaataata 540cagcatcctt cagcaaaggg
taacctgtgt cccccaacaa atgagacaag aaagtgtaca 600gtgcaaagga
agaagtgtca gaagggagaa cgaggaaaaa aaggaaggga gaggaaaaga
660aaaaaaccta ataaaggaga aagtaaagaa gcaatacctg acagcaaaag
tctggaatcc 720agcaaagaaa tcccagagca acgagaaaac aaacagcagc
agaagaagcg aaaagtccaa 780gataaacaga aatcggtatc agtcagcact gtacactag
81923672DNAHomo sapiens 23atgcgggcgc cactctgcct gctcctgctc
gtcgcccacg ccgtggacat gctcgccctg 60aaccgaagga agaagcaagt gggcactggc
ctggggggca actgcacagg ctgtatcatc 120tgctcagagg agaacggctg
ttccacctgc cagcagaggc tcttcctgtt catccgccgg 180gaaggcatcc
gccagtacgg caagtgcctg cacgactgtc cccctgggta cttcggcatc
240cgcggccagg aggtcaacag gtgcaaaaaa tgtggggcca cttgtgagag
ctgcttcagc 300caggacttct gcatccggtg caagaggcag ttttacttgt
acaaggggaa gtgtctgccc 360acctgcccgc cgggcacttt ggcccaccag
aacacacggg agtgccaggg ggagtgtgaa 420ctgggtccct ggggcggctg
gagcccctgc acacacaatg gaaagacctg cggctcggct 480tggggcctgg
agagccgggt acgagaggct ggccgggctg ggcatgagga ggcagccacc
540tgccaggtgc tttctgagtc aaggaaatgt cccatccaga ggccctgccc
aggagagagg 600agccccggcc agaagaaggg caggaaggac cggcgcccac
gcaaggacag gaagctggac 660cgcaggctgg ac 67224732DNAHomo sapiens
24atgcagtttc aactcttttc attcgccctg atcatcctga actgtgtgga ttacagtcac
60tgccaagcct cccgctggag acggagcaag agagccagct atgggaccaa cccgatatgc
120aaaggttgcc tgtcctgctc aaaagataat gggtgcctcc gctgccagcc
aaaactgttt 180ttctttctgc gaagagaagg tatgaggcag tatggagagt
gtctgcagtc ctgccctccg 240ggatactatg gagtcagagg acctgatatg
aacaggtgtt ccagatgcag aattgaaaat 300tgcgactctt gttttagtag
agatttttgc ataaagtgca aatcgggctt ttactccctc 360aaggggcaat
gctttgaaga atgcccagaa ggatttgcac cactggatga taccatggtg
420tgtgtggatg gctgcgaagt agggccatgg agtgaatggg gcacatgcag
ccgaaataac 480agaacgtgcg gtttcaaatg gggcctggag accagaacgc
gacaaattgt gaagaaacca 540gcaaaagaca ccatcccctg cccaactatt
gctgaatcca gaagatgtaa gatggcaata 600agacactgcc ctggaggaaa
gagaactaca aagaagaagg acaagaggaa caagaagaag 660aaaaagaagt
tactggagag ggcccaagag cagcacagcg tcgtccttgc tacagaccgg
720tctagccaat ag 73225262PRTHomo sapiens 25Met Arg Leu Gly Leu Cys
Val Val Ala Leu Val Leu Ser Trp Thr His 1 5 10 15 Leu Thr Ile Ser
Ser Arg Gly Ile Lys Gly Lys Arg Gln Arg Arg Ile 20 25 30 Ser Ala
Glu Gly Ser Gln Ala Cys Ala Lys Gly Cys Glu Leu Cys Ser 35 40 45
Glu Val Asn Gly Cys Leu Lys Cys Ser Pro Lys Leu Phe Ile Leu Leu 50
55 60 Glu Arg Asn Asp Ile Arg Gln Val Gly Val Cys Leu Pro Ser Cys
Pro 65 70 75 80 Pro Gly Tyr Phe Asp Ala Arg Asn Pro Asp Met Asn Lys
Cys Ile Cys 85 90 95 Lys Ile Glu His Cys Glu Ala Cys Phe Ser His
Asn Phe Cys Thr Lys 100 105 110 Cys Lys Glu Gly Leu Tyr Leu His Lys
Gly Arg Cys Tyr Pro Ala Cys 115 120 125 Pro Glu Gly Ser Ser Ala Ala
Asn Gly Thr Met Glu Cys Ser Ser Pro 130 135 140 Ala Gln Cys Glu Met
Ser Glu Trp Ser Pro Trp Gly Pro Cys Ser Lys 145 150 155 160 Lys Gln
Gln Leu Cys Gly Phe Arg Arg Gly Ser Glu Glu Arg Thr Arg 165 170 175
Arg Val Leu His Ala Pro Val Gly Asp His Ala Ala Cys Ser Asp Thr 180
185 190 Lys Glu Thr Arg Arg Cys Thr Val Arg Arg Val Pro Cys Pro Glu
Gly 195 200 205 Gln Lys Arg Arg Lys Gly Gly Gln Gly Arg Arg Glu Asn
Ala Asn Arg 210 215 220 Asn Leu Ala Arg Lys Glu Ser Lys Glu Ala Gly
Ala Gly Ser Arg Arg 225 230 235 240 Arg Lys Gly Gln Gln Gln Gln Gln
Gln Gln Gly Thr Val Gly Pro Leu 245 250 255 Thr Ser Ala Gly Pro Ala
260 26243PRTHomo sapiens 26Met Gln Phe Arg Leu Phe Ser Phe Ala Leu
Ile Ile Leu Asn Cys Met 1 5 10 15 Asp Tyr Ser His Cys Gln Gly Asn
Arg Trp Arg Arg Ser Lys Arg Ala 20 25 30 Ser Tyr Val Ser Asn Pro
Ile Cys Lys Gly Cys Leu Ser Cys Ser Lys 35 40 45 Asp Asn Gly Cys
Ser Arg Cys Gln Gln Lys Leu Phe Phe Phe Leu Arg 50 55 60 Arg Glu
Gly Met Arg Gln Tyr Gly Glu Cys Leu His Ser Cys Pro Ser 65 70 75 80
Gly Tyr Tyr Gly His Arg Ala Pro Asp Met Asn Arg Cys Ala Arg Cys 85
90 95 Arg Ile Glu Asn Cys Asp Ser Cys Phe Ser Lys Asp Phe Cys Thr
Lys 100 105 110 Cys Lys Val Gly Phe Tyr Leu His Arg Gly Arg Ser Phe
Asp Glu Cys 115 120 125 Pro Asp Gly Phe Ala Pro Leu Glu Glu Thr Met
Glu Cys Val Glu Gly 130 135 140 Cys Glu Val Gly His Trp Ser Glu Trp
Gly Thr Cys Ser Arg Asn Asn 145 150 155 160 Arg Thr Cys Gly Phe Lys
Trp Gly Leu Glu Thr Arg Thr Arg Gln Ile 165 170 175 Val Lys Lys Pro
Val Lys Asp Thr Ile Pro Cys Pro Thr Ile Ala Glu 180 185 190 Ser Arg
Arg Cys Lys Met Thr Met Arg His Cys Pro Gly Gly Lys Arg 195 200 205
Thr Pro Lys Ala Lys Glu Lys Arg Asn Lys Lys Lys Lys Arg Lys Leu 210
215 220 Ile Glu Arg Ala Gln Glu Gly His Ser Val Phe Leu Ala Thr Asp
Arg 225 230 235 240 Ala Asn Gln 27272PRTHomo sapiens 27Met His Leu
Arg Leu Ile Ser Trp Leu Phe Ile Ile Leu Asn Phe Met 1 5 10 15 Glu
Tyr Ile Gly Ser Gln Asn Ala Ser Arg Gly Arg Arg Gln Arg Arg 20 25
30 Met His Pro Asn Val Ser Gln Gly Cys Gln Gly Gly Cys Ala Thr Cys
35 40 45 Ser Asp Tyr Asn Gly Cys Leu Ser Cys Lys Pro Arg Leu Phe
Phe Ala 50 55 60 Leu Glu Arg Ile Gly Met Lys Gln Ile Gly Val Cys
Leu Ser Ser Cys 65 70 75 80 Pro Ser Gly Tyr Tyr Gly Thr Arg Tyr Pro
Asp Ile Asn Lys Cys Thr 85 90 95 Lys Cys Lys Ala Asp Cys Asp Thr
Cys Phe Asn Lys Asn Phe Cys Thr 100 105 110 Lys Cys Lys Ser Gly Phe
Tyr Leu His Leu Gly Lys Cys Leu Asp Asn 115 120 125 Cys Pro Glu Gly
Leu Glu Ala Asn Asn His Thr Met Glu Cys Val Ser 130 135 140 Ile Val
His Cys Glu Val Ser Glu Trp Asn Pro Trp Ser Pro Cys Thr 145 150 155
160 Lys Lys Gly Lys Thr Cys Gly Phe Lys Arg Gly Thr Glu Thr Arg Val
165 170 175 Arg Glu Ile Ile Gln His Pro Ser Ala Lys Gly Asn Leu Cys
Pro Pro 180 185 190 Thr Asn Glu Thr Arg Lys Cys Thr Val Gln Arg Lys
Lys Cys Gln Lys 195 200 205 Gly Glu Arg Gly Lys Lys Gly Arg Glu Arg
Lys Arg Lys Lys Pro Asn 210 215 220 Lys Gly Glu Ser Lys Glu Ala Ile
Pro Asp Ser Lys Ser Leu Glu Ser 225 230 235 240 Ser Lys Glu Ile Pro
Glu Gln Arg Glu Asn Lys Gln Gln Gln Lys Lys 245 250 255 Arg Lys Val
Gln Asp Lys Gln Lys Ser Val Ser Val Ser Thr Val His 260 265 270
28224PRTHomo sapiens 28Met Arg Ala Pro Leu Cys Leu Leu Leu Leu Val
Ala His Ala Val Asp 1 5 10 15 Met Leu Ala Leu Asn Arg Arg Lys Lys
Gln Val Gly Thr Gly Leu Gly 20 25 30 Gly Asn Cys Thr Gly Cys Ile
Ile Cys Ser Glu Glu Asn Gly Cys Ser 35 40 45 Thr Cys Gln Gln Arg
Leu Phe Leu Phe Ile Arg Arg Glu Gly Ile Arg 50 55 60 Gln Tyr Gly
Lys Cys Leu His Asp Cys Pro Pro Gly Tyr Phe Gly Ile 65 70 75 80 Arg
Gly Gln Glu Val Asn Arg Cys Lys Lys Cys Gly Ala Thr Cys Glu 85 90
95 Ser Cys Phe Ser Gln Asp Phe Cys Ile Arg Cys Lys Arg Gln Phe Tyr
100 105 110 Leu Tyr Lys Gly Lys Cys Leu Pro Thr Cys Pro Pro Gly Thr
Leu Ala 115 120 125 His Gln Asn Thr Arg Glu Cys Gln Gly Glu Cys Glu
Leu Gly Pro Trp 130 135 140 Gly Gly Trp Ser Pro Cys Thr His Asn Gly
Lys Thr Cys Gly Ser Ala 145 150 155 160 Trp Gly Leu Glu Ser Arg Val
Arg Glu Ala Gly Arg Ala Gly His Glu 165 170 175 Glu Ala Ala Thr Cys
Gln Val Leu Ser Glu Ser Arg Lys Cys Pro Ile 180 185 190 Gln Arg Pro
Cys Pro Gly Glu Arg Ser Pro Gly Gln Lys Lys Gly Arg 195 200 205 Lys
Asp Arg Arg Pro Arg Lys Asp Arg Lys Leu Asp Arg Arg Leu Asp 210 215
220 29262PRTHomo sapiens 29Met Arg Leu Gly Leu Cys Val Val Ala Leu
Val Leu Ser Trp Thr His 1 5 10 15 Leu Thr Ile Ser Ser Arg Gly Ile
Lys Gly Lys Arg Gln Arg Arg Ile 20 25 30 Ser Ala Glu Gly Ser Gln
Ala Cys Ala Lys Gly Cys Glu Leu Cys Ser 35 40 45 Glu Val Asn Gly
Cys Leu Lys Cys Ser Pro Lys Leu Phe Ile Leu Leu 50 55 60 Glu Arg
Asn Asp Ile Arg Gln Val Gly Val Cys Leu Pro Ser Cys Pro 65 70 75 80
Pro Gly Tyr Phe Asp Ala Arg Asn Pro Asp Met Asn Lys Cys Ile Cys 85
90 95 Lys Ile Glu His Cys Glu Ala Cys Phe Ser His Asn Phe Cys Thr
Lys 100 105 110 Cys Lys Glu Gly Leu Tyr Leu His Lys Gly Arg Cys Tyr
Pro Ala Cys 115 120 125 Pro Glu Gly Ser Ser Ala Ala Asn Gly Thr Met
Glu Cys Ser Ser Pro 130 135 140 Ala Gln Cys Glu Met Ser Glu Trp Ser
Pro Trp Gly Pro Cys Ser Lys 145 150 155 160 Lys Gln Gln Leu Cys Gly
Phe Arg Arg Gly Ser Glu Glu Arg Thr Arg 165 170 175 Arg Val Leu His
Ala Pro Val Gly Asp His Ala Ala Cys Ser Asp Thr 180 185 190 Lys Glu
Thr Arg Arg Cys Thr Val Arg Arg Val Pro Cys Pro Glu Gly 195 200 205
Gln Lys Arg Arg Lys Gly Gly Gln Gly Arg Arg Glu Asn Ala Asn Arg 210
215 220 Asn Leu Ala Arg Lys Glu Ser Lys Glu Ala Gly Ala Gly Ser Arg
Arg 225 230 235 240 Arg Lys Gly Gln Gln Gln Gln Gln Gln Gln Gly Thr
Val Gly Pro Leu 245 250 255 Thr Ser Ala Gly Pro Ala 260
30243PRTHomo sapiens 30Met Gln Phe Arg Leu Phe Ser Phe Ala Leu Ile
Ile Leu Asn Cys Met 1 5 10 15 Asp Tyr Ser His Cys Gln Gly Asn Arg
Trp Arg Arg Ser Lys Arg Ala 20 25 30 Ser Tyr Val Ser Asn Pro Ile
Cys Lys Gly Cys Leu Ser Cys Ser Lys 35 40 45 Asp Asn Gly Cys Ser
Arg Cys Gln Gln Lys Leu Phe Phe Phe Leu Arg 50 55 60 Arg Glu Gly
Met Arg Gln Tyr Gly Glu Cys Leu His Ser Cys Pro Ser 65 70 75 80 Gly
Tyr Tyr Gly His Arg Ala Pro Asp Met Asn Arg Cys Ala Arg Cys 85 90
95 Arg Ile Glu Asn Cys Asp Ser Cys
Phe Ser Lys Asp Phe Cys Thr Lys 100 105 110 Cys Lys Val Gly Phe Tyr
Leu His Arg Gly Arg Ser Phe Asp Glu Cys 115 120 125 Pro Asp Gly Phe
Ala Pro Leu Glu Glu Thr Met Glu Cys Val Glu Gly 130 135 140 Cys Glu
Val Gly His Trp Ser Glu Trp Gly Thr Cys Ser Arg Asn Asn 145 150 155
160 Arg Thr Cys Gly Phe Lys Trp Gly Leu Glu Thr Arg Thr Arg Gln Ile
165 170 175 Val Lys Lys Pro Val Lys Asp Thr Ile Pro Cys Pro Thr Ile
Ala Glu 180 185 190 Ser Arg Arg Cys Lys Met Thr Met Arg His Cys Pro
Gly Gly Lys Arg 195 200 205 Thr Pro Lys Ala Lys Glu Lys Arg Asn Lys
Lys Lys Lys Arg Lys Leu 210 215 220 Ile Glu Arg Ala Gln Glu Gly His
Ser Val Phe Leu Ala Thr Asp Arg 225 230 235 240 Ala Asn Gln
31272PRTHomo sapiens 31Met His Leu Arg Leu Ile Ser Trp Leu Phe Ile
Ile Leu Asn Phe Met 1 5 10 15 Glu Tyr Ile Gly Ser Gln Asn Ala Ser
Arg Gly Arg Arg Gln Arg Arg 20 25 30 Met His Pro Asn Val Ser Gln
Gly Cys Gln Gly Gly Cys Ala Thr Cys 35 40 45 Ser Asp Tyr Asn Gly
Cys Leu Ser Cys Lys Pro Arg Leu Phe Phe Ala 50 55 60 Leu Glu Arg
Ile Gly Met Lys Gln Ile Gly Val Cys Leu Ser Ser Cys 65 70 75 80 Pro
Ser Gly Tyr Tyr Gly Thr Arg Tyr Pro Asp Ile Asn Lys Cys Thr 85 90
95 Lys Cys Lys Ala Asp Cys Asp Thr Cys Phe Asn Lys Asn Phe Cys Thr
100 105 110 Lys Cys Lys Ser Gly Phe Tyr Leu His Leu Gly Lys Cys Leu
Asp Asn 115 120 125 Cys Pro Glu Gly Leu Glu Ala Asn Asn His Thr Met
Glu Cys Val Ser 130 135 140 Ile Val His Cys Glu Val Ser Glu Trp Asn
Pro Trp Ser Pro Cys Thr 145 150 155 160 Lys Lys Gly Lys Thr Cys Gly
Phe Lys Arg Gly Thr Glu Thr Arg Val 165 170 175 Arg Glu Ile Ile Gln
His Pro Ser Ala Lys Gly Asn Leu Cys Pro Pro 180 185 190 Thr Asn Glu
Thr Arg Lys Cys Thr Val Gln Arg Lys Lys Cys Gln Lys 195 200 205 Gly
Glu Arg Gly Lys Lys Gly Arg Glu Arg Lys Arg Lys Lys Pro Asn 210 215
220 Lys Gly Glu Ser Lys Glu Ala Ile Pro Asp Ser Lys Ser Leu Glu Ser
225 230 235 240 Ser Lys Glu Ile Pro Glu Gln Arg Glu Asn Lys Gln Gln
Gln Lys Lys 245 250 255 Arg Lys Val Gln Asp Lys Gln Lys Ser Val Ser
Val Ser Thr Val His 260 265 270 32224PRTHomo sapiens 32Met Arg Ala
Pro Leu Cys Leu Leu Leu Leu Val Ala His Ala Val Asp 1 5 10 15 Met
Leu Ala Leu Asn Arg Arg Lys Lys Gln Val Gly Thr Gly Leu Gly 20 25
30 Gly Asn Cys Thr Gly Cys Ile Ile Cys Ser Glu Glu Asn Gly Cys Ser
35 40 45 Thr Cys Gln Gln Arg Leu Phe Leu Phe Ile Arg Arg Glu Gly
Ile Arg 50 55 60 Gln Tyr Gly Lys Cys Leu His Asp Cys Pro Pro Gly
Tyr Phe Gly Ile 65 70 75 80 Arg Gly Gln Glu Val Asn Arg Cys Lys Lys
Cys Gly Ala Thr Cys Glu 85 90 95 Ser Cys Phe Ser Gln Asp Phe Cys
Ile Arg Cys Lys Arg Gln Phe Tyr 100 105 110 Leu Tyr Lys Gly Lys Cys
Leu Pro Thr Cys Pro Pro Gly Thr Leu Ala 115 120 125 His Gln Asn Thr
Arg Glu Cys Gln Gly Glu Cys Glu Leu Gly Pro Trp 130 135 140 Gly Gly
Trp Ser Pro Cys Thr His Asn Gly Lys Thr Cys Gly Ser Ala 145 150 155
160 Trp Gly Leu Glu Ser Arg Val Arg Glu Ala Gly Arg Ala Gly His Glu
165 170 175 Glu Ala Ala Thr Cys Gln Val Leu Ser Glu Ser Arg Lys Cys
Pro Ile 180 185 190 Gln Arg Pro Cys Pro Gly Glu Arg Ser Pro Gly Gln
Lys Lys Gly Arg 195 200 205 Lys Asp Arg Arg Pro Arg Lys Asp Arg Lys
Leu Asp Arg Arg Leu Asp 210 215 220
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